CA2437250A1 - Microencapsulation of living cells in covalently crosslinked alginate-poly-l-lysine-alginate membranes - Google Patents
Microencapsulation of living cells in covalently crosslinked alginate-poly-l-lysine-alginate membranes Download PDFInfo
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- CA2437250A1 CA2437250A1 CA 2437250 CA2437250A CA2437250A1 CA 2437250 A1 CA2437250 A1 CA 2437250A1 CA 2437250 CA2437250 CA 2437250 CA 2437250 A CA2437250 A CA 2437250A CA 2437250 A1 CA2437250 A1 CA 2437250A1
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- alginate
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5005—Wall or coating material
- A61K9/5021—Organic macromolecular compounds
- A61K9/5036—Polysaccharides, e.g. gums, alginate; Cyclodextrin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/20—Polysaccharides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3895—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/43—Hormones, e.g. dexamethasone
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
- A61L2300/62—Encapsulated active agents, e.g. emulsified droplets
- A61L2300/622—Microcapsules
Abstract
Immunoisolation of cells in semipermeable membranes has been proposed as a means to prevent their immune destruction following transplantation[1, 2].
However, several fundamental issues remain to be addressed before considering clinical application of this method. One such issue is the resistance of microcapsules to chemical and mechanical degradation. Strong microcapsules will obviously increase the durability of the transplant. It is also likely to improve long-term biocompatibility of microcapsules, since a strong pericapsular reaction always develops around broken or damaged capsules[3, 4]. Moreover, the strength and durability of microcapsule membrane are critical issues for the transplantation of virus-transfected bioengineered cells. These virus-transfected bioengineered cells potentially include insulin-producing cells as well as cells producing molecules, which promote islet cell survival/replication/transdifferentiation or insure islet and microcapsule immunoprotection. Finally, considering the limited supply of human islets, the most promising approach to .beta.-cell replacement is the transdifferentiation of stem cells into islets[5] (not necessarily embryonic stem cells, which arise controversy, but stem cells of many other origins, including autologous cells).
Note that even autologous cells would require immunoprotection against the recurrence of the autoimmune process. The major concern related to this approach is the risk of malignant transformation of immature stem cells. Microcapsules that can hardly be destroyed in conditions compatible with life would provide a safe method of transplanting stem cell derived cells, bioengineered cells or immortalized cells.
Complexation between alginate and polycations such as poly-L-lysine(PLL) is the most widely used method to microencapsulate cells[2]. Microcapsules are constructed by a simple three steps procedure. First, the calls are entrapped in a bead formed by the ionic cross-linking of alginate by a divalent ration such as calcium[6].
The second step comprises coating the alginate beads with a polycation such as PLL, which forms a peripheral membrane ensuring a good control of the molecular weight cut-off and increasing membrane stability. Finally, the third step consists to coat the microcapsules with a dilute alginate solution for insuring biocompatibility.
The two latter reactions rely on the ionic interactions between the polyanionic alginate and the polycationic polymer of lysine[7, 8].
In order to improve microcapsule strength, we[9] and others[7, 8, 10, 11], have evaluated the effect on microcapsule strength of modulating intrinsic parameters PLL
molecular weight, concentration and incubation time and the mannuronic acid/guluronic acid ratio of alginate. The formation of neutral capsules by the introduction of a new coating agent[12] has also been investigated. Following these experiments, tighter binding between PLL and alginate was obtained. Nevertheless, poly-L-lysine still competes with other positively charged molecules in the environment to bind to the alginate beads. A prolonged incubation in solutions with high concentrations of Ca2+ or Sr2+ has showed a displacement of the alginate-poly-L-lysine bounds[13, 14].
In addition, Thu et al. have observed the progressive loss of the external sheet of alginate from microcapsules within days or weeks[7].
To prevent this competition with charged molecules in the environment, a new concept has been proposed: the introduction of covalent links into the membrane of the microcapsule[15-17]. Covalent links are known to be more stable than electrostatic interactions. The challenge is that most methods to create or to break a covalent link are incompatible with the survival of living cells. The objective of the present work was to develop and validate a method, compatible with encapsulated cell survival, to create a covalent link between the PLL layer and both alginate from the microcapsule core and from the outer sheet. It would be unlikely that such a microcapsules would be destroyed in conditions found in the living body.
Here we show that it is possible to microencapsulate living cells in membranes composed of covalently linked alginate-PLL-alginate membranes, without affecting cell viability. The formation of a covalent link between PLL and alginate from both the alginate core and the outer alginate shoes considerably increased microcapsule resistance to chemical and mechanical stress. Standard APA microcapsules dissolved within seconds when they were incubated in an alkaline solution whereas covalently linked APA
microcapsules remained unaltered after two years in the same alkaline solution Covalently linked microcapsules were 22 times more resistant than standard microcapsules when they were submitted to a mechanical stress. The process is based on the chemical derivation of poly-L-lysine (PLL) with a photoactivatable heterobifunctional cross-linker, N-5-Azido-2-nitrobenzoyloxysuccinimide (ANB-NOS), which is able to covalently bind PLL to alginate when energized by UVA light illumination[18, 19]. The N-hydroxysuccinimid of the cross-linked was fist covalently linked to PLL in conditions that would damage living cells, but before the colts are involved in the procedure. Then islet cells encapsulated in calcium alginate microcapsules were incubated with the arose-linker derived PLL, than in alginate again to form the outer biocompatible sheet. When the preparation is illuminated with a UVA
lamp the photoactivatable residue reacts with the phenyl azide residue on alginate creating a covalent link between PLL and alginate. The latter reaction is not harmful for living cells. All reactions involving the crosslinker-derived PLL were performed in a dark room, until UVA illumination, to prevent activation of the photoactivatable residue.
We, herein, present a proof that a covalent link was formed providing considerable improvement of microcapsule resistance to chemical and mechanical degradation.
The results of the study showed that the procedure did not modify microcapsule membrane permeability and did not affect in vitro and in viva encapsulated cell survival.
However, several fundamental issues remain to be addressed before considering clinical application of this method. One such issue is the resistance of microcapsules to chemical and mechanical degradation. Strong microcapsules will obviously increase the durability of the transplant. It is also likely to improve long-term biocompatibility of microcapsules, since a strong pericapsular reaction always develops around broken or damaged capsules[3, 4]. Moreover, the strength and durability of microcapsule membrane are critical issues for the transplantation of virus-transfected bioengineered cells. These virus-transfected bioengineered cells potentially include insulin-producing cells as well as cells producing molecules, which promote islet cell survival/replication/transdifferentiation or insure islet and microcapsule immunoprotection. Finally, considering the limited supply of human islets, the most promising approach to .beta.-cell replacement is the transdifferentiation of stem cells into islets[5] (not necessarily embryonic stem cells, which arise controversy, but stem cells of many other origins, including autologous cells).
Note that even autologous cells would require immunoprotection against the recurrence of the autoimmune process. The major concern related to this approach is the risk of malignant transformation of immature stem cells. Microcapsules that can hardly be destroyed in conditions compatible with life would provide a safe method of transplanting stem cell derived cells, bioengineered cells or immortalized cells.
Complexation between alginate and polycations such as poly-L-lysine(PLL) is the most widely used method to microencapsulate cells[2]. Microcapsules are constructed by a simple three steps procedure. First, the calls are entrapped in a bead formed by the ionic cross-linking of alginate by a divalent ration such as calcium[6].
The second step comprises coating the alginate beads with a polycation such as PLL, which forms a peripheral membrane ensuring a good control of the molecular weight cut-off and increasing membrane stability. Finally, the third step consists to coat the microcapsules with a dilute alginate solution for insuring biocompatibility.
The two latter reactions rely on the ionic interactions between the polyanionic alginate and the polycationic polymer of lysine[7, 8].
In order to improve microcapsule strength, we[9] and others[7, 8, 10, 11], have evaluated the effect on microcapsule strength of modulating intrinsic parameters PLL
molecular weight, concentration and incubation time and the mannuronic acid/guluronic acid ratio of alginate. The formation of neutral capsules by the introduction of a new coating agent[12] has also been investigated. Following these experiments, tighter binding between PLL and alginate was obtained. Nevertheless, poly-L-lysine still competes with other positively charged molecules in the environment to bind to the alginate beads. A prolonged incubation in solutions with high concentrations of Ca2+ or Sr2+ has showed a displacement of the alginate-poly-L-lysine bounds[13, 14].
In addition, Thu et al. have observed the progressive loss of the external sheet of alginate from microcapsules within days or weeks[7].
To prevent this competition with charged molecules in the environment, a new concept has been proposed: the introduction of covalent links into the membrane of the microcapsule[15-17]. Covalent links are known to be more stable than electrostatic interactions. The challenge is that most methods to create or to break a covalent link are incompatible with the survival of living cells. The objective of the present work was to develop and validate a method, compatible with encapsulated cell survival, to create a covalent link between the PLL layer and both alginate from the microcapsule core and from the outer sheet. It would be unlikely that such a microcapsules would be destroyed in conditions found in the living body.
Here we show that it is possible to microencapsulate living cells in membranes composed of covalently linked alginate-PLL-alginate membranes, without affecting cell viability. The formation of a covalent link between PLL and alginate from both the alginate core and the outer alginate shoes considerably increased microcapsule resistance to chemical and mechanical stress. Standard APA microcapsules dissolved within seconds when they were incubated in an alkaline solution whereas covalently linked APA
microcapsules remained unaltered after two years in the same alkaline solution Covalently linked microcapsules were 22 times more resistant than standard microcapsules when they were submitted to a mechanical stress. The process is based on the chemical derivation of poly-L-lysine (PLL) with a photoactivatable heterobifunctional cross-linker, N-5-Azido-2-nitrobenzoyloxysuccinimide (ANB-NOS), which is able to covalently bind PLL to alginate when energized by UVA light illumination[18, 19]. The N-hydroxysuccinimid of the cross-linked was fist covalently linked to PLL in conditions that would damage living cells, but before the colts are involved in the procedure. Then islet cells encapsulated in calcium alginate microcapsules were incubated with the arose-linker derived PLL, than in alginate again to form the outer biocompatible sheet. When the preparation is illuminated with a UVA
lamp the photoactivatable residue reacts with the phenyl azide residue on alginate creating a covalent link between PLL and alginate. The latter reaction is not harmful for living cells. All reactions involving the crosslinker-derived PLL were performed in a dark room, until UVA illumination, to prevent activation of the photoactivatable residue.
We, herein, present a proof that a covalent link was formed providing considerable improvement of microcapsule resistance to chemical and mechanical degradation.
The results of the study showed that the procedure did not modify microcapsule membrane permeability and did not affect in vitro and in viva encapsulated cell survival.
Description
DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTE PARTIE I)E CETTE DEMANDE OU CE BREVETS
COMPRI~:ND PLUS D'UN TOME.
CECI EST ~.E TOME 1 DE 2 NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadien des Brevets.
JUMBO APPLICATIONS / PATENTS
THIS SECTION OF THE APPLICATION / PATENT CONTAINS MORE
THAN ONE VOLUME.
NOTE: For additional vohxmes please contact the Canadian Patent Oi~ice.
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a _ ., ~~~._ ____-._____ ~trlscaz-~n.a~fe ite,ns recei~,Ted ~,%i~.il this ap~Iica~ion (Request original docun2ents ill File prep. Section on the I~'' door) Locuments recu avuc cette aeniande ne pouva:~t ~tre 'r;alay°es (Commander les documents or!wgi~:~au~~ duns Ia section de prv°paratzon des dossiers aLI
~ hem a ~.tage,~
~_~e uSe o~rrlICrOencapSL:~ated cells or enZ~'lheS 3n blOreaC.i:OeS
ra,preS'ntS a Set ~f potential industrial applications for extremely resistalnt r~icrocapsuies.
~i'hese include microencapsuiation of hybridoma dells. Gvi~icl~ col~sf;~erablyl enhances tire production of monoclonal antibodies. of lactic acid bacteria vor the ferr~entatio=.~ c~f dairy products, a~~d of bacteria or yeast for the industrial production of a large number cf drugs by ~I~I~
recombinant technologies. zvVhe~~ the desired ;rod~acis or reactions are obtained, cell co~:tainirig microcapsules are easily separated from -0:~.ze solution. 1-lc;~~avever, the strong shearing forces encountered in bi"reactcrs br::ak the microcapsu~es, releasing: r~icro-erganisrns or cells, this contamination decreases vliP purity of tlm prodr.acts and may jeopardise the safety, efficacy and LisefSalness of the method ~, efforts have been made to in~~rove rnicrocapsule streng~:l~. ~ orr~ple~cation between tile negs:.tlVely .~'-~"larged poi5'anror'i u.l'~,JIIIate ~iT~ poSl~:',.Vel'V
~el2ar~?~:(~, ~~JL'(.~at?43~tS SL1C7 'ilS
pol'v-L-=ysine ~~I,L~j to form alginate-po?yW -t~~sir~e-~~lgir~ate (~.~b~'~~
rrricrocapsules is, fi3e most u-r_dely used rr~ethod to rnidroencapsuiate cells ~:2, %, and ~j. C;rhers ['?, F~, i ~, and l ij, and wTe [9j hare evaluated tie effect can :,;Zicroc°apsule strer~gtia of modulating 1~~.~~, molecular weight, concentration and incubation tune and the rF°~annL~rov~ic acid/guluronic acid ratio of alginate. New coating agents gave also been investigated X121.
~~~evertheless, with these methods, alginate and ~L,~, still c~mpetes with other 'charged molecules in the envirorrrnent ~i ~, 1 =j and ahe resistance i.ar~~rovent is iimitec~..
~7e~e developed a rr~ethod to covalently link FI~L with al gnates z~orn both the alginate core and the outer sheet of rnicroc;apsules ~pendir~g patent;. The challenge was that most methods cased to create gas well as to lest=voY> covalent links are incompatible ~;!~L:;roeifr.a~oSLtlatlol2 oI C'a:ii; :i St~;i"~I:~Je~'~~neat?LoI'!'leaT'~bral~.d:S ~'?s~.S ~C%eC;Ii 131."opoS:vCi ~
~., ~'; to prevent/ t'_~eir iz~~mune ciestr3~cti,~n ~ol~ew~,-~ng tra:sspOantatioru eiimi-.~atins~ the r~.:add f'or tdsir~~g topic imrr~~nostippressive ~rtys. 's hi; a~~api~oae,~ could be sued fod' replacing i:;set:; eo Y
lJar~geri~args (islets}, whicb contain lrssusan~~rodt~c;iay; cells, for she treat~ncnt of diabetes9 and for delivering other natural or s~~nthetic tl~erape~z~ic rr:olecta~es Ao4~
tl~e treatmea~t o~
i'~t~nlc%'~tlS d3SC~3Ss,S. ~~11e rT'L2~or lS5?.ie IS ~:1"~lc:'Cia.%;k:g;St3lC
reSIStanCC 't;d~ ~a'1_~.,'ilCal ell'ld rnecnan~val c~eg~adataon. :~l~crcc~~:,~~,e:es ~na~ ~:a~~ ~<ar~~ly ~w destrc~~~~ec.~ ::~ co~~c;~~zons corrspatible ~r'itl~ life ~v'ould proir~s~~ she d~az~~tic~n of ef-fictive trc~rc ~entc. I'~ost:
inporta~~tl~-, the~;~ ~~r>uid inipro~-e ehe sa~~eaq,~ o traas~ aarltin~T ;-ir3~~_t.'ans'ected bioeng3r~es~red cells, immortalized t;e;lls or stern cell ~~erived cell s.
'I~~is ~nel:hod ~ot~ld dccrCaSe _t~rle r~Sl~ O~ ~%lral dlSSC'rrll.t cai::;«n ~~i16~ 1~'i~egratW'~l~
lt"ato the Y'~~Ci~'.C.'~'~~:'S ~ei2OZTle 1i1 Elle ~rn~.er and o~ malignant cell trans:fo ~n~.tiosE an d clis';s~,mination ir;.
tree t~,ree cascs. '~~~e~~
would ailo~a,~ safer gene therapy.
with Ii\ring eeli szlr~.-i~-~1. ~t~Tsin<~'-~~-rw:~i~io-«-rzixro-heflzoyioxy'stacr~il~7c-nis:~e (~'~r~l~-I~I~:~~~ a p~'~utor~z.~',tiv'~t~'E3I~,' hetel'L?~ii'tllCt1()axui i~?'~sSIII's.ast '~~~
ti, .~3~, VJe vx''6~Yd', s~IJ'iE"' tCr 2IIe~.').~S1'sI%:~~e is?ets iI-I covaIeIItlv linked A~'.=~ rrbE,~I~ral~es, v.-i~h<~ut aI=feeting isl~w°t cell via'oii~t~'. Tr; thc~
present ar~:ic:ie, ~ISing a I~zodel of isle: ;::~Iicroezlcr~psulxition, ~~~e d~:s~°zv~e r?le method alld its ezFect on rnieroeapsLlle eharacterisa~ s end I'~Inctions.
~hl~e ~1-h ~'dro:~ys~;~ceil'IiIZIid residLh ;~ ~?~~~~-1~~~;~ v~'as f'trsi ~vo~'aI~-.zlt3 J' iiuilsed to ~°L.~,, ~zsiil~ an exoti'Ier.~Iic cher~Iica? z'eae:?iorz haa: ~-o~uid d~irrla~~e ~i ink; ceps, I~zzt be~ore t~~I~~
~e!Is ale Iilv'oI~'ed IIi t~le pI'OCs::ji~T'~:. ~~eil Islets.
'I:~.I.YI~JhIilSed In ~:<sIC_:ii~ aIt?Inate C.~Y°fa, '~.Eere inea~ated with cross-Ii~n:er ~b:rived I°I.~,, then ~aI al7inate a~ail3 to io=zn Edhe o~z~~e~' I7iC76;oIlapa~Ii_9Ie sheet. lq~llieil th a j7i'~,'~;tb;fc'~tiiirt is atl~3b Fir:l_atei~ ~~Ii~h a '~~~rf~ 's.~T3a~, -~:L2C' phot:oactivata~le phen~'I aZide I'esa«e «i~ tl a~ cross-lisrke~ I°eacts rvpidl~l ~~~ithiz~ a cahool~-~,:c'~I°'oon J~ F.~ eaT'~«n-u~'CiI-~'~en'~7'v'II~;~ I~~tC? the ;7l~;nate C.~efLa.Ca~ StxliCi3.~is:, ~,Yeatlll~ r'~
co~;'ale:It link oetw'een t Liu and aI<~ir~ate i~-o~~ ~~oth t~~e, micz'ocaj~si.ue eo:~e and oLlter sheet.
~lY'<e Ic'it~er I'L,'2cC~P6?n Is ilot I2~11'iY'~IL'I ~C)r 'Iv'112,~'.~;
~v~CLis.
t h.e presence o-~cov'alent I~oz.Inds ~~~-~~s conf~°rn_e d tay zrcuhations irI ~: str::~ng alhaiine sJILItIoI'I 'p"~ i~~. ~n standitT'd 1111CE3C~~Sl~f5, LI~e IrlteraCtIOI?s ~e~~'i'eei~. the ~C3si.tl~Jel~,' ~.ix~i~e~ ~~3~~, ally the I'ie~Tll ~~el.b' Cf:~I'~ec~ cz~i~Ili~te e~~i ~3e ~~"rt'~~iel~I ~y' nei~':al3ZrII~ OY'i~; s~~ the Ch aI'bed ,~~I'otaps. ~Il ti'l~; pI"eSeYIt Case, '.iSlr1~~ G.~~ ~ilt~Iln~w ~LI~~eI"., Vii(: flrs',i~~.raaslZed the h~~_, esIdLIe ~,%~hlCh h%~s ~?i ISOeICCtI°1e ~:~~~I~1~ _ ~ ~',.~4-. ~
~ile~~iC~psLL~eS '~~'k 1e IY°W'd.II3wzEeCi In c. GL~
Iv'~.;IIiC ~itlli~eT' 2~ p~ ~ ~ Cd.~'. a ~7~i ~~L .'.gin 'zt;~1 I-~ost sfde~~yhal~2 c'3YnIIIC~- ~I'~a,~I~~ aI'e 22e>~~tl'f~kls?e~~.
standard rnicroeapsules, zlsed as coll-c>ols ~~~li~b~re I ~a~9 I~apadly dissolved and disappeared a'I the SLO~LtIoi< W'Ithirl :.~J seC. ~C'~I~LI:t"C i ~~~ and ~~j. in t~Jt?YYr.'iS29 dlllt~'o-vapS?.saes pYepa_"ed ,~~'ivh ~I'1~-->I~~ derived i IJL did :'es~st to a~ in cFalaation iz< chis ~II~°alil~e solz~tion, v~i~~h s~,-e~l_izz~ percentage of approxirnavwf% ~~% ~;~ ~z~.re 1 ~ az.d 1~~, z3.}-.d a rnieroeapsule reeo~'ery rate of '~6.5 °,% ~4.3. %af~ez- % Fears rz the al~~:.li~e ~;~.zffec. -they ~.z-e rez-r~airli~I
rztaet. with a rzorinal round shape alzd a svn~;o-tia srzrfac;e.
,.
~eYlatl~ Y)I ~~?e er oSS-1112ker S~aeer ar'~ ~. ~22Y t°sa~< ~', ldSlr~
th6: ,~f~l<c'~ llr a CY ~SS(3lLxtlY'szI test, t"~~~'o SI?'Miiar'"~31i~toaeta'atatl3e eY'aiSS-t:!:it~erS ~:~~:~re e~'a~3.i~ted: a'~.~eT°~i:'~~~ s~re'ld ~z:ifos~zecinirrzid~rl ~-(~-°-azido-2'-.~?it~opner~srYaI~iY~.o~
;~exanoate ;,'~zt~.f~-'~~l~l.~iT~. The orli~ difference between these tv~ro ~c~Ieez'l~;s s tg~e length of the c~Aai;z _joirzin~; ts'~e t~~~
reaetli.''e ~?rC3llpS. ~UI'IO-~t~~~_~i',.~-1 ';°7CiS:'i~.',SSf:S 7 ~~r'~~'I1 ',.rlld eil~~. altrisge't'i ~~'~rrlS LtrJriI2Yr°~~; a S°1~5a~er artY"a Ol' t ~.~ l~ . ~li'1Ce t' I!~ ~-'.°~7 ~~ ~laS, ilC:r Spaef.~" arrYZ. tA''9e ~!'~tStEil7.eC; bei,~~s'f~erl take tw~lf9 reaetive t'esidues is orzy 7.7 t~. ~;'vi,_e:= zrzi~:rcea~szzies Yna~ie with sv~rlfo-~~,1~'~~.-den°ved ~~z i~'ere suhazitted -to the aiiCal:z~c ci=ssc~d.~av~voYf test, ti a;' complev~=~i'~' ~i~sappeal°ed ~~~.~Y~ the solutiarz ~vit~it a few rrzirzutes I~teehaaieai stress test ~9~. Mieroea~szzies u~°re a~i~ec:i v~~itiZ 3 rrzYm diarrzeter glass beads iI°~
J Yril p3o>~StVY°e_~e, tLl~7eS, ~I'~fyd ~I1 ~. 'v";',:.°tie~~
'~~'ll~,el Y''~t's:EItIYL~?,? ai .~9J "~3T'Y? It(9I" 7~ 11i3L1rS. l~t t~'IIS
sloe' speed, tr°~e eentrifuge foree is v~~'eak, arid v~rl~Iile tize tubes turn ~.~~side dog*Y~, tl~e glass beads anez rrzieroeapsules I~o~,'e rapidv i'oI-i o~~e end <zf the tube t~>
tiZe orbs:. s,u~orrzivairz;g rYE~ieroeapsules to thozzsands of strobes arid ~;z'*~zsi~es 'oe~wee:~ beads arid the t~zb~°, wail.
b'~urirzg tl~zis test, r°rzicroeapsules, ~.oYetwiaing ~? l~.l: ~ iTt_.' l,~befed d~;~~ararze are sLzbrrzitted tc~
rrzore arid stronger meeharzieal stresses tl~zan i:~Ie;~ are lilcel'' to enec~uater duriYrg a lifetirrle irI ara. irrzpiantatiorz site suei~ as the peritoz~zeuYr~ ~.rzd irI ~
iorea~tors. 'the pereerrtage of broken rrlier~eapsuies'~~igtzre 2 !-~) was ~Z fold loaa~el' fcr r ~ieroe;~.~~szzles eross--iiahed usi~~zg ~.~-~-i'd~~ ('~.4%~ than for starzdarcl rr~ieroeaps~les ~~3.3%',.,.
fs~l eorgtreis she~,~~rve ~~'igazre 2 ~ aad ~'~ negligible arr~oz!I~ a of dextrarin the stzpernata~:~It.
~;onzlrrrziY~g that ~,.F y -/ A , x "f ~~~crc '~~as n~ de~tr~a~ leal4irg ~~t :~~c~~ un~r~k~,z~ r~~ic:;=c~caps~:les.
~~.~,~~,~~,~~~r ,~ ;x ,,..
.-~r. > , e° ~~a .,r ,. ~ - ~ ,. - , ~ ~ x ;:
tr , r 1T .fFxy.a. .:
' '!, ~ ~,: ; ., H
r ~~ ~~' ~ ~ ~ ~r ~'~t~
c~' w ~~~~~N_w,~~o. _ ~_w. ~. _,~ .~,w,_.:
_~~r_h~__ ~ .~~ a.3 ~~~.~~, v~~ -~ ~ ~~ ~ a gas do r dI_F~~r~'Tl~.eS clltrc acs ~1~11trOl~s et %'s ~~~ i~._~1 ~'=;ij p;~~IhIadJ'd'.r i ~ c'~vaYlt-~'~~;~°Yy zur~ pllraSc pa" ~~ '~1~:
str~.llg$~'1 3~the sta.rl~ard l'YlICrr,3~a~.''s:cs tl:i~~. 1~:. t~1=s C,ssay b~'cts p~"I~:eJICi~I"S~j~' v,~t1I211Zf.,'<~ 3a ~tner c:~~e:lrner~ a ~zsing ~raeta~ds ~c~~:~ era t~~ ~rrprc~ ~'e .~ ~c~-~c~ps~.~.~ ,'~sasranc~: ; ~, ~. ~, I :~, ..~s 3 ~ , t x:.,. ,s~~., ~7v r~
.,.4 '~'". ~ a,m,..,.., ~~ ~ ~,k s ~.~~. ... z, ~. , :~~'9-~," w -:_ 3 ~. ~ , ~. ::~e w,.,: ~ E~ r ,.,.-q ~ 7.... .~ ~..~,..~°~~ ~ .,r» ~ ;.
~ :f=~. ., J -x ~~ , is T l'" h :. gv .~a.~, - "? <r~ ._ .n.~P . ., ( ~, - n ...its .. ,r,."s w , ,~:.~, C"s>... «.~.:~;;,~..~t a"-'a...s - f..,2, ,s~ ~p.-s. z,. ..a.:
~'x~~,'ez. L S" s , W~; y _ ~'s.~E, _.s f .' ~' ,. ~S~ ,'~,~r !d 2 ':x~r. : J' x~, ,.~L, L 3 ,. ,~' rs ,...>°~, . F -.
~,~ a ~~: : .>e;y..,z . ~u " .... 1 rt ....x s 5° . , :;. a ~'. :'e ;,. ~ r,., I . .r. 'ø S/, .~ ~'"(, x~ ,. ~ ~, t.
"9 . , ~ ': a' . vT, ... ~~ 1 .. e. ' ~ '_ ~ i~ , , c t < " .~' ., :' . -r ~~ - e~. . t ' -.. , ~. ~ .
, ~ , t. , '.
-. r , ~.: ; ... <, "u s :' ':1t3 a~ . d ~ ~~~~~r s~~~., .~.~o.~~~.~~r~ .~~ ~ ~~~,>~. b~~r~ ~r.~ ~, ~r ~x~..6~.A~
~~,~.~.~.~~~a~,~,~~«a ~t'adics ~f rrzic~-c~caps~ie mcmi7rane, r~olecu~aa ~,rcig~t coat-off (1'~I'~pJ~~)9 ~aiz~g a prP<~i~7asiy p~ablishcd rneth~d. ~~, « , sl~~v,-c~ treat c~w~aPc,~~
cr~ss._Ii~3~~ir~g ~~~~.~~~
a~~~zbrar°p~s has ~~~ signi'icant e~~cct ~r~ r~~c_~a~ra~~ p~,rea~ii2ty.
both c~valcr°,tly cr~ss-ii~,kcd a~~d standard f~icx~capsulcs cxcl~,~d~d ~part~ti~a chr~~atc~g~'api~i~:
cc~c,ffic_~er~t (i~$~,~~
~.I~ dc~trar~s ~F~~ith a.'~t~i >_ I9 i~~~. ~'~~isc'v~sivy radi~~~s ~I~~>_ ~.~~
~~:~;
a ~~~~"~~~~~ ~~~~ ~ ~lYl.:~ ' T~~t~krlS V~Jtd'? ~' ~,~
>W5 _I~~Sa (R~ > 2.~~, v~~hcreas do ~ty~arzs r~aith a a~,~i'~%t~ < ~-.'~ k~a ~;~~~~ < I. i~ a9~d pr~ieir~s =v~~ith a i~<~J < ~.~.2 k~a ~I~r; _< ~.'7~w di~I'~s~~d i~~-~øIy ~ k~sr' =
i.t~~ txv~gh 6:~~th t~rpcs ~
a~crn'~rar~~s. .~ he apparcnt discrcpar~ci?s ~ct~~c~r~ the .~~crri sari~a ~;~
carl,~hydrat~ aad p~°ctcir~s arc daze t~ the fact that: dc~traa~s are !iz~car x~c:at~°aI r~:~~Iecx~s~~~s ~rh~,reas prctevas arc gl~hular charged rr~~lec~zlc.s. It is n~tc~~rti~~y v'~at tg~e ~! pr'c~~idc.s a he~aer relative esilrriatso%i OI rr3,oleC:.lle d~ar2leterS t~Ic2~ the '~~J':r.
Lell 41a43111ty. ~i"xe 1<c~l SteCS of the yrocess S td~e E~t.~:IC:'ata~i?
ei'rf CCn,%aiel'2t ilrll~:S lay ju''a~ ~~
il:LiiT'nTlatIOrT. ~-a 'tlv'a5~'f:Ic~gtl2 Of ~L;i-J~~~ n1T-.., sCCio~T.°rl t; lil~t.3~e I2l~rx.irY'~~1 dell dc3l'TlagC" YN~S
selected. f~0ier~t:al cell darTlage.s 'v~'er~c~Ia~L~aie~ ':is2r'ag% c C$~il~~e STa~~i ~Jia~"Jlllt~.' test at"~d rite obs~:r~ation of ~ecro~ic centres or fr°sh islets ii:nder a stes-eo~nicrcasco~c,. i~f zi:itial studies, a 2~ kJ%rn- ~~f~ dose, ~vit..2 ~~.~ ~~%it=u.o~a ~~a~~-i'~~~~, decr~::ase.d i.~let crdll via~ility (~~igurc J), whereas ~~'~'~i3-I~t(~~ aloe had ~~o direct ~;~~tovoxicity.
''~l~e cell dare°_a=4e is partially e~~lair~ed by a thermal e~i'c~::'~, since tie ~rei~aratio-r~
wa~~a~a~°d ~zv~ despite t'rac fact it ~yda.s ors ice. ~~$~ ir~vestigaie the hypotvesis t;r-xat the ha~~ful effect s dose relatL:d, ~:o~alerg~~~ly linked rnicrocapsules ~~~~~.c ~roc~zced9 ~~sing ~ large rai~~~.: of il~%~~~. doses. ~
dose of ~ k3/rrA2, 11.5 fold s~nallc~ ih~=z the =ritiaa dose (?~ :c.l/rr~?'Y :
as -e~uall,~~ effe~i~c :n ger~er=~t~ng co~'aaent links and aF=~~rob'~.~~g r~~croc~~~sute resista~!c,e. ~ :ors small dose ~~~as used (anl cor~y~red ~%ith the high dose wvherc i~die~.ted) for all t~~c e~peri~_et~ts re~or$ed is~ the present article, except for tl~e ~~wg~v~~c~ st~,~dics. The latter wvere ~~ov repeated, since e~era the high dose had rzo effect oAx Leis pararnetcr. ~~he srr°zaller dose was as effcctizm as the large dose ire improving cl~e;~3;~~i ar?d ri2et~har?:~c~:.i resistance.
~i~hc e: fact does r~c~t se,err~ progressi' a (~'igure ~; therefore, vt s~wggest5 t~:att the srr~allf:i pose ir~duees the ;same lc~-el of covalent links as the largez c.osc. ~l"gym results :~f the ~Jia~i:ty tests (hi'uie ~~ arid the e~,~als~~atior~ of necrotic centres (~i:~ure ~-j sho~-e~that tire ill~a~nination of er~ca~sulated islets With a 2 1cJ/rn~ dose of 1_;'~'~'~, ~,-al~~ or ~'iv'no~~t z%~.Nl~-~~3~, c~.~.d «ot affeci islet ::x,11 ~~iahility as compared with s~ar~d~.rd ~:~icroca~;s~les (-d ~~~ ~/~ ~ria~ili~y and l S~/t necro'ric centres f or eac~ of the 3 conditions;.
a ne cbjeeti~'e of tti~s ~,~ac:~i,: ~r,;«s ru ,we~r~~ls; ~ a ~:~iet~u.3d it~r ;~r~dus~irlg Fiery StrOrl~ rillCr~Cc'L~7SLaleS, by Crec,'itli~L"T '~'(3:'aiei2s'1?:s2~~C~1 i7et"v'v'een ITLICrC3C;'c'a~S~,.i~.E~ mf:lT'e(3rc'ine c~m~onents. The chalien~e vas Lhav: host r~le-t~ods si-iat il~d~~ce s6~c;k~ a link v:,-c~~~d se-vereiy atl~eC~ Cø~~ vla~)l~lt'EI. ~~10$~~CL1L'eitaC?iw-' CrC;SS-illrlG~.;rS
~reSel~ltt,S~ a ~K;t~:'n ~i'c,'~ SC~iL!flQn ~OeC;a ISC
the reaCEion indL:ced by Ll~ia~ ilhLiiIaarion oi'tire ~h~~I~y~ a~::de ~~l°onE~ ~~s Iesf; Iikeiv~~ to ridi_1Ce C;e~l Ciarl2'cl~?E;. r~~W~(3 ~7h~JtJi3<,iL~'~''=tc'L~lF,: ~~rCSS-IlrlY~;.rS. ~.~'t'I1C~I C 1~ ~a,;" C'n ~~J ~'~' i:2EI ~1~.'.li~~I~"1 o~ t he spacer arm included in the~I~ 5~lvzcture. ~ ~'er.u i .~~esti~ated.
~'~aI 4sA~~ropi"iate iel~~~~i :.~b tt'~C S~aC:i,r arrll redllC;CS SI r1C i?in ~e~:iCf: ~e~:'v~h.'~,Ii ~:~~If~tc'7tC ai2~ ~'~~~ ~C~iyirLezS, Vd~'tILG
c~~3O~v'1?1~ therd~'1 t'3 1~51r'zd tCJ~Ct~ICr c'~.'~ Flle~ %ime .9h~~J~,T
CrisSS-lirl~'sln~. ~',~hC :Ceiid~tS S~i05~If;Ci ti2a~C
sL;i~~-~«iJ~~~~-deri~:'~~.d ~'Li~ ~vres ;r=ef~ecti,'e ~~~~ ~~roc~ucin<,~ ri co~;al~;IaL rink ~.~'~th aigiflate ~>v~ld,reaS SL:C~1 c'1 I?I7.k LVaS II1C'iLICed ll:il:a~ .~'il~~-~''~~~.
i~~Il:S SLt~beSt~ xca ~:~le S~?a".Cer c'i"r-3~
~en~t~'i tS CrItlCa~.
x'~ie evaiu~2ion con~il-rr~ect 'h~~t s' ~i~_~S~J de:i~~cd ~r,icr;~c;a~sL~lc~s arc: ~.;'ery resistanto r~iechanicai and che:ricastresses a~W ua~.iil<_ei~~ ?o be ~~~~ecte~~ by com~aetitiorl L'wlt~l C~lsyi'~ed ril i~CCLIleS lr2 C~1C f.i'Lt,~ii~:>:~11~1Cr?!:. 3~a2t;
E7r4~'~~.k~i~.?C :~T CC)ti~z'vnt '.aII~GS i~LSLIalgy io~'.CaiTIrCS e.:~'treIY'SC. C~tlC~altlOIiS C'lt ~~I O~ Lvi2"l~Cra~:~iE' i~lt aEe znCt7r'z1~°lail~S'~E,'vY2~~~ ~~e~l ~IL.C.°.
.~2E;rei~r°, t~'leSLi rTllC$'OCd=5S'~'ie5 ~rl"C=, '..-'l:~t't~~~i~:i' In ~CStr~Yv;,~lblaC: I~1 t~lC; CGg~C'~'~I°1~riS <:11COLLYi~CrC'~a.
n L~le il'JIi'la b~dy.
~.'~~C'i~5~,° t31 e'i'=3~i0tflclCtl~Je'9~E~~'I~C~ u:C3SS-vzr"aGCr SLE;~l e'iS f°'b~~--I'~YC~~ ~;i CrC:e'3tC COV'c'~.~erl.t Ii~'alcs ~,'ithin trie lnicrc~ca~sL~~e rr~en,b~~arle is aCis~anta~~:Caus. ~'he ~~~-ocednre does not rewired l:arsii conditions such as drastic te~ler~ttlr w chari~es, .~tvong s~i ~~ar.atiol~s o. tile use of organic solvents. It does not produce toxic byproducts.
~iydroxysuc.oinimide is easily and simply removed by a dialysis step. Short wavelengths in ultraviolet radiations such as ~'~~13 and U :%~ can provoi~:~ rapid and severe damages in living cells. The classical aryl azide ~..znctionai grois~ used ie~r ~~~ for.r~ation of covai~,nt bonds is generally acti vateci vaith light possessing this hind of sh~~-t ~%va~~~I~ngtl-As (~~~-~~~ nrn). Ther~for~, a modified aryl aide groLrp ;~~as pref°~°re~. '~'~'~e ~rosen~e of the nid:ro group on the aryl aide group ailovved the use o~'sa~~~ long ivav~l~ngths ~orrespor~cii~y tc~
~J'VA and ~~mn visible iiglzt for activating the cross-iinl~in~, re~c~:ioni l ~, 1~~. The viability experiments showed r~o cytotoxic effects of the Total proced~.~r~, e:~_cept Lor mi.~brc~ncapsulatod islets Lxposed to higb doses of IlVA radiations.
ThP covalent cross-linking betv~-eer the t~~~o r~~mbrane components, alginate and ~l,L, insures a proper long-term control of the meanbrane 1~I'~'~C~. ~Iitl2 the conditions used in tl~e present studt~, the i~l~'~~) ~r-as such that =aextrarts and proteins. with I~~% >_ 1 ~ kl7a ~ I~rl % 3.4) and >_ 4~ ltl~a ~f~p > 2.9) respectively, cannot pass through the ~n~cnbra~=e. The latter uI'W corresp ands t,~ a n-~cleic ~bLid fragrnen_ off'-. ~ bases and. a peptide f~-agm~,r~-c of ~.pproxirr~ater~ ~ °, a~nin~~ aoi.d:~. It is not ko.~J~~t~ i'~ a virus derived fragment of this size could be inco~-porat~d intro a corr°~plete virus i~°~zoGting the recipi~n~
and ~~nodify its pathogenicity. T ho risk that ;~u~ix an e~a~nt o~~~~rs so~a~s minimal but cannot be completely excluded. Tlbe ~:nicrocaps~zl~ ~V~~ ~rouls~ certainly pr~;v~nt complete virus, viral proteins or virus release tc ~rosr= micro~aps~wi~
rn.ernbranes. Tlm donor cells could not be infected since the viral vectors are deprived of vhe components re~uirod for penetrating cell I)1VA and the recipient ~riruses cannot penetrate into rr~iorocapsules. T herefore, the additional safety provided by these microcapsules for the ~.~se of viral vectors is consider4zble, althoug~~ t~eoretically not a~solate.
It is n3tes~orthy that we ~.av~= pr~vaous~y found that. a~: hgh ~.:onc.e~-~trations. such as ~.0~%,1'f,l. leas a direct to~;i; efi~:rct on ideas ~:~~hedded in al~in~~e beads_ ~L~t not at sow concentratiox°a {unpublished :late).
The availability of these extrerrAely strong nzicrocaps;a.les -will h~,~
i~,seiul for many applications. In tl~e field of islet tra~~spian ration, which is our privnary=
interest, it gill allo;~~ a safer use of stem cell devived islets. a,~mortalized ins~xl;r~-pt~oclF~zcing cell lines or 'oioennineered irasuliz;-produci~~~ cells. It tviial;',~ allow a sales v~s~ov islets or eels co-encapsulated ~~ith islets, trans~'e.f_tecwith tl~e gerv~°s of molecules that pro2l'lote ales cell survival or irnmur°~oproteetio~~. for example, ~,~ve ~°ecently sho°:ved '~ ~N.~ r. z <,~~,~'~.r~~~that pre-zz~a~splant4xtion insulin E~ro«~th factor-II
{I~~-11) ~~ 11. incubations allo>>=~ prolongeu ~~orr~~alisation ot~ Jlood =~Iucose os diabetic mice, using a reduced islet cell ~r~ass {< 5~E%) transplantatiorg, ~~p~e also developed a method to transfer the l~I~-Il gene into islet cells or into nor_ islet cells that can be co-encapsulated dvith islets {pending peter=t). 'fhe goal is to provide continuous i~~ v~vo 1GF'-iI supply to islets, preventi~~g islet cell apoptosis and necrosis.
~.rev~ertl~eiess. to obtain stable gene expression into islet cells, retroviral-:used vectors are -=.equired, ~~r~icr~ present sigr°~ifrcant risl~s. Using non ~eira.l vectors, vve heave also Iran:»_ecaed Ih~-l:l into cells fro~~
an irr3~nortalized cell line. l~y seective elirnir.~ati.or3 of non transt'ecGed cells, m-~ich cannot be done with a rrrulticellular st-r~cture such as islets, a cell life with a stable l~~-11 gene expression was developed. T he use of~o~~ viral vectors erlls.c~nces the s~.frtn of the method. ~'ever'eless, immortalized cells also present a rich of ~maiigrla~it tra=~sformation and dissemil:atior~. '~'lle mieroellcapsu'ation of i.llese e.ells in eovalently linked API
mieroeapsules Mould improve the safety of tr~es~; treatments.
Tile same principle may de pplied $o numeroas applications ire tt,e ~hcle field of gene therapy. In recent years. :gene ~~~erap~,~ l;as r aised great entllusiasrrl, rein ~ perceived as a revolutionary technology ~~all tile potential to cure almost any diseases, provided tile Genetic and molecular basis is k~~o~t~rl. t ~ta~.z~e~-er, tl?e entiliasi~.srls vanished ~~;~hell it vaas realized that several obstacles =_a.Illper elir~ieai applications. It evas recen$I~,~ reported that reirovira-Based veetors, uses to permaallerltly tr~:nsi:er genes i~Ito cells, rr~av pr°f~rentially be inserted illio or near an onc~ger~~:, alld cause a, le~l~aerrli~,-iik~
disease. Otllcr types of potentially safer vectors, sue: as aaenovi~~as, adenov:$rus-assc:~ciated and ~~on viral vectors, are Being developed. Ilo~eve~~, they us~_aally provide only a rtrar~saent gerl~; expression. In addztiorl, many of the imrnunc iobicai d~;f.~Ilcc systegns., ~~~hi:;.~~
prote;ct the: host against ~al~Ci~tVpe InfeCtlC)rlS, I'ec'3.Ct il~~,I~7S~t tile veCtJr allt~Oz neW
~eI't'~; ~;Ai:)d ~CtS e~;.pre'SSs;L1 a$ tile surface of transplanted cells itc~:° recent review ~~1 vectors for w~,r~e tllera~y see rei;.# ~..
1 j. 'ref ~-1 = Thomas ~~, ~~~a~.ardt A, l~~y, _~. ~'ra~ress anti prable~-~is with the use af~ ;ral vectors for ber~e aherapy.
1'~at l~.ev ~'rerlet. 2~~3 l'~ay;4~~~:3~~-5. ~aviev.~.
ulicroelzct~.llsatat~oll of these ~io-engineered cells i~. almost inc~escructibie i~Iieroea.ps~le,s v,~oLu.d ~~fe~~
a proteetioll against i~~I~unologieal reaction and ~isserz~inaaio~ o~'I~aliir~aant cells, aiio~~ing safer g~:~Ie therapy. ~'hatever tile progress tvaliserl. Tai viral vector safet5r7 it is utllil~ely tile adsolute elimination of any risk can ~e .~-~tair~~;~.. ':~?le ad~itiorzal safety provided by microeneapsulation in cowalelltly li~~~et~. r~ieroc:~psuies vrovid er~ai~le the etllic;al use of gene therapy in mal~fy clinical applications.
~i~ 4',(3YlC~LISivI'1. (Ile iiSL. ~ A"-''~~~; d'=:1_"iT ~,C'v"v'lia~i iIZG
t3kt~~Cet.'',t3'~''l~ei7~C CTt~ss-~i?ihe~'"
=~~~-'~~~. associated w'it~l ~,;_.~' dc~s~, ~qv ~~, iiaur::,~r~at~cY&, i~ld,.~~~:-.~! a ~~v'a~~erf~ lv:~~, q~,r '{J°~$~,~'<'a',C, 1 ~ '~.1~ ~'-"~~~~~,.alld e'i g=Yiu$y. ~"~:'als aYl~:~~'16)L~: CC~i'lsltli'.T'a~~~~:~ 'a',Yl~'l.aPle'.."~JLi 11i1Cr~3C;a.'I'an'~'SLI~(:
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~_a'l~r~'y'~~J. °~~?f.',1~$~re, ~%:''~ are ~Y~ )~'11~;?, a ,~t~~:v' ,rr~~e :s)r ilOl~:,r~r~YIC:o3~lS~~nGit~::. ~i ~1~15 W;',~'ii LISe '~c~ ~~rc~tne~ trans~~arlted Bells ~ac~~lj t:~le, nc~s~ zwl~~r~r.~.
s~'s~0..rrl. ~JcrYhrers~ui~', we pro~~,~se ~~s LlSe «) ICrC~eC:$ t~t~: ~l(3st i'ri'3Y't2 ~:rC T"a#?s~)~~a~.
~~~~~~°i~l ca~~ Y~c~~ent~
~~d~~m a~g~rzate ~~G~~~l~le '_,~,~~~~"- ~~_% 1~~,~,~~rc~l-~~~ ae~d~~~'a;>
~~~airaed ~t~~l ~S~
~v~ ~I~~~~~ ~Yl~. (~aYl ~~eg~;9 ~=:r='~i, ~~~)y...~_:-~,ysi.~le ~r~~ ~i~;r:~la ~~~~erl~i~wl ~'o. {~~-~,~Lhs, i~~~~) alld SLII~~o-~l~l~~.''~~~ algid :~~i~~.-i'~1~:J~ ~1°vm ~ieree ~~~e~c':~rd, ~~,~. ~s~ets wrxe ?s~ia~~~,d ~r~Yr= ~a~-'~Ot~g ~Nis~ar s~a~s (v:~e~rl~ -~~~;..9 ~~--~:c~rlsE.aE~S~, ~.~~'~ ~ar~ada~
~t°~~at~oi'io~r of covc~let~t'ly-li~tie~c~'.~oolv-l:-z;.~irc~ c~r~C
ct~o,~a~-lit~~;~~°s.
~:Cpet'IinentS LiSIn~~ p~:L7~Cs?L~1~-cli.%3~~~' c:i'GSS-ii~iICe:S "~~e'~1'' -,;~t'Ie~rYn~~ in a !i~.I'~ idJ~IJE'7 L~neii ~~'!'~ iilLlminatic~n. ~L:l~e~-~f~~.T°~~ f'~ ~~ cY a=~i'o ~-~'~s~ fuisscfived i:~I dil"rE,t~v~s:~lf~%ide ~~%i~~~ v~'~re rrai~ed qavitlu i~~ ~ ~~~s~c~Ivvd I~~ (t,2~~~~ b~~a~e ~L~i ~e'~
pA °: ~,~., ~~~..~, af. a ra~i~
CI'~SSII17~~~YS:~~,SI3P J-s~ 1:~~, I"e~nL=ilill's1~ ~ ~"!vLlsS a~ "~',.r'~'i1 get'Tlpez:ai ie ~n'~,1' ~~Srp-t G~''~I'~l~~Ifi ~,~
-°~. ~'~,I_,-Crc'3SS°il:PiCvT' ~,'daS pe: lsaed ~I'C3;;T"~
>:~SF~i.R~.,n. I~~u~Ll~u pI',~~al~~ ~5 ~~y' r~lal~Si,~'s ~'.~ainJ$
saiil~e, usil~~ a 6t3(~~-~0(~(~ ~a I:Ea~ie~L~ia~ v~e~~~t ~~La~-~~lCmer~:u'~~'an ~ E~pecttp~I~
~'O~~Y'~a$O:"3~:S, E.'l'.%~ ~, a~ ~r«~ f('~ ;~;',i da~~c ~°vi~r"''2 ~~'il:i' It2~C'~ILti'1_ C,~L~I1~C;S. ~~ia,e 'AilOC~It~~f~ ;:>~_,~
vas qiaantii:i~;d ~~'3~h i:fi~~cW~.:~~ ~ rc~ei°, ~~,ssa~' ~i~ze~'~~~~;, i~c~~~u~'r~., ~~~ ~:nd d~IL~~=~c'~ ~,~ith Seri?n2 a~ °iiie appYOpaia~t; e~IICC'ilv.'zT~'Gn.
.as!~i~t~o~'~~,~~i'p tt~re~ct~°Ltt'iot~
1~/~icr~capsLlles vmp'e ptwpv°.~'ed a~co:3-dng j.~ a p~~.~:~Lt"zy isLl~:iisim~ I~I~rs~z~~d 2i, ~~
d'v'?~'~a f~'?f,' ~~~iL)VJln~ Ifl~dlrle~~Io 'I;~. ~., S~E:IIi~I Sv~Ll'r:~:Olx L~'~~ i.t~RO~> 3~LI1'll_Ie d SC~~rl.Yn c ~~lYla~~: 'vvaS
e~~sL~ded ila~C~ a ~a~il c~5~ IJ~3nn~i t~;~.i~i~anr. i~~~~.re, ~~,~f~'e~'~d ~~i~~~ fp~-~~. a;'h:, cae~:iLt~a c'i~.,~,''lFi.alG, '~,~iC;d iSP't3fS ~'e~~ In 4'.~~;' ~i~~ '=_7:i:~t3 ~
i>.'dS''~ vc%%'V ~rL~ a~Jt~~il~:x ZC7I' :°.- Y~IIrIL~ceS, ~jIaSF~~d v~r'ILii SaII'iI~ and 1'e.'ar,%Ll~?t ::~>:'.t. 5 YiaII~ILI?~S
PI:~~C; ~.s ~°~Q -~~t%'~' c.~3Cl~I~z'Qø~IT'a~E.. :~'":,~''a~t~~~, rnir°°r~~aps~a~es -ere wrasl~ed ~ ~~Ice v°;j~n saail~e.
~..:ps :w~ivi~ f~ ~;vus:~lin~~;,o de~iv~,d ~p,~.
'~~ieef; ~9eI'~CB.I'~neC~. °,lndG,i CIIIII Il~~~~.
i'~~~IC;iCi°,a;:~SL~eS 'v'~'ei'L, ~zlL.'iI Sl.i.'~,Snlli~~d ~~ ~
~°t~f~? ?'clt:i>qy5 (~iacs~.iig~gc 25~,' ~',-'~25~'~-~~,, ~ ~hr nroda~,~. ~,pg~~~d, ~:f'~.~ a~ _?
i~virr~'. ~i~i~~c~c;apsR~Ies pi~p~.Y~G 't~'t$?2Cil,l~ ~f~SS-~II'I~C~1" aI'z~ ~~~f-h Ia~Ld~aF:tnG'~,~1&3II
"/~1'~~~: F.'3;~~~C;c %.?.S (;e)n~I°C~~S.
~~;~zot~s~~cztiot~ of~~h~~t°ps~~a~~ Er'a cr.~~%~E'~P~t~ link c~;'t9~~~~~ ~~~rt~ ~~~tcl ~lg~t~rt~
A suspension of.niczocapsules in Bali a a%as ad~~tsted to ~l-1 J % l~~v adding volume oi'2~1 glycine buffer l~~ ~ i2 ~2r~ ylycinc:. 14 mi~~ IVa~I, '?~ ~~ia~I-'y.
~Iicrocapsules were incubated in this soluior ~cr at least ~ n~inEates.
ivlorphological chanbes were follovaed by rnicz~oscopic examination.
Lr'VC1i21L1~1LJi2 G'~~YYll~C'~'()CCl~JSldl2 ~~'1J>.'~iCCII fl'."~1~
~llGlC'tlLJ;2C~~ Cl2Cl3~Cd'CI'f'i~'lSit'~;;,~.' i~/:icrocapsuie mechanical resi,st~r~cc: u'as ~.val~.~ar:cd ..sing a ~lv4aa~~tita3i~';: ~~xtethcc~
previously reported ~9j u;itr~ tl~~e lollowin=~ n~odil~cations. ~fc ca'=_=_caz~ate tl~e ~erwentage og broken microcaps~ales, the an~o~:r~t or ~'I ~'~~ iahe~led dextran ~r~ the supernatant and in intae,t znicrocapsules is quaz3tified. ~ta~.dard rnicr°ocapsules ~~rre dissolved in 2() r~~I of ~.R°~~ ~la~l buffered to p~-lI % ueh C~. i,~ ~tycine ~s described ~i~'.
since ti~is rrrethod could not break cross-linked micro~rapsules, tl~e ;after u-ere digested by the addition ot~ 1~% v/v xmy~F _ c..$ .. R ~~ \ °ff.~ .~--,~. w$"~r..
of kip 1 i ~T°~ and incaabated at I ~~°C~ <o:r I ~ rrsirt~ctes.
'~ or nornnali~arion, t~~e sa~~~ples of the standard curve were digestev~ in same co~~ditons.
Evaluation of rnicrod.apsule mes~~hrane molecular ~.~'ei~ht cut-off (~'G~~C~) ~,~aas performed, using sire e~clvsi;~n ch~'c~n~~to~raphy as preE~io~~aly described {R, ~3). .l~
col;<<n~r~ was rr°bounted using the n~icroc,aL7sules under study.
;~~L'r'~-de~tran {'~.~~fl13 kl'~) ~:~ras used to measure 'the column dead or void vo<urne {tJo) and glc~cose {1~~.3 da) u~as used to dete~'~nine the column total vourn.e ~~Tt). l~~arh<ers of diffe~~-ent rraolecuiar u.~eight {l~~) and viscosity radius (~r~) w~.re sec~xaentially run on the co:(~~mn : de~trans '~'~li~ : 2,~0~
i~d, 19 1:~, 4.~ l~; ~;~ ~4.'~, ~.4, I.';~, c-espectively); proteins : bovine seru~~nA aiburr~ine (,I~~GJ 66 k~; ~~ ~.~), ovalFournine {~~~: : ~~ kl~; R~ 2.~ø~ and carboxipeptidase (~.~~1 .~.~ ice: Rri 2. %). for each ~~°aar.~er, ~ pnrtirio~~
cbro~~~ato~;~a~~ic coe~~icierlt {-~~e~) was ~aiculate~ ~~.sir~g the formui~: }~:,~~ _ {~:re - "EI~,>) I ('gin--Vo;, ~~~he;re v'c: is '~~m mariner retention volume {correspondiraaT to the ~3eak of the elution ~sraz~le of the marker). ire°ly.
permeable mariners have a T~S~~ value near I.~. E~cl~a~.e~ mar~ers have a ~,~~
value near ~.
Fffect o~'~the ~nicroer~ccapsi,rlca~~or~ ~neil~c.~cl on ih;~ ~~zcrwivc~l of'e:r2c.~b,sralcxter~ islets ~f L~:~gerl?r~rs islets from 'V~%istar rats {3~JC~-=~-3~~, ~'haa:~a-I~ive3., St-~ or~sstana, w~~:. Canada) ~.z-ere isolate tasin~ a ~ublisbed method [2~=t~~~ci en:~ a~suiate~in stan~ar~l or eovalenti~;r lini~~:r~ l~P~.
_micr~ca~sules. 'i'he survival of :~-~icroez~caps~:lated islet cells ~~ms evacuated i.=r ~itYO usin dual staining vith ara_nge acridine anc prapidiu-_~z iadide and observation under a iiuorescent microscope f,S-~ ='i ~~oresc~:~~ce spec:~~ophoe:orneter {"~e~win-hlrraer ~',~nada, Travel, ~Cy c;xcitation ~9~ nr~: <<3:d emisson ~?~~ nmj as prc,vzo~~siyv d~;scribeci -='~-~~~~
f=resh {unstained) islets were cxa~rined under a stereomicrasccpe {Labavert i~S, Leizt.
Leica Canada. St-Laarent, Vii:, Canada;, anci ihu nurt~e~~:.r {°r<;) a~~ci size of~islets ~%ith necrotic centres mere recorded ~s ~vel~. as t;he di:~aneter of the necrotic ar~;a[~Sj.
~'~a~isPicc~l cancad~~is Resets are expressed as mean = S~'~t. The differences t>etwLen experitnetaai ~ro~ps mere analyzed by ~.~npaired student's t-test ~~ati~ ~ va~ne5 less tP~=~n i~.05 ~oz~~sidered si~nii'ic;an~..
~'~ UI~E: li~~~IMU~ 50 ~~a irE~~~~S
z iba~~°~ 1 : Standard arid :~:\'~~-i~~~S cr;~s5-fin<ced nlic;rocapsuies vvel~~~ irzct~batc;ci ~Ilto strozlg alkaiille pH (p~=1'?~. .~. =~~tal~dard ~icroct3psules_ f ~:~
b°~orL tile illcubatioll. I3.
~tar~u2rC1 mlC;roCapSLlleS IIliO ~~t;'F~ir'1C; b~~~:l~~f~,' p=-; ~.~~, O~Si,.
a f~~u~J St'rC:I2dS u~tel' c'LddItIC3I1 0%<
the ~fl~aliz~ze buffer ~'. Standard ~:~.crocaps~~les i9~t,o ~lyc:ile L~;.xff~~r pE:Il2 ruiaer ~-~ s~:corzds.
. ~~~13-~1~5 cross-linked rr~icr~iaps~les bei'ore. vhe ir~cubatior:. 1(~~~.
~.. cross-finked Illicrocapsule into ~lycine buffer ply I'~ after t~,~,'o years, ld~'~. Since the ph ~-~aizle of the side chain amino ~~roup of~tiza~,~ 1~;'sizle is a~~g~ro~xr'uatcly f J,j~,. ai i~i-~ i 2 the ioll«, liitelai.tlOr'a C77(~tZJv'C,'CTl l:~~ ~, alld i,li':Ilelt~ l.;i I_illtfe?~I:i~C~z, f~IIIS e.""iaJtrirYle~L ~~Oi~~IrrI~S tlli~ the use :~IV~-~1~5 azld I~..i-'~~ illlzl~~~~atio_1 il~l~~na:e t'e:~: formation of co~-Giellt finks t~lat are resistant to ionic competitions.
~i~w~r'e ~ : l~iechanical ~'esisv~rr~::w c~i~n~ic~'ocapslales were rl~:«na~ied has the ps:rcentage of ~oroken zlicrocapsuies after a i"~ i~ours of agitation whiz borosiiicate beads. ~. Standard microcapszzles as well as the ao~:~e azld Vigil do;ses for cross-iizhec>
microc~psuies ~x-ere s tudied.
~. ~ozltroas cortairzirl~~ z~~i~~rocap:7zxles and borosaiicate beads without a~~itation is sho-~-~n as a control of tile Inicroc:a.psule ~reparatiorfs. ~.
~or<trols of a~i~ated microcapszHies wit:llout borosilicate beads irs izlcluded has a control of the agitation. C~ata are preserli:c d b~,s a~-~,ra~e ~ S~'I~. * ~~~-' s~-e~ 1 Vin= % E
~~~a~~ ~ : Isolated pancreatic islets ~~erc encapsulated into sta.~~ldard and cross-ir~lced ;~icrocapsules and subn~zitted to L~, 2 arid i~, IC~/z~~ of ~~~J~ ix-radiaLion. ~=~t 1, u., i ~zad i 4 days after rrlicroeneapsuiation, islets were double- stained dvitr= t~.01 ~rg~'rrlt orange acridirle (iivng sells, green) algid ~ l~ngi,a~I prop idvurn odide ~;~'iet~d c~~Ils, re;d~, anc~~ ~rere classified ur~zder ~luorescerlce r~ir~ ~oscoy. h~ata are presented a.s average _~ ~E~. ~~ p<
0,~~ ~~s sr_az~dard el:capsolated isvets (10'.x- ;~~'=~z dose), ~~r p<(~.dj5 vs cross-finked encapsulated islets (IO'zJ U v'~. dose) ar._d d p<s;~.G~ vs stands-rd encapsulated islets without UV~ irradiations.
n ~,-~aEues are represented k~~y llurr~~ers ir:'co~cs. T ~~ese data sl~c~-than ~l~e nletllod does not ai'te~;t islet sell viability, e~:cept ,-Yi~en ~. urge ~2~'~ k.l/lzl' j ~'°n,~z ~iosG~: i~~ used.
Flg~x~°e ~: Isolated pancreatic islets t,~~ere enc~ps~alated into standard rrlicrocapsules or covalently ~~ross-linked microcapsuies using io=.~-~ U~~ dose or~iy. at 'i, 4~. 7 arid I~ days a2ts',L' 1Y11C0eIlCapsLllatl0n, tile llr:sellC~' f~~' pleCrOCI~: (:eTllres In ~~~~;r~f:rrOapS'~lated lSlels 'Vm'aS
o'oserved Ltnder optieal n~icl°osc:c~py. Ti3~°
c°.~toto;~~city mss de.vern~lined as the perc;e~~~;age of islets ~r~itla a necrotic centre. .Results are ,~reserlted as the average ~
~F~.
~~ ~i'~~'v~I~:EL~~EI'r~IETdT
This work was supported by a grant i-om tl~e ~aradian oiabetes ~ssociaiion in =~or~our of the late t:mily C.
I-(av~~es.
/J..'~, _.. , To prevene this corz~apetitiora v,.jith chargc;c~ 3a~oi~ye~ales in thf°
a=m~iri~nmea~t, a ne~v concept ha.s been proposed: the irar~roductiogr ox co~,~~l~;nt tini;s into t~:~e ra~emda'ane oi' the rnicrocapsule~ l ~-17~.
~'or instance, microencapaLaat~~~ islets oi~ i=,~.a.~agerhans (~~Liets;, ~;~hici; coma=
insulin-proda,acing ~-cells. could he ~transpiantcd into type 1 ~ins~aiia~-dependealt; dia~etic patients. ~~icrocapsules intro~Lac~= a ~ap~r~er bc:r~~,eer vhe transpla ~t ~.ntif45en presenting ;ells and the host T Iympl~oc~-te a'eceptorsa prevea~siahg (hindering?) antigen recognition by the imnsune system. This a~erhod pz°otects islets against allogenic reactions anti recurrence in the transplant oi'tl~G ~~.rtcira~ra~Lxa~e tv:co~~ ;ss that has nitiaiiy caused dia~etes.
Therefore. anicroencapsulation T~~La- elimaaje tl~~e r~ee~ uor the ease of toxic ii~ramunosuppressive drugs that coaa:dera~ly iia~its the indications o~~c;ell traztsplan~~~ations.
~yloree~ver. the strength and diara~ilit~~ of ~nic;rocapsule men:hranes are.
critical issues for the safety ~~t~'arlSp~arltal~orl Of' ~JaV?a~J'tr~ii"J~eC.t'w'.~
~laoeyl,.~~'.I!leeru.Cl c4~.ls. lnCiLidahlg li'aJLllr~' producing cells as well as c°ljs prodaxcing a~caolecules, v,hic;~
prorrzote islet cell s~arvival~replication%trar,sdifferentiatiora ~~~a~' ~~'fR~~~T i~ i_,i=,, ~~.~i~
E~1~~~~IhiC~L~C~i~~ or insure islet and ~,raica°ocapsnl=~.p ~a~raear~oprotectio:~ ~,~~~.
~~~U~E L'~'~~i~_~1'i'~~~t ~IJi~ ~:~~,~, ~:~~, ~'~~'T~~,~~). ~zna.ll~.r. tae rraost promising approach ro ~-cell replacement is the transdif~erea~tiation of stern cells unto iS~etS~~~ tnOt neCesSuriiy' err ~'?r'?;QI~iC s'ea'a'I CP'ilS, th~'illCli arise ContrOYrerSf, C)ut stem calls of many other oriius, ncl~zdin~y autologovs oells~. Joie that even autologous cells would re~uir.: ir:~rr~u.,o~rote~iion against the r ec;trrenee of ths:
autoirnmnne process. the ~xra~or ;.ona~r_:~ related to this a~~roac~, is the risk of malignant transformation ar,d dissemznLaion o~~immature stem cells ~ ~ .
Tl~e process is based on the chemical derivation of ~irL ~~.-ith a photoactivatable heterobifunctional cross-linker, ~-~-r=~zi~io..2_nitrobenzoylc~cyst~c~~inimide (~~~11~-I~~S), which is able to covalentl bind i~~,:~. to alginate ~,~:mr~ er~er(~izec~ icy ~JVI~ light illurninazion~L~, i9~. 'hhe ~i-hydroxysuccir.i.mid ov~tlic: cross-linked ~:~ras fist c:ovalently linked to Pd,lr. in conditions that ~,~ oul;~ ciana.a~e ii~,iy-~:~ cellss but ils~tore ~~he c;ells are irtvol'-ed ir3 tre procedure. Then islets enia~s~alated. vr~ algirFate microcapsules ~~e.re inaubate~W vitl~ the oross-'inkc~r deritmd ~Ll~. ~~ll~c~rt ~:lne pr~~arati.,~n is ill~tmir~ated v~=itl~ a L1~'~ ian~p, the photoactivatabk; residue reacts ~.vitl~ the phenyl azide residue on al~inat~, croati:~~ a covalent link. 'fhe latter reaction is ~~ot har;~ful for livn~ ;
veils. ~'e, herein, present ~ proof tihat a covalent linlr, v;~aras fo~r~~d ~~~ovidira~
consid~.rable improvement ~~
microcar~sule resistance to chemical and trsec;i~anieade~radatior_. i'~~e _~~,sults of th~~ study sho~~~e~3 ~dhat the procedure did not modify Ynicrocapsule m~n2br~rie permeability and did not affect i~ vi~v~ and zn ~~iv~ encapsulated cell survival, I~II~I~~~~ICt~P~UL~'l l~i~T C~_~ L_~V k~~ ~~.I~~J~ I:~, C..~~~~-'~Llrl~ :Y
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Dote that .1. ~usseault aald F.:~. IJeblc-~:ad lza~,~e øqually contrabazte~ to $.lAi.= ~-or~.
~:enzre de ~eche_rcbe CJuy-~eraier. ~~pitai °~aisonraeuve-Is.osem~3nt_ 54-15 foul de 1' ~ssofnptioa~, i~Iontreal ~czebec, Ca',ada, I ~ I 'h 2I'~~i4.
e-avail : ~allejp'~.videotroal.ca ~lzoit title : Covaler~~tly lra~i<ed al~i~ate pol~aalvsinc: ar~icr~capsuleJ.
I~e~~~~ords: Pl~otoactivatable crossiirrlcers, i:~m~i~a~ois;~Iatioa~, cell '~ra'~~sp'~aratatir~rz, bioreactors, islets o~ I aayerhaals ~~ ~ s~, p,~ . .,.~~,~~A ~y . . ~~.a~ ~, M
~j.: :~
'~~orrespc~azdence should be addressed to ~1PI-l i~Ioratreal, rebruar~r, i5'~'. ?OU3 i ,J
I-ialle, .lean-Pierre -'r~._.~..._,....-..~_ Pa a IVum6ers ~ Ad~nspeei~ta~~e~~ a~ad ~a~naf~r~ ~~~~
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Htmlformat _ TI~ f --.~_ _. ._ .~..._. _ ...~ _ ~~~ _-Detailed 8ud9et for Initial ~udpe_t Period 2 Detailed Consultant or Co~atractual Costs for Initial Budciet Period _ Budget for entire Project Period 8udget Justification ~~er Support g Resources ~--- __. _.~_.~ Ig _...-.
~~~~1 LI.
Principal Investigator (Nod to exceed twe asses)lI
_ _ _ ~3 ~.
AI! other key personnel listed in the "'Applicant and iCey Personnel"
! html section of this applicats'oaa (blot go exceed two pac7es t'or.eao~) a rah ~~~ L9 Copy of summary statement from original application-~
(if appiicablej Introduction to revised ao licationtif a licabie riot t~o exceed t~ir> pa es) A. Specific Aims (Items.-D rat ~o exceed ~?
sages.) B. Background and Significance of this ~vorl~ 2g to ~' pe f Diabetes C. Preiiminary StudiesJProgress P.eport (fo~ ~ 2,3~
campetitive rer~ewal) ~~ ~6 D. Research Desi n and Methoes ~. literature Cited (no page limit; ~ ~ 30 ~
11'~9~ I~ ~ IVor tc= exceed ZO ~2 es exc!udirr mar~~rscrit~t~s Collaborative arrangements if an ) 4Q
_ ~2 IRB andJor ACLiC a rovai forms Abstracfi a es Trom LL other soLrces of suc~~or-~t~current~:l & ~endin~, Other Page 1 of ~ ~' i-iaife, Sean-Pierre ~E3'~~~EL~ S~I~E~' ~~ ~a ~. ~~3~Ei' PER,~~
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aec~g~t ~a~~-'~~~r ~. ~~~ ~eg~rii~~ r~~a~t~~ra~ s~l~re~u~srsa~~~ ~ ~c~al~~~:dr ~e~sonne~ ~~e ~~ ~~'e i ~ s~~ai~~9a~reat 9a~ ~rire~~ ~°~T'~ri.~
i ~a~a~~ ~r~,pe~~ ~G~s~ a~~, E~r~ gee et~~~e~e~i s _ ~~o"e~a~a Sean-Pierre Hall2 Principal 3v 10 % $43,205 ~~,320 ___-_a. pt,320 Investi a*~r (~at~ralie ~ienie~r Research ~ 35 ~ 100 % $28,800 ~2t3 800 X32,255 X3,455 a~ssis~an~ _;
Markin Menar~ ~ Research 35 ~ 100 % 26 640 '2C~~~ ~3 in7 29 837 . . $ , ~ssis~a r~~:~
i 1 _.
f i i e,. -. _ i _ ~ - ~_ -._. ._ - S~S'~~3'~~iLS _-. $58,760 '~ $6,653 66 413 5~~~e~ ~~~es~z~ ~ ~2~~t~ ~n s~t~~~,~~~~a~~~~~~~~ ~E'r'1' ~. 81,715 i ~ti»f ~~s~ (Sp~c~~ - ~e~6ze ~~ ~~r~ g~~~~ ~ _~~~~~ ~ 1,500 ~~s~tif~atei~~~ s~~m - dc'~b ~n Eq~aipm~~~ (~escrab~ ins ~a~dge~ ,~e~~~ra~o~~~ ~____ _~ ~,~ii' ~ 0 ~~ns~~t~e~~/Cc~~tr~cta~~~ ~~~i~ ~Se~ s~ a~~ ~_~~.~'r '~T i9UE { i ~ Ii ~ ~ ~ 1" 149,628 f'ape w c~f 47 Halle, Jean-Pierre ~E'~LE~ ~~~atlL1'~.~~ ~~ ~C~i~' ~C"i'~l~t ~t~ FC3R API L ~It~~~'i' pE~i~~
Fii1 out this gage only it c:~nsuftar~c or oo~ tr ac-~ual coals are request-vd.
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~rovi~e a jus~.ifica~ion for each i~er~ listed in the ~ud~e~ including ~:on~rac~ual cots. ?ro~ride i~s5~titutior~al fringe be~efu rates t~or ail persc~r~ne6' DIr~'A~1J~~5 ~tJD~I=,'1?
AID II~A~,B
~xp. # sulDjeets~r'at~3rrtap~ lr~e''._.~otasz~glar~./elaya~taI
~~a~siu~g'Total # tai --WistarRatsD.1 200 $1.20 $15,45 -~ $777,60 x3866,89 $0,26 C57B116 D.2 48 $10,75 $11,69 $0,20 $14x,00 $705,36 rnicc C57131/6D.2 48 $10;75 $11,59 $0,20 $5 376,00 $5 937,36 mice V37istarRatsD.3 360 $14,20 $15,45 $0,26 $1399,68 b6960,40 C57~I/6 D.5 108 $1u,75 $11,69 $0,20 $324,00 $1 587,05 mice C57i31.~6D.5 108 $10,75 $1'!,6980,20 812 096,00~i3 mice 359,05 I-IaanstersD.6 270 $1 l,St $12,52 $fl,23 $928,96 $~ 309,83 V~IistarD.6 180 $14,20 $1 S,4S$0,26 $699,84 $3 480,20 Rats T~-lamstersD.7 4 $11,51 $12,52 $6,23 $13,76 $63,85 C57131l6D.7 48 $1w,75 $11,69 $0,20 $144,00 $705,36 mice C57~116 D,7 48 $1fl,75 $1 i,6980,20 $5 376,00 85 937,36 mice CS?HIl6 D.9 108 $I0, $i 1,6380,20 $324,00 $1 587,05 mice 75 IrrOD D.9 108 $20,55 $22;36 $0,20 $12 096,00$14 mice 510,35 TD'L'A~ ~.~ I~IAt.,S (3 ~'E~iZ.S~ x;63 0111,13 ')i'~TA~ AI'~1~IAT,~ 3~h,I2 '1'~A~. ~~'21003,3~a li~I~IDA~~,ES
~:~p. # # a~iaaxa9$ia~uisrraa$1~-sc9.'~~tal R:a~
Isict isotatiora200 819,60 $21,32 $4 264,57 D.1 and culture 48 $19,60 $21,32 81 023,50 D.2 D.3 360 $19,60 $21,32 $? 676 22 ~.5 108 ;~I9,60~~'21,32$2 302,87 ~.6 180 ;,19,60$21,32 $3 838,11 D.6 270 $19,60 $21,32 $5 757,17 D.7 4 w19,60 $21,32 885,29 D.7 48 819,60 $21,32 81023,56 D.9 108 839,60 821,32 $2 302,8 ~' 828 274,'9 Page S of 47 Halle, 3eari-Pierre ~'' ~,d ~ICaaC.~ ~9SC~. ~C9V:a~
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Encaps=a!ationD.l 60 881,00 $88,12 85 281,19 D.2 4 S81,fl0$88,12 8352,48 D.3 150 ~8a,00 x88,12 $13 2:11,99 13.5 9 881,00 $88,12 8793,68 D.6 60 881,00 888,:2 85 26'7,19 D.7 4 $8:x,00$88,12 $352,~~8 D.9 12 881,00 888,12 $105'7,44 X2634'7,85 ~xp. ~ samples$ls~~apie$ na~cl. 'y'rs~a:a ~ tax FluorescenceD.1 300 $0,15 $0,16 $48,95 microscopyD.'? 96 $0,15 $0,16 515,6'7 D.3 960 $0,15 $0,16 $156,66 D.5 216 $0,15 $0,16 $35,25 D.6 270 $0,15 $0,16 X44,06 D.7 96 $0,15 $0,16 $15,67 D.9 216 $0,15 80,16 $35,2.5 $351,50 ~xp. # samples$!ssmple$ a~a.l. 'T~~:~1 ?# ~ ax TtJNEL D.1 300 $6,84 87,44 82 232;37 assay -D.2 96 $6,84 $7,44 $7IG;,36 D.3 96C 86,84 57,44 $7 243,:9 D.5 216 $6,84 $7,44 $1 607, 1 D.6 270 $6,84 $7,44. $2 009,13 D.7 96 $6,84 ,x',7,44 8714,36 D.9 216 86,84 87,44 81607,~~-I $16028,4'?
Exp. ~' saanples$,~sa~rplen i,sa;h ~"~ea:E
# taa:~
FlistologyD.1 900 $5,23 $5,69 85 120;15 and immuno- D2 288 85,23 $5,69 $1638,54 cytochemistryD.3 2880 $5,23 85,69 $16 386,38 D.5 648 $5,23 $5,69 $3 686,94.
D.6 810 $5,23 $5,69 $4 608,67 D.7 288 $5,23 $5,69 $1638,64 D.8 936 $5,'23 $5,69 $5 325;38 D.9 648 $5,23 $5,69 $3 686,94 $42 092,3 ~"~ge 6 of ~
t~a~fe, 3eart-Pierre EXp.'#~F' $I~a,b s ~mcl. '~'cf~
~,u~~ ~.ax Growth D.1 1500 $3,60 $3,91rv $5 $67,74 Factors D.2 180 $.3,60 $3,91 $704;,13 D.3 1800 $3,60 $3,91 $7 041,29 _ D.5 540 $3,60 $3,91 $2 212,39 D.6 900 $3,60 $3,,91 $3 520 b5 D.7 240 $3,60 $3,91 $938,84 D.9 540 $3,50 !63,91 $2 112,35 X22 29T,4~~
Exp. ~ sa~~ples$~saple$ emcl. '1'ota~.
# tai:
~r~ ~;a~ilzt~D.1 90e X0.16 $0,17 $1x6,66 assay D.2 288 $0,15 $0,17 $50,13 D.3 2880 $0,16 $0,17 $501,30 D.5 648 $0,16 $0,17 $112, 7=:
a .6 810 $0,16 $0,17 $140,9~r D.7 288 $0,16 $3,17 $50,1:?
D.9 648 $0,16 $0,17 $112,79 $~ 1249$0 ~xp. # sars~ples$lsa~plc$ n~cl. "1'ofak # tars Callprol:ferationD.1 900 $1,09 $x.,19 $1067;23 assay D.2 288 :1,09 $1,19 $341,51 D.3 2880 $1,09 $i,19 $3 415,'! T
D.5 648 ~~1,09 $1,19 $768,41 D.6 810 N1,09 $1,19 $960,51 D.7 288 X1,09 $1,19 $341,51 D.9 648 $1,09 X71,1.9 $768,41 $766297 ~xp. # samples$lsa~~ple$ i~cl. Togal # rFa~
131oad D.2 4080 $0,92 $1,00 $4 083,5 glucose ~
D.5 9180 $0,92 $1,00 $'~ 187,91 D.7 4080 $0,92 $1,00 $4083,54 D.9 9180 $),92 $1,00 $9187,97 $26543,02 l;,up.# saxraples$Isaple$ l~7cl.:focal # aax 1;L1~A D.1 75 $8,83 $9,61 $720,4ei D.3 270 $&,83 $9,61 $2 593,E~~i 1J,6 27 $8,83 $9,51 $253,37 $3 573,49 '~r~e :~ o~E =1~
Hale, Jean-Pierre Micelaneous~~~. # sara~~~es $ ir~ct.~'ce~al # ~~is~p~~ ~Gx Str;:~tozotocinn/a 1 b67i,$1 $757,39 $73'7,:9 IvIotecularen/a 1 $2 Oi~~,QQ $2 i $2 175,84 7,80 Siology , regents l~t~TA~ n/a i7 :~~C6,40 $'?89,8:2$4 926,8 $7 ~~.a,07 'h~'I'~1~.. ~~h~,PII~~~I~~S (3 ~'~~IoS} 512 I3a,9I
'I'~T~I.'E~I'~1~1I~~~L~S )1'~It ~'~A~. ~6~ 7p1,97 'I'~7C~9y. ~~IIIi~I~P~S,1~I~'I'~I2hI. ~~ID X245 I~~,03 ~~I~'~g,II;~ (3 '~'~~~~) 'I'I~'I'~tl. ~I~IiVIAL,S91~A'I'~~I~&, ~1~~ ~~17~ 5,~4 S3l~IsI,I~S I~I~ A~Ik ~FFII~ICpE ~~Po'~:~y~~. Lt~.'I'E.St I~e~~I'~~~~~ ~~rl~T~~7~~ o ~L~~O
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h~t~2 ~ 07~ °~ ~' E cake, Jean-Pierre ~~ ~~~rE ~~r ~~s ~~~,~eb~e~rg ~s~~~6~~ ~~e~~~~r~~ l~es ~ f~!o ~o yes, list the funding agency, the title cf the project, year one and total fundir3g received/requested.
Specify areas of scientific and budgetary overlap if not obvious from the aims and abstract.
"I~~ ~~~ ~~~~ ~~~~4$9~i9 ~~ ~fC~IL~E'~1~~ Ef ~'I~~~fi"~ 6~ r~3I~'~8~~~ ~PC9I~r ~~"~~° ~~8~l~iL~".~ ~~~J ~ #~LG
ist ~.~. ~a~e~ sources of support ~et~rr~ent and pending j. List funding agency, complete title, total award, yearly ward, inclusive funding dates, the rose of the appiicar~t, and percent of tirrse devoted to each grant.
he specific airr~s and abstract for each grant should be app'nded to the supporting Docu~enla suction of this application. (~~ra~ecutf~ely rs~ra~~er ar~y ~ttac,~rs~errt.s P'car t°~a~s~ section.) ~~J~'~'(~F~.~' ~~T~~'~"~.~
__ Bi~-artz~aciai ~~~dacrix~e~2~~~24'7.63~1$t 9b,249.15$~4/~l/2t~fDi Pancreas : Tire mole o "~
~F~ 2 to '. Pancreatic ~7nct fv'edis and f~4 999.24 C"srowth i actors :~ i~Iai ~ter~ar~ce3~ X4,999.2,4~3/~ 1/2i~04 and Promotion o~
T~iicroencapsuiated ~sle~ ~eia ~aSS aYlC1 ~11I1Cti45i~.
i r ~2C?I~E t~~' l-~PP~,~~~'~' :
~'rincipal lnwesti~atox /o ti ~e = 2~fQ
.
, ~~.fi ~ ~~'an$plarltitBG~$ O~ IS~~tS 4,1"' ~/'/~i~/?~'~~
~ ~J,~.L~~S ,~ ~C7~;~ 1 encapsulated irs ~nofi:r~ed (coValently Aiaed~ alginate ~~/3/2004 i poly-h,-lysine n~iLroeapsules _ ~~~,E ~9~ ~..~~~,.~~f~~ : ~'?'413~11?a~gLi~~t~T, ~ $~OJ~
~22Ve; 4/n t3lT"e =
Page g s~f ~-7 I iaiie, .lean-Pierre ~i~~~
~~~~ll~~:~e~: specify the facilities to iae. used f or the conduct of the proposed research. indicate the perfor~-rtance sites and describe capacities, r~er~inent capabilities, relative: proximvy, and extent of availability to the project. Under '~~ther", identify support services such as machine shop, electronics shop, and specify tire extent to which they will be available do the project, iJse continuation !sages if necessary, ~ aboratory:
Most of the proposed work will be conducf.ed in the laboratory of ~r dean-~Haife Haile at the Guy-t~ernier Research Center of Nlaisonneuve-Rosemont Hospital, This X00 square foot laboratory is fully equipped for tissue culture and cell isolation (three Foods, two incubators, a table top centrifuge, a microcentrifuge, a COO 292 centrifuge, and three inverted microcospes) and for moleoufar biology and biochemis°try experiments (two balances, 6 micropipettes, PC'R, horizontal arid vertical gel systerns with power supply, a blotting system, a spectrophometer and a spectrofluorometer. ?n the same:
area, the Research Center common facilities also include a cold room, Cs dark rr~orn, -20(~C and -80CC
freezers, a variety of cen"trifuges, an automatic sequences, a microtome, irnrnunofluorescence microscopes, floor cytometers and high perFormance cell sorter. Facilities and eqt.aipments for developing transgenic animals are also available.
Clinical: Nlaisonneuve-i~osemont Hospital has very active diabetes clinical research unit and transplantation programs (bone marrow, kidney, eye bank).
.~nimai:
~lnimai housing is equipped to received a large quantity and variety of species (mice, rats, hamsters, rabbits, dogs, pigs and cats) in stesile or non-sterile environment. The animal housing contained ac'. a!I ecuipped surgical room and large quantity of hood and bench used for animal manipulation. animal care technicians services are also available for ail the experiments in~,olving animals and hare considerable expertise with immunodeficient animals.
Computer:
The laboratory is equipped with a PC and a Mac c~arrputer. The PC is connected with the Nlaisonneuve_ Rosemont Hospital I~etworh that gives us access to all the common lab computers of the research center.
Moreover, this network give us Internet access to the ~niversityy of Mantreai library ~rhere eve can fnd a large variety of on-line electronic journals, O;fFce:
Dr Halle have is personal offsce located in ~zhe laboratory area (at ~0 feet).
t:r l~afle clinical and research activities are supported by a secretarial star or: sit: providing f ufl secretarial support.
Gther:
~9~~~~ t~ta~pett~~t: List the most important equipi~nen'c items :already availaL~le Eor this project, noting the location and pertinent capabilities of eacF.
Pace 1t~ of ~7 i-laiie, Jean-Pierre I~I~,. ~C"~
Provide the foiiovvincJ information for ~~.~ key personnel in the order listed in sec~ion "Applioar~t arsd i<ey Persortnei".
Foifova this format for each person. L~upiicate biasketch form as needede ~~' ~~~ ~A~S r e°~~~.
-"r,",. ~~~.
i ~~9~~g ~~a~a-~~~re~ r~oi~~9 I~n~~~~r~~~
~.__ .._.._ ~i ~D~~'iI~pT II9~~S (Begin ~vifh baccalaureaW rar ofher lrrlfial professional education, s uoh as nursing, and y include pos~doetoral training.
IP~~ l~°9~~AL~7~~'~I_ ~~~~R~~'~~ i° I~ ~~ Y
Liniversite de Montreal, avlontreal, Qc, Canada ivi<CJ. Medicine i lJniversite de Montr~a3, Montreair Qc, ~.anada ~ CSPQ _~.9?9 Internal Medicine tJniversite de Montreal, Montreal, Qc, Canada 2 i"RCP (~) ~3?9 Ii7ternai 1'dedicine ~niversite de Montreal, f~orttreai, Qc, ~arsar~a ~ L~~PQ 3.980 ~ndocr inoiogy ~,. i~~~i~9~ts~ ~~ae! N~~. List in chronoioe;icai order previous positions;
conoiudinc~ with y°our preses7t position.
List any honors, Includ~ present membership on any pubiie or private advisory Lomi nittee.
~d~aEYT~~~.5 E,It'l~E~'~~EI~ITy 3~'u~~EId~EI~T~~i t~l~Y~.~. ~'~CS1~1d3hsf Endocrinologist, Sacre-Coeur I-Iospital, lVLont~~.al ('1980-138b) assistant-Professor, Department oFi'~edicine( 1980-I98b) Univet-sity of Montreal (Sacre-Coeur hospital;
Chief, Endocrinology Division (T987-1986) Sacre-Coeur Hospital, Montreal Founder of the Diabetes ~ducatian anel'freatment(:t98~-1986) Canter (Day care) Sacre-Coeur I~ospital, Director of this Center .
Chief, Endocrinology Division {1.986-1995) MaisorFneuve-Rosemont Flospital, ~;=orareal Founder Diabetes Day Care Center. '~aisonneuve-Rosernora(1988-2990;
piospital Medical 37irector of this center Director of clinical research, Maison.neuve-I~.oserasor~t(1995-2oU~) Ylospital PR~3~'ESS~#31'~.~L, E~~'E~2IEi~I~E
P~tE~EIT'3' ~~5~1!'IDhT
Director Research Laboratory on Islet (158-Now) transp'antation associate F'roFessor, Department of (1986-hlov.~) h~FoL~icine University of Montreal Page ~.~ of 4?
Halie, Sean-Pierre ~~~~~ ~5~~~-r~~ae~~~~ ~~ebl~~~e~~ ~~~ ~a~~~a~il~ii~~~ ~~a~a~~3. List i:he tide and compete reference to alt pubiications during the past three years and to representaciye eariiPr pubiications pertinent to ti~is application.
Do nor inciude publications submitted ;~r in prepay°ation. ~~~ ac~h ~~e~~~eJ ~~~~ ~~~ ~u~ ~~s~ a~~~inta~~a~e~
~ge< ~~~;~~~~~i~~i~ s~c~~a~~~ ~~~~~rte~~s~n ~~~ f~a~ vas ~~~~s~ea , 1. Gerstein HC, Mann 3FF, Pogiaa; J, Dipuieen Sr, iialle ~ er rah The prevale~de and characteristics of patients with atbuminuria and diabetes in the HesPE and MICRC'HOPE study.
i:~ialaetes t~aøo 23 :835-1339, 2000.
LA PRESENTE PARTIE I)E CETTE DEMANDE OU CE BREVETS
COMPRI~:ND PLUS D'UN TOME.
CECI EST ~.E TOME 1 DE 2 NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadien des Brevets.
JUMBO APPLICATIONS / PATENTS
THIS SECTION OF THE APPLICATION / PATENT CONTAINS MORE
THAN ONE VOLUME.
NOTE: For additional vohxmes please contact the Canadian Patent Oi~ice.
~LIyIi:;.MtiOiI IILIIIl~»:'~' 121211I:-i0 do d~.Iil~ltli_~: ;~~_~~~~~'~~-°f__._-__-_.
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,~c:~fYC~s. ~~~~~~~f~,.
a _ ., ~~~._ ____-._____ ~trlscaz-~n.a~fe ite,ns recei~,Ted ~,%i~.il this ap~Iica~ion (Request original docun2ents ill File prep. Section on the I~'' door) Locuments recu avuc cette aeniande ne pouva:~t ~tre 'r;alay°es (Commander les documents or!wgi~:~au~~ duns Ia section de prv°paratzon des dossiers aLI
~ hem a ~.tage,~
~_~e uSe o~rrlICrOencapSL:~ated cells or enZ~'lheS 3n blOreaC.i:OeS
ra,preS'ntS a Set ~f potential industrial applications for extremely resistalnt r~icrocapsuies.
~i'hese include microencapsuiation of hybridoma dells. Gvi~icl~ col~sf;~erablyl enhances tire production of monoclonal antibodies. of lactic acid bacteria vor the ferr~entatio=.~ c~f dairy products, a~~d of bacteria or yeast for the industrial production of a large number cf drugs by ~I~I~
recombinant technologies. zvVhe~~ the desired ;rod~acis or reactions are obtained, cell co~:tainirig microcapsules are easily separated from -0:~.ze solution. 1-lc;~~avever, the strong shearing forces encountered in bi"reactcrs br::ak the microcapsu~es, releasing: r~icro-erganisrns or cells, this contamination decreases vliP purity of tlm prodr.acts and may jeopardise the safety, efficacy and LisefSalness of the method ~, efforts have been made to in~~rove rnicrocapsule streng~:l~. ~ orr~ple~cation between tile negs:.tlVely .~'-~"larged poi5'anror'i u.l'~,JIIIate ~iT~ poSl~:',.Vel'V
~el2ar~?~:(~, ~~JL'(.~at?43~tS SL1C7 'ilS
pol'v-L-=ysine ~~I,L~j to form alginate-po?yW -t~~sir~e-~~lgir~ate (~.~b~'~~
rrricrocapsules is, fi3e most u-r_dely used rr~ethod to rnidroencapsuiate cells ~:2, %, and ~j. C;rhers ['?, F~, i ~, and l ij, and wTe [9j hare evaluated tie effect can :,;Zicroc°apsule strer~gtia of modulating 1~~.~~, molecular weight, concentration and incubation tune and the rF°~annL~rov~ic acid/guluronic acid ratio of alginate. New coating agents gave also been investigated X121.
~~~evertheless, with these methods, alginate and ~L,~, still c~mpetes with other 'charged molecules in the envirorrrnent ~i ~, 1 =j and ahe resistance i.ar~~rovent is iimitec~..
~7e~e developed a rr~ethod to covalently link FI~L with al gnates z~orn both the alginate core and the outer sheet of rnicroc;apsules ~pendir~g patent;. The challenge was that most methods cased to create gas well as to lest=voY> covalent links are incompatible ~;!~L:;roeifr.a~oSLtlatlol2 oI C'a:ii; :i St~;i"~I:~Je~'~~neat?LoI'!'leaT'~bral~.d:S ~'?s~.S ~C%eC;Ii 131."opoS:vCi ~
~., ~'; to prevent/ t'_~eir iz~~mune ciestr3~cti,~n ~ol~ew~,-~ng tra:sspOantatioru eiimi-.~atins~ the r~.:add f'or tdsir~~g topic imrr~~nostippressive ~rtys. 's hi; a~~api~oae,~ could be sued fod' replacing i:;set:; eo Y
lJar~geri~args (islets}, whicb contain lrssusan~~rodt~c;iay; cells, for she treat~ncnt of diabetes9 and for delivering other natural or s~~nthetic tl~erape~z~ic rr:olecta~es Ao4~
tl~e treatmea~t o~
i'~t~nlc%'~tlS d3SC~3Ss,S. ~~11e rT'L2~or lS5?.ie IS ~:1"~lc:'Cia.%;k:g;St3lC
reSIStanCC 't;d~ ~a'1_~.,'ilCal ell'ld rnecnan~val c~eg~adataon. :~l~crcc~~:,~~,e:es ~na~ ~:a~~ ~<ar~~ly ~w destrc~~~~ec.~ ::~ co~~c;~~zons corrspatible ~r'itl~ life ~v'ould proir~s~~ she d~az~~tic~n of ef-fictive trc~rc ~entc. I'~ost:
inporta~~tl~-, the~;~ ~~r>uid inipro~-e ehe sa~~eaq,~ o traas~ aarltin~T ;-ir3~~_t.'ans'ected bioeng3r~es~red cells, immortalized t;e;lls or stern cell ~~erived cell s.
'I~~is ~nel:hod ~ot~ld dccrCaSe _t~rle r~Sl~ O~ ~%lral dlSSC'rrll.t cai::;«n ~~i16~ 1~'i~egratW'~l~
lt"ato the Y'~~Ci~'.C.'~'~~:'S ~ei2OZTle 1i1 Elle ~rn~.er and o~ malignant cell trans:fo ~n~.tiosE an d clis';s~,mination ir;.
tree t~,ree cascs. '~~~e~~
would ailo~a,~ safer gene therapy.
with Ii\ring eeli szlr~.-i~-~1. ~t~Tsin<~'-~~-rw:~i~io-«-rzixro-heflzoyioxy'stacr~il~7c-nis:~e (~'~r~l~-I~I~:~~~ a p~'~utor~z.~',tiv'~t~'E3I~,' hetel'L?~ii'tllCt1()axui i~?'~sSIII's.ast '~~~
ti, .~3~, VJe vx''6~Yd', s~IJ'iE"' tCr 2IIe~.').~S1'sI%:~~e is?ets iI-I covaIeIItlv linked A~'.=~ rrbE,~I~ral~es, v.-i~h<~ut aI=feeting isl~w°t cell via'oii~t~'. Tr; thc~
present ar~:ic:ie, ~ISing a I~zodel of isle: ;::~Iicroezlcr~psulxition, ~~~e d~:s~°zv~e r?le method alld its ezFect on rnieroeapsLlle eharacterisa~ s end I'~Inctions.
~hl~e ~1-h ~'dro:~ys~;~ceil'IiIZIid residLh ;~ ~?~~~~-1~~~;~ v~'as f'trsi ~vo~'aI~-.zlt3 J' iiuilsed to ~°L.~,, ~zsiil~ an exoti'Ier.~Iic cher~Iica? z'eae:?iorz haa: ~-o~uid d~irrla~~e ~i ink; ceps, I~zzt be~ore t~~I~~
~e!Is ale Iilv'oI~'ed IIi t~le pI'OCs::ji~T'~:. ~~eil Islets.
'I:~.I.YI~JhIilSed In ~:<sIC_:ii~ aIt?Inate C.~Y°fa, '~.Eere inea~ated with cross-Ii~n:er ~b:rived I°I.~,, then ~aI al7inate a~ail3 to io=zn Edhe o~z~~e~' I7iC76;oIlapa~Ii_9Ie sheet. lq~llieil th a j7i'~,'~;tb;fc'~tiiirt is atl~3b Fir:l_atei~ ~~Ii~h a '~~~rf~ 's.~T3a~, -~:L2C' phot:oactivata~le phen~'I aZide I'esa«e «i~ tl a~ cross-lisrke~ I°eacts rvpidl~l ~~~ithiz~ a cahool~-~,:c'~I°'oon J~ F.~ eaT'~«n-u~'CiI-~'~en'~7'v'II~;~ I~~tC? the ;7l~;nate C.~efLa.Ca~ StxliCi3.~is:, ~,Yeatlll~ r'~
co~;'ale:It link oetw'een t Liu and aI<~ir~ate i~-o~~ ~~oth t~~e, micz'ocaj~si.ue eo:~e and oLlter sheet.
~lY'<e Ic'it~er I'L,'2cC~P6?n Is ilot I2~11'iY'~IL'I ~C)r 'Iv'112,~'.~;
~v~CLis.
t h.e presence o-~cov'alent I~oz.Inds ~~~-~~s conf~°rn_e d tay zrcuhations irI ~: str::~ng alhaiine sJILItIoI'I 'p"~ i~~. ~n standitT'd 1111CE3C~~Sl~f5, LI~e IrlteraCtIOI?s ~e~~'i'eei~. the ~C3si.tl~Jel~,' ~.ix~i~e~ ~~3~~, ally the I'ie~Tll ~~el.b' Cf:~I'~ec~ cz~i~Ili~te e~~i ~3e ~~"rt'~~iel~I ~y' nei~':al3ZrII~ OY'i~; s~~ the Ch aI'bed ,~~I'otaps. ~Il ti'l~; pI"eSeYIt Case, '.iSlr1~~ G.~~ ~ilt~Iln~w ~LI~~eI"., Vii(: flrs',i~~.raaslZed the h~~_, esIdLIe ~,%~hlCh h%~s ~?i ISOeICCtI°1e ~:~~~I~1~ _ ~ ~',.~4-. ~
~ile~~iC~psLL~eS '~~'k 1e IY°W'd.II3wzEeCi In c. GL~
Iv'~.;IIiC ~itlli~eT' 2~ p~ ~ ~ Cd.~'. a ~7~i ~~L .'.gin 'zt;~1 I-~ost sfde~~yhal~2 c'3YnIIIC~- ~I'~a,~I~~ aI'e 22e>~~tl'f~kls?e~~.
standard rnicroeapsules, zlsed as coll-c>ols ~~~li~b~re I ~a~9 I~apadly dissolved and disappeared a'I the SLO~LtIoi< W'Ithirl :.~J seC. ~C'~I~LI:t"C i ~~~ and ~~j. in t~Jt?YYr.'iS29 dlllt~'o-vapS?.saes pYepa_"ed ,~~'ivh ~I'1~-->I~~ derived i IJL did :'es~st to a~ in cFalaation iz< chis ~II~°alil~e solz~tion, v~i~~h s~,-e~l_izz~ percentage of approxirnavwf% ~~% ~;~ ~z~.re 1 ~ az.d 1~~, z3.}-.d a rnieroeapsule reeo~'ery rate of '~6.5 °,% ~4.3. %af~ez- % Fears rz the al~~:.li~e ~;~.zffec. -they ~.z-e rez-r~airli~I
rztaet. with a rzorinal round shape alzd a svn~;o-tia srzrfac;e.
,.
~eYlatl~ Y)I ~~?e er oSS-1112ker S~aeer ar'~ ~. ~22Y t°sa~< ~', ldSlr~
th6: ,~f~l<c'~ llr a CY ~SS(3lLxtlY'szI test, t"~~~'o SI?'Miiar'"~31i~toaeta'atatl3e eY'aiSS-t:!:it~erS ~:~~:~re e~'a~3.i~ted: a'~.~eT°~i:'~~~ s~re'ld ~z:ifos~zecinirrzid~rl ~-(~-°-azido-2'-.~?it~opner~srYaI~iY~.o~
;~exanoate ;,'~zt~.f~-'~~l~l.~iT~. The orli~ difference between these tv~ro ~c~Ieez'l~;s s tg~e length of the c~Aai;z _joirzin~; ts'~e t~~~
reaetli.''e ~?rC3llpS. ~UI'IO-~t~~~_~i',.~-1 ';°7CiS:'i~.',SSf:S 7 ~~r'~~'I1 ',.rlld eil~~. altrisge't'i ~~'~rrlS LtrJriI2Yr°~~; a S°1~5a~er artY"a Ol' t ~.~ l~ . ~li'1Ce t' I!~ ~-'.°~7 ~~ ~laS, ilC:r Spaef.~" arrYZ. tA''9e ~!'~tStEil7.eC; bei,~~s'f~erl take tw~lf9 reaetive t'esidues is orzy 7.7 t~. ~;'vi,_e:= zrzi~:rcea~szzies Yna~ie with sv~rlfo-~~,1~'~~.-den°ved ~~z i~'ere suhazitted -to the aiiCal:z~c ci=ssc~d.~av~voYf test, ti a;' complev~=~i'~' ~i~sappeal°ed ~~~.~Y~ the solutiarz ~vit~it a few rrzirzutes I~teehaaieai stress test ~9~. Mieroea~szzies u~°re a~i~ec:i v~~itiZ 3 rrzYm diarrzeter glass beads iI°~
J Yril p3o>~StVY°e_~e, tLl~7eS, ~I'~fyd ~I1 ~. 'v";',:.°tie~~
'~~'ll~,el Y''~t's:EItIYL~?,? ai .~9J "~3T'Y? It(9I" 7~ 11i3L1rS. l~t t~'IIS
sloe' speed, tr°~e eentrifuge foree is v~~'eak, arid v~rl~Iile tize tubes turn ~.~~side dog*Y~, tl~e glass beads anez rrzieroeapsules I~o~,'e rapidv i'oI-i o~~e end <zf the tube t~>
tiZe orbs:. s,u~orrzivairz;g rYE~ieroeapsules to thozzsands of strobes arid ~;z'*~zsi~es 'oe~wee:~ beads arid the t~zb~°, wail.
b'~urirzg tl~zis test, r°rzicroeapsules, ~.oYetwiaing ~? l~.l: ~ iTt_.' l,~befed d~;~~ararze are sLzbrrzitted tc~
rrzore arid stronger meeharzieal stresses tl~zan i:~Ie;~ are lilcel'' to enec~uater duriYrg a lifetirrle irI ara. irrzpiantatiorz site suei~ as the peritoz~zeuYr~ ~.rzd irI ~
iorea~tors. 'the pereerrtage of broken rrlier~eapsuies'~~igtzre 2 !-~) was ~Z fold loaa~el' fcr r ~ieroe;~.~~szzles eross--iiahed usi~~zg ~.~-~-i'd~~ ('~.4%~ than for starzdarcl rr~ieroeaps~les ~~3.3%',.,.
fs~l eorgtreis she~,~~rve ~~'igazre 2 ~ aad ~'~ negligible arr~oz!I~ a of dextrarin the stzpernata~:~It.
~;onzlrrrziY~g that ~,.F y -/ A , x "f ~~~crc '~~as n~ de~tr~a~ leal4irg ~~t :~~c~~ un~r~k~,z~ r~~ic:;=c~caps~:les.
~~.~,~~,~~,~~~r ,~ ;x ,,..
.-~r. > , e° ~~a .,r ,. ~ - ~ ,. - , ~ ~ x ;:
tr , r 1T .fFxy.a. .:
' '!, ~ ~,: ; ., H
r ~~ ~~' ~ ~ ~ ~r ~'~t~
c~' w ~~~~~N_w,~~o. _ ~_w. ~. _,~ .~,w,_.:
_~~r_h~__ ~ .~~ a.3 ~~~.~~, v~~ -~ ~ ~~ ~ a gas do r dI_F~~r~'Tl~.eS clltrc acs ~1~11trOl~s et %'s ~~~ i~._~1 ~'=;ij p;~~IhIadJ'd'.r i ~ c'~vaYlt-~'~~;~°Yy zur~ pllraSc pa" ~~ '~1~:
str~.llg$~'1 3~the sta.rl~ard l'YlICrr,3~a~.''s:cs tl:i~~. 1~:. t~1=s C,ssay b~'cts p~"I~:eJICi~I"S~j~' v,~t1I211Zf.,'<~ 3a ~tner c:~~e:lrner~ a ~zsing ~raeta~ds ~c~~:~ era t~~ ~rrprc~ ~'e .~ ~c~-~c~ps~.~.~ ,'~sasranc~: ; ~, ~. ~, I :~, ..~s 3 ~ , t x:.,. ,s~~., ~7v r~
.,.4 '~'". ~ a,m,..,.., ~~ ~ ~,k s ~.~~. ... z, ~. , :~~'9-~," w -:_ 3 ~. ~ , ~. ::~e w,.,: ~ E~ r ,.,.-q ~ 7.... .~ ~..~,..~°~~ ~ .,r» ~ ;.
~ :f=~. ., J -x ~~ , is T l'" h :. gv .~a.~, - "? <r~ ._ .n.~P . ., ( ~, - n ...its .. ,r,."s w , ,~:.~, C"s>... «.~.:~;;,~..~t a"-'a...s - f..,2, ,s~ ~p.-s. z,. ..a.:
~'x~~,'ez. L S" s , W~; y _ ~'s.~E, _.s f .' ~' ,. ~S~ ,'~,~r !d 2 ':x~r. : J' x~, ,.~L, L 3 ,. ,~' rs ,...>°~, . F -.
~,~ a ~~: : .>e;y..,z . ~u " .... 1 rt ....x s 5° . , :;. a ~'. :'e ;,. ~ r,., I . .r. 'ø S/, .~ ~'"(, x~ ,. ~ ~, t.
"9 . , ~ ': a' . vT, ... ~~ 1 .. e. ' ~ '_ ~ i~ , , c t < " .~' ., :' . -r ~~ - e~. . t ' -.. , ~. ~ .
, ~ , t. , '.
-. r , ~.: ; ... <, "u s :' ':1t3 a~ . d ~ ~~~~~r s~~~., .~.~o.~~~.~~r~ .~~ ~ ~~~,>~. b~~r~ ~r.~ ~, ~r ~x~..6~.A~
~~,~.~.~.~~~a~,~,~~«a ~t'adics ~f rrzic~-c~caps~ie mcmi7rane, r~olecu~aa ~,rcig~t coat-off (1'~I'~pJ~~)9 ~aiz~g a prP<~i~7asiy p~ablishcd rneth~d. ~~, « , sl~~v,-c~ treat c~w~aPc,~~
cr~ss._Ii~3~~ir~g ~~~~.~~~
a~~~zbrar°p~s has ~~~ signi'icant e~~cct ~r~ r~~c_~a~ra~~ p~,rea~ii2ty.
both c~valcr°,tly cr~ss-ii~,kcd a~~d standard f~icx~capsulcs cxcl~,~d~d ~part~ti~a chr~~atc~g~'api~i~:
cc~c,ffic_~er~t (i~$~,~~
~.I~ dc~trar~s ~F~~ith a.'~t~i >_ I9 i~~~. ~'~~isc'v~sivy radi~~~s ~I~~>_ ~.~~
~~:~;
a ~~~~"~~~~~ ~~~~ ~ ~lYl.:~ ' T~~t~krlS V~Jtd'? ~' ~,~
>W5 _I~~Sa (R~ > 2.~~, v~~hcreas do ~ty~arzs r~aith a a~,~i'~%t~ < ~-.'~ k~a ~;~~~~ < I. i~ a9~d pr~ieir~s =v~~ith a i~<~J < ~.~.2 k~a ~I~r; _< ~.'7~w di~I'~s~~d i~~-~øIy ~ k~sr' =
i.t~~ txv~gh 6:~~th t~rpcs ~
a~crn'~rar~~s. .~ he apparcnt discrcpar~ci?s ~ct~~c~r~ the .~~crri sari~a ~;~
carl,~hydrat~ aad p~°ctcir~s arc daze t~ the fact that: dc~traa~s are !iz~car x~c:at~°aI r~:~~Iecx~s~~~s ~rh~,reas prctevas arc gl~hular charged rr~~lec~zlc.s. It is n~tc~~rti~~y v'~at tg~e ~! pr'c~~idc.s a he~aer relative esilrriatso%i OI rr3,oleC:.lle d~ar2leterS t~Ic2~ the '~~J':r.
Lell 41a43111ty. ~i"xe 1<c~l SteCS of the yrocess S td~e E~t.~:IC:'ata~i?
ei'rf CCn,%aiel'2t ilrll~:S lay ju''a~ ~~
il:LiiT'nTlatIOrT. ~-a 'tlv'a5~'f:Ic~gtl2 Of ~L;i-J~~~ n1T-.., sCCio~T.°rl t; lil~t.3~e I2l~rx.irY'~~1 dell dc3l'TlagC" YN~S
selected. f~0ier~t:al cell darTlage.s 'v~'er~c~Ia~L~aie~ ':is2r'ag% c C$~il~~e STa~~i ~Jia~"Jlllt~.' test at"~d rite obs~:r~ation of ~ecro~ic centres or fr°sh islets ii:nder a stes-eo~nicrcasco~c,. i~f zi:itial studies, a 2~ kJ%rn- ~~f~ dose, ~vit..2 ~~.~ ~~%it=u.o~a ~~a~~-i'~~~~, decr~::ase.d i.~let crdll via~ility (~~igurc J), whereas ~~'~'~i3-I~t(~~ aloe had ~~o direct ~;~~tovoxicity.
''~l~e cell dare°_a=4e is partially e~~lair~ed by a thermal e~i'c~::'~, since tie ~rei~aratio-r~
wa~~a~a~°d ~zv~ despite t'rac fact it ~yda.s ors ice. ~~$~ ir~vestigaie the hypotvesis t;r-xat the ha~~ful effect s dose relatL:d, ~:o~alerg~~~ly linked rnicrocapsules ~~~~~.c ~roc~zced9 ~~sing ~ large rai~~~.: of il~%~~~. doses. ~
dose of ~ k3/rrA2, 11.5 fold s~nallc~ ih~=z the =ritiaa dose (?~ :c.l/rr~?'Y :
as -e~uall,~~ effe~i~c :n ger~er=~t~ng co~'aaent links and aF=~~rob'~.~~g r~~croc~~~sute resista~!c,e. ~ :ors small dose ~~~as used (anl cor~y~red ~%ith the high dose wvherc i~die~.ted) for all t~~c e~peri~_et~ts re~or$ed is~ the present article, except for tl~e ~~wg~v~~c~ st~,~dics. The latter wvere ~~ov repeated, since e~era the high dose had rzo effect oAx Leis pararnetcr. ~~he srr°zaller dose was as effcctizm as the large dose ire improving cl~e;~3;~~i ar?d ri2et~har?:~c~:.i resistance.
~i~hc e: fact does r~c~t se,err~ progressi' a (~'igure ~; therefore, vt s~wggest5 t~:att the srr~allf:i pose ir~duees the ;same lc~-el of covalent links as the largez c.osc. ~l"gym results :~f the ~Jia~i:ty tests (hi'uie ~~ arid the e~,~als~~atior~ of necrotic centres (~i:~ure ~-j sho~-e~that tire ill~a~nination of er~ca~sulated islets With a 2 1cJ/rn~ dose of 1_;'~'~'~, ~,-al~~ or ~'iv'no~~t z%~.Nl~-~~3~, c~.~.d «ot affeci islet ::x,11 ~~iahility as compared with s~ar~d~.rd ~:~icroca~;s~les (-d ~~~ ~/~ ~ria~ili~y and l S~/t necro'ric centres f or eac~ of the 3 conditions;.
a ne cbjeeti~'e of tti~s ~,~ac:~i,: ~r,;«s ru ,we~r~~ls; ~ a ~:~iet~u.3d it~r ;~r~dus~irlg Fiery StrOrl~ rillCr~Cc'L~7SLaleS, by Crec,'itli~L"T '~'(3:'aiei2s'1?:s2~~C~1 i7et"v'v'een ITLICrC3C;'c'a~S~,.i~.E~ mf:lT'e(3rc'ine c~m~onents. The chalien~e vas Lhav: host r~le-t~ods si-iat il~d~~ce s6~c;k~ a link v:,-c~~~d se-vereiy atl~eC~ Cø~~ vla~)l~lt'EI. ~~10$~~CL1L'eitaC?iw-' CrC;SS-illrlG~.;rS
~reSel~ltt,S~ a ~K;t~:'n ~i'c,'~ SC~iL!flQn ~OeC;a ISC
the reaCEion indL:ced by Ll~ia~ ilhLiiIaarion oi'tire ~h~~I~y~ a~::de ~~l°onE~ ~~s Iesf; Iikeiv~~ to ridi_1Ce C;e~l Ciarl2'cl~?E;. r~~W~(3 ~7h~JtJi3<,iL~'~''=tc'L~lF,: ~~rCSS-IlrlY~;.rS. ~.~'t'I1C~I C 1~ ~a,;" C'n ~~J ~'~' i:2EI ~1~.'.li~~I~"1 o~ t he spacer arm included in the~I~ 5~lvzcture. ~ ~'er.u i .~~esti~ated.
~'~aI 4sA~~ropi"iate iel~~~~i :.~b tt'~C S~aC:i,r arrll redllC;CS SI r1C i?in ~e~:iCf: ~e~:'v~h.'~,Ii ~:~~If~tc'7tC ai2~ ~'~~~ ~C~iyirLezS, Vd~'tILG
c~~3O~v'1?1~ therd~'1 t'3 1~51r'zd tCJ~Ct~ICr c'~.'~ Flle~ %ime .9h~~J~,T
CrisSS-lirl~'sln~. ~',~hC :Ceiid~tS S~i05~If;Ci ti2a~C
sL;i~~-~«iJ~~~~-deri~:'~~.d ~'Li~ ~vres ;r=ef~ecti,'e ~~~~ ~~roc~ucin<,~ ri co~;al~;IaL rink ~.~'~th aigiflate ~>v~ld,reaS SL:C~1 c'1 I?I7.k LVaS II1C'iLICed ll:il:a~ .~'il~~-~''~~~.
i~~Il:S SLt~beSt~ xca ~:~le S~?a".Cer c'i"r-3~
~en~t~'i tS CrItlCa~.
x'~ie evaiu~2ion con~il-rr~ect 'h~~t s' ~i~_~S~J de:i~~cd ~r,icr;~c;a~sL~lc~s arc: ~.;'ery resistanto r~iechanicai and che:ricastresses a~W ua~.iil<_ei~~ ?o be ~~~~ecte~~ by com~aetitiorl L'wlt~l C~lsyi'~ed ril i~CCLIleS lr2 C~1C f.i'Lt,~ii~:>:~11~1Cr?!:. 3~a2t;
E7r4~'~~.k~i~.?C :~T CC)ti~z'vnt '.aII~GS i~LSLIalgy io~'.CaiTIrCS e.:~'treIY'SC. C~tlC~altlOIiS C'lt ~~I O~ Lvi2"l~Cra~:~iE' i~lt aEe znCt7r'z1~°lail~S'~E,'vY2~~~ ~~e~l ~IL.C.°.
.~2E;rei~r°, t~'leSLi rTllC$'OCd=5S'~'ie5 ~rl"C=, '..-'l:~t't~~~i~:i' In ~CStr~Yv;,~lblaC: I~1 t~lC; CGg~C'~'~I°1~riS <:11COLLYi~CrC'~a.
n L~le il'JIi'la b~dy.
~.'~~C'i~5~,° t31 e'i'=3~i0tflclCtl~Je'9~E~~'I~C~ u:C3SS-vzr"aGCr SLE;~l e'iS f°'b~~--I'~YC~~ ~;i CrC:e'3tC COV'c'~.~erl.t Ii~'alcs ~,'ithin trie lnicrc~ca~sL~~e rr~en,b~~arle is aCis~anta~~:Caus. ~'he ~~~-ocednre does not rewired l:arsii conditions such as drastic te~ler~ttlr w chari~es, .~tvong s~i ~~ar.atiol~s o. tile use of organic solvents. It does not produce toxic byproducts.
~iydroxysuc.oinimide is easily and simply removed by a dialysis step. Short wavelengths in ultraviolet radiations such as ~'~~13 and U :%~ can provoi~:~ rapid and severe damages in living cells. The classical aryl azide ~..znctionai grois~ used ie~r ~~~ for.r~ation of covai~,nt bonds is generally acti vateci vaith light possessing this hind of sh~~-t ~%va~~~I~ngtl-As (~~~-~~~ nrn). Ther~for~, a modified aryl aide groLrp ;~~as pref°~°re~. '~'~'~e ~rosen~e of the nid:ro group on the aryl aide group ailovved the use o~'sa~~~ long ivav~l~ngths ~orrespor~cii~y tc~
~J'VA and ~~mn visible iiglzt for activating the cross-iinl~in~, re~c~:ioni l ~, 1~~. The viability experiments showed r~o cytotoxic effects of the Total proced~.~r~, e:~_cept Lor mi.~brc~ncapsulatod islets Lxposed to higb doses of IlVA radiations.
ThP covalent cross-linking betv~-eer the t~~~o r~~mbrane components, alginate and ~l,L, insures a proper long-term control of the meanbrane 1~I'~'~C~. ~Iitl2 the conditions used in tl~e present studt~, the i~l~'~~) ~r-as such that =aextrarts and proteins. with I~~% >_ 1 ~ kl7a ~ I~rl % 3.4) and >_ 4~ ltl~a ~f~p > 2.9) respectively, cannot pass through the ~n~cnbra~=e. The latter uI'W corresp ands t,~ a n-~cleic ~bLid fragrnen_ off'-. ~ bases and. a peptide f~-agm~,r~-c of ~.pproxirr~ater~ ~ °, a~nin~~ aoi.d:~. It is not ko.~J~~t~ i'~ a virus derived fragment of this size could be inco~-porat~d intro a corr°~plete virus i~°~zoGting the recipi~n~
and ~~nodify its pathogenicity. T ho risk that ;~u~ix an e~a~nt o~~~~rs so~a~s minimal but cannot be completely excluded. Tlbe ~:nicrocaps~zl~ ~V~~ ~rouls~ certainly pr~;v~nt complete virus, viral proteins or virus release tc ~rosr= micro~aps~wi~
rn.ernbranes. Tlm donor cells could not be infected since the viral vectors are deprived of vhe components re~uirod for penetrating cell I)1VA and the recipient ~riruses cannot penetrate into rr~iorocapsules. T herefore, the additional safety provided by these microcapsules for the ~.~se of viral vectors is consider4zble, althoug~~ t~eoretically not a~solate.
It is n3tes~orthy that we ~.av~= pr~vaous~y found that. a~: hgh ~.:onc.e~-~trations. such as ~.0~%,1'f,l. leas a direct to~;i; efi~:rct on ideas ~:~~hedded in al~in~~e beads_ ~L~t not at sow concentratiox°a {unpublished :late).
The availability of these extrerrAely strong nzicrocaps;a.les -will h~,~
i~,seiul for many applications. In tl~e field of islet tra~~spian ration, which is our privnary=
interest, it gill allo;~~ a safer use of stem cell devived islets. a,~mortalized ins~xl;r~-pt~oclF~zcing cell lines or 'oioennineered irasuliz;-produci~~~ cells. It tviial;',~ allow a sales v~s~ov islets or eels co-encapsulated ~~ith islets, trans~'e.f_tecwith tl~e gerv~°s of molecules that pro2l'lote ales cell survival or irnmur°~oproteetio~~. for example, ~,~ve ~°ecently sho°:ved '~ ~N.~ r. z <,~~,~'~.r~~~that pre-zz~a~splant4xtion insulin E~ro«~th factor-II
{I~~-11) ~~ 11. incubations allo>>=~ prolongeu ~~orr~~alisation ot~ Jlood =~Iucose os diabetic mice, using a reduced islet cell ~r~ass {< 5~E%) transplantatiorg, ~~p~e also developed a method to transfer the l~I~-Il gene into islet cells or into nor_ islet cells that can be co-encapsulated dvith islets {pending peter=t). 'fhe goal is to provide continuous i~~ v~vo 1GF'-iI supply to islets, preventi~~g islet cell apoptosis and necrosis.
~.rev~ertl~eiess. to obtain stable gene expression into islet cells, retroviral-:used vectors are -=.equired, ~~r~icr~ present sigr°~ifrcant risl~s. Using non ~eira.l vectors, vve heave also Iran:»_ecaed Ih~-l:l into cells fro~~
an irr3~nortalized cell line. l~y seective elirnir.~ati.or3 of non transt'ecGed cells, m-~ich cannot be done with a rrrulticellular st-r~cture such as islets, a cell life with a stable l~~-11 gene expression was developed. T he use of~o~~ viral vectors erlls.c~nces the s~.frtn of the method. ~'ever'eless, immortalized cells also present a rich of ~maiigrla~it tra=~sformation and dissemil:atior~. '~'lle mieroellcapsu'ation of i.llese e.ells in eovalently linked API
mieroeapsules Mould improve the safety of tr~es~; treatments.
Tile same principle may de pplied $o numeroas applications ire tt,e ~hcle field of gene therapy. In recent years. :gene ~~~erap~,~ l;as r aised great entllusiasrrl, rein ~ perceived as a revolutionary technology ~~all tile potential to cure almost any diseases, provided tile Genetic and molecular basis is k~~o~t~rl. t ~ta~.z~e~-er, tl?e entiliasi~.srls vanished ~~;~hell it vaas realized that several obstacles =_a.Illper elir~ieai applications. It evas recen$I~,~ reported that reirovira-Based veetors, uses to permaallerltly tr~:nsi:er genes i~Ito cells, rr~av pr°f~rentially be inserted illio or near an onc~ger~~:, alld cause a, le~l~aerrli~,-iik~
disease. Otllcr types of potentially safer vectors, sue: as aaenovi~~as, adenov:$rus-assc:~ciated and ~~on viral vectors, are Being developed. Ilo~eve~~, they us~_aally provide only a rtrar~saent gerl~; expression. In addztiorl, many of the imrnunc iobicai d~;f.~Ilcc systegns., ~~~hi:;.~~
prote;ct the: host against ~al~Ci~tVpe InfeCtlC)rlS, I'ec'3.Ct il~~,I~7S~t tile veCtJr allt~Oz neW
~eI't'~; ~;Ai:)d ~CtS e~;.pre'SSs;L1 a$ tile surface of transplanted cells itc~:° recent review ~~1 vectors for w~,r~e tllera~y see rei;.# ~..
1 j. 'ref ~-1 = Thomas ~~, ~~~a~.ardt A, l~~y, _~. ~'ra~ress anti prable~-~is with the use af~ ;ral vectors for ber~e aherapy.
1'~at l~.ev ~'rerlet. 2~~3 l'~ay;4~~~:3~~-5. ~aviev.~.
ulicroelzct~.llsatat~oll of these ~io-engineered cells i~. almost inc~escructibie i~Iieroea.ps~le,s v,~oLu.d ~~fe~~
a proteetioll against i~~I~unologieal reaction and ~isserz~inaaio~ o~'I~aliir~aant cells, aiio~~ing safer g~:~Ie therapy. ~'hatever tile progress tvaliserl. Tai viral vector safet5r7 it is utllil~ely tile adsolute elimination of any risk can ~e .~-~tair~~;~.. ':~?le ad~itiorzal safety provided by microeneapsulation in cowalelltly li~~~et~. r~ieroc:~psuies vrovid er~ai~le the etllic;al use of gene therapy in mal~fy clinical applications.
~i~ 4',(3YlC~LISivI'1. (Ile iiSL. ~ A"-''~~~; d'=:1_"iT ~,C'v"v'lia~i iIZG
t3kt~~Cet.'',t3'~''l~ei7~C CTt~ss-~i?ihe~'"
=~~~-'~~~. associated w'it~l ~,;_.~' dc~s~, ~qv ~~, iiaur::,~r~at~cY&, i~ld,.~~~:-.~! a ~~v'a~~erf~ lv:~~, q~,r '{J°~$~,~'<'a',C, 1 ~ '~.1~ ~'-"~~~~~,.alld e'i g=Yiu$y. ~"~:'als aYl~:~~'16)L~: CC~i'lsltli'.T'a~~~~:~ 'a',Yl~'l.aPle'.."~JLi 11i1Cr~3C;a.'I'an'~'SLI~(:
r~sis~ai~c~ ~~ seve,re chem~~al r;:~c~ a~f~~,~E~n~~a! si:rcrsses. ~w~il~: , r~s~.r;in:~ i3.~ des~.recd :~mlr~rar~~ I~ls~~'~~. T'rlese r~u~rc;Mas)sues ~.re, ~H-°e;~°y L:rn~d<~~~' LE_; ~~.~ damaged: cr d~str~~r~d ~~
i~L~ e~~l:~'lr~rlfiY'~21$ ~.''aiLl2d :r' i~le ~ L?~=n ~O~s.~' Jr is ~7?53r~~C°~7rS. '~'.ed~i 1"~':S'~~,d. ~ur ()v'~;r $'~i'~
'.'ei~r:i, 1S e'CtrelTle C:12C'1111Cai C'~'C~°t~C)t2s, ~Li~'~.~i ~S
'~~~~~'.'u'd'i's.eri;,~..p ,~,iii.Lld~rC~ llliCl"C3C~3.~)SLe~to',~,5 diSSC~'>Ct W'ii~illi ~ C SCE:. iIi IiIG ;ei~Tli: (~~iCiii a()I1S. i ~LC;'y is,'1"L)~'e l!~ 'C(: ~.3$rei~l'O:il' r~SYS$al"iL 'i~'u lili.C~'ta:CIlCa~ seI'eSSes. i ~e T3ler~a3ta CiS~rE~:7C ~I_~~,Ci :1'e.elT1(JraYIC OE'~Yil~:,a~%ill$~' s')r 1311f:ra'3eTl~c';~9;;Ll~c'3$Cd Cei~ ~~'iui)I~iz~. i': ~~~l~I ~:'~G list Ill' ~C,'r $~$W sc')~FC. Yi.=;7.Sp3~1~17~LaLiiTi1 C~" sezl~ ~e~~
dC:riw'a:,d, i!2~lliC)r~~l~lZe.'~. ~f' ~10e,?'.=s,?iiC>',ied ~elz~s, aYi4w Ids"' 1'l~.irfle~~°t;'sJs t~~iIC~3$1(uI2S lii OICe?'eai;$drs. ~'lil'sl:~~', fflls 1'T'Te$~b~>~ ~'elc~.~' a°.Y>a~i~
$ile ~c'11~:Y Lfi_Se flI <,'ir~il ~':wCLC7rs ~C~r g0?t~
~_a'l~r~'y'~~J. °~~?f.',1~$~re, ~%:''~ are ~Y~ )~'11~;?, a ,~t~~:v' ,rr~~e :s)r ilOl~:,r~r~YIC:o3~lS~~nGit~::. ~i ~1~15 W;',~'ii LISe '~c~ ~~rc~tne~ trans~~arlted Bells ~ac~~lj t:~le, nc~s~ zwl~~r~r.~.
s~'s~0..rrl. ~JcrYhrers~ui~', we pro~~,~se ~~s LlSe «) ICrC~eC:$ t~t~: ~l(3st i'ri'3Y't2 ~:rC T"a#?s~)~~a~.
~~~~~~°i~l ca~~ Y~c~~ent~
~~d~~m a~g~rzate ~~G~~~l~le '_,~,~~~~"- ~~_% 1~~,~,~~rc~l-~~~ ae~d~~~'a;>
~~~airaed ~t~~l ~S~
~v~ ~I~~~~~ ~Yl~. (~aYl ~~eg~;9 ~=:r='~i, ~~~)y...~_:-~,ysi.~le ~r~~ ~i~;r:~la ~~~~erl~i~wl ~'o. {~~-~,~Lhs, i~~~~) alld SLII~~o-~l~l~~.''~~~ algid :~~i~~.-i'~1~:J~ ~1°vm ~ieree ~~~e~c':~rd, ~~,~. ~s~ets wrxe ?s~ia~~~,d ~r~Yr= ~a~-'~Ot~g ~Nis~ar s~a~s (v:~e~rl~ -~~~;..9 ~~--~:c~rlsE.aE~S~, ~.~~'~ ~ar~ada~
~t°~~at~oi'io~r of covc~let~t'ly-li~tie~c~'.~oolv-l:-z;.~irc~ c~r~C
ct~o,~a~-lit~~;~~°s.
~:Cpet'IinentS LiSIn~~ p~:L7~Cs?L~1~-cli.%3~~~' c:i'GSS-ii~iICe:S "~~e'~1'' -,;~t'Ie~rYn~~ in a !i~.I'~ idJ~IJE'7 L~neii ~~'!'~ iilLlminatic~n. ~L:l~e~-~f~~.T°~~ f'~ ~~ cY a=~i'o ~-~'~s~ fuisscfived i:~I dil"rE,t~v~s:~lf~%ide ~~%i~~~ v~'~re rrai~ed qavitlu i~~ ~ ~~~s~c~Ivvd I~~ (t,2~~~~ b~~a~e ~L~i ~e'~
pA °: ~,~., ~~~..~, af. a ra~i~
CI'~SSII17~~~YS:~~,SI3P J-s~ 1:~~, I"e~nL=ilill's1~ ~ ~"!vLlsS a~ "~',.r'~'i1 get'Tlpez:ai ie ~n'~,1' ~~Srp-t G~''~I'~l~~Ifi ~,~
-°~. ~'~,I_,-Crc'3SS°il:PiCvT' ~,'daS pe: lsaed ~I'C3;;T"~
>:~SF~i.R~.,n. I~~u~Ll~u pI',~~al~~ ~5 ~~y' r~lal~Si,~'s ~'.~ainJ$
saiil~e, usil~~ a 6t3(~~-~0(~(~ ~a I:Ea~ie~L~ia~ v~e~~~t ~~La~-~~lCmer~:u'~~'an ~ E~pecttp~I~
~'O~~Y'~a$O:"3~:S, E.'l'.%~ ~, a~ ~r«~ f('~ ;~;',i da~~c ~°vi~r"''2 ~~'il:i' It2~C'~ILti'1_ C,~L~I1~C;S. ~~ia,e 'AilOC~It~~f~ ;:>~_,~
vas qiaantii:i~;d ~~'3~h i:fi~~cW~.:~~ ~ rc~ei°, ~~,ssa~' ~i~ze~'~~~~;, i~c~~~u~'r~., ~~~ ~:nd d~IL~~=~c'~ ~,~ith Seri?n2 a~ °iiie appYOpaia~t; e~IICC'ilv.'zT~'Gn.
.as!~i~t~o~'~~,~~i'p tt~re~ct~°Ltt'iot~
1~/~icr~capsLlles vmp'e ptwpv°.~'ed a~co:3-dng j.~ a p~~.~:~Lt"zy isLl~:iisim~ I~I~rs~z~~d 2i, ~~
d'v'?~'~a f~'?f,' ~~~iL)VJln~ Ifl~dlrle~~Io 'I;~. ~., S~E:IIi~I Sv~Ll'r:~:Olx L~'~~ i.t~RO~> 3~LI1'll_Ie d SC~~rl.Yn c ~~lYla~~: 'vvaS
e~~sL~ded ila~C~ a ~a~il c~5~ IJ~3nn~i t~;~.i~i~anr. i~~~~.re, ~~,~f~'e~'~d ~~i~~~ fp~-~~. a;'h:, cae~:iLt~a c'i~.,~,''lFi.alG, '~,~iC;d iSP't3fS ~'e~~ In 4'.~~;' ~i~~ '=_7:i:~t3 ~
i>.'dS''~ vc%%'V ~rL~ a~Jt~~il~:x ZC7I' :°.- Y~IIrIL~ceS, ~jIaSF~~d v~r'ILii SaII'iI~ and 1'e.'ar,%Ll~?t ::~>:'.t. 5 YiaII~ILI?~S
PI:~~C; ~.s ~°~Q -~~t%'~' c.~3Cl~I~z'Qø~IT'a~E.. :~'":,~''a~t~~~, rnir°°r~~aps~a~es -ere wrasl~ed ~ ~~Ice v°;j~n saail~e.
~..:ps :w~ivi~ f~ ~;vus:~lin~~;,o de~iv~,d ~p,~.
'~~ieef; ~9eI'~CB.I'~neC~. °,lndG,i CIIIII Il~~~~.
i'~~~IC;iCi°,a;:~SL~eS 'v'~'ei'L, ~zlL.'iI Sl.i.'~,Snlli~~d ~~ ~
~°t~f~? ?'clt:i>qy5 (~iacs~.iig~gc 25~,' ~',-'~25~'~-~~,, ~ ~hr nroda~,~. ~,pg~~~d, ~:f'~.~ a~ _?
i~virr~'. ~i~i~~c~c;apsR~Ies pi~p~.Y~G 't~'t$?2Cil,l~ ~f~SS-~II'I~C~1" aI'z~ ~~~f-h Ia~Ld~aF:tnG'~,~1&3II
"/~1'~~~: F.'3;~~~C;c %.?.S (;e)n~I°C~~S.
~~;~zot~s~~cztiot~ of~~h~~t°ps~~a~~ Er'a cr.~~%~E'~P~t~ link c~;'t9~~~~~ ~~~rt~ ~~~tcl ~lg~t~rt~
A suspension of.niczocapsules in Bali a a%as ad~~tsted to ~l-1 J % l~~v adding volume oi'2~1 glycine buffer l~~ ~ i2 ~2r~ ylycinc:. 14 mi~~ IVa~I, '?~ ~~ia~I-'y.
~Iicrocapsules were incubated in this soluior ~cr at least ~ n~inEates.
ivlorphological chanbes were follovaed by rnicz~oscopic examination.
Lr'VC1i21L1~1LJi2 G'~~YYll~C'~'()CCl~JSldl2 ~~'1J>.'~iCCII fl'."~1~
~llGlC'tlLJ;2C~~ Cl2Cl3~Cd'CI'f'i~'lSit'~;;,~.' i~/:icrocapsuie mechanical resi,st~r~cc: u'as ~.val~.~ar:cd ..sing a ~lv4aa~~tita3i~';: ~~xtethcc~
previously reported ~9j u;itr~ tl~~e lollowin=~ n~odil~cations. ~fc ca'=_=_caz~ate tl~e ~erwentage og broken microcaps~ales, the an~o~:r~t or ~'I ~'~~ iahe~led dextran ~r~ the supernatant and in intae,t znicrocapsules is quaz3tified. ~ta~.dard rnicr°ocapsules ~~rre dissolved in 2() r~~I of ~.R°~~ ~la~l buffered to p~-lI % ueh C~. i,~ ~tycine ~s described ~i~'.
since ti~is rrrethod could not break cross-linked micro~rapsules, tl~e ;after u-ere digested by the addition ot~ 1~% v/v xmy~F _ c..$ .. R ~~ \ °ff.~ .~--,~. w$"~r..
of kip 1 i ~T°~ and incaabated at I ~~°C~ <o:r I ~ rrsirt~ctes.
'~ or nornnali~arion, t~~e sa~~~ples of the standard curve were digestev~ in same co~~ditons.
Evaluation of rnicrod.apsule mes~~hrane molecular ~.~'ei~ht cut-off (~'G~~C~) ~,~aas performed, using sire e~clvsi;~n ch~'c~n~~to~raphy as preE~io~~aly described {R, ~3). .l~
col;<<n~r~ was rr°bounted using the n~icroc,aL7sules under study.
;~~L'r'~-de~tran {'~.~~fl13 kl'~) ~:~ras used to measure 'the column dead or void vo<urne {tJo) and glc~cose {1~~.3 da) u~as used to dete~'~nine the column total vourn.e ~~Tt). l~~arh<ers of diffe~~-ent rraolecuiar u.~eight {l~~) and viscosity radius (~r~) w~.re sec~xaentially run on the co:(~~mn : de~trans '~'~li~ : 2,~0~
i~d, 19 1:~, 4.~ l~; ~;~ ~4.'~, ~.4, I.';~, c-espectively); proteins : bovine seru~~nA aiburr~ine (,I~~GJ 66 k~; ~~ ~.~), ovalFournine {~~~: : ~~ kl~; R~ 2.~ø~ and carboxipeptidase (~.~~1 .~.~ ice: Rri 2. %). for each ~~°aar.~er, ~ pnrtirio~~
cbro~~~ato~;~a~~ic coe~~icierlt {-~~e~) was ~aiculate~ ~~.sir~g the formui~: }~:,~~ _ {~:re - "EI~,>) I ('gin--Vo;, ~~~he;re v'c: is '~~m mariner retention volume {correspondiraaT to the ~3eak of the elution ~sraz~le of the marker). ire°ly.
permeable mariners have a T~S~~ value near I.~. E~cl~a~.e~ mar~ers have a ~,~~
value near ~.
Fffect o~'~the ~nicroer~ccapsi,rlca~~or~ ~neil~c.~cl on ih;~ ~~zcrwivc~l of'e:r2c.~b,sralcxter~ islets ~f L~:~gerl?r~rs islets from 'V~%istar rats {3~JC~-=~-3~~, ~'haa:~a-I~ive3., St-~ or~sstana, w~~:. Canada) ~.z-ere isolate tasin~ a ~ublisbed method [2~=t~~~ci en:~ a~suiate~in stan~ar~l or eovalenti~;r lini~~:r~ l~P~.
_micr~ca~sules. 'i'he survival of :~-~icroez~caps~:lated islet cells ~~ms evacuated i.=r ~itYO usin dual staining vith ara_nge acridine anc prapidiu-_~z iadide and observation under a iiuorescent microscope f,S-~ ='i ~~oresc~:~~ce spec:~~ophoe:orneter {"~e~win-hlrraer ~',~nada, Travel, ~Cy c;xcitation ~9~ nr~: <<3:d emisson ~?~~ nmj as prc,vzo~~siyv d~;scribeci -='~-~~~~
f=resh {unstained) islets were cxa~rined under a stereomicrasccpe {Labavert i~S, Leizt.
Leica Canada. St-Laarent, Vii:, Canada;, anci ihu nurt~e~~:.r {°r<;) a~~ci size of~islets ~%ith necrotic centres mere recorded ~s ~vel~. as t;he di:~aneter of the necrotic ar~;a[~Sj.
~'~a~isPicc~l cancad~~is Resets are expressed as mean = S~'~t. The differences t>etwLen experitnetaai ~ro~ps mere analyzed by ~.~npaired student's t-test ~~ati~ ~ va~ne5 less tP~=~n i~.05 ~oz~~sidered si~nii'ic;an~..
~'~ UI~E: li~~~IMU~ 50 ~~a irE~~~~S
z iba~~°~ 1 : Standard arid :~:\'~~-i~~~S cr;~s5-fin<ced nlic;rocapsuies vvel~~~ irzct~batc;ci ~Ilto strozlg alkaiille pH (p~=1'?~. .~. =~~tal~dard ~icroct3psules_ f ~:~
b°~orL tile illcubatioll. I3.
~tar~u2rC1 mlC;roCapSLlleS IIliO ~~t;'F~ir'1C; b~~~:l~~f~,' p=-; ~.~~, O~Si,.
a f~~u~J St'rC:I2dS u~tel' c'LddItIC3I1 0%<
the ~fl~aliz~ze buffer ~'. Standard ~:~.crocaps~~les i9~t,o ~lyc:ile L~;.xff~~r pE:Il2 ruiaer ~-~ s~:corzds.
. ~~~13-~1~5 cross-linked rr~icr~iaps~les bei'ore. vhe ir~cubatior:. 1(~~~.
~.. cross-finked Illicrocapsule into ~lycine buffer ply I'~ after t~,~,'o years, ld~'~. Since the ph ~-~aizle of the side chain amino ~~roup of~tiza~,~ 1~;'sizle is a~~g~ro~xr'uatcly f J,j~,. ai i~i-~ i 2 the ioll«, liitelai.tlOr'a C77(~tZJv'C,'CTl l:~~ ~, alld i,li':Ilelt~ l.;i I_illtfe?~I:i~C~z, f~IIIS e.""iaJtrirYle~L ~~Oi~~IrrI~S tlli~ the use :~IV~-~1~5 azld I~..i-'~~ illlzl~~~~atio_1 il~l~~na:e t'e:~: formation of co~-Giellt finks t~lat are resistant to ionic competitions.
~i~w~r'e ~ : l~iechanical ~'esisv~rr~::w c~i~n~ic~'ocapslales were rl~:«na~ied has the ps:rcentage of ~oroken zlicrocapsuies after a i"~ i~ours of agitation whiz borosiiicate beads. ~. Standard microcapszzles as well as the ao~:~e azld Vigil do;ses for cross-iizhec>
microc~psuies ~x-ere s tudied.
~. ~ozltroas cortairzirl~~ z~~i~~rocap:7zxles and borosaiicate beads without a~~itation is sho-~-~n as a control of tile Inicroc:a.psule ~reparatiorfs. ~.
~or<trols of a~i~ated microcapszHies wit:llout borosilicate beads irs izlcluded has a control of the agitation. C~ata are preserli:c d b~,s a~-~,ra~e ~ S~'I~. * ~~~-' s~-e~ 1 Vin= % E
~~~a~~ ~ : Isolated pancreatic islets ~~erc encapsulated into sta.~~ldard and cross-ir~lced ;~icrocapsules and subn~zitted to L~, 2 arid i~, IC~/z~~ of ~~~J~ ix-radiaLion. ~=~t 1, u., i ~zad i 4 days after rrlicroeneapsuiation, islets were double- stained dvitr= t~.01 ~rg~'rrlt orange acridirle (iivng sells, green) algid ~ l~ngi,a~I prop idvurn odide ~;~'iet~d c~~Ils, re;d~, anc~~ ~rere classified ur~zder ~luorescerlce r~ir~ ~oscoy. h~ata are presented a.s average _~ ~E~. ~~ p<
0,~~ ~~s sr_az~dard el:capsolated isvets (10'.x- ;~~'=~z dose), ~~r p<(~.dj5 vs cross-finked encapsulated islets (IO'zJ U v'~. dose) ar._d d p<s;~.G~ vs stands-rd encapsulated islets without UV~ irradiations.
n ~,-~aEues are represented k~~y llurr~~ers ir:'co~cs. T ~~ese data sl~c~-than ~l~e nletllod does not ai'te~;t islet sell viability, e~:cept ,-Yi~en ~. urge ~2~'~ k.l/lzl' j ~'°n,~z ~iosG~: i~~ used.
Flg~x~°e ~: Isolated pancreatic islets t,~~ere enc~ps~alated into standard rrlicrocapsules or covalently ~~ross-linked microcapsuies using io=.~-~ U~~ dose or~iy. at 'i, 4~. 7 arid I~ days a2ts',L' 1Y11C0eIlCapsLllatl0n, tile llr:sellC~' f~~' pleCrOCI~: (:eTllres In ~~~~;r~f:rrOapS'~lated lSlels 'Vm'aS
o'oserved Ltnder optieal n~icl°osc:c~py. Ti3~°
c°.~toto;~~city mss de.vern~lined as the perc;e~~~;age of islets ~r~itla a necrotic centre. .Results are ,~reserlted as the average ~
~F~.
~~ ~i'~~'v~I~:EL~~EI'r~IETdT
This work was supported by a grant i-om tl~e ~aradian oiabetes ~ssociaiion in =~or~our of the late t:mily C.
I-(av~~es.
/J..'~, _.. , To prevene this corz~apetitiora v,.jith chargc;c~ 3a~oi~ye~ales in thf°
a=m~iri~nmea~t, a ne~v concept ha.s been proposed: the irar~roductiogr ox co~,~~l~;nt tini;s into t~:~e ra~emda'ane oi' the rnicrocapsule~ l ~-17~.
~'or instance, microencapaLaat~~~ islets oi~ i=,~.a.~agerhans (~~Liets;, ~;~hici; coma=
insulin-proda,acing ~-cells. could he ~transpiantcd into type 1 ~ins~aiia~-dependealt; dia~etic patients. ~~icrocapsules intro~Lac~= a ~ap~r~er bc:r~~,eer vhe transpla ~t ~.ntif45en presenting ;ells and the host T Iympl~oc~-te a'eceptorsa prevea~siahg (hindering?) antigen recognition by the imnsune system. This a~erhod pz°otects islets against allogenic reactions anti recurrence in the transplant oi'tl~G ~~.rtcira~ra~Lxa~e tv:co~~ ;ss that has nitiaiiy caused dia~etes.
Therefore. anicroencapsulation T~~La- elimaaje tl~~e r~ee~ uor the ease of toxic ii~ramunosuppressive drugs that coaa:dera~ly iia~its the indications o~~c;ell traztsplan~~~ations.
~yloree~ver. the strength and diara~ilit~~ of ~nic;rocapsule men:hranes are.
critical issues for the safety ~~t~'arlSp~arltal~orl Of' ~JaV?a~J'tr~ii"J~eC.t'w'.~
~laoeyl,.~~'.I!leeru.Cl c4~.ls. lnCiLidahlg li'aJLllr~' producing cells as well as c°ljs prodaxcing a~caolecules, v,hic;~
prorrzote islet cell s~arvival~replication%trar,sdifferentiatiora ~~~a~' ~~'fR~~~T i~ i_,i=,, ~~.~i~
E~1~~~~IhiC~L~C~i~~ or insure islet and ~,raica°ocapsnl=~.p ~a~raear~oprotectio:~ ~,~~~.
~~~U~E L'~'~~i~_~1'i'~~~t ~IJi~ ~:~~,~, ~:~~, ~'~~'T~~,~~). ~zna.ll~.r. tae rraost promising approach ro ~-cell replacement is the transdif~erea~tiation of stern cells unto iS~etS~~~ tnOt neCesSuriiy' err ~'?r'?;QI~iC s'ea'a'I CP'ilS, th~'illCli arise ContrOYrerSf, C)ut stem calls of many other oriius, ncl~zdin~y autologovs oells~. Joie that even autologous cells would re~uir.: ir:~rr~u.,o~rote~iion against the r ec;trrenee of ths:
autoirnmnne process. the ~xra~or ;.ona~r_:~ related to this a~~roac~, is the risk of malignant transformation ar,d dissemznLaion o~~immature stem cells ~ ~ .
Tl~e process is based on the chemical derivation of ~irL ~~.-ith a photoactivatable heterobifunctional cross-linker, ~-~-r=~zi~io..2_nitrobenzoylc~cyst~c~~inimide (~~~11~-I~~S), which is able to covalentl bind i~~,:~. to alginate ~,~:mr~ er~er(~izec~ icy ~JVI~ light illurninazion~L~, i9~. 'hhe ~i-hydroxysuccir.i.mid ov~tlic: cross-linked ~:~ras fist c:ovalently linked to Pd,lr. in conditions that ~,~ oul;~ ciana.a~e ii~,iy-~:~ cellss but ils~tore ~~he c;ells are irtvol'-ed ir3 tre procedure. Then islets enia~s~alated. vr~ algirFate microcapsules ~~e.re inaubate~W vitl~ the oross-'inkc~r deritmd ~Ll~. ~~ll~c~rt ~:lne pr~~arati.,~n is ill~tmir~ated v~=itl~ a L1~'~ ian~p, the photoactivatabk; residue reacts ~.vitl~ the phenyl azide residue on al~inat~, croati:~~ a covalent link. 'fhe latter reaction is ~~ot har;~ful for livn~ ;
veils. ~'e, herein, present ~ proof tihat a covalent linlr, v;~aras fo~r~~d ~~~ovidira~
consid~.rable improvement ~~
microcar~sule resistance to chemical and trsec;i~anieade~radatior_. i'~~e _~~,sults of th~~ study sho~~~e~3 ~dhat the procedure did not modify Ynicrocapsule m~n2br~rie permeability and did not affect i~ vi~v~ and zn ~~iv~ encapsulated cell survival, I~II~I~~~~ICt~P~UL~'l l~i~T C~_~ L_~V k~~ ~~.I~~J~ I:~, C..~~~~-'~Llrl~ :Y
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~:enzre de ~eche_rcbe CJuy-~eraier. ~~pitai °~aisonraeuve-Is.osem~3nt_ 54-15 foul de 1' ~ssofnptioa~, i~Iontreal ~czebec, Ca',ada, I ~ I 'h 2I'~~i4.
e-avail : ~allejp'~.videotroal.ca ~lzoit title : Covaler~~tly lra~i<ed al~i~ate pol~aalvsinc: ar~icr~capsuleJ.
I~e~~~~ords: Pl~otoactivatable crossiirrlcers, i:~m~i~a~ois;~Iatioa~, cell '~ra'~~sp'~aratatir~rz, bioreactors, islets o~ I aayerhaals ~~ ~ s~, p,~ . .,.~~,~~A ~y . . ~~.a~ ~, M
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'~~orrespc~azdence should be addressed to ~1PI-l i~Ioratreal, rebruar~r, i5'~'. ?OU3 i ,J
I-ialle, .lean-Pierre -'r~._.~..._,....-..~_ Pa a IVum6ers ~ Ad~nspeei~ta~~e~~ a~ad ~a~naf~r~ ~~~~
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Htmlformat _ TI~ f --.~_ _. ._ .~..._. _ ...~ _ ~~~ _-Detailed 8ud9et for Initial ~udpe_t Period 2 Detailed Consultant or Co~atractual Costs for Initial Budciet Period _ Budget for entire Project Period 8udget Justification ~~er Support g Resources ~--- __. _.~_.~ Ig _...-.
~~~~1 LI.
Principal Investigator (Nod to exceed twe asses)lI
_ _ _ ~3 ~.
AI! other key personnel listed in the "'Applicant and iCey Personnel"
! html section of this applicats'oaa (blot go exceed two pac7es t'or.eao~) a rah ~~~ L9 Copy of summary statement from original application-~
(if appiicablej Introduction to revised ao licationtif a licabie riot t~o exceed t~ir> pa es) A. Specific Aims (Items.-D rat ~o exceed ~?
sages.) B. Background and Significance of this ~vorl~ 2g to ~' pe f Diabetes C. Preiiminary StudiesJProgress P.eport (fo~ ~ 2,3~
campetitive rer~ewal) ~~ ~6 D. Research Desi n and Methoes ~. literature Cited (no page limit; ~ ~ 30 ~
11'~9~ I~ ~ IVor tc= exceed ZO ~2 es exc!udirr mar~~rscrit~t~s Collaborative arrangements if an ) 4Q
_ ~2 IRB andJor ACLiC a rovai forms Abstracfi a es Trom LL other soLrces of suc~~or-~t~current~:l & ~endin~, Other Page 1 of ~ ~' i-iaife, Sean-Pierre ~E3'~~~EL~ S~I~E~' ~~ ~a ~. ~~3~Ei' PER,~~
- ~~~a~~~: 04 01 i~-~ ~r~~h~ o~~~ o~
aec~g~t ~a~~-'~~~r ~. ~~~ ~eg~rii~~ r~~a~t~~ra~ s~l~re~u~srsa~~~ ~ ~c~al~~~:dr ~e~sonne~ ~~e ~~ ~~'e i ~ s~~ai~~9a~reat 9a~ ~rire~~ ~°~T'~ri.~
i ~a~a~~ ~r~,pe~~ ~G~s~ a~~, E~r~ gee et~~~e~e~i s _ ~~o"e~a~a Sean-Pierre Hall2 Principal 3v 10 % $43,205 ~~,320 ___-_a. pt,320 Investi a*~r (~at~ralie ~ienie~r Research ~ 35 ~ 100 % $28,800 ~2t3 800 X32,255 X3,455 a~ssis~an~ _;
Markin Menar~ ~ Research 35 ~ 100 % 26 640 '2C~~~ ~3 in7 29 837 . . $ , ~ssis~a r~~:~
i 1 _.
f i i e,. -. _ i _ ~ - ~_ -._. ._ - S~S'~~3'~~iLS _-. $58,760 '~ $6,653 66 413 5~~~e~ ~~~es~z~ ~ ~2~~t~ ~n s~t~~~,~~~~a~~~~~~~~ ~E'r'1' ~. 81,715 i ~ti»f ~~s~ (Sp~c~~ - ~e~6ze ~~ ~~r~ g~~~~ ~ _~~~~~ ~ 1,500 ~~s~tif~atei~~~ s~~m - dc'~b ~n Eq~aipm~~~ (~escrab~ ins ~a~dge~ ,~e~~~ra~o~~~ ~____ _~ ~,~ii' ~ 0 ~~ns~~t~e~~/Cc~~tr~cta~~~ ~~~i~ ~Se~ s~ a~~ ~_~~.~'r '~T i9UE { i ~ Ii ~ ~ ~ 1" 149,628 f'ape w c~f 47 Halle, Jean-Pierre ~E'~LE~ ~~~atlL1'~.~~ ~~ ~C~i~' ~C"i'~l~t ~t~ FC3R API L ~It~~~'i' pE~i~~
Fii1 out this gage only it c:~nsuftar~c or oo~ tr ac-~ual coals are request-vd.
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~~g~°~~~ ~e~r ~ i I~_ ~~g~ 4 ~'F 4:g Half, Jean-Pierre ~' 3~'~'~~~~~J~~P~
~rovi~e a jus~.ifica~ion for each i~er~ listed in the ~ud~e~ including ~:on~rac~ual cots. ?ro~ride i~s5~titutior~al fringe be~efu rates t~or ail persc~r~ne6' DIr~'A~1J~~5 ~tJD~I=,'1?
AID II~A~,B
~xp. # sulDjeets~r'at~3rrtap~ lr~e''._.~otasz~glar~./elaya~taI
~~a~siu~g'Total # tai --WistarRatsD.1 200 $1.20 $15,45 -~ $777,60 x3866,89 $0,26 C57B116 D.2 48 $10,75 $11,69 $0,20 $14x,00 $705,36 rnicc C57131/6D.2 48 $10;75 $11,59 $0,20 $5 376,00 $5 937,36 mice V37istarRatsD.3 360 $14,20 $15,45 $0,26 $1399,68 b6960,40 C57~I/6 D.5 108 $1u,75 $11,69 $0,20 $324,00 $1 587,05 mice C57i31.~6D.5 108 $10,75 $1'!,6980,20 812 096,00~i3 mice 359,05 I-IaanstersD.6 270 $1 l,St $12,52 $fl,23 $928,96 $~ 309,83 V~IistarD.6 180 $14,20 $1 S,4S$0,26 $699,84 $3 480,20 Rats T~-lamstersD.7 4 $11,51 $12,52 $6,23 $13,76 $63,85 C57131l6D.7 48 $1w,75 $11,69 $0,20 $144,00 $705,36 mice C57~116 D,7 48 $1fl,75 $1 i,6980,20 $5 376,00 85 937,36 mice CS?HIl6 D.9 108 $I0, $i 1,6380,20 $324,00 $1 587,05 mice 75 IrrOD D.9 108 $20,55 $22;36 $0,20 $12 096,00$14 mice 510,35 TD'L'A~ ~.~ I~IAt.,S (3 ~'E~iZ.S~ x;63 0111,13 ')i'~TA~ AI'~1~IAT,~ 3~h,I2 '1'~A~. ~~'21003,3~a li~I~IDA~~,ES
~:~p. # # a~iaaxa9$ia~uisrraa$1~-sc9.'~~tal R:a~
Isict isotatiora200 819,60 $21,32 $4 264,57 D.1 and culture 48 $19,60 $21,32 81 023,50 D.2 D.3 360 $19,60 $21,32 $? 676 22 ~.5 108 ;~I9,60~~'21,32$2 302,87 ~.6 180 ;,19,60$21,32 $3 838,11 D.6 270 $19,60 $21,32 $5 757,17 D.7 4 w19,60 $21,32 885,29 D.7 48 819,60 $21,32 81023,56 D.9 108 839,60 821,32 $2 302,8 ~' 828 274,'9 Page S of 47 Halle, 3eari-Pierre ~'' ~,d ~ICaaC.~ ~9SC~. ~C9V:a~
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Encaps=a!ationD.l 60 881,00 $88,12 85 281,19 D.2 4 S81,fl0$88,12 8352,48 D.3 150 ~8a,00 x88,12 $13 2:11,99 13.5 9 881,00 $88,12 8793,68 D.6 60 881,00 888,:2 85 26'7,19 D.7 4 $8:x,00$88,12 $352,~~8 D.9 12 881,00 888,12 $105'7,44 X2634'7,85 ~xp. ~ samples$ls~~apie$ na~cl. 'y'rs~a:a ~ tax FluorescenceD.1 300 $0,15 $0,16 $48,95 microscopyD.'? 96 $0,15 $0,16 515,6'7 D.3 960 $0,15 $0,16 $156,66 D.5 216 $0,15 $0,16 $35,25 D.6 270 $0,15 $0,16 X44,06 D.7 96 $0,15 $0,16 $15,67 D.9 216 $0,15 80,16 $35,2.5 $351,50 ~xp. # samples$!ssmple$ a~a.l. 'T~~:~1 ?# ~ ax TtJNEL D.1 300 $6,84 87,44 82 232;37 assay -D.2 96 $6,84 $7,44 $7IG;,36 D.3 96C 86,84 57,44 $7 243,:9 D.5 216 $6,84 $7,44 $1 607, 1 D.6 270 $6,84 $7,44. $2 009,13 D.7 96 $6,84 ,x',7,44 8714,36 D.9 216 86,84 87,44 81607,~~-I $16028,4'?
Exp. ~' saanples$,~sa~rplen i,sa;h ~"~ea:E
# taa:~
FlistologyD.1 900 $5,23 $5,69 85 120;15 and immuno- D2 288 85,23 $5,69 $1638,54 cytochemistryD.3 2880 $5,23 85,69 $16 386,38 D.5 648 $5,23 $5,69 $3 686,94.
D.6 810 $5,23 $5,69 $4 608,67 D.7 288 $5,23 $5,69 $1638,64 D.8 936 $5,'23 $5,69 $5 325;38 D.9 648 $5,23 $5,69 $3 686,94 $42 092,3 ~"~ge 6 of ~
t~a~fe, 3eart-Pierre EXp.'#~F' $I~a,b s ~mcl. '~'cf~
~,u~~ ~.ax Growth D.1 1500 $3,60 $3,91rv $5 $67,74 Factors D.2 180 $.3,60 $3,91 $704;,13 D.3 1800 $3,60 $3,91 $7 041,29 _ D.5 540 $3,60 $3,91 $2 212,39 D.6 900 $3,60 $3,,91 $3 520 b5 D.7 240 $3,60 $3,91 $938,84 D.9 540 $3,50 !63,91 $2 112,35 X22 29T,4~~
Exp. ~ sa~~ples$~saple$ emcl. '1'ota~.
# tai:
~r~ ~;a~ilzt~D.1 90e X0.16 $0,17 $1x6,66 assay D.2 288 $0,15 $0,17 $50,13 D.3 2880 $0,16 $0,17 $501,30 D.5 648 $0,16 $0,17 $112, 7=:
a .6 810 $0,16 $0,17 $140,9~r D.7 288 $0,16 $3,17 $50,1:?
D.9 648 $0,16 $0,17 $112,79 $~ 1249$0 ~xp. # sars~ples$lsa~plc$ n~cl. "1'ofak # tars Callprol:ferationD.1 900 $1,09 $x.,19 $1067;23 assay D.2 288 :1,09 $1,19 $341,51 D.3 2880 $1,09 $i,19 $3 415,'! T
D.5 648 ~~1,09 $1,19 $768,41 D.6 810 N1,09 $1,19 $960,51 D.7 288 X1,09 $1,19 $341,51 D.9 648 $1,09 X71,1.9 $768,41 $766297 ~xp. # samples$lsa~~ple$ i~cl. Togal # rFa~
131oad D.2 4080 $0,92 $1,00 $4 083,5 glucose ~
D.5 9180 $0,92 $1,00 $'~ 187,91 D.7 4080 $0,92 $1,00 $4083,54 D.9 9180 $),92 $1,00 $9187,97 $26543,02 l;,up.# saxraples$Isaple$ l~7cl.:focal # aax 1;L1~A D.1 75 $8,83 $9,61 $720,4ei D.3 270 $&,83 $9,61 $2 593,E~~i 1J,6 27 $8,83 $9,51 $253,37 $3 573,49 '~r~e :~ o~E =1~
Hale, Jean-Pierre Micelaneous~~~. # sara~~~es $ ir~ct.~'ce~al # ~~is~p~~ ~Gx Str;:~tozotocinn/a 1 b67i,$1 $757,39 $73'7,:9 IvIotecularen/a 1 $2 Oi~~,QQ $2 i $2 175,84 7,80 Siology , regents l~t~TA~ n/a i7 :~~C6,40 $'?89,8:2$4 926,8 $7 ~~.a,07 'h~'I'~1~.. ~~h~,PII~~~I~~S (3 ~'~~IoS} 512 I3a,9I
'I'~T~I.'E~I'~1~1I~~~L~S )1'~It ~'~A~. ~6~ 7p1,97 'I'~7C~9y. ~~IIIi~I~P~S,1~I~'I'~I2hI. ~~ID X245 I~~,03 ~~I~'~g,II;~ (3 '~'~~~~) 'I'I~'I'~tl. ~I~IiVIAL,S91~A'I'~~I~&, ~1~~ ~~17~ 5,~4 S3l~IsI,I~S I~I~ A~Ik ~FFII~ICpE ~~Po'~:~y~~. Lt~.'I'E.St I~e~~I'~~~~~ ~~rl~T~~7~~ o ~L~~O
~~~~ T~~~~: ~z~~Q
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h~t~2 ~ 07~ °~ ~' E cake, Jean-Pierre ~~ ~~~rE ~~r ~~s ~~~,~eb~e~rg ~s~~~6~~ ~~e~~~~r~~ l~es ~ f~!o ~o yes, list the funding agency, the title cf the project, year one and total fundir3g received/requested.
Specify areas of scientific and budgetary overlap if not obvious from the aims and abstract.
"I~~ ~~~ ~~~~ ~~~~4$9~i9 ~~ ~fC~IL~E'~1~~ Ef ~'I~~~fi"~ 6~ r~3I~'~8~~~ ~PC9I~r ~~"~~° ~~8~l~iL~".~ ~~~J ~ #~LG
ist ~.~. ~a~e~ sources of support ~et~rr~ent and pending j. List funding agency, complete title, total award, yearly ward, inclusive funding dates, the rose of the appiicar~t, and percent of tirrse devoted to each grant.
he specific airr~s and abstract for each grant should be app'nded to the supporting Docu~enla suction of this application. (~~ra~ecutf~ely rs~ra~~er ar~y ~ttac,~rs~errt.s P'car t°~a~s~ section.) ~~J~'~'(~F~.~' ~~T~~'~"~.~
__ Bi~-artz~aciai ~~~dacrix~e~2~~~24'7.63~1$t 9b,249.15$~4/~l/2t~fDi Pancreas : Tire mole o "~
~F~ 2 to '. Pancreatic ~7nct fv'edis and f~4 999.24 C"srowth i actors :~ i~Iai ~ter~ar~ce3~ X4,999.2,4~3/~ 1/2i~04 and Promotion o~
T~iicroencapsuiated ~sle~ ~eia ~aSS aYlC1 ~11I1Cti45i~.
i r ~2C?I~E t~~' l-~PP~,~~~'~' :
~'rincipal lnwesti~atox /o ti ~e = 2~fQ
.
, ~~.fi ~ ~~'an$plarltitBG~$ O~ IS~~tS 4,1"' ~/'/~i~/?~'~~
~ ~J,~.L~~S ,~ ~C7~;~ 1 encapsulated irs ~nofi:r~ed (coValently Aiaed~ alginate ~~/3/2004 i poly-h,-lysine n~iLroeapsules _ ~~~,E ~9~ ~..~~~,.~~f~~ : ~'?'413~11?a~gLi~~t~T, ~ $~OJ~
~22Ve; 4/n t3lT"e =
Page g s~f ~-7 I iaiie, .lean-Pierre ~i~~~
~~~~ll~~:~e~: specify the facilities to iae. used f or the conduct of the proposed research. indicate the perfor~-rtance sites and describe capacities, r~er~inent capabilities, relative: proximvy, and extent of availability to the project. Under '~~ther", identify support services such as machine shop, electronics shop, and specify tire extent to which they will be available do the project, iJse continuation !sages if necessary, ~ aboratory:
Most of the proposed work will be conducf.ed in the laboratory of ~r dean-~Haife Haile at the Guy-t~ernier Research Center of Nlaisonneuve-Rosemont Hospital, This X00 square foot laboratory is fully equipped for tissue culture and cell isolation (three Foods, two incubators, a table top centrifuge, a microcentrifuge, a COO 292 centrifuge, and three inverted microcospes) and for moleoufar biology and biochemis°try experiments (two balances, 6 micropipettes, PC'R, horizontal arid vertical gel systerns with power supply, a blotting system, a spectrophometer and a spectrofluorometer. ?n the same:
area, the Research Center common facilities also include a cold room, Cs dark rr~orn, -20(~C and -80CC
freezers, a variety of cen"trifuges, an automatic sequences, a microtome, irnrnunofluorescence microscopes, floor cytometers and high perFormance cell sorter. Facilities and eqt.aipments for developing transgenic animals are also available.
Clinical: Nlaisonneuve-i~osemont Hospital has very active diabetes clinical research unit and transplantation programs (bone marrow, kidney, eye bank).
.~nimai:
~lnimai housing is equipped to received a large quantity and variety of species (mice, rats, hamsters, rabbits, dogs, pigs and cats) in stesile or non-sterile environment. The animal housing contained ac'. a!I ecuipped surgical room and large quantity of hood and bench used for animal manipulation. animal care technicians services are also available for ail the experiments in~,olving animals and hare considerable expertise with immunodeficient animals.
Computer:
The laboratory is equipped with a PC and a Mac c~arrputer. The PC is connected with the Nlaisonneuve_ Rosemont Hospital I~etworh that gives us access to all the common lab computers of the research center.
Moreover, this network give us Internet access to the ~niversityy of Mantreai library ~rhere eve can fnd a large variety of on-line electronic journals, O;fFce:
Dr Halle have is personal offsce located in ~zhe laboratory area (at ~0 feet).
t:r l~afle clinical and research activities are supported by a secretarial star or: sit: providing f ufl secretarial support.
Gther:
~9~~~~ t~ta~pett~~t: List the most important equipi~nen'c items :already availaL~le Eor this project, noting the location and pertinent capabilities of eacF.
Pace 1t~ of ~7 i-laiie, Jean-Pierre I~I~,. ~C"~
Provide the foiiovvincJ information for ~~.~ key personnel in the order listed in sec~ion "Applioar~t arsd i<ey Persortnei".
Foifova this format for each person. L~upiicate biasketch form as needede ~~' ~~~ ~A~S r e°~~~.
-"r,",. ~~~.
i ~~9~~g ~~a~a-~~~re~ r~oi~~9 I~n~~~~r~~~
~.__ .._.._ ~i ~D~~'iI~pT II9~~S (Begin ~vifh baccalaureaW rar ofher lrrlfial professional education, s uoh as nursing, and y include pos~doetoral training.
IP~~ l~°9~~AL~7~~'~I_ ~~~~R~~'~~ i° I~ ~~ Y
Liniversite de Montreal, avlontreal, Qc, Canada ivi<CJ. Medicine i lJniversite de Montr~a3, Montreair Qc, ~.anada ~ CSPQ _~.9?9 Internal Medicine tJniversite de Montreal, Montreal, Qc, Canada 2 i"RCP (~) ~3?9 Ii7ternai 1'dedicine ~niversite de Montreal, f~orttreai, Qc, ~arsar~a ~ L~~PQ 3.980 ~ndocr inoiogy ~,. i~~~i~9~ts~ ~~ae! N~~. List in chronoioe;icai order previous positions;
conoiudinc~ with y°our preses7t position.
List any honors, Includ~ present membership on any pubiie or private advisory Lomi nittee.
~d~aEYT~~~.5 E,It'l~E~'~~EI~ITy 3~'u~~EId~EI~T~~i t~l~Y~.~. ~'~CS1~1d3hsf Endocrinologist, Sacre-Coeur I-Iospital, lVLont~~.al ('1980-138b) assistant-Professor, Department oFi'~edicine( 1980-I98b) Univet-sity of Montreal (Sacre-Coeur hospital;
Chief, Endocrinology Division (T987-1986) Sacre-Coeur Hospital, Montreal Founder of the Diabetes ~ducatian anel'freatment(:t98~-1986) Canter (Day care) Sacre-Coeur I~ospital, Director of this Center .
Chief, Endocrinology Division {1.986-1995) MaisorFneuve-Rosemont Flospital, ~;=orareal Founder Diabetes Day Care Center. '~aisonneuve-Rosernora(1988-2990;
piospital Medical 37irector of this center Director of clinical research, Maison.neuve-I~.oserasor~t(1995-2oU~) Ylospital PR~3~'ESS~#31'~.~L, E~~'E~2IEi~I~E
P~tE~EIT'3' ~~5~1!'IDhT
Director Research Laboratory on Islet (158-Now) transp'antation associate F'roFessor, Department of (1986-hlov.~) h~FoL~icine University of Montreal Page ~.~ of 4?
Halie, Sean-Pierre ~~~~~ ~5~~~-r~~ae~~~~ ~~ebl~~~e~~ ~~~ ~a~~~a~il~ii~~~ ~~a~a~~3. List i:he tide and compete reference to alt pubiications during the past three years and to representaciye eariiPr pubiications pertinent to ti~is application.
Do nor inciude publications submitted ;~r in prepay°ation. ~~~ ac~h ~~e~~~eJ ~~~~ ~~~ ~u~ ~~s~ a~~~inta~~a~e~
~ge< ~~~;~~~~~i~~i~ s~c~~a~~~ ~~~~~rte~~s~n ~~~ f~a~ vas ~~~~s~ea , 1. Gerstein HC, Mann 3FF, Pogiaa; J, Dipuieen Sr, iialle ~ er rah The prevale~de and characteristics of patients with atbuminuria and diabetes in the HesPE and MICRC'HOPE study.
i:~ialaetes t~aøo 23 :835-1339, 2000.
2. The HOPE study investigators (w~°itir4g ca~mraaatte~, Gerstain HC, ~''usuf S, Mann ~'FE, Hoo~;werf B, Zinman Ii, Held C, Fisher G~, Wolffenbuttel 13, ~osch 3, P.ichardson ~., Pogue J, Halls ~P).
Effects of rarnipriI on cardiovascular and microvascnlar ouza;~mes in people with diabetes mellitus:
results of the HOPE study and MICItOi-IOPE subsi:udy. Dancet 3:95: 253-259, 200(t.
Effects of rarnipriI on cardiovascular and microvascnlar ouza;~mes in people with diabetes mellitus:
results of the HOPE study and MICItOi-IOPE subsi:udy. Dancet 3:95: 253-259, 200(t.
3. The HOPE Study Investigators-. l3ffects of an angiotensin-converting-on~yme inhibitor, ran~ipr:l, on death from cardiovasc;ilar causes, myocardial infarction, and stroke in high-risk patients.
P~ ~ngl ~ I~e~. 342:145-53; 2000.
P~ ~ngl ~ I~e~. 342:145-53; 2000.
4. Robitaille R, Leblond FA, L3ourgeois Y, Henley N, Loignon M, iia116 .iP.
Studies on small (<350u.m) alginate poly-L-lysine n~icrc.~capsuies: V- Determinatian of microcapsule permeability to carbohydrates and proteins by reverse sire exclusion chromatography. ~
~iorra~d Mat R.ars 50 (3):420-427,2000.
". Robi.tailie I2, Desbiens K, ~ienley I~~ and JT~1P~ . Time course of trans9vormi.ng growth factor - ~a (TOF-(3,) mI2NA expression in the host reaction to alginate poly-L-lysine mierocapsules following impiantations into rat epididymal fat pads J ~ioma;d lflat ices 52 (1 ) :18-23, 2000.
6. Lacourciere Y, Belanger A, Godin, iiaile Vii', Ross S, $~Uright N and Marion d. Long-term comparison of losartan and enalapril on kidney function in hypertensive type' diabetics witr~ early nephropathy. rlnov int. 58 (?) :762-~'69; 2000.
'7. Malone 3K, Woodworth .llt, ..~~aarora V, Yang &I, Carnpaigne vhT, HaI~
~i', Yaie 3F, and Crrossman L. Improved postprandial glyceanic control with insulin Lispra Mix2ST~' foliewing a standard lest meal in patients with type 2 diabetes. Oliraieai 'i'Iaerapeutins 22 : 222-230, 2000.
8. Malone JK, Yang H, Wood:vorth Jh, Hexang 3, campaigns 13N, iia9tal iii', Yaie ,IF, Orossman LD.
I-lumalog Mix25 offers better mealtime glycemic control in patients v~itl!
type 1 or type 2 diabetes.
Diabetes l~~otals. 26(6):481-fi81, 2000.
9. hang SL, Chen X, Lec:icre lvi, llenley tl, Allidina A, iialle ~>P, G.-I3rune;te vl, Filep ICr, Tang SS, Ingelfinger JIt, Chan 3SD. I~x:yperglycernza induces insulin resistatnce on a~zgiotensinogen ge ne e;cpression in dialaetic rat kidney pro:cimal tubular cells. J
i;~naiccri~~a~a.1 '2(2):333-3~6, 2002.
10. Robitaille 12, Dusseault x, Henley bT, I:osenberg L and I-iall6 ~iD.
Insulin-like growth faotor II
allows prolonged blood glucose normalization with a reduced islet cell mass transplantation.
~rsd~c~an~logy 144 (7) 303-3045, 2003.
Pi$PS~hi'i't~°i'iV~ EA~LiPig i°rJ~I ,itr ATI~'T~S P~i2TIiid~N'i' 'i'~ TiiiiS tsLDPk.iC A'FiOl~l 1. Robitaille R, Pariseau JF, Leblorxd Ft~, Lamoureux M, Lepage ~.'', iialla;
,~. Seudies on small (<350p.m) alginate poly-L-lysine microcapsuIes. III-8ioeompatibility of small versus standard microcapsules. ~ 1'3aonaed l~at has. 44:11ei-120, 1991.
2. Robitaille R, Leblond FA, l~eniey i~,,T, Frud'homme tr3, Drobetsky E, iiallaa 3i'. Alginate-polyL~~
lysine biocompatibility: a novel Is'T-PCR :.zethod for eytokine gene e>~~ression analysis in pericapsular infihrates. .i T~icme~ fiat bars 45:223-230, 1999.
3. Leblond FA, Simard Cy, Henley Tai, Rocheleau I3, Huet °M, i~all~ ~.
Studies on smaller (315 lxPvl) microcapsules: IV. Feasibility and safety of intraportal microcapsule implantation in the rat. CeI)i Te-amsplantati~a~ 8:327-337, 1es99.
I Dr 3P l3alle is a FIOPE investigator.
i3ag2 i2 of ~y P-laiie, Sean-Pierre Iii l'iI ~ irCT~
provide the foilowfing information for ~.~. icey personnel in the order Listed is~ section °'Applic~:nt and i<ey personnel".
Follow this format For each person. ~upiicate bios~ctch form as needed. ~& ~' ~E~t~ ~ ~~~~r' eso ~1~~ '~'E.
~~~~~b~~~, ~.~r~~a~ ~ml~o~~s~~~~.r lP>l '~~/~" i~l~ ~8egin with baccatac!reaøe ar at~her initial pro>='essia3ai educatian, such as nursing, ~:r~d include postdoctarai ~-raining,) _,.. ~ _ -.~..._ I~ tJ'~'I~rJ ~~ !.~~~ ~~9EIIt~ '~~~~ __~_.it~ ~~ ~'fll~~' IVIcGiil University ~i.P~., I:.M. 1.979 Medicine .~
i i~cGi91 IJniversit'y ~ ivl.Sc. ! m982 Q:::xperimentai 8urc~e~~r ~IcGiPi L3niversify ~ :.985 General Surgery ~icGilf University i''s-~.. ~ :.985 h~perimonta3 Surgery University of i~iohigan ~ post-graduate ~ _~~9_87 'f~ranspPar~ta'cian University of ~Nestern CSntario i Post-graduatq 1987 Er<doscopic pancroatico-biliary surgery i Dice University j ' _ 1998 Tissue EEnginaerinr~
~, iP~~sf:g~ns ~~d ~rt~e~. List in chronological order previaus positions, cot~c9udin_e, with your prøser~t position.
List as~y honors. Include present membe~-s~tip on any public cr private advisor~~ coms-nitlee.
APi~~l~r°~M~i9~'S
Uraiversi~J
inept. of Surgery, NDc~'rill A ssistant ProfessorD987-91 University T~ept.ofMedicine,McCaiIlIJni~,~e.rsityAssistant Professor1987-91 Section of Transplant Surgery,Chief 1996-98 McCyilD L~nive'a~sity~
l7ept. of Surgery, MeGill UniversityProfessor 1996-present Uept. ofMedicine, McG'ill UniversityProfessor 1996-present division of SurgicaD Research L~ir~ator $997-preserve l:Iospttal dept. of Pathology, Montreal .~.ssociate Surgeon798'7-98 C3eneral Hospital dept. of Surgery, Royal VictoriaAssistant Surgeon1988-97 Hospital Dept. of Surgery, Montreal Assistant Surgeon199(3-99 Children's fIospitai dept. of Surgery, Royal VictoriaSenior Surgeon x996-99 hospital ~3ept. of Surgery, Montreal Senior Surgeon 1998-99 General I-lospital Dept. of Surgery, MeGill Jni~ers2ySenior Surg~or~ #.999-present Health Center I3ept, of Surgery, SMBIJ-JewishSenior Surgeon 1999-preseaat ~'aeneraD Hospital NIcGill University I-lealth A.C'a, Tl~ompsor~
Center C'nair of Surgical Research20(31-present 3f~RF Center for Beta-cell Director 2002-present Replaaerxent in Montreal SPFC.xaAlr. »~I~T~ItS, t~'9VAIg~S. EZ~,~:t3GlVi'f'~CSI
McLaughiin Foundation i~ellowship 1985-87 American Surgical Association Pour~dation FeDDowship 1488 (farstcanadian >ecipient I988-90 Medical Research Council of Canada- Scholarship I989-94 Senior Clinician-Scientist Award (P'F~SQ) 1997-Ol Nesbitt-McMaster Award for ExceDlence in Medicine and Surgery 1997-OD
hlational Scientist Award (FRSQ) 2001-09 ~at~c: I;~ of 47 Halle, .3ean-pier re Da lead ~~r~r~~le~d g~~~dl~l~a~~ ~~r~ ~9~r~Ia~~ll; ~rd~r~. i-ist the title and Gon~plet~ -~~ference ~~o all publications durinc the past three years and to representative earlier publicat'sons pertinent to this application.
Do not include publicafiions submitted or in preparationo ~~~ a~~~~~~xV ~hG~ ~
~Ie~~ ~~t~ r~t~~te~~a~n ~~~o ~~ra~~~st~~~~~ ra~~at~~r ~~n~ia~~t~e~~a ~~~ ~~~ ~e~"s~ s~s~~~s~o articles in Deer-reviewed joiarrzr~ls ('~= first auti~or is a graduate student ox fellam) i. Paraskevas, S, i~Jfay siz~ger D, VVarcg .~N, l~osenberg L. Cell Loss in isolai:~d hurnsAn islets occurs by apaptosis. Pancreas 20:2%0-2?fi, 2000*
2. IvIacDonald N, Ayouli JI', 8ark~:rt 1~, Dalzell IVIA, Gagnon E, Rosexiberg L.
Carcinoma of the pancreas- :4n integrated programane. ~an.~:er Strategy 2:1?..2y, 2000.
3. v1'ang G-S, Gruber ~,. Sanyth'4'; Pulicio ~, Rosenaerg L, Duguid WP, ScottF'~7.
Hydrolysed caseim diet protects diabetes-prone I3B rats'. 'rare developing diabe3:es by promoting islet neogenesis. J Ausoim~~nity 15:407-16, 2000*
4. Aiki.n It, Rosenberg L: Maysi~~.ger D. Phasplxatidylinosiitoi 3-kinase sigr~aaing to Aht mediates survival in isolated canine islets of Lar~gerhar:~. . ~I~I~c G
277:465-461, 2rf00.''~
S. Paraskevas S, Aiken R, IViaysinger, l.akes~ jR i', Cavanagh T;f, Wang RN, Roser~berg L . IVlodulation of ~I~TI4: rbnd p38 stz°ess acti~rat~d proteins.
kiraases xW solaced islets of Langerhans: Insulin a.s an auto;.rine survival signal. Aura Sur g 2.33:7.24-.i33, 2001 k 6. Tang RN, Li J, Rasen:i3erg L. Factors mediating the transdifferentiation of islets of Langerhans to duct eI>ithelial-like s'erracL,.~res. J o~ Endocrinology 1?1:309-328, 2001*
7. Tam J, Rosenberg L., ?t~laysing~r D. Islet-'leogenesis-Associated Protein.
enhances neurite outgrowth t=ram DING ~eurans. E-SRC 293:&49-654, 2002.*
~ Tcherven.ivanov 1~', '"!;aan S, Lipsett ill, A japitos D, Ros,et~besg L.
Morphological and functional studies ansubznucosal ish:~t transplants r_orynal and diabetic hamsters. Dell TraS~Esplantation I'I:529-538; 21702''°
a Petropavlovsi:aia fib, P~osez~bexg h. Identification and chrs.racteri~atiar, of srnaii cells in the adult pancreas: Potential progenitors? Cell ~ Tis y~ae Research. 2002 (in pressJ.
itl. gang ~-S., Karamo=iar>dari l., I'~:lieie D,, Rasenberg L., ScottF.~.
""Altered is3et homeostasis before c:lausic insuiitis in El3 rats". Diabetes ~: Iv~etaboliszn, Dec,;28(G 1't 2):3590-7; discussion 3SI08-12.20i?2.
L~.. Robitaille R, Dusseault 3, Fle:~le~r ~, Icosen'oerg L, Flaile 3Fn.
Insulin-like growth factor II allows prolonged blood gluoose; ~~ormali~ation with a redG.ced islet cell mass transr~lantatian.
Endocrinology. 20433 ,T::1;I44(?):3Q37-45.
1'.vIID: I28:0~ 60 1"ac~e 1~ of 4y i-iaHe, ;ean-Pierre Provide tile following information for ~L~. ~e~~ personnel in the order listed in section ".Applicant and fey personnel".
Foiiow this format for each person. Duplicate biosketch form as needed. ~ ~'~' ~~E~ ~ ~~t~~ ~n N~ ~ ~°ff~T~.~:
~s~~~~,, at~ala~ ~~r~6~ ~lsn~~
~/'~ BiI~~i (begin with bar~ca.'aureate car ether fnieial ~r~'ession al educ~attan, such as nursag, arid intf;as~e ~ostc~ottoral trainin~.~
i yll°~~~ ~.~~6>aGE~~ ~~.~~
k~niversite de Montreal 3.Sc. ! V 1991 ~iochernistry s t~niversite de ~iontreai s~.~c. ~.~94 Biochemistry Universite du t~uebec ~ Montreal ~er'cficate 1394 Education ___~__ ~ P~~~~ii~n~ ~e~d l~o~~r~a i_ist in chcrnoiog'scai order previous positio:ts, Col~~auuing witi~ your present/ position.
i_ist any honors. include present merrrbersgip on any public or pri~rate advisory ccarr~rr~it'cee.
~hT~~S
~~nada's ~ovemment Scholarship it s9~8 ~°or outstanding grades in eollc~e.
P~a~~~,SS~~~rA;i, ~'~E'~~~
PR~~11~US EPsiIP~~YlVIENT ,~i~iaJ P;~_P~P.I~s'~C:
'Technician / ~ustorr~er Service Soviets d'Ariaiyses ~iophaxxnacenti~nes ins.
388 bout. ~ndnstriei, ~ava$
~Summcr 199) B.Sc. Stage fir. Grilles lean dray-~ernier Research renter ~aisonneuve-Rosemont os~ita?, i~iog~txval (~.~Tinter 1991) ~~aChin~ ASSiStant biochemistry DePartrnent ~3ni~rersite de t'i~ontreal ~Sept. 1992-April 1993) S~~"~' ~'~SI'T~Ol'~
Research Assistant fir. ~Lan-Pierge ~IaIIe ~auy-33err~icr Research renter I~aisonneuvc-~Zosernont ~ospita, ~~or~trcai ( 199-hour) ~a~e ~.~ of ~7 ir9aile, :~eara-Pierre ~e~~S~~r-re~~e~~ ~c~~lis~~9~s ~ie~ cc~~~~~~~ or~~r~~ fist th W title and corrpiete r eference ~~o ai1 publications during the past three years and to represer~tati~re earlier publicai:'sor~s pera~in2nt to this application.
Jo rot include pubiicatsons submitted cr in preparation. ~~e~ ~~t ~o~~d( ~iba~
~~~ p~~~ ~n~ ~;~~~~~~r~~u~~
~~~. ~~~~~oa~~~~e~~ r~~~er ~~~afil~a~~t°i~n ~a~fir ~~i~ ~~~a~~a.~
~'L~~~'~'~~l~IS
~.rticles Sirraard ~'a., IJehlond I~.A.., ~~ale-~.~, Vie, ~:.oci~eleaa. ~., H~et ~'.IVi., I~all~ 3.f'.:
Studies on smaller (~~ 15=tAn~ ~nicrocaps~.zies I~-p'easibil.r.ty and safety of intrahepatic implantation of small alginate poly-~.-lysine icrocaps~les. ~e ~ransptantatic~~a ~:~2?-3s7, 199.
I~obitaille l~, ~,ebloncl ~a~, Henley l~; i~rod';~ornx3~e C~J, ~robetsl~y IJ, i~all~ J~.
alginate-poly-i.-lysine bioco~rpatuility: a =.~ovel l~.'1~-P~i~ method for cytolar~e gene expression analysis in pericaps~.Iar infi~trates. J ~is~ed i~~t ~.es 45:223-230, 1993.
I2obitaille ~.., IJebIond F.~..., ~orirgeois ~., e~aley ~o, ~.,oignon '~d., ~a.lle J.~.;
Studies on small (<35C~nz~ algi~~te-poly-~._lysirse n~icrocapsnles ~j_ ~3etermi~~ation of n~icroc;aps~le perr~~ealiilit~; to carbohydrates and proteins by reverse size exclusion ~;I~romatograpl:y. ~P ~aomel~at ~;es 50 (~~ : 42~-42?, 2~~~.
F~obitaille I$, l~esbier~s ~, eraley .~i a~~cl ~palle Jl~. T'irrie c;o7arse of transfo~in~;
groe~rth faetor - ~ I ('T~r-~31~ ~~ expression ir3 the host rEaction to alginate poly-L-lysine micxocaps~ales follo~vi~g inaplax3tatiens infra rat epirlidyrr.al fat pads ,3 lmecl ~.t ~.es 52 (I: 1 ~-2v, 2I30~.
hang 5~.., when X, ~ecierc , ltenley ~, ~llidina ~., :~:all~ 3h, ~.-brunette lvi., 1~ilep JCS, Tang SS, Iaagelfnger Jib., r!uhan JSI~. ~~ypergiycenaia attenuates iras~zlir~
action on a~giotensinogef~ gene expression :Ire diiabetic rat i~ifxnney proximal tubular cells. ~e~rnal ~rf end~c~°inology 1?2 : 3~3-:~~~~, 2fl~2.
I~obitaille IZ, I~~sseault 3, ~~tley I~, ;l'~osenberg ~, any =~all~ 3~'.
Insulin-li~A
gro~rth factor lI allo~hls prolonged blood. gl~.tcose normalization ~jtl~ a ~°ed~ac:ed islet cell mass transplaratati:or~. ~ndoc~°lrac~logy 1~~~ (?) 3~'~?-~~4~, 2GJ3.
#~a~e ~.~ of ~7 i-la(le, Sean-Pierre I~~I~"~ ~KE~'~
Provide thte following infarrnation far A~.~. key persor~nei in the prder listed in secticr °'~pp9icant and fey Person:°~el".
1=oilow this format for each person. i~uplicate biosketch form as needed. ~' ~t~E~ ~ A~:r ~~
~~3~ "~~'~.
~~~r~~ ~r~n ~ ~e~e~re~ ;~i~~~a~ r ~~°PI/T II~~ Begin ~vii~h hec~:aiaarreetc or other iriitiai prafessir~rtei ødt~ca~ion, such as e~ursing, and include ~c~sto"octorai tt~air?ing.) I,t4~3 ~~~°~I~~e ~~'~"va E~.~ ~ 13C8'~
ilniversite do Quebec a Montreal i3.~c. -~ l.gg6 Sioc6~ernistr°~
iJniversite de Montreal t~.Sc. iggg i3ioci'aear?istr~~
Universite du Quebec a Montreal CLrtific~!te 1~9~ general ~cco~ntar~cy ~, l~~~g~i~r~s a~~ e~r~~. List ire chrr~noogicai order previous positions, conciudinc~ with your present position.
List any i~o~aors. Include present members'~ip on any public or private adviscsr gr committee.
~~~3~~SSI~1~1r1.~.. ~~~'~E~'~
r REVIC~~.TS E~PI,~3~~E:~3~' l~~'~E~.~E.I~IE1~7~:~
E.Sc. Stvge I~r ~u~taswa~~y ~Iau,~uuati~
buy-Eerr~ier research ~etater ?~aisonneuve-I2ose~nout I-laspvta~., ~~ontrea.i (Su.~r~xner I996~
I-~esearc~ Assistant I~r I~uttaswan~y I~a~ju~~ati~
~~y-Eemier IZesearcl3 ~'er~tei Maisorc~euve-~.osemar~t I-lcrspitah o~treaY
(1999-20flI) 1'~.ESENT ~C~SI°~I~:c~t ~esearcl~ Assistant I~r ~ ear-Pierre I-Iall~
C~uy-Beruier I2esearcY~~ ~er~ter l~Iaise~n~xeuve-~.oserr~o~<t ~fospitaP, I~Ion~eal (20C~ I -avow j Page ~ > of 4~3 ~iai(e, ~~an-Pierre ~ pr~~6e~~~ pa~~Z~rr,~tl~~~ ~u ~~r~~a~~u~~~ ~r~~r~~ i ist the title and complete reterence to ail publications during the past three years and to representative earlier pu'~iicatlon;s pertinent to tY~is application.
~o not include publications submitted or in preparation. ~~~ ra~~ ~~c~~c~ ~~~a p~~ p6~s~ ~~a~~~s~~etu~~tiun p~~~a ~s~~~castive~~ s~aa~~r ~~ra~an~~~~~ ~~ar ~~1~ t~~s.~ _ ~~.3~~I~.~'I~I~~TS
~~~11~.~~~
~on~plementarry deoxyribon~,~cleic acid cloning and tissue expression of BSI'-~~ and I3Sl' ~0-lc~a: ~hosphatid~lcholine a~nd'_nepari:r~-bi~~d<ng proteins o:i'oovine seirsal plasma. ~alois, I~., l~~xtar°d, h~'., F'aduettc, i'°. and lVxanjunatl~, ~'. l~k~logy of eep~°esduction 6t?, 2gg-2~7 ~ 1 g~9).
Major proteins of bovine sen~ir~ai plas~~a Bind to Ion-density lipoprotein fractioal of hen's egg yolk. l~lIanjunath. :~.*, i~larsic, ~~" ~e:rgeron; ~. and ~~~na~°d: ~ ~1~10~~ ~f rep~oductlr~n. ~7, 125Jd12Sg ~2~C~2) ~lovel pnri~'lcation method for n2a~rmalian ~em_ina?. plasma pl~o ph~iipid-l~ir~dh3g protei~~s reveals ~.~he presence of no~r~l r~~err~ber of this Tamil°y of proi:ein in stallion seminal fluid. l~~nar~i, lea, :~Tauc, ~~~., IJazure, ~., Vaillar~co~~ri,1~, and lVlanjua~atl~, ~'.
'~Ioleeula~- reps~da~ctls~n and devel~pr~aent~ septern'oer (2~JV3) ~~ p~~.~
Al~st~°a.c~a presented fps i~~fe~uational ee~:ang ~na~d, lei. arad l~Iarljunath, ~.: ~usi~lc;ation ai° hu~mart serni~~ai plasma proteins analogous to choline phopsholipid-~uind:lng protiens o~C bovi~:e smninal r~uid, gel' International ~yrz~~osium o~.~ ~pea-~natolog~, ~or~treal, ~ana.~way ~.ug~ast 17-22, 199.
ienaE el, ice. and Manj~znaih, p. purification of human seminal phospholipid-binding proteins closel.~ related to tl<e maior proteins o:~bovine seminal plasma. III
Interrm2ional Congress or .~~ndrolcgy, I~orrtrea: bane 15-2C, 2Qs 1.
lvianjath, 'a~~., IVauc, V. and ~Ieuar; u~a 7~3o~rel Mechanism of'sp~.rna protecticrher egg-folk. VIII International Congress or''.~.n.drc~logyr, Montreal vu~ae 15-2a, 2Q01.
h~anjur~ath, h., Il~~nap ~, I~o, i~auc, '~. arqd Lazure, C. ~lovep p~~rlfi~ation method for mammalian seminal plasma phospholip3d-binding proteins. ~ociet~ for tl~e study of r,~production, 2?Q1 Annual ~ivetir~g, l.,rniversit~r of~3tta~ura, Ontario, Canada. Jul~r )g-~~g~.~t 1, 2iIVl.
f~C~e f ~ Ot ~i 1-ialle, Jean-Pie: re ~~~°~~~ic~~ ~~ai~eai A~9i~~~i~~ ~nciude a letier sum~ar izing al!
deletions, additions, and other changes, and responding to al! aspects of the cr itique4 Reference should he made to an~r nevv ore9i~~ninan, data included in the application. ~~ ~ ~~~~~si ~ ~g~~~
~~E.C~
See 3~RF guidelines for specific instr:~ctic~ns and page lirnita.j ,a ~~~if~ Air ~~~l~~r~a~~~ ~r~d iw~io~t ~~i~ ~r~ ~ ~ii~~te~
~ r~tli!a~i~~~°c~di~sJ~r~r~~~ err ~~~r z:~~a~~~i~i~~ r ~:e~~~il~
~~rci~ ~si~ra ~~t~~th~~
~irare iteci (~a~ ~~ge 6ie~i~~
Page 1~ of -=~7 Halfe, Jean-Pierre ~,on;-t~rF~ prevenf'smn ot° ~-cell ap~ptosls and necroses key ~,oratenuc~~FS i~~ via~~ s~ppig~ of I~F'-~ to ~ansplat~ted Tslets, nsirFg I~Ii gerFe transfer ~~dt~, non viral v~~°ts~rs, s~a° cap-on~aps~.xlatgon ofaslets auFd pancreatic duct sells.
~. Shl;~I~'IC S ~C~~B. "~~ ~E~sI~ ~~ l~~S~'~1~ SL1~~~:T' A.g. ~b,~ecteve: To develop and. evaluate methods for Iong-term zraintenance aF~d promotion of the tra_nspianted a-cell anass~
~.2< I~ypotresgs. Ira viva I~.F-lI conti~2~aoaa supply tc Fslets Can provide syupport to transplanted islets, leadiFZg to the maintenance and prorrrotion of tl2e a-cell mass, ~.~. Specific alms.
~,~Y2~' ~ . ~'o sfady, in vi~rc~ (aim T~, and iaz v.rivo (a.am ~, tl~e effect, on Eros and mieroencapsulated islet cell surzrival, of transf F~in~ IC3F sense into islet oe~ls.
~~.'~ 2. ~'o siudy the effect' un islet cell survival, of eo-encapsulating islets and cells with a stable ICiF Gene expression, including, (~1~} i~ vi~o studies, (A~l~), developing a rrretlFOd that allows turning on-off ire viva the I~F genes, and (~~am ~} in viva studies, including the timing and duration of effective trra~nvnt and possibility of failed islet rescue.
~~' ~. To study tlFe effect of co-encapsulating islets aid rsaneroatic duct falls (which secrete 1~F-II, no ICF-I, and contain beta-cell precursors) on islet yell sLFrvival, in vi~o (t~i:~ G} anal i~ viv,~ (~irn 7. I'~~I~'d' 4.
(fix ~). the mechanisms (signali_rig pathways) of I~~F protective effects.
PAI~'J' ~. (F ~).
St~zdies in autaimmune diabetes (~T~I~ FniGo), ~. ~~1C~G~Z~'I~ SI~~'I~~f ~~ ~F" ~'I'~"~f ~ '~ ~E I ~I~.~E'I'~~
~.I. 3~atlar~al~ lveltind tl~s prc~psasaL Islet transplantation has induced insuin independence in patients with type 1 diabetes. I~owover, this tzLat~nent has se6~eral limitatioFis, irlcludir~g requirement for immunosuppressio~~; r~.~ee foa~ rt~.~cltiplo donors, rr~arginal w-cell reserve, vahic:~h may lead to recurrence of it;satin depend,nce, snd i_ixnited supply of allogeneic islets. ~~r r~sear°~I°~ focuses ors I) eliF~~:.inating the hoed for iF:zmunosuppressior~, using islet microencapsulation, 2) pa°~mots.~Fg ;oaFg-t~r-rxa sr~aiFrt~:rcara~e of ~-cetl mass, 3) ixnprovir~g t~'~e safety of gene therapy, immortalized cell lines and stern cell derived islets, and ~.l) scaling=~Fp microencapsulation technologies :roc application in large animal arFOdels.
°~'lt~ p~°es~nt prc~posat pcr°taaars to ~rar second prirari<ty sect stews i°~°r~rn tlxe ianclings~I~ oaf a~e~r- crarrent g~°ant, ending fl~/2t)04.
araipot~ant tissue , relevant to n aicroencaps~alated as ~,voll as to free.
isle, is flee rnaimtena:~FCe of the islet dell mass. ~ne of us (L .I~..) has proposed that relevant pancreatic components would provide trophic support to islets aF~d prort~ote surviva~~:~. I-Ie (L.I~.} has isolated and characterized Islet NeoGenesis Associated t rotein (II~iGAF)[.~-~;. ~ngoing phase ?
c;linical trials as°e evaluatiF~Fg ta~.e use of I~TGAF' in t~,~pe I and type 2 diabetic patients. Ire has als~~
sl-m~n that L~Fwubations of islets with pancreatic duct polls, decd conditioned rrFedium (width was slFova~~ to contain mainly I~aF-II, but no IC'rF-I) or IGrI~-II prevent both apoptosis and necrosis i~
vi~-~.~6;~ This work on islet biology is very relevant to islet ~trar~splantaGion. ~~ar cFSxrent Tf~I~' gran$ aimed at applying tlz~_s new knowledge to islet transplantatik3n. In the Iast 2 ~~~arsg we oor~fr-rr~~d a vi~rQ that Ice: -TI
prevents both apoptosis and necrosis in a dose-dependent fashion, acrd w~° ~~rst evaluated the use of I~F-II in a transplantation setting. ~r~-transplaa~tatla~n f~F'-i~ ear a~i~x°a 4~ 3~oa~r era~aahassa~s allowed prolonged blood glucose rzorFnalizauon in a higher proportion (~~00°,'°) of diabetic trice, using reduced islet cell mass (~tI%} tt~ansplantations.(i' 't"lais 4fl(1%
effscacy improvement encouraged us to pursue in this di~:eotion. Islet neogenesis and a-cell veplication ratE; have been shown to be normal in transplanted encapsulated ideas, suggesting tlxe decrease of the a-cell mass is caused by uzcreased apoptosis and necrosis.f;) ~'lFerefore, ~rao hypothesaze that sc~ntan~a~us d.~
va<v~ supply ~f ~~~-~ (andJor IGs -I) would prevent apoptosis and necrosis, and promote longw terse islet survival. This may be achieved, using gene therapy or co-encapsu.ation of islets and duct Bolls (IGF-II). ~lowever, several obstacles haFnper clinical appiicadons of gzne therapy.
I~etrovirus-based vectors, the only doctors t:~at d~rectl~ induce a stable gong expression, have huge 2t: of ~'~
Hake, 3ear~-Pierre caused a leukemia-like disease. ,~,d~nov~~-us-based c~e~tors provide a nigh transfectio<~ rate but induce a ~ansient gene expression a_°ad are the most irnmunogez~ic.
They have recently been reported to pause fulxnirzant disseminated ~ztravasC;ular coagulation c'isease resulting in rapid death. Note that IGF-I gene has been transfected into islet cells, using an adenoviral vector[gj, and has snor~rn a protective effect on islets in riiro. 'Ibis method, useful for physiological studies, has not'oeen used for in vav~ st~~rsies {probably because it is too irrarriurxogenic), and, considering the side effects mentioned above, is not applicable for human isl;t ~ansolantation (for recent review on vectors for gene therapy see ref~9j).
In the p~~ser~t prop~sal, 3 strategies, appl.icaiale to l7i~maz~s, for i~z viva IGl continuous s~__=pply to islets will be evaluated (see C. Progress repor~,~preliminary st~~xdies).
IGF-II was selected as a patentiai protective molecule because it v~~:~s found to be a compo:~mnt of the normal islet microenvirora.:~nur~t, and to reproduce the effect of duet cell incubations. Ti2ree categories of molecules promotit°zg islet cell survival could bo con.sidered: 1) peptides, such as IGFs, released in the environrn°nt and having a paracr~,ne and auto;;rine action, and ~) intracellular molecules inhibiting or stimulating pro- cr° anti-apoptotic path~~ays or ~) nezztrali...ing free radicals. ~n advantage ot°° ti~o first one is the paracr",ne effect on cells other than the transfected cells. Therefore, a l~C~/a cell trar:sfection rate is not required for full protection {we airn at transfectizig 10~% of islets but even a low ~Jo of cell irz each islet znay provide full protection). In addition, the strategy used rn I":~R~' ~, of co-encapsulating islets and cells from a well line with a stable gene exprr,ssion, is applicable only with n.°.ol~~,~ule~ having a paracrine action. t~mong intracellular rrzolecules, those bloc;cing one signaling pathway may induce potentially harmful hyperactivwtion of alternate ps.thways. Enhs~.~ing antioxidant defense mechanisms of the cells is an int~restii~g alt~_rnstiv~ approach, sirlae Eros radicals are the end-point effectors of several patlz~,vays. [~llothods for transf<drz-ing genus of such anti-o~~idant molecules have been developed .:,rr~ shovrn to protect islet cells ire ~eerc by I'r p- ~' ~enh~~nou's group, in Grezaoble, France[lCj, and ~vrll be evaluated in ou:= laboratory (sea letter of collaboration) using in viv~ models, by a student mom Grenol3lej.
Intrace3aular rrzoleczzlc;s vrili probably require the high transfection and stable gene expression provided only by viral vectors, which may be possible and safe ~:~rith our Govalently lirsked znicrocapsules (see bolo=N). ~sli of these approaches deserve to be i~n;rest3gated. Nevertheless, ~vo sele;cted ItIF-II, because of its paracrine action a,nd extremely pova~erful effect of in vitro incu'oations,.
I! is noteworthy that 1 or approaches could be combined.
~.z ~u~~-~nt stataas of r~es~a~~I~ iii whe pa°op~sed .r'xeld of s,ta~cly~
~.:~ol. 'fhe ~°a~lQ of the ~nec~a~-en;d~~onent in Islet ~esu~~ri~ml.
Growth factors {GFs) act in an autocrine and/or paracrine manner~l 1,12j to mediate a broad range of oellula~
responses[2~j. The role of GFs in islet cell sun.~ivsl remains to be clarified. IGFs affect cell growth and differentiation in both the fetal and adult paflcroas~l4-~'~j I~~ariiig induced islet cell neogenesis in the adult hamster, one of us (f,.~.) has found that t'3.cc~1 differentiation foam the dusa epi'~eliunn uras associated with a ~ansier~~c increase in IGF-II exprossion~3~;, recapitulating the fetal environment as characterized by I~~ill.;3l, ~2,j ~'hzs s~zggosts a direct role for IGF-II ir. the differentiation process in the adult. Fvidenc°° for IGFs as sur~i~,ral factors has also been reported.[33j IGFs are able to (i) reduce cell deaf=~~"T,27,2g] and {~,) protect islets i~rom autoimmune cytokines.~7j IG.F-IE[7,2'7,2~j and du~;z: cell-conditioned medium ~C~) can promote islet cell surviva~6j. h9CN~' is con~po5ed of Ir~F-II, but not IGa-L~6j IGF-I stirrzu3:ates insulin secretion in isolated marine L~4j and laurna~~~35 j islets and can antagonize inhibitory and cytotoxic effects ofcytokines in rat islets.{35j. IGF-?~ and II elect on apoptosis has been shov~n to be mediated by the same r~ccpto~~. I~ec~,nt fandings support a rol.;
fc°r I~1GF" in norrxaal developrrzent, morphogenesis and fznctionai rnat-uratia:rz of islets~3'T-~1 j.
I-Iepatio growth motor ((IiGF) has been detected in aeirzawr~~2j anal islet cells[4:3j, can promote.
the formation of insulin-producing cells frown acinar cells[~j and is znitogen to ~-cells[4~,46j acrd duct cells.[47j beg, is a gene that is overexpressed vvr~zin par~a;reatic cells after pancreatitis or resection[~.8j , and dzzring ~'a~o ~~ of 4f ~ialle, ~ea7-Pierre islet cell regeneration. Cne of us (a .~.)[49i and others[Sli] have found that the IZeg gene Es induced ire islet neogenesis. beg may be an. °irnportant paracrine factor in the maintenae~ce and growth of !3-cells[49-5~]. ~ther CFs reported to irr#luence islet :yell survival, replication and function include transforming ~.sF (TGF}- ~. [SS_SF~, 'f~aF-l~ [3~,5~-c~i], vascular endothelial growth factor (~C'aF)[62,63] at.d parathyroid hormone-related protein (fTHrP)[~.]. C.rFS may also be inhibitory in almost all situations n. which apoptosis is positi~Tely stimidated. This suggests that regulatien o~ CF levels is not oni;f important for ~.eli.
proliferation but also in n7aintainirzg viability of cells susceptible to apoptosis.[65-67]
Aitoget'_~er, these data suggest That the activity of CaFs may'oe fundamental to tl~e long tezrn maintenance of gram mass and f~u~ction.
.2~2 islet ~ramsptan~atl~n. Islet isolation; [68-82] ~::~~d transplantation hake been succ4ssful in several animal models.[83-51] Islet transplantation has now become a clinical reality [2,93], using imrnunosuppression.[91,9] :alternative approaches iznclude irnznurae-alteration of the g~°aft [9S-1t35] and induction of tolerance. X1136-1CJ9] o2a3d ~s~tan~-itolatlon by ~eBermencapSnlatiaa~a[lIO-11S] anc. macroencapsulation [116-118] are alternative approaches.
There are now clear evidences that ?~icroencapsulavon offers an adequate protection against allorejectiora and the recurrence of the auto-irmrrsune disease. [119] There is controversy on the efficacy of microencapsulation alone it disv°ordant xenotransplantation. [120] In the past, a much larger number of encapsulated tha>n non encapsulated islets were req~gired to normalize olood glucose. This low efficacy may be due to ;1) the use of large (> 7019 p,.n) ~raicrocap~sules, (i) tire poor biocompatibility of the capsules, (3) the inadequacy of the peritonemn as a transplantation site, (4) the host reaction to encapsulated islets, (5) an inappropriate ~ t~l cut off, and (6) tie removal of islets from their natural envirozur~ent. Ctrr past and current work has .addressed all these issues. ~iocompatibilit; of rnicrocapsnles with the host reFUai~~zs a poorly understood problem. fast laboratories have observed, in vi~ro, a pericapsular r.~e,action or ~siM.[115,12i-12~] There is an inverse correlation bet~~ree~~ the intensity of the ~Ii~I~
and the sun~ival of encapsulated xenogeneic islet's.[12i.,12~-1;1,122] ~3ther important para~reters are rnicrocapsule permeability and strength. We ha=re establisl<ed expertise in investigating and optimizing ail these issues. [i32-140]
~.~ ~edevaz~t pu°e~Ita~s w~~~ of ~e applicaa~~o :~ lei ic~°~e~casrala~f~~. For over 1S
years, we have strongly supporded the idea that fundamental issues iriust ba addressed before clinical application of rnicroencaps~..alatao~z. ~~7e systematically designed in vitra an.d/or i~.vivo methods to evaluate each microcapsule characteristic, and used these methods as end points to optimize the technology. [133-13?,1'?~-143] I~lc~°c~caps~(e see ;'s a c~°itlcal Tsar e. In most tissues, the maxarnusn diffusion distance for effective oxygen and nutrient transfer to cells is 200pm.[l4~-146] The absers.ce of convection Ynovement within a cass~~.le induces an importaf~t C32 gradient from the capsule s'~u-face to the center of the oxygen-censurrzir.g islet.~_147,:48]
reduced capsule size therefore allows for a better oxygc=n and aautrient supply to cells, and oifhrs many other advantages such as: (1) an expor°.ential decrease of the Total iraaplant volume, (2) improved insulin kinetics [149], (3) capsule resista3~ce[i.Sf3,], (~r) and biocompatibility[13~], and
Studies on small (<350u.m) alginate poly-L-lysine n~icrc.~capsuies: V- Determinatian of microcapsule permeability to carbohydrates and proteins by reverse sire exclusion chromatography. ~
~iorra~d Mat R.ars 50 (3):420-427,2000.
". Robi.tailie I2, Desbiens K, ~ienley I~~ and JT~1P~ . Time course of trans9vormi.ng growth factor - ~a (TOF-(3,) mI2NA expression in the host reaction to alginate poly-L-lysine mierocapsules following impiantations into rat epididymal fat pads J ~ioma;d lflat ices 52 (1 ) :18-23, 2000.
6. Lacourciere Y, Belanger A, Godin, iiaile Vii', Ross S, $~Uright N and Marion d. Long-term comparison of losartan and enalapril on kidney function in hypertensive type' diabetics witr~ early nephropathy. rlnov int. 58 (?) :762-~'69; 2000.
'7. Malone 3K, Woodworth .llt, ..~~aarora V, Yang &I, Carnpaigne vhT, HaI~
~i', Yaie 3F, and Crrossman L. Improved postprandial glyceanic control with insulin Lispra Mix2ST~' foliewing a standard lest meal in patients with type 2 diabetes. Oliraieai 'i'Iaerapeutins 22 : 222-230, 2000.
8. Malone JK, Yang H, Wood:vorth Jh, Hexang 3, campaigns 13N, iia9tal iii', Yaie ,IF, Orossman LD.
I-lumalog Mix25 offers better mealtime glycemic control in patients v~itl!
type 1 or type 2 diabetes.
Diabetes l~~otals. 26(6):481-fi81, 2000.
9. hang SL, Chen X, Lec:icre lvi, llenley tl, Allidina A, iialle ~>P, G.-I3rune;te vl, Filep ICr, Tang SS, Ingelfinger JIt, Chan 3SD. I~x:yperglycernza induces insulin resistatnce on a~zgiotensinogen ge ne e;cpression in dialaetic rat kidney pro:cimal tubular cells. J
i;~naiccri~~a~a.1 '2(2):333-3~6, 2002.
10. Robitaille 12, Dusseault x, Henley bT, I:osenberg L and I-iall6 ~iD.
Insulin-like growth faotor II
allows prolonged blood glucose normalization with a reduced islet cell mass transplantation.
~rsd~c~an~logy 144 (7) 303-3045, 2003.
Pi$PS~hi'i't~°i'iV~ EA~LiPig i°rJ~I ,itr ATI~'T~S P~i2TIiid~N'i' 'i'~ TiiiiS tsLDPk.iC A'FiOl~l 1. Robitaille R, Pariseau JF, Leblorxd Ft~, Lamoureux M, Lepage ~.'', iialla;
,~. Seudies on small (<350p.m) alginate poly-L-lysine microcapsuIes. III-8ioeompatibility of small versus standard microcapsules. ~ 1'3aonaed l~at has. 44:11ei-120, 1991.
2. Robitaille R, Leblond FA, l~eniey i~,,T, Frud'homme tr3, Drobetsky E, iiallaa 3i'. Alginate-polyL~~
lysine biocompatibility: a novel Is'T-PCR :.zethod for eytokine gene e>~~ression analysis in pericapsular infihrates. .i T~icme~ fiat bars 45:223-230, 1999.
3. Leblond FA, Simard Cy, Henley Tai, Rocheleau I3, Huet °M, i~all~ ~.
Studies on smaller (315 lxPvl) microcapsules: IV. Feasibility and safety of intraportal microcapsule implantation in the rat. CeI)i Te-amsplantati~a~ 8:327-337, 1es99.
I Dr 3P l3alle is a FIOPE investigator.
i3ag2 i2 of ~y P-laiie, Sean-Pierre Iii l'iI ~ irCT~
provide the foilowfing information for ~.~. icey personnel in the order Listed is~ section °'Applic~:nt and i<ey personnel".
Follow this format For each person. ~upiicate bios~ctch form as needed. ~& ~' ~E~t~ ~ ~~~~r' eso ~1~~ '~'E.
~~~~~b~~~, ~.~r~~a~ ~ml~o~~s~~~~.r lP>l '~~/~" i~l~ ~8egin with baccatac!reaøe ar at~her initial pro>='essia3ai educatian, such as nursing, ~:r~d include postdoctarai ~-raining,) _,.. ~ _ -.~..._ I~ tJ'~'I~rJ ~~ !.~~~ ~~9EIIt~ '~~~~ __~_.it~ ~~ ~'fll~~' IVIcGiil University ~i.P~., I:.M. 1.979 Medicine .~
i i~cGi91 IJniversit'y ~ ivl.Sc. ! m982 Q:::xperimentai 8urc~e~~r ~IcGiPi L3niversify ~ :.985 General Surgery ~icGilf University i''s-~.. ~ :.985 h~perimonta3 Surgery University of i~iohigan ~ post-graduate ~ _~~9_87 'f~ranspPar~ta'cian University of ~Nestern CSntario i Post-graduatq 1987 Er<doscopic pancroatico-biliary surgery i Dice University j ' _ 1998 Tissue EEnginaerinr~
~, iP~~sf:g~ns ~~d ~rt~e~. List in chronological order previaus positions, cot~c9udin_e, with your prøser~t position.
List as~y honors. Include present membe~-s~tip on any public cr private advisor~~ coms-nitlee.
APi~~l~r°~M~i9~'S
Uraiversi~J
inept. of Surgery, NDc~'rill A ssistant ProfessorD987-91 University T~ept.ofMedicine,McCaiIlIJni~,~e.rsityAssistant Professor1987-91 Section of Transplant Surgery,Chief 1996-98 McCyilD L~nive'a~sity~
l7ept. of Surgery, MeGill UniversityProfessor 1996-present Uept. ofMedicine, McG'ill UniversityProfessor 1996-present division of SurgicaD Research L~ir~ator $997-preserve l:Iospttal dept. of Pathology, Montreal .~.ssociate Surgeon798'7-98 C3eneral Hospital dept. of Surgery, Royal VictoriaAssistant Surgeon1988-97 Hospital Dept. of Surgery, Montreal Assistant Surgeon199(3-99 Children's fIospitai dept. of Surgery, Royal VictoriaSenior Surgeon x996-99 hospital ~3ept. of Surgery, Montreal Senior Surgeon 1998-99 General I-lospital Dept. of Surgery, MeGill Jni~ers2ySenior Surg~or~ #.999-present Health Center I3ept, of Surgery, SMBIJ-JewishSenior Surgeon 1999-preseaat ~'aeneraD Hospital NIcGill University I-lealth A.C'a, Tl~ompsor~
Center C'nair of Surgical Research20(31-present 3f~RF Center for Beta-cell Director 2002-present Replaaerxent in Montreal SPFC.xaAlr. »~I~T~ItS, t~'9VAIg~S. EZ~,~:t3GlVi'f'~CSI
McLaughiin Foundation i~ellowship 1985-87 American Surgical Association Pour~dation FeDDowship 1488 (farstcanadian >ecipient I988-90 Medical Research Council of Canada- Scholarship I989-94 Senior Clinician-Scientist Award (P'F~SQ) 1997-Ol Nesbitt-McMaster Award for ExceDlence in Medicine and Surgery 1997-OD
hlational Scientist Award (FRSQ) 2001-09 ~at~c: I;~ of 47 Halle, .3ean-pier re Da lead ~~r~r~~le~d g~~~dl~l~a~~ ~~r~ ~9~r~Ia~~ll; ~rd~r~. i-ist the title and Gon~plet~ -~~ference ~~o all publications durinc the past three years and to representative earlier publicat'sons pertinent to this application.
Do not include publicafiions submitted or in preparationo ~~~ a~~~~~~xV ~hG~ ~
~Ie~~ ~~t~ r~t~~te~~a~n ~~~o ~~ra~~~st~~~~~ ra~~at~~r ~~n~ia~~t~e~~a ~~~ ~~~ ~e~"s~ s~s~~~s~o articles in Deer-reviewed joiarrzr~ls ('~= first auti~or is a graduate student ox fellam) i. Paraskevas, S, i~Jfay siz~ger D, VVarcg .~N, l~osenberg L. Cell Loss in isolai:~d hurnsAn islets occurs by apaptosis. Pancreas 20:2%0-2?fi, 2000*
2. IvIacDonald N, Ayouli JI', 8ark~:rt 1~, Dalzell IVIA, Gagnon E, Rosexiberg L.
Carcinoma of the pancreas- :4n integrated programane. ~an.~:er Strategy 2:1?..2y, 2000.
3. v1'ang G-S, Gruber ~,. Sanyth'4'; Pulicio ~, Rosenaerg L, Duguid WP, ScottF'~7.
Hydrolysed caseim diet protects diabetes-prone I3B rats'. 'rare developing diabe3:es by promoting islet neogenesis. J Ausoim~~nity 15:407-16, 2000*
4. Aiki.n It, Rosenberg L: Maysi~~.ger D. Phasplxatidylinosiitoi 3-kinase sigr~aaing to Aht mediates survival in isolated canine islets of Lar~gerhar:~. . ~I~I~c G
277:465-461, 2rf00.''~
S. Paraskevas S, Aiken R, IViaysinger, l.akes~ jR i', Cavanagh T;f, Wang RN, Roser~berg L . IVlodulation of ~I~TI4: rbnd p38 stz°ess acti~rat~d proteins.
kiraases xW solaced islets of Langerhans: Insulin a.s an auto;.rine survival signal. Aura Sur g 2.33:7.24-.i33, 2001 k 6. Tang RN, Li J, Rasen:i3erg L. Factors mediating the transdifferentiation of islets of Langerhans to duct eI>ithelial-like s'erracL,.~res. J o~ Endocrinology 1?1:309-328, 2001*
7. Tam J, Rosenberg L., ?t~laysing~r D. Islet-'leogenesis-Associated Protein.
enhances neurite outgrowth t=ram DING ~eurans. E-SRC 293:&49-654, 2002.*
~ Tcherven.ivanov 1~', '"!;aan S, Lipsett ill, A japitos D, Ros,et~besg L.
Morphological and functional studies ansubznucosal ish:~t transplants r_orynal and diabetic hamsters. Dell TraS~Esplantation I'I:529-538; 21702''°
a Petropavlovsi:aia fib, P~osez~bexg h. Identification and chrs.racteri~atiar, of srnaii cells in the adult pancreas: Potential progenitors? Cell ~ Tis y~ae Research. 2002 (in pressJ.
itl. gang ~-S., Karamo=iar>dari l., I'~:lieie D,, Rasenberg L., ScottF.~.
""Altered is3et homeostasis before c:lausic insuiitis in El3 rats". Diabetes ~: Iv~etaboliszn, Dec,;28(G 1't 2):3590-7; discussion 3SI08-12.20i?2.
L~.. Robitaille R, Dusseault 3, Fle:~le~r ~, Icosen'oerg L, Flaile 3Fn.
Insulin-like growth factor II allows prolonged blood gluoose; ~~ormali~ation with a redG.ced islet cell mass transr~lantatian.
Endocrinology. 20433 ,T::1;I44(?):3Q37-45.
1'.vIID: I28:0~ 60 1"ac~e 1~ of 4y i-iaHe, ;ean-Pierre Provide tile following information for ~L~. ~e~~ personnel in the order listed in section ".Applicant and fey personnel".
Foiiow this format for each person. Duplicate biosketch form as needed. ~ ~'~' ~~E~ ~ ~~t~~ ~n N~ ~ ~°ff~T~.~:
~s~~~~,, at~ala~ ~~r~6~ ~lsn~~
~/'~ BiI~~i (begin with bar~ca.'aureate car ether fnieial ~r~'ession al educ~attan, such as nursag, arid intf;as~e ~ostc~ottoral trainin~.~
i yll°~~~ ~.~~6>aGE~~ ~~.~~
k~niversite de Montreal 3.Sc. ! V 1991 ~iochernistry s t~niversite de ~iontreai s~.~c. ~.~94 Biochemistry Universite du t~uebec ~ Montreal ~er'cficate 1394 Education ___~__ ~ P~~~~ii~n~ ~e~d l~o~~r~a i_ist in chcrnoiog'scai order previous positio:ts, Col~~auuing witi~ your present/ position.
i_ist any honors. include present merrrbersgip on any public or pri~rate advisory ccarr~rr~it'cee.
~hT~~S
~~nada's ~ovemment Scholarship it s9~8 ~°or outstanding grades in eollc~e.
P~a~~~,SS~~~rA;i, ~'~E'~~~
PR~~11~US EPsiIP~~YlVIENT ,~i~iaJ P;~_P~P.I~s'~C:
'Technician / ~ustorr~er Service Soviets d'Ariaiyses ~iophaxxnacenti~nes ins.
388 bout. ~ndnstriei, ~ava$
~Summcr 199) B.Sc. Stage fir. Grilles lean dray-~ernier Research renter ~aisonneuve-Rosemont os~ita?, i~iog~txval (~.~Tinter 1991) ~~aChin~ ASSiStant biochemistry DePartrnent ~3ni~rersite de t'i~ontreal ~Sept. 1992-April 1993) S~~"~' ~'~SI'T~Ol'~
Research Assistant fir. ~Lan-Pierge ~IaIIe ~auy-33err~icr Research renter I~aisonneuvc-~Zosernont ~ospita, ~~or~trcai ( 199-hour) ~a~e ~.~ of ~7 ir9aile, :~eara-Pierre ~e~~S~~r-re~~e~~ ~c~~lis~~9~s ~ie~ cc~~~~~~~ or~~r~~ fist th W title and corrpiete r eference ~~o ai1 publications during the past three years and to represer~tati~re earlier publicai:'sor~s pera~in2nt to this application.
Jo rot include pubiicatsons submitted cr in preparation. ~~e~ ~~t ~o~~d( ~iba~
~~~ p~~~ ~n~ ~;~~~~~~r~~u~~
~~~. ~~~~~oa~~~~e~~ r~~~er ~~~afil~a~~t°i~n ~a~fir ~~i~ ~~~a~~a.~
~'L~~~'~'~~l~IS
~.rticles Sirraard ~'a., IJehlond I~.A.., ~~ale-~.~, Vie, ~:.oci~eleaa. ~., H~et ~'.IVi., I~all~ 3.f'.:
Studies on smaller (~~ 15=tAn~ ~nicrocaps~.zies I~-p'easibil.r.ty and safety of intrahepatic implantation of small alginate poly-~.-lysine icrocaps~les. ~e ~ransptantatic~~a ~:~2?-3s7, 199.
I~obitaille l~, ~,ebloncl ~a~, Henley l~; i~rod';~ornx3~e C~J, ~robetsl~y IJ, i~all~ J~.
alginate-poly-i.-lysine bioco~rpatuility: a =.~ovel l~.'1~-P~i~ method for cytolar~e gene expression analysis in pericaps~.Iar infi~trates. J ~is~ed i~~t ~.es 45:223-230, 1993.
I2obitaille ~.., IJebIond F.~..., ~orirgeois ~., e~aley ~o, ~.,oignon '~d., ~a.lle J.~.;
Studies on small (<35C~nz~ algi~~te-poly-~._lysirse n~icrocapsnles ~j_ ~3etermi~~ation of n~icroc;aps~le perr~~ealiilit~; to carbohydrates and proteins by reverse size exclusion ~;I~romatograpl:y. ~P ~aomel~at ~;es 50 (~~ : 42~-42?, 2~~~.
F~obitaille I$, l~esbier~s ~, eraley .~i a~~cl ~palle Jl~. T'irrie c;o7arse of transfo~in~;
groe~rth faetor - ~ I ('T~r-~31~ ~~ expression ir3 the host rEaction to alginate poly-L-lysine micxocaps~ales follo~vi~g inaplax3tatiens infra rat epirlidyrr.al fat pads ,3 lmecl ~.t ~.es 52 (I: 1 ~-2v, 2I30~.
hang 5~.., when X, ~ecierc , ltenley ~, ~llidina ~., :~:all~ 3h, ~.-brunette lvi., 1~ilep JCS, Tang SS, Iaagelfnger Jib., r!uhan JSI~. ~~ypergiycenaia attenuates iras~zlir~
action on a~giotensinogef~ gene expression :Ire diiabetic rat i~ifxnney proximal tubular cells. ~e~rnal ~rf end~c~°inology 1?2 : 3~3-:~~~~, 2fl~2.
I~obitaille IZ, I~~sseault 3, ~~tley I~, ;l'~osenberg ~, any =~all~ 3~'.
Insulin-li~A
gro~rth factor lI allo~hls prolonged blood. gl~.tcose normalization ~jtl~ a ~°ed~ac:ed islet cell mass transplaratati:or~. ~ndoc~°lrac~logy 1~~~ (?) 3~'~?-~~4~, 2GJ3.
#~a~e ~.~ of ~7 i-la(le, Sean-Pierre I~~I~"~ ~KE~'~
Provide thte following infarrnation far A~.~. key persor~nei in the prder listed in secticr °'~pp9icant and fey Person:°~el".
1=oilow this format for each person. i~uplicate biosketch form as needed. ~' ~t~E~ ~ A~:r ~~
~~3~ "~~'~.
~~~r~~ ~r~n ~ ~e~e~re~ ;~i~~~a~ r ~~°PI/T II~~ Begin ~vii~h hec~:aiaarreetc or other iriitiai prafessir~rtei ødt~ca~ion, such as e~ursing, and include ~c~sto"octorai tt~air?ing.) I,t4~3 ~~~°~I~~e ~~'~"va E~.~ ~ 13C8'~
ilniversite do Quebec a Montreal i3.~c. -~ l.gg6 Sioc6~ernistr°~
iJniversite de Montreal t~.Sc. iggg i3ioci'aear?istr~~
Universite du Quebec a Montreal CLrtific~!te 1~9~ general ~cco~ntar~cy ~, l~~~g~i~r~s a~~ e~r~~. List ire chrr~noogicai order previous positions, conciudinc~ with your present position.
List any i~o~aors. Include present members'~ip on any public or private adviscsr gr committee.
~~~3~~SSI~1~1r1.~.. ~~~'~E~'~
r REVIC~~.TS E~PI,~3~~E:~3~' l~~'~E~.~E.I~IE1~7~:~
E.Sc. Stvge I~r ~u~taswa~~y ~Iau,~uuati~
buy-Eerr~ier research ~etater ?~aisonneuve-I2ose~nout I-laspvta~., ~~ontrea.i (Su.~r~xner I996~
I-~esearc~ Assistant I~r I~uttaswan~y I~a~ju~~ati~
~~y-Eemier IZesearcl3 ~'er~tei Maisorc~euve-~.osemar~t I-lcrspitah o~treaY
(1999-20flI) 1'~.ESENT ~C~SI°~I~:c~t ~esearcl~ Assistant I~r ~ ear-Pierre I-Iall~
C~uy-Beruier I2esearcY~~ ~er~ter l~Iaise~n~xeuve-~.oserr~o~<t ~fospitaP, I~Ion~eal (20C~ I -avow j Page ~ > of 4~3 ~iai(e, ~~an-Pierre ~ pr~~6e~~~ pa~~Z~rr,~tl~~~ ~u ~~r~~a~~u~~~ ~r~~r~~ i ist the title and complete reterence to ail publications during the past three years and to representative earlier pu'~iicatlon;s pertinent to tY~is application.
~o not include publications submitted or in preparation. ~~~ ra~~ ~~c~~c~ ~~~a p~~ p6~s~ ~~a~~~s~~etu~~tiun p~~~a ~s~~~castive~~ s~aa~~r ~~ra~an~~~~~ ~~ar ~~1~ t~~s.~ _ ~~.3~~I~.~'I~I~~TS
~~~11~.~~~
~on~plementarry deoxyribon~,~cleic acid cloning and tissue expression of BSI'-~~ and I3Sl' ~0-lc~a: ~hosphatid~lcholine a~nd'_nepari:r~-bi~~d<ng proteins o:i'oovine seirsal plasma. ~alois, I~., l~~xtar°d, h~'., F'aduettc, i'°. and lVxanjunatl~, ~'. l~k~logy of eep~°esduction 6t?, 2gg-2~7 ~ 1 g~9).
Major proteins of bovine sen~ir~ai plas~~a Bind to Ion-density lipoprotein fractioal of hen's egg yolk. l~lIanjunath. :~.*, i~larsic, ~~" ~e:rgeron; ~. and ~~~na~°d: ~ ~1~10~~ ~f rep~oductlr~n. ~7, 125Jd12Sg ~2~C~2) ~lovel pnri~'lcation method for n2a~rmalian ~em_ina?. plasma pl~o ph~iipid-l~ir~dh3g protei~~s reveals ~.~he presence of no~r~l r~~err~ber of this Tamil°y of proi:ein in stallion seminal fluid. l~~nar~i, lea, :~Tauc, ~~~., IJazure, ~., Vaillar~co~~ri,1~, and lVlanjua~atl~, ~'.
'~Ioleeula~- reps~da~ctls~n and devel~pr~aent~ septern'oer (2~JV3) ~~ p~~.~
Al~st~°a.c~a presented fps i~~fe~uational ee~:ang ~na~d, lei. arad l~Iarljunath, ~.: ~usi~lc;ation ai° hu~mart serni~~ai plasma proteins analogous to choline phopsholipid-~uind:lng protiens o~C bovi~:e smninal r~uid, gel' International ~yrz~~osium o~.~ ~pea-~natolog~, ~or~treal, ~ana.~way ~.ug~ast 17-22, 199.
ienaE el, ice. and Manj~znaih, p. purification of human seminal phospholipid-binding proteins closel.~ related to tl<e maior proteins o:~bovine seminal plasma. III
Interrm2ional Congress or .~~ndrolcgy, I~orrtrea: bane 15-2C, 2Qs 1.
lvianjath, 'a~~., IVauc, V. and ~Ieuar; u~a 7~3o~rel Mechanism of'sp~.rna protecticrher egg-folk. VIII International Congress or''.~.n.drc~logyr, Montreal vu~ae 15-2a, 2Q01.
h~anjur~ath, h., Il~~nap ~, I~o, i~auc, '~. arqd Lazure, C. ~lovep p~~rlfi~ation method for mammalian seminal plasma phospholip3d-binding proteins. ~ociet~ for tl~e study of r,~production, 2?Q1 Annual ~ivetir~g, l.,rniversit~r of~3tta~ura, Ontario, Canada. Jul~r )g-~~g~.~t 1, 2iIVl.
f~C~e f ~ Ot ~i 1-ialle, Jean-Pie: re ~~~°~~~ic~~ ~~ai~eai A~9i~~~i~~ ~nciude a letier sum~ar izing al!
deletions, additions, and other changes, and responding to al! aspects of the cr itique4 Reference should he made to an~r nevv ore9i~~ninan, data included in the application. ~~ ~ ~~~~~si ~ ~g~~~
~~E.C~
See 3~RF guidelines for specific instr:~ctic~ns and page lirnita.j ,a ~~~if~ Air ~~~l~~r~a~~~ ~r~d iw~io~t ~~i~ ~r~ ~ ~ii~~te~
~ r~tli!a~i~~~°c~di~sJ~r~r~~~ err ~~~r z:~~a~~~i~i~~ r ~:e~~~il~
~~rci~ ~si~ra ~~t~~th~~
~irare iteci (~a~ ~~ge 6ie~i~~
Page 1~ of -=~7 Halfe, Jean-Pierre ~,on;-t~rF~ prevenf'smn ot° ~-cell ap~ptosls and necroses key ~,oratenuc~~FS i~~ via~~ s~ppig~ of I~F'-~ to ~ansplat~ted Tslets, nsirFg I~Ii gerFe transfer ~~dt~, non viral v~~°ts~rs, s~a° cap-on~aps~.xlatgon ofaslets auFd pancreatic duct sells.
~. Shl;~I~'IC S ~C~~B. "~~ ~E~sI~ ~~ l~~S~'~1~ SL1~~~:T' A.g. ~b,~ecteve: To develop and. evaluate methods for Iong-term zraintenance aF~d promotion of the tra_nspianted a-cell anass~
~.2< I~ypotresgs. Ira viva I~.F-lI conti~2~aoaa supply tc Fslets Can provide syupport to transplanted islets, leadiFZg to the maintenance and prorrrotion of tl2e a-cell mass, ~.~. Specific alms.
~,~Y2~' ~ . ~'o sfady, in vi~rc~ (aim T~, and iaz v.rivo (a.am ~, tl~e effect, on Eros and mieroencapsulated islet cell surzrival, of transf F~in~ IC3F sense into islet oe~ls.
~~.'~ 2. ~'o siudy the effect' un islet cell survival, of eo-encapsulating islets and cells with a stable ICiF Gene expression, including, (~1~} i~ vi~o studies, (A~l~), developing a rrretlFOd that allows turning on-off ire viva the I~F genes, and (~~am ~} in viva studies, including the timing and duration of effective trra~nvnt and possibility of failed islet rescue.
~~' ~. To study tlFe effect of co-encapsulating islets aid rsaneroatic duct falls (which secrete 1~F-II, no ICF-I, and contain beta-cell precursors) on islet yell sLFrvival, in vi~o (t~i:~ G} anal i~ viv,~ (~irn 7. I'~~I~'d' 4.
(fix ~). the mechanisms (signali_rig pathways) of I~~F protective effects.
PAI~'J' ~. (F ~).
St~zdies in autaimmune diabetes (~T~I~ FniGo), ~. ~~1C~G~Z~'I~ SI~~'I~~f ~~ ~F" ~'I'~"~f ~ '~ ~E I ~I~.~E'I'~~
~.I. 3~atlar~al~ lveltind tl~s prc~psasaL Islet transplantation has induced insuin independence in patients with type 1 diabetes. I~owover, this tzLat~nent has se6~eral limitatioFis, irlcludir~g requirement for immunosuppressio~~; r~.~ee foa~ rt~.~cltiplo donors, rr~arginal w-cell reserve, vahic:~h may lead to recurrence of it;satin depend,nce, snd i_ixnited supply of allogeneic islets. ~~r r~sear°~I°~ focuses ors I) eliF~~:.inating the hoed for iF:zmunosuppressior~, using islet microencapsulation, 2) pa°~mots.~Fg ;oaFg-t~r-rxa sr~aiFrt~:rcara~e of ~-cetl mass, 3) ixnprovir~g t~'~e safety of gene therapy, immortalized cell lines and stern cell derived islets, and ~.l) scaling=~Fp microencapsulation technologies :roc application in large animal arFOdels.
°~'lt~ p~°es~nt prc~posat pcr°taaars to ~rar second prirari<ty sect stews i°~°r~rn tlxe ianclings~I~ oaf a~e~r- crarrent g~°ant, ending fl~/2t)04.
araipot~ant tissue , relevant to n aicroencaps~alated as ~,voll as to free.
isle, is flee rnaimtena:~FCe of the islet dell mass. ~ne of us (L .I~..) has proposed that relevant pancreatic components would provide trophic support to islets aF~d prort~ote surviva~~:~. I-Ie (L.I~.} has isolated and characterized Islet NeoGenesis Associated t rotein (II~iGAF)[.~-~;. ~ngoing phase ?
c;linical trials as°e evaluatiF~Fg ta~.e use of I~TGAF' in t~,~pe I and type 2 diabetic patients. Ire has als~~
sl-m~n that L~Fwubations of islets with pancreatic duct polls, decd conditioned rrFedium (width was slFova~~ to contain mainly I~aF-II, but no IC'rF-I) or IGrI~-II prevent both apoptosis and necrosis i~
vi~-~.~6;~ This work on islet biology is very relevant to islet ~trar~splantaGion. ~~ar cFSxrent Tf~I~' gran$ aimed at applying tlz~_s new knowledge to islet transplantatik3n. In the Iast 2 ~~~arsg we oor~fr-rr~~d a vi~rQ that Ice: -TI
prevents both apoptosis and necrosis in a dose-dependent fashion, acrd w~° ~~rst evaluated the use of I~F-II in a transplantation setting. ~r~-transplaa~tatla~n f~F'-i~ ear a~i~x°a 4~ 3~oa~r era~aahassa~s allowed prolonged blood glucose rzorFnalizauon in a higher proportion (~~00°,'°) of diabetic trice, using reduced islet cell mass (~tI%} tt~ansplantations.(i' 't"lais 4fl(1%
effscacy improvement encouraged us to pursue in this di~:eotion. Islet neogenesis and a-cell veplication ratE; have been shown to be normal in transplanted encapsulated ideas, suggesting tlxe decrease of the a-cell mass is caused by uzcreased apoptosis and necrosis.f;) ~'lFerefore, ~rao hypothesaze that sc~ntan~a~us d.~
va<v~ supply ~f ~~~-~ (andJor IGs -I) would prevent apoptosis and necrosis, and promote longw terse islet survival. This may be achieved, using gene therapy or co-encapsu.ation of islets and duct Bolls (IGF-II). ~lowever, several obstacles haFnper clinical appiicadons of gzne therapy.
I~etrovirus-based vectors, the only doctors t:~at d~rectl~ induce a stable gong expression, have huge 2t: of ~'~
Hake, 3ear~-Pierre caused a leukemia-like disease. ,~,d~nov~~-us-based c~e~tors provide a nigh transfectio<~ rate but induce a ~ansient gene expression a_°ad are the most irnmunogez~ic.
They have recently been reported to pause fulxnirzant disseminated ~ztravasC;ular coagulation c'isease resulting in rapid death. Note that IGF-I gene has been transfected into islet cells, using an adenoviral vector[gj, and has snor~rn a protective effect on islets in riiro. 'Ibis method, useful for physiological studies, has not'oeen used for in vav~ st~~rsies {probably because it is too irrarriurxogenic), and, considering the side effects mentioned above, is not applicable for human isl;t ~ansolantation (for recent review on vectors for gene therapy see ref~9j).
In the p~~ser~t prop~sal, 3 strategies, appl.icaiale to l7i~maz~s, for i~z viva IGl continuous s~__=pply to islets will be evaluated (see C. Progress repor~,~preliminary st~~xdies).
IGF-II was selected as a patentiai protective molecule because it v~~:~s found to be a compo:~mnt of the normal islet microenvirora.:~nur~t, and to reproduce the effect of duet cell incubations. Ti2ree categories of molecules promotit°zg islet cell survival could bo con.sidered: 1) peptides, such as IGFs, released in the environrn°nt and having a paracr~,ne and auto;;rine action, and ~) intracellular molecules inhibiting or stimulating pro- cr° anti-apoptotic path~~ays or ~) nezztrali...ing free radicals. ~n advantage ot°° ti~o first one is the paracr",ne effect on cells other than the transfected cells. Therefore, a l~C~/a cell trar:sfection rate is not required for full protection {we airn at transfectizig 10~% of islets but even a low ~Jo of cell irz each islet znay provide full protection). In addition, the strategy used rn I":~R~' ~, of co-encapsulating islets and cells from a well line with a stable gene exprr,ssion, is applicable only with n.°.ol~~,~ule~ having a paracrine action. t~mong intracellular rrzolecules, those bloc;cing one signaling pathway may induce potentially harmful hyperactivwtion of alternate ps.thways. Enhs~.~ing antioxidant defense mechanisms of the cells is an int~restii~g alt~_rnstiv~ approach, sirlae Eros radicals are the end-point effectors of several patlz~,vays. [~llothods for transf<drz-ing genus of such anti-o~~idant molecules have been developed .:,rr~ shovrn to protect islet cells ire ~eerc by I'r p- ~' ~enh~~nou's group, in Grezaoble, France[lCj, and ~vrll be evaluated in ou:= laboratory (sea letter of collaboration) using in viv~ models, by a student mom Grenol3lej.
Intrace3aular rrzoleczzlc;s vrili probably require the high transfection and stable gene expression provided only by viral vectors, which may be possible and safe ~:~rith our Govalently lirsked znicrocapsules (see bolo=N). ~sli of these approaches deserve to be i~n;rest3gated. Nevertheless, ~vo sele;cted ItIF-II, because of its paracrine action a,nd extremely pova~erful effect of in vitro incu'oations,.
I! is noteworthy that 1 or approaches could be combined.
~.z ~u~~-~nt stataas of r~es~a~~I~ iii whe pa°op~sed .r'xeld of s,ta~cly~
~.:~ol. 'fhe ~°a~lQ of the ~nec~a~-en;d~~onent in Islet ~esu~~ri~ml.
Growth factors {GFs) act in an autocrine and/or paracrine manner~l 1,12j to mediate a broad range of oellula~
responses[2~j. The role of GFs in islet cell sun.~ivsl remains to be clarified. IGFs affect cell growth and differentiation in both the fetal and adult paflcroas~l4-~'~j I~~ariiig induced islet cell neogenesis in the adult hamster, one of us (f,.~.) has found that t'3.cc~1 differentiation foam the dusa epi'~eliunn uras associated with a ~ansier~~c increase in IGF-II exprossion~3~;, recapitulating the fetal environment as characterized by I~~ill.;3l, ~2,j ~'hzs s~zggosts a direct role for IGF-II ir. the differentiation process in the adult. Fvidenc°° for IGFs as sur~i~,ral factors has also been reported.[33j IGFs are able to (i) reduce cell deaf=~~"T,27,2g] and {~,) protect islets i~rom autoimmune cytokines.~7j IG.F-IE[7,2'7,2~j and du~;z: cell-conditioned medium ~C~) can promote islet cell surviva~6j. h9CN~' is con~po5ed of Ir~F-II, but not IGa-L~6j IGF-I stirrzu3:ates insulin secretion in isolated marine L~4j and laurna~~~35 j islets and can antagonize inhibitory and cytotoxic effects ofcytokines in rat islets.{35j. IGF-?~ and II elect on apoptosis has been shov~n to be mediated by the same r~ccpto~~. I~ec~,nt fandings support a rol.;
fc°r I~1GF" in norrxaal developrrzent, morphogenesis and fznctionai rnat-uratia:rz of islets~3'T-~1 j.
I-Iepatio growth motor ((IiGF) has been detected in aeirzawr~~2j anal islet cells[4:3j, can promote.
the formation of insulin-producing cells frown acinar cells[~j and is znitogen to ~-cells[4~,46j acrd duct cells.[47j beg, is a gene that is overexpressed vvr~zin par~a;reatic cells after pancreatitis or resection[~.8j , and dzzring ~'a~o ~~ of 4f ~ialle, ~ea7-Pierre islet cell regeneration. Cne of us (a .~.)[49i and others[Sli] have found that the IZeg gene Es induced ire islet neogenesis. beg may be an. °irnportant paracrine factor in the maintenae~ce and growth of !3-cells[49-5~]. ~ther CFs reported to irr#luence islet :yell survival, replication and function include transforming ~.sF (TGF}- ~. [SS_SF~, 'f~aF-l~ [3~,5~-c~i], vascular endothelial growth factor (~C'aF)[62,63] at.d parathyroid hormone-related protein (fTHrP)[~.]. C.rFS may also be inhibitory in almost all situations n. which apoptosis is positi~Tely stimidated. This suggests that regulatien o~ CF levels is not oni;f important for ~.eli.
proliferation but also in n7aintainirzg viability of cells susceptible to apoptosis.[65-67]
Aitoget'_~er, these data suggest That the activity of CaFs may'oe fundamental to tl~e long tezrn maintenance of gram mass and f~u~ction.
.2~2 islet ~ramsptan~atl~n. Islet isolation; [68-82] ~::~~d transplantation hake been succ4ssful in several animal models.[83-51] Islet transplantation has now become a clinical reality [2,93], using imrnunosuppression.[91,9] :alternative approaches iznclude irnznurae-alteration of the g~°aft [9S-1t35] and induction of tolerance. X1136-1CJ9] o2a3d ~s~tan~-itolatlon by ~eBermencapSnlatiaa~a[lIO-11S] anc. macroencapsulation [116-118] are alternative approaches.
There are now clear evidences that ?~icroencapsulavon offers an adequate protection against allorejectiora and the recurrence of the auto-irmrrsune disease. [119] There is controversy on the efficacy of microencapsulation alone it disv°ordant xenotransplantation. [120] In the past, a much larger number of encapsulated tha>n non encapsulated islets were req~gired to normalize olood glucose. This low efficacy may be due to ;1) the use of large (> 7019 p,.n) ~raicrocap~sules, (i) tire poor biocompatibility of the capsules, (3) the inadequacy of the peritonemn as a transplantation site, (4) the host reaction to encapsulated islets, (5) an inappropriate ~ t~l cut off, and (6) tie removal of islets from their natural envirozur~ent. Ctrr past and current work has .addressed all these issues. ~iocompatibilit; of rnicrocapsnles with the host reFUai~~zs a poorly understood problem. fast laboratories have observed, in vi~ro, a pericapsular r.~e,action or ~siM.[115,12i-12~] There is an inverse correlation bet~~ree~~ the intensity of the ~Ii~I~
and the sun~ival of encapsulated xenogeneic islet's.[12i.,12~-1;1,122] ~3ther important para~reters are rnicrocapsule permeability and strength. We ha=re establisl<ed expertise in investigating and optimizing ail these issues. [i32-140]
~.~ ~edevaz~t pu°e~Ita~s w~~~ of ~e applicaa~~o :~ lei ic~°~e~casrala~f~~. For over 1S
years, we have strongly supporded the idea that fundamental issues iriust ba addressed before clinical application of rnicroencaps~..alatao~z. ~~7e systematically designed in vitra an.d/or i~.vivo methods to evaluate each microcapsule characteristic, and used these methods as end points to optimize the technology. [133-13?,1'?~-143] I~lc~°c~caps~(e see ;'s a c~°itlcal Tsar e. In most tissues, the maxarnusn diffusion distance for effective oxygen and nutrient transfer to cells is 200pm.[l4~-146] The absers.ce of convection Ynovement within a cass~~.le induces an importaf~t C32 gradient from the capsule s'~u-face to the center of the oxygen-censurrzir.g islet.~_147,:48]
reduced capsule size therefore allows for a better oxygc=n and aautrient supply to cells, and oifhrs many other advantages such as: (1) an expor°.ential decrease of the Total iraaplant volume, (2) improved insulin kinetics [149], (3) capsule resista3~ce[i.Sf3,], (~r) and biocompatibility[13~], and
(5) the possibility to use extra-~iaeritoneai implant;~tion sites.[l~] due to the sphey-oid cona~'rguration, the volume of 3019 ;.crr°g diameter capsules is 4.6 and IS fold s7xraller than tl~rat of SQO p.rn and 8110 ~,rn diameter capsr:les respectively. Capsule size rsepends upon the size of alginate droplets. Ii is reduced to '7J1~.-12Q0 laen distracter with an air-bet impinging on the needle, and to 500 ~,n~ with a rrrodified air jet system developed by ~A~olters et al.
[ 129.,151] used on the principles set forth by I-Iorrrnel et al. [113,152], ~je have designed and huil~ in collaboration vrith the Hydro-C~uebec lZesearch Instit'ate on electricity (IF.F~), an electrostatic pulse generator to produce alginate beads as small as 18S p.rn diarneters.['13~,135] Much effort was devoied tcs developing and characterizing these smaller znicrocapsules.[133-1'~'T,i39,140]
~icrocapsule resistance and membrane l~r' cut-of~ (Ni~C;C9) are other important parameters.
'techniques for evaluating capsule resistance have been rather prim~itiv,,.[ISd,153] ~~Je have developed simple and reliable methods to evaluate rnicrocapsule resistance[133] and me:rrzbrane 1CC.[I37] 'The Page 22 of ~~~' ~1a11e, .7ea;~-Pierre large size of existing microcapsules has izrnited their implantation to tire peritoneum.. which presents rnar_y limitations. ~'e h~avo been investigating the liver as an implantation site for encapsulated islets.[I4tl] ~~e havv developed methods and invesr~gatod t:'xe important issue of Ynicrocapsule bi.ocompatibility[a:~~,l~-i], including tl:e role of different cytokines.[~36,15~] fore relevant to the proposed studies, methods were estab:ished[l ] to evacuate islet cell viahilitv[15.~], apoptosis, necrosis, replication as well as iraulixa secretion (perifusion sW
dies).~l] fee also progress report for our studies of islet cell Survival and methods to irxrprove .capsule resat~.nce.
~.4 Significance and iporta.r~oe a~f tl~ls wo~Ir ~:o hrarraan ~iai~~t~so ~1~~ fia~~iiangs of ~nr° e~r~~-ent g~ant~~] is ~~aclily ~pli~a~h °te~ dli.a~a~~l is~ot tr°a~spl~rtataon~ a.nwc~~Id have ~ axrajor ip~~t. since the efficacy oiC islet transplantation was increased by 4;)1?a/o [1], ~ to ~ fold more diabetic patients would bene~'.-'zt from a~n islet graft, and the likeliness of diabetes recurrence world be decreased.
'The present proposal is the extension of this research. ~:.ansidcri.z-rg the mrery powerful effect observed with ire vit~d IGF-Il 48 hour incubations, investigatiarg the eff act of iz~ vivo I~~°-II
eortinuous delivery is a must.
Another advantage of sel~ct~.ng a peptide (I~'rf'-~) with a paracrino zff~ct, as a protective molecule, is a potentially easier approvs.l for clinical applications. Ir3 addition, tlr;, present proposal methods for gene therapy are applicable to humans. This i;~ it contrast with the use of other vectors for gene transfer (sT2ch as adenovirus), wl~ic~, are appropriate for physiological st~kdies, but not for clinical appli~ati.oz~. Fear methods described in fart 2, human poll Iines, such as I3U~-~~ (I~uman CTmbilical ~or~l), ~°ould be used.
'V~le have access to human islets from f,. Rosenberg, co-applicant, ~fvlc~ill ~~. I-Iealth ;:enter, 3~3 min. drive from our centre), who has initiated h~r.~na~i islet isolation and will be ready for clinical trials soon. dither sor~rcos of ~sulir~-producing cells arL being investigated. In our perspecti-Je, the most promising approach is tlz~ use of stags cell derived islets. I_..
Rcsenberg''s grotEp has pioneered the study of islet neogei*esis arid is pursuing research en stern cells. since type 1 diauetic patients would develop ar~ i~xurnrrne reaction against even airtolobous insulin-prodcrcirrg oohs, an irrununoprotection method, such as n ~ioroencapsralatiota, will still b~ required.
~,.ar novel covalently linked microeapscrles, visually ~~destructibie in conditions fi~iuad in the living body (see ~. Progress report, 'cast paragraph), will provide additional sar~ety, and enable the safe use of gene therapy, imrnortaliz~°d cell lines and stare cell derived islets.
'Therefore, the eolia'ooration betwecri ouf.° 2 laboratories ~:uith complementary o;~periise will retrain (and even become increasingly more) reaevant ire duo future.
~~ :i'Rt3~lt~SS t'~12'~'lh~ r 1' S'~'~'~I~S
As mentioned above, during pair craxrent JD~F' grant, we showed that I~F -L:r~
va~ca incuhations prevent islet cell apoptosis and necrosis and allow the ;~oz~nalization of topic (~20~~/0) diabetic mouse blood glucose with reduced (~~~alo) islet call mass transplantation: (1 a0 vrs 3~~' islets;. "f"his work Ied to a publication in ~ndocr~'nologly.~l] Irr the past, a much larger .~aenber of encapsaalated than non encapsulated islets has been rewired to induce insulin independence.
In this work=1], racing IC1F-~ incu'aations and ~.3C~ ~zm n~icrocapsules, norrrioglycemia was induced with less encapsulated isletslkg than with free. ;slats in laun~a.n trials.~3~,156~
Idowever, rising this minimal mass model, normoglycemia Iasted ~ months, wl~i: h is g~aod witlj dais m;~dul but is not a cured.
In other ongoing studies (unpublisl-ied data), blood glucose was nom~alized for morn than one year in 17 diabetic mice. In several mine, diabetes wary cured as dof~red by continuous blood glucose normalization, wi~hout any food restriction, irrusr~anosrrppressiorr or other i~nte~~-ferenee in.
the recipient immune system, without the use of any drag (oral or injectable), arid persisting until tire animal die from causes) other 'rkian hyperglycemia (i.e. ageing), at approxixrzately 1 ~ month of age (mouse lifespan is about 18 to 2Ii months). Borne of t;~ese normoglycemic mice were sacrificed 'oefore they die in order to perform an, autopsy on fresh ~isssros.
~ base autopsies revealed no significant insulin production by the recipients' own pancreas.
'f1°iese results were Palo 2~ ot' 47 Naile, .7ea~°t-Pierre obtained using heierogeneous conditions, because of ongoing investigations on the effect of dif~erent parameters. We will perf~qrn a st~s~iy on 2~. rni~~e using unifc>r.~.
These results wore obtained us~.~a~; xenografts of 11)0 or ~0~
:n~icroencapsulatc;d islets (not incubated in IGF-II], the number used i:-~ most publications on encapsulated islets, wi3ich is approximately 2 to 4 fold the nugabor/kg used in clizZical (non encapsulated) islet trarusplantation.
The goal of this proposal, using IGF in viva supply, is to cure diabetes (as defined above) i~n m ce, using the number of encapsulated islets used irr clinica? (non encapsulated) islet transplantation.
The methodologies that will be z~sed, for in vitro and i~' viva islet surs~ival evaluatior;s; are similar to those developed, validated, and published during our carrot=t ~DFF
grant.~l]
Three strategies :for ire viva IGF-lI supply will bo evaluated. ??) IGf gone transfer into islet tolls, using non viral vectors 2) co-encap, ulatior~ of islets and culls with a stable ~~rF gene expression, developed using a non viral vector , and 3~ co-encapsulatian of islets and duct culls.
All gone trairsfers were (and will be) performed using the liposorne rc~utliod (-.~.~G TAIS), a safe non viral vector. Non viral methods allow transient gene expression i.e. tl-rat is not always transmitted to both daughter colas of dividing cells. ~o~~sidoring that the lifespan of bate-Loll is 3 months, IGF
will be supply to islets during a critical 2-3 mor°~tlr post-transplantartion period. Sirrwe pernnanent IGF-II supply might provide beater very Ion;-term su.Yvival, a cell line with a stable gene expression (transmitted to all daughter cells or dividing cells; was devel.opod, usilrl; a neon~ycua resistance gone, and co-encapsulated with islets.
erring the Last year, we designed four di~orent AIRES plasmids coding for GFP, IGF-I, IGF-II
and IGF-I+II proteins. To do so, IGF-"~ and IGF-II mvzrine coding s~,quences were produced by IZT-P~1~ amplification of mIZNA ~-orr~ hepatic tissue extraction. These mI'cIvTA sequenc,,s were tra!LSlated in ~Nl~ by reverse transcriptase anu' c.lonec: in a pCR2.1 .~lasmid for arrrplifrcaiion in ITl-'VtxF' Ir.~oli. Then, these IGF ceding secluene es wc~e ctrt and included in ::lze pII~ES eulcaryote plasmids to generate three diffe_ront plasmids: pIGF--I, pIGF-II, are d pGF-I+II. Note that the plasn~id of expression, AIRES, a.llcws the expression c~F two differera proteins at use saax~e level and tlme. The identity of the transgenes was oonfnrned using restriction enzymes and sequencing.
I3ur:rrg the same period, we esta151ished the paran-reters for islets transfection by pIP~S plasmids using IJ(~TAP liposomes.[1S7-a5~~ Isolated nxouse and rat islets wore transfocted vfith DGTAP/phRES-GFP used as a reporter plasmid with different pre-cult=ure t"lrnes, I~N?Jliposorcre ratios, I3'~<A conoentrat~ons and transfoctior. duration tunes. ~C~a:-~sfec~:ion efficiency was quantified joy microscopic eval:xation of GfP gene expression. The g'aigher transfection rate (73 %), 5 days post-transfection, worn obtainec~l following a protocol of ~ hours of culture orz fresh isolated islets before 24 hours :ncr<bations wiii~ 6 ~,glrnl :1~NA and 3~
~tl/anl of 1:7()TAF. islet viability was similar for GFP-transfected and not transfected islets (data not shown). The transfection efficiLncy allowed us to pursue o~~:.r study using the therapeutic ge:~re plasidse pIl~=.ES- IGF-I+ II, AIRES-1GF-I grad AIRES-IG.F-II (fi~;~~ro 1).
Fig l: Observation at day 3 of encapsu?ated transfected rat's is;zets by pGFb and BOTAP on f~uores~ence microscope (A) and white light (B). 'the transfectior~
rate was evaluated at 63%.
Page ;~~ of ~-~
~a~le, Jean-Pierre Jsing the sane protocol, rat islets were transfected with 1~"xF plasnzds and microGncapsalated.
These islets were then incubated ~:t 37 ~ i~~. free-sorur~mediut~rs (C~/.
'C~f,-?~5~j or i ,LJitra~"~zltu~~e medium for ~ to 10 days. The presence of the IGF-1 or IGF-II proteins irz the sorespondzng medium was confim3ed by imt~nurtoblottirtg (anti-I~F-l, anti-IGF-II).
ivloreover, these IGF
proteins were shown to be biologically active, since, irz all expo~iments., IGf-Z and i~'rF-Z
transfected islets had a better viability than non ztansfected islets anal FGF-I+II transfected islets (fig=are 2j.
Fig. 2: Evaluaticn of microencapsalated rat islets transfected with plash id pl~sF'-a -t- II, pI sF-Ia antl pIGF-I viability (not transfected islets, C-). A) Islets were :;ultured in minimally nutritive CMR~-1066 serum-free) medium for a 6 days ~~~:ricd. Islets Friability urns analysed by Isropidiun ~ iodide and orange acridine staining. Experiments were performed on 2 different islet preparations ~n=;~). The viabilii-~~ was evaluated by two different observers. I~) Islets wore cultured in LsitraCult~>re medism for 3, 6 and 10 days period. Islets viability was analysed by propidium iodide and orange a.cridine staining. :Exper=i:~ents v~ere performed on I islet preparatians . Tho viability was ev~aIuatod by two different observers.
Prelirrainary works were also initiated on the second ap=sxoach. A cell I"_ne ~:aitit a stable IG~' gene expression was developed. F?rst, rnonolayer cells Tlilf~., at ~0% confluence wore transfected with
[ 129.,151] used on the principles set forth by I-Iorrrnel et al. [113,152], ~je have designed and huil~ in collaboration vrith the Hydro-C~uebec lZesearch Instit'ate on electricity (IF.F~), an electrostatic pulse generator to produce alginate beads as small as 18S p.rn diarneters.['13~,135] Much effort was devoied tcs developing and characterizing these smaller znicrocapsules.[133-1'~'T,i39,140]
~icrocapsule resistance and membrane l~r' cut-of~ (Ni~C;C9) are other important parameters.
'techniques for evaluating capsule resistance have been rather prim~itiv,,.[ISd,153] ~~Je have developed simple and reliable methods to evaluate rnicrocapsule resistance[133] and me:rrzbrane 1CC.[I37] 'The Page 22 of ~~~' ~1a11e, .7ea;~-Pierre large size of existing microcapsules has izrnited their implantation to tire peritoneum.. which presents rnar_y limitations. ~'e h~avo been investigating the liver as an implantation site for encapsulated islets.[I4tl] ~~e havv developed methods and invesr~gatod t:'xe important issue of Ynicrocapsule bi.ocompatibility[a:~~,l~-i], including tl:e role of different cytokines.[~36,15~] fore relevant to the proposed studies, methods were estab:ished[l ] to evacuate islet cell viahilitv[15.~], apoptosis, necrosis, replication as well as iraulixa secretion (perifusion sW
dies).~l] fee also progress report for our studies of islet cell Survival and methods to irxrprove .capsule resat~.nce.
~.4 Significance and iporta.r~oe a~f tl~ls wo~Ir ~:o hrarraan ~iai~~t~so ~1~~ fia~~iiangs of ~nr° e~r~~-ent g~ant~~] is ~~aclily ~pli~a~h °te~ dli.a~a~~l is~ot tr°a~spl~rtataon~ a.nwc~~Id have ~ axrajor ip~~t. since the efficacy oiC islet transplantation was increased by 4;)1?a/o [1], ~ to ~ fold more diabetic patients would bene~'.-'zt from a~n islet graft, and the likeliness of diabetes recurrence world be decreased.
'The present proposal is the extension of this research. ~:.ansidcri.z-rg the mrery powerful effect observed with ire vit~d IGF-Il 48 hour incubations, investigatiarg the eff act of iz~ vivo I~~°-II
eortinuous delivery is a must.
Another advantage of sel~ct~.ng a peptide (I~'rf'-~) with a paracrino zff~ct, as a protective molecule, is a potentially easier approvs.l for clinical applications. Ir3 addition, tlr;, present proposal methods for gene therapy are applicable to humans. This i;~ it contrast with the use of other vectors for gene transfer (sT2ch as adenovirus), wl~ic~, are appropriate for physiological st~kdies, but not for clinical appli~ati.oz~. Fear methods described in fart 2, human poll Iines, such as I3U~-~~ (I~uman CTmbilical ~or~l), ~°ould be used.
'V~le have access to human islets from f,. Rosenberg, co-applicant, ~fvlc~ill ~~. I-Iealth ;:enter, 3~3 min. drive from our centre), who has initiated h~r.~na~i islet isolation and will be ready for clinical trials soon. dither sor~rcos of ~sulir~-producing cells arL being investigated. In our perspecti-Je, the most promising approach is tlz~ use of stags cell derived islets. I_..
Rcsenberg''s grotEp has pioneered the study of islet neogei*esis arid is pursuing research en stern cells. since type 1 diauetic patients would develop ar~ i~xurnrrne reaction against even airtolobous insulin-prodcrcirrg oohs, an irrununoprotection method, such as n ~ioroencapsralatiota, will still b~ required.
~,.ar novel covalently linked microeapscrles, visually ~~destructibie in conditions fi~iuad in the living body (see ~. Progress report, 'cast paragraph), will provide additional sar~ety, and enable the safe use of gene therapy, imrnortaliz~°d cell lines and stare cell derived islets.
'Therefore, the eolia'ooration betwecri ouf.° 2 laboratories ~:uith complementary o;~periise will retrain (and even become increasingly more) reaevant ire duo future.
~~ :i'Rt3~lt~SS t'~12'~'lh~ r 1' S'~'~'~I~S
As mentioned above, during pair craxrent JD~F' grant, we showed that I~F -L:r~
va~ca incuhations prevent islet cell apoptosis and necrosis and allow the ;~oz~nalization of topic (~20~~/0) diabetic mouse blood glucose with reduced (~~~alo) islet call mass transplantation: (1 a0 vrs 3~~' islets;. "f"his work Ied to a publication in ~ndocr~'nologly.~l] Irr the past, a much larger .~aenber of encapsaalated than non encapsulated islets has been rewired to induce insulin independence.
In this work=1], racing IC1F-~ incu'aations and ~.3C~ ~zm n~icrocapsules, norrrioglycemia was induced with less encapsulated isletslkg than with free. ;slats in laun~a.n trials.~3~,156~
Idowever, rising this minimal mass model, normoglycemia Iasted ~ months, wl~i: h is g~aod witlj dais m;~dul but is not a cured.
In other ongoing studies (unpublisl-ied data), blood glucose was nom~alized for morn than one year in 17 diabetic mice. In several mine, diabetes wary cured as dof~red by continuous blood glucose normalization, wi~hout any food restriction, irrusr~anosrrppressiorr or other i~nte~~-ferenee in.
the recipient immune system, without the use of any drag (oral or injectable), arid persisting until tire animal die from causes) other 'rkian hyperglycemia (i.e. ageing), at approxixrzately 1 ~ month of age (mouse lifespan is about 18 to 2Ii months). Borne of t;~ese normoglycemic mice were sacrificed 'oefore they die in order to perform an, autopsy on fresh ~isssros.
~ base autopsies revealed no significant insulin production by the recipients' own pancreas.
'f1°iese results were Palo 2~ ot' 47 Naile, .7ea~°t-Pierre obtained using heierogeneous conditions, because of ongoing investigations on the effect of dif~erent parameters. We will perf~qrn a st~s~iy on 2~. rni~~e using unifc>r.~.
These results wore obtained us~.~a~; xenografts of 11)0 or ~0~
:n~icroencapsulatc;d islets (not incubated in IGF-II], the number used i:-~ most publications on encapsulated islets, wi3ich is approximately 2 to 4 fold the nugabor/kg used in clizZical (non encapsulated) islet trarusplantation.
The goal of this proposal, using IGF in viva supply, is to cure diabetes (as defined above) i~n m ce, using the number of encapsulated islets used irr clinica? (non encapsulated) islet transplantation.
The methodologies that will be z~sed, for in vitro and i~' viva islet surs~ival evaluatior;s; are similar to those developed, validated, and published during our carrot=t ~DFF
grant.~l]
Three strategies :for ire viva IGF-lI supply will bo evaluated. ??) IGf gone transfer into islet tolls, using non viral vectors 2) co-encap, ulatior~ of islets and culls with a stable ~~rF gene expression, developed using a non viral vector , and 3~ co-encapsulatian of islets and duct culls.
All gone trairsfers were (and will be) performed using the liposorne rc~utliod (-.~.~G TAIS), a safe non viral vector. Non viral methods allow transient gene expression i.e. tl-rat is not always transmitted to both daughter colas of dividing cells. ~o~~sidoring that the lifespan of bate-Loll is 3 months, IGF
will be supply to islets during a critical 2-3 mor°~tlr post-transplantartion period. Sirrwe pernnanent IGF-II supply might provide beater very Ion;-term su.Yvival, a cell line with a stable gene expression (transmitted to all daughter cells or dividing cells; was devel.opod, usilrl; a neon~ycua resistance gone, and co-encapsulated with islets.
erring the Last year, we designed four di~orent AIRES plasmids coding for GFP, IGF-I, IGF-II
and IGF-I+II proteins. To do so, IGF-"~ and IGF-II mvzrine coding s~,quences were produced by IZT-P~1~ amplification of mIZNA ~-orr~ hepatic tissue extraction. These mI'cIvTA sequenc,,s were tra!LSlated in ~Nl~ by reverse transcriptase anu' c.lonec: in a pCR2.1 .~lasmid for arrrplifrcaiion in ITl-'VtxF' Ir.~oli. Then, these IGF ceding secluene es wc~e ctrt and included in ::lze pII~ES eulcaryote plasmids to generate three diffe_ront plasmids: pIGF--I, pIGF-II, are d pGF-I+II. Note that the plasn~id of expression, AIRES, a.llcws the expression c~F two differera proteins at use saax~e level and tlme. The identity of the transgenes was oonfnrned using restriction enzymes and sequencing.
I3ur:rrg the same period, we esta151ished the paran-reters for islets transfection by pIP~S plasmids using IJ(~TAP liposomes.[1S7-a5~~ Isolated nxouse and rat islets wore transfocted vfith DGTAP/phRES-GFP used as a reporter plasmid with different pre-cult=ure t"lrnes, I~N?Jliposorcre ratios, I3'~<A conoentrat~ons and transfoctior. duration tunes. ~C~a:-~sfec~:ion efficiency was quantified joy microscopic eval:xation of GfP gene expression. The g'aigher transfection rate (73 %), 5 days post-transfection, worn obtainec~l following a protocol of ~ hours of culture orz fresh isolated islets before 24 hours :ncr<bations wiii~ 6 ~,glrnl :1~NA and 3~
~tl/anl of 1:7()TAF. islet viability was similar for GFP-transfected and not transfected islets (data not shown). The transfection efficiLncy allowed us to pursue o~~:.r study using the therapeutic ge:~re plasidse pIl~=.ES- IGF-I+ II, AIRES-1GF-I grad AIRES-IG.F-II (fi~;~~ro 1).
Fig l: Observation at day 3 of encapsu?ated transfected rat's is;zets by pGFb and BOTAP on f~uores~ence microscope (A) and white light (B). 'the transfectior~
rate was evaluated at 63%.
Page ;~~ of ~-~
~a~le, Jean-Pierre Jsing the sane protocol, rat islets were transfected with 1~"xF plasnzds and microGncapsalated.
These islets were then incubated ~:t 37 ~ i~~. free-sorur~mediut~rs (C~/.
'C~f,-?~5~j or i ,LJitra~"~zltu~~e medium for ~ to 10 days. The presence of the IGF-1 or IGF-II proteins irz the sorespondzng medium was confim3ed by imt~nurtoblottirtg (anti-I~F-l, anti-IGF-II).
ivloreover, these IGF
proteins were shown to be biologically active, since, irz all expo~iments., IGf-Z and i~'rF-Z
transfected islets had a better viability than non ztansfected islets anal FGF-I+II transfected islets (fig=are 2j.
Fig. 2: Evaluaticn of microencapsalated rat islets transfected with plash id pl~sF'-a -t- II, pI sF-Ia antl pIGF-I viability (not transfected islets, C-). A) Islets were :;ultured in minimally nutritive CMR~-1066 serum-free) medium for a 6 days ~~~:ricd. Islets Friability urns analysed by Isropidiun ~ iodide and orange acridine staining. Experiments were performed on 2 different islet preparations ~n=;~). The viabilii-~~ was evaluated by two different observers. I~) Islets wore cultured in LsitraCult~>re medism for 3, 6 and 10 days period. Islets viability was analysed by propidium iodide and orange a.cridine staining. :Exper=i:~ents v~ere performed on I islet preparatians . Tho viability was ev~aIuatod by two different observers.
Prelirrainary works were also initiated on the second ap=sxoach. A cell I"_ne ~:aitit a stable IG~' gene expression was developed. F?rst, rnonolayer cells Tlilf~., at ~0% confluence wore transfected with
6 ~g plasmids and 18 ~l Fugcne ~ during 2~ hours, in a medium with senrrn.
.ells veers ror~~wod with basal r3~edium during one day for recovery prior the additionx of Geneticin (G418) for selection of neomycin-resistant cells. From this p~in~;, resistar!t clones were harva~sted fox an additional 4 -weeps in presence ot' i ~ 18 and tlmn agaiA_. in basal medium.
"t he establishment of a GFP stable cell line generated in the: earns condition ay the IGF cell lne eras used as a control.
Rat islets were co-encapsulated with cells transfected ve~th IGF-II and GFl' genes using different concentration of'f2VI4 cells (~xIC~, l.~e, and ~ ~ 3~' oellules/mi algixtute) (figure 3). ~Y~ese islets were then incubated at 37 ~ inl~ltra~ult~re cdi~.aan ii;r 3 days. ~Ie observed at a;!1 doses that a.~~Cer three days the islet viability v~~as bettor witl.~ IGF-Ix co-encapsulated cells than w.'tth G'=_~'-cell or with islets encapsulated alone ~ftgare 4). for the dov~~lopment of i~ie methods, a ~'i~lI4 cell line was used because it is hnom~ to I~avo the maohinc_ry to secrete (minimal atnosznt of) IGF, vfluLh avoided problems in interpreting negative monditsor~s as potentially duo to the cell ty);e. III
experiments included non transfected TM~ cells as controls, and showed differences. 'The experimental conditions being well established, we will now use, for t~~e proposed studies: a cell line that does not produce any IGF', i.e. Chinese I-Ia_mstet- C)~rarian cells ~~;~~) Page 25 0~' ~7 Na34~, Jean-P~prre d -_~ - , .~_._ ., r ,. ; .: .~ ~ b ig 4: evaluation of rr~rtcroanca~suIed rat islets o!r Fig 3: coencapsuled rat's islets wiih Tl~l~ . ~ coencasulcd with Tlt~li~-~1'P
ce'11s or Ti~%P~.-I~F -2xIO6 cellules/mI d'alginate ~ ~ II cells. Viability. Islets urere cultured ir:
------i ~ ~ltracultnre C~hl2L-I(3~6 medi~~rrrE for a 3 days peQ°iod. Islets viability =Jas analysed by propidiun iodide and orange acridine staining. "tlroe; viability b were evaluated by twt~ di~~eren'~ observers.
f~ other st~diesg ~°-~:nded by the ~aradian ISiabetes ;~~.sscciation, t~~E; developed a rnetbod that allows rr;icroencapsulation of livity ces.ls i~. covalently linked algiruzte poly-I,--lysine- aigi;za:~e (~P~) membranes. The extreme conditions ~~equired to produce and 9;o destroy coval,:nt IiPn:s are usually incompatible with living veils. fbereFore, these e:~treely ;~tt-o~
nicr°oes~ps~le.s are vi~~aally indestr~cti~Ie ire tlae ~~etieal and pl~~~sieal o.oraditac~ns yo~~rad its tire ivvin~:~a~dy.
They remained intact aver 2 years xn a pI-y I2 all~al~.~~ bufFe:°, wlaer-eas standard microcapsules completely dissolved af$er 45 sec. itz the same conditions, and they resist to a ineclr.anical stress test that implies stronger a9orasioz2, crusbi3~g and shearing forces tiara they are likely to be submitted during a lifetime in tl~e Lzving body. I~ ote ~f~a~ ~it~ his te~It~i~~y we pt°°~p=rn;~e ~
new ~°~Ie fir ~t4~re~~:r~~~~sulnf~osrx addition t~ ;.jrotecting the tr°ansplant (islets; from tfze recipient irrtune system, it will inversely pvoteet t-he recipient fr~trt t~~e tr~raspp~n t, en~bl~r~
the safe nse of gene ther~py~ irarart~I~esi oell i~-,e ~:nai step sail ~lari~r~;~ islets.
~. i'~I~'I""~I~S ~~ f3~~~~3~J~°~T':i~~,d ~w~' ~''~'~ ~~~~ f~~~PC9~~.I~
~~~;~.
~~~"I' ~. ~'l~e effect, ~n fx°ee end t:~iet°oe~t~~ps~late~3~:
islet sell snrp.Ya~r~h or t~°~nsfer~°in~ li~~' genes into islet oePls, usi~a~ non ~riral veet~~c°s. mote that ~~nicrocapsales ~vitb covalently h~~ked ~I'~mebranes, as described in i=~e p:i°ecediaag parag~°apl2, will be use3~, ira aII st.~dies.
~~. ~n vitr°~ stndi~s, f~~taonale. ~r~ vi~-r~ studies gill allow evaluations and ecastomi~ations of many parameters, and establish a dequate experigner3~aal conditions.
~'~°eli~a~inar°yr d~t~. I'n tb~e ver~r t'.rst ia~ vitro experiments (see prerimir~ay dataj, _~~-I and IC~I~-II
(but not I~:~ -I+Lt) gene transfer improved islet Bell v~.abiliiy at 3, ~ and l il days in culture.
~:~pe~°ie.~tal stesi~~a< ~listar rat islets eo-tranfected ~~~ith ~-reen ~ l;aerescena l~roteir~ (a.~FPj, l~~-I, A~~b~'-II or IC3~-I+TI and zeal ve islets will be compared. por all ~°eatrnr;nt groups, free and rnicroencapsulata~d will becopred.
Idea will be cultured for 30 days arzd stuciies will bt; performed at 3, ~, 1 ~, ~ l ~.r~d 313 'icays.
culture media will be changed ~; 2 days. Iaietl~ods briefly described.
prei~rtinary data and in more details in our resent papers l~, as well as zew methods will b a ~:.sed.
This include tlae stczdy of transfection rates, using C'aF~~, Imp secretid~n in the supernatant usin;;
westein bhts and ~.~,I~t~~, islet cell viability studies, using ~~zciescenc;~: microscope with propicl~strr"~ iodide (dead cell.sj and orange acridine (living cells), ~', scud measu.~°errtent ~md qua~~~tificaa;~n of islet nets°otic centers.
I-~garose blocks will be prepared fog irrnnu~M~obistocbe~~nfcal studies irzcl~.ading apop8:osis studies, P~~ ~~ o= g.-~ts~~ ~~,~~~ ~,m ~e~o ~:n t~~~~e, JC't3il-p12C1's'~.
usi2; the 'pIJ assay and ICI protein rr~;asu~~exnents. In order to identify ~r~l~ich t~rpe(s) of islet cell undergo into apoptosis, successive sli; Ls ~~11 be studied wi'ali the ~"UIr-EL as;yay at3d ar~ti-ans~lin and anti-glucagon antibodies, as p<<evio~csly :~~scr°ibed.~I~.
~-yell replication aid islet neogenesis will be studied using hrdLT incorporation end immunopero~~ida y~ co-localization of insulin and cytolceratin 20. A ~~1~ pre-inc~:~a2ion with ~0(lng/ml Iuf'-II wig also be studied to investigate a potential additive ~~~ct tc~ IG~ -II gwne ~-a°~sfer.
Anficipated results and ~ignif~can~~ . ~h'e anticipate tl~~ ccz~,~.rmat~on of preliminary data.
I~~:.er optimization of ti_e method and longer duration in ~~~Iture, the positive efff.ct on islet saarviv°al st~ouid 2~e even snore important. In addition, for airn 1 to 7, vie anticipate that pre-ixansplantation ire vitro I~F
incubations and I~F gene transfer will have additive ef"ects, since th.e for7ner have s,n irn.~nedito eff"~ct and prevent the important damages ir~duoLd by the isolation and oncapsulat~~oa procedures, whereas the transfected genes tame a few days to effecrvely produce I~~p s. '~
he islet transfecticn rate, currently at 63%, a good result with this method, as von~pared to published data~l5'7.,15~], can be fur~.her improved, sine some p~am~t~rs ~.~»re zd~nt~sfi~d as important but not 4.~ot optimized. ~~n~gen~y sta~ez~~~nts. fee a~.n ~, Alns . Its viva. atia~~ale.
~~~e pote;ntial clinical significance of the preceding studies will be e~~aluate~l. Fcr studies in ~Llaer animal model:; see airr~ ~. ~r~i$arainary data. Aithoa.~gh we have only irc vii-~~o data yet ors I~v° gene transfer, we hare shown(1) that pre-transplantior~ ICI'-II in~-~:batiors Iza~ro a powerfi;l effect ~~n tr~~;nplant~,d islet survival. ~~Iaerian~utal design. If pr~limd~ar'~ data ere ccnfirrned, or~dy I~F-TI v~ill ho study initially (unless other studies shovr a stronger effect ef hrrF-I er I+II~. A
ninir:~E~,I islet mass model, based on the results of gar published s~.'-crdy[1;°s will 'o~;
used. I=~tra-pcriton~:al allografts (n=12 per conditions) of lj~ or 75 ~57I31/f mouse rnicroencapsulated iss.ets, L~-ansferted with I~F
gene or not, will be performed into ~571~1.~6 ~~'~ diabetic mice. I're ~'annsplantaticsn I~a~-II it vita~o incubations will be performed in all conditions :~. none, d~pendin~p upcan ia~ °~i~o st~~dies (aian 1). controls will include free islet 'Tx. 'fhe prep~.ration and follov:~-up of diabetic ar.~nals will be as previously described. i 1) Briefly, animals ~;nill have free access to food and blond glucose will be measured at defn-zed~ til~aes. "3'~°o successive blood glucose >_ 11 ml; (~#IO rng%) will sign a recurrence of diabetes. In addition, at tl~e spontaneous dea.tl~
(if and°) or at the ss.crifi~e of the recipients, microencapsvlated islets will b,, retrieved for the evaluation of apoptosis and necrosis and the presence of ins~.~iic~. The follow-up c~f diabetic mis~c will ~,o on until diabetes rec3~rrence occurs or animal die. Tl!e ~ last animals s>~11 nerrnoglyce.~nic in each ~~oup will be sacrificed when presenting severe signs of agoiz~g arid autopsied obi ia°esh tissue to eliminate residual endogenous insulin secret.ionEi,l6t~) (has never ~Oeen positive in the lab). End-points v~°ill anclude: number of recipients maintaining ,~orsnoglyc~,:ania, average I°gloeJd glucose and glucose tolerance tests (insulin and glucose) at d, 1? and l8. ~.~~tieipat~d resa~its and sig~ilacanoo. die anticipate that arx effective ;stet cull protectiw:n dL~ring tl;.e critical post~~tfansplant poric~d will af.low prolonged normoglycemia, using a reduced islet cell mass. Cura~gotZ~:y stato~no~~ (~srns I +~).
'This method does not induce a stable ge ~w o~pressioz~, and efFect7ve hGF
produ~;tion should slo~Arly decrease over 3 months. froteating islets duriLag a critical post-transplantation tnay be sufficient. It is possible that optnmal long-;verr~ results will require;
pe~man~nt IC'gf-II supply.
Alternative strategics to achieve our goal are described in part 2 and j. ~A'~
2. 'I°lae efI°eet ~n islet cell survival of ~a~m~ncapsulaiiug islets a~~d cells with a. sta~l~ g~n~
expressi~n of h~~-~G~'-~ (~sr ~ ~r ~-Y-~ ). ational~ grad pr~l~auary data fir ~O;~rt ;~. Using multi~;ehular structures, such as islets, only retro~Jiral vectors could dn°' ~ctly induce si:ab'.e gene e~cpressior~.
Although new very effective tra~fection methods involving viral vectors ofaer exciW g scientific opportunities for physiological s~.tdi~s, their use i~7 cli:?ical applica?io~as remains anc;ertairt, c;v~.n for rapidly fatal genetic diseases.~~] Using th~;; non v al Izposorr~e nWtlaod (l~t~fAF') and a neomycin resistance gene, we v~rere able to ,~rodu~e mc~~aonuvlear co1',1.
lii=es v~ith stable ICIf-I, II
and I+II gene expressions. ~f~ese cells were cc-wncapsulated vdi~, i islets (see Progress repart/preliminary data). ~~z vit-r~a studies will allow the evaluation of G
laa°ge nutnbex of conditions. .Paa viva studies will cor~~rnz the potential clinical signiric~-~c;e of i~a va~o findings, ~ac~~~ ~y of 4i Heiie, Jean-bier re determine the timing of effective il~.t~i dontion, arid evsiuate a possib.te rescu;, effect. r~~° Ira vit;~c studies. Exper~~nentai design. islets will be co-Pracapsulated with Clzin=wse Hamster ~varian (CH~; cells transfected with 1GF ~, II, or I-~ 3I genes btsis~;
different eoncentratiom of CHC cells. The number of co-°ncaps~.zlated islets she==wing ~:he best results (viabilitt~~ =~s .veils concentration) will 'be incubated ~~~8 h. (24 f~. before arid a~:r encapsulation) in 50~ ng ml cf ho IGF-II under study as described ixz (l~. Controls will include islets co-encapsulated with non trar;sfected CHC3 cells, encapsui~ted alone and Trot can capsulated.
Additional expeziments wl~
CH~ cells and islets encapsulated separately and cuit,~red together will include a dose-response with increasing proportion of CHG veils. Tl°_rroar~e7~, cultures and follow-up v~ili be Iseri°orzxzed, as described ira aims 1. Andlcapated ~°ests and signi~a~ano~. ~Je anticipate t°zat tile method ~Ffill prevent apoptosis and necrosis foV~ a very prolonged period, sumo the iGF
genes will be permanently expressed in all cells of tlae successive pe.nerations. Th.e study will determine, tl~e amount of CHG veils and IGF required for an optinxai effect, and establish ti~ze best oxperimer~tal conditions. As explained in airn l , ~~e anticipate an additive effect; of in vzt~o ir°~cubations and gene transfer. Cor~tengen~y state.rnent. An excessive amount of ~:EIG cells may provice more IGF supply but be potentially harrrrful to encapsulated =gal suz-vival. Tk-ds study will ~,eranit to fz:~d tile optimal (approximate) amo.r:~t of cell ,or >maic;rocspsules. If oux~ goal is not a~,hieved, this proposal contains alternative z~ethods. Aaxn ~.° To devoi..~p a method ixzat allovds turning on/ofF z'n vivo, 1GF gene expression a.tional~. ~eirig able to tzrrn onloff, ira viva, transfected IGF gene expression will provide an interesting tool to address several ~~;u°stions, part:~c.ularly those described iz~z aizrs 5. In addition, it will provide ideal controls: exacdy tile same cells will be transplanted, but the activating drug will not be injec°ttrd. ~ruli ° ~z~y data. Using; the pL~S
vector, ~ genes would be transfe~zed into the sarrze cehi, in addition to he ~eornyoin resistance gene that alio~~;s the development ona stable liz~~. ~xp~rixnontal de:slg~.
Ush~g a Tetracyclir<<o-Regulated expression systez~. (T-Vie:; System), p~i~..I~A~ITG vector, ~:HG
cells will'oe trar~sfected with IGF-I1 (or I or I+II) gene us,ng a tetracycline responsive promoter '~lzat ran be trur~ed on (or offj by admizxistezing (or stoppinJ) Lotracyeli;~ze ~.o the z~ccipient, and wilt oe co-encapsulated whiz islets. 1'relirninary i~ vitra tetracycline dose-response study will be aer i°orn-~~~d z~o;zsuring only IGr protein concentrations in t_~o supernatant. Antlcgpated ~°os~&ts° ~o anticipate that this study, combined with the results of foregoing studies, =will a11~3w to ~sta'olislti~zg the r,onditions for ahr~ 5 studies. Alan ~. T o study, in viva, '~.hc; effect of c;o-encapsulating islets and eelts wi,.h a stable IGF
gerfe expression, including the tinning and da~atioz~ of r:l°-fective treataoent avd possibility of failed islet rescue. ~atl~nale. Same as =or airn 2. ~~z addition, the tinning acrd duration of IGF delivery required for optimal protection ~:uill be e~aaluatod. I3~~~dilaaar°y e3at~° S=.raze as fen ainz 'i3.
~xpa~°atnental deslgn° The same ani_~~zal model as in aii~zs ~
will be used. All recipients (n=1 ~ per groups) will be transplanted with the; sab~ne P°~.II~3 c°lls tranfected with IGF'-IT (or I or I+II;s genes using the tetracycline responsive "on off'"' Te: T ~ sequence and c~-encapsulated with islets.
Controls ~.lill include recipients transplanted with tlae sazrze cells but will not receive tetraeyclir?e and transplanted with og~capsulated islets mono. I?.xpe,r irnentai groups will receive tetracycline during 1, 3 months or indef~nitel~~. Follow-up aced shady methods wilt, g~se as ir= ain.~ %. In addiv~~ion, after 4 days of hyperglycemia rev°urrence of anirrzals off the c~ug, tetracycline ;rill be reir_troduced to evaluate whether islet cell r~,scue is possible. Anticipated r°esults arid sagnlf°bean~~. ~e anticipate that oz~;, irzorzt_~ treatments rnay provide signi~~~,ant protection, b~.at rev;zrrences may occur earlier than ~~~th 3 mouth or szerri~zancnt trE;aWerzts, and that islet poll rescue is possible. Contu~ng~raey staternents° All of the zvurrrerovs quost~ions ay not °~se answered.
hut, with this flexible model, other experirnerzts rr?ay be designed to ac~.rlress other issues.
~A~ 3. Tlae effect on Islet sell surwlval of so-~e~oapsula~ng negate and duct cells°
Alaxr ~. .fin vitro studies. Ratlorrale° °lze advantages of this approach r~r~ that it does not rcquu:~~
gene therapy, and provides, in ad~sition 'S~c~ ILxF-II, other components of islet physiological nucroenvironment (GFs, extraceilular $natrix rraolecules, pluripotential ~-cell precursors).
~~°eligna~y data. inuring the last 6 rr3onths, we began ~o develop and evaluate different duct cell farts 2~ of ~;~
'~~8'2, 3ean-Pierre isolation methods in hamsters, rats and mice. ale established the tech3~iques reduired far tl°ss project, including iznm-.moperoxidase for cytoke~°at~an 20 {a rr~arker for duct cells; and IC~F-II
(main duct cell secretion product), as well as western'olot and (ixz development} ELTSA. for I~aF-II. F,xperimentai design. In ~ iirss se's of expE;riments, the survival and fzmctiozl of mieroencapsulated or free duct cells v~riii be evalF'ated. IGF-II -aaill be rn.eas°~zred in the supernatant, and im~znunocheznistty for cytokeratiln 2(I and IC:rF-Il 'c~:~ll be pert"orr~~ed ors slides.
Islets will be co-encapsulated vritla different conce~tratzc~ns of duct ce;:i s a3zd cultured for 3~ da~,~s.
'The experimental design and metl3ods will be as in aim i. A~aticipate~~"
results and sigearaee.
Islets co-encapsulated with duct cysts sh.e~,zlcl prese~zi decreased ;apoptosis and necrosis a=~d improved viability. Cus~tingency statenaertts. See aim 7. ~irn '~. In v~vo studies. ~a.tie~nale" See a~~m 2 and ~. Experimental design. Islets, cc-encapsulated ~~ith ~
concentrations of duct cysts or alone will be transplanted LF. 'The experimental design and znethosis will be as in aizn 2. In.
addition, rrzicrocapsules retrieved at (a} the spontaneous death or (b) sacrifice of the animals gill be studied for evidence of fe.~nctic~ing islets and duct cells and it, let :neogenesis and a-cell replication, using immu_noperoxidase with ar=ti-i<ns;ilin, anti-glucagon, anti-cytolteratin 2t7, anti_ I~'r~-II as well as the ~I. assay fnr apoptosis and ~rL'3U
izxcorp~>x°atio~~. ~reticip~ted results anti signi~sance. ale anticipate long-term norrnoglycemia in more recipients with islets co-encapsulated with duct cells tha;A alone. ~~le expec~. to obsezved signs ox islet neogenesis.
~~mtingea~c~% statements. Duct cells grow more slowly theta cell lines such as Th~4 and ~I3D.
Ideal culture media for islets and ciazcts diffez-. ~oine questions remain to be answered, such as the need to wait several passages before encapsulat';ng d:~.et cells. Thw potential ~s,dvantages {see rationale} justify to pursuing our prelimina°_y efforts tr> answer then; qupstzons. This research proposal contains several altez-nat»~~m app~:oaol~es if tire ex-petted results are v~ot obtained.
~'I' 4. ~'~te rnechaaaisms ~t '~~F's p~-ntY'tive effect. ~.im ~. ~ better understanding of tl-3e intracellular rneCharisms will 'de useful fer fiztare developments, lExpe~inAent:al ~lesigza. "fl~is study will use material from studies described in part 1 to 3, mostly from in vitro sti;dies, but on recovered islets also. Microerzcaps~zlatec? islets, san-zpleci at different tirmes pre and post treatment, will he fixed and slides ~,vill be done as previously descbed, j 1~. Fffe:ct of the treatments on pro-apaptotic and anti-apoptotic pathvdvays and anti-oxidara~ systems, ush~zg immunoperoxidase with antibodies to molecules involved. ir~ these pathways, szzch as T anti-apoptotic;,, ~aspase-3 (pro-apoptatic}, ~u,Zn-St3D (anti-oxidant systenx,~ will be studied.
PAS' S. Studies in an nut~i~3mnn.e model of diabetes. ~xrt :~. 5~:~dies in iVDD zr~c.e.
ttatinnale. the use of the same anirxzal model for aims ?, 5 and 7 will allow comparison between the different approaches proposed. 'The efFzcacy or" tie z~~ost effective :method will be e.oz~~zrnaed in autoirnmune diabetes. experimental ciesigxz. Allografts will be performed into Nt'~D mice. ~tucy design and methods will be as in airrz ~, 5 0< 7 dependizzg upon the selected rr~ethod. .~~aticigated results and sign'afaeance. since bath n~ier~;encapsulat.on and I~F have been shov~rz to ;'rotect agaizzst immune attacks, the results sl°zould be sin~Iar to those obtain in the foregoing experiments.
~:(~1~~~.,~IS~~~S
her marFy years of sustained worlc_ the fields of islet microencapsulation and islet biology, our combined laboratories can provide a zzr-~que. anti signizvcant contribution t-o die development of treatments to cure type 1 diabetes, based on ~;1 } ability i;o transfer genes, using viral and non ~,~iral vectors, (2} ability to produce; cell l irves with a stable, gene expression, usiz.g non viral vectors (3}
ability to co-encapsulate such cells and islets {4} ability to prodazce microcapsazles wirln covale~ztly linked membranes, virtually ir_desi~~.ctible witlsixz the living body {5}
ability to produce ~Jt3 p.m :<~cdoral" microcapsules with o~ery uniform size azid rnorpl?olo~;~, {6} long ex.periezzce of original methods to measure and control microcapsule characteristics, (7}
ability to predeterroir_e nucrocapsule size with high precision, (8} expertise an~e. facilities to work ~with imrn~.~rtodefzciezat mice (~} original fiecl~niques to isolate and culture duet cells, (lt7}
expertise in the investigation of the z°o1e of the exocrine pancreas, Ca.'~s a~~zd duct cells iii islet rzeogenesis; and a-cell replication, apoptosis and necrosis, and {l l} access to hurrzan islets.
Pale ~'? et 4~
i~i~lle, dean-~ierr2 ~ ~~~~t~~t~E , ~°~., t-icntø~y ~, ~.~sG,a~~cr~ , i~al~~ ~Y'~' (2~J~~
i~~-ii ~tto~~~s prolonged blood glucose nor~n~a~~oa~ v~t~ a rer~eed Rslet cell mass traa~spt~ntat~~n.
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~2 ~ R~scn~e~rg9 L. (1~9~) ~~a wivo cell f~~a$asfor.r~ataon~ l~c~a~;c~~ais of data ceps frog pa~creati~ ductal cells. ~e:~l 'F'raatslalantation 4, 3'7i-3~4.
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islet cell rcgc~aeratio~~ a~a tE~fe dl~t~etic ~,stc~° par~s~~~eas ~-gt~
re~~toratxoa~ oi°
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[~~ G~~nnoul~a~s, TEL, I~t,'., L-~..~ade~~t, ~~~<~,., ~a.~a~otto, t~., °~~tr?~cs:o, I'.~Y. and o~~sxr~ps, ~.
(2~~0) reventiora of l~e:a Deli dysi'~~~~~aio~~ a~~d alaotosis activation in ~Snra~a~~a ~siets by aaiea~ovi~°al gene trarssfer of the iarsulin-Bite gro~rth factr~r i.
gene .'her i, ~~i~-[9~ Tlxornas, ~°, ~hrhard*, ~.. sand a~y .~i° (~Q~~) . progress ~s~~ profiles ~~~atl~ tl~~ nse of vnral vectors for gene they°apy. l~at i~ev genet 4, X45-35~.
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[2~j ~etriic, ~.,1'ell, ~.I~.,r~~~n~, ~., ~lconald, ~i'.~., ~aar~, °~'.b~., .ails, ~. aar~d ill, lb.~>
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[~~j Rabi~o~itcia, A., a"~a~igley, ~., I~o.ssell, '~., ~~atai, ~'. and lisei.~, 1J~. ~1'~82~ ~sulie~
and ~n~tiplisation stiana~iating actie~i~ tan insa~lin-lilza grr~~tl~ factory sti.ul~ta islet bets-caU. replicatioza iaa a~aoraatal rat pancreadc ~o~aola~er ~.ult~ares.
I3iahetes 31, 16x-164.
[~Cj ~afaeloff, ~.e.a. ~199.~~ diabetes 42, i~ i~.
[~1 j ~iil,1).3., ~etrils, .~. and ~r~any, ~.. ~1998~ ~ro~vtli 1°act~rs anal Mite relatis~n of fetal grorvti~. diabetes. Care. 21 Suppl 2, f~6i3-X69.
[~~j ifl, ~.~. end n~illia, . (2iii)0~;~n~,re~tic da~raloy~naent e~t~~d ada~it diabE;tes. ?~ediatr yes 48, 269-'74.
[~~_~arras, ~.A.., ~Ia~°t,1.., Colas, .5.,1-3~a-ue., ~.~'., ~oy~odic, .1.'~'., l3icliardson. '~~L~.
aa~d gaff, .C. ~1.99'~~ Call death and ce~ntrol oz call sar~i~raf i~~ tl~a oligodendrrc=Lyre li~aaa~e. CaD1'0, .31-46.
[34j ~l~een, S.°1'., ltajln3ar, . and l~lurpy, ~.,~. ~I996~ ~ffe~.ts of ins ° -lira groevtt~
factors {1~~ on pancrawdc islet ft~uctio~a i~ 1~~ ~indi~ag protein-1 transgenic mice.
l5iabatologia. 39, 124'-12 s4.
[3~j ~,i~irii~, ~.L.., Sl~ottaer, ~" aid ~ellerstro, C. (1990 ins e~.li~n-life gro~~tta factor 1 does not aadyil~it ins sac~°~tioa i~~ adult ~u~an pancreatic islets in tissue culture.
Eu~°. ~. ~e~docrinol.133, ~4~-.25.
~ac~e ~a cat' ~~~
Hale, .lean-r~erre [36~ ~=~Iahley, 3.~., ~elao, ~7., .Tga2, ~T. ~ ~~ ~-ce~a; LC. (1987) I~soilr~..l°~.~c gro~tla factoe° I
~°c~crses °ar~terleui~~n-Ibeta. ix~~d~it~ooi° ~osdio sccrctior~, io~c~ctin~a of nitric oxide syntlzasc and r.ytoltir~c-~~iatc~ aaoptosis i~x rat islets oa :~~a~ag~erlaaa~s. I~~S i.Ettcr 417, 235-235.
[~'~Pictct, ~..~.. aid ~aatte~°, ~~r.,I. (~9i~~ Ccll ao~~ractio~s io E~tifi'crcutiatioo. (~barl~oe.o-~aas~edaia~eo, Ii4. et al., c~s.~, pp. 337, ~caelcic Press, ~os~.~3o~°.
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a~a~ Potter, . (1993) l~TerF=%e g~°ossvv~l~ fa~eor in~~la~ces en~r~r~-lf:~~, dit'feretiati~n ~f ao 's~~-secretgng pancreatic, hate ccline. Pr oc. TgiFatd. .~ca~l. Sci. LT. S. ~.
90, 5'75~-~755.
[~9~ Scdaarfanann, 12., 'I'a~i, .., Ps~la, ~., aal~a7 ~. a~s~:~er:~aic~.o~,;
P. (L99~~
~xpressiaa~ of functional ~°~brv~, g~°o~t~ t'actor rccepta~rs iy~ paocrcatAC ~~;ta-cep dines and fetal rat islets in prff~ea~ry ct~lta~re. l~na~et~ 4291529-~5~6.
[4~~ Snyder, .~.e.a. (1994) Cell '~"~, 62'7.
[~:~ _ "~Jir~il~, ~<:d., ~~ewloo, P<~.y ~~a~terirs, ~".:1. and ah, e.
(1.99s~° 7C~iau~etes esea:a-cl~ 3, 39, [42s ~aroegar, lte, l~~ga, S., al~ija, I2. anal i~ae~alopox~los, ~. (1990 'd'iss~ze slistri~.~tio~a of hepatopoietin-~d a lxepar~-bi~i~~g polylxvpticle gro~:-ti~ factod°
for d~epatocytes.
Pros. Natl. Aced. Sci. ~" S. ~. 5~, ;.252-L2~6.
[431 '~s~x~a, ~., Iswase, ~., ~ats~oto, ,~.~., Lto, l~Ly ~ ono, ~., i~eshi~a, ~., ~'ae~a~aaoto, Ice., '~ate~atso, . and ~al~~o~~~w, '~'. (~992~ gaa~~olafst:~acl~ci~a~ lo~,adi~atdoo of l:epatocyte growtda factor protein in pancreas islet ~-celFs of are asad rats.
,~pa~. ~.
Caocer. ~8.es. 53,1262-1266.
[44~ :~Jlashia, ~., Shi'oata, .j ire, ~. ~~al ~'~~,[d~-~a, ~. (1996) P'or~~~eatioo e~f ia~;~~li~-prodncirgg cells from panc~°eatic ;~ca.r 423 cells by hepatocy to gra~tlZ factor.
~~a~~crioology 13 i, 3969-~~~'76.
[4~~arcia-~cana, fl.., '~aliane, :~.~., Syed, T~a~.A.9 I'lailhricds, ~~~%.1~~.9 Vasa~ada, It.C. aa~~
~te~a.~art, A..:~. (2~0~y~ l~e~t~cr~tc gro~~tla i~tcto~° o~erc~prcssio~
in. the islet of tra~sgeoic mice increases ~et~ cexl prolifer~.tio~a, enhancaes islyt ~-oass, anal i~a~xces mild hypogdyceania. ~l~e ~o~arnal of i~iodogica~! Chemistry 2 i~,122ti-1232.
[45~ ~to~dCOSI~i.,'1'., Cir uldi, ~., ea~xxe, ~~~f.y i~~ddy, D.1., Soto, C., obis, ~.~. ,aa~~l ayel~g A. (~996~ A role for bepatocyte growth factoe/scatter factor in fetal mese~acl-~yrne-:ual~cecl pancreatic hate-~;~.il grs~w~t~~. ~'.o~ocrioology ~~7, 3131-3139.
[47~ Lefehvre, ~.., ~tonl~osl~i, '6_'., ~sti~o~7 ,~., ~notari, 1F~:,~.~,., Pi;~edeers, ~.~. asarl ovens, L. (1995 Cnlt~~°e ~f a~halt 1?nn°.an islet preparations with l~es~atc~cyte gro~~th fact~~° and 5~4~ r~~ata°i~ i~~ ~nivoge~~, fo~°
doct ce;I!ls ant ~~ot i°oa° beta-cellw.
~iad~etes 4'x,134-137.
[45~ ~ e~°a~o~ae~, I~., ~a~aaoto, ~'~., "~'al~asa~.~a, S., Shiga, R., ~'o~aea~ra, ~'., ~'oclaino, '~.
and H3d~amoto, i-I. (1955) r~ ~ao~el gene activ~aed in ~-eger3e3°ati~g F,sdets. '~'he ~o~~~°nal of ~iodogicad Chemistry 26J, 2111-2114.
[49$ ~afaeloff, ~.e.a. (1992 ~'~°~c. ~~aadoc.. Sac. , 5.~~,.
[.~'sa~ ~enigamaea, D.E.e.a. (I993~ Surg. Forum. 44, I'r~.
[5~~ ~'ra~cis, ~'.,~., Soa~tltgate, ~.~., ~Iil~.a~a, '1".f. and ~a~re, ~..~.
(~.992~ ~~pressi~an ~1° are islet rege~teratix~g (reg~ gene i~a isolated rmit islets: effects of a~otrient and ~ao~-nnt~°ient gro~~~tda factors. ~iabetologia 3S, 235-242.
[~2~ ~to~~lcosl~, ~.9 Dally, 1V.1. aosl ayel~, ~~ (~g94~ ~phositc effects of het~.-celi slfffere~atiation anrl gro~~th o~ rag expression in haanaan xetad ~a~acreatic cells.
~ial~etes 43.1164-1166.
[53~ atanadaa, '~., '~'onemnraA ~., ~.'ooe~°a, FI.» S isi, Y.%
I'~ii;~asFdta, ~L, Sngiya~na;
~., ~torii,~z~a.i, S>, ITnrao, ~~.1>, "~a~a~l~a, ~., Rondo, . and al., e.
(1994 Pancreatic beta-cell relalication and. anneliora~-ioo of surgical diabet~;s lay leg p~°~~tehe. ~~~oc.
~latl. .flcad. Sci, al. S< 1~. 91, 3559-392.
~a~e :3~ ~f ~T
i~~~~~; ~G81?-~12i'r2 ~~ ~i~atanahe, 'I'., ~.'o~ed~~ara..,, °~>; ~or~~f~nra, I:i:., ~odci9 ~'., ~~iyas~dta, II., ~ugiyar~-~a, ~.., orii~~i, ~., I3nno, i>, ~'aa~aka, ~> a~~d I~o~ado, ~. tI99~) :d~atacrcatic beta-cell rcp#scatior~ and aaoeddot°atd~o of s~rgicad diad~etcs ~y ~.eg p~°~tcir~. ~~°occcding of the i~latio~tad r~.cadey oi° Sciences of t3S~ 9'~, 3~~9-3592> , ~5~~ ~laappar~, ~., Stahle, ~., ~ard~it~s. ~.f~>, ~'a~~to, ~.~ ~~aaitd~.;
~'.I~. a~sd ~~erlino, ~°'~.'.
(1990) 'I'G~ alpha over e~pryssio~a ~-ao.sgea~ic naive induces diver neoplasia a~ad a~~sorad ~levelopnsent off the rrca~aj.a~~ gland aa~d pancreas. held 61, 1137-114P6.
~5~i$ I~orc,11~., ~taandrasedc~r, i~., '~'aauactadca, '~", ~'riess, IL, d~a.~cdnFe~-, > seed ~ege~-, .~.
~I992) ~vere~pression of the epideYad grow~~td~ factor reseptc~~- ha dxua:~
panc~°eatac eance~- is associated ~zth coucoitat increases in the levels of epidermal ~~-owth factor and traa~sfore~~g gro~~th factor alpha. ~ou~°~ead ~di~aicad Investigation 90, I3~2-1360.
~5 f~ aa~g, ~'.C., onx~er-ei~-, ., dates, .~>, t:h~adad~, .~ ;~i~ou, :~., ls~rdir~o, ~ ~>, ~cd?~cicdt, ~.~1> and randn ~.~> (1993) P'az~creatic gastrir3 stiudates islet differeattiation of transt'or~ai~ag grovv~tth fazvtor aldaha-induced ductanlar pryc~~rsor cells [see eoraxs~ents]. >Iourual ~dis~icad Investigation 93, ~ 3~9-d 3~6.
~~~ Song, ~.~L, ~annoo., ~L.y ~~'asI°.angtou, ~I.., Sc~gge~es~ ~.It.;
es~oedy, ~~>~.~., ~oddeaar~ng, ,I.~r, i'~ar~ooy ~.~a, Saaadgreu, ~.~.9 ~offey, ~3a~.~ fir., ~ragdit, C.~. and Ileact~s S.I~. (1999) E~pansiou of d~I-e~pressi~ag pancreatic epitheli~r~.and islet neogenesis ate transgenic ruice o~°ere~press~.ng ~-ansforiog gro~vtd°~ faetor adpd~a.
~ast~°oenterodogy.11 t, L~:d.6-126.
~~9_~ ~'austo, ~> (I99Q) I-Iepatoiogy~ .~u te~thood~ of diver disea se ~ad~-a~e, ~. and I~~ayer, 'I'.~., eds.), pp. ~9,'f6'~ Sa~::nders., ~daidadedpdaia.
~6p~ ~daea-laaaner, ~., ~u~-sc~a, ~~'., ~iefen~achery It>, I'roschl, g.;
iPaveldsa, ~d.., ~~a~~:d~~o, ~. and Sch~zdte-~Iles°an~s, ~. (d993) ~~opte3is fs ioduce~~ day trausforu~ia:g g~°o~wth factor-beta ~. within S d~~o~ s i~a re~~°eusia.~~ di~>er g~ritdaout sTyaai gcaa~t f~°a~~nenta~~ra~ o~
the~I~~. elaatology> d~~ ~'-23S-Id~o.
~61a ~,i~~ia~o, ~. (I9S~) ~'a°anufornaing growth factrc~r-betao xr~~adtipte ef~ects o~. veil dafferea~tiation and e~traced3udar matrices. ~~.v. ~iod" i3d, ~~.&-X22.
[62~ ~o:-de~e, .~., l~and~-iota, 5.~., i~rlontesano, lt., ~~°ci, L,.
~~nd ~e~oper, I~.S. (199'%) ~asc~ada~- e~adothediad gr o~~~td~ facsor is iucx yase~d is sie~~ascr~dav°ized rat ishas oi°
IJau.gerdaaz~s in ~~itro.'i'raaspd~ntatior~ 63, ~3fi-4~.3>
~63~ ~berg-~Iedsh, C., Sa~addedfg S., t~ndersson, ~. and ~'el~h, T~. (L99'~)~
~i'fe~as of ~~ascudar eaadothelial gro~;~rth factor oar pancre~.tic da~ct cell krepdi~.atro~a a:ad the irzsu~a p~-od~ection of feted isdet~lid~e cell eluster~ in vitro. .~~Iolecuiar anal ~eddular ~aadocrinology 126, ~ ~a~133.
~6~ ~ ~og°ter, 5.~., Sore~xsoo, .~,., ~~~s~~, ~., a~°cia-scans,,, Vim., Stewart, ~>~'. arad ~asavada, It.~. (1995) g°o~~°essive pa~acreata~ islet l~yperdadasNa io the islet-targeted, paratdnyroid horo~ae-~°edared pr6.tei~a-overe~p~-essing u~o~~se.
~~~docrg~~oloby> 139, 3'743-375I"~>
[6~~ ~od~ns, 1~.~>, ~erd~ins, v., I~odrigue;~-'Tarduchy, G., I~ieto, ~'~f.~.
and I~:~pe~._ Divas, ~" (1994) ~rog~tl% facto~°~ ~s s~ar~evad facto~°sa rega~davion of apolatosis.
~foessays 16, L33-~>
~66'~ Arato, hT>, Roaa~o, ~'., ~_~.ndgwis, ~>~., ~~.c;°,aguedla, ~,.
a~~d l~hax°chesini, ~> (1~~6) ~ro~tTa staulatiou and apoptosis induced i~.~ codtaa°es of r~eonatai rat liver cedes day repeated e~p~sures to e~ider~aaai grola fa.ctorlanrogs.strone with or ~arico~at associated pancreatic h~ra~os~es. ~edl. 'Tissue. rtes. 3~~, ~.71w4~~.
~67~ I~aff, l~'L~;. (199'3) Social c~y~r~tra~is eon cell sur~ri~al anal cell.
death. l~ata~re 3~6, 39""r'-~ac~e 33 of ~T
Hafi~, .lean-Pierre z6~~ ~rneG, ~,., ~stcban, r~.ir~~., ~ia~, ~'., ~e,~ac~a, ~'., ~arcia, ~, attd lariat, ~. (1996) ~~stttpariss~tt of varkc~~a~ ~.c~.t~tti~nt:u fot° rat islet pt°~Epa~°atimn. '~"rangplarttati~tt ~roccedin~s 22, "~77-'~~~.
~69~ Don~, ., ~otot~,1., ~~t~v~ai~, '~'., ~h~a.t~, ., a~aai, ~.., I~~~nndcn, 1.~, and I~set-i. ~'.
(I992) ~'itc role of calcitttn ita c~iia~cttasc di;estiott ~~~p~-eser~s~atiur~
oi' islets.
"I'ransplatttatiott ~roceadin~s 2~., ~.dt~fl-~.di0f.
~'7fl] group, 'f'.d.~.a.C.t.~. (:1993) °f't~e. effect of atntettsi~e treatt~tettt atf diabetes ott the de~eloptrtent attd pro~t°essiott of tott~-terttt cotttplic~atiotts itF
instZ.li.tt-tlepøndettt diabetes tttellittts. bite i~~e~~ Ettglattd :~ottrttal of ledicitte :~25~, 97'x-9~~.
a'~1~ ~neteatt, I~d.ll~L, ~e~root, ~"..~., ~lar ttocic, .L,. attd ~~otte, R"~~I. (I990) Pancreas pt°eset~ratiota prior to islet isolatitttf a ~~ainatiott of attar. ale solutions in a rodent artoslel. 'f'rattsplatttatio~t ~~°c~ceedit'<~s ~2, S~~-542.
~~2.~ ~raetero~n, ~.I~., ~e~r~ai;,'f'.J., ~~'ar~toc~, ~.~, attcl ~'~a~tav~te9 ~..'~l. (L99fl; Islet recover; front rat pancreas is ~:ziteh ed by iartactt~°erttertl tecFtttiae fit~sik sointion.
'I°ransplantation Pr~ceettitt~s 22, '~'79~
~'l3~ Lacy, P.~., Wager, I.~~, and ~'iak, ~.J. (I9?2) Periftt~;itD~ of isolate rat islets itt ~ita°~. Diab°tes 2~, 9~7a99~.
x7~~ ~a~e, ~.I~., Jatttes, ~.~'.t;., ~.ttdet°sott, .~., iJitastt~eriair~, J., far doer, ~. attd ~e~, P.f3.~°. (~ 9~6) E1n irrspro~ed isolation ttaetlt~d f~r rat pattct°eatic aerate.
'f'ratnspgantatiott f'roceedin~s ~~, m~1 ~-I~f~.
~75~ ~alce, ~.l'., Anderson, J., ~~attxbet°laitt, J., ~"~arrdtter, S.J., eii, P..F'. atttl Jatttes, f~., PL (~9~'7) ~3o~itte sertttn ~iibattttit3 density ~Aradietat isolatitytt of rat pancreatic islets.
~'rattsplantation 43, ~flS-~fl~.
~~~ Iae~t~ttarc3t, ~., 'l'ytl~~, r~.,'~,xnca, i~,, ~at°tlael, T~., ~topcls;.ttt~tt~, ~'., ~oltlet-, ~., ~ie~ei, ~.~''~.,1'~el~ettd~ltl, ~. aotl ~:~°eeat~f~:idt., '< (i.993) 'tae effe~~t of vet°ettt ;~olatiotts gor o?°~an preser~ration otz ittt~ed:iatc postis~laetic pantwcati~
~tat~ctitsv~. itt ~itt°o.
Trattspiaattatao~ SS, f ~ -~ ~.
(7~_' l~eilsen, IJ.J. att~ ~,ernarl~, ~. (~ 3~~) Pttrificatiott of Islet att~
c~Ils froth Islets. Ina deli separation tttethods atts~ selected application pp. 99-I2.~g "
~7fl~ ~icmrdi, ~., dray, D., ~ieritt~,1~.J.5 attfartan, D.D., ~~arttocls~, ~.Ia., ~atetetrtat~, ~.I~~., Y.ake, S.P., ~.ottdtan, I~.J.I~., ~occi9 ~.. ~A,le~atttiro, ~., ~eng, 'i'., Scltarp, ~.~., Viviatti, G., Paldtti, L., ~~al~s, ~., ~ref.~e~, ~..~., Pederlitt, ., I°o~za, G., Jarttes, .P.'~,., ajotte, ~t.~., I~i~arlo, ~%., T'~dittrris, P.J., ~ntlterlattd, I~.~.I~., ~tarzl, ~<~., l~int~, ~8.~, and Lacy, ~.~. (I99a) Islet isolation assesstnettt ita than attar lar;e aatitt~als. ~~,ta ~ialtetc~lo~it:a Latdr~;~ 2'~, IBS-y9S.
~~'9~ 5ctaarp, D.W., ~ettt;a, ~'., ~, ~nige~a, I~~d.J., ~allin~er, "4~.p'.
attd Lacy, '.L. (L9?3) '~tt~
nee of ftcoll itt the pr epat°atiott atf ~riabie islet,H taf f.ao~erl~atts f~°otn tl~e r;~t pa~tc~~eas.
~'rattsplatttation I6, 6~6-~~9.
~~fl~ltibata, ~.~, I~,ttsi~i~sett, ~.~~1., ~taber, ~.P., :~Ic~attiel, 1~~.L.
a~td Lacy, P.~.. (197th) Stattdardi~atiettt of a sli~estion-filta atic~n ntetlaod fear ist~latiott ttipancreatic islets.
DiabQtes 25, 667-672.
~fllx 'I"alsal;i, ~, attd ~tto, .f. (x9~S) Pt°eparatEt~tt anti ctiltttre of isolated islets attd dissociated islet cells froth rodent patter eas. :Yoorttal of ~.'asstte ~~tltatre l~et&ts~tls 9, 6I-66.
(~2~ Lacgr, P.~. and ostianet~sy, 1. (I96~~Ietitod fctr the isolaa~otr of intact islets of Latt~crl~atts front tie rat pancreas. Diabetes i 6, 3S-9.
~~3g Farttey, ~..~., hIa,~at°iatt, ~a~'.~., ~'altl~iie, .P. and al., e.
(1990 f~ntotransplantatiot~ of dispea°sed laancreatic islet tisane cottabittetl ~etlt total or tllnar-ttatr~ pattcreatect~stny for ta°eattrtettt of clxroxtic pa~creatitis. Sn~~~er;~ I~ fl, ~2~~&3'~.
Pale 3~ ~f ~7 H~!le, J~~n-Pierre ]~~] r~ctenaar~,~.I~.,~~a~°$s,~lf,, ~l~scaa, . end ~~.j~ttc., ~.~'.
~~9~9) 1'roic~ngcd i"a~~lctiora of i~t~°aspicnic ca~ac paa~c~°catic ~ra~cnts autcgr~fts.
'f'cv~spia~atation ~roce~d~ags ~1, a619w2620.
~~5] ~iiriCO~itcd~, V. and ~apic~c, Ice. X1976; S~.ccesfaaii intrasiaic~°~ic ~ototrs~nspiantatio~a of pancreatic tissue aa~ tetaily iras~~s:re°~.tec.to~~cd dogs.
T'ra~aspi~~~tatio~ ~3,> ~6~>~~69.
]~6] ~aI~ingcr, ~.F. and l,~c.~; ~.~. ~:i97~-) '1'ra~xispiant~tio~a oii° intact panc~°catae ish;ts ire rats. Surgery X2,175-1~6.
'~~ ~ra~, ~.'6~LI~., '~ar~aoc~, ~.1J., Sa.~tto~, ~.., ~cters; i~L, cStsanc, _~. and l~orrig, ~.3.
~19~6) Successfa~l a~ata~t~°anspiant-atior~ of isolated islets of ~sam~gcr~ans i~-~ tlae cynomolgus a-~ao~tl~ey. ~ritxsl~ ~ou~-oal of Surgery "73x X50-~5~.
arnoc~ ~.3.. aced ~a,]otte,1~.'~'" ~19~~) f;ritical mass of pa~ritled islets that ia~~luced norogl;~ cecxaia after iplantatio~~ into dogs. ~iahetes 3'7, X67-~7~:~.
~~~] nester, 5.~., Seelig, ~.~~. and Sta~ach, wl. ~g97~) lte~rersihiity of function and o~°pholog'sc glower ~ata~ lesio:as ~y islet transplantation are long term diahe~c rats.
lJiahetologia 13, 4iD9.
]9~] i~ane~°, 5.1~., Sntlaerland, ~.~.~., steffes, l~T.~~., ~.eonard, i~.~'., lea,]ar~a~~, .1.5., ichacl, ~..~°. a~ad ~ro~~n, D.1~. ~19'~4; 1'ao.~:reatic islet transplantation. Effects on the gloernlar lesions of c~pe~°i~;ntal aliai~:tes i~a tree rat.
:l~iahetes 23, ~'~~-753.
~91~ S~athea°land, ~.E. ~19~1~ ~~ncreas arid isieg. l;ransplacatatiou~.
~. ~~perie~atal st~~dic,s.
~a~etologia 2~,161m~.~5.
]9~] Shaiai~°o, ~.., g.al~ey, .1.~~,., yarn, ,..~., ti~rhutt, t~.u._f '~or'~a, E., arnocis, ~.~.., netean, l~T.l~I. and ajotte, ~,3~. ~~~OQ~ islet '~~°aospiantation i~a se~~e~a patients with type 1 dialaetes epitas =~si~~g a giaaca~co~-ticoi~h~re~
~nu~~aesa~iaprcssi~e ~-egan~eaa. l~' Eogl .lied 3~.,~, 23ti~~:
[93~'o-'aa-oc~cl~, ~.~1., ~aaztea~~, ~1.~:.~ .ya~ay E., S~pelis, 12.~;.,..~,., ~~,.~ino~~u;~cii% ~. ~~ac3 ~a,]otte, fl.~. ~~.991) i'~orogtycapirs aftcr ~~-anspia~atatro~ ~m f~°esfaiy i.sol~ted a~~d cryopreservad pancreatic islets in '1"ype 1 ~iz~suliHy-~depe:~dex~t) diaietes cii.itos.
~iaietologia ~~., 55-S~.
]9~] Yasunami, ~., yu, S, and ~anaei, '. X19911) char acterization of the iunos~appresside activity of ~Sdi6 iy!i rat islet allo~,r~fts.
~'~°ar~splaa~iatioo roceedangs 22, b~5.
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]96~ ~"orsen, ,1.'~1., l~iu, E ~,., l~ret~~:l, t~.~. and Y~ ederlin, K. ~19~~) fsiet tr ar~splat~tation in e~periental diai~etes oi° the rat. . fn vlitro culture ~°eda~c~;s la~l~earing cells in rat islets. ~orones anal T~teholic f~esearch 16, 6~1-~'7~.
~9'1~ ~ ac;~, E.lr,., Davie, .1.1'1. end ~ inise, E.~. ~19~9) ~rolonga~~ion of islet allsugraft survival following i~a vitro .~,ultn~~e ~2~a~) anti a single iojeaaio4~ of ,~5. scie~ace ~0~, ~1 ~~313.
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~retreatir~ent of rat pancreatic islets with ~~ ~~~, onocio~.al a~.tiioslye ire vitro and in vivo efffects on islet aa~tigec~icatg~. liorar:~oa~es sand I~~aai~tic l~eseax~wh 23, 'x,57..
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]lOd] 1~'lescla,1~.I~., Entenu~ana~91~., ~~ilde, .% ~ren-:~el, R... aid Eed.erlia~, ~~. !1991; islet transplantatioa~ in experimental daai~etes of the rat. . ~Cryopreservataon redances I1~~ class f~ ant not class 1 antigens of rat pancreatic a.slets. oro~aes and I~et~holic l~esea~°°cl2 23,1-6.
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j103~ L,an, ~>, ~eeot~sxna, K. ax~~l hardy, .~i>A.. (~9~4r~ I'rolon,gati~x-~
csf rat i,~Iet ae~g~°a~
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i'a~e 3~ of 47 Hafie, )can-Pierre ~~~fl~'~;~'~ i~~~~ll~E~l'~~
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die lave established oollaboratiorrs according th.e requirerraenda of ~~ur differer_t projects. In addition, we have responded positively to requests Ior collaborations.
t3~r ourrent JD' grant and tl~e present proposal sterns from a::oll~boratiora with ~r ~,awrer~ce Rosenberg, from l~Ic~ill University, co-applicant.
~e are currently collaborating vTicl~ ~r hierre-~7ves ~enl~a~~nc~u (see letter of collaboration), from tlrenoble, 1~rance, wlo is invol~!ec4 in clinical islet transplarJtaticsr~. a is sypeawising the Frenc;h-Swiss group ~~tt~.CiIL, one of the rrrost active groups worldwide in clinical islet transplantatio:~.
'his collaboration has involved the participation of t3uillaume Jourdan, a graduate student fram Clrenoble, who did his F.Sc. a~~d ~,~as'~er studies iri Clrenoble, is tu~rer~tly dewelopi~~g new islet research tools in Tray laborator<l for one year and w~sll of~acially begin his hl:l;~ in September, in co-direction with both laboratoriPS. 1-Ie wzll spend about ~t~~/o off' his l~h.~. work ira each laboratory. FIe will be in a position to faoilitate the transfer to human islet studies of the tecllraologies we have developed ir~s l~Iontr~al.
~iVe lave recently accepted a proposition for collaboration frorr_~ l~r 1'aui ~e Vos from C'rroningen, 'the Netherlands, one of the most respected experts, worldwide, on islet rnicroencapsulation. ~Je have accepted to load l~airn our device and teaol hirer our technique for rrraising small ~3~(i ~.rra) sxncrooapsules. Ur ~De Vc~s wa.ncs to evaluate or;r teclnologies and compare it to other technologies recently dev~~:Eoped ~y lis group. ~l~r I5e Vos is currently is holiday, therefore we have no letter frorrr lira but he can be contacted: ~~r lie Los coordinates,: ~ a ~ : t ~ 1 ) ( 5 G ) 3 6 ~. ~. 0 ~ 5 fax: (31) (5a) "~51~a11 mail: P.D~.VCSSC~N;;ji7.:~TJG.NL) fast collaboratians include .T'.~r Gerald ~nxd'hon~rne, from ~r~eC7i11 University, patlology depar-i:rnent, a JI3~'-funded researcher, -for the investigation of the: role of cytokines in the hosi:
reaction to rnicrocapsules, ~r 1?i.erre-l0/:ichel Duet, director oi" the .~~ndr~ ~,~iallet :i~esearcl~
centre, Centre Hospitalier de 1'Uriversit~ de IVfontreal-St ~uc, a leader in portal lyp~ertensio~
research, for studies on intra-hepatic rr~~crocapsule irnplantatiorøs aa~d the h3ydro-Quebec electricity research Institute, <or the development of the high voltage electrostatic pulse generators to produce smaller rnicrocapsrrles.
Page 40 of 4y -~alie, Jean-Pierre ~'~~ bmt~ p,ar I B$~1i~6~6$2~~. .._''~OCR9~OTy~G;E 6~'f~:$ fs;9? ~,a= f~2 ~,.°.~_$.~. :v ~~tCF ~'~wLa7CFtlNOl.Ortg_L~tlf3WOl~.'~fC-t~Us°FIYtO~f s!e ~ffltraesur Ssrge HALC~s~
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'ar'txdee dt r~hCtehe Guy_T3eri,Ser 349 5 bttul. a3e f~4sstsltiptauts, pUtautrdal, ~, Cacouda i-i t "a~' 2?ai i FI4TF:3-SIB-25~-343pr Re : our collnbaretion dear Jean~Pietre, 'l~ti~ 9ntteT 3~ L3esr~ied c~ noaiirm shut our two iat~oratca:ries lv~ve dcvclopc4l a callsbsaratiowort i:~suss xelflted to sxiet trEnsplantatioft. X13 you krlatv, islet ttans~kafttetion stalld9 as ills attain trccgeaeels Loøic tff tiu J~urtment ui'Em3owrint9tagy, Univer>ify Hospital, ~eref~o6te. ror 4 P~ca:~des our i;raup Saes hc~n attvoivcd in both preelitvcal sand ciifrical tc-se~nch inn this aiem. ~linien3 research cor~istc of a rr~alticefatric is3et ts3tL2Dt.~ttlFian pmgrtfrra ~8ih.~r'ing ? Freach tvfiv~~sity hospitals d~r~sstoPsie, Lyon, 9ttasbssurg, ~es~st~tin, LSijnn, Fviarseiitc, ~aatcy, llhonipeSlinr} sort thf: aztftlcrsity hospital of Clcxtcva. Tftis pm~a°am, sailed CiR~GIL and rf:r e:ftear my supcrrision, t<as ~sn cane the tttost ectivc islet txar~pfantstion program in ihc world for slue qast 3 years. '~reclinicat r2:.turcRa :~raa ~ocu~d ssta ties dvag in imprmve isfct viability, mairaty through liar cniuutccm~nt o' aatiinxsdaHat dcfcl9sc mechanisms of the czlls. C3ur grOnyz has teen developing gene tr~P'as~r avc;(tssotp$ics tt9isag 'vitas aftd nnrt vire6 vectors, hue' tin applying il«f: tools fo rite trasfer oFsnii~Oxidetat cnzyiries into ?aumeft pertcr~atic islets. ~Vc llavz, pubiisite'd data ebouC the ~f':.~cs.~ aaF°r_ats~asr. sn~ctoxid.;
disrncatasc attd ~hatcihion 1'~~'oxiciese terse iran~,frr an islet viability.
~.s you koow, we ltavc: rPaare T~ently de~relop~i a coSla~azativn w~itia yuur Icborhtory, through widah we hope to demnewttate the tclev5flcm of such ~~t appresaah of scavenging;
s;r~yrne gene trans~a sat irrrptaving rhea visiritiiy of Fr~icroercepsuteBe~i rstcts, ar9 itf viva stradcls esg aaetyltiaata~2~r. 77tis c.-o112boration has involved the pasticipativn of ene of ou~ bifs'ogy sBudent, ~rraso ttt~tercd in Gr~,nb~, devcloired .sea~erxf 3sPtt freta.,~~alntafiun tools in your L~boratcary i'nt khc iSasE ~~x, and f~c abocit to inltl~ie iris PhD in 6oG'i =escaxcli a~cfisties. considering our experience v~fih Ciinicai islet taarbpigntatian, aFus cu~lavora2ifan wilt agars aacilita4e the transfer to htamesa isntx tsFit~ ;ws~cht,iques yott 3save devalo~d in Montr~ai.
I look forwntd to eat ongoayg olosc coilaboratioa ;with you and your cratlcagues.
~iiaes::,tya ~Y~.fw .~~.~.=.~-r ~rofes.5cur Pierre ~ ø~es Fscatharuau. taifa; Phi:
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Page ~~. as ~7 H~lie, J~an~~ierre ETIiICAI ACCEPTANCE &rF A1VlfhALvOft!FCtRMf'r~ i~ L'~TH1t3t3E
ItESA8Cf3 E'3 P~AY3eRE 13E
D<te.~OGtT OF T~9~ AtiIIIUIALAEC3fEIit:HE SUR LES
~tiiie CC31LSN14TYc__ &lfsdBfMAeSIC
s~As F~Cf?f Dl! C~9f~l f~ Df: IrP,C'1"E~~1'1~'N
~eCS ANiIWAUae Statement atom the fnstitutioar2~elcrestinn de 1'v~tabllsncmenE
in w6iah the reseaecft mriilau so d3rouieta to recheecfac~
kro partcranod The ,4nfmei Care Committee .atairBisfted hv: i~u Cramitd de protection dea arairnaux r°stablie pear:
~n~re a~~ R~~~f~~~i~~ ~~fy°er~R3er d~ ~'tir~i~ai 9~miso~Rrl~tasre-~%ca~esr~mr!'~
has eramined the research lrrotocal antitied; a ~tudiw .e protocols de rec:herahe intitul~:
Lotrg-rescues press~tean cef' ~-SC1B apoptania and xtessrai>r sty continuous ire vatns sttpp'sy oIP'sF-3Y to tfisnsplanted L,feta, uetng t~~ gene tratss~er v~94h soar a°irra4 rectrcrg~ or co-ertcaspsssiatsors sa9'iafets acrd rsancssatsc daeet Belts.
(Use d~ sumo title as nn the apPlicationi -(Utifkser 1s me9me 2l;ra que nelaSS Indigos sur la demands) submitted by; prc~sent~e par;
i3r' ICiit7-S' iUS i E Y~BIIt' 'blame{51 of aRpiicantlsl as appearing on the a;aplicatianl ti~lnmlsf duldesl c andidattsl tal qu'ils apparaissant sur fa demandef and found the proposed protocolet a oonvenu qua la protoc:n'ta involving proposf: rApond aux animals to meet the standaedsnormes dtablies par to of the Conseil canadien de pro-zection Canadian Council on Animal des animaux (CCPA7, et Cara'GCAC1, que fos installations qui and that the facilities abriteronf tea animaux in which the animals qui sarvirant 8 1'axp6rimantaiion wilt he i,oused and used, cant conformer aux exigerrcos comply uvith the CCAC du CCPA.
requirements.
Date ~ ~~r . ~ , iSignature _...._ .. _~-. 1 ,g at _.
I5r Jean-Pacers Mails __ __ __ _ Dr. Pierre! t7uhe 4AppiicantlsilCandidattsi ~ ~~ linstitutianlsllisp-esantativef {D~l~gu~ de i°Eitabiissementf Pavilron R9ueexanneas~e 5415, bnul. do fAtsompr=.on, 113onlrkat, Qc H7T 2~R~i Tel.: (5147 25?-3dlHl~as4: 3329,'i'ciewpieur: (513) r52~36?0 ~PuvilfonRusetnnnr 5699,bou1.8oscmont,btonetel,Qc HtT 2FI1 Tc.aSrd)2i-3a00poste4959,TEIEcepiGUr:(5143252-3S2!
~~ ~3~ ~~
Naile, Jean-Pierre ~~~fr~p~~~~ fr~rr~ ~~~ ~~~r ~~aaar~~~ a~f ~~r~acs~~aersr~~~ ~~~~n~ung~
~W. ~I~~ S~TP~'~~~'o a~~,~enale I~n~~~~e~ ~'~~a~~~~pa~~g ~~~~~a~~~~~~~~
~~4/~31/2001 Co ~?3;'~I/~~0~
~~~d~~~ ~~~~~~~ ~~s~ac~~~~~~e 0'~/~1/Z~OZ to Q~i:~~r/20~'~
~ag2 4~ c~~ ~?
HQile, ~ear~-Pierre Crxrront grant ~~~eng~~ ~ia~~tes ~o~r~da~ia~a ~rat~rna.~ioua~
~410i12~~I to 03;3112004.
~ic~-artiticial endocrine pancreas: Tie raie a~'panLreati~, ci~ot cc;iis anti grawt~~ ~'acts~rs can maintenance ari~~ prorraatza~a a~r3xicroeraaapsulatesi. islets veil mass arid v~ur~ctis~n Summary Clinical application of microencapsulated islet transplantation remains elusive. One of us (L.~..) has recently shown that incubation of islets witt-a ductal calls, ductal cell conditioned media, or IGF-II has an inhibitory action on ~i-ceil susceptibili~ to apoptosis and necrosis. The nb,~~cfive of this proposal is to design and evaluate methods for improving encapsulated islet cell mass"
survival ana function. Our hypotheses are: (l ) Growth factor (GF), particularly ICF-II, incubations with islets, :z~ould pa~ovide suppert to islets during ex-viva manipulatio:~s, and improve long-term survival. (~) 'I"ransplantation of ductal cells with islets will supply a balanced amount of different GFs :md a limited amount of ~-cell progenitor cells, and positively affect islet call survival and proliferation.
~r°olirnir~ary cpa via',r~a grad it3 viva dai;a support the hypothesis that microencapsulation incuces apoptosis and has a negative effect on islet cell survival and function, that IGF-II incubations have a rescue effect on these parameters, and allow suceess_Ful encapsulated islet transplantation in mice, using a smaller number of isleis.
It was also shown that ao-encapsulation of islets and ducts is feasible. The specifre airres and related rneflaorls are: 1'AItT I. fhe effect of ~F' ~x-vava incubations on eiaa°.alasraIazed islet cell survival aaa,I fuaa~~tior<. Airn 1. To evaluate the °ffect of rat islet encapsulation on the induction of apoptosis, necrosis, cc.Ii survival, and maintenance of the phenotypes and function of islet ce,Ils. At different times before and after encapsulation, apoptosis (TIlNFL assay and other complementary methods) and necrosis (rniv.roscep~y and histologica.I studies) ~~~ill be evaluated. Maintenance of islet poll phenotypes will be evaluated with irnmunohistochemistry targeting islet hormones. Islet viability and function will'be sradied. Aien 2. To evaloa.te the effect of incubations of isleis with C:la, including IOF"-II, IOF'-YS i~IGF', HGF'9 beg gene farot~lr~
as~d 'y'lfF-~z in decreasing susceptibility to apoptosis and necrosis, and improving survival parameters described in Aim 1. Islets will be incubated with the GF under study at different periods during ex-viva manipulations. ~utcome measures: as in Aura 1 + incorporation of thimi~~ine H3 to evaluate cell proliferation:. Alsn 3. To evaluate interactions of GFs with the mic:rocapsule matrix, using reverse; size exolusios, chromatography. f~im ~. To evaluate the effect of ductal cell secretions on islet .suYVival p~aranzeiers, by incubating encapsulated islets with free ductal epithelial cells. Outcome sh;dies: as in dim 2. Airn S. To evaluate, an viva, the effects of GF incubations, using transplantations in non-diabetic and STZ-induced diabetic acid mice. Outcomes measures: 1- metabolic (% mice normalizing blood glucose etc.) 2- survival parameters on recovered islets as in Aim 1. ~A~'T 2. The effect of co-~ne.apsuiatiaan of ducts and islets ors nreeapsulated islet st~r-vival ant function. Usirdg the methods and outcome measures described above, we will rim 6 evaluate the effect of encapsulation on ductal cell, including .viability tess:ing and secretion of IGIr-II and NGF, ~,im 7 optianize the method to encapsulate isieia and ducts, t~irnn 3 evaluate and corr.pare, in vitYO, the effect on surS.~ival parameters, of islets and ductal cysts encapsulated separately vs co-encapsulated, .~i~n 3 evaluate, in viva, transplantation of islets and d;.rcts into non-diabetic and STZ-induced diabetic said mine (to evaluate survival). ~itra 11I evaluate, i3a viva, the methods developed. using transplarktations unto lVOIl mice (to evaluate survival and immunoproteation). in ~onclusio~, the proposed studies will impreve the understanding of the mechanisms invo2ved irr the survival o: encapsulated islets, and will contri'oute to design methods to improve Ieng-term islet cell zr~ass and function.
Papa ~. of 47 ~leii~, .lean-Pierre ~~grent grant ~anadaan ~ial~etes ~ssociativn 117107.1200:; to 0513012~~4 Transplantation of islets encaps~~ated in modified alginate poly-~.-lysine rnicrocapsules The goal of our research is to op'~irr°~ise islet m.icro~-encapsulation tec~~nology to evenarally transplant islets without in~~ur~osuppression. 'tee have developed ed~ods to evaluate the different rr~icrocapsule characteristics and n~ctions. urge sus;lz irrnportant parameter is rnicrocaps=ale resistance. Stror~ge~ rr~icrocapsules will improve the duration of the graft. In addition, broken rnicrocapsules, as ~vel~ as the detachment of the outer alginate sheet, elicit a pericapsular immune reaction. In addition, micxocapsule resistance is a critical issue for tlae encapsulation of virus-tray=_sfected b~.oengineered cells, ~s~hich may prod~zce insulin or rnoleeules designed to promote islet cell survival, as well as fox stern cell derived islets, which may present a risk of malignant transformation.
i~reIlnannary data.
~Ic have developed a novel method for producinb covalently cross~linked alginate-pol;r.-s,-lysine- alginate {cov~l~l~) z~i.ciocapsules compatible with. cell life.
~'he chemical conditions required to break or to create a covalent link are usually ~:r~corrpati~sle with cell life. ~Lecl~anical resistance tests indicated that the neua mic:aocapsulc were 22 fold resistant tlxan standard capsules. The objective ~cas to evalu,~.te isle'a encapsulated ir, these covt~l'~. ale hypothesl~ed that tl~e ne~~~ method vain not negatively affect a~icrocapsule perrrseability arz~i biocornpL~.ti'cility, ~v~~ile rs~aintain ~ng and ever prolonging encapsulated islet cell viabilit~~ and func3:io~-~, a~zd that rnicrocaos~~le str~;ngtl~ will prevent the dissemination of {potentially) rnalin ant cells. The spcvi~c afros and related methods are: to investigate f~a vitro tllae effect of the new n~etl~od ora. b j a~~:rnbr~ne permeability and 2) islet viability and function, ~~.si~~g techz~.idue established in our laboratory, and ~) g~ viva -rnicrocapsule bioeornpatibility. 4.) To evaluate the effect of the :aew x~ethod on i~ vivo isle~~ viability and function by t3~a,nsplancativ~n studies of nncapsulatcd rat islet into :rcl~
{imrnunodeficient) strepto~,otocin-induced diabetic mice {~TZ-soir~). ~) To e~,raluate the effect of the new method on rr~ vivc3 ale; survival ar°ad imrr~~unoprotecrtion, usi~-~g transplantation of encapsulated rat islets into ~'f~-induced ~5'~L~,/6 {~nxnunoco.mpetent) nZice, and ~j on the proteetio~-z againsv'~ dissenainatior; of encapsulated (potentially)malignant cells, by twansplartir~g ~L-~ thyoma cells from C5713i,16.~i'l~
1,2 mice, encap5alated encapsulated in standard and rzew ~:~icrocapsules and not encapsulated, into 1~6.f'L.Tr l . L mice.
~a~nclnsfr~n. encapsulation of islets irz rrcrocapsules that axe virtually indestxarctible within an animal body would alloy,=,~ safe ~°a~'isplantatiori ot° virus--transfected cells as well as stem cells derived islets, urhich rnay present a xisl~ of o malignant transformation.
Page 4S or ~~
f~aa~e, Jean-Pierre a ~i~~r ~c~ ~°y d~~~r~~~a~~~ s~a~a~~rscrlp ONLY I~"-FR~55 ra7ar~uscr ipts ~na~e suber~itted.
r Mer7uscricats afread~ p~~iished sfao~rfd b~ r_toterJ gut caoies shoufd ~~It3T'b~ su~rrsit~ed.
D Manuscripts in~r~~r~tfon or ct~rron~lv rir~tder review ~~r~ ~JQ~' ~cce~tabf~.
L'~'~'i° A~'C3.~~s I~obitaille I~, ~usseaui~.: .~, Flenl~:y 1~1, Rosenberg L arid :~ialle .IF.
Insulin-life growth factor iI allows prolonged i~lood glucose normalization ;pith a rwduced islet cell mass transplantation. ~ndocrgna~I~gy 144- ('1} X1:3"7-3IY~~, 2f~U:3.
Illieva f°~., ~'uan S, ~Varg I:I°~~, Agapitos ll, Hill ~~3 and.
~oser~'i~erg ~,. ~'ancreatic islet cell survival following islet isoiavicn: the role of cell°alar uxteractions in the pancreas. Ja~urnal e~fendacrl~aol~gy 161 : 3~7-36~-, 195~~.
IJeblond Fps, Sirr~ard ~G, F~er~ey 2~, Rocizeleau ~, I-Iuet FNt, ~3~.11~ ~~. -~easibil.ity and safetzJ of intraportal microcapsole irr~plantation ~1 the rat.
~el?L'~'ra~splantatl~n~a ~ : ~'27-337, 1999,.
I~obitaille R, Leblond F~~, Bourgeois Y, FIenley l~T, I,oignon i~T, I-Ialle I~. St~z~dies on small (<35U~,m} alginate prai~~-~,-lysine microcal~sules: ~T-a~eter°ir~ation of microcapsule permeability to carbohydrates and proteins'oy reverse size exclusioa~
c~'zroanatc~graph~.
~ ~I~ed bates SU : ~.2C~~427, 2Gt~U.
Robitaille ft., Leblond F:P., I~Pnley i~I,1'rcd'hon 3rle ~J, I3robet slcy E, I~~,ih 3h. Algiriate-poly-L-lysine biocorrtpatibili~y: a novel f~~'-~'~'I~. method for ~,ytokine gene expression analysis in pericapsular infiltrates. ..~ ~iaeal l~at yes 45 : 223-23U, I999.
Leblond Fry., 'fessier ;~, Iialle 3F'. A quantitative rnetbod for the evaluation of biornicracapsule resistance to rnechanicai stress. ~iaar~ateraa~s 1'~(21~ :
2U9'7-2IU2, 1996.
Robitaille ~:~, ~esbiens K, I~erdey ''~, k-Ialle sue. :~ hne co~°se oI' transforming gt-owth.
factor-~, (T~F-Vii, } mI~T~ e~~pressior: in the host reaction to alginate poly-L-lysine microcapsulesfoliowing irnplantatians into rat epididyn~al fat pads. ~ ~foe~
~I~t yes 52 : I8-23, 2UUU.
Faraskevas S, htaysinger l~, '~l'ang Z., ~uguid , Iiosenbf;rg 1J. dell loss in isolated human islets occurs by apoptosis. ar~creas 2U : 2?U-276, 2UUU.
Rose~nberg L, Wang 12, ~'aras~:evas S, Waysinger ~. Stnzctur~l and functional changes resulting from islet isolation lead to islet cell death.
Snr°ge~°y I2.~ : 33-~g8, I999.
Valang IZ, Rosenberg s ., ~a;rltenance of beta-dill function arid survival folla,~ring i;>let isolation requires re-establisl~zment of the islet-n Matrix :relatianship.
~~~aa~°naI of endocrin~angy 163 : I81-I9U, 1999.
fang I~, ParaslCevas S, liosenberg ~,. ~haracters.satior~ of integrin expression in islets isolated fram hamster, canine, porcine, and h~ama_n pancreas. ~e ,~o~rnal ~sff hflstoclresn.
~ eyt~chexrt. 47 : 4.99-~Of~, I999.
~'aqe ~~ of ~~
i~a!!~, 3ean-P'serre t aras~evas S, Aiki~ ~., ~/tavsnger ~, I.akey I'i'i,, ~avatiagl~. ~w, I-Iering ~, ~t'ang ~., Itosenberg L. Activat~otz and expr~,ssio~ of l:t~K, .l~ anti p38 li~I-l~inase ire isolased islets of IJangerhans : is-:iplications for cultured islet survival. 3"~~~
tet7te~s 455 :, ~03-2~8, 1999.
i~uan S, ~etrakos T', IJugald ~.~~, ~~osenberg f . Isolation an.~~ culture, of intraloba:lsr d:.~cts from the hamster pancreas. il~ vat~-o ~eII a~ae~ ~evet~gnaent~t ~iols~d A,ml~sea~ 31 : 77-80, i995.
P~,~e ~~ raF ~'7 E5~
:5 i~
Zi I, ~,
.ells veers ror~~wod with basal r3~edium during one day for recovery prior the additionx of Geneticin (G418) for selection of neomycin-resistant cells. From this p~in~;, resistar!t clones were harva~sted fox an additional 4 -weeps in presence ot' i ~ 18 and tlmn agaiA_. in basal medium.
"t he establishment of a GFP stable cell line generated in the: earns condition ay the IGF cell lne eras used as a control.
Rat islets were co-encapsulated with cells transfected ve~th IGF-II and GFl' genes using different concentration of'f2VI4 cells (~xIC~, l.~e, and ~ ~ 3~' oellules/mi algixtute) (figure 3). ~Y~ese islets were then incubated at 37 ~ inl~ltra~ult~re cdi~.aan ii;r 3 days. ~Ie observed at a;!1 doses that a.~~Cer three days the islet viability v~~as bettor witl.~ IGF-Ix co-encapsulated cells than w.'tth G'=_~'-cell or with islets encapsulated alone ~ftgare 4). for the dov~~lopment of i~ie methods, a ~'i~lI4 cell line was used because it is hnom~ to I~avo the maohinc_ry to secrete (minimal atnosznt of) IGF, vfluLh avoided problems in interpreting negative monditsor~s as potentially duo to the cell ty);e. III
experiments included non transfected TM~ cells as controls, and showed differences. 'The experimental conditions being well established, we will now use, for t~~e proposed studies: a cell line that does not produce any IGF', i.e. Chinese I-Ia_mstet- C)~rarian cells ~~;~~) Page 25 0~' ~7 Na34~, Jean-P~prre d -_~ - , .~_._ ., r ,. ; .: .~ ~ b ig 4: evaluation of rr~rtcroanca~suIed rat islets o!r Fig 3: coencapsuled rat's islets wiih Tl~l~ . ~ coencasulcd with Tlt~li~-~1'P
ce'11s or Ti~%P~.-I~F -2xIO6 cellules/mI d'alginate ~ ~ II cells. Viability. Islets urere cultured ir:
------i ~ ~ltracultnre C~hl2L-I(3~6 medi~~rrrE for a 3 days peQ°iod. Islets viability =Jas analysed by propidiun iodide and orange acridine staining. "tlroe; viability b were evaluated by twt~ di~~eren'~ observers.
f~ other st~diesg ~°-~:nded by the ~aradian ISiabetes ;~~.sscciation, t~~E; developed a rnetbod that allows rr;icroencapsulation of livity ces.ls i~. covalently linked algiruzte poly-I,--lysine- aigi;za:~e (~P~) membranes. The extreme conditions ~~equired to produce and 9;o destroy coval,:nt IiPn:s are usually incompatible with living veils. fbereFore, these e:~treely ;~tt-o~
nicr°oes~ps~le.s are vi~~aally indestr~cti~Ie ire tlae ~~etieal and pl~~~sieal o.oraditac~ns yo~~rad its tire ivvin~:~a~dy.
They remained intact aver 2 years xn a pI-y I2 all~al~.~~ bufFe:°, wlaer-eas standard microcapsules completely dissolved af$er 45 sec. itz the same conditions, and they resist to a ineclr.anical stress test that implies stronger a9orasioz2, crusbi3~g and shearing forces tiara they are likely to be submitted during a lifetime in tl~e Lzving body. I~ ote ~f~a~ ~it~ his te~It~i~~y we pt°°~p=rn;~e ~
new ~°~Ie fir ~t4~re~~:r~~~~sulnf~osrx addition t~ ;.jrotecting the tr°ansplant (islets; from tfze recipient irrtune system, it will inversely pvoteet t-he recipient fr~trt t~~e tr~raspp~n t, en~bl~r~
the safe nse of gene ther~py~ irarart~I~esi oell i~-,e ~:nai step sail ~lari~r~;~ islets.
~. i'~I~'I""~I~S ~~ f3~~~~3~J~°~T':i~~,d ~w~' ~''~'~ ~~~~ f~~~PC9~~.I~
~~~;~.
~~~"I' ~. ~'l~e effect, ~n fx°ee end t:~iet°oe~t~~ps~late~3~:
islet sell snrp.Ya~r~h or t~°~nsfer~°in~ li~~' genes into islet oePls, usi~a~ non ~riral veet~~c°s. mote that ~~nicrocapsales ~vitb covalently h~~ked ~I'~mebranes, as described in i=~e p:i°ecediaag parag~°apl2, will be use3~, ira aII st.~dies.
~~. ~n vitr°~ stndi~s, f~~taonale. ~r~ vi~-r~ studies gill allow evaluations and ecastomi~ations of many parameters, and establish a dequate experigner3~aal conditions.
~'~°eli~a~inar°yr d~t~. I'n tb~e ver~r t'.rst ia~ vitro experiments (see prerimir~ay dataj, _~~-I and IC~I~-II
(but not I~:~ -I+Lt) gene transfer improved islet Bell v~.abiliiy at 3, ~ and l il days in culture.
~:~pe~°ie.~tal stesi~~a< ~listar rat islets eo-tranfected ~~~ith ~-reen ~ l;aerescena l~roteir~ (a.~FPj, l~~-I, A~~b~'-II or IC3~-I+TI and zeal ve islets will be compared. por all ~°eatrnr;nt groups, free and rnicroencapsulata~d will becopred.
Idea will be cultured for 30 days arzd stuciies will bt; performed at 3, ~, 1 ~, ~ l ~.r~d 313 'icays.
culture media will be changed ~; 2 days. Iaietl~ods briefly described.
prei~rtinary data and in more details in our resent papers l~, as well as zew methods will b a ~:.sed.
This include tlae stczdy of transfection rates, using C'aF~~, Imp secretid~n in the supernatant usin;;
westein bhts and ~.~,I~t~~, islet cell viability studies, using ~~zciescenc;~: microscope with propicl~strr"~ iodide (dead cell.sj and orange acridine (living cells), ~', scud measu.~°errtent ~md qua~~~tificaa;~n of islet nets°otic centers.
I-~garose blocks will be prepared fog irrnnu~M~obistocbe~~nfcal studies irzcl~.ading apop8:osis studies, P~~ ~~ o= g.-~ts~~ ~~,~~~ ~,m ~e~o ~:n t~~~~e, JC't3il-p12C1's'~.
usi2; the 'pIJ assay and ICI protein rr~;asu~~exnents. In order to identify ~r~l~ich t~rpe(s) of islet cell undergo into apoptosis, successive sli; Ls ~~11 be studied wi'ali the ~"UIr-EL as;yay at3d ar~ti-ans~lin and anti-glucagon antibodies, as p<<evio~csly :~~scr°ibed.~I~.
~-yell replication aid islet neogenesis will be studied using hrdLT incorporation end immunopero~~ida y~ co-localization of insulin and cytolceratin 20. A ~~1~ pre-inc~:~a2ion with ~0(lng/ml Iuf'-II wig also be studied to investigate a potential additive ~~~ct tc~ IG~ -II gwne ~-a°~sfer.
Anficipated results and ~ignif~can~~ . ~h'e anticipate tl~~ ccz~,~.rmat~on of preliminary data.
I~~:.er optimization of ti_e method and longer duration in ~~~Iture, the positive efff.ct on islet saarviv°al st~ouid 2~e even snore important. In addition, for airn 1 to 7, vie anticipate that pre-ixansplantation ire vitro I~F
incubations and I~F gene transfer will have additive ef"ects, since th.e for7ner have s,n irn.~nedito eff"~ct and prevent the important damages ir~duoLd by the isolation and oncapsulat~~oa procedures, whereas the transfected genes tame a few days to effecrvely produce I~~p s. '~
he islet transfecticn rate, currently at 63%, a good result with this method, as von~pared to published data~l5'7.,15~], can be fur~.her improved, sine some p~am~t~rs ~.~»re zd~nt~sfi~d as important but not 4.~ot optimized. ~~n~gen~y sta~ez~~~nts. fee a~.n ~, Alns . Its viva. atia~~ale.
~~~e pote;ntial clinical significance of the preceding studies will be e~~aluate~l. Fcr studies in ~Llaer animal model:; see airr~ ~. ~r~i$arainary data. Aithoa.~gh we have only irc vii-~~o data yet ors I~v° gene transfer, we hare shown(1) that pre-transplantior~ ICI'-II in~-~:batiors Iza~ro a powerfi;l effect ~~n tr~~;nplant~,d islet survival. ~~Iaerian~utal design. If pr~limd~ar'~ data ere ccnfirrned, or~dy I~F-TI v~ill ho study initially (unless other studies shovr a stronger effect ef hrrF-I er I+II~. A
ninir:~E~,I islet mass model, based on the results of gar published s~.'-crdy[1;°s will 'o~;
used. I=~tra-pcriton~:al allografts (n=12 per conditions) of lj~ or 75 ~57I31/f mouse rnicroencapsulated iss.ets, L~-ansferted with I~F
gene or not, will be performed into ~571~1.~6 ~~'~ diabetic mice. I're ~'annsplantaticsn I~a~-II it vita~o incubations will be performed in all conditions :~. none, d~pendin~p upcan ia~ °~i~o st~~dies (aian 1). controls will include free islet 'Tx. 'fhe prep~.ration and follov:~-up of diabetic ar.~nals will be as previously described. i 1) Briefly, animals ~;nill have free access to food and blond glucose will be measured at defn-zed~ til~aes. "3'~°o successive blood glucose >_ 11 ml; (~#IO rng%) will sign a recurrence of diabetes. In addition, at tl~e spontaneous dea.tl~
(if and°) or at the ss.crifi~e of the recipients, microencapsvlated islets will b,, retrieved for the evaluation of apoptosis and necrosis and the presence of ins~.~iic~. The follow-up c~f diabetic mis~c will ~,o on until diabetes rec3~rrence occurs or animal die. Tl!e ~ last animals s>~11 nerrnoglyce.~nic in each ~~oup will be sacrificed when presenting severe signs of agoiz~g arid autopsied obi ia°esh tissue to eliminate residual endogenous insulin secret.ionEi,l6t~) (has never ~Oeen positive in the lab). End-points v~°ill anclude: number of recipients maintaining ,~orsnoglyc~,:ania, average I°gloeJd glucose and glucose tolerance tests (insulin and glucose) at d, 1? and l8. ~.~~tieipat~d resa~its and sig~ilacanoo. die anticipate that arx effective ;stet cull protectiw:n dL~ring tl;.e critical post~~tfansplant poric~d will af.low prolonged normoglycemia, using a reduced islet cell mass. Cura~gotZ~:y stato~no~~ (~srns I +~).
'This method does not induce a stable ge ~w o~pressioz~, and efFect7ve hGF
produ~;tion should slo~Arly decrease over 3 months. froteating islets duriLag a critical post-transplantation tnay be sufficient. It is possible that optnmal long-;verr~ results will require;
pe~man~nt IC'gf-II supply.
Alternative strategics to achieve our goal are described in part 2 and j. ~A'~
2. 'I°lae efI°eet ~n islet cell survival of ~a~m~ncapsulaiiug islets a~~d cells with a. sta~l~ g~n~
expressi~n of h~~-~G~'-~ (~sr ~ ~r ~-Y-~ ). ational~ grad pr~l~auary data fir ~O;~rt ;~. Using multi~;ehular structures, such as islets, only retro~Jiral vectors could dn°' ~ctly induce si:ab'.e gene e~cpressior~.
Although new very effective tra~fection methods involving viral vectors ofaer exciW g scientific opportunities for physiological s~.tdi~s, their use i~7 cli:?ical applica?io~as remains anc;ertairt, c;v~.n for rapidly fatal genetic diseases.~~] Using th~;; non v al Izposorr~e nWtlaod (l~t~fAF') and a neomycin resistance gene, we v~rere able to ,~rodu~e mc~~aonuvlear co1',1.
lii=es v~ith stable ICIf-I, II
and I+II gene expressions. ~f~ese cells were cc-wncapsulated vdi~, i islets (see Progress repart/preliminary data). ~~z vit-r~a studies will allow the evaluation of G
laa°ge nutnbex of conditions. .Paa viva studies will cor~~rnz the potential clinical signiric~-~c;e of i~a va~o findings, ~ac~~~ ~y of 4i Heiie, Jean-bier re determine the timing of effective il~.t~i dontion, arid evsiuate a possib.te rescu;, effect. r~~° Ira vit;~c studies. Exper~~nentai design. islets will be co-Pracapsulated with Clzin=wse Hamster ~varian (CH~; cells transfected with 1GF ~, II, or I-~ 3I genes btsis~;
different eoncentratiom of CHC cells. The number of co-°ncaps~.zlated islets she==wing ~:he best results (viabilitt~~ =~s .veils concentration) will 'be incubated ~~~8 h. (24 f~. before arid a~:r encapsulation) in 50~ ng ml cf ho IGF-II under study as described ixz (l~. Controls will include islets co-encapsulated with non trar;sfected CHC3 cells, encapsui~ted alone and Trot can capsulated.
Additional expeziments wl~
CH~ cells and islets encapsulated separately and cuit,~red together will include a dose-response with increasing proportion of CHG veils. Tl°_rroar~e7~, cultures and follow-up v~ili be Iseri°orzxzed, as described ira aims 1. Andlcapated ~°ests and signi~a~ano~. ~Je anticipate t°zat tile method ~Ffill prevent apoptosis and necrosis foV~ a very prolonged period, sumo the iGF
genes will be permanently expressed in all cells of tlae successive pe.nerations. Th.e study will determine, tl~e amount of CHG veils and IGF required for an optinxai effect, and establish ti~ze best oxperimer~tal conditions. As explained in airn l , ~~e anticipate an additive effect; of in vzt~o ir°~cubations and gene transfer. Cor~tengen~y state.rnent. An excessive amount of ~:EIG cells may provice more IGF supply but be potentially harrrrful to encapsulated =gal suz-vival. Tk-ds study will ~,eranit to fz:~d tile optimal (approximate) amo.r:~t of cell ,or >maic;rocspsules. If oux~ goal is not a~,hieved, this proposal contains alternative z~ethods. Aaxn ~.° To devoi..~p a method ixzat allovds turning on/ofF z'n vivo, 1GF gene expression a.tional~. ~eirig able to tzrrn onloff, ira viva, transfected IGF gene expression will provide an interesting tool to address several ~~;u°stions, part:~c.ularly those described iz~z aizrs 5. In addition, it will provide ideal controls: exacdy tile same cells will be transplanted, but the activating drug will not be injec°ttrd. ~ruli ° ~z~y data. Using; the pL~S
vector, ~ genes would be transfe~zed into the sarrze cehi, in addition to he ~eornyoin resistance gene that alio~~;s the development ona stable liz~~. ~xp~rixnontal de:slg~.
Ush~g a Tetracyclir<<o-Regulated expression systez~. (T-Vie:; System), p~i~..I~A~ITG vector, ~:HG
cells will'oe trar~sfected with IGF-I1 (or I or I+II) gene us,ng a tetracycline responsive promoter '~lzat ran be trur~ed on (or offj by admizxistezing (or stoppinJ) Lotracyeli;~ze ~.o the z~ccipient, and wilt oe co-encapsulated whiz islets. 1'relirninary i~ vitra tetracycline dose-response study will be aer i°orn-~~~d z~o;zsuring only IGr protein concentrations in t_~o supernatant. Antlcgpated ~°os~&ts° ~o anticipate that this study, combined with the results of foregoing studies, =will a11~3w to ~sta'olislti~zg the r,onditions for ahr~ 5 studies. Alan ~. T o study, in viva, '~.hc; effect of c;o-encapsulating islets and eelts wi,.h a stable IGF
gerfe expression, including the tinning and da~atioz~ of r:l°-fective treataoent avd possibility of failed islet rescue. ~atl~nale. Same as =or airn 2. ~~z addition, the tinning acrd duration of IGF delivery required for optimal protection ~:uill be e~aaluatod. I3~~~dilaaar°y e3at~° S=.raze as fen ainz 'i3.
~xpa~°atnental deslgn° The same ani_~~zal model as in aii~zs ~
will be used. All recipients (n=1 ~ per groups) will be transplanted with the; sab~ne P°~.II~3 c°lls tranfected with IGF'-IT (or I or I+II;s genes using the tetracycline responsive "on off'"' Te: T ~ sequence and c~-encapsulated with islets.
Controls ~.lill include recipients transplanted with tlae sazrze cells but will not receive tetraeyclir?e and transplanted with og~capsulated islets mono. I?.xpe,r irnentai groups will receive tetracycline during 1, 3 months or indef~nitel~~. Follow-up aced shady methods wilt, g~se as ir= ain.~ %. In addiv~~ion, after 4 days of hyperglycemia rev°urrence of anirrzals off the c~ug, tetracycline ;rill be reir_troduced to evaluate whether islet cell r~,scue is possible. Anticipated r°esults arid sagnlf°bean~~. ~e anticipate that oz~;, irzorzt_~ treatments rnay provide signi~~~,ant protection, b~.at rev;zrrences may occur earlier than ~~~th 3 mouth or szerri~zancnt trE;aWerzts, and that islet poll rescue is possible. Contu~ng~raey staternents° All of the zvurrrerovs quost~ions ay not °~se answered.
hut, with this flexible model, other experirnerzts rr?ay be designed to ac~.rlress other issues.
~A~ 3. Tlae effect on Islet sell surwlval of so-~e~oapsula~ng negate and duct cells°
Alaxr ~. .fin vitro studies. Ratlorrale° °lze advantages of this approach r~r~ that it does not rcquu:~~
gene therapy, and provides, in ad~sition 'S~c~ ILxF-II, other components of islet physiological nucroenvironment (GFs, extraceilular $natrix rraolecules, pluripotential ~-cell precursors).
~~°eligna~y data. inuring the last 6 rr3onths, we began ~o develop and evaluate different duct cell farts 2~ of ~;~
'~~8'2, 3ean-Pierre isolation methods in hamsters, rats and mice. ale established the tech3~iques reduired far tl°ss project, including iznm-.moperoxidase for cytoke~°at~an 20 {a rr~arker for duct cells; and IC~F-II
(main duct cell secretion product), as well as western'olot and (ixz development} ELTSA. for I~aF-II. F,xperimentai design. In ~ iirss se's of expE;riments, the survival and fzmctiozl of mieroencapsulated or free duct cells v~riii be evalF'ated. IGF-II -aaill be rn.eas°~zred in the supernatant, and im~znunocheznistty for cytokeratiln 2(I and IC:rF-Il 'c~:~ll be pert"orr~~ed ors slides.
Islets will be co-encapsulated vritla different conce~tratzc~ns of duct ce;:i s a3zd cultured for 3~ da~,~s.
'The experimental design and metl3ods will be as in aim i. A~aticipate~~"
results and sigearaee.
Islets co-encapsulated with duct cysts sh.e~,zlcl prese~zi decreased ;apoptosis and necrosis a=~d improved viability. Cus~tingency statenaertts. See aim 7. ~irn '~. In v~vo studies. ~a.tie~nale" See a~~m 2 and ~. Experimental design. Islets, cc-encapsulated ~~ith ~
concentrations of duct cysts or alone will be transplanted LF. 'The experimental design and znethosis will be as in aizn 2. In.
addition, rrzicrocapsules retrieved at (a} the spontaneous death or (b) sacrifice of the animals gill be studied for evidence of fe.~nctic~ing islets and duct cells and it, let :neogenesis and a-cell replication, using immu_noperoxidase with ar=ti-i<ns;ilin, anti-glucagon, anti-cytolteratin 2t7, anti_ I~'r~-II as well as the ~I. assay fnr apoptosis and ~rL'3U
izxcorp~>x°atio~~. ~reticip~ted results anti signi~sance. ale anticipate long-term norrnoglycemia in more recipients with islets co-encapsulated with duct cells tha;A alone. ~~le expec~. to obsezved signs ox islet neogenesis.
~~mtingea~c~% statements. Duct cells grow more slowly theta cell lines such as Th~4 and ~I3D.
Ideal culture media for islets and ciazcts diffez-. ~oine questions remain to be answered, such as the need to wait several passages before encapsulat';ng d:~.et cells. Thw potential ~s,dvantages {see rationale} justify to pursuing our prelimina°_y efforts tr> answer then; qupstzons. This research proposal contains several altez-nat»~~m app~:oaol~es if tire ex-petted results are v~ot obtained.
~'I' 4. ~'~te rnechaaaisms ~t '~~F's p~-ntY'tive effect. ~.im ~. ~ better understanding of tl-3e intracellular rneCharisms will 'de useful fer fiztare developments, lExpe~inAent:al ~lesigza. "fl~is study will use material from studies described in part 1 to 3, mostly from in vitro sti;dies, but on recovered islets also. Microerzcaps~zlatec? islets, san-zpleci at different tirmes pre and post treatment, will he fixed and slides ~,vill be done as previously descbed, j 1~. Fffe:ct of the treatments on pro-apaptotic and anti-apoptotic pathvdvays and anti-oxidara~ systems, ush~zg immunoperoxidase with antibodies to molecules involved. ir~ these pathways, szzch as T anti-apoptotic;,, ~aspase-3 (pro-apoptatic}, ~u,Zn-St3D (anti-oxidant systenx,~ will be studied.
PAS' S. Studies in an nut~i~3mnn.e model of diabetes. ~xrt :~. 5~:~dies in iVDD zr~c.e.
ttatinnale. the use of the same anirxzal model for aims ?, 5 and 7 will allow comparison between the different approaches proposed. 'The efFzcacy or" tie z~~ost effective :method will be e.oz~~zrnaed in autoirnmune diabetes. experimental ciesigxz. Allografts will be performed into Nt'~D mice. ~tucy design and methods will be as in airrz ~, 5 0< 7 dependizzg upon the selected rr~ethod. .~~aticigated results and sign'afaeance. since bath n~ier~;encapsulat.on and I~F have been shov~rz to ;'rotect agaizzst immune attacks, the results sl°zould be sin~Iar to those obtain in the foregoing experiments.
~:(~1~~~.,~IS~~~S
her marFy years of sustained worlc_ the fields of islet microencapsulation and islet biology, our combined laboratories can provide a zzr-~que. anti signizvcant contribution t-o die development of treatments to cure type 1 diabetes, based on ~;1 } ability i;o transfer genes, using viral and non ~,~iral vectors, (2} ability to produce; cell l irves with a stable, gene expression, usiz.g non viral vectors (3}
ability to co-encapsulate such cells and islets {4} ability to prodazce microcapsazles wirln covale~ztly linked membranes, virtually ir_desi~~.ctible witlsixz the living body {5}
ability to produce ~Jt3 p.m :<~cdoral" microcapsules with o~ery uniform size azid rnorpl?olo~;~, {6} long ex.periezzce of original methods to measure and control microcapsule characteristics, (7}
ability to predeterroir_e nucrocapsule size with high precision, (8} expertise an~e. facilities to work ~with imrn~.~rtodefzciezat mice (~} original fiecl~niques to isolate and culture duet cells, (lt7}
expertise in the investigation of the z°o1e of the exocrine pancreas, Ca.'~s a~~zd duct cells iii islet rzeogenesis; and a-cell replication, apoptosis and necrosis, and {l l} access to hurrzan islets.
Pale ~'? et 4~
i~i~lle, dean-~ierr2 ~ ~~~~t~~t~E , ~°~., t-icntø~y ~, ~.~sG,a~~cr~ , i~al~~ ~Y'~' (2~J~~
i~~-ii ~tto~~~s prolonged blood glucose nor~n~a~~oa~ v~t~ a rer~eed Rslet cell mass traa~spt~ntat~~n.
~,ndocranology 144("T):~~3~".~4~. .
~2 ~ R~scn~e~rg9 L. (1~9~) ~~a wivo cell f~~a$asfor.r~ataon~ l~c~a~;c~~ais of data ceps frog pa~creati~ ductal cells. ~e:~l 'F'raatslalantation 4, 3'7i-3~4.
~~ I~o~er~~e~rg, L., ~ ° ~~.i., P'l~tcngc~-y ~.~., iiafacloff3 ~, ~.~.r~d ~ug~afd, ~'.~. ('i~9~
islet cell rcgc~aeratio~~ a~a tE~fe dl~t~etic ~,stc~° par~s~~~eas ~-gt~
re~~toratxoa~ oi°
nor anogiycca can be lnducc~l by a local growth factor(s). ~aal~ebologia 39, 2~~-2~2.
~~ 3~afaeloff, it., ~ir~, X°~~., a~°lo~~, S.'J., osenbcrg, ~.
and '~llt, A.i. (i996) identl~ca~o~a seed dlffere~~~ally r:xpressed genes induced l~~a g~~na~rcatlc islet ncoger~esis. ~'ES Letter ~~~~, ~~9-~~3.
~~ ~at°aeloff, ., ~nttenger~, ~.i..,., ~rloev, 5~~~., 1n3 ~.~°, ~a~~r~, :., i2o:~enb~:rg, L °, Dugati~l, W.i'. and ~lir~ai~, <~..i. (1997) ~l~aiing aa~d sequ.c~~.cg of the p~aacre~t~c Islet neogencsis associated protein (~"j gene and its expression in islet neogenesis in lxaste~°s. ~o~arnal ~lingca~. investigation 99, :~R~~-~:~9.
~~ Ilieva, ~., ~a~an, S~, ~~ag,. R.I~T., ..~gapitos, ., dill, 1~.~° cud i3osenherg, L° (i999) Par~erea~c islet cell sur~eval following islet isolation: the rage of cellular interactions an tl~e pancreas. J ~~ado~.rgnol 1~1, ~~'7-~4.
[i~ 1'etrik, ~°, Arany, E., l~c~onald, ~'.~~ arid X111, Ib.~. (i99~) ~p~sptosis in the pancreatic islet cells of the neonatal rat is ass~scia~:e~l with a redraced expression of insulin-lal~e growth factor ~ tl~~t nea.y act ~.~a a sug°vival :factssr. iJndoc~°nology 1~~, 2994-34304.
[~~ G~~nnoul~a~s, TEL, I~t,'., L-~..~ade~~t, ~~~<~,., ~a.~a~otto, t~., °~~tr?~cs:o, I'.~Y. and o~~sxr~ps, ~.
(2~~0) reventiora of l~e:a Deli dysi'~~~~~aio~~ a~~d alaotosis activation in ~Snra~a~~a ~siets by aaiea~ovi~°al gene trarssfer of the iarsulin-Bite gro~rth factr~r i.
gene .'her i, ~~i~-[9~ Tlxornas, ~°, ~hrhard*, ~.. sand a~y .~i° (~Q~~) . progress ~s~~ profiles ~~~atl~ tl~~ nse of vnral vectors for gene they°apy. l~at i~ev genet 4, X45-35~.
[iiDj i3e~hauao~~, i'°~'., ~~~riscc~t, ~'°,, I~c~ard, :.~°, ~eatrixy ~., ~a~let, L., ~'attou, ~"., i~e~°r-~orste, ~°, Clarohc~~~,1~, ~.; Lenaarclaar~~, ~°
and alia~i, S. (~9~g) ,~denovi~°ns ediated catalase gene tr~.nsf~r reduces oxnda~tt stress in hnrnan, poreine axed rat pancreatic islets. ~iahetologia ~I, ~ ~9~-i lDdi.
[ii A ~~~astiae, ~..5., Leof, ~°~,, Shipley,, ~.~. and I~I~ases, ~.L° (~~~6~ growth factors arad cancer. ~ar~cer F~esearch 4~, iii s-~~~~.
~i~~ ~Jnderwood, L.E,, l~P~rcolv, ~.,i., C;lern~ans, ~.~° and ~ar~
'yls, ~..i. (~ ~~6) ~aracrine fo~nctions of sonxatornedins. ~lir~. i;ndocria~ol. etah° i~, ~9-7 i.
[i~~ Sporn,1~:.~. aid i~~l~er ts, ~.~. (i~~g) peptide growth factors are rna~l~.-if~anctional°
Nataare ~3'~, 2i 7-9~
~i4~ ~illestraap, h1. and l3~artau, .i.lVi. (19~~ ~ ~rovvth horrrqone iahacltng tai specific receptors stixn~at.;s growth arad function. of cloned insulin-producing rat iaasulin:~~na -Sf~.~ cells. Er~doc~°i~aoiogy. i.I~, i.i'~~-~i~l.
~i~~ ~ryson, J.i~~., ~'e~ch, ~°',. axed axaer. .~. 0.989) ~roda~ction of ins~lan-lxl~e gr~ovvtl~
fscto.r-iI ~y lau~nan fatal _:~ancrea;~ ire cogtu~-e. ~on.rnag of ~~ad~acrinology i~~, ~6'~-3'~~.
[iC~ Mill, ~..i., Fra~er, r~., S~venue, i., ird~mru, P.~. and T~illner, .~.
(~9~
Soatonaedin-~ in ha~ar~ fetal pane°eas. ~rellula~° 1~ca~i~atiorx a!nd release drrriug organ ca~li~~°e. I~ialaetes 3~., ~r6~-4i-l..
(~7~ itornanus, .f.A., ~abhrrsvitvh, ~. at~d ieechle~°, l~.i'vi.
(19i3~) l~eonatal rat islet cell cultures syuthesaze insuiira-~ilse gro~~tth facto~° i. l~ia.~etes ~~4~F
~9~-~~~~
?sae 3p of '~~
Halle, Jean°Pierre [dl3j S~c~aa~e, ~., ~eidso, ~.'.<, .~Iiti,; i3.~. and ~~il~rst~°~, ~>
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~i'fe~as of ~~ascudar eaadothelial gro~;~rth factor oar pancre~.tic da~ct cell krepdi~.atro~a a:ad the irzsu~a p~-od~ection of feted isdet~lid~e cell eluster~ in vitro. .~~Iolecuiar anal ~eddular ~aadocrinology 126, ~ ~a~133.
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instZ.li.tt-tlepøndettt diabetes tttellittts. bite i~~e~~ Ettglattd :~ottrttal of ledicitte :~25~, 97'x-9~~.
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~'l3~ Lacy, P.~., Wager, I.~~, and ~'iak, ~.J. (I9?2) Periftt~;itD~ of isolate rat islets itt ~ita°~. Diab°tes 2~, 9~7a99~.
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~fllx 'I"alsal;i, ~, attd ~tto, .f. (x9~S) Pt°eparatEt~tt anti ctiltttre of isolated islets attd dissociated islet cells froth rodent patter eas. :Yoorttal of ~.'asstte ~~tltatre l~et&ts~tls 9, 6I-66.
(~2~ Lacgr, P.~. and ostianet~sy, 1. (I96~~Ietitod fctr the isolaa~otr of intact islets of Latt~crl~atts front tie rat pancreas. Diabetes i 6, 3S-9.
~~3g Farttey, ~..~., hIa,~at°iatt, ~a~'.~., ~'altl~iie, .P. and al., e.
(1990 f~ntotransplantatiot~ of dispea°sed laancreatic islet tisane cottabittetl ~etlt total or tllnar-ttatr~ pattcreatect~stny for ta°eattrtettt of clxroxtic pa~creatitis. Sn~~~er;~ I~ fl, ~2~~&3'~.
Pale 3~ ~f ~7 H~!le, J~~n-Pierre ]~~] r~ctenaar~,~.I~.,~~a~°$s,~lf,, ~l~scaa, . end ~~.j~ttc., ~.~'.
~~9~9) 1'roic~ngcd i"a~~lctiora of i~t~°aspicnic ca~ac paa~c~°catic ~ra~cnts autcgr~fts.
'f'cv~spia~atation ~roce~d~ags ~1, a619w2620.
~~5] ~iiriCO~itcd~, V. and ~apic~c, Ice. X1976; S~.ccesfaaii intrasiaic~°~ic ~ototrs~nspiantatio~a of pancreatic tissue aa~ tetaily iras~~s:re°~.tec.to~~cd dogs.
T'ra~aspi~~~tatio~ ~3,> ~6~>~~69.
]~6] ~aI~ingcr, ~.F. and l,~c.~; ~.~. ~:i97~-) '1'ra~xispiant~tio~a oii° intact panc~°catae ish;ts ire rats. Surgery X2,175-1~6.
'~~ ~ra~, ~.'6~LI~., '~ar~aoc~, ~.1J., Sa.~tto~, ~.., ~cters; i~L, cStsanc, _~. and l~orrig, ~.3.
~19~6) Successfa~l a~ata~t~°anspiant-atior~ of isolated islets of ~sam~gcr~ans i~-~ tlae cynomolgus a-~ao~tl~ey. ~ritxsl~ ~ou~-oal of Surgery "73x X50-~5~.
arnoc~ ~.3.. aced ~a,]otte,1~.'~'" ~19~~) f;ritical mass of pa~ritled islets that ia~~luced norogl;~ cecxaia after iplantatio~~ into dogs. ~iahetes 3'7, X67-~7~:~.
~~~] nester, 5.~., Seelig, ~.~~. and Sta~ach, wl. ~g97~) lte~rersihiity of function and o~°pholog'sc glower ~ata~ lesio:as ~y islet transplantation are long term diahe~c rats.
lJiahetologia 13, 4iD9.
]9~] i~ane~°, 5.1~., Sntlaerland, ~.~.~., steffes, l~T.~~., ~.eonard, i~.~'., lea,]ar~a~~, .1.5., ichacl, ~..~°. a~ad ~ro~~n, D.1~. ~19'~4; 1'ao.~:reatic islet transplantation. Effects on the gloernlar lesions of c~pe~°i~;ntal aliai~:tes i~a tree rat.
:l~iahetes 23, ~'~~-753.
~91~ S~athea°land, ~.E. ~19~1~ ~~ncreas arid isieg. l;ransplacatatiou~.
~. ~~perie~atal st~~dic,s.
~a~etologia 2~,161m~.~5.
]9~] Shaiai~°o, ~.., g.al~ey, .1.~~,., yarn, ,..~., ti~rhutt, t~.u._f '~or'~a, E., arnocis, ~.~.., netean, l~T.l~I. and ajotte, ~,3~. ~~~OQ~ islet '~~°aospiantation i~a se~~e~a patients with type 1 dialaetes epitas =~si~~g a giaaca~co~-ticoi~h~re~
~nu~~aesa~iaprcssi~e ~-egan~eaa. l~' Eogl .lied 3~.,~, 23ti~~:
[93~'o-'aa-oc~cl~, ~.~1., ~aaztea~~, ~1.~:.~ .ya~ay E., S~pelis, 12.~;.,..~,., ~~,.~ino~~u;~cii% ~. ~~ac3 ~a,]otte, fl.~. ~~.991) i'~orogtycapirs aftcr ~~-anspia~atatro~ ~m f~°esfaiy i.sol~ted a~~d cryopreservad pancreatic islets in '1"ype 1 ~iz~suliHy-~depe:~dex~t) diaietes cii.itos.
~iaietologia ~~., 55-S~.
]9~] Yasunami, ~., yu, S, and ~anaei, '. X19911) char acterization of the iunos~appresside activity of ~Sdi6 iy!i rat islet allo~,r~fts.
~'~°ar~splaa~iatioo roceedangs 22, b~5.
X95; ~retael, 12.G., 1$hz, ~.E.< ~~ 11, E. and al.., e. :1~ 9~6) ~,at islet allograft su=wival foliovving different i~unouzc~rl:dati~e and irnu~a~suppressive treatnea~at.
Esc.
~led.1~S '71 °7, ~~1.
]96~ ~"orsen, ,1.'~1., l~iu, E ~,., l~ret~~:l, t~.~. and Y~ ederlin, K. ~19~~) fsiet tr ar~splat~tation in e~periental diai~etes oi° the rat. . fn vlitro culture ~°eda~c~;s la~l~earing cells in rat islets. ~orones anal T~teholic f~esearch 16, 6~1-~'7~.
~9'1~ ~ ac;~, E.lr,., Davie, .1.1'1. end ~ inise, E.~. ~19~9) ~rolonga~~ion of islet allsugraft survival following i~a vitro .~,ultn~~e ~2~a~) anti a single iojeaaio4~ of ,~5. scie~ace ~0~, ~1 ~~313.
]9~~ pretzel, .~., ~lescln,1~.1~., ~~illig:: ~., -~Joel~ri:~, . and Federl~-~, . ~1991i) Effects of gaa~glioside ~~-~~aassial~ tree en* oo I~1~~' ~~ antigen ~~p~°es;~ion a~ad alls~graft sor~rval of paa~creatic islets i~a da.alsetic ~°ats. ~ial~et~logia ?u, 1.1 ~~11.~..
(99? E3esela,1~.K., Zlret~el, 1~.,~., ~tto, ~3e~., Ernst-Scl~egel, h,~. ~r~~l ~'ederlio, . X1991;
~retreatir~ent of rat pancreatic islets with ~~ ~~~, onocio~.al a~.tiioslye ire vitro and in vivo efffects on islet aa~tigec~icatg~. liorar:~oa~es sand I~~aai~tic l~eseax~wh 23, 'x,57..
3s1.
]lOd] 1~'lescla,1~.I~., Entenu~ana~91~., ~~ilde, .% ~ren-:~el, R... aid Eed.erlia~, ~~. !1991; islet transplantatioa~ in experimental daai~etes of the rat. . ~Cryopreservataon redances I1~~ class f~ ant not class 1 antigens of rat pancreatic a.slets. oro~aes and I~et~holic l~esea~°°cl2 23,1-6.
F~aci~ 35 of ~7 H~112, Je~r~-die; re [I~1.~ ~ca~vex~, ~..,~ndr~s, ~.. and L.affertyj ..1. (I9~0~ ~nccess~ol allcstransrolantation ~f xnonse pancreatic. islets vc~ a~n~xri$nnx-~os~ppressed recipiexxts. Diabetes ~~, ~~-I04.
[dd~F l~ax~ai, ~('.,1'~~tex°, ~<, ~~atx~I-~, l~/~., I~ea~a~;~, x.18, axx~I I~IaIsi~ 'I'. (~9~~~ ~i'i"ect of gaxna ir r~diat~aon ou axe~axse p~~ax~~ eatic ~~slet--allog~alt sux°vival<
1~~~L~e~:es 3~y 1~~-156.
j103~ L,an, ~>, ~eeot~sxna, K. ax~~l hardy, .~i>A.. (~9~4r~ I'rolon,gati~x-~
csf rat i,~Iet ae~g~°a~
saxrvival by dix°ect ultraviolet 3rradi~~.~on of ~.hP graft. Science ~~33 607-~~)9<
[I04[ ~anstnzaxx, D.I,., ~:ang~t~eid, ;~., Lacy, 1~.E. and Davie, .I<~'~.
(1.~~1) ~rc~longatror~ of mnx°ine islet allograsnr~~ivai by pretreatment of islets ~wxtb aa~~i~ocly directed to Ia deterinaaats. ~x°oc< l~~tl ~~~,ad. ~~:i. ~'~~ ~~_, ~IS~-SI59.
d~Os~ ~eras~Isa, R.9 l:,acy, ~<~.., a~aptffeld, ~., ~ncy, F3<h. aH~al D~vi.e, ~<l~!.i< (~~~~~ 'I'lae effect of cycl~sporin-~$ Ie~v terxvperat~are9 axed ante-Ia an~tibc~~lies on ~srewex~tiox~ of rejection of rat isht allogx°afts. l7iahetes ~s, ~~-Rli.
[106j lax°racls, ~., I,,o,1~., leg insist, I~. and al., e. i~9~~~ .~
Ixe effect of tbyn~as en~iroxarnent ef ~° sell development and tolerav~:e. deli ~~, ~'~'7.
[~~7~ ~~ssselt, ~.1VL, l~arl~er9 ~.~< a..o.~~ ~'oa~as~e~sld, ~.~. (71~~0~
I~~dncti~n of dcx~ox°
specific ~znrespoaesi~eness by intratyxra~~; islet traxxsplaxxt6tti~xx.
science ~4~'g I~~:i.
[10~j ~o~tisl~ova,11~t. and Lexagex°ova, .~. (I9&~~ 'L'Ixyneus xraedaated t~leraa~ce. to cellnla~
alloantigens. °I'rarxspEantaa:~on 6, =l~.
jI09~ dames, ~'., Jin,1.~., ~l~o~abnry, I~L~.< and ~la~wt~le, ~.la. (~39~~ .x olerance induction t~ a at islet allogs~as ~y ixst~~ati~yxnic i~naoc~alati~ax ~f do~~aaor spleen cells.
~ransplaotatioxa ~6, ~5.~'~~-I1.52.
jI~O~ ~Ixang, '1'<1~.~., l~aclxatosla9 I~.~< axed. l~as~xc, S<~. (I9~6~
~e~nil~erx~ae~~ule a~~aeeaxs micgocapsxales, I. Preparation aa~.d pr~opex~es. Iranaslian .~oax~-~aal of Pla;~si~Io~ a~ad ~laaracotogy 44, IIS-L'~~.
(xl,~~ 6~:Iaang,'F.~L~. (39'2) ~.~~ti~~cial calls ~Ix~.rle,~'~'bo;~s, Spr~ag~:xelc~,1~.
[ti2~ Clxang, 'L.I~.~. (1990 i~tecl~aei~gic,al aid ax~edic~iu appL:~.ation., ;bas~.d. oxa ixn~~obili~atic~n of ~Wp;~~tocytes, snicra~~a>ga,sr~s, ~u,> ~u~~yrx~e ;~yster~s ~Sy micr~encaps~alati~c in artificial c~:Ils. nnals ~f tlxe ~ie~ ~~ ~~-l~
~cadean~r of ~cien~.es jli~~ I,i~, li. and ,~vn9 ~..I~I. (~~$0} I~Iicroencapsulated islets as bioartificiai extdocrine pa.nc.reas. science CIO, 90~~NIO.
[~:~4~ haax, I1~<~'.., L.~zrx~, ~<~.p ~'~.~g ~.t~i., i~e~aes~oe, 1;<, ~'~.f, I.'~. and Suxa, ~.l'~I. (1.99~~
l~e~mrsal ~f diabetes iax ~ls rats Icy tx axaspla~xrati~axa cf encapslat'~d paz~~:r>eatic islets<
Diabetes 39, 51.9-~~<
(I ~ ~j L,uxn, ~.3P., ~'ai, I.'I'., esta~~.v, lax., i~drsrtorr" J., 'Vacelc, ~.~ aaxsl Sean, ~.~I. ( I99I~
~rola~ragecl x°evex°sal ~f dial7erc s"ate Jin ~~D pace by ~enografWs of micx°oexacapsaxlated rat isle~~s. Diabetes ~0, I51~-~.
[~ ~.6~ ~'lii~lC, ~~'.~.., L.ilce, l~.r~<, Lan~~is, ~i'., ~alletti, ~~.l~l<, clxarelsox~,1~.1., I'axaol, ~.,1 ' , '1'<~i< and ~oltc~n, ~.I~. (~~7~' A Iqybrid ax°tificial pancreas.
°~~a~~s. ~~axx. ~axc. ~~taf Inter~a. ~rgans< 2a, ll-~~,"
[Ii.7~ ~hicli, W.L,. and 1'erna, ,.;.~. dI97:~;~ °xdcial pancreas using living beta cells: effects ~a~a glucose laorxxeostasis ixe dialxetic rats. ~ciem~:e f~°~, 7~0-7~I.
j~~~~ ~Iaicl~, ~J.L<, C.il~e, A ~~. axed L,dn~is,, '~< (39'7~~ beta ~~ell c~!taxx°e ~xt syuvluetic capillax°iesa axe ax-ttiificial ez«a~ocu ins panc~e;ss. ~cieace ~.~7, fi~7-~~.
[1I9~ I3uvivier-Bali, V.1?<, l~na,e~, ~., ~;~rent~ 1~..I., ~'~deil, ~.~5, axgd ''heir, ~.~. (20~I~
~oanplete protection of isEets against allox-e~ectioxa and aoto:xn~anity by a simple barium-alginate xnembr ane. Diabetes 50,169-7f35.
[~~0~ ~ra~, D.J. (I99'~ l;xxsa.p~~ll~at:~d islet cellsa tlxe role of direct:
and indirect presentati~xa and tlxe relevaaYCe tea :~enot~ansplaxgtati.~xd axan~
autoi~;~une recacr~ence.
l~r l~/Ied I~uII ~3, T77-~~.
Page ~6 ~f 4?
Hake, J~anm~ierr~
[tai] ~ielcer, ~.'~., Stocl~berger, S.I~ff., ~Iallson, h..., ~isenbar, ~.5.
and ~o~er-leis, S.
(1986 hyper°p~~a~ne response to rnlc~°oencpsu~ated xept~~gen~c tissa~e in non obese diabetic mice. fn: ~'lae ~rn~nunology of I3iabetes l~ellitocs (~av~orsl~l,1.~.
et al., esis.~, pp. 193-200, ~~cepta ~dica, sterd~~a~t.
[122] ~nrn, ~.~., I~restow, ~., '~ ai, LT., Vacels, ~. anal Sun, ~.l!~. (~992~
~enograffis of ~-at islets into diabetie mice. ~.n e~al~:ataon ofn~~ sa$le~° ca.~sr~les.
~franspzantat~~s~ 5'~, 180-3.
[123] Soon-Slriong, ~., ~ttea°~~, i"~., 5;~,]alc~~~:ae~~~, G., Sansdsrv~d, ., l~einta;, t~., ~"~n~a9 ft.l'. and Espevilc, T. (1991 A.a~ a~~aanunologic basis for the i"abrotic reaction to xpianted naicrocapsules. '~ransp~ant ~~°oc 6a~, 7S8-9.
~12~j sVeber, ~.J., ~abinsk~, S., l~ose.l~$t~l~yy°, 'T.,: ~3el~er, 1.., ~ayotte, ., ~'Agat:, ~~., Peterson, f.., Gorton, ,f. ~r~d ~eer~~tsrna, ~. (1.990 The rolls of C~4+
helper T cells in the ~lestcti~n of pica°oencapsealated islet ~-enografts in °eod mice. ~raansplar~tatnon ~.9, X96-404.
]i.25] Dar~uy, S., ~hichepo~iclae, 13., apron, '., ~oita~°d, ~:'".. and l~eacl~, ~. ~;~ 990 ~oparati°ve steady off microencapsulated ~°at islets implanted ii~~ different diabetic models in nnice,. ~rrn i~vletab ekes S~appl ZS, 209-f ~.
]126j Eritschy, ~.1~., Strublae. .3.~., otters, .H. and var~ Schilfgaarde, 1~.
(1991 ~lncose tolerance and plasma ir,snlin respo:~se trs ie:tra~enoa:s glucose ~nfa~svon and test meal in rats with microencap~aalated islet allografts. Iliabetologia ~~, 542-?.
~f ~'7] IVIa~aherg, ft.; Atlas~~5 '., St~ller°, ~'., ~tapre, .1., Vose, ~. and ~°Slae,~, ~o ~991~
Transplantati~n of encapssalated allogeneic islets into diabetic »13/i~l rats.
Effects of imrnunosnppressi~n. 'Transplantation Sl, 7S~-4.
X128] 'S~'eber, ~., byres-Price, .3., ~ostan~cs, ., ~ecl~er, t~. and Stall, ~.
(199~~ t~~1~
rnonse peritoneal cellai~r response tai poly-.~~-l~yslne-algirt°~ to micr°oencaps~alat~,d rat islets. Transplant f'roc ?~, 1116-9.
~f~9] ~e '~os, p., ~e aan, ., hater, ~. and Van Schilffgaarde, . (199G~
~sso~:aation bet~veer~ capsaale diameter, adesinacy of encapsulation, and su~°vl~al.
of rnicroencapsulated rat islet allogr afts. Transplantation 62, b93-9.
]1~0] ~e ~los, ~., ~e I3aan, ., olteg~~, ~.~. ar~tl Vary Sc1731fgaarcie, lt.
(19~9C~ Factors influencing the adequacy of rxricroencwpsnlation of° rat pancreatic islets.
Transplantation 62, 888-9~.
]l~l] ~$orcher, ~.., ~ekarat, "~., Siebers, iJ., l~iocl~, ~., ~'raatlc, ., ~ioubean, R., i3retzel, ~irraeranann, ~'J. and ~eerl.~~a, ~~. (19~~4~ T'ranspla~ntatior~ rsf neicroencapsulated islets in rats: °~idence for indarction of fibrotic overgrowth by islet alloantigeras released fr°orn mierocapsnles. ~'ransplant roc 26, ~84..~~a ~1~2] Henley, I~., )ltobitaille, ~. arad ~alle, .f.~. (1999 ~'i~nne coarse of peritoneal cellular hosi: reaction against alglraate-pa~ly-~-lysine nDlcroeapsctlbs. ~~anadian ~'oanr°rcal of ~labetes dare 23, 98.
~,eblond, ~.~.., Tessie~~, ~. a~cd ~~alle, d.l~. (1996) ~uantdtati~re e~etlLOd for the ewalnation of hioicrocapsanle resistance r.o mechanical stress. ~iomat~rials 17, 209'~102 ]f ~4j »alle, ~.~., Lebloa~x~3, ~'.r~., ~andru.~" l~<, ~oaart:ier, ~. and ~'lrewa~lier, S. (~.992~ S~-~.dies of X00-microns rnicr ocaps:ales: 1. l,Tse of arginine esterase release by gnicr°oencapsulated prostatic cells pus a measure of membrane permeablli.
Transplant roc 24, 290-2.
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Pale '~7 ~sf 4a ~alle, .7e~n-Fierre (136] ~obitaillc:, h., Leblor~d5 ~>~,., lieukey, ~~., ~ rod~li~nlarae, ~..1., ~robctsl~~, ~. a~~d ~alle, J.1'. (1999) Alginate~poly-~-lysine ~nicrocapsule biocorn~aatibility~ a novel l~~' 1~CI~ xnetkrod for ~ytolrir~~: gene cxprcssior. auakysis in pericapsrtlar infiltrates. J
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~137~ Robitaikke, R, Lebkonci, ~._~., ~oa~rgeois, ~., I:Ienkey, 1~., t,oignon, Ice. and ~allc, 3.~.
(2090 St-a,~dies o~ srnail '~'3~1i anic~°oro) a~gir~ate-poly-1~-.lysxaac icroa~~ps~lcs. ~.
Determination of carb~layd~-ate and protein pcra~aeatioa~ tltroangl~
a~aic~°ocapsnlcs by reverse-sine exclusion ci~roatr~grapby. .i io~ne~l I~'later l~cs 5il, ~~0-'~.
(13~~ Robitaikle, It., erelcy, ~3., ~ro~P°l~~nzc, ~..~V, ~~°obctsl~y, ~. and akle, J.I'. ;19g~) ~'i~ne course of T~~'-beta 1 ~~ expression in tl~c least reaction to alginate-poly-~-iysine icrocapsnles. ~arradi~n ao~ar~.al ot° Diabetes C~.~rc ~3, 98.
(139 csbitaille, ~., Parisea~a, J.E ., lJcblo~d, E.A., l.,a.onrcnx,1~., i.,epage, "~. and llc, .1.. (1999) Studies on small (<3SG nlic~-orn) alginate-poly-I~-lysine crocapsufes.
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and dap" J°uP. (199 '1'iac r~.t epidialytnal flat pad as an i~npiantation sitQ for the stud3~ of icrocapsaale biocouDpatnbility. ~alidati~n of tlec n~cth~d. Jour~eal of ~ioranedical I~~tcriais ~es~arcl~ X9,1331-1335.
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~14~.~ uellcr- 'escr, '.E. a~ad Sutlaer~aa~d, 1~.I~. (f982~ fnfl~iu~~a~;c of couac~aion is tl~c growth cdiaaon oxygen tension iu arctlticell~alar ta~ar~r spheroids. ~areaccr escarch 42,, X37-X42.
14~~ ~aeller- 'seer, '~T., Er ayer, ,1~. a~~d Sutlae,rland, ~.Y~I. (1986) fr~tluencs~ of glaxcose and oxygen supply conditions on tl~e oxygenation of ~n~alti.cellular spheroids. Dritisla Joa~rraal of dancer ~3, 34~-353.
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Eresence of irnannnogkob ° ~s l~ ~vlde;li bind to iyphocytes.
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~148~ Schre~emaeir, J., i~ircl~gessncr, .i., hero, E., Kuct:c, 1...A., ~eyer, J. anal ~~~elle~--l~licscr, . {1994) Effect of ~ic~°~sena;apsuiati~n ou ox~yge~
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(149 ~hicbeportiche, D. and each, ~. (1988) i~~ vitro kinetics of insulin release by icroencapsulated rat iskcts : effecv~ of the s~~y of the enicrecapsules.
:l~iab~~tologia 31, 54-5 t.
~15C~~ Ira, ~., ~acel~, 1, and Su~r, A. (199.) generation of akgir~ate-poly-k-kysine-alginate (APA) bionaicrocapsaylesm tl~e relationship bei~een tl~e ~:n~bran~: stren~~th arid the reaction conditions. ° elks Mood Sa~bstit hnr~aobil ~ioteehnol ~~, 43-69.
~a~e 3~ et' ~7 ~iaaie, Jean-Pierre [~~i] ~~oit~rs, s~.., ~rits~y, ~°~.i0~., ~errtts, ~ and van ~s~;i~~~aa~rde, t~. ~$9~I1~ ~
versatile alginate droplet generator appiicahte liar rnacr~cncapsniation of pancreatic islets. .~ A.ppi i3iornatcr ~, ~~1-~.
[t5~; ~orreanei,1.~., Sun, A.I~I. and loosen, T~.~.~,. ~~9~~j ~atcnt ~ugiisiaer, Canada.
[~~~] Wing, G.~., Daa~g~tiis, A.J., ~°~eai%n.~s, P. and ~oose~a,, ~/~.~.~,. ~~9~'>'~ ~gi~~at~_ pa~iylysc mierocapsuies of cor~irocd rs~~~n~ran~ rnolecuiar ~weigl~t cutoff for an:naa~an cell cuitaare crag~neeriog. ~i~~ee~nalogy t'r~grEas :~s 2~~-X40.
[t5~] .obitae, ., ~cs~iens, >~., eniey, ~~. and sac, J.7P. ~~J00~ 'dime course of transforming growth fact~~°-~eta~ j ~~'F-~eta~~~o xra i~ ~~pressioo ia~
tE~e least r section to alginate-posy-~,-lysine mice ocapsnies folyo~~ iu~plaotatious ia~to rat eisidzdyanai fat pads. J ~eonacd later rtes 5~.,1~-2~.
[a5~1 a~n~:, .f.. (~~ggj I~~pid assessr=~eg~t of isia.t viability w~it~a acnidine grange arid pr~pidiaam iodide. yn'~Iitro ~etl 33ev ioi 24, 2b6-73.
[-fs~] yau, ~,.~., ~..a%ey, J.., ~~]otte, ~ ~.~'., or~ott, Via., f~, 'Y'., ~er~os, ~., ~abinovitc~~, ~., Eiliott, J.ia'., ~igam, ., eteruao., ~T.l., ~~a~rnoeR~, ~.i~., i=,arson, I. and 5iaapiro9 :~.1!'I. ~200J) Clinical 0~tcoes aaud Yosnii~a s~creteor~ after islet trauspla~~tatis~~it the ivdmonton protocol. f?iahetes 50, CIO-9.
[~~~] enhaenou, .'~., I'~oriscot., ~., prevost, ~'., ~oiiar~d, ~., (alii, ~.
and t~iaro~~ac~e%, J. ~l~g'7~ ~tandardizataon of proceda~rc for ef>Ficie~at e~ viva gene transfer into porcine pancreatic islets evith eati~c~afic iiposorr~es. ~'ransiai~.atat~on ~3, &"I~~i#-~0~.
[i5~j f..,akey, J. X2001) Non viral traosfectior~ of intact pancreatic islets.
~e~
'~'rauspiantatior~ ~.0, 6~~-70~<
[~59] ~eigner, ~., ~ade%, ~'., ~cem, Ice'., Ionian, ;v2., khan, ., i~wz. ire, I~Tortiarop, .~., ~.ingoid, ~. aria ~ae~iciser~, l~. (1~~~T~ i~ipof;~ctio~, a ~igi~.i.y ~t'~gci~,nt, lipid-rra~diat°d ~f~~ transfection procedaxr e. roc Nati cad ~ci SSA g4, ~ ~.~-"74'7.
[~~0] ~izicheportiche, ~., I)ar~aa~~, ~., ~cpein~t~-e, J., ~apra~ra, ~.9 ~ai~ara; ~< aunt Reacia, ~.
~I9~0~ ~igix-performance i°~a~id c.hromatog~°api~~~ anaEysis of ci~°cu~athag insulir~s distinguishes iaeta~ree~a ea~dogenoos insulin proda~ctioa~ ~a potential ipitfai;i with streptozotocin diabetic d ats~ a~ad islet ~eraograft ffunction. ~iahetologia ~~, 457.-~~J.
i'a~e 3~ of 47 Hafie, )can-Pierre ~~~fl~'~;~'~ i~~~~ll~E~l'~~
Please attach your supporting documents belovu i~ the order listed. the supporting docurnQnts seci.ion of your application may ~i1' exceed iD pages, e~ceiuding qn-pr~manuscripts. ~!n addit~On, provide original signed copies of #1-2 via mail (rant Administrator, 3~PPP v~0 Waii St. igr"
Fiooi°J Nevi 1"'ork, ~'Y' 10005-4001, USA).
If you do not have a hard copy of a certain supporking document you must =can the document and paste it in the appropriate section below. ~ti~~w~t~~~~se~s~a~~ ~~~ ei~~t i~~~~aeb~~~~~E, o~iisE~~
~riit9 't' b~ ~ ~te~ ~s,~a~redi~an ~~fi~~bmr~~e~~ ~er~ng~r~ra~~f as~~
die lave established oollaboratiorrs according th.e requirerraenda of ~~ur differer_t projects. In addition, we have responded positively to requests Ior collaborations.
t3~r ourrent JD' grant and tl~e present proposal sterns from a::oll~boratiora with ~r ~,awrer~ce Rosenberg, from l~Ic~ill University, co-applicant.
~e are currently collaborating vTicl~ ~r hierre-~7ves ~enl~a~~nc~u (see letter of collaboration), from tlrenoble, 1~rance, wlo is invol~!ec4 in clinical islet transplarJtaticsr~. a is sypeawising the Frenc;h-Swiss group ~~tt~.CiIL, one of the rrrost active groups worldwide in clinical islet transplantatio:~.
'his collaboration has involved the participation of t3uillaume Jourdan, a graduate student fram Clrenoble, who did his F.Sc. a~~d ~,~as'~er studies iri Clrenoble, is tu~rer~tly dewelopi~~g new islet research tools in Tray laborator<l for one year and w~sll of~acially begin his hl:l;~ in September, in co-direction with both laboratoriPS. 1-Ie wzll spend about ~t~~/o off' his l~h.~. work ira each laboratory. FIe will be in a position to faoilitate the transfer to human islet studies of the tecllraologies we have developed ir~s l~Iontr~al.
~iVe lave recently accepted a proposition for collaboration frorr_~ l~r 1'aui ~e Vos from C'rroningen, 'the Netherlands, one of the most respected experts, worldwide, on islet rnicroencapsulation. ~Je have accepted to load l~airn our device and teaol hirer our technique for rrraising small ~3~(i ~.rra) sxncrooapsules. Ur ~De Vc~s wa.ncs to evaluate or;r teclnologies and compare it to other technologies recently dev~~:Eoped ~y lis group. ~l~r I5e Vos is currently is holiday, therefore we have no letter frorrr lira but he can be contacted: ~~r lie Los coordinates,: ~ a ~ : t ~ 1 ) ( 5 G ) 3 6 ~. ~. 0 ~ 5 fax: (31) (5a) "~51~a11 mail: P.D~.VCSSC~N;;ji7.:~TJG.NL) fast collaboratians include .T'.~r Gerald ~nxd'hon~rne, from ~r~eC7i11 University, patlology depar-i:rnent, a JI3~'-funded researcher, -for the investigation of the: role of cytokines in the hosi:
reaction to rnicrocapsules, ~r 1?i.erre-l0/:ichel Duet, director oi" the .~~ndr~ ~,~iallet :i~esearcl~
centre, Centre Hospitalier de 1'Uriversit~ de IVfontreal-St ~uc, a leader in portal lyp~ertensio~
research, for studies on intra-hepatic rr~~crocapsule irnplantatiorøs aa~d the h3ydro-Quebec electricity research Institute, <or the development of the high voltage electrostatic pulse generators to produce smaller rnicrocapsrrles.
Page 40 of 4y -~alie, Jean-Pierre ~'~~ bmt~ p,ar I B$~1i~6~6$2~~. .._''~OCR9~OTy~G;E 6~'f~:$ fs;9? ~,a= f~2 ~,.°.~_$.~. :v ~~tCF ~'~wLa7CFtlNOl.Ortg_L~tlf3WOl~.'~fC-t~Us°FIYtO~f s!e ~ffltraesur Ssrge HALC~s~
~~RtC3$L.~" FrfePB$BBUP Ft2tfEY~VeS ~~f.~HAItl .. . w._......r Pt~ABt&Eut CINf~r CiIBHR~ 4~~. y ~reffo:~fe, ~egu~r 5~. 2Q~3 I'r 3cun-Pieef~ ~taeBG
'ar'txdee dt r~hCtehe Guy_T3eri,Ser 349 5 bttul. a3e f~4sstsltiptauts, pUtautrdal, ~, Cacouda i-i t "a~' 2?ai i FI4TF:3-SIB-25~-343pr Re : our collnbaretion dear Jean~Pietre, 'l~ti~ 9ntteT 3~ L3esr~ied c~ noaiirm shut our two iat~oratca:ries lv~ve dcvclopc4l a callsbsaratiowort i:~suss xelflted to sxiet trEnsplantatioft. X13 you krlatv, islet ttans~kafttetion stalld9 as ills attain trccgeaeels Loøic tff tiu J~urtment ui'Em3owrint9tagy, Univer>ify Hospital, ~eref~o6te. ror 4 P~ca:~des our i;raup Saes hc~n attvoivcd in both preelitvcal sand ciifrical tc-se~nch inn this aiem. ~linien3 research cor~istc of a rr~alticefatric is3et ts3tL2Dt.~ttlFian pmgrtfrra ~8ih.~r'ing ? Freach tvfiv~~sity hospitals d~r~sstoPsie, Lyon, 9ttasbssurg, ~es~st~tin, LSijnn, Fviarseiitc, ~aatcy, llhonipeSlinr} sort thf: aztftlcrsity hospital of Clcxtcva. Tftis pm~a°am, sailed CiR~GIL and rf:r e:ftear my supcrrision, t<as ~sn cane the tttost ectivc islet txar~pfantstion program in ihc world for slue qast 3 years. '~reclinicat r2:.turcRa :~raa ~ocu~d ssta ties dvag in imprmve isfct viability, mairaty through liar cniuutccm~nt o' aatiinxsdaHat dcfcl9sc mechanisms of the czlls. C3ur grOnyz has teen developing gene tr~P'as~r avc;(tssotp$ics tt9isag 'vitas aftd nnrt vire6 vectors, hue' tin applying il«f: tools fo rite trasfer oFsnii~Oxidetat cnzyiries into ?aumeft pertcr~atic islets. ~Vc llavz, pubiisite'd data ebouC the ~f':.~cs.~ aaF°r_ats~asr. sn~ctoxid.;
disrncatasc attd ~hatcihion 1'~~'oxiciese terse iran~,frr an islet viability.
~.s you koow, we ltavc: rPaare T~ently de~relop~i a coSla~azativn w~itia yuur Icborhtory, through widah we hope to demnewttate the tclev5flcm of such ~~t appresaah of scavenging;
s;r~yrne gene trans~a sat irrrptaving rhea visiritiiy of Fr~icroercepsuteBe~i rstcts, ar9 itf viva stradcls esg aaetyltiaata~2~r. 77tis c.-o112boration has involved the pasticipativn of ene of ou~ bifs'ogy sBudent, ~rraso ttt~tercd in Gr~,nb~, devcloired .sea~erxf 3sPtt freta.,~~alntafiun tools in your L~boratcary i'nt khc iSasE ~~x, and f~c abocit to inltl~ie iris PhD in 6oG'i =escaxcli a~cfisties. considering our experience v~fih Ciinicai islet taarbpigntatian, aFus cu~lavora2ifan wilt agars aacilita4e the transfer to htamesa isntx tsFit~ ;ws~cht,iques yott 3save devalo~d in Montr~ai.
I look forwntd to eat ongoayg olosc coilaboratioa ;with you and your cratlcagues.
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Page ~~. as ~7 H~lie, J~an~~ierre ETIiICAI ACCEPTANCE &rF A1VlfhALvOft!FCtRMf'r~ i~ L'~TH1t3t3E
ItESA8Cf3 E'3 P~AY3eRE 13E
D<te.~OGtT OF T~9~ AtiIIIUIALAEC3fEIit:HE SUR LES
~tiiie CC31LSN14TYc__ &lfsdBfMAeSIC
s~As F~Cf?f Dl! C~9f~l f~ Df: IrP,C'1"E~~1'1~'N
~eCS ANiIWAUae Statement atom the fnstitutioar2~elcrestinn de 1'v~tabllsncmenE
in w6iah the reseaecft mriilau so d3rouieta to recheecfac~
kro partcranod The ,4nfmei Care Committee .atairBisfted hv: i~u Cramitd de protection dea arairnaux r°stablie pear:
~n~re a~~ R~~~f~~~i~~ ~~fy°er~R3er d~ ~'tir~i~ai 9~miso~Rrl~tasre-~%ca~esr~mr!'~
has eramined the research lrrotocal antitied; a ~tudiw .e protocols de rec:herahe intitul~:
Lotrg-rescues press~tean cef' ~-SC1B apoptania and xtessrai>r sty continuous ire vatns sttpp'sy oIP'sF-3Y to tfisnsplanted L,feta, uetng t~~ gene tratss~er v~94h soar a°irra4 rectrcrg~ or co-ertcaspsssiatsors sa9'iafets acrd rsancssatsc daeet Belts.
(Use d~ sumo title as nn the apPlicationi -(Utifkser 1s me9me 2l;ra que nelaSS Indigos sur la demands) submitted by; prc~sent~e par;
i3r' ICiit7-S' iUS i E Y~BIIt' 'blame{51 of aRpiicantlsl as appearing on the a;aplicatianl ti~lnmlsf duldesl c andidattsl tal qu'ils apparaissant sur fa demandef and found the proposed protocolet a oonvenu qua la protoc:n'ta involving proposf: rApond aux animals to meet the standaedsnormes dtablies par to of the Conseil canadien de pro-zection Canadian Council on Animal des animaux (CCPA7, et Cara'GCAC1, que fos installations qui and that the facilities abriteronf tea animaux in which the animals qui sarvirant 8 1'axp6rimantaiion wilt he i,oused and used, cant conformer aux exigerrcos comply uvith the CCAC du CCPA.
requirements.
Date ~ ~~r . ~ , iSignature _...._ .. _~-. 1 ,g at _.
I5r Jean-Pacers Mails __ __ __ _ Dr. Pierre! t7uhe 4AppiicantlsilCandidattsi ~ ~~ linstitutianlsllisp-esantativef {D~l~gu~ de i°Eitabiissementf Pavilron R9ueexanneas~e 5415, bnul. do fAtsompr=.on, 113onlrkat, Qc H7T 2~R~i Tel.: (5147 25?-3dlHl~as4: 3329,'i'ciewpieur: (513) r52~36?0 ~PuvilfonRusetnnnr 5699,bou1.8oscmont,btonetel,Qc HtT 2FI1 Tc.aSrd)2i-3a00poste4959,TEIEcepiGUr:(5143252-3S2!
~~ ~3~ ~~
Naile, Jean-Pierre ~~~fr~p~~~~ fr~rr~ ~~~ ~~~r ~~aaar~~~ a~f ~~r~acs~~aersr~~~ ~~~~n~ung~
~W. ~I~~ S~TP~'~~~'o a~~,~enale I~n~~~~e~ ~'~~a~~~~pa~~g ~~~~~a~~~~~~~~
~~4/~31/2001 Co ~?3;'~I/~~0~
~~~d~~~ ~~~~~~~ ~~s~ac~~~~~~e 0'~/~1/Z~OZ to Q~i:~~r/20~'~
~ag2 4~ c~~ ~?
HQile, ~ear~-Pierre Crxrront grant ~~~eng~~ ~ia~~tes ~o~r~da~ia~a ~rat~rna.~ioua~
~410i12~~I to 03;3112004.
~ic~-artiticial endocrine pancreas: Tie raie a~'panLreati~, ci~ot cc;iis anti grawt~~ ~'acts~rs can maintenance ari~~ prorraatza~a a~r3xicroeraaapsulatesi. islets veil mass arid v~ur~ctis~n Summary Clinical application of microencapsulated islet transplantation remains elusive. One of us (L.~..) has recently shown that incubation of islets witt-a ductal calls, ductal cell conditioned media, or IGF-II has an inhibitory action on ~i-ceil susceptibili~ to apoptosis and necrosis. The nb,~~cfive of this proposal is to design and evaluate methods for improving encapsulated islet cell mass"
survival ana function. Our hypotheses are: (l ) Growth factor (GF), particularly ICF-II, incubations with islets, :z~ould pa~ovide suppert to islets during ex-viva manipulatio:~s, and improve long-term survival. (~) 'I"ransplantation of ductal cells with islets will supply a balanced amount of different GFs :md a limited amount of ~-cell progenitor cells, and positively affect islet call survival and proliferation.
~r°olirnir~ary cpa via',r~a grad it3 viva dai;a support the hypothesis that microencapsulation incuces apoptosis and has a negative effect on islet cell survival and function, that IGF-II incubations have a rescue effect on these parameters, and allow suceess_Ful encapsulated islet transplantation in mice, using a smaller number of isleis.
It was also shown that ao-encapsulation of islets and ducts is feasible. The specifre airres and related rneflaorls are: 1'AItT I. fhe effect of ~F' ~x-vava incubations on eiaa°.alasraIazed islet cell survival aaa,I fuaa~~tior<. Airn 1. To evaluate the °ffect of rat islet encapsulation on the induction of apoptosis, necrosis, cc.Ii survival, and maintenance of the phenotypes and function of islet ce,Ils. At different times before and after encapsulation, apoptosis (TIlNFL assay and other complementary methods) and necrosis (rniv.roscep~y and histologica.I studies) ~~~ill be evaluated. Maintenance of islet poll phenotypes will be evaluated with irnmunohistochemistry targeting islet hormones. Islet viability and function will'be sradied. Aien 2. To evaloa.te the effect of incubations of isleis with C:la, including IOF"-II, IOF'-YS i~IGF', HGF'9 beg gene farot~lr~
as~d 'y'lfF-~z in decreasing susceptibility to apoptosis and necrosis, and improving survival parameters described in Aim 1. Islets will be incubated with the GF under study at different periods during ex-viva manipulations. ~utcome measures: as in Aura 1 + incorporation of thimi~~ine H3 to evaluate cell proliferation:. Alsn 3. To evaluate interactions of GFs with the mic:rocapsule matrix, using reverse; size exolusios, chromatography. f~im ~. To evaluate the effect of ductal cell secretions on islet .suYVival p~aranzeiers, by incubating encapsulated islets with free ductal epithelial cells. Outcome sh;dies: as in dim 2. Airn S. To evaluate, an viva, the effects of GF incubations, using transplantations in non-diabetic and STZ-induced diabetic acid mice. Outcomes measures: 1- metabolic (% mice normalizing blood glucose etc.) 2- survival parameters on recovered islets as in Aim 1. ~A~'T 2. The effect of co-~ne.apsuiatiaan of ducts and islets ors nreeapsulated islet st~r-vival ant function. Usirdg the methods and outcome measures described above, we will rim 6 evaluate the effect of encapsulation on ductal cell, including .viability tess:ing and secretion of IGIr-II and NGF, ~,im 7 optianize the method to encapsulate isieia and ducts, t~irnn 3 evaluate and corr.pare, in vitYO, the effect on surS.~ival parameters, of islets and ductal cysts encapsulated separately vs co-encapsulated, .~i~n 3 evaluate, in viva, transplantation of islets and d;.rcts into non-diabetic and STZ-induced diabetic said mine (to evaluate survival). ~itra 11I evaluate, i3a viva, the methods developed. using transplarktations unto lVOIl mice (to evaluate survival and immunoproteation). in ~onclusio~, the proposed studies will impreve the understanding of the mechanisms invo2ved irr the survival o: encapsulated islets, and will contri'oute to design methods to improve Ieng-term islet cell zr~ass and function.
Papa ~. of 47 ~leii~, .lean-Pierre ~~grent grant ~anadaan ~ial~etes ~ssociativn 117107.1200:; to 0513012~~4 Transplantation of islets encaps~~ated in modified alginate poly-~.-lysine rnicrocapsules The goal of our research is to op'~irr°~ise islet m.icro~-encapsulation tec~~nology to evenarally transplant islets without in~~ur~osuppression. 'tee have developed ed~ods to evaluate the different rr~icrocapsule characteristics and n~ctions. urge sus;lz irrnportant parameter is rnicrocaps=ale resistance. Stror~ge~ rr~icrocapsules will improve the duration of the graft. In addition, broken rnicrocapsules, as ~vel~ as the detachment of the outer alginate sheet, elicit a pericapsular immune reaction. In addition, micxocapsule resistance is a critical issue for tlae encapsulation of virus-tray=_sfected b~.oengineered cells, ~s~hich may prod~zce insulin or rnoleeules designed to promote islet cell survival, as well as fox stern cell derived islets, which may present a risk of malignant transformation.
i~reIlnannary data.
~Ic have developed a novel method for producinb covalently cross~linked alginate-pol;r.-s,-lysine- alginate {cov~l~l~) z~i.ciocapsules compatible with. cell life.
~'he chemical conditions required to break or to create a covalent link are usually ~:r~corrpati~sle with cell life. ~Lecl~anical resistance tests indicated that the neua mic:aocapsulc were 22 fold resistant tlxan standard capsules. The objective ~cas to evalu,~.te isle'a encapsulated ir, these covt~l'~. ale hypothesl~ed that tl~e ne~~~ method vain not negatively affect a~icrocapsule perrrseability arz~i biocornpL~.ti'cility, ~v~~ile rs~aintain ~ng and ever prolonging encapsulated islet cell viabilit~~ and func3:io~-~, a~zd that rnicrocaos~~le str~;ngtl~ will prevent the dissemination of {potentially) rnalin ant cells. The spcvi~c afros and related methods are: to investigate f~a vitro tllae effect of the new n~etl~od ora. b j a~~:rnbr~ne permeability and 2) islet viability and function, ~~.si~~g techz~.idue established in our laboratory, and ~) g~ viva -rnicrocapsule bioeornpatibility. 4.) To evaluate the effect of the :aew x~ethod on i~ vivo isle~~ viability and function by t3~a,nsplancativ~n studies of nncapsulatcd rat islet into :rcl~
{imrnunodeficient) strepto~,otocin-induced diabetic mice {~TZ-soir~). ~) To e~,raluate the effect of the new method on rr~ vivc3 ale; survival ar°ad imrr~~unoprotecrtion, usi~-~g transplantation of encapsulated rat islets into ~'f~-induced ~5'~L~,/6 {~nxnunoco.mpetent) nZice, and ~j on the proteetio~-z againsv'~ dissenainatior; of encapsulated (potentially)malignant cells, by twansplartir~g ~L-~ thyoma cells from C5713i,16.~i'l~
1,2 mice, encap5alated encapsulated in standard and rzew ~:~icrocapsules and not encapsulated, into 1~6.f'L.Tr l . L mice.
~a~nclnsfr~n. encapsulation of islets irz rrcrocapsules that axe virtually indestxarctible within an animal body would alloy,=,~ safe ~°a~'isplantatiori ot° virus--transfected cells as well as stem cells derived islets, urhich rnay present a xisl~ of o malignant transformation.
Page 4S or ~~
f~aa~e, Jean-Pierre a ~i~~r ~c~ ~°y d~~~r~~~a~~~ s~a~a~~rscrlp ONLY I~"-FR~55 ra7ar~uscr ipts ~na~e suber~itted.
r Mer7uscricats afread~ p~~iished sfao~rfd b~ r_toterJ gut caoies shoufd ~~It3T'b~ su~rrsit~ed.
D Manuscripts in~r~~r~tfon or ct~rron~lv rir~tder review ~~r~ ~JQ~' ~cce~tabf~.
L'~'~'i° A~'C3.~~s I~obitaille I~, ~usseaui~.: .~, Flenl~:y 1~1, Rosenberg L arid :~ialle .IF.
Insulin-life growth factor iI allows prolonged i~lood glucose normalization ;pith a rwduced islet cell mass transplantation. ~ndocrgna~I~gy 144- ('1} X1:3"7-3IY~~, 2f~U:3.
Illieva f°~., ~'uan S, ~Varg I:I°~~, Agapitos ll, Hill ~~3 and.
~oser~'i~erg ~,. ~'ancreatic islet cell survival following islet isoiavicn: the role of cell°alar uxteractions in the pancreas. Ja~urnal e~fendacrl~aol~gy 161 : 3~7-36~-, 195~~.
IJeblond Fps, Sirr~ard ~G, F~er~ey 2~, Rocizeleau ~, I-Iuet FNt, ~3~.11~ ~~. -~easibil.ity and safetzJ of intraportal microcapsole irr~plantation ~1 the rat.
~el?L'~'ra~splantatl~n~a ~ : ~'27-337, 1999,.
I~obitaille R, Leblond F~~, Bourgeois Y, FIenley l~T, I,oignon i~T, I-Ialle I~. St~z~dies on small (<35U~,m} alginate prai~~-~,-lysine microcal~sules: ~T-a~eter°ir~ation of microcapsule permeability to carbohydrates and proteins'oy reverse size exclusioa~
c~'zroanatc~graph~.
~ ~I~ed bates SU : ~.2C~~427, 2Gt~U.
Robitaille ft., Leblond F:P., I~Pnley i~I,1'rcd'hon 3rle ~J, I3robet slcy E, I~~,ih 3h. Algiriate-poly-L-lysine biocorrtpatibili~y: a novel f~~'-~'~'I~. method for ~,ytokine gene expression analysis in pericapsular infiltrates. ..~ ~iaeal l~at yes 45 : 223-23U, I999.
Leblond Fry., 'fessier ;~, Iialle 3F'. A quantitative rnetbod for the evaluation of biornicracapsule resistance to rnechanicai stress. ~iaar~ateraa~s 1'~(21~ :
2U9'7-2IU2, 1996.
Robitaille ~:~, ~esbiens K, I~erdey ''~, k-Ialle sue. :~ hne co~°se oI' transforming gt-owth.
factor-~, (T~F-Vii, } mI~T~ e~~pressior: in the host reaction to alginate poly-L-lysine microcapsulesfoliowing irnplantatians into rat epididyn~al fat pads. ~ ~foe~
~I~t yes 52 : I8-23, 2UUU.
Faraskevas S, htaysinger l~, '~l'ang Z., ~uguid , Iiosenbf;rg 1J. dell loss in isolated human islets occurs by apoptosis. ar~creas 2U : 2?U-276, 2UUU.
Rose~nberg L, Wang 12, ~'aras~:evas S, Waysinger ~. Stnzctur~l and functional changes resulting from islet isolation lead to islet cell death.
Snr°ge~°y I2.~ : 33-~g8, I999.
Valang IZ, Rosenberg s ., ~a;rltenance of beta-dill function arid survival folla,~ring i;>let isolation requires re-establisl~zment of the islet-n Matrix :relatianship.
~~~aa~°naI of endocrin~angy 163 : I81-I9U, 1999.
fang I~, ParaslCevas S, liosenberg ~,. ~haracters.satior~ of integrin expression in islets isolated fram hamster, canine, porcine, and h~ama_n pancreas. ~e ,~o~rnal ~sff hflstoclresn.
~ eyt~chexrt. 47 : 4.99-~Of~, I999.
~'aqe ~~ of ~~
i~a!!~, 3ean-P'serre t aras~evas S, Aiki~ ~., ~/tavsnger ~, I.akey I'i'i,, ~avatiagl~. ~w, I-Iering ~, ~t'ang ~., Itosenberg L. Activat~otz and expr~,ssio~ of l:t~K, .l~ anti p38 li~I-l~inase ire isolased islets of IJangerhans : is-:iplications for cultured islet survival. 3"~~~
tet7te~s 455 :, ~03-2~8, 1999.
i~uan S, ~etrakos T', IJugald ~.~~, ~~osenberg f . Isolation an.~~ culture, of intraloba:lsr d:.~cts from the hamster pancreas. il~ vat~-o ~eII a~ae~ ~evet~gnaent~t ~iols~d A,ml~sea~ 31 : 77-80, i995.
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~,e diab~te, d~, typP ~ est one afrecticrn drz systv~~e errdec.rirrier°h se. c~rvct~;risa~~t par ~~ d~reg-ierreent du cc~r~tr~ie et de 1'~.ztitisati~r~ daa glU.cese par 1'~srg~~isr~e. ~.ile tc~~ch~; esserrtieiier~~erit ies ~e~rres avec ~rre incidence de 7 no;~veavx cas par ar°; s~.~r t~C ~~;~ ~;ørsor~r~xes de ~mi~~.s de, 2~ arrs. ~~e~t~; patbelegie est respensabie de ccrrrrplicatierrs cor~rrie : ?'acid~c~t~se, la u~cit~, les nerarepathies p~ripb~riques, is rieplrrepathie et ies cardi~r~y~pathies. ~ es car,rses rural c:.orrrraxes de is p athelegie; irrrpliqra.errt des r"'acte~rs enviror~rrerr~errtaaa;~ et gdn~tiqraes. ~,a pirysi~p~ h~si~gie dv ~ia's~~te de ~~p~; ~ est ~rre destcti~~ des filets de ~,a~gerh~ns paracr~atiq~es par le s~s~w~rrre i~a~~~rritaire sraivie par ~rre ~°~rrnati~~r~ de centres gerrrrinatifs de rn~.t~rati~r~' des i;~r~rphcrc~tes ~ pr~~i~~.ite. ~.es nets ~e ~,angerha;r~s seat des a~~~ras ceia~la.ires c~rrstit~es ~ ~~ ~/~ de ceii~Ies ~ q~i s~rzt resper~sa~~ies de is predr.~ctien d'irrsr;~~ia~e et de celLl~.ies ~x c~a~< pred~ise~rt le gi~cageiz. ~,a dest~~ctierr des ~lo-ts par to sys~:~~v ia~°arrr~r~itaire ird~~it :~a ~erte she sdcrdtierr de ces dea.rx herones i~rp?iq~~es dar3s le c~rrtr~ie ~~e is g3re~r~rie.
~'irrs~iinetherapse est le principal tr-~.itenrer~t a~ ciiab~te ~e type ~.
~ierr q~de p~i~.iati~, ii permet err cerrtrdle de la giyc~r~rie darns la g~r~~errt~ ties cas. ~e;~errdai~At, ii ~~ss~d~: bea~cr~~p d'incerrv~~ients. Il irrrpiiq~e des cerrtr-~.intes q~i a~'fecter~t la q~~irte de vie d~a patient:
des injectier~~o q~et?die~rrres d'irlsrxlirre, iarr cerrtrale de l'alirrrerrtatierr et de p'activht~ p~°~ysiq~e. ~~e p(~.rs, i'ir~s~ii~c~th~rapie rr'arrive pas a~er-g~rasiser la giyc~z~rie de terns les diab~tiques. F~ °est p;~~rq~e:i is trrnsplarrtatitra~ d'~tets de ~,a~gerhar~s ~
ate develeppee err alternative ~ i'ins~~iirreth~rapie. ~,ile a pear ~~ju de precrarer a~;~ trarlsplantes err cerrtrs~ie de is giycer~rie a.d~,pt~e ~.~ bvseirr de i'~ar-g~.rrisrrre, ~~~rrs irr~ectir>rr d'xr~s~iirte, ~,vec. ~rre a~~iieratieal de i~, q~atite de vie ter.~t err spy ~prx.~argt ies cer~pli~;atie~s de sa ~,ia~sie. ~eperadarrt; yes obstacles bieqa~errt is g~r~eraiisatic~n de is tr-arhspiar~tatierr d'ilets cc:~~~rre traiter~rrerrt d~ diab~~te de type ~.
err effet, ~rre trarrsplar~tatierr d'ilets rr~cessi-~e ~ nrogr~s cie~x p;~rrcr~as par receve~r ~~t 1e rrombre de pancreas disperrible est iirrrite par le derr d'ergar~.e. err traiterrmxzt irYar~urres~appresserrr est teu~cr~rs rr~cessaire near s~pprinler la rip~r~~se irrrrrrrarritair a centre Ies 'nets ~;t le risq~e de rech~t.e est rrrecerlrru par le rrrarlq~e de recd des ~t~des. ~e~r ces r~.is~rrrsH bya~cenp de gre~;~ps de recherche travaillerrt sir l' ~;rrr~iier Mien d~ i~retecole de trarrsp i~;r: tatier~ d' T lets.
~,e iab~rateire d~ ~r .~earr-Pierre ~all~ d~.r centre de r~echerche ivy-yer:~ier d~~ i'h~pital l~aisenrre~ve-~eserr.~rrt de l~~errtr~al err fait ~artie. ~e labc~rata~i3e ~t:~die is rrric.reerrc;apsulatierr et i'aa~~Iaer-atien de, to srzrvie des filets peer preteger les nets de -~~angerbar~s centre l~:
systerr~e in~rrxr~rritaire et dirnirruea° le rrerrrbre d'ilets par tr~.rcsplarztatic~rr. ~n~err s~~et s'irrsc~:it darn ce de~~r~i~r~e acre de :~echerch a seit : la protection des hots do ~,a~ager~ans cogR~ur ~ ~'apo~o~-os~ et fa n~c.rosc i~~.r 6a t~ansfcctior~ d~,s ~~;nes de 1'IC~F-I et ~~~~'-I~ darts la transplantation d'~io~a rr~icroer~<apsulds.
~e ~~~emoire expii~ue I'approche rr~~;-ti=odoio,~i~ue et i°s resL~~tats o&~ten~.s au seirl d~j l~.boratoire du ~r ai.ie sur ce suet. ~3ans urge prer~_i~re ~>ar~ie, a r~s~rrlca-ai, par 1'irrterr:~ediaire d'urze rovue de litt~rature, les causes de I'utiiisatior~ d'un aussi ,fir ~.r~d r~c;~~n~re d'iiots par trara::piant~.tie~ra et ies raisons du cl~roix de i'i:~~'-~ et i~-~'-~~ coz~~ne rr~ol~cules i3~-otectrices des ~~oia. Je d~cdr<~i er~su.te ies rechr-~i~iues utiiis~es pour synt~~tiser Ies plasrnides perrnettant i'e~iaressiori des trans~Pnes dares ig~s hots et is wise au point du protocoie de transfection. ~ar~s une tr~isi~r~e; panic, je pr°serr~~,rai ies r~s~zltats ~~tenues dens Ia pr°paration des transg~nes et des protocoies de trarasf°ectioides dots awan~: de discc~ter des tec~r~iques utilis~;es daA=_s cette ~,tude, d~ i'irr°apacz. e.k de ~.a i~oursuite de cute ~tr~de par une i~ese e~~ co~t~telle entre ie Iadoratoire d~. pr I~/Iarie ~'a~arot de i'~ ~~J~~~'~~ er ceiui du :fir ails.
z I
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~,a transplarztatior d'ilots est ur;e alter~~ative a I'i~.s~li~~otl~~rapie dares le traitex~~er~t day slia'~~te de type ~.
far rappor'~ a la ~reffe de pancreas, ells, r~~c;essite ~~e ~; fair~zr~ie zno~~s ionrde sans a~est'r~esie ~~~~rale et a~n protocoie d'irr3rnursEosrappression n~rns fort e~. lirr~iea~t le risqv~~e de a~eactior~ I~~te vo~-sus ~reffon et ~re~~0i1 vers~.IS ~lOt~, C~, °~il~ Cc?I7.Stl~al~e3t Lay ~e~S
avarPtabes. ~.a ~;1.'a~'PSpla~tc'd.txC9(~ d'Ilc~iS ~St d~~lellLle ~.l~e reality clini~~e ~r~.ce aux r~s~altats de 1'~c~uip~ de ~hapiro ~«). ~ls o~~
oi~ter~.us 7 raorn~o~iyc~rraies sur '7 paøients era wtilas~,:~t un protocols d'iig~~~u~osupp~-essior~ r~or~
diab~;a:o~~ne sans corticost~ro~de) et 11~~~3 dots ~qui~.ralents par kilo du receveur. o~epuis, de ~orr~l~re~a~ ~rotoc~les scant ~, I'~tude ~, travers le ~or~de, co~rne cel~ai dca troupe C~«CBI~, ~r~.r~~co-suisse ~~). i~«ais, -la tr~,r~sp«an~:atao~ d'~lots possede ea~core de noaia.hrecases lirnites qui er~p~c~e sa ~;~~~~ralisation, prixmip~,le~~er~t un r~ornbre d'ilots ~lev~ et la r~~cesszte d'ran protocols d'irn~ur~osuppressio~.
~~z pr~leverrFent du pa~cr~as a~.~ co~~tact avec; les tissr~s du recevv;~gr, les dots s~bissent des stress aussi ~cier~ physiques clue chirr~i~ues ~ui yes cond~isent ~ lei moat par 1'apoptose et la n~crose. «_.a coru~a~ssar~ce de ees stress et des g~~o~~~ens poor les di~x~inuer est ,~sser~tielle pour dimiri~aer le rzo~~l3re d'alois par t~~anspla~tatior~.
«.2. «. Stress lip a I'iso«~Ltion et la p~z~-i~c;:~,tioa~ des il~~~rs de I_Aarl~erlZans:
l~'isolatior~ d'ilots de ~,ari~er~ar~s d'~n pa.ncr~as r~~ce ssite des ~t:apes do digestion du pa~.cr~as par la collar ass p~.ais de purificatiorts des Blots. yes grapes ;~or~t respor~:».bles ~;l'apoptose et de r~~crose. ~Jr~e etude realise par 1'~~nipe day r c~s~;n~e-r~; rc~ontre cl:ze 1'~rape ~~I'isola~:ioa~ et de pe~ri~'~catior. des dots conduit ~ I'apoptoso par 1'alteration do ~'int~~;rit~ de lay r~atrice c~~trace«Iulaire ento~raz~t les dots. ~e~:e parts de rnatrice ir~daait i'apoptose de c~x~;~-c:i via les iraf:~;gria~es et is voie G~es ~ ~1~~ ~t p~~ {3) ~4).
'irateractior~ irttercellulaire est aussi i~°,.porxar~te spar la a=ria'oi.Iit~ de;> ilo~a. ~iasi, 1'inter action des rellules d~~ctales pr~sentes dens I'enviro~a~e~n~4;x~t des 3.lots leer procure ~~r~e r~;sistance ~. i'apoptose via des facteurs l~o~-rr3or~au;~ cornrrae 1'~~~-~« ~;t le 1~~~°~~ ~~). ~'a~tres ~ta~.des rr~ette~t erg e~lider~ce i'eff'et pr~-apoptotique et pro-necrotique de 1'isclt=:rnie ~roi~«e et d~ stress oxydar~t au rraomer~~c de 1'isolation des dots par une production de radicaux oxyn~ys~s et de ~rzo~~o~.yde d'azot~; ~T~~ (~~.
~I est irziportant de souli~ner qua le protocols de rr~icroencaps~.l~:~io~ affe.cte aussi la ~iabilit~ cl~;s Mots corr~e (e su~~ere des observations faites dens Ie Iaboratoire du p~- ~--~<~II~. :~, pr~paratio~~
a~:.s dots en vue d.e la transplantation est une steps critique pour Ia viabili.v ~~es ?lots. ~,°~,r~i~Iioration des techniqr;.es de pr~:l~werr~ents et de pr~paratior~s des transplantations ne cease d'~tr~; waiuwe.
~.~.'~. ~,'apoptose et is n~crose dea plots apr~s ~~rar;:aplant~:::ion ~rne fois lee dots transplantes, ce~x~cv writ subir ~.iff~:~ents stress P~espor~sables d'apoptose et de n~crose.
~,a transplantation d'ilots s'accompa~ne d'un pro~:oc:~le ~'irn~n~.~riosuppression, air; on ne peat wiser une ~-~action inflarnrnatoire au site de rrar~splantation. y~ette r eactis;~n ir~~flarnrnatoir a produit des cytokines responsables d'apoptose dens lee flogs. _~es etudes d~nnorttrent qua Ie '~v~,, 1'~~~~~~ et 1°~,-~~ son~t cyto~.oxiques pour lee calluses ~3 (~~. ale ph~sy lee Iyr~~pl~oc~tLs '~~
c~otE~xiques pei~~er~t reconnaitre les slots co~3~rne non soa reacr~on auto-~~~n~~ur~e,~ e~ ~ew~ detr~~~re par le s~rsterc~e fas~ras~. I=,e scherrda =epr~sente lee diff~rentes ~~oies de si~;nalisa~:on. i~~d~:ites par le ::~~st~~ru i~;~-euni~:a=re respo:~s~~bles d~;
necrose et d'apoptose (~~ (~~. ~n pest ~aoir q~~e deux principales voies de si~nalisation sons irnpiiqu~es :
les i~~I~inase (p~~ et .~) qui sons res~or;s~ble de, ~.a syr~these de 1'e:~z~~~te I~~ s~nih~tase et Ia voie des caspases via Ie cytochron~e ~. fans Ie cas d'ilots rnic~-oer~c~~p~ules, il semblerait qua lee rr~acropha~es ~ouent un brand rc~Ie dens la s~.~rv:~~; des dots. ~n effet, une ~t~zde parut clans ~enotransplantation en 2003 rt~ontre des trar~$splantations d'ilots de ~orc encapsules ch:~~ des rats exempts en :macrophages produisent to;~:~o~~~rs de 1'ins;~line apr~s ~i fours, alors qua Ies rats aver rr~acrophages n'en produisent plays de~s i~ fours( I ~;~.
~Jne sutra source de stress est la prod~~ct:Ion de ra~ic~~~x ox~~~ru;y lips a la reperfusion d.es 'ilot:~ ce qui conduit ~~~lerr~ent a de 1'apoptose et de la n~c,rose.
~orrirne noes venous de le voir, Ies ;I~~ts s~a~issent ~r~or~~e~~nent de stress ce q~ai entrains la destrLaction partielle else calluses ~ d~ transplant. ~=~la explique le r~c~g~bre ~Iev~d'~lot;s n~cessaire par transplantation.
i=,es recher~hes act~aelles visent a aa:~cliorer la <<zia'oilit~ et la for!utaonn4~Iit~ en prot~°~ea..~t lee dots de 1'apoptose et de la necrose par la theral~ie ,~~nique po=as permettre I°~~xpression par lee ~;ellules de I'~lor de prgt~ines protectrices. ~l exists diff~rencs choix de prot~ines po~ar,~~hot~~e~- lee dots.
ohm ,~~ ~°~~~~n~~ ~~~x~~~~p~~~~~ t~~~s~s ~s ~~~ ~°~~e~~~ ~s ~~~
~~~~~°
s~~~ ~.~
~,a proteins I~-20 est raaturellernsnt pr~sen~e da~~s les ~~etlules. Soy:
expression est augrr~er~tee par 'I'NI~'-~.
Vaa '_~- . ~.,Tlie ~ftl~e a deinontr~ e;°s~,.~e sa. sL~rexpreSSt~3r1 ai:! Ili~r'ea~ ~.~5 3loCS Ct3x'~i2~"e a ceLtx-C~ ~dl~:
protection fi~,onire I'apoptoss induite ~~:r ~i-I~ et ~'I~i-y. I~~ais ells rye ~on~;~re pas acne resistance contra lc nivea~: ~asaI de I'apoptose {I1).
~a proteir~e ~cl-~ connue po~.ar so~~ b~cti its anti-apo~toti~~e cv~::~I°~r a aux cellulss ~ une resistance i~
I'apoptose induite par les cytolsines Ii-:., '-~ et I~'~1-y et la pe~roxyda~bion lipid.icius (~2).
a proteins ~~~s-3 ciui est respot~sa:~ie ~~a r~crocontrc~le negatil' E~~
signal propag~ par T'~-y et I~-I~3 con~ere uns resistance aux c.slluies ~. I'ax~op~rose induce par ces d~~~ax car,.<ol~ines (1~,~.
I,a prot~ir~e p35 i~.l~ibe les caspases ~ of ~ et possede ~~r~e ar,ti~~it~;
anti-oxy~an~e intrir~s~ci~e. IJJne etude realises sur des cellules ~ rnontre I'ac,ti~,~i~t~ anti-apopt.oticlue de a:e'ce prot~ir~e, mais aussi sore ac~ti~it~
anti-oxydante pa: la diminution L.~ rgc~rn~l~re d'sspeces rec~wti~res de I'oxygene ger~eree par i~~apoxis/reperf~sion (i~~).
iJes enzyrr~es anti-oxydantes et anti-ir~~arnrn~toirvs ~c~,'~ala.se, ~~~':~i~i/:~ ~~I~) haisssr~t la c;ytotoxicite ~~~2I~2 sur les dots et diminuent le cl~~e~otax;srr~e ass x~~acrop~~agss (i ~;.
~s scl~err~a I represents les dif~erer~tss cihles o~~ L'acti~nte de ces proa:eines se si'eue.
~,es ~acte~rs de croissants cor~arne Ies I~~s ont rr~o~~tre acne actin°ite ~~rotectrice anti-apoptotid~ae des $iots. IJ'I~~-I ~loq~ae ?'e~fet deietere cl' i~-I~3 sur la secretion ti'ins~r.~~bne et e~~~eche I'ir~dt~c~:ion de la ~~s et 1'apoptose ~l~j ~.~.. ~. I,e syster~ae des I~~~~s st ses j=onctioras I~iologici~aes ~a r~aille des I~~s est constitues ~,s cleux p rotei~~us i~~-I et I~l~'_I~
d'~~nviron y.5 I~~ cl~ac~ane, de six prote:ines sericiues ~,IC.3-~'~~~y ds haute a~ ir~ite poor les ~~~'s et ds cie~..~x rvcepte~,ars I~~'-l: et I~~'-II ~~7) ~ s ~). L,es I~~'-I et ICIh-II joL~et~t ~r~ ~~le predoa~inar~t dens le cr~pn.tr~le; la proliferation cel.lulaire, de I'apo~atose st de la ~li~f~rentiatior~. ~liea agisscr~t de y=a~;or~
a~xtocrir~.s st p~.racrine sur des differsnts types cell~laires corns : les osteo~lastes ~~~~), ies cellules c~.r~ce3-euses, les cellules ~~yocardidues, ~øs reIIL~les nerveuses et les ~Iot.s de Langer~ans ~~°~y. _~:~es proteines ds liaison ~~es I~l_~s rnodulent I'activite des IC~~'s par ~ar~e action sur la ~ua~?tite d'I~~~b lure:. sanguine et elles p~~:ssedent urge activite intrins~~ue de r~,~zalation de 1'apoptose nor! lick a. la ~zxatiorz des I~:~"s (~J~. I'ar~
oxcl~l~le, sans I~~ lip a lour surface respective, i'I~$F~p-~ augr:aente 1'apoptose alors qz.:e 1'3CIF~~--~ I~, dirninve ('~fl).
f,a I;roduction des I~~s est dep~,z~d~,ntc do 1'~gc. I,'I~'-II cs~: prodz.~ite au rnoz~ent intra-zzteria~ et ~z~:onatal sa production baisse q~elqu~~s fours apr~s la rzaissance. Ellw est rcsponsat~le do la croissance des dots au stack c~'c~ryonnairc, par I'aui;~°ntatiok~ du nonabz-c do ccllul~;s ~ et la s~pprcssio~ de lour apoptose. plusieurs travaux ont decri'~ wes propri~t~s do x'I~~-II
IJes hots des souris transgeniq~.zcs q~,i s=,~rcxpr~rnerat 1'I~~~-.~:I ont ur5e a~agzncr~tation do taill~ de qua'~rc ~ cinq fois ~ la naissaz~~~,c (~ 1 j LTrze ~tzxdc rdalis~e par 1'dqu>.p~~ diz Iyr I~os~,z:~~,r,~ a d~r~csrard q,~c to r~lo ~,n~i-apoptoticlue des cellules ductales sur ies hots do L,angerharts est prir:cipalcz~.c~a d~ ~ la s~cr~tior~ d'I~-~-II (~~.
IJne etude realises par ol~c°z-°~ obitailac au r.aboratozre du I~r ~Tcan-Pierre I-Iall~ rnontrc que 1'a~o~.zt d'I~a~-II au milieu de la culture des slots perrrzeT~: de dir~zin~aer de ~oitr~ 1~:, noz~bre d'ilots rz~cessaire ~ la restauratior d'~ar~e norrr~oglyc~zr$ie (2~.
~.'I~~-I est produ.ite rnajoritairerrzent ~:pr~s Ia rzaissance. Sa prodzzction E;st pul~iquitaire eme si ells est znajoritairen:ent produite air niveau ~~pavique wt ciza'elle est depen~ant~~
notazner~t de 1'l2orrrzone de croissance (Cz~~. Mlle a oirzs d'efn~et: rnitogvne, zxzais possede rz~~
eI°fev anti-apoptotique. I~iffdrerztes etudes mettent en d~~idence les propridtes cle 1' ~_c~-1~-w IJ'I~r-I sti~ule la prolif~ratiof~ cellulaire et la ~L9fferentiatic~:~ du tissu pancz-~atique (2 i)o IJne expression locale d'IC~P-I ~.IZe~ l~;s souris trsnsg~rziques darts ass cellules ~ r~~~rz~;re Ies hots de Iaangez-~arzs et contrecarre ~e ~~ial~ute d~ ?ype I (>4~
IJ'I~~~-I prot~ge les cellules cc~ratre 1~ or'~ celluIaire causc~e pa-r l'absez~ce des nutrients (25;3 .
1.4.2. ~,es ~ecanrsrnes zz~ol~ca~iaiz-es de ~'activitd bioncsgique des I~~' IJes activites biologiqzxes d'I~P-I et d':~P-II soot prirtcipalerrzcnt issues cle leer izxat=on s~;~,r Ie r~cepteur I~F'-I~.J. L,'I~~-Ia une affrzite dia 'ois sup~rieure r~our 1'I~~'-hL.
IJ'It.~~'-Ifs re sernble- pas jozaer un r~sle pans 1'apoptose, il sedlerait qz:~'il contr~~~c ie ~iveau plasz~~sique d'ICI'-II par sa capacitd i~
degrader au. rzi~reau des lysosornes les I~:r~ ix,~s ~ sa su~wace 1i4~ ( a ~y.
IJ'I~-fib. a azne afilrzit~ plus forte pour IMP-I clue pour I~~-II rrernc si Ic,~ deux ~.frzni'v~s du r~ceptezar pour ses ligarzds sort Ple~~es, lr~T°~
et ~nl~~I I~d respecti~errzent. I_,'I~i -lest de t~~pc t~rros~ne kinase et il est actif sons forrr~e de dirr~re de deux lz~terodi~neres (clzalrz.es ~ et ~3~ corzznc le :c~ec;epteu~~ ~
1'i~~sugin~;. IJe sc~~a 2 z-epr~serzte les ~oies de signalisation celhzlaire induite par i'a~,~:iwat<or~ da~z r~ceyteur. ~n c~onstatw qu'il exists trois principaies voies de signalisation corzduisarzt ~ I'inl"~i'~ition de I'apo~~tose et ~ Ia r~ultiplicatioz? cel~~laiFe:
~"~ premiere voie est I'activation de is ~~-3~i~ase et de :~~~ prot~ine ~kt/~h~~. ~et~e vole de sigrialisatiorx inni~ae par p~osp~~or~,-Iatior~ d;~ diff~;rentes prot:~i~e~
;pro-apoptotiques co:nme E,ad, caspase ~ et ~~3 via l'activatiort a -~. ~e pi.~s, i'activati~sn de ~kt/Pir~~duit i'acti~ratior~ de proteines anti-apoptotiques cornb~ae ~ci-~; ~cl~ et des proteinr~s e~Atraceiluiaires ci'adhesion avec is ~ratrice comrr~e ies ir~t~grires.
~,a deuxi~me voie est cede des I~/~W'~iz~~.se via is ~irlase ~1~~~-i/~. ~ette voie est irnpliquee daps di~ferentes ~or~ction s bioiogiques cornrrie is ~~igr;~tio~ celiule~.ire, is dicf~re~-~tiatior~~., a~a.is aussi is protection. coritre 1'apoptose. ~or~ action est lire ~. ~d rr~ais suss ~~
i'i~zni~ait3o~ de Ea voie ,i=qui est pro apoptotiquP via l'activatior~ yes fact°urs de transcription c-:~~~~~, ~~.'i:"~'-~; ~1~-i.
~.-"~ troisie:me voie de sig~,alisato~~z ir~pi-_'que la ~roie caiciurr:
d.~p~;ndante. ~.e~~e voie conduit ~
l'activation de ~acteur de trar~scri,~tio~t Evrxti-apoptotique con~.~x~~e i~C~'~'~ et C~:E~ qui augments d'e~pression de ~cl-2.
Les id's poss~de~t arse actavite a~~ti-apoptotique irxdependante du r~~.epte5ar ~~i~-~~. ~ette activite provier~t du clivage de I'T~~'-i; par use protease s~~°ique qui p~wd~azt ~r~ tri peptide aux propri~t~s protectrices sur les raeurories rlotarrme~-.-~ daps ies ~~od~':es des mata.dies de; ~r~~atirbg~orz et de ~a~~cirao~.
~a~m ,~s ~~~~~s d~ ~e~°~~~.
es celiuies de ~ertoti sort 1es cei"s_ui~~s p~~og~~.itnic.es des sperrnr~=o~o~c.es. ~~ies soot recor~~ues prsur secr~ter des facteurs de croissance corr~rn~° I~~-~; ~'~~~-~ et ~~~j, ~:~ais aussi corn~~~ae prod~ctrice des fac~te~:rs id~~ri~nomoduiate~rs comrrm gas ligar~d. yes etudes orn i~vestig~~~
leur utiiisation dares is trar~spiar~tatior~ d'iiots en pr~parant uvt site irn~~uraoi~a~ivil~gi~ pcq~~r is traa~spiantatio~ des i'iots. yes approc~es ont montre que ie pr~traite~~~;~~t par des ceiluies de ~ertc~:':a du siE:e ~$e trar~splar~tation augrrmnte de de~x a quatre ~ois is dares de is sur~,rie des hots vrar~spiant~;~ (v-nod~ie de ror~geur aver 500 dots ~quivaients par transpiantatioi~~ ~2d~. ~.~ co-cuaure de c;°Iiuies de ~~ertoli g~vec; des Hots de rats augmer~te de hu=t ~ois is proli~'~ration des celiuies ~ e,t de deu~ ~'cg~s is produs~tis~r~ d'i~zsuline ~~'~~. 'iJ~.e autre ~t~gde rnor~tre, que la co-encapsuiatiorl de c°i uies de ~ertoli avec dots ~~e poissor~g augmeb~te de dcux sois Ia sur~ne des dots transpiant~s erg ai~SSer~ce d'irnm.ur~osuppresseur ~trarxspl~ntatiorl x~rzog~niqice cilez ie rongeur~ ~2~~.
~ar~s noire ~eude, nous alior~s travaiiier aver is iign~e ~M~ de ceii~ie.. de Sertoii recor~r~u pour ~tre r~oa~
turr~orai et produire ies facteurs protec,te.rs et imr_rur~o~~~odulateur ~1~~.
~.. ~~~~~~ .~ ~.~~~~ ~~~
~,~, dut do ootte etude ~,st ~'a°cnelior~.ti~:~~ du protooole; d~
tr~.z~spi.~i~t~tion d'itots per l~ dim<~~tior~ du norc~'c~re d'iiots tr~raspiantds r~e~ess~ir~: ~ 1'o~t~r~t4o~ d'u~a~
norruo~I~c,~i~. ~ous penso=~s riue i~, trar~sf~ctio~i des hots ~vec ~~~'-I ou ~~:.~~-~~ oL~ t~us d~,~~ ~,o~f~r~ra ~~~~ xlots urre prot~otio~ ~or~.tre les stress induits pa.r i~ r~i~roencapsuiatio~~, ins ~~~to~~r~es ~t i~
r~per~~~sio~c. ~,~s o~jectii"s 4r,~is~s dares c~tto etude s~roa~t:
L~ ~re~,tior~ d~ voctoui-s euc~r~~otos c~p~bi~s d~ tr~.rlsferer ses ~eA~es murirss d~s ~.~~s dins los ooliui~s dos dots do ~a~~er~~r;f,.
~,'~taade do i'~xpressioa~ du tr~,r~s~~r~~ ~t do i'ide~~tit~ d~ is p~°at~sr~~ socr°~td~.
~~. rrlis~ ~.u poi.rgt d'ura proto~;o=o cio tr~rzsf~~tior~ dos hots ngo ~~.rrr~~rh~r~~s de ~~or~~our perrr~ett~rrt ur~o vxpressi~r~ dos prot~it~os su.~~as~rit~, pour pr~~t~~~r ins iic~t s.
~.I.I. ~yntiZesc do I' ~~c de i'I~;x~-I a-0; IC~~-Is.
2.1. i.l. ~'xtraction d'~;~i total (T~Tt) do :~oi~ do so~ris f,e prete~r~~ner3t d'un fragment d° ~~ic do sourrs se fait irna-~a~,diatc~r~cnt apres cxsang~~ination do i'arlirnal. L.e. tissu est rapidemer~t cor~ge~ dares de 1'~~ote iiq~iq~e.
~,'E;~traction d'.I~t des cell~ales ~~pati~~es se fail a froid par ho~~og~ ~~isatio~f dens i rni cie 'f~.~~al_, (Tr~~itrogen ~~ ) pour I~7~ ng de tiss~. ~,'ho~Aog~nat est incul?e ~ :~:r~an~~tes ~ ternp~r~trare pied: a~~ant i'e~traction de i't par piaenoi/c~Iorofarme (ratio 25 :~~ :i). I_' 't est precipite pay- un uoiur~e d'isopropanci pals-ia~e dens de i'ethanob 7~%. T,'.~Tt est ~r~aiem ent dissout dens de 1'~~ ~:raitee au I~~~C et conserve ~ -20''x.
IJa cor~cuntratior~ et la pa,arete do i'l~Ta; do i'~,c~aantiilo~ sons ar~aly~.ees p6~,r spectrop:~oto~~etrie ~ 260 nn (~~I, ~T~I) et ~~~ nm (proteincs) et par eiectropi~orese sur geI d'garo~;e I
~/o. ~,a cualite de i'isolatio est caracter~see par is presence des ~an~Ies des r''~Tr ~.~s et ~.~s sur gel d'electrop~orese.
~,. i.I.~. ~ynti~ese de l'~~~';.~~ rornpiez~aPnt~=ire (~~~I~TC;~ par ~:~-~~R
~'~lt est trar~scrit en ~c par u~ie, reaction de n~'i'-~~ su~~rar#t i~~
protocolc d.'Iz~~ritroger~~. ~n bre 1.5 ~g d'~t est denature pe?~dac~t I~ r~~ir~utes ~ 6~°~ en presence de IQ nT~l~ de dI~T~'~ et des a~~orces poly 'T'. I_,'appariernent des a~~~~rces s~ar i'~;~t se fait en ~;iiieu tamponne ;pendant ~ n-~inutes a 4~°~. ~,a reaction de poiyrnerisation esr catalyses par ka ~up5:,r~~It~-T(Invitroger~~~ 5~ Minutes a ~-2°~. ~,'arret de is reaction se fait par i'~nacvi~rat~on de ~a poiymera.:;e ~ 7C? °~ pendant I5 r~j.nutes.
2. i.2. selection et ampii~~,a:~io:~. des t=~:L~T~Ic :~e i'I~-~~'-I eg II par ~~~
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~,e diab~te, d~, typP ~ est one afrecticrn drz systv~~e errdec.rirrier°h se. c~rvct~;risa~~t par ~~ d~reg-ierreent du cc~r~tr~ie et de 1'~.ztitisati~r~ daa glU.cese par 1'~srg~~isr~e. ~.ile tc~~ch~; esserrtieiier~~erit ies ~e~rres avec ~rre incidence de 7 no;~veavx cas par ar°; s~.~r t~C ~~;~ ~;ørsor~r~xes de ~mi~~.s de, 2~ arrs. ~~e~t~; patbelegie est respensabie de ccrrrrplicatierrs cor~rrie : ?'acid~c~t~se, la u~cit~, les nerarepathies p~ripb~riques, is rieplrrepathie et ies cardi~r~y~pathies. ~ es car,rses rural c:.orrrraxes de is p athelegie; irrrpliqra.errt des r"'acte~rs enviror~rrerr~errtaaa;~ et gdn~tiqraes. ~,a pirysi~p~ h~si~gie dv ~ia's~~te de ~~p~; ~ est ~rre destcti~~ des filets de ~,a~gerh~ns paracr~atiq~es par le s~s~w~rrre i~a~~~rritaire sraivie par ~rre ~°~rrnati~~r~ de centres gerrrrinatifs de rn~.t~rati~r~' des i;~r~rphcrc~tes ~ pr~~i~~.ite. ~.es nets ~e ~,angerha;r~s seat des a~~~ras ceia~la.ires c~rrstit~es ~ ~~ ~/~ de ceii~Ies ~ q~i s~rzt resper~sa~~ies de is predr.~ctien d'irrsr;~~ia~e et de celLl~.ies ~x c~a~< pred~ise~rt le gi~cageiz. ~,a dest~~ctierr des ~lo-ts par to sys~:~~v ia~°arrr~r~itaire ird~~it :~a ~erte she sdcrdtierr de ces dea.rx herones i~rp?iq~~es dar3s le c~rrtr~ie ~~e is g3re~r~rie.
~'irrs~iinetherapse est le principal tr-~.itenrer~t a~ ciiab~te ~e type ~.
~ierr q~de p~i~.iati~, ii permet err cerrtrdle de la giyc~r~rie darns la g~r~~errt~ ties cas. ~e;~errdai~At, ii ~~ss~d~: bea~cr~~p d'incerrv~~ients. Il irrrpiiq~e des cerrtr-~.intes q~i a~'fecter~t la q~~irte de vie d~a patient:
des injectier~~o q~et?die~rrres d'irlsrxlirre, iarr cerrtrale de l'alirrrerrtatierr et de p'activht~ p~°~ysiq~e. ~~e p(~.rs, i'ir~s~ii~c~th~rapie rr'arrive pas a~er-g~rasiser la giyc~z~rie de terns les diab~tiques. F~ °est p;~~rq~e:i is trrnsplarrtatitra~ d'~tets de ~,a~gerhar~s ~
ate develeppee err alternative ~ i'ins~~iirreth~rapie. ~,ile a pear ~~ju de precrarer a~;~ trarlsplantes err cerrtrs~ie de is giycer~rie a.d~,pt~e ~.~ bvseirr de i'~ar-g~.rrisrrre, ~~~rrs irr~ectir>rr d'xr~s~iirte, ~,vec. ~rre a~~iieratieal de i~, q~atite de vie ter.~t err spy ~prx.~argt ies cer~pli~;atie~s de sa ~,ia~sie. ~eperadarrt; yes obstacles bieqa~errt is g~r~eraiisatic~n de is tr-arhspiar~tatierr d'ilets cc:~~~rre traiter~rrerrt d~ diab~~te de type ~.
err effet, ~rre trarrsplar~tatierr d'ilets rr~cessi-~e ~ nrogr~s cie~x p;~rrcr~as par receve~r ~~t 1e rrombre de pancreas disperrible est iirrrite par le derr d'ergar~.e. err traiterrmxzt irYar~urres~appresserrr est teu~cr~rs rr~cessaire near s~pprinler la rip~r~~se irrrrrrrarritair a centre Ies 'nets ~;t le risq~e de rech~t.e est rrrecerlrru par le rrrarlq~e de recd des ~t~des. ~e~r ces r~.is~rrrsH bya~cenp de gre~;~ps de recherche travaillerrt sir l' ~;rrr~iier Mien d~ i~retecole de trarrsp i~;r: tatier~ d' T lets.
~,e iab~rateire d~ ~r .~earr-Pierre ~all~ d~.r centre de r~echerche ivy-yer:~ier d~~ i'h~pital l~aisenrre~ve-~eserr.~rrt de l~~errtr~al err fait ~artie. ~e labc~rata~i3e ~t:~die is rrric.reerrc;apsulatierr et i'aa~~Iaer-atien de, to srzrvie des filets peer preteger les nets de -~~angerbar~s centre l~:
systerr~e in~rrxr~rritaire et dirnirruea° le rrerrrbre d'ilets par tr~.rcsplarztatic~rr. ~n~err s~~et s'irrsc~:it darn ce de~~r~i~r~e acre de :~echerch a seit : la protection des hots do ~,a~ager~ans cogR~ur ~ ~'apo~o~-os~ et fa n~c.rosc i~~.r 6a t~ansfcctior~ d~,s ~~;nes de 1'IC~F-I et ~~~~'-I~ darts la transplantation d'~io~a rr~icroer~<apsulds.
~e ~~~emoire expii~ue I'approche rr~~;-ti=odoio,~i~ue et i°s resL~~tats o&~ten~.s au seirl d~j l~.boratoire du ~r ai.ie sur ce suet. ~3ans urge prer~_i~re ~>ar~ie, a r~s~rrlca-ai, par 1'irrterr:~ediaire d'urze rovue de litt~rature, les causes de I'utiiisatior~ d'un aussi ,fir ~.r~d r~c;~~n~re d'iiots par trara::piant~.tie~ra et ies raisons du cl~roix de i'i:~~'-~ et i~-~'-~~ coz~~ne rr~ol~cules i3~-otectrices des ~~oia. Je d~cdr<~i er~su.te ies rechr-~i~iues utiiis~es pour synt~~tiser Ies plasrnides perrnettant i'e~iaressiori des trans~Pnes dares ig~s hots et is wise au point du protocoie de transfection. ~ar~s une tr~isi~r~e; panic, je pr°serr~~,rai ies r~s~zltats ~~tenues dens Ia pr°paration des transg~nes et des protocoies de trarasf°ectioides dots awan~: de discc~ter des tec~r~iques utilis~;es daA=_s cette ~,tude, d~ i'irr°apacz. e.k de ~.a i~oursuite de cute ~tr~de par une i~ese e~~ co~t~telle entre ie Iadoratoire d~. pr I~/Iarie ~'a~arot de i'~ ~~J~~~'~~ er ceiui du :fir ails.
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~,a transplarztatior d'ilots est ur;e alter~~ative a I'i~.s~li~~otl~~rapie dares le traitex~~er~t day slia'~~te de type ~.
far rappor'~ a la ~reffe de pancreas, ells, r~~c;essite ~~e ~; fair~zr~ie zno~~s ionrde sans a~est'r~esie ~~~~rale et a~n protocoie d'irr3rnursEosrappression n~rns fort e~. lirr~iea~t le risqv~~e de a~eactior~ I~~te vo~-sus ~reffon et ~re~~0i1 vers~.IS ~lOt~, C~, °~il~ Cc?I7.Stl~al~e3t Lay ~e~S
avarPtabes. ~.a ~;1.'a~'PSpla~tc'd.txC9(~ d'Ilc~iS ~St d~~lellLle ~.l~e reality clini~~e ~r~.ce aux r~s~altats de 1'~c~uip~ de ~hapiro ~«). ~ls o~~
oi~ter~.us 7 raorn~o~iyc~rraies sur '7 paøients era wtilas~,:~t un protocols d'iig~~~u~osupp~-essior~ r~or~
diab~;a:o~~ne sans corticost~ro~de) et 11~~~3 dots ~qui~.ralents par kilo du receveur. o~epuis, de ~orr~l~re~a~ ~rotoc~les scant ~, I'~tude ~, travers le ~or~de, co~rne cel~ai dca troupe C~«CBI~, ~r~.r~~co-suisse ~~). i~«ais, -la tr~,r~sp«an~:atao~ d'~lots possede ea~core de noaia.hrecases lirnites qui er~p~c~e sa ~;~~~~ralisation, prixmip~,le~~er~t un r~ornbre d'ilots ~lev~ et la r~~cesszte d'ran protocols d'irn~ur~osuppressio~.
~~z pr~leverrFent du pa~cr~as a~.~ co~~tact avec; les tissr~s du recevv;~gr, les dots s~bissent des stress aussi ~cier~ physiques clue chirr~i~ues ~ui yes cond~isent ~ lei moat par 1'apoptose et la n~crose. «_.a coru~a~ssar~ce de ees stress et des g~~o~~~ens poor les di~x~inuer est ,~sser~tielle pour dimiri~aer le rzo~~l3re d'alois par t~~anspla~tatior~.
«.2. «. Stress lip a I'iso«~Ltion et la p~z~-i~c;:~,tioa~ des il~~~rs de I_Aarl~erlZans:
l~'isolatior~ d'ilots de ~,ari~er~ar~s d'~n pa.ncr~as r~~ce ssite des ~t:apes do digestion du pa~.cr~as par la collar ass p~.ais de purificatiorts des Blots. yes grapes ;~or~t respor~:».bles ~;l'apoptose et de r~~crose. ~Jr~e etude realise par 1'~~nipe day r c~s~;n~e-r~; rc~ontre cl:ze 1'~rape ~~I'isola~:ioa~ et de pe~ri~'~catior. des dots conduit ~ I'apoptoso par 1'alteration do ~'int~~;rit~ de lay r~atrice c~~trace«Iulaire ento~raz~t les dots. ~e~:e parts de rnatrice ir~daait i'apoptose de c~x~;~-c:i via les iraf:~;gria~es et is voie G~es ~ ~1~~ ~t p~~ {3) ~4).
'irateractior~ irttercellulaire est aussi i~°,.porxar~te spar la a=ria'oi.Iit~ de;> ilo~a. ~iasi, 1'inter action des rellules d~~ctales pr~sentes dens I'enviro~a~e~n~4;x~t des 3.lots leer procure ~~r~e r~;sistance ~. i'apoptose via des facteurs l~o~-rr3or~au;~ cornrrae 1'~~~-~« ~;t le 1~~~°~~ ~~). ~'a~tres ~ta~.des rr~ette~t erg e~lider~ce i'eff'et pr~-apoptotique et pro-necrotique de 1'isclt=:rnie ~roi~«e et d~ stress oxydar~t au rraomer~~c de 1'isolation des dots par une production de radicaux oxyn~ys~s et de ~rzo~~o~.yde d'azot~; ~T~~ (~~.
~I est irziportant de souli~ner qua le protocols de rr~icroencaps~.l~:~io~ affe.cte aussi la ~iabilit~ cl~;s Mots corr~e (e su~~ere des observations faites dens Ie Iaboratoire du p~- ~--~<~II~. :~, pr~paratio~~
a~:.s dots en vue d.e la transplantation est une steps critique pour Ia viabili.v ~~es ?lots. ~,°~,r~i~Iioration des techniqr;.es de pr~:l~werr~ents et de pr~paratior~s des transplantations ne cease d'~tr~; waiuwe.
~.~.'~. ~,'apoptose et is n~crose dea plots apr~s ~~rar;:aplant~:::ion ~rne fois lee dots transplantes, ce~x~cv writ subir ~.iff~:~ents stress P~espor~sables d'apoptose et de n~crose.
~,a transplantation d'ilots s'accompa~ne d'un pro~:oc:~le ~'irn~n~.~riosuppression, air; on ne peat wiser une ~-~action inflarnrnatoire au site de rrar~splantation. y~ette r eactis;~n ir~~flarnrnatoir a produit des cytokines responsables d'apoptose dens lee flogs. _~es etudes d~nnorttrent qua Ie '~v~,, 1'~~~~~~ et 1°~,-~~ son~t cyto~.oxiques pour lee calluses ~3 (~~. ale ph~sy lee Iyr~~pl~oc~tLs '~~
c~otE~xiques pei~~er~t reconnaitre les slots co~3~rne non soa reacr~on auto-~~~n~~ur~e,~ e~ ~ew~ detr~~~re par le s~rsterc~e fas~ras~. I=,e scherrda =epr~sente lee diff~rentes ~~oies de si~;nalisa~:on. i~~d~:ites par le ::~~st~~ru i~;~-euni~:a=re respo:~s~~bles d~;
necrose et d'apoptose (~~ (~~. ~n pest ~aoir q~~e deux principales voies de si~nalisation sons irnpiiqu~es :
les i~~I~inase (p~~ et .~) qui sons res~or;s~ble de, ~.a syr~these de 1'e:~z~~~te I~~ s~nih~tase et Ia voie des caspases via Ie cytochron~e ~. fans Ie cas d'ilots rnic~-oer~c~~p~ules, il semblerait qua lee rr~acropha~es ~ouent un brand rc~Ie dens la s~.~rv:~~; des dots. ~n effet, une ~t~zde parut clans ~enotransplantation en 2003 rt~ontre des trar~$splantations d'ilots de ~orc encapsules ch:~~ des rats exempts en :macrophages produisent to;~:~o~~~rs de 1'ins;~line apr~s ~i fours, alors qua Ies rats aver rr~acrophages n'en produisent plays de~s i~ fours( I ~;~.
~Jne sutra source de stress est la prod~~ct:Ion de ra~ic~~~x ox~~~ru;y lips a la reperfusion d.es 'ilot:~ ce qui conduit ~~~lerr~ent a de 1'apoptose et de la n~c,rose.
~orrirne noes venous de le voir, Ies ;I~~ts s~a~issent ~r~or~~e~~nent de stress ce q~ai entrains la destrLaction partielle else calluses ~ d~ transplant. ~=~la explique le r~c~g~bre ~Iev~d'~lot;s n~cessaire par transplantation.
i=,es recher~hes act~aelles visent a aa:~cliorer la <<zia'oilit~ et la for!utaonn4~Iit~ en prot~°~ea..~t lee dots de 1'apoptose et de la necrose par la theral~ie ,~~nique po=as permettre I°~~xpression par lee ~;ellules de I'~lor de prgt~ines protectrices. ~l exists diff~rencs choix de prot~ines po~ar,~~hot~~e~- lee dots.
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~,a proteins I~-20 est raaturellernsnt pr~sen~e da~~s les ~~etlules. Soy:
expression est augrr~er~tee par 'I'NI~'-~.
Vaa '_~- . ~.,Tlie ~ftl~e a deinontr~ e;°s~,.~e sa. sL~rexpreSSt~3r1 ai:! Ili~r'ea~ ~.~5 3loCS Ct3x'~i2~"e a ceLtx-C~ ~dl~:
protection fi~,onire I'apoptoss induite ~~:r ~i-I~ et ~'I~i-y. I~~ais ells rye ~on~;~re pas acne resistance contra lc nivea~: ~asaI de I'apoptose {I1).
~a proteir~e ~cl-~ connue po~.ar so~~ b~cti its anti-apo~toti~~e cv~::~I°~r a aux cellulss ~ une resistance i~
I'apoptose induite par les cytolsines Ii-:., '-~ et I~'~1-y et la pe~roxyda~bion lipid.icius (~2).
a proteins ~~~s-3 ciui est respot~sa:~ie ~~a r~crocontrc~le negatil' E~~
signal propag~ par T'~-y et I~-I~3 con~ere uns resistance aux c.slluies ~. I'ax~op~rose induce par ces d~~~ax car,.<ol~ines (1~,~.
I,a prot~ir~e p35 i~.l~ibe les caspases ~ of ~ et possede ~~r~e ar,ti~~it~;
anti-oxy~an~e intrir~s~ci~e. IJJne etude realises sur des cellules ~ rnontre I'ac,ti~,~i~t~ anti-apopt.oticlue de a:e'ce prot~ir~e, mais aussi sore ac~ti~it~
anti-oxydante pa: la diminution L.~ rgc~rn~l~re d'sspeces rec~wti~res de I'oxygene ger~eree par i~~apoxis/reperf~sion (i~~).
iJes enzyrr~es anti-oxydantes et anti-ir~~arnrn~toirvs ~c~,'~ala.se, ~~~':~i~i/:~ ~~I~) haisssr~t la c;ytotoxicite ~~~2I~2 sur les dots et diminuent le cl~~e~otax;srr~e ass x~~acrop~~agss (i ~;.
~s scl~err~a I represents les dif~erer~tss cihles o~~ L'acti~nte de ces proa:eines se si'eue.
~,es ~acte~rs de croissants cor~arne Ies I~~s ont rr~o~~tre acne actin°ite ~~rotectrice anti-apoptotid~ae des $iots. IJ'I~~-I ~loq~ae ?'e~fet deietere cl' i~-I~3 sur la secretion ti'ins~r.~~bne et e~~~eche I'ir~dt~c~:ion de la ~~s et 1'apoptose ~l~j ~.~.. ~. I,e syster~ae des I~~~~s st ses j=onctioras I~iologici~aes ~a r~aille des I~~s est constitues ~,s cleux p rotei~~us i~~-I et I~l~'_I~
d'~~nviron y.5 I~~ cl~ac~ane, de six prote:ines sericiues ~,IC.3-~'~~~y ds haute a~ ir~ite poor les ~~~'s et ds cie~..~x rvcepte~,ars I~~'-l: et I~~'-II ~~7) ~ s ~). L,es I~~'-I et ICIh-II joL~et~t ~r~ ~~le predoa~inar~t dens le cr~pn.tr~le; la proliferation cel.lulaire, de I'apo~atose st de la ~li~f~rentiatior~. ~liea agisscr~t de y=a~;or~
a~xtocrir~.s st p~.racrine sur des differsnts types cell~laires corns : les osteo~lastes ~~~~), ies cellules c~.r~ce3-euses, les cellules ~~yocardidues, ~øs reIIL~les nerveuses et les ~Iot.s de Langer~ans ~~°~y. _~:~es proteines ds liaison ~~es I~l_~s rnodulent I'activite des IC~~'s par ~ar~e action sur la ~ua~?tite d'I~~~b lure:. sanguine et elles p~~:ssedent urge activite intrins~~ue de r~,~zalation de 1'apoptose nor! lick a. la ~zxatiorz des I~:~"s (~J~. I'ar~
oxcl~l~le, sans I~~ lip a lour surface respective, i'I~$F~p-~ augr:aente 1'apoptose alors qz.:e 1'3CIF~~--~ I~, dirninve ('~fl).
f,a I;roduction des I~~s est dep~,z~d~,ntc do 1'~gc. I,'I~'-II cs~: prodz.~ite au rnoz~ent intra-zzteria~ et ~z~:onatal sa production baisse q~elqu~~s fours apr~s la rzaissance. Ellw est rcsponsat~le do la croissance des dots au stack c~'c~ryonnairc, par I'aui;~°ntatiok~ du nonabz-c do ccllul~;s ~ et la s~pprcssio~ de lour apoptose. plusieurs travaux ont decri'~ wes propri~t~s do x'I~~-II
IJes hots des souris transgeniq~.zcs q~,i s=,~rcxpr~rnerat 1'I~~~-.~:I ont ur5e a~agzncr~tation do taill~ de qua'~rc ~ cinq fois ~ la naissaz~~~,c (~ 1 j LTrze ~tzxdc rdalis~e par 1'dqu>.p~~ diz Iyr I~os~,z:~~,r,~ a d~r~csrard q,~c to r~lo ~,n~i-apoptoticlue des cellules ductales sur ies hots do L,angerharts est prir:cipalcz~.c~a d~ ~ la s~cr~tior~ d'I~-~-II (~~.
IJne etude realises par ol~c°z-°~ obitailac au r.aboratozre du I~r ~Tcan-Pierre I-Iall~ rnontrc que 1'a~o~.zt d'I~a~-II au milieu de la culture des slots perrrzeT~: de dir~zin~aer de ~oitr~ 1~:, noz~bre d'ilots rz~cessaire ~ la restauratior d'~ar~e norrr~oglyc~zr$ie (2~.
~.'I~~-I est produ.ite rnajoritairerrzent ~:pr~s Ia rzaissance. Sa prodzzction E;st pul~iquitaire eme si ells est znajoritairen:ent produite air niveau ~~pavique wt ciza'elle est depen~ant~~
notazner~t de 1'l2orrrzone de croissance (Cz~~. Mlle a oirzs d'efn~et: rnitogvne, zxzais possede rz~~
eI°fev anti-apoptotique. I~iffdrerztes etudes mettent en d~~idence les propridtes cle 1' ~_c~-1~-w IJ'I~r-I sti~ule la prolif~ratiof~ cellulaire et la ~L9fferentiatic~:~ du tissu pancz-~atique (2 i)o IJne expression locale d'IC~P-I ~.IZe~ l~;s souris trsnsg~rziques darts ass cellules ~ r~~~rz~;re Ies hots de Iaangez-~arzs et contrecarre ~e ~~ial~ute d~ ?ype I (>4~
IJ'I~~~-I prot~ge les cellules cc~ratre 1~ or'~ celluIaire causc~e pa-r l'absez~ce des nutrients (25;3 .
1.4.2. ~,es ~ecanrsrnes zz~ol~ca~iaiz-es de ~'activitd bioncsgique des I~~' IJes activites biologiqzxes d'I~P-I et d':~P-II soot prirtcipalerrzcnt issues cle leer izxat=on s~;~,r Ie r~cepteur I~F'-I~.J. L,'I~~-Ia une affrzite dia 'ois sup~rieure r~our 1'I~~'-hL.
IJ'It.~~'-Ifs re sernble- pas jozaer un r~sle pans 1'apoptose, il sedlerait qz:~'il contr~~~c ie ~iveau plasz~~sique d'ICI'-II par sa capacitd i~
degrader au. rzi~reau des lysosornes les I~:r~ ix,~s ~ sa su~wace 1i4~ ( a ~y.
IJ'I~-fib. a azne afilrzit~ plus forte pour IMP-I clue pour I~~-II rrernc si Ic,~ deux ~.frzni'v~s du r~ceptezar pour ses ligarzds sort Ple~~es, lr~T°~
et ~nl~~I I~d respecti~errzent. I_,'I~i -lest de t~~pc t~rros~ne kinase et il est actif sons forrr~e de dirr~re de deux lz~terodi~neres (clzalrz.es ~ et ~3~ corzznc le :c~ec;epteu~~ ~
1'i~~sugin~;. IJe sc~~a 2 z-epr~serzte les ~oies de signalisation celhzlaire induite par i'a~,~:iwat<or~ da~z r~ceyteur. ~n c~onstatw qu'il exists trois principaies voies de signalisation corzduisarzt ~ I'inl"~i'~ition de I'apo~~tose et ~ Ia r~ultiplicatioz? cel~~laiFe:
~"~ premiere voie est I'activation de is ~~-3~i~ase et de :~~~ prot~ine ~kt/~h~~. ~et~e vole de sigrialisatiorx inni~ae par p~osp~~or~,-Iatior~ d;~ diff~;rentes prot:~i~e~
;pro-apoptotiques co:nme E,ad, caspase ~ et ~~3 via l'activatiort a -~. ~e pi.~s, i'activati~sn de ~kt/Pir~~duit i'acti~ratior~ de proteines anti-apoptotiques cornb~ae ~ci-~; ~cl~ et des proteinr~s e~Atraceiluiaires ci'adhesion avec is ~ratrice comrr~e ies ir~t~grires.
~,a deuxi~me voie est cede des I~/~W'~iz~~.se via is ~irlase ~1~~~-i/~. ~ette voie est irnpliquee daps di~ferentes ~or~ction s bioiogiques cornrrie is ~~igr;~tio~ celiule~.ire, is dicf~re~-~tiatior~~., a~a.is aussi is protection. coritre 1'apoptose. ~or~ action est lire ~. ~d rr~ais suss ~~
i'i~zni~ait3o~ de Ea voie ,i=qui est pro apoptotiquP via l'activatior~ yes fact°urs de transcription c-:~~~~~, ~~.'i:"~'-~; ~1~-i.
~.-"~ troisie:me voie de sig~,alisato~~z ir~pi-_'que la ~roie caiciurr:
d.~p~;ndante. ~.e~~e voie conduit ~
l'activation de ~acteur de trar~scri,~tio~t Evrxti-apoptotique con~.~x~~e i~C~'~'~ et C~:E~ qui augments d'e~pression de ~cl-2.
Les id's poss~de~t arse actavite a~~ti-apoptotique irxdependante du r~~.epte5ar ~~i~-~~. ~ette activite provier~t du clivage de I'T~~'-i; par use protease s~~°ique qui p~wd~azt ~r~ tri peptide aux propri~t~s protectrices sur les raeurories rlotarrme~-.-~ daps ies ~~od~':es des mata.dies de; ~r~~atirbg~orz et de ~a~~cirao~.
~a~m ,~s ~~~~~s d~ ~e~°~~~.
es celiuies de ~ertoti sort 1es cei"s_ui~~s p~~og~~.itnic.es des sperrnr~=o~o~c.es. ~~ies soot recor~~ues prsur secr~ter des facteurs de croissance corr~rn~° I~~-~; ~'~~~-~ et ~~~j, ~:~ais aussi corn~~~ae prod~ctrice des fac~te~:rs id~~ri~nomoduiate~rs comrrm gas ligar~d. yes etudes orn i~vestig~~~
leur utiiisation dares is trar~spiar~tatior~ d'iiots en pr~parant uvt site irn~~uraoi~a~ivil~gi~ pcq~~r is traa~spiantatio~ des i'iots. yes approc~es ont montre que ie pr~traite~~~;~~t par des ceiluies de ~ertc~:':a du siE:e ~$e trar~splar~tation augrrmnte de de~x a quatre ~ois is dares de is sur~,rie des hots vrar~spiant~;~ (v-nod~ie de ror~geur aver 500 dots ~quivaients par transpiantatioi~~ ~2d~. ~.~ co-cuaure de c;°Iiuies de ~~ertoli g~vec; des Hots de rats augmer~te de hu=t ~ois is proli~'~ration des celiuies ~ e,t de deu~ ~'cg~s is produs~tis~r~ d'i~zsuline ~~'~~. 'iJ~.e autre ~t~gde rnor~tre, que la co-encapsuiatiorl de c°i uies de ~ertoli avec dots ~~e poissor~g augmeb~te de dcux sois Ia sur~ne des dots transpiant~s erg ai~SSer~ce d'irnm.ur~osuppresseur ~trarxspl~ntatiorl x~rzog~niqice cilez ie rongeur~ ~2~~.
~ar~s noire ~eude, nous alior~s travaiiier aver is iign~e ~M~ de ceii~ie.. de Sertoii recor~r~u pour ~tre r~oa~
turr~orai et produire ies facteurs protec,te.rs et imr_rur~o~~~odulateur ~1~~.
~.. ~~~~~~ .~ ~.~~~~ ~~~
~,~, dut do ootte etude ~,st ~'a°cnelior~.ti~:~~ du protooole; d~
tr~.z~spi.~i~t~tion d'itots per l~ dim<~~tior~ du norc~'c~re d'iiots tr~raspiantds r~e~ess~ir~: ~ 1'o~t~r~t4o~ d'u~a~
norruo~I~c,~i~. ~ous penso=~s riue i~, trar~sf~ctio~i des hots ~vec ~~~'-I ou ~~:.~~-~~ oL~ t~us d~,~~ ~,o~f~r~ra ~~~~ xlots urre prot~otio~ ~or~.tre les stress induits pa.r i~ r~i~roencapsuiatio~~, ins ~~~to~~r~es ~t i~
r~per~~~sio~c. ~,~s o~jectii"s 4r,~is~s dares c~tto etude s~roa~t:
L~ ~re~,tior~ d~ voctoui-s euc~r~~otos c~p~bi~s d~ tr~.rlsferer ses ~eA~es murirss d~s ~.~~s dins los ooliui~s dos dots do ~a~~er~~r;f,.
~,'~taade do i'~xpressioa~ du tr~,r~s~~r~~ ~t do i'ide~~tit~ d~ is p~°at~sr~~ socr°~td~.
~~. rrlis~ ~.u poi.rgt d'ura proto~;o=o cio tr~rzsf~~tior~ dos hots ngo ~~.rrr~~rh~r~~s de ~~or~~our perrr~ett~rrt ur~o vxpressi~r~ dos prot~it~os su.~~as~rit~, pour pr~~t~~~r ins iic~t s.
~.I.I. ~yntiZesc do I' ~~c de i'I~;x~-I a-0; IC~~-Is.
2.1. i.l. ~'xtraction d'~;~i total (T~Tt) do :~oi~ do so~ris f,e prete~r~~ner3t d'un fragment d° ~~ic do sourrs se fait irna-~a~,diatc~r~cnt apres cxsang~~ination do i'arlirnal. L.e. tissu est rapidemer~t cor~ge~ dares de 1'~~ote iiq~iq~e.
~,'E;~traction d'.I~t des cell~ales ~~pati~~es se fail a froid par ho~~og~ ~~isatio~f dens i rni cie 'f~.~~al_, (Tr~~itrogen ~~ ) pour I~7~ ng de tiss~. ~,'ho~Aog~nat est incul?e ~ :~:r~an~~tes ~ ternp~r~trare pied: a~~ant i'e~traction de i't par piaenoi/c~Iorofarme (ratio 25 :~~ :i). I_' 't est precipite pay- un uoiur~e d'isopropanci pals-ia~e dens de i'ethanob 7~%. T,'.~Tt est ~r~aiem ent dissout dens de 1'~~ ~:raitee au I~~~C et conserve ~ -20''x.
IJa cor~cuntratior~ et la pa,arete do i'l~Ta; do i'~,c~aantiilo~ sons ar~aly~.ees p6~,r spectrop:~oto~~etrie ~ 260 nn (~~I, ~T~I) et ~~~ nm (proteincs) et par eiectropi~orese sur geI d'garo~;e I
~/o. ~,a cualite de i'isolatio est caracter~see par is presence des ~an~Ies des r''~Tr ~.~s et ~.~s sur gel d'electrop~orese.
~,. i.I.~. ~ynti~ese de l'~~~';.~~ rornpiez~aPnt~=ire (~~~I~TC;~ par ~:~-~~R
~'~lt est trar~scrit en ~c par u~ie, reaction de n~'i'-~~ su~~rar#t i~~
protocolc d.'Iz~~ritroger~~. ~n bre 1.5 ~g d'~t est denature pe?~dac~t I~ r~~ir~utes ~ 6~°~ en presence de IQ nT~l~ de dI~T~'~ et des a~~orces poly 'T'. I_,'appariernent des a~~~~rces s~ar i'~;~t se fait en ~;iiieu tamponne ;pendant ~ n-~inutes a 4~°~. ~,a reaction de poiyrnerisation esr catalyses par ka ~up5:,r~~It~-T(Invitroger~~~ 5~ Minutes a ~-2°~. ~,'arret de is reaction se fait par i'~nacvi~rat~on de ~a poiymera.:;e ~ 7C? °~ pendant I5 r~j.nutes.
2. i.2. selection et ampii~~,a:~io:~. des t=~:L~T~Ic :~e i'I~-~~'-I eg II par ~~~
retie reaction est catalyses par i'e~r~~e ~1e ~autp f'ideiite, la ~r,~ p;~iy~-nerase (T:r~v~.trogen z~;. L.es sec~c~ences des arnorces sort dessinees ~ par$ir des se~~ances coda~o,des proteines I~~' r~vrir~es p~;~bliecs sur z< gcnbank » (~aexe ~ et 2). ~,a pr~;gra.rr~J:~~ation de i'appareii ~'~est is sui~antc:
9 c"~~~~A~ ~~
I
~Wre~.l~~..Il~~.il~.~ ~ 1'l~l~a~"d.~~'~' '~ ~lp~'~G.S '~, _--I .~''~~ ~ __- '~~ "r,~.
-rp~~~W~.~ . ~ ~ ~ s~~ond~J ~. ~~~. ~vJ~nd~ S ~,. .__ ' x l, r ~,~ ~Lt~~~' .
i ~~ ~~~~~5 I iTliil~k~~ ~ ~~J.~%~. ~ ~'S~ s~,-"..~'oil~~5 ~~p~~ri~rtl~~~~~ ~, ~~ .~a''~.
I
1 rCtlni.HCe c7 'L~~'~ ~ ~ 12zy3':Y~~~
~~IO~yJr~~2"lS~$loll'~ c3 ~~~
__--_ _ 'i ~Q ~~
3~~ ~ ' r~f:~IS ~ ' ~i~ii ~ ~
~~ ' r ~ YY2i31L3 e ~ r :~
. 2s c~ Yi~n~x~L
e x-- ,s ~
<~
- ~
~ ~~.i?le c~~s prod~its d~ ~~~s~ ~o~i~~-~le~, par ~;l°~;~wrop~orese d~
~/~~ d'~i ~rod~~t de ~~sir rev d'~.~arose ~~/~.
. ~~on~.~~ da,ns <o ~~~~~~i:~ ~'~~p~ess9on pro~~,r~~c~~~ p~"~~ ~.~ (v~ ono shot ~~,~' d'~iiv~tr~~en~):
~,~ ~~c~e~r p~~. i poss~dk; ~n ~~n~ d~ rcvsay~n~o ~ ~'~~~i~i'~io~:Aq~~
l~~~i~n~~r~ino y~ ~n ~~ne d~: ~
ba.lactosadase. ~,~ proto~o~~ so fait erg ~:r~~xs ~~~~;es ~r~e posy ad~n~~a3~or d~ ~'o~~rka~r~i~e 3' ct~s proda~irs de ~'~J. per t'kr~:~zyrn° '';"aq polY~~n~r~se ~~n~,~i~rogenC~~ p~nd~.~~ I~ rrait~~~~~s ~ i'2°~ s~a~o~i~ d'~..~~~o p~ra~~~~~ion par p~~e>~olloloro~or~e ~2~ :~~ :l~ e~ d'~.~n~ rnise ~n s~sp~~~s;o~~ d~ '~.~1~dins aE ~'z~~~a~~~.
~~ ~t~~~iC9i'i e'~~ ~~ins~~ ~~°~ ~e SWC~~~,~~'° S~ 1°Si~
~~1 r~~.li: ~ :i a~(a ~°~ _~u : ~r~~~~tir~ ~ 1~°~ p~nd~n9. i6~7 ~t ~s~ ca~~~ys~~ par ~~ '~4 ~1~1~ ~~~~~s~.
ro- ~ ~S ~7rot~LlItS Ci~ ~1~a~2~n song '~i"~'~S~'~i3r~'n~S k's~elS ~~5 ~~~~~;rl~,s ~~i:I'i~9v~6~n~~a ~~~~fle.~~'' par ~r1 G~1C5C
~a~ern~~q~e d~ 3C se~ond~s a ~~2°~:. ~z:ir~s dep~~ ~~~ ~0 ~,~ ~~a ~,~
d~: ~~.o~er~~s ~r~.nsfor~~~os es~t r~~~is~ sa:~r des p~tr~s d'~bar ~ont~~°~an~ d~: rr~k~~~~ d~ ra~~aN~z-~ s~~~,~~~~~~~~
~~~n~.~~~d;gr~~ (~~~ritro~~nJ) ~~ gala~tos~
~~~i'ri~ro~~ri~~. ~,es p~~r~s sore gn~~ cps p~nd~~~~ ~ ~ ~°~~~r~s ~ ~ 7 '~ ~~ra.~~t 1e ~gr~l~~r~i~nont dos oolc~nies ~a~~;~ri~'iTl~'S ~7~an~~eS ~u~Cpl2~S ~3 ~~,~~C$:C~Sia:aas~ C~,~ICI~"n~S ~'~:
f~~3'1a~'~~Cir9k~ rk~S>s~~a'i~s~ q~i 5~,1'~311~
~m~a~i~ies par l~r~~na ~r~ps et l~a~~ ~'~ps.
~.~.~. ~~p~if~c~~ion ~~ ~,~~r~o~ion Los ~T p~asrna~iq~~,s ~~o~~r~ens 1_~ri~ pr~~
Ps co~on~~s b~an~h~s pre~~v~es d~ ~~~:xi ,sor~,~ r°z~~s~n~ ~n ~o~~~r~
~;~ns ~ n~~ d~ ~~ ~o~~ta~i~ 5~ ~,~/t~a~ cue ~anarnycin~ perd~.n~ ~~ ~i ~ 37°~ soaps ~~~ita~~o~i. ~Jn ~~oa~.irne do ~..~ z~i~ :~~ ~;~~~~xre: b~.~~~,r~o:~n~ est r~c~~illi pod.dr edgy oxtrairo i'I'~ s~ivad~t le prc~to~oi~ do ~~i~i ~r~p ~~~~3~.~~.
~~~s serios d.o di~estifld~s par les enzydnos do restriction sordt realises sur los _''.~I'~ oxtraits air do selecticsnnor les ~Ior~es posiri~s ~''~'2?l;~IdaF3t -~'ddls~'r'~ d~ bO~lrl~' tadll~ ~.~: a:~~ l>°, ~ioz'?
s~'TdS. ~,C,s Clo;~z~~ r~~.orEl.?;~ ~C3i i~~'tlo p~3sdtd~s Serollt arr~pli~ es par i~faxi Prep. ~,e ~ol~dne ~.~ ba~ter3e; r~stad~;: sera ~on~~~ze ~. -~~°~ dens 1 ~ '% dp ~ly~,erol.
l~.~axi Pr op ~.J~a ~rolmn~ do o~lt~ar~ hacteri~d~ard~ do i~!~iG~i Prop do 5(~~ p,l sort d'~r~oc~l~i~ ~C~O r~l de ~~ I~ardam~JGirde 50 p,~/dnl. t~.pres ~ 6 l~ ~. +3 i'°~ sops a~itatiot~ ~'~~fl r p:~r~), l' ~ plas'rrdariyao baot~raod~ ost extrait s~zivad~t to protocol:. Q~~ ltor 1'~axi Prop do ~~~~~,I'~ ~z . ~,a pu~-ete of ?~w ~~.~ad~~rite d' 1'~T sort a~.alyseos par spectropllotometrio ~ 26th dzdn of 2~~ r~3r ~. ~,a r~at~.dre dos plasr~ides ,rut ardalyseo par ~ar~o serio do digostioTds avoc les eydnos ~coof P~~~.
~. ~.~. ~dzso~qor~ dos sed~,ao3~~;os c=~dad~t d'~_~P-~ of d'~_~~~°-~~
daTds rare octo~r d'oxprossion c~caryoto (pS ;,lontocl~0~
-~o ~octo~.dr p~ZE~ possedo t~d~ ,keno do re,sistad~c;o ~, l'a~~dpic~lii~do ~c~df'~~ro bacted-ionrdo~ o>~ ~ ~a ~eodnycidm ~c~lt~dro daa~ds ceil~go od~caryote;. f~v ~~octo~.d~ a la partic~larite dE~
possedor ~d~o sed~eco h~ ~~d~tra ~~bosodne ~r~tor ~o~~er°bco~ ~t~i porrr~er~ l'oxp-f ossiod~ d~: doddx ~ed~os poor r d1 ~ne:rd~e pr ord~ote~r. Plusze:~rs plas~~.idos sort cod~stridats poor cotta e~:9~.do : p~C.ap'-~~-, p~~~-~:~, p~,~ ~:~~-3~--.~~iF'-~~ of p~ ,~ ~~'P. Los protocoies utilises poor lo~dr c~~dstr~ctior~ sort los r~~dn4s, solos ios di~,osriod~s ~.i~~°erod~t.
'~x~tractiorz dos l'~c dos ~~~~~s d~ oc~o~xr ~~y~;~. ~ :
~,a prodnie~~o di~estior~ a lio~~ ~. 37°~ roT~dardt ~ lao~.dro dads ø~
~l ~bo ~olE~rr~o -g;actio>~~el avec i'o.r~~ydne ~.~i d~ecessiro to tadnpor~ sali~ to pl~.d. ~aiblo. ~,'i~<activatior~ do l'or~mydno ~~st rea?iseo ~c~ ~drd~~tos ~ ~5°~.
~,a do~z~err~e di~ostiod~ se Fait da.ns ~drQ vol~~d~do do l~ ~l po~~ddarft I
~o°~~-e ~ 3~°~' a~oc l'ajo~xt do ~ gz1 d~
tadnpon do la d~o~~ello or~ydne do eli~os~ioy~ ~ ~'abloa~~ 1 ~.
P~ri~ catior~
es prodcaits do dibost:iords soot: sepa~:es par eloct3oporese spar Col d'a;arosv ~~/~. ~,os brigs d'I~
c~rrospod~dad~dt a~ax seer erdces d'idZteret sort ~:=ra:.lo~es sops ~.T~~.
~.',~~I ost p~rifde do l'a~arose par des billos I!-X s~i~ra~~: to protocolo do ~'~~~~-~~I'~r,~. -~,'~~.i3~d est ea~~c~~iLo el;~e par ea~~or~odzt d~ pdens d~ iris i<-~~~~ p~.~.
Il ~lgaG~ora ut arr~pltfaoat~~~~ ~~a~~tor~or~r~o ~o ratio voof~~r irss~r~ est cle ~:3 po~~r 30C~ ~~ cl~, voo~~:~r dares 20 ~,1 ~c, vol~~~o reactionnol (~cP~erna 3).
~,a r~a~tior~ ~~rP I~ 11 ~. +4°~ avar~~ ~'~tro ar=rates liar ina.ctiva~iori ~o Ia ~1'~-~~ Pigas~ 'T~ (.Invitrogen~) por~da~~t ~(3 rr~ir~a~tes ~. 65°~. Un p~oto:,c~3o clc; t3 ar?sforr~:a~cio~ pui=P clo l~:ar~i ~t Maxi i~rops rlar~s 3~~P et 2~OmP de ~,~ asnpicilline 3Ofl ~~,~r?~l (Ir°avitro~~rC~) gist realise ~f~~ d'obteynr yes q~.~arltit~s ~'h~
s~f~sar~tes ~o~r son anaPysc et soy vtri~isatio~ a~~s los protooc~ies ~.~~
trar~sfaoti~~r~. ~,a naturo tPos insorts ,gist v~rif~~~ par dos serios de digestioi~~s av~c l~s ~a~~~~rr~~a ~o rostrio~c~or: l~o~W, ~fiair~t seq=~~~n~ago.
~.~.6. ~eq~or~~a~~ par a~roato SIC&~~:
~,a pr~paratior~ ci~ 1'~ p~azr is s~;q~otl~a.gp r~~oo~~-sito ~~o a~r~:~.plif~oation p ar ~~C. d~ ~~4~ r~~
pPasi~e. r.Jno ~~~ ost realiseo po~zr c;P~aqi:o grin pa.r l~s a~~orc;o se:r~s et rov ~'~~~Ii-~ of ~G~-I~. ~ os conditions sort fixos : 2 rnirr~t~s ~, q~°~~; ; ~~ fois :~~ s~oor~~os ~
q~~°~, I5 seconcles ~, ~0°~ ; ~ a~inut~s ~.
6~°~. ~:,o pro~~it clc P~~ est ens~zco pr~oipit~ ~.ar~s ~e '''~t~~anoP
~:~ ~/~ + acetate; ale so~i~.~rr~ p~~ ~.6 avargt ~'etre sol~~ilise ~aras c~~ tapo~ for~~.arr~ide ~~~ r~~ so~-rant a~.z secl~,:>~.:~;~a~o. ~e seq~on~~.bo ost realise a 1' ~xteri~ur ~~u. Pal~or atoir~.
~~G ~~~~v~~ ~e~~~~~°~
2.2. I. isolation d'ilots ~o ~:ar~~orlxa~s ~~ so~~ris °t clo raga 2.~. ~ . I. ~PZir~.r~i~
~~s s01?r~S sol~$ SavrIfI~~S avaY'at P~ p~'~rW '~~~~rxv ~~i~ ps°s.~~r ~e~s. ~~r~ r a'~s sO~l~: o~'~" owls aed Sot~~~iO~o~
saori~es pa.r iraoisior~ ~~.~ cc~~r apres a.voir c;a~~~i~, P~
ohoPe~oq~°. ~,d~s ar~ia~ka~~: so~~: incises ~. partir d~ leas 0 1'aucl~rr~o~. p~is en <t ~I ,> j~scP~'v~. ~aa~~: ~::~ =u~l~~i-c~ ~~
rr~ar~ie;~-o ~ acc;eclor a~.~ foso, a.~ panoreas ~t 1'ostoir~ac. IJcs visoeros sort alors r~froi~ios oar ~~~o solution d'~a~~ffore~ ~alir~~; ~ol~tior~ (I~,~3~5) froicic avart la Pi~at~ro (so~zris) o~ io '~~.Ia~.~p" (rats) d~ oPzoPf~oq~o a~
~~ivoa~ c~~ duocler~~a~. ~,e ~P~ole~oq~o gist alors oar~~~le ~. P'a.iclo '~rto aiill~ ~o ~~p (so~aris) o~.
cl'~ne oa~~~le (rats) pc~~r p~rrr~octro la p~rf~sion par ~n~ sohxtion de colia~e,nas~ ~ ~ 1 tn~,In~P ~~ ~~~ p'~.4 f~-oicie ~~ pancreas j~sq~'~, sa.
~.istznsion. lv.e pancreas cst aloes, prelo-ve ~r i~nr~~e~zatc:~~er~t ~~is ~a~zs ~:~~~~ soP~.~ior~ froido de ooPla~er~aso ~:~ sir blacc. ~,a c~irur~ie ~oit etro fait t~ plus rapi~e.~~onv possib~~ poor obtenir ~rgc i~onne vialailite des IPots (cn rnoyonrle s~ so~ris o~ ~ rats).
l 2.2.1.2. ~i~estion du ya~~~;r~as ~~. di~estiora est rea.lisee pendant 3C~ rr~in3~tes ~. ~?°~ ~,~~ivie dune agitation d~ l~ seoonde sons vortex.
Mlle est arr~t~e par une serge de lava.~.l dens du a'SS ~roid pule :°~..SS + IO °/a ~~tax ~ovi~ae S~r~~rr~
~~~5~ ~roid a 3~0 x g pendant 2 rr~ir~~e~,s. ~,e di~estat est e~s~ite ~l~zd ~
travers deux tarr~is de ~QO~.rr~ et %St~~m.. ~ a fltrat eontenant Ies ~lc~s est a~ors p~arif.~ ~~ tiss~.as aoir~e~ax payf ~r~ Uradient de F'-:eoll d' ~uroeollir~ (~. t~~, I.~95, 1.Q69, ~.~~~°~~. .tea co~.e~3e eontena~zt Ies dots est ens~;ite Iav~e du ~ieoll par du.
ASS +IQ ~/~ ~'~S event d'~tre rngse e~ ea~ltawre dares ~i~ Ci.~ 1~~~ +y~.fl ~/~ I:~~S ~'~~co~~npl~ment~ (3 t1 rr~irg~tes a -_~~°~~. ~,a suspension d'~~ots est eo~t°e e~s.
r~o~n~re d'~lots ~~ival~~nts ~~ ie~ = aS~J ~,rn de dia.etre). ~,a pLaretd peat-~tre ar~~rne~~t~e par seleetion ruar~oeile po~~r atte5.ndre plus de ~3~~/~ ca'llots.
2.2.2. ~~r~eranon de celi~~es ~ :
yes hats sort r~d~iLs en cellules par iner~l~ation pen8aant ~. rninvtes ~
~'~°~ a~reo ~r~e solution de ~_~S
~. ~ I~ eontenant ~.2~°/a (v/v~ de t~-~aps:~ne et 2?~ d'~I:~~'~..
t,'arr~~t de Ia ~°~aot~.c;n es'~ r~~lasde par I'ajout dune solutiorg froide de ~~S content ~.6 rngi~n~ de Soyl~ea~~ ~.r;rps~ne ~~al~i~ite~r ~S~'~I;~. ~a suspension eeilulaire est a.lors vortexes pendant ~ seoorades avan't iw lavabe et ~rriise en oulta.~re a 3'~°~':, 5~/~ C~~2 dons du ~'i~ tt365 eo.plet + ~~' ~/Q ~~S d~oo:~ple~~~:ent~.
2.2.x. ~ult~re de Ia Ii ;nee u°~ de eellvle sie Sertoti (~~ ~~'~~;~
sell lys-~e~
~,es eellules 'I'~~t de Sertoli sons ouit~v~es clans d~.~ r~~~Iieo ~-l:i~I~ ll +~ °~3 horse Serc~r~z + 2.5 ~/o _~5. ~,e rn~p.ieu est eha.rPbe tC3di5 lee ddux a, arro~s o~.=rs po~~
~err~e~Lre pan paSSa~e y~a.r Sea~ne. ~.,eS
oelluges sont tr~sir~~es et reensernene~es lorsciu7elles ~:.ttei~net~t ~~
~/° de oonfg~~enee. ~La dilution est de 1:25 entre Ia r'lasq$.~e rr~~re et Ies r'~as~ues ~Ines. ~,e surplus de o~:~I~~ies est oo~npt~ sun one eellule de ~ala.ssei~ au ~Ieu de trypan et eorP~eid dens use solut:ion de ~iliev~
os~n~~Iet eonter~ant ~ ~/~ de ~1~5~
dens I'a~ote. ~,a d~oon~~latior~ est rapide par 1'~,~out des ~,eFlutes dee.c,r~~elees ~. +:~7°~ dens un volurrle de r=~aliea~ ec~~nplet pr~al~blernent ?neub~ ~. :''~ ~ 5~/a ~~'2 p,u~darPt ~~
~ni~~utes.
l~
2.2.x. i°rotocole de tr~.r~s;~.~:.=.ioi: des spots ~a transfection est r~alise~ avec di.i I~a~ ' et l~, plasrnidc pCly~i~ po~:x~
la i~:rs~ a~ point. I~i~~ereratos C~~%r7,C~i~c''S~i2s d~:. trar&S~a°,C$iorl sOnw. ~.'SS~e)!C'.~."S:
'~'tdl~~C dE", la trailsl'~.'~t.tl4Jl~ ~J~, ~., ~~.; 'G~. 11~.L"si'..r-'S~, rr'3ti0 Z~1/s~~~' ~ ~i:3, ~:~, I:~, ~:~, 1::~ :2~, ~~~.ra~rt~ d'I~a ~:~, ~, ~, ~ 2 ~~i;, pry incubation ~:ies dots event Ia traiasfection ~fl, 3, 9, 2~- hearse). Iso~r ~flfl icy dens ~n nolo.rrie de 1.
~~i dry rr~ilif~ia de c~lt~are corriplet, tee conditions id~ales sort ~r~ ration 4: i '~~t : I~~ ~ ~~~y ~~bd' preparation I~~~~~'~a' F h~~,~r~ a~.%a~at la e:~ansl'cctioii ~l~i~s d~
tr~ili~v~ corr~pL~t sans s~n~ir tJrse p cV'.battor~ ~t~ ~ ~.~~r°s, d~~ ~~~<~ts ap-~Y~,s ~so~aLz~a~., ~~ar~t le ~~~~~t de la trai~s~'~c-tio~
J11C tr ai~s~C..Cti(7r1 i$~ G~ ~~lCka.~'.rCs C%l ', C.-'sCl'~;:a:, ~, SCr~~li ~ tau;~ de trar~s~ection est anal~~s~ par a~rr~pt~:~~c de l~~aocscoi~co d~
~~'~ a~ i~~roscop~ a.
~h~or~sc~nc~. i=,a ~ia~ilit~ doe dots tra~s~~ct~;~ gist oar~ti~~c par ~.i~
~t~st a 1'acridiiz~ ~ran~e ~r iodurc do ~ropidi'~,3?~.
2.2.x. ~'ransF~ction des c~ll~~ics ~'1~~~ de ~e~-~oli :
~,~s celluieu 'T1~~~. de Sertoli sons trarss~cct~es ~ j'fl ~i~ de eor~~i~ci~cc per d~~~ ~~~E~r~~6/pI~-~~-II. i~~~rcr~tes conditions oi1$ dte cssay~es : co~accr~tratior~s en ~~< ~'~ ratio l~/'~j~~onc&. I.,e protocolo do tr~.~s~cctiora a.~a.ret donne lee rneillc~rs r~s~lt~.ts ost : ~~:r~ ratio i:6 1~T/~'~~~rk~d pour 1 1.~~ d'r~~~t dens 2 gel de ~~ili~~.~ do c~lt~.rc a~ec du ser~~i~ p~r~da.rt 2~ ~~c~.tr~ . ~'~.nal~s~ d~
lctrans~'~ctic~:!~ so pit a~ microscope a ~l~orescence. pour 1'expression de ~~~~~. ~'~t~.de de la ~x_-ot~i.no I~~'.-II
est r~alis~e par I~ot ~3lot si-it 20~3~.1 de 2 i~l de rnilie~ de culture corr~p:et l~r~le~~ ~ u~ours a~:~~s la pransl~ctio~..
2. i .7. got plot ~,e rriilie~ de cu?t~re des c~ll~les tra.~°~.s~ec;$~es esi cei~tri~.z~~. ~. i 2 ~~~~flfl ~ ~. i=,e s~arria~ea.r~t ~sr recueilli et cc~nser~~ a, - ~fl ~. 2t3t~ ~,l de milie~~ es~~ d~pos~ s~,ar one r~~~rnlsranu d~ nitroceilulc~se. IJ~. i~,em~rane est traitee pendant ~ l~oure 30 r~zinutes a~ec. r~ anticorps ~~riinaire anti-I~~-I
o~ a~~ti-I~~'--II d~ iapin, dilu~
au z : iflOfl. ~~is, on ~~azt one it~c~x~atiox~ p~:ndan$ i ~uxv ~fl rr~inut~e.s a~rec ~r~ a.n.ticorps s~cor~daire (dil~.e x:iflOflfl~ couple ~. ~~r~e pert~~~%ds,se. ~a~tre c:da.p~e ir~c~l~atior~ p~zr .l~s anticorps pri~~iaires ~t l~
s°cor~dair~s, des series rte ia~a~e so~~ r~alis~°~~ ~~,r ci~.~
~~~~ ~.G:~'%., ~.C~~~ % '~~~ox~, ~.~ ~~o ~~f~. ~,~.
r~v~iatio~ se fait far o~ia~iiur~ines~,er~oe aveo to pit ~I'~~r~3ers~a~~~. ~ a si~~ai i~ir~eux est er~svite ~~tect~ far ia~~ressio~ sir zz~ ~il~~ ~~oto,~ra~i~i~,~v s~.iva~t ~iffs~~er~t s ~e~s pie ~or~taot. ~Jne a~aa~yse ~ensitornetzic~ue est faite poor a~~ro~<i~~~er fes ~,~a~tit~4~ ~e g~roteine,s.
~5 o~~ ~~tes~ d~s ~~v d~s s~~~~;~~ ~~d~~~~s df ~~~ ~~-:~"~:~
~.l.l ~xtractior~ de 1'1~~~L d;.~ ~oio dsouris ~'o~jcctif set 1'~.xtractior~ d'!=~.~'~'~ t co~tc~,~r~t lcs ~rt~ d~ 1' i:~~-~
Lt ~~~F'-~i ~srese~~ts au a~ivcaaa taepatidus. ~cux-ci r~ou.s scrvir~o~~s do ~asc ~ la sy~tla~;so do 1' I°~c ~,s s~q~c~ccs cod~.r~t dcs ~~F. ~,a fi~ur~ l ~-epr~sente lc r~s~ltat do #'~lcctropl~or~s~ ds dsux prod=nits d'cxtr:~.cti~r~s d'2~Tt fans sn parail~le. ~r~ cor~state la pr~so~cc d~. ;~ou~T prig-~cip~les l3ar~dos d'a~I~
~, ~ Pt 2 ~l~ ~corriparaiso~ avcv ~arq~~ur ~~ twills) qui c~rr~spor~d~r~t resp~~;~~iwor~crat ~~ux 1~-~lT
r:ic~c~s~ar~x ~fjs ct ~~3s. La pr~senc~ d~
css 'oandss proaa~c le b~~-~ stet d~; ~' ~~ _: ~~pr es cJxtr action. 1=a p~~°ct~ d~ws cxtraits ar~alysec par spectrophotorn~trc est sic ~.'~~ st ~.~~ ~~-atio ~~~°~~,/~,~6~) ~o~r dss ~~~.~a~°rtit~s d'At ds K.~ ~.b%~.l st 1.~7 ~~/#~l rsspsctivsrrasnt. l~,'~xtractfor~ d':~lTt set do:~c; bones sa~u d~bradatio~ do c~l~n-cl pendant is protocols d'sxtraction. ~'t set traaascrx= sn I~Tc =~otal par l~~'..p ~1~..
>~ s but cst ds s~lsctioransr et d'amplifs: iss r=i°~TC ds s~q~sr~ces ~~odan~s;s 1'~S.~F-~ s-' 1'~~-F-~~ obtsn~zss par 1~.~'-~'~~ sir 1' t. ~ a ~gurs ~ or~trs bus is protocols ds ~~;aplifis ~$ra ~~lc ayant sans tailje approxir~nat~vs ds ~S~ ~aircs do bases ~,pl~) pour 1'~~s~-~~ st ~0~ ply poaxr i'~~JF-~ sn co~parai,son au ~r~arqusur ds twills ~ ~0 pb ~~ssxs ~-~3. ass ta~ilss csr~respondsnt au.x s~q~zsrtcss ~~odantss ds l'~~F-I et de 1'~~~-~~ p~ubliess (~nsxs ~ st 2) et s~~scti~n:c~~ss par iss a~~o~-ces ds is r~actlo~~ ds ~~J~~. ~.Tr~e dibsstion par 1's~yz~e ds restriction ~~~des prod~i~s ds 1~~o~n~rs dss i~a~~~snts ds taillss ids-tiqucs a~a patron tl~eoriqa,~s ds di~sstiora ~d~rra~ss ~~cr~ p~r~ser~tess). ~l y ~ do~nc ds f~rtcs ~~roba'dilites pou~z- q~s tee ~I~c aplaf~~s par l~~~Z co~-espor~der~~ ac~x s~~iuences codar~tss d~I' ~~~-~~
et ~fe 1'~ ~h-:~.
~m ~~~ d~s ~~~~~~° p~y~~ .~ ~~~~~o~~d~ ~a~. ~d~s ~ ~~ ~t 1'~~~°~p~
~.a synt~~ss d'~n ~rectezar procar;~ots p~.~ _aoss~dar~1'~ ~'-~ st 1'~~~-~~
r~ou.s psrettr~a d'it~s~rsr pus facilsgnsnt st rapidsr~nent lee I~~ da.ra ~~~ ~cctcux~ e~car;ots er~'~tillsar~t ces sites poly i~:r~lcsr. f,a selection dss clor~ss b~.ct~rlsns positlfs set felts par cul~:urs dares ~~ra ~r°sii~s~ s~;secti~des l~ac,'~~racs transfsct~ss st par I'analyse den produits d'exs~ractio~ ~.es l~~:ir~i Preps par ~a~e c~iges~ioa~
e~~~~natiq~e aver I~v~~~ et ~ccWl.
1_,'arnplificatio~ ales clones positifs par ~~~.~i :~r4ps :~~o~s perrr~e~~
I'ob$:e~tior~ de plas~~ide:~ de ptz:ete ele~,~re et erx q~~ar~tite s~~~'ISaa~te po~.~r is pos~rs~i~.e ~~~ ~:~-ot~scole. yes prod~its de ~~axi Preps so~1-~ ~, r~o~veau sournis a ~r~e aerie de digestion par ~~v~~l~ et ~~coqaai co~~'zr~e Ies r~s~Itats den l~fi~~~i Preps. ~,e sequeriqage de p~~.1--1~F-1 r~o~s r~v~Ie I'1-~~Jrr~ologie ~. I~a~/fl aver la s~q~aence publiee de I'I~F-I rnais sors ir~ser~ior~ dean Ie rna~vais serfs ~donr~ee r~on rreserztee}.
~~rc~;~~ p~
~.~.1. ~~r~tl~~se de p~I~~l'~~h I~~~-I_ ~p~~~'-1}; p~=~-I~s~l~-II: ~pl~~~-1I;
et p~:i~~!-~Z~~-I~~-I1 ~p~-I~~'-II}:
~,'objecti~ eat la ~;r~atio:~ d'~.n plasr-ode d'e.xpressio~~ e~car~rote exprir~~a~t a'IGF-I o~ <'I~F-II o~ Ies deazx ~ partir ales plasrr~ides d'expression~ pr~,cai~ote. ~~o~ar ce faiwb, no~.~s ~.vor:s ~,xtilis~ I':~oologie den ext~-~~~it~s cohesives ties I~ apres Iei~r dig,estio~ far certaizxes e~.~~rnes de r~;strictio~ ~scla~r:~a 3}. yes c~~igestions vont orienter et azag~~enter- Ie re:~~err~er~t rIe I'ir!ser~:ion den s~qa~~,rdces codar~tes des l~~' dens le vec~eur en elir~ir~ar~t lea rr~a~vaises ins~r~ior~s sans steps de depl!~~ospl~orylati~~~s den extrerr~it~s ~'. ~,a i<gure ~ rr~c~r~tre I'electrophorese den prod~its ~e digestions avar~t ~;t apr~;s prelcwezr~e~t. bra ~.'~oit gets lea digestions opt biers e~ lier.~, car Ies tailles den prod~its de digestion correspor~denr~ a~zx ~~-agnrer~ts t:~eoriq~es attendees ~anr~exe 2). I,es ~~-~~~s d'i~~~:~.r~t se:~or~t pr~lev~;s:, soi~ : 57f~ pb po;~r ~'I~~'-I ; 7I3 pb poaxr I'I~P -II; 5~'~~ pb poa~r ie p r;~ P~r~pa~°~ pour i'vrasertior~
d'I~~~'--1; ~~-3C~ pb po~~r Ie p~ prepare po~~r d'I~F-II saran Ia s~que~~ce :~ ~r ~~7~:~ pava~.~r Ie ply pr~~;ar~ po:.ar I'ir~ser~iorø d'i~F'--II aver; Ia s~que~ce I~. I=.a selection den clones positi~s pea digestion den prod~.~its de l~~lirci Preps est ,T~alisee par Ies e~,~~es de ~-estricti.on ~coRl, arr:ydo~~:~~ees n~~n presentec;~s}.
1_,'arnplil°ication den ces clo~~es eat realises par ~,~Q rnl de Maxi Prep.
~,'ar~alyse den prod~zits d'extractior°z de;s a:~i P;-eps acs spect~'ophotor~evre ~ ~~Ci r~rn Ft 2~ nr~~ d.~z pl~'r~'-1, pIC~P-~I et p ~,~ LAP -II. or~tre ~~m c~JLgcea~~rati~r~ de 1.7~
rr°~g/~~~I, l.~~f gI~~~I et x.93 g/~nl respecciver_~erat. ~,e ratio de p,~arer~e .~.'~b~~m,'' '~'~,~C3nrr~ donrte respective~r~~~t~t 1.:~2, 1.~9 et 1.~:~
respective~-ne~t poor 1es train plasrrrda:,s, ce qua cor~-esp~~d ~. ~r~e excellertte p~rere. L,a digestion den prod~ats de Maxi Preps co~~rrr~e I'ir~se~-tior~ den irbse~~s provenant ~~e Ia digesaiors de p~;,.1. I_,e res~gtat d~ se~~en~age r~o~s apporte to pre~ave de I'ic~er~tite des inserts corxme et~s~t IC'-~1?-?~ et i~~'_II dens pS
avec Brie born~IOgie proche de 10~°,% den sequences co~a~tes p~blaees ~er~ reco~zpar~t ie sequer~~age den i~riris seas et anti-sans). I=,es s~cluen~a,~es novas ~;orl~r~~~ent ~~ssi li'inse~ion duns le ~o~~ sans des ~1~T
c~.ns le vecteur ~~'i~-~res 4 et ~).
3.4.2. ~yntlaese de p~~ I~~~-I +I~I~'-I
ve ~recte~~- ply a I'~jrantage de po~vc~ir exi::rier deux ~~ries di~'~'~rents ~~~~.oe ~ sa sdduenee I~~~
sons oor~firdle d'ura rn~~~e prorno'ceur : ~'~I. ~~ela noun perrnettra ti'~c~dier I'i~~ter~otion des deux I~~ et de eomp~.rer leer ef~°~ca~.it~ dens i~. protec-tior~ dcs ;ilc~ts. ~Jn p~rotooole sirnil~ire est r~alis~ pour I~, s~rnt~~se du pl~srnide ply I~~'-I + ~~"~~_II. ~..a digestion dpi pl~~~'-a se I'ait pa.r I~tl~e~/ gall pour Iibdrer ~~~'-I et ma digestion du p ~ I~~-~ par :~~I~eI/~aho~ pour prelparer le ~eote~ur a I'insertion d'IG~-I.
~~nnexe 3). ~pres oon~irrnati~n des ::;i~~nes posixil='s par digesfiion d~~s prodazits de ~~ini prep par ~co~ et ~arr~, ceux-ei sont an~plii~es par ~fl~xi r re,p et .na~~s~s par le speetrop~otornetre et des dibestions ~fa,gure n). ~Jes produits de digestions so-nt ide~~ti~ues a~~ patrons t~~~ori~ues ~r~,~~raexe 3), ee tlui ~;on:~yr~~e la tonne orienfiation des inserts dens a ~recreur. ~e sdsluen~a~e c~.es inserfis r~'dst pas g~~~.lis~, oar noes s,vor~s utilises les ~n~~nes 1°~ cque pour Ies plasr~-~ides pI~-~'-I et ply-II ~ui onfi ate s~~eenc~s.
~e resv~ltat noes ~:~ont~e due les pr~-~fi~~;oles c~e ~iolc>gie ~raol~eulai<v rfi,=alisds sort efldx:,°aces et ont per~uis la erdation de plasides d'expressio~~ cr~ea~-yote poss~dant les s~~raer~ees eodar~t de i'~~'~~-I, de I'I~~-IlL
ec de I'I~F'-~ + I~~'-II.
mn s~~~r~ d~~~~~ ~ p~~~:~°~~~ d~ ~~t~~~s ~d~ ~~~s ~,e protoeoie d'isolation d'~I~ts de rats et so~.~ris est ~tnw tape i~npc~r'~~.r~te d~.r~s 1'.~ tr~,r~splantation d'ilc~ts.
~ette tape izaer~ee directerne~~t Iw~~ ~rial~ilyt~ et le~f:~°
~'~~°efiion~~~.lit~. L,'o~je~~ti~' est d'o~ten.ir a ~?us d'llots possi~le a~eo la ~~eilleure via~ilit~ possil~ie. i°~o~s ol~te~~ons des Aei~deents de I'c~rdre de 300 ~ec~.
par par°~oreas de souris et 1200 ~ec~ per p~ncr~~s de raga. ~~~
~r~,hilit~, r~est~e p~.r I~ efi ~.~, des dots est tcsu~ours sul~erieure ~ ~~ ~/o dens les eontrcsies n~~,~.til"s des firar~m~eotions, soft de 2 ~ 5 fours apx~s I'isolation.
l~
~u I~~~ ~~~~~~ ~°~~~~~ ~ ~~°~s~:~~~ p~~ ~~~~~ ~s ~~~~t~ ~
~~~~°~s ~ ~°~~~~
~,e but est d'obtenir un taux d'exr~ressior~ du tr~,vs~.ene suftlsa~n~~°~er~~: di~vc~ poor acciu~~-ir u~ e~fet protecteur le pl~zs important possible s~.~r les dots de I,s,~a~erh~.ns. f~-r~°~~re;~~:es condftior~s. de miss au point soot testees : eluaratite d'II'~, radio liposon~es/~~~'.%-~~, terr~ps d'g~~cubatgon, terr~ps de culture apr~s l'isol~taor~ et event is trans~'ectio:~a. ~,es r~sutt~.ts de cette noise au poi~~t pd~ur les dots de rats sont repr~s~r~t~s ~ i'a~~nexe 6. retie f~ise ~.u rorat a p°r~~is po~:r 1.
~~milleure cor~~'ition ~7r~ tau~ de transfectidn cue ~~%. ~.:es ~~~urES 7 et ~ r~ao:ntfen$w des dots d°
rats ~:::~ans~e~,t~s; o~:~ .constate la presence a celluies et/ou d'arnas cellulairo expri~~ant Ie :~e. ~n peer voir c~~se po~~r ce protoco:~e_ la ~r~ajorate des Mots soa~t tr~nsfPct~s au rnoir~s anti fois ct ~u~ cert~.ins dots sort vra.nsfect~;~ ~lzas de cin~ ~'ois. t~es r~,sultats ~ta'~leau ~~ des trar~sf~ctior~s corresponda~-~t ~~~a~ p~oto~~-;:~pl~ies don~zt~nt 6~a °~~ Pt ~~°'° d'ilots transfectes respectivernent a. cinci et sip ~o~ars. ~a ~~ure ~ .rw~Ie la pr~se~~ce de poirns l~.~mineu~ de plus iaible intensite pouvar~t titre des ceil~~ies tra~sfect~es. ~,'~tude de xa via;bili_t~ par pp et ~~ r~alisee en parallels a i'~t~de du ~~~.a~ de r~-c;.r~s~'e~,tio~z r~.ontrc ~~.~e le protocols d~~ tra~.;~fectgo;c~ n'affecte pas la viabglit~ des zlots ~tab~eau 4). n ez~ cor~~;Iut c~~ae le protocols de tra~~s~ecti~r~ r~'ai~ecte pas la vzabilite des dots et pernaet i'e~g~ression day tr~.nsg~ne dens plus de 6a~ ~r~ % ~le.s dots aver anti ~~aa~or=°.t~ de ce~~-ci transfect~s plus dune foil.
i=,~ transfection des cellules ~I4 est =~mporeante co~~~me outil pour i'~t~dc de la production des prot~ines ~~~~mI et I~~~-~ par dot blot, ~r~ais ~.ussi po:.~r des ap~l3catior~s iztL~resS comrne la cr~~:~ion de lign~;es stables e~prrr~tant ~~--~ ou ~~~-i~ ou l~~s deu.~. i~es prearniers r~sultats rnr~i~trent anti t~~~icite irtaporta~~te du ~~'~'1~P ~ la difference du ~'ubene6 ~~i a2e s~;mble pas aecter ies cellul~es ~I~~~. ~,a toxicity du ~~~'~~ se traduit par anti inort inapox-~~.nte des eelx3.~les vasibles so~.~s ~~r~ic~ oscop~e et par ~a co~.oration au bleu de ~ou~~assie. ~.,es r~s~itats pr~~irrir~aires donnena: ,des ta~~:~ de transfection de l'ordre de 30 % pour le ~~~avec ura ratio de ~ :6 pour ~ ~,~ iii et ~~3 ~ifl a~rec un ratio de ~d :6 pour ~ p,~ d'2~ po~.~r le ~u~er~e6 trois ~oa~rs apres la fin de la tra:~s~'ection.
~~ ~~s~ ~ ~~~~~~~~~ ~: ~a p~~~:~~ ~'-~~r L~ ~~t gist ~~ v~rifi~r s> les cell-~l~s tr~nsfoctees paY~ pT~~-II s~cr~t~a~t i~, tr~.ns,°~~~ne ~t si ool~i-of est dens one oonfarrnation r~can~~~ao par ~.a~~ ant-ioarps ~a~~~~or~;iaa anti-I~IF'-Ih.
T~Ta~s ~~rons ~aur vela realise pan pat riot aw~c le rr~ilie~ de oultur~ o~ ~oll~.~~~s '~T~I~ ~rar~sfoot~~;> par ~a,~ rn~l<~n~e pF~~-Ilf~~ageneE et plop'-~IW~'f. I~~ f~b~re ~ montr~ i~, roa~altas dv I~o~ ~Ios:. I)n ,b~z~t cansta~~c.r ~~o los ~~~za canrrales positifs rdalises av~c TO~r~n d'ICIf-II a~p~raiss~r~3t ~.~~sss iaion ~~n~s 3~
~ ~~ ~~o aans io rnilie~ de ~,ult~r~
~a~plot. F,a lirr~ito ~e deteoti~a~ vst a L~into ar~ro~ 1~ oantr~lo pasitif r~aiis~ a~m~, 1J nsi'I~F-II dens du ~~3~ ~i~i n'apparait pas. Las oontr~l.~s ~~~~atil~ ~~~eevt~nt: en ~vv~on~;~;
l~. prdsonoe: ~'I~-f~-II dens le mil~ou de o-ulture ocsanpl~t des ~,eli~ies 'fT~I~- ~v~c ~~~ s~~ ~~n rnais pas ~t~.ras to rniii~~_a ~l~ o~~alt~re s~r~s s~rurn (~r~sonc~ ~a'I~f-II ~a~as le sdra~rn~. ~.-~ c;onstat~ ~~:a.~;s~ ~an~
pracl~~;tiar~ ~~as~.le ~~'I~~-II par l~s o~ll~l~s ''~T~~ s~rva~°~t clo ~antrs~l~ r~d~atif a is t~-~.nsf~~=iar~.
L_'~t~.~lo donsita~-n~tri~~e rdvelo o~~; 1'imtar~site est d~
d~~x fois s~~perie~~r~ ps~~ar l~s ~oll~i~,s '~'~'~:4 rra.nsf~ct~;os par pI~~T'-:~.i elve pour les c~~l'~~~les 'fI4 non transf~ct~~s (-~aialea~ 5~. I~o plays, 1'x~2to~sitd r~ fat I~lot pair les ~,oll~los transfoot~os ost s~,~p~rie~ur~ ~
1'int~nsit~ d~ oontr~le positif (lOt~ n~ ~'I~~~'-'-l dens d~ iii~~ a~ro ~~~
se:~~a, ~e p~zi signi~e d~.ie los coll~l~s ~'I~ transr'°~otdos pra~l~aisor~t pies ~~° TC~~~ n~
~i'I~,~~~-II. u. p~~Lt dano c,oncl~r~ ~aae los o~ll~les 'fc+ do ~o_~ali trar~sfeoteos par pl~~'.-I~k. prc~d~:zs~nt plr~s d'I~~-II
cIE~~ l~~s ooll~~l~s nova transfe~t~os ~ (yes co~contrat:ons approximativos s~zp~r~~~,s.res ~. 5~~ n~l~l ~~~ barr~p~~~'aison a~.~ oe:ntrt~lo pasitif (I~at blot realise a'm~; 2~i3 ~.l d~ ~. ml tatal do ~~aiii~La. cl~ c~lt~ro c~c~rnpar~ ~, Tp~ ng d'Ii~I~~II). ~;e r~sazltat gist or~co~:~ag~ant of ~~orstre ~a~e io plasan:~~ pI~-p'-I ~ p ~rFc~~E
1'o~:pr~ssia~'< d~~ trnns~~rke I~~I'-II.
'?.0 s ~~t~to etude se sitrie dais to cadre des r~c~°~~ro~iio;~, a~aor~~~s sri r la prop octiorF des ''°iiots do f,a.rager bais corers I'apoptose of is necrose porir 1'ain~iior~.tior~ des pi°otocoles de trarr.spiar~t~aioi o~e~ ie diabetiqa:~~ do tape i=. ~oilri~e ions I'a~ora v~~ dens i~. ~eev~~e nee iitt~rar:~re, differs fits ia'ooratoir; s ~ tr~~rers ie rriorde, ~tiiisait differente.s approc'ies, ~ta~~~~e~~t ie. ~=~o~~ei de diriinrier i~=
r~~oi~bre d'ilots yar transplantation, i'objectif ~tai~t d'obtenir rin pancreas po3~z ririe transplantation.
f~e~~eo~ap de pietas sont ir~~,~estigu~es dens rafts res;~iero~ie : differeites prot~i~es a~~x capacia:es proteotris;e, diffe,rents r,~o;~eizs de traisfection, is i~ioroe~oaps~iatioi, ies trai~splar~tatiori d'~øovs aiio~;diiq~es on ;c~nog~r~iq~~~,s. l~o~.~s a~or~s cboisi de trawaiiier dens Sri prerr~ier temps a~~ee ies proteines de is fa~~iaie des ~~~p" et ~n ~rotooole de transpiantation par liposoi3es por r pi~.~.se~rs ~-~isoras.
m I6 ~~°~~ ~~~
~orrie ions i'avons dens 1'ii~trod~otio:~, ies I~~ ont rii bait ~o~:vc:~ir de i~roteoti.o~ pour dimin~aer i'apopto,se et is necrose s~.zr ies dots oo~r~nae srzr d'aritres t~pvs oeil~iaires. f.'aaantage de cette stride par r~.ppoa~ ~. ri'arirres tztilisant des proteiries aiti-apoptotiqries coiri~~e ~~ci-2, p35, ~'~,0, ~~~s3 est i~otai~ii~erat ie rriode d'a~,tior~. ~i effet, s.'aoti~~ation d'I~'-~~ indriit is rx~obiiis~:~ioi de differents senders celia~laires, a~ssi b~er~ a~ ii~ea~ oytoi~ias~~iqa~e q~i'a~~ iivea~
rr~~i.~og~naq~,ae. p a fonctionnerrient d'riie ceilrale est coa~iple~e et 1'ii~bibition on F'aeoengz~atio~i d'~.an sei~itier s~~uli~iaire coridriit dens is i~ajorite des ~aS ~ ~'aCtd~%ati012 ~1'i..~llC', joie Cse 53gia~isatlo~.'i N~aia~I~l.~' de i~o~tlpeu'is~tloi~i '.old d, i'l~a %bitloYi d'riii effet v~i~eessaire art fonctionierg°aent de is ~,eiric~ie ~art~eie~. g'ar exeizpie, b~oqa:er ies effete des e~to~ines aia.
' ~~i~; sari de ses effecterirs e~coplasrniq3ues pe~~ s~~~~ria~~;r ~i effet '~;~,ri~f~q ve dø c.e?lemc~i - ~ r , ;~: ~a . ie~ei~pie oaf'-.
,J;se ~~. i_,es ~C~~' sont secretes et or~t ~i~e aot=~rit° ~.~ssi bie.n paraerin~; q~~°a~~:~orir~~; du i~ is ~~atior~ sur des '.~' r~,cepte~rs extraceiirilaires. ~,e fonctior.rierxeit ~~e is c.e Ii~ie i~'est do~~~c; has r~°~odifi~, ii s'agit j~~iste de i'activation d'rine joie bioiogiqrae iri~i~cei~te ~, is celli~ie az~ ni~ea~
cy~topias~~iiq~ze er ivcieaire. I~Tous pori~ao~is faire 1'~oi~~ologie eitre i'acti~fit~ proieccrice i:;~diaite par yes vr~nsg~nas exprirnait ies ~~-~ et i'activite protectrice indriite par lee ~_~' ari coi~rs dye ~,~veioppPe~it ei~br~oinaire, ~~oc~s n~iriois rive activite protoctrice endogene sacs rnodi~ne~° ie foi~ctionnei~er~t iori~mi des celi~tes Vii.
~,a deu~ieir~e raison est i'utilisation d~~Es g.e laboatoir~; de is i~icr~~yeicapsriiatioi. ~'n eft=et, is poursuite de cette stride a tree co~i~ terrine porte:~~-~ sr$r i~. tarispla:r~-~:ation d'~lo~tv r~iicroericapsraies transfectes par ies plasi~ides d'expressioi ci'x~~. ~3r, dew. et-aades oi~t r~~o~~tre qrie la ~:~~icroei~icapsiilation geiere rin rnic;ro ~i er~viror~rler~er~t autour des hots. ~J'ir~t~rvt de rravaiiier ,~,vec des z~~>i~~cu:a~s protectrices secretes Pst donc i'ohtention d'ur~ ~radie~t de cor~ce~~traticr~ au~c~~~r de i'~iot. ~.d~rsi;
~~~r~~e si i.e norrihre de ceiiuies tra.~sfectees, dens un giot est faihie, <'activite prtctectri~:e pour~-a ~~re pari:a~~e par toutes les ceiiuies de i'~iot. ~e qui noun a~nene a discuter d~~ c~oi~ de is trar~~sfectiora.
~v ~u~°~~ u~~ ~-r ~~~~~~~~~ ~r ~~~~~
~.or~~~e nous venous de is voir, l~ r~icroeracaps~viatio~ perr~~et is b~~~ratao~~ d'ur~ ~radier~t de corcerntration des ~~~' secr~t~es pa:r ies ceiiuies tr~~sfectdes. ~~~ous :,~e so~nt~es done pas obii~~s d'obterzir url taux de transfectsoi~ de ~~0% ~u sein des ceiiules de i'iiot.
yes hots soa~t r~aturelie~ner~t diffciies ~ trarisfecter, ~ sont des ~~as~ ceiiuiair~ er~tour~ d'u~~e capsule clui f~~~j~ obstacle a. is tra~~si"ectior~. ~a~~s cette etude, noun ~~~orrtror~s due :~oa.~s sornrnes capahic;s de transfecter des hots anec plus de is rr~ajorit~ des dots trar~sfect~s ~u ~t~ir~s une fois. yes r~sultats sprat proette~~~-s e~~ cor~paraxsor~
avec d'autres ~t~des utilisargts des !iposves ~so~~~~ne ~thodes de t~wr~sfectiorz cles dots. ~i_n_si une etude utiiis~r~t du ~,ipofecta~ine et p~~'-~:~'~~ ou p~'~~f obtient d~~:~ tau~ de taarisfectior~ faiblc sur des hots i3urnain tout erg ohse~var~t une au~fr~eratatio~~ de is production .de ~~:~~ de; 6 fois par ceux-ci {30~.
~r~e autre ~~~ude ~vec ie ~ ~ ~..~ svr d~~s dots de pore ~btie~~t un -~au:~~
de rra~sf~ctiorA des hots de 25 %
{3 ~). dais ce protocols Boat pouvc~rr ~tre ar~eEio~~c~, des tau~ de ~~-~0 ~/~
d'~lots tra~sfectes aver ie ~~~'1~..~ et is ~-naiactosidase corn~~e ~e~~e rappor~eur out d~j~. ~~t~ obtenu {32~. ~i est ir~tEressant de cc~~~stater clue l'on sous-estirne ie rende~er t de is transfection. ~r~
~ffe~;; certain:3 ps~i~ts lurr~arse~~ molds i~ter~ses ~or~ prig' en vorr~pte dens le ta~:~x de ~:rarafecti~r~~ peL~venr ~tre de s ceii~xies trar~sfect~es dons ie sibriai est a~~a~u~ par la faille de i'~loi. ~T~a~ai~oit~s, avec ce bia;is positif, ~oo_gs ohter~or~s u.r~ -~au~ de traa~sfectiorl de ~~ ~ 7C3 % des dots tr~~~sfectvs avec ~=ius de is ~~oiti~ de ceux-ci au .pins une fois cor~r~e dit pr~c~derr~rr~ent, ~e slui Esc sup~rAeur ~ d'~r.atres ~tude;s fa5tc~s aver des ii~osoa~~es. l~Tous anor~trc~ns aussi, ~r~e sl ii s'abit d'~tvde pr~iir~ir~air~;, sue is t~-ansfectior~ de ce:iiute de ~ertoii ~'1~4 par p~:~f-~~ conduit a is production d~, plus de ~ ~lrc~I d'~Cl~ -~~ {caicul~sur i'~tude demirog~~~triciue). ~r, ie ta~~ de trar~sfectior~ des ceiiuies de hex loll ~,st de '~(~ ~% ~.°t ies yravau;~ de ~oberi .obitaxiie prat o~tr~ que i'ajout de SQ~ri~/rni d'~~~'-~~ dares ie _r~ilieu de c~.iture perr~zet de b<~isser de r~oi~:ie is ~uantit~ d'~iots par trarlspiantation n~cessaire a. i'obtention dune ~~~rr~~ogl~rc~rr~ie. Sri peut donc s'atter~ci.ro ~ observer un effet protecteur des dots g~icroer~capsui~s transfectes par p~~~"-~~ ~.
:i'aade d'ur~ lipos~~e.
~J~p autre raison est i'absence d'itrzur~c~~~~~c~it~, de is transfectio~r par iiposor~~e. ~r~ eff°t, i'e~p~rience ~~nor~tre que i'utiiisation de vecteurs viA~au~, bleu ~~.e ~:~.pab;e d'iruduire urn ta~~:;~ de ~~ ansr'°ection ~iev~, induit aussi uz~e fore r~ponse irnrr~u~~it~~rgre, tableau :~. fir, ii a ciairerr~~r~t ate d~~montre due ie s~ster:e irn~nitaire per I'ir~ter~~~diaire des ~v~tol~ines ii~flar~ar~~~toires esfi, er~'~re autre, respor~sable d~. ~ornl:~re ~lev~ d'ilots par crar~splanfiafiion. "'e pl~.s, I'~fiztisation de prornotc~~~rs vir~~a~ coi~~e Ie ~l pest voir leer efflcacit~ di~ni~.uer par E'IF'~T-~ se~:r~fi~ ate ,ours d3~ la r~porse;
ir~rn~nitaire anti-virale. ~lotre etude fait abstracfiion de ce probl~me par Irailisatio n de Iiposon°xe. ~I
est vrai s.i~e la capsa.~.Ie poss~de ~n mile protec;teur contre la, r~acLion I~6te ~rers~s ~ref~~sn, a~ssb biers qLze d~~s l~rrr~phoc~ces c-~fiot~xiq~~°s qve des anfiicorps, rna.is poor I'instant auc~r~e ~~.~de n~o~~fi~-e I a r~Ic pro~t~cfie~.~r des r~~icroc~ps~,~les contra Ies particles v hales, a:~ssi bier ~~te vwr3~S brel ~>n q~e ~reffor~ v~:i-~~s I~i~te. ~e iaoi~~fi est d'a.i3le~rs en ~-°'~'"'- ir~vest:i~~tion dens le I~,borafioire d~r f'r ~ea~~-~iWrre ~~Ile. oar cefi~e ~visor~, noes n'~cartons pas. la possil~ilafie de firansf~rer les bones des Iw~~ par des vectr~vrs vir~~a~. ~-~
contraire, Ies ~;t~des faites sir Ies plasrrlides pIS I~~'-I efi I~~-II efi l.'expressior des pro~dir~xes no~ss facilifiero~fi la ~~n~r~.fiion de plas~~ides vira~x exprin~anfi I~~-I, I~"~_II o~z I~~s dc~.~.
~ma ~~ ~-~~~~~~~su~~ ~~
3e tiers ~ disc~fier de I'~tilis~,;~ior~x de is r~icz-oenca.ps~~latic~~, car na~:~x~e si ~e re I'ai has encore realise dens le cadre de r:~on ~t~de dv ~~~icroerrcaps~i~,t3or~, ~e devr~.i, ~.~~ v~~e des r~sa~afi;~cs, c-ornrnencer les protoccles d':ici peg. ~,a rnicroencaps~l,~fiio~ cor~siste ~, isoler les ~Ic~~s d; I_,an~er~~.ns d e~ ap~ticorps et des I~~~pi'~oc~tes c~fio'~oxiqnes to~~: en p=~~L:er~rar~t l.e pass~~e des n~firi~~e~~fis et de I'irs~.~I_~.ne. Le bit est de s~pprie.r la r~ponse irnn~~znit~.ire do I'I~c~ve verses ire-I'fon pr~ser~~:wt dens is firarzsplan'cati~r:. et I~ r~ponse ~~.fio-i~n~~une responsable de is desfi~action des ~=ots de ~.,ange:r~ars ~Iaras le diab~fie de type ~. f,e ~aboratoire d~.a I~r I~all~ fait pai~ie des I~~ior~aiers d~.~.s l~;
d~veiopper=~er~t de ces fiechniques. II travaille sir g~. ~~ise a~ poinfi de rnic~-ocaps~Ies ~~~;~, s~~r la biocorr~patibilite ~~4~~, s~.~r I~, protecfiion apport~e par les ~~~icrocaps~Ies cor~~tre Is; s~sfi~me i~r~-~~r3taire ~3~e;> spar I~, ir~.r,~sp>antation de ces rricrocapsvles a~
nivea.n h~patig~e ~~6).
L,es firava~~ r~alisds depuis ~oiobre ;~af~2 s'ir~scrivenfi d~~:ls r. pro~et de doctorafi e~~ co-fi~ztelle entre Ie l~.b~r~.toirv d~z ~r alle et le ~I:orafioire d~.~ ~'r ~svr~t de I'-~Jr3t~ :i~te de ~'h~r~.pie ~ell~laire et ~°ass~,Iaire de ~reroble, ~°rxertar~t ainsi ~: prof~~ Ies ~:onr~~3issances d~es de~a;~ laboratoires d~,r~s la recherche sir I<z transplantation d'ilots. yes r~s~stats obfie~a~s d~r~s ce firava~.i or~fi ~~era~~is la raise a~z point d'ouatils c;ssuntie's a l~. r~alisafiior°x de la d~.~xi~~~e ~:fiape de cefifie ~~~:a~de, snit Ia transplantation d'~lots atl2cg'oE,'Ylc~,pSL$iL'S ~r~3nSf~Tt2l~~",s ~eT'xeiT.t'~~.41'T''aa.°.,:~'3t. "a~.~.°li:a°', ~$3."sdE.' 'a'%a'~''~-~ c°3i.A6'sS! :~s~rlMlt~.$°'~S'~e a'STC:~;
I'~'.Z'I:~r13~1EJ11$afilOr.
2' sp..~r les ani~na~x et is ca~lt~xre cel~~laire des dots ~.e -~,an~g rhan.s °t de la ligzree ~'~. de cellules de er~~li.
I~io~s po~ve~ns aar~tenant comrnence~- ;'~t~de ~. proprernent pane cbe proi:ecta~c~ des dots de ~,an~er~ar~s par les proteines I~~'. ~o~.r cela, des prt~tocole9 de ~.ra~splantati~~n d'i~.ots rnicrc~er~caps~,al~s (ou non, transfectes par les plasrnides I~~ se~-o-l~t realises c~~e~ la so~:~-is e~
:;1~~,~, le rat en cornparaison aver des clots non transfectes. IJe model ani~nc:i ~~tilise ;~er4~ ron~e~zr (s~~ris ~~
et rats ~~''i tart'; ies ar~irr~.a~~ seront rend~.rs diab~ticl~es par le strepto~ot~ci-r~, ~~~ ~y~e~~t o~dan-t capable ~'ir~tiuire ie diabete par destruction des il,~ts de ~,angerl~ans. yes pr otoco~~es z~o~as ypb~or~eror;s des in3aor3:~a~io~as s~.nr la pro~~ection apportee par les IC~~, naais aussi la comparaison ~'activit~ entre des de~~x I~~i. ?~o~as devrions e~~ ool~plea~ent des etazdes ~z~ -~x~~~ etudier ~~ va~~ la protection, Ia fiabilite et la forktio~;~.lit~~ des dots icroer~capsi~les transfectes par ies plasrnides. rmJn a~ ~tr~~ axe f~'~tude s~;r a la raise ~r~
poinr d' urge li~ne° stable de cell~ale de ~ertoii ~~~~ exprirna~at de z~ani~.r~; cE~nstit~ti~~e 1'~_~~-I, I~~'-I~ et I~~~-I-~-II. IJna felts 1-ibnee gurus perm~ttra de realiser des co--c~tt7~res et des c~~--er9caps~s~,tions entry:
les Il~sts et cette li~n~e. I~'objectif est d'evaluer les benefices d'~ne tells app,.-oche par rappor~ a~ ~~ransf~;; ~ des ~~nes dens ies hots. ~e rnir~i-projet a 1'a~,~a.ntage d'~tre rapidement rnis en r lace en par~ant des ~~reiE~rs resultats de transfectiorg des celiui.es '~i~~4 et de I'expression des ~~o~.~$s~enes dens c~.s cell~les. ~Lrn int~;~~~t secondai.re, non fe :noindre, est la anise au point d'un protocols ds~ rr~icroer~capsu~ation de c~::~:i~les adlaere~~tes ~~~orpi~oic~giduerraent tres dif'ferer~te des dots. ~n tel protoc;.~~e de rr~icroenca~;sulat~r~n poa.~rrait ~~tre transpose ~ d'autres iig~ees cell'~laires et tro~ver d'avtres applicati~~ns, notarnr~ent en car~cerolc~gie.
2~
~.e b~~t pre>~ier de cet~e ~t~de est a~,ar?t ts~~~t ~.e ~-e~,lierc:~e.r des ~
°~s~~e~s c~r~cret pc~~ar a~~~li~rer ae preto~.ole de la tra~splantataorl d'~lc~ts. ~~ es de~.~;~ stapes li~~itar~tes de la trarasplantati~~ d'ilots sat la via~ilite des slots q~i se trad~it per ~~~ ~~r~~~~re d'-elots r~~cessaire ~le~re ~ t,'c~hter~tierz d'F~r~e ~~rrn~~lrc~.ie et la ~aladie auto-i~:~~~n~r~e irzl~~re~te ~ la ~~aladie.
~.'~~rp~tl~~se de d~~ar~t de cette et~rde est la capacity de 1'~C~~'-~~ aj~~tee ~a~~s le r~ile~~ de c~lt~a~:e a c~irr~i~~r par de~x le r~e~~'cre d'alots r~~cessaire ~ 1'c~~ter~ti~r~ d'~~e r~c~r~r~~~l~c~~na° via la p3.~tecti~r~ ~.~;,~ ~x~ts w~r~tre 1'ap~ptose. ~.,'ir~t~ret de cette ~t~de est la ~~r~eratic~~ d'c~~tils c~.pa~~e de rras~s~rrer 1'~~~-l:l, ~
ais a~ssi 1'~~~~-:~ s~~ lee de~tx dais yes ilC~tS. ~..e 1?Yit a°,st d'&5'~te31~.3' d~S r~s'~~~t='zt5 ~3r~7~~,1r'~~.5 ~. ~c~~C3~"t ~'~'~~.~;V-~~ a~? r~lll~lt, 112aas V~51A"e peLEt°vtl'e de ~a~eiilea~rs r~s~ltats d~ Fait de la d~r~e s~;ipe~~e~re a 1'ea~pressis~n de tr;~r~s~~~e. ~,rr~ a~tre ~.tc~~at est la p~ssi~ilite de ct~parer lee e~°~'ets des d~~x ~~_~~' sir ~a pr~tecti~,~,r~ des dots. ~r~~r~; lee ~a~tils ~en~r~s ~~vre~t le chaps d'u~~ larbe ~~er~tail d'~t~des sir is pr~tectic~r~ d~e.s dots via la tra~s~ecti~p~ des dots r~~
la co--e~caps~lati~~°~ a~ec ~tr~e lib~ee ~,erl~aaaire a x~ra~~a~~t des pr~t~i~es prcstectrices ~~~ la c~o-c~zlt~re avec one li~r~ee exprir~ant des pr~t~i~es p'~otectrices. ~'ei a per~~~ d'~t~dier dit°fere~tes appr~cl~es dares la protection des dots. ~ette dt~.~de ~~vre ~~~ lar~~, c~a~~p ~~'applicati~r~ par l~;s d:u~~rerates tec~~r~i~iaaes mise~t erg ~e~a, ~~e ce snit erg biotogie mcj~~~Iair~;; e~ c~lt~~re cell~~lairy, la arlar~ap~latio~ d'ar~i~~~a.nx o~~ la I:ficroencaps~latzo~.. vela reed cette ~~:~de tr~s znorivr~~jte et tree v~t~ressar~te, ~~.ai pivs est, cette ~t~.z~e s'i~scrit dares Ie ca.dre d'~r~e co-t~te.Ie er~tre de~x lai~c~rat~ires, ~. ~a co~~pi~~~e~~tarit~ certai~ze9 ~a~e colia=~oratio~a no~~relle et pro~~ette~se.
S~q~enee ~ocJar~t ~ø~F-~ rnuri~
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~haaager~er~t ci';~r~e g~a~it~e p~~~° :,wre a~~r~i~e poor ~~irrar~~er ie:> st~~act~.~res seeondaires ~'apparie~ent ~e l'arnoree.
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~e i'a~o~ ~e seas a, anti-seas snit ~ a is base 59 ~, j4fl : 4~ ~ pb fl ~e~°
a ,~Qq~~e~cc,~ ~,~ Ztv~i° c~'~~~s ~~c~~'t~~~ ~~a ~~tf~'c~ca~eF ~~~~
t~~a~liF~-l:~k~ ~~~a~th ~'~cto~° 1 ~pwc~.c~.~~~~o'~ei1 ~~, St<~~~ien ig.'~, ~ o:vg ~il~~~., a.i~,~, l~I~eleic ~_eic~s yes ~~~6 bet 2~-; ~4~20~:?'~'Z~--~2 o ~ns~.iin-like growth fa~,tc~r ~e~~iates ,~e~.ibone ar~u~rai ~ensivy ~r~
~~i~.e by ~ot~ grovath ~s~r~~or~e-deper~~e~t any -i~~depe~ent ~~ec~anisz~s4 I~~oi~a~. S, ohrna~a ~;
~.~uo ~, !~rnaar , ~onah~e ~..~, ~~~e.~;eci~.i ~, ~~a~~iir~: ~~~ ~,r~~~oerxrfotogy 2~G3 ar;~.44~~~:!~29.~36 S°quu~ce codar~~t ~~ ~'~~:F~_iC'~: ~~:~~:~~~.:
~~~:~ge~.~c: ~tt~~~c.~~.e t~~~~gt~~~: ~.~'ggg~a~t~ ~~~gtggg~~~ ~g~~g~'~g~v gg~g~tt~~~
~.t:'3G~'~8 Se~S ,'_~C3E~~Ii St~ti ~.~~~~_~~tgg ~~~~~g~~a~ gtg~~g~:~':~w g~~g~tt~.~~ g~~.~gg~.g~ g:~.~~~.tgtg~
~g~ggg~9~
ttgttg~~~~: g~tt~~.g~~~ g~~~g~~:~g~~ :~.~~g~gg~tt ~~a~~~~~g~~gc~.
~~t~~~.~g~~c~'tg~~~~~~g~~gc:r~g~~gtgc>~~t~~~~gg~ag~.gtg~tg~ttc~gc~
~.g~~g~g~~~.~g~c~~~~c~g~g~.~~_~~~..~g~.g~e~..a~.~.~.~~gc~.ia~.~g~~u~.~g~.g~ggg~
,'r~~g~~~~~~t ~~6~~S~g~~J~~~'L~.~~gg~.~g~.~lwt~~.~.~~~~~~~~~.a..g~ggg~.~~gt~~t~~~~.~~~tg~~~~~
.~g~d~g ~~~g~c~g~g~g~~9~~~g~g~~ggg~~t g~~tg~~~~t~~~g~g~g~~~::g~c.g9~g~~g~~tg~~tg~~
~~~.~~~g~t~.~~.g~~~t~~g~g~gg,.~~.~~.~~g~~~t~.~g~~~~~~~~.t~gf~g~~~~~~.~.~~:~.~:~
g~.cc~:~g ~6..~a~gggg~~-~~~~~~~~gg~g~,~3~~~~~.g~~.~~~r~~~:g~g~~$.~.f~.~.~~~~g~~~~ ~-F~t~iSg~~~~
~~~~a3 ,~t~p giag~~.~~~.~ ~~~~
~~.~.~i.tg~g~ ~
~~~~~..~~~~.~
~. ~~~
~ '~<
~~~~
~.
~~ ~,~tli~~~
~~C!~t~~'tc~ ~~S ~.iTlQiccS
~~uS : bc~~cctc~Lt~.cc~LC~a~cttc~
~~~,~;~err~er~t d'~~c g~~~inc ~c~~r ~~~ ~.~~~r~~~~m ~~~~r ~it~~r~~a~r ~~S
St~~~ct~rea scc~r~da.~r~s ~'~~rp~r~er~cr~t de ~'~~sorcc ~°~t~-Ser~S : ctct~ga;c~.g~~~c~~,~a~;,c~.tc ~ec°~~"r~c~ c~cla.r~tc d~ ccad~~ Step ~. St~~, ~c ~~. ~~5~ ~'~ ~. ~7~
Sc~~t ~~~2 ~~. r~c~ ~'!-~I~i~yf ~.a~~~a~ per :w~
e~c a~'~~SC 3 ~ ~4~ spit ~~5 p Re~°
~'~c;a~~;~cc~~ ~e~l~c~~~~c~~ c.~l~T~z ~.°~~cr~r~~~~ ~.' e~ ~cer.~w ~~~~'~.~~z~-~apk~: ~°r)s~r~~a~°c~c~r~a ?~~~E~c~c°~~~~a ~~'~~
~tc~~~3i~~ i~~., ~o~~, ~~~ ~,~, ~~ ~xo ~~~~ ~ct;~j~;5~:~~°~-6t ,~n,s~~i~~-t~~Ce ~a-~w~~ae,~e~~~~~ Y~~~~~~z~~.~~ ~~~k ~~~~~ ~~~~~~~~~~
de~.s~Ey ~aa c~i~~ ~~ ~~~,~ ~°~~v~~ ~~~~r~~a~-d~~e~~~~~ ~~~~' -~~ac~'e~a~~~~~~ ~r~~~c~~~;;~~:~~ ~~~~3~~~.r: ~, c:~b~~,n ~;', a:~~c. ~~., ~~.r~~~,r ~', ~oel~.h~e t,, ~~r~~c~al ~, a~~~r~~~~~a ~~~~cr~~~~=~g;~ 2G~~~3 ~r;~~~~;~~:~~9-3~
~7 ~,~r~~ ~i~~~riciu~ ~.~,s di~e~ii~l3s ~r~~~~.~~ v /i~zs~;~ ~~ T~~~~i ~reia:~~
~~~-~ + ~~~-~~
spel, l~hes :. I~--! (5no ~asj I~l~~: ~."°e. ":, ,J ~, 'e ~;
~Cl~.'! a~~-! ~: ~vo ~ 8~P=-a Y~~~
~. :3a ~$3 ACS ~~~~ ~~. ~(..~~
'~ ...a ~~c:.S
r~ -._.ysho!
''~V~aP
~..,-'~~.~°'""'.
igesiicn lOigestion Ciges:ion i bands 3 5?U pb (IGF-I) + 1 bands a 71U pb {iGF-i.) + 1 bands: a 810 pb (GFh) +
1 bands a 3830 pb 1 bands a 3840 pb (~CR2.1) (pCR2.1) 1 bands: a 3900 p!> (vecteur) 1CS r.Xho! F~C5;,~l~lee! A
~/~
!P''a!e a ~z I$s"S ra I~~,S
i ~l~~S
~~9G ~b ~u1~4 ~~ q~ ~ '~E9~~! ~b ~.~ba8 CS B vICS S ~~
'''~Xlaa! P'i5of~ ~' '~CS 8 °~..'..-_....~ ~~~_,_..... "~o~&
Cigestior:. ~ digestion Cigesfion 1 bands a 650 pb (II~FS) + 1 bands a e,'6U pb ((F:rS) - 1 oande a ?0 pi; +
1 5ande a 3250 pb (vecteur) 1 bands a : 24U pb (vecteur) 1 bands a 3880 pb (~,recfeur) ~h I ~~'~~ CS B CS..t~31t2! !RE ~c:~~~!
EcoPi! -E\d?le~,~'~Sln '~ Ses~,~6 PdlCS
!CF-!1 ...-._ , ,MCS .~ ~~CF"i1 a ,~t:a~S !~',,~-i! ° ~ 1n &CF-!
&8003 ~b ~WF-j I~F- ~l~ir~~S !~~h_! .=-!CF-!!
t ' 658PJ ~h ~~ ~ r4~61 ~t~
IVlCS S ~~''~CS S ~:W .~"' y JlCS A ;~C& .~ ~
...-,~ ~..-..'~Saii -.~.~Rd SercB~!
---~-~ ~ __ i7igestion iUigesfion 'Digestion t t~ande a 515 pb (IGF=-i)+ i bands ~ 650 pb (IFtES) ,~ ~i bands a 6fiU (IGF-II) +
1 bands a 10 pb + 1 bandP i 59U pb (tCF-i) j- 1 bands ~ 5490 pb (ve:cfeur) + 1 hands is 30 pb 1 bands d 6780 pb {vecteur) 1 bands ~; 5520 pb (vec".ur) i~~S i~~~ 8~d~
Sarn~ll ~, I=ar I
hACS iviCS '° ~C~;
Ear llfiCS A g,-CvICS A ~ CF-!9 ~~lC F-!! r~CS A ~" ~ wF-f!
!CF-~ PIF~~S ~~F-! ~IGF-!! g~p.~' plf~~S fi~"~F-9 +l~fi'-!! ~ &~F-~9~t~S iGE-I
+l~f~-I
~s~00 pb a ~~UO )3tz p Ear'r~' 7400 !s!c CS >~. I~ICS CS A iViCS =a:r ! ?CS A !tJ! ~,S , 5sp! ~~ ~'~ p~ ,. Ssp! ~--. Ear !
i ~ ~~anai-!! (~a30~) '~Sspl ar 1 °° .
i7igestion Cigest'son' Digesfi ~ ar , far I
1 bands a 2970 pb {iGF-I!) + 1 bands a 373U ;~'L (1C=s, IRES) +
i hands a 4380 pb (vecteur) 1 bands a 185U pc + 1 bands d 1?15 pb+ 2850 pb +
1520 plo ;- 1260 p'o +
1 bands a 50 et 2°~ pb T00 pb + 630 pb +. 'Z10 pb + 500 pb :
~dqtaez~rage p~ar~-~
~E~'Cil~C2lCp CflC'ari$C ~,'-~,7LtCe'~,CCtC ~~CC$Ca$~.~~, aCC~;aC~Ci,g aCC$gCtg$g' ~~aaaCgaCCC ~pc'la:'Cl,at;~~.a aaa$gaCCgC as.CtgCusat~
grin ~erss ~ppariemen$ ar~s~rce ~~~s aacgaccc F3;gav,cta~~;ca aa'-~~:gaccgc acctgcaata a~rIi1 i~l~$=-~CI15 gtgttCaCCtC agCCtCa$Lagt a~'Ca~~,r',aCtCtgaCC$gCGgt~~øa'r~aCgr.'~.CCC apg~,CCt~!~Ca aa~$gaCC~~,=L' ~'..;C?CgCsx.a$a aaga'~tc~tCaC~i $C~~;~ Cg~C $tCaCc,~.CC$C ttC$aCC$gg C~C$4;$gC~L gC$Cs~aCC$x~C
~ia~CagCtCCa CCGS.Cc,gCi.ff;g aCCagagaCC
a~lt~'~tt931'~C$ $C~~~~CT~~T$ ~2$CaCc~.CCtC $tCtaCCtgg '~:,gC'.'.CtgCt$
~>CtvaCC'LLC ~a.CCc"2gC$CCa Cd,FaCagCt~g aCCimgagaCC
aaga$acaca $ca~~ cgtc tt~cacacctc $tctacctgg cgcs:c~atgc$t ,~~~cacc$~~;
accagctcca c;cacaget,~g ac.°°cagag~.cc ctt~gcgggg ctgagctggt gga$gctct$ cad tcgtgE g:~ggaccgag gggct~.~ta!;
ttcaa~caagc c,cacaggwta c$ttgcgggg ctgagc$ggt gga~~gctctt cagttcgtgt gtggaccgag gggc$t$tac $tcaacaagc c,cacagg to c$ttgcgggg c$gagc$gb ggatgctctt cag~tcgtg~ g$ggaccgag gggc~.~$a~; ttc;aacaagc c .: acagg;~ta $ggc$ccagc: ai~tcggaggg cacctcaga., aggcat~tg~g g~:.$gagtgt~;, gcttccgg~,g ctgtgatc$g .ggagactgg $ggctccagcv a$tcggaggg caccteagac aggcat$g~tg gr tgagtgtE gct$ccggag crgtga$c$g ~:ggagactgg $ggctccagc a$tcggaggg cacc$cagac aggcattgtg g~,tgagtg~~ gct$ccgg.~ag ctgtgatctg rxggagactgg agatgtactg tgccccactg aagcctacaa aagcagcccg c~c:$a$ceg-v gcc.°cagcgtc acactgacw gccca;~gact aga$gtactg tgccccactg aagcc$acaa aagcagcccg c$ctavcc~~ gcccagc~;cc acadtgaca'.
gccc~;~gact agatg~:ac$g tgccccactg aagcctacaa aagcagcccg c'.:ctatcggaaccagc' ~~c acac$gaca ~ g~~ccate.~t Cagaaggaag $aCc'3$~$ga~i, gaaCc'3Caa~'~ a~aggaag~~ C:3ggaaaCa~. gaCC$aCaga a$g$d8~g~~.,~~,~ ;gCCtCCCa~
Cagaagga2g $aCa$~'$gaa ge:aCc'3.Cac'1~~~'e agaggaagtg Ca2g,~~.'lac'~C~7s~
gsaCCtaCaga a$g~;'c$~g~g~ ~gCa:.$CCCaC
c~i~aaggaag ta-~aaariaa gaacacaagt agaggaag~tg caggaaac~.a gacctacag~a, a ~.~. .aacaaaaa a$~ccaca~tc gga~agcag~.aa acgcca ~pp~rie~er~$ a~~or~e as~$i-sera ~~as$ seq~ei~ee a~d:r~g i~~-~ ~~~c~c~r~ s~;~~°t-s$~*~) i seci~:ea~eir~~; r~~ s~~a~s:
~eore = ~ i7 bi$s ~~7~), ~~pec$ _ ~.0 ~der~3:i~~c;s = ~'~4/3'7~ ~99~/~) ~tran~. _ ~i3~s / ~I~s~
~iasv seci~e~ee ec~dircg I~~-~ ~ec~~~r1 s~ar$...s$~p) ,' seq~etrein'; ~rir~
a=~$i-seas:
~cere = ~C~G bits ~3 ~ 2), ~.xpec$ = e-~ ~9 i~er~$iries = ~6213~ i ~9~°/m), raps = jal~~~ i ~ i~'i~) ~$ra~d = ~'i=.~s / Ii~~s r ~~~7~.er~rage ~~~~-I~
~CL$ilsrlCC CC5da~2tC ~'~C,~~CCtC CttaCCCaaC ttC~'$~i,CC, aQ$~g~~a$C
CC~,~~'~.'gggga agtCg~~gtt ;~~gCttCtC r~.-~CvCtt~,~'~~.g CCttCgCCtt ~5'~~rl~&1'?~Ilt ~113OTC~ ~øk'~s gtgctgcatc gctgcttacg gccecggaga gactctgtgc gg~,~~ggagc $tgtvgacac g~.,tLcagt'~ g~ctg~.'tc.gg accgcggct~~
tacg gcc.ceggaga gactctgti~..r, $~.',c,t~gggagc ttg? ~ga~.a~zi~~cag~i.~i:
gvctgttEagg acegcgge~'~
tgctgcate gctg~7tttaeg gccec~ggagagactcttgcg~,~aggggagc$~gt$g_=~c cacgcttca~;~'~tg~~ctgL~cgg accgcggctt etacttcagc aggcet~bcaa gcegtgceaa ccgtcgcagc c~tggcatcg tggaag~gtg ctgcttccgc;
agetg<gacc tggccctcct etacttcagc aggccttcaa gccgtgceaa ccgtcgcagc cgtggcateg $ggaagagtg ctgcttcegc;
agetgc:gaec tggccctce$
ctacttcagc aggecttcaa gccgtgccaa ccgtcgcage cvtggcatcg $ggaagagi:g ctgcttccgc ~.gc$gc,gacc tggecctcct ggagacatas; tgtgceaccc ccgceaagtc cgagagggac ~'.gte~aect cteaggccgt ~:~~,'tc;cggae gacrødccca gataccccgt ggagacatac~ tgtgccaeec ccgccaagte egagagggac ~gtc.t~acct cteaggocg$
aettceggiac ga'cv~dceea ~atacc.cegt ggagacatac tgtgccaece ecgccaagtc egagagggac s;tgtctace$ ctcaggccgt ae$tccggae gact$~~ccc~ gataccccgt gggcaagttc; i-tccaatatg acacctggag ac.agtrcgcg ggaegcctgc gcagaggect gcctgcccS9: c~Wge~;cgccc gccggggtcg gggeaagttc ttceaatatg acaectggag aeagteegcggg~~e.gccctgegeagags;ec$g~ee$gccc;$eet:gcg,~ gcccgceggggtcg gggcaag~$c; viceaa atg ~cacctggc~gacagtcegcg ggaegcctgc gcagaggect gectgccce:c ctgc~ ~gccc gccggg~,~tcg C~ztgC$~:gCC aaagagCtCa aagag$;~Cag agaggCCaaC. ,ri~CC~l,$e~~:, C~.C~gc'~.tegt g$$aCCaCCC; ~a~gaCCCCg CCCaCggggg Ca$gCttgCC aaas3gagetCaaagag$tc~yl~~gaggCCsl~~ ~,~tCf~tC~:CC~°Ctg~tC;;~
gt~3.CCaccC awaa4~aC.Ce,Cg C~~'~~,ggg~g CatgCttgCC aaagagCtCa adgagttCag ~gaggCCai"~~ C~7 Cc'~t~glC d~el~$Y~at2~~gt gi~aCCgllg,:.Ela~ags~.eCCCgeCCc~Cggggg agcctcttcg gagatgccca geaaccatca gtgaatca~a =~~tgtggta attctgcaat ,gtagtaccat ca~atctgtg~ cctcc$c$~
agcctct$cggagatg~cc ~ri~~ sc~~s agcnt~itc ~r$rs ar~~~ s;~s a~ca~~~aca_ ceatc ~,~~ar~er~ea~~t a~c~rce a~c~-sins east ~e~~e~~~ ~.~da~at~ ~~~d~r~ Mart-~c~~~~ st;~~u se~~~r:~ag~; saris ~;~r~-~s~ssrl~~.~,t ~c~re = ?3 3 tits ~3 ~ ~ ~, ~~~e~t = ~. Q
3si~~$iti~;s = 4~J7,~~~~ X95%~9 has = ~/~~~~~%~
~tra~~ -_ ~'l~s / ~l~s Mast sec~,~a~r~ce ~~dar~t (sari-st~~~/ s~~~~r~c,ag~ a~~ti-s~~.s ~~~~ - 7~4 bats ~~~7;9 ~X~~~$ -- k~.~
~~~~3$itze;s = ~r~~/46~ ~~~°/'m~; ~'~~3s = ~1~~5~'~ ~~%) ~trarc~ = Plus / i~~s 3~
~su~~~~s d~s rnis~s au ~c~i~t du ~r~tcsv~~~ d~ ~r~~s.~~~tic~~~ pay ~~'~~~Ip~F~' suø d~s a~ws d~ r~~.s.
~ ~ ~ I ~ I - I
' ~. ~ LEI ~ ~I _ I i _. ~ _ I ~__ ~
~~~ ~o~l ~ ~ + ' ~ ~- ; ~ ~ ~ ~ ~~ aa~ S~
i~_ i~ ~_ i ~ -~ ~ ~- ~ ~ ~a~ ~ ~~ ~~~ ~~~
s ~~~/~ ~ ~~ ~~~ ~~~
~ ~l~ j I i, ~ ~U~~°~ ~ ~~ ~~~ ~~~
t~ca ~ o~ ;~~J ~ ' ~ __~I ~U~/~a ~ ~.~ ~~~ ~S~
=~U~, ~3 ~ ,3,~ ~~~ 5S~
- j S~~f~ a~ ~,~ a~~ ~s _ ' ~_~ _ j ~g~ ~ n~ ,~ , ~U'~r~. ~~ ~~ aye ~U~ ~
j_ _ ~~~ ~ el ~- ~ ~- I ~ .~~'~/~ ~ :~~ ale 5~~
I
6 ~ 1~
tip ~ ~~ ! , ~ _~. _. ~ - ~~~~~ ~ S~ aa~ ~7 i ~~,~ t' I ~ ~ ~~
~ ~ E ~~ i I _ ~~~,~ ~ ~~I ,U~ S~ p~
i ' I _ i ~_.
I 1 -~ -' SU~f~ ~ ~~ axis ~0~
i ~a~~e~ -~ I ( ~r _ ~~ ~/~~4~ai<~4~c I
II ~~~ ~ ~ __~ -G l~ I
I I
6 ~ 1~ j ~ , ~ ' ~-~~~ 5 e~ I ~ ~ ~ ~ ~_v. ~' ~ ~~°l~ ~ S,~ eke ~~~
I ~ .a- ~ ',,- ~ -a- 4 6~f%~ ~ S,~ ~~e ~2~
~ 6ys,~ i'~T i ~ i i i ~ __ j S~~/~ :~ ~a~ ale ~US~ _ __ ~f~ Sri I I _~. -_~~ ~ ~~ ~ h ~. S~ Q~e ~U~a ~, 1 ~
?~~a~ 4 ~~ ~ ~ ~.- ~ ~ ~~/~ ~. S~ a~a; X33 ~ ~ IbT~d ~ i _~_ f 3~
.c'~, T~~~r~u.e~ar ~~ ~~,ilg~ F ~~ ~~ ~=v!u ~~ ~e'~r~vi~rc~b~~. a ~tiEis~s d~.a~~ ~e5 ~~e~tr~phor~ses ~2 ~i~re~ra.tior~s utiiis~~s dens ~o ~~~rr:oy ~~~'~'~ ~ : ~r~it~ ~~f~o de ~'h~ra~i~ ~'.°xaTai~,ir~ ~t '~iss~z'c:ire ~~.ci : ~ ie~ est ~n hots dont i° da~r~Wtrv os~r~~ ~~~
-cc : ~u~no~~ ~i~cr~c~sis ~~oto~--a ~'~.1-~~: Inter~°orc~r~ ~~.mr~n~~, ~i~I~: ~nreri~gakir~o-~.~
~~F: ~ndot~~ii~r~~ ~~-ow-th z~~,ot;~r ~~~-~ et ~: -ons~iir~ ~ro~~~ia ~~,ctor~ ~ ~
~~F~~: ~ns~aiin (~ro~th ~'aoto~- ~ir~din~ ~rotoi~~
~~~-7 ~~~-: ~~~' ~oo~~tor typo ~ ~~: ~
l°~'~'->~: l~T~oi~~,r ~'~otor-~~~~x: ~ro~wth ~orone l~t~: rnor3oxido dy~.~oto i~~s: 1~r~ s~~nthdt~,so :~: ~ratra ~-i~oso~e ~ntor se~a.~~r~oe ~~u/~.n,-'r~ ~~~: ~~:por tyxyd~ ~is:~Y~,.so ~~ ~~ai~~rw o~.~ ~in~, ov T~~y~r~.~sia~~
~~: : ~etl~zl~ d~ So~to~i ~~~ : ~ree~ ~lao~-oscent Protein ~~1~~ : ~~~ol~~~n~.~~ ~~2'L~s E~~Tc: ~ oornrlerr~~ntaar~
~Tt: ~d total fir: 1 ri~oso~nio~~~
3~
?. Shapirca ~,1~. :~~ax>ey ~;. ~~yar~ ~~g., ~~b~.~t t4iS, '~~th ~,, ~~~lar~m~~
~a-~,; r~~te~~an Iii, ~ajott~ : '~I. 2000 s~i. Is~°t transp~a~aatic~~ in sv~~r! patents ~~it~~type 1 ~.iab~t~s ~~eliitus ,sing a ~i~~nc~3-tic~id-free i<~~~~n~°s~~~pr~ssiv~ regirner4. 1~1 sngl ~ ~d. ~%;3~r3~~-~:23~-2. ~r°~ac~a~, ~.~~'., ~. ~b~rb~i~~r, ~.. ~'~s~, I=,. ~ssi~r, ~.
~~~f~rr~is, F'. ~ayi~, ~.
'~biv~I~t, ~~. t~~artin, f. ~~is, L-.. ~ad~t, ~. ~~ii~P, and L~. i?~~~r~~.
2C~OI . I~~?an isles: trar~sp?antatir~ra r~~t~~c~rk for the treat~~er~t c~3_'°'Typ~ 1 diabetes: ~~r st data ~-crr~ the Spas-Fr~r~~;i~ ~~I~, ~:~~s~r~:i~rr~ (1999-~~0~~.
l~~c~~~~o~og~~. 4~: X59-~4.
R.esenberg, ~., b~.. $Jarxg, S. aras~nas, arid ~. ~:aysin~~r. 1999.
Strri~t~rai and fian~ti~nai ehanbes rest~itir~g fr~~xa islet Isoiati~n lead t~ islet sell d~:at~~. ~~~~~r~. 126::~9~-~.
~iiwa, ~., S. ~a~~t, .~~. ~~~ang, . ~gapit;L~s, ~~..1. ill, ~r~d ~.
R~ser~berg. 1999.
~ar~~r~ati~ islet cell s~r~i~aai ~~il~~ir~b asset isc~iati~n: tb~r~i~ ~f ~~li~iar ir~t~ra~ti~ns in the pan~r~;as. .~~r~r~oori~aod. 16l :~~"~-6~.
~~obr~aeier ~-, ~'~gigperz ~~, '~'rar~. ~J'~, a~ri;~ ~; ~I~~garc~ ~. 199 I~uay. Stabh e:~p~~essic~~ c~f rnar~ga~ese s~.~p~.rq~~id~ ~ist~tas~ (l~~S~~~~ in ir~s~.l~in~t~a cetis ;~re~rer~ts ~~.-lbeta-in~~=ced eytot~~sieity arfd red~~~s r~itria; ~~~i~e pr~~~t,ti~r~. a ~~"'i~:~
Lr~aest. ~;101~;9):~.~11-2~.
~,i~iri~, f~.L,., a~~:c1 ~. ~~~rdr-~p-f~e~isen. ~~I~. ~~ eb~i~,e ~f death-~~~e sig~~al-rransd~cti~n c~fir~~r~e-a~ediatecl beta-c;eøl apc~ptc~sis. ~')i~a~»~o~o~~~~.
~~.:2zI~-~3.
9. fhQmas I~ar~dr~p~~~~.;ise~~. 2C~~1. ~-eeli I-~p~p':~sis StirraF~ai and Signa1png.
~ia'~etes 5C :~~~-S6~
1~3. ~bd~l~cadir ~~~s:r~, i~iu~:shell ~~.eegaz~, ~-ra ~~is:a~adia, ~~aazl ~e ~'~s, I~l~ce 2~ar ~~~i~er~, S~asar~ ~r~r~e~=~eir arid ~'~rd~n C. ~~eir. ~~0?. l~aere~~~a~age ~epietis~~ ipr~~es s~rviwal ~1°
pc~rcirce r~e~r~atai par~ereatie sail cla~st,c~rs c~ntair~ed i~~ alginate ~~~aer~~,aps'~i~~s %ra~spianted irit~ rats.
~~er~c~trartsplantatic~r~; 1° ~; Z~f~-251.
11. grey, S~~'., ~. ~ ~ek, ~.~-~. ~ae~, arid ~. ~err.~ar~. ~~c~s.
,~_den~~Erir~s-mediated ~e~e transfer ~f ~2C~ ire a~rine islets i~bibirs gas-irl~~vee~ ap~pt~:~sis.
~~~~~,~~~~~~c~ ~r°o~. ~3:5'~7-~.
1~. d~abiz~~~itel°g 1~~ S.-~.L, to ~r~a~.ica , ~~ a~, ~~elsrei~~ f.~.;
brc~~~anl~:~ , ~~rb~tiv ~, aj~tte . 1999. tra~sfeeti~r~ ~f l~t~rfi~an. panc::~°eatie isleta>
~ait~~ are a~~ti-ap~ptc~tie gene (gel-~) ~r~tects beta eelis ~r~a~ ~ytel~~r~e-~nd~eed I~estr~etic~r~. t~~c~b~~~,~. 4S:
I~~2~-1'~~9.
1~. ~laa~ E. arise~~. Sif ~y. .e~~, ~~arer~ ~~,indbe,rg., ;lesper ;ic~l~an~~ese~~, Elisabth D;
~alsgaard, ~lernrr~ir~g l~oe~~:, yens ~I. i'~ielsea~, ~lZ~mas ?~a~~c.'~r~.~p-I~cs~ls~;~, 3c~rr~ I'~erc~~p, _'i°~ils iiiestr~~. 2~0 i . ~~~~ressor of ~y'~olci~e si~L~alik~L~ ~ ~~~C~,-~~ ~rc~teets ~~-cells a'ains-e inter~e~a~in-~~ arLd Interferon-~,_r~ediated to:~icity. I~tf~.~; vo~9~; '~I;
~2~9a-12i9G.
date, 'f., ~..~ s=~eia~;~er, ~. _'~a;e~i.~iai4~, ~.~. ~;axiiiva~; ~.... ~,iaz, ~. ;~earia, 5.~.
~~e3z~, ~...~. ~re~ory, and ~;. ~~a._a~. '~~(~~. ~de~ovir~as-~nedia~rod ex~ressior~ of~~~ prevents ypoxialreo~y~;er~ation ira~~ry bar red~x~;~rL~ reaoti~e o~y~~L~ se~;ies and caspase activity.
C~c~;~~a~vc~sc Re.~. 55:09-19.
~ 5. ~eL~i-~al~o~, .~., ~. i~~o:iscot, ~.~~.~. x~~ieixard, ~. ~eatri~_ ~,.
cadet, ~'. ~~a&~to~, 1. ~err-~'on-re; ~. ~iLroboe~ek, ~ . ~,err°aarehar~d, ~Y~d ~. ~~aairgai.
~.~39~. ~de~~~~~°ir~gs-r~ediat~~d c~ta~ase gene transfer rPd~ees o~idar~t stress in ~~arL, porcine ~L~d L~at par~ere~ti~, islets.
~~~~abeto~.o~ia. Vii.: ~~93-~~0.
Mann~~c~a~is, i~., ~. ~~i, °i.~. ~.v~der~, l~_. ~a~ottc~; ~~. ~fv~eco, arLd ~. ~.obbira.s. X000.
re~lerLtion ofbeta cell dysf~r~ction a~~d apoptcbsis ~.o~i~mtion irL
da~~r~~~.r~ asie°a by aderLt~nirai ;~er~e transfer of tie ia~suls~a-liife ~ro~t~L factor 4. ~<:~~~; I~~°. '~::~~~~-~~'.
17. ~~drea Ice, z~iir5ce~~r arid ~.~:~ ~~,. ~'cidmarg. 2~~~:. control o~'ceil s~rvi~al ~y ~~F
si~~aallir~~ pat~~aays. ~ro~%vtf~ ~orrrLO:~e and ~~~-~ r~,sear~;~~. $ 5.~~-19°?
~alCae ~, ~ido ~I, ~~cciii ~. ~~~~ sec . istir~ct aL~d o~rerlappinn ~.~~nctio~~s of insrair~ and ~a~p'-~ receptor so ~,v~ciocr .c~v.;~2~~):~?I~-~~.
19. ~!ioan S, f~ic,~~~a~~.r~ ~ , ~7-~0 ., ~a-~:~aar ~, or~4~~~~e ~,_~, w,r~ecal :~, aylin~ ~.~.
~~~~ ~~ai. ~a~Sdz~LLi-~1~CG' ~r~~~~ i~.~ti~r re:.~t"rla~~"s ~~;a~ ~~Ile rLILFL~za~ C~etISL$y l~L ri~i~e by ~3Gt$k ~rO~Jv~i1 horz~one-deperLdent and -indepe ndent ~nea,~La_~isn~s.
~,r~docriL<olo~y.;~~4~:~):9i9-3~i 2~;. ~a:~ter ~. 2~~ ~ ~~~;~t ~~~s~Ik~a-ii~e ~rov~~t~ factor ,;~~~~'~-bin~iiL~~
proteins:
z~ateractions ~~aitl~ ~Gfs arLd intrinsic bioactivities. ~L~ 3 ~I~ys~ol ~L~docr,not Ietab.;27~~6~:96 i-7~.
Zi. ~'etri~ ~, ~elj ~'~~, ~~-any ~., i~~c~o~~aid'f~,, f~ear~ a,%~,; ~~;ii~ , ~:.i11 .~. 1999 ~a» .
erexpressiorL o~°ir~s~iirL-ii~e ~~w~~factor-%~ ire transbenis; :i~.;e i.s associated ~.~it~ pa~mreatic islet cell ~y;~Prplasia. ~~~docriroso~y.;l~~m;~):~~~~~-~3.
'~2. obitaiile F~, ..1., .eL~le~ 1~, .~ser~berb ~, 1-~afz~ J~ . ~~~~. i~f'-il allo~%~s pro~o~°~ged blood ~l~cose norL~alization. ~~it~ a red~,~ced islet cell ~~ass trarsspia~nati;~r~. ~~~c~~~~aa~~~~~%.
2.j. ~rrLit~, f.~., ~.. oseL~, ~,. Vila-:oa~-off, ~.~. ~~F.eir, any ~.
~~:~raner-~.~leir. 1991.
vla~i~.FIGeC~ L3~sLFl~ii1-illCe ~roaL? factor ~ ~~I~e e34~3ressloll L~2 rC~,4~Tt,Izt,rat:~?'L~ ~c"~~ ~=ai'L;Lrz;s'~.S. ~v'ad~ l~,ICL'~~'.~~C'lt~~'CZ
2~. ~vorge, 1~., ~,. :'~r~~.LSO, ~~:. ~ aseilau, ~. ~:osta, .;a.~::.
~e~ed~iaa~, arLd ~'. ~osc~a. 2~G2.
:~e~da veil e.xpressi~~z s~f~~f-~ leads t~ re~,o~er~~ fr~~~ type 1 diabetes. ~
~'1~~~ I~vc,~t. 109:1 ~ ~~-~4 .
~5 '~5. ~.i~, ~~L, ~. ~~air-~'~a~ce, ~. ~. ~.,ee; and ~~%~~.~,. l~o~~re, Jr.
:~~~0~,. ~ctivatior~ of phosph.atidylinositol ~-lCir~ase cor~~t=h.utes t:~; 3~s~li_:~-like grow=el~
fac~.tor ~-media~ed ir~hibitior~ of pancreatic beta-cell deatl~..~'~~'~cr°i~~i~,~%. 1~.~:~~(~~-1>.
~ir~ ~', f~~~otte ~ ~~, ~~~~ c~~r ~iV~, orb~att ~a~. 2J~2. e~r{~lcc~~er~t of are irrgrr~~noprivileged site tc prolong islet allograft s~ar~rivaa. ;ell 'frargsplant.;i ~~~~:~~.'~_~;~_ 27. I~~ca C~, ~alvitti I'~, I~Ie~ i ~,~~i, eccl~etti ~, ~~,pitarn ~, ~,~sta ice, ~rageletti ~, fanelli ~, ~ru~etti ~, ~alaf~ore ~.0 2f~01? ~Vov. Sertoli cell--ir~d~ced reversal of ~.d~~ia rat pancreatic islet hate-cells into fetal-li4ce status: potential i~plic:~~or~s foi° islet tra:
spl~.r~tatior~. ~:~ type ~ diahetes r:riellitus. 3 ~nvestig 1'~~ed;~~~~~;:~~1~-~.
2~. ~ ar~g I~. ~lrigl~t fir. -~ X99. ~ome~caps~~lated of ~~~~oii enriched ~estic~ziar cell fractions f~r~-~°~c;r prolongs fis3~~-to-mouse islet ~erograft s~~~-vival.
'~'rar~splai~trrLior~ ~',~; 6'~~r~): ~~~-~Zi~.
29. ~ngeliqne ~~cray, Iic~,~le ~3locl~.el, etsia ~:~ess, ~~~~offi~e~r ~.
~~.arr~~or~d, ~~ubert ~erard, ~e ~~rard_ ~~9~. ~stablisl~ent of ~~ o~ase Sertoli well line prodvcin;g rat arsdrogeta-hir~ding po~:ein ~~~. Steroids ~~: ~~~-2~'~.
3~. F~arr~ ~. ah~.to, saes ~~enry, r~~it S. i~~rar~g, ~~ai;rr~a Sa~~e,k, ~~.a~iel f raga, ~al~.l~ I~ctb, ~sa~na gaher. 20G~. cationic ~,ipid and ~s~iy~~r~e~~-used gene ~~~li~re~~r to ~-~urnar~ ~~.r~cre~tic islets.
P~xoieccalar ~'herapy ~?~; ~9- l ~G.
Pierre ~. ~erL~ao~, ~'wisLit~e I~:ao~~isc~i~, f'hilippe l-~re~aost, ~;rac olla~ad, Serge ali~~.i, ~ad~wiga ~~rol~ocsek. ~9~7. ,Star~dardi~atiov of pr ocedur° for er'~aci~::r~t ex viva gene tr~.nsfer into porcine pancreatic islets with catio~iv liposo3~~e,~~ ranspl;~~tati~~a~
(~3~7~'<~~~1f~3.
32. ~--~ '~ ~,akey. '~0~ ~ . ~o~x ~iiraa tr ar~sfec'~io.r~ of intact lF~~,ncr eratic islets. ~ r~.nspl~.r~tation cell;
i(~:697-7~3~
3~. Le~ic~nd, ~'..A., ~. ~'essie~, a~~d ~.t~. :alle. 1~~~. ~ar~titacive rc~ethod for tl~e eval~atioa~ of bioicrocapsvlo resistance to rnecl~a~.ic~.l stress. ~~~c~tw~~~c~i'~.
t7:~;~'~'~~-1(a~.
3~. t~obitaillc, ., 3,f. ~a~~a~~~~:~, F'.~. ~ ehlc~nd, lh~w. ~,ayr~o~.~re~x, "~~. ~,epage, ~~d :~.. I-~alle.
19~~. Studies ors small ~<~~~ ~icrorr~,~ alginate-poly-~~,-lysine rrai~:,roc,ap~~ales. ~~~. ~ioc;oa~apati'oility of smaller versus standard g~nicrocapsuleN. ~~~'~~~r~!~l~~e~~ ~~.~. ~~:1 l~-~~_ 35. ~alle, J.~., S. ~o~zrassa., ~.~. ~ebls~nd, S. ~h.evalier, . e~.udry, ~..
~~apdelair~e, S.
~oa~si~eau, w. Sai~tor~ge, and s. f : a'ah=.. 1 ~~~. .~rot.ectior~ of islets of iJ~.ngeri~a.r~s from antibodies by ~~acroencapsulatic~r~ wit~°~ alginate-poly-~ mlysia~e ~er~brd~anes.
~'~~,x~soc~~~~c~~~~~a~. 5~:35(~-~.
3C~. ~.ebi~~ad, ~.~., ~. Si~~ard, ~~1. ~enavy , t~. f~ochele~.~~~, ~.T~i.
~.et, ar d ~.~. ~~;lle. 199.
Studies on s~aaller (approxirr~ately ~ ~ ~ ~ic~-oa~~ r~.icr oc~.psoles: iV.
~e~.sit~ility and safety of ir~tra~~epatic irr~plant~.tior°~s of srr~all alginate poly-~~ ,-l.~sine t~~icr.o aps,~~les. ~~~1 ~~~,~~.~,~a~~~s~~. ~:3~ f-3 ~.
~~c~~e~aca~~ p~~~' i .~~tt~a~~t~ ~~~t~atagt a~~~aw~~:.tg a~~tg~tgtg taaa~ga~:~~ gga~~t°a~~.
aaatga~~g~
a~~tg~aata aa~c~~a~~~ gga~~ta~~a aa-tga~~:g~
a~<=tg~aata gtgtt~~.~~t~ ag~~t~attagt ~.~~~~w:.~t~tg a~~tg~tgtg taaa~a~a~~~. gga~~ta~~~~
aaatga~~g~
~i~c~tg~~.ata ~.ac~ata~~~~a t~~gt~gt~ vt~a~a~~.~~ tt~ta~~agg ~ug~t~tg~tt g~:~a~~tt~ a~~ag~-~~~.a ~~z~~agctgg a~~agaga~~
~~a~~ttttr~&~t t~tgt~~ar~ ~'t~~~~~~~ ~t~.~~itr~~g~~ ~g~a~.tg~i.,s ~~~~.~4ma~~tt.~ a~~ag'.t~~~
~~~~~~.g~tgg a~~~gaga~~
aa~~at~c~.~a t~~~gt~:gt~ ttt~a~a~.~~t~ tt~~~~:~w~~tgg ~.g~t~g~~wt g~tca~~.tt~
a~r:ag~t~~~.
~~~L~ag~tgg a~~agaga~~
~~.t~:g~ggg9 ~tgag~tggt ggatg~.t~~tt ~:agtt~gtgt gtgga~~gs~~g gg~tttta~
tt~a~.~aag~
~ca.~agg~ta ~tttg~gggg ~tg~.g~tggt ggatg~t~ tt ~~gtt~.gvgt gtgga~~g~g gg~tttta~
tt~a~.~aag~
~~~.~agg~ta ~tt~:gcgggg ~tgag~tggt ggatg~tfwtt ~agtw~:gtg~ gtgga~cg~.g ggg~tttta~
tt~aa~aag~
~~~.~agg~ta tge~~t~~~.r~~ att~gg~.gg~a~~t~aga~ a~ng~ attgv:g g~.tgag~:,:tt gcat~~ggag ~tgtg~at~tg aggaga~mgg ~~gg~t~~ag~ att~ggagg~~.~~t~:a<~a~ agg~artg~~:~g~.tgagt:gtt g~at~~ggag ~tgtgat~.tg agc~~:ga~tgg tgg~t~~ag~ att~ggaggg ~a~~t~a~~a~ agg~attg~:g gatgagtgtt g~:tt~~ggag ~vgtga~c~tg aggag~.~tgg ag~agta~tg tg~~,c~a~tg aag~~t~.~~a a~.g~ag~~~g ~t~t~.t~~gt g~~:~ag~g~~
~~a~°tg~.~at g~~~aaga~t ag~.tgta~tg tg~~c~actg aag~~t~~~.a aag~ag~~~g ~t~tatc~~~t g~~~~ag~g~~
~~:a~tg~.~.at g~~~aaga~t aga.tgta~ttg~~~~a~tg aagc~t.~.~ ~ag~~g~~~g ~t~tat~~aa~:~ag~gc~ a~:a~tg~.~at g~~~-!tit ~~g~.~gg~~g ~~~~~~~g~.~ c~~,~.~~.~~~.g~ ~g~gg.~,g~c~. ~~gg~,~~.~~ g~~~~~~~g~.
~.~gt~gg~9g ~g~W~~~~~~~
~.~c~~~.gga~g ~~~~.~~tg~~ g~.~~~.~~~s~~ ~.gg,g~~.~~6~~~.gg~.;~~~~~ g~::~~~~~g~
~.~g~gag9 ~g~~~~~~:.~~
c~~~~~ ~~g~~~c~~~
ggc~g~~ga~~ ~~g~.~~
~~,si s~c~~~r~~~ c~darice I~~~~~ ~d~~ sv~, ~-s~~~) r s~c~~e~~~;~~~ ~~ir~
s~r~s:
~co~~ _ '~~'~ bits ~3'~~), ~xpe~~ = f~.~'~
~d~r~~a~ta~s = 374r~75 (9~%) ~~r~r~.d = ~~~s / ~l~~s~
~ ~~t se~~.~err~~ ~~dar~~e ICS-~<-li ~~~d~~ s~~dr~~s~~~.~) r s~q~~~~c~~~ ~rvr~
~~~~4s~~s:
~c;or~ = 500 bits (3I2), ~xp~c~ _ ~- ~ ~~
~de~~i~i~s = 3~~r3~ i X95%), ~a~s - ~r3~ i '~ ~'~) ~~~~.r~d = ~~~~ r ~r~v~
,~~~~e~a~~ ~od~~:~ I~~-I:
t~~c~.~g~t't ~~~a~.z'~~~~~ ~~~~~.~~a.s~.~cs. ~a~~~~.~tgt n~.~t~s~~g~~:
t~~g'~t~t ~~ I c~ttcac~t~g ~~~t~at~.~t ~.~~:a~t~tg a~t~tg~ttg b ~4I tga;~tgg~ ga.t~t~t~ .~~'~~e~tt tc~~.c~a~ c~.~vtt~ta~
~tc°~~r~~~~c~~~~~c~~s ~~I
~,~1 ~o~~~ s~or~
~eI .gc~t~~~ac'~ac~agaa ~~~.~:~~~:. ~.~~~~aggat ~:~y~~tttg~tg~ ttgag~a~.~.~:
a ~~~I tg~~.a.a~at ~gaaa~a~~t ~c~~aata~~ ~~.t~.taagt ~~s~.ataa~:at t~.~a~~gatg ~~~ I ggeat'~t~~~. ~~aatgaaat a~~~~.~gv~ a~ ato:y:.
~~q~~~~~ ~~~~t~ dI ~~-~
~,ef o ,~~~~~ea~~es ~~'l~we~ ~~3~~~',s ~~e~~z~a~ ~"~~r~ ~a're~~e~f ~~~~s~v i~asa~l~a~-~z~~ ~~~~th f~~~'oa°
~~r~e~a~~sn~se'~oii ~~, ~torra~~r~ I~~.~, ~~oi~g ~-, I~~ii ~,~~, l~v~i~i~
~a.hds ~.os 1~~6 ~~~ ~~;~~.t~o~: ~~~~-~s~
o ~~s~iir~-Iiic~ ~ro~rt~b I'~~c~or r~~~i~~~s ~~~is ~or~~ ~~i~~~r~i d~~sr~Jr i~
r~~~~ by both bro~v'ch hor~o~e-.d~pvr~d~r~~~ ~~d -i~gd~;~~r~d~~~ rr~~;i~h~.r~iss~ I~,~ohar ~'t, oha~~~~ ~, ~o ~, l~.z~~ar ~, I~or~~h~ao ~,~, l~y~~~dai ~, ~ayi~nic ~:~p ~r~do~rir~oio~~
~0~3~
~~/Ia.x; I ~.~.(~ ):92~-3 DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVETS
COMPRI~:ND PLUS D'UN TOME.
CECI EST L,E TOME 1 DE 2 NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadien des Brevets.
JUMBO APPLICATIONS / PATENTS
THIS SECTION OF THE APPLICATION / PATENT CONTAINS MORE
THAN ONE VOLUME.
NOTE: For additional valumes please contact the Canadian Patent Office.
I
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~/~~ d'~i ~rod~~t de ~~sir rev d'~.~arose ~~/~.
. ~~on~.~~ da,ns <o ~~~~~~i:~ ~'~~p~ess9on pro~~,r~~c~~~ p~"~~ ~.~ (v~ ono shot ~~,~' d'~iiv~tr~~en~):
~,~ ~~c~e~r p~~. i poss~dk; ~n ~~n~ d~ rcvsay~n~o ~ ~'~~~i~i'~io~:Aq~~
l~~~i~n~~r~ino y~ ~n ~~ne d~: ~
ba.lactosadase. ~,~ proto~o~~ so fait erg ~:r~~xs ~~~~;es ~r~e posy ad~n~~a3~or d~ ~'o~~rka~r~i~e 3' ct~s proda~irs de ~'~J. per t'kr~:~zyrn° '';"aq polY~~n~r~se ~~n~,~i~rogenC~~ p~nd~.~~ I~ rrait~~~~~s ~ i'2°~ s~a~o~i~ d'~..~~~o p~ra~~~~~ion par p~~e>~olloloro~or~e ~2~ :~~ :l~ e~ d'~.~n~ rnise ~n s~sp~~~s;o~~ d~ '~.~1~dins aE ~'z~~~a~~~.
~~ ~t~~~iC9i'i e'~~ ~~ins~~ ~~°~ ~e SWC~~~,~~'° S~ 1°Si~
~~1 r~~.li: ~ :i a~(a ~°~ _~u : ~r~~~~tir~ ~ 1~°~ p~nd~n9. i6~7 ~t ~s~ ca~~~ys~~ par ~~ '~4 ~1~1~ ~~~~~s~.
ro- ~ ~S ~7rot~LlItS Ci~ ~1~a~2~n song '~i"~'~S~'~i3r~'n~S k's~elS ~~5 ~~~~~;rl~,s ~~i:I'i~9v~6~n~~a ~~~~fle.~~'' par ~r1 G~1C5C
~a~ern~~q~e d~ 3C se~ond~s a ~~2°~:. ~z:ir~s dep~~ ~~~ ~0 ~,~ ~~a ~,~
d~: ~~.o~er~~s ~r~.nsfor~~~os es~t r~~~is~ sa:~r des p~tr~s d'~bar ~ont~~°~an~ d~: rr~k~~~~ d~ ra~~aN~z-~ s~~~,~~~~~~~~
~~~n~.~~~d;gr~~ (~~~ritro~~nJ) ~~ gala~tos~
~~~i'ri~ro~~ri~~. ~,es p~~r~s sore gn~~ cps p~nd~~~~ ~ ~ ~°~~~r~s ~ ~ 7 '~ ~~ra.~~t 1e ~gr~l~~r~i~nont dos oolc~nies ~a~~;~ri~'iTl~'S ~7~an~~eS ~u~Cpl2~S ~3 ~~,~~C$:C~Sia:aas~ C~,~ICI~"n~S ~'~:
f~~3'1a~'~~Cir9k~ rk~S>s~~a'i~s~ q~i 5~,1'~311~
~m~a~i~ies par l~r~~na ~r~ps et l~a~~ ~'~ps.
~.~.~. ~~p~if~c~~ion ~~ ~,~~r~o~ion Los ~T p~asrna~iq~~,s ~~o~~r~ens 1_~ri~ pr~~
Ps co~on~~s b~an~h~s pre~~v~es d~ ~~~:xi ,sor~,~ r°z~~s~n~ ~n ~o~~~r~
~;~ns ~ n~~ d~ ~~ ~o~~ta~i~ 5~ ~,~/t~a~ cue ~anarnycin~ perd~.n~ ~~ ~i ~ 37°~ soaps ~~~ita~~o~i. ~Jn ~~oa~.irne do ~..~ z~i~ :~~ ~;~~~~xre: b~.~~~,r~o:~n~ est r~c~~illi pod.dr edgy oxtrairo i'I'~ s~ivad~t le prc~to~oi~ do ~~i~i ~r~p ~~~~3~.~~.
~~~s serios d.o di~estifld~s par les enzydnos do restriction sordt realises sur los _''.~I'~ oxtraits air do selecticsnnor les ~Ior~es posiri~s ~''~'2?l;~IdaF3t -~'ddls~'r'~ d~ bO~lrl~' tadll~ ~.~: a:~~ l>°, ~ioz'?
s~'TdS. ~,C,s Clo;~z~~ r~~.orEl.?;~ ~C3i i~~'tlo p~3sdtd~s Serollt arr~pli~ es par i~faxi Prep. ~,e ~ol~dne ~.~ ba~ter3e; r~stad~;: sera ~on~~~ze ~. -~~°~ dens 1 ~ '% dp ~ly~,erol.
l~.~axi Pr op ~.J~a ~rolmn~ do o~lt~ar~ hacteri~d~ard~ do i~!~iG~i Prop do 5(~~ p,l sort d'~r~oc~l~i~ ~C~O r~l de ~~ I~ardam~JGirde 50 p,~/dnl. t~.pres ~ 6 l~ ~. +3 i'°~ sops a~itatiot~ ~'~~fl r p:~r~), l' ~ plas'rrdariyao baot~raod~ ost extrait s~zivad~t to protocol:. Q~~ ltor 1'~axi Prop do ~~~~~,I'~ ~z . ~,a pu~-ete of ?~w ~~.~ad~~rite d' 1'~T sort a~.alyseos par spectropllotometrio ~ 26th dzdn of 2~~ r~3r ~. ~,a r~at~.dre dos plasr~ides ,rut ardalyseo par ~ar~o serio do digostioTds avoc les eydnos ~coof P~~~.
~. ~.~. ~dzso~qor~ dos sed~,ao3~~;os c=~dad~t d'~_~P-~ of d'~_~~~°-~~
daTds rare octo~r d'oxprossion c~caryoto (pS ;,lontocl~0~
-~o ~octo~.dr p~ZE~ possedo t~d~ ,keno do re,sistad~c;o ~, l'a~~dpic~lii~do ~c~df'~~ro bacted-ionrdo~ o>~ ~ ~a ~eodnycidm ~c~lt~dro daa~ds ceil~go od~caryote;. f~v ~~octo~.d~ a la partic~larite dE~
possedor ~d~o sed~eco h~ ~~d~tra ~~bosodne ~r~tor ~o~~er°bco~ ~t~i porrr~er~ l'oxp-f ossiod~ d~: doddx ~ed~os poor r d1 ~ne:rd~e pr ord~ote~r. Plusze:~rs plas~~.idos sort cod~stridats poor cotta e~:9~.do : p~C.ap'-~~-, p~~~-~:~, p~,~ ~:~~-3~--.~~iF'-~~ of p~ ,~ ~~'P. Los protocoies utilises poor lo~dr c~~dstr~ctior~ sort los r~~dn4s, solos ios di~,osriod~s ~.i~~°erod~t.
'~x~tractiorz dos l'~c dos ~~~~~s d~ oc~o~xr ~~y~;~. ~ :
~,a prodnie~~o di~estior~ a lio~~ ~. 37°~ roT~dardt ~ lao~.dro dads ø~
~l ~bo ~olE~rr~o -g;actio>~~el avec i'o.r~~ydne ~.~i d~ecessiro to tadnpor~ sali~ to pl~.d. ~aiblo. ~,'i~<activatior~ do l'or~mydno ~~st rea?iseo ~c~ ~drd~~tos ~ ~5°~.
~,a do~z~err~e di~ostiod~ se Fait da.ns ~drQ vol~~d~do do l~ ~l po~~ddarft I
~o°~~-e ~ 3~°~' a~oc l'ajo~xt do ~ gz1 d~
tadnpon do la d~o~~ello or~ydne do eli~os~ioy~ ~ ~'abloa~~ 1 ~.
P~ri~ catior~
es prodcaits do dibost:iords soot: sepa~:es par eloct3oporese spar Col d'a;arosv ~~/~. ~,os brigs d'I~
c~rrospod~dad~dt a~ax seer erdces d'idZteret sort ~:=ra:.lo~es sops ~.T~~.
~.',~~I ost p~rifde do l'a~arose par des billos I!-X s~i~ra~~: to protocolo do ~'~~~~-~~I'~r,~. -~,'~~.i3~d est ea~~c~~iLo el;~e par ea~~or~odzt d~ pdens d~ iris i<-~~~~ p~.~.
Il ~lgaG~ora ut arr~pltfaoat~~~~ ~~a~~tor~or~r~o ~o ratio voof~~r irss~r~ est cle ~:3 po~~r 30C~ ~~ cl~, voo~~:~r dares 20 ~,1 ~c, vol~~~o reactionnol (~cP~erna 3).
~,a r~a~tior~ ~~rP I~ 11 ~. +4°~ avar~~ ~'~tro ar=rates liar ina.ctiva~iori ~o Ia ~1'~-~~ Pigas~ 'T~ (.Invitrogen~) por~da~~t ~(3 rr~ir~a~tes ~. 65°~. Un p~oto:,c~3o clc; t3 ar?sforr~:a~cio~ pui=P clo l~:ar~i ~t Maxi i~rops rlar~s 3~~P et 2~OmP de ~,~ asnpicilline 3Ofl ~~,~r?~l (Ir°avitro~~rC~) gist realise ~f~~ d'obteynr yes q~.~arltit~s ~'h~
s~f~sar~tes ~o~r son anaPysc et soy vtri~isatio~ a~~s los protooc~ies ~.~~
trar~sfaoti~~r~. ~,a naturo tPos insorts ,gist v~rif~~~ par dos serios de digestioi~~s av~c l~s ~a~~~~rr~~a ~o rostrio~c~or: l~o~W, ~fiair~t seq=~~~n~ago.
~.~.6. ~eq~or~~a~~ par a~roato SIC&~~:
~,a pr~paratior~ ci~ 1'~ p~azr is s~;q~otl~a.gp r~~oo~~-sito ~~o a~r~:~.plif~oation p ar ~~C. d~ ~~4~ r~~
pPasi~e. r.Jno ~~~ ost realiseo po~zr c;P~aqi:o grin pa.r l~s a~~orc;o se:r~s et rov ~'~~~Ii-~ of ~G~-I~. ~ os conditions sort fixos : 2 rnirr~t~s ~, q~°~~; ; ~~ fois :~~ s~oor~~os ~
q~~°~, I5 seconcles ~, ~0°~ ; ~ a~inut~s ~.
6~°~. ~:,o pro~~it clc P~~ est ens~zco pr~oipit~ ~.ar~s ~e '''~t~~anoP
~:~ ~/~ + acetate; ale so~i~.~rr~ p~~ ~.6 avargt ~'etre sol~~ilise ~aras c~~ tapo~ for~~.arr~ide ~~~ r~~ so~-rant a~.z secl~,:>~.:~;~a~o. ~e seq~on~~.bo ost realise a 1' ~xteri~ur ~~u. Pal~or atoir~.
~~G ~~~~v~~ ~e~~~~~°~
2.2. I. isolation d'ilots ~o ~:ar~~orlxa~s ~~ so~~ris °t clo raga 2.~. ~ . I. ~PZir~.r~i~
~~s s01?r~S sol~$ SavrIfI~~S avaY'at P~ p~'~rW '~~~~rxv ~~i~ ps°s.~~r ~e~s. ~~r~ r a'~s sO~l~: o~'~" owls aed Sot~~~iO~o~
saori~es pa.r iraoisior~ ~~.~ cc~~r apres a.voir c;a~~~i~, P~
ohoPe~oq~°. ~,d~s ar~ia~ka~~: so~~: incises ~. partir d~ leas 0 1'aucl~rr~o~. p~is en <t ~I ,> j~scP~'v~. ~aa~~: ~::~ =u~l~~i-c~ ~~
rr~ar~ie;~-o ~ acc;eclor a~.~ foso, a.~ panoreas ~t 1'ostoir~ac. IJcs visoeros sort alors r~froi~ios oar ~~~o solution d'~a~~ffore~ ~alir~~; ~ol~tior~ (I~,~3~5) froicic avart la Pi~at~ro (so~zris) o~ io '~~.Ia~.~p" (rats) d~ oPzoPf~oq~o a~
~~ivoa~ c~~ duocler~~a~. ~,e ~P~ole~oq~o gist alors oar~~~le ~. P'a.iclo '~rto aiill~ ~o ~~p (so~aris) o~.
cl'~ne oa~~~le (rats) pc~~r p~rrr~octro la p~rf~sion par ~n~ sohxtion de colia~e,nas~ ~ ~ 1 tn~,In~P ~~ ~~~ p'~.4 f~-oicie ~~ pancreas j~sq~'~, sa.
~.istznsion. lv.e pancreas cst aloes, prelo-ve ~r i~nr~~e~zatc:~~er~t ~~is ~a~zs ~:~~~~ soP~.~ior~ froido de ooPla~er~aso ~:~ sir blacc. ~,a c~irur~ie ~oit etro fait t~ plus rapi~e.~~onv possib~~ poor obtenir ~rgc i~onne vialailite des IPots (cn rnoyonrle s~ so~ris o~ ~ rats).
l 2.2.1.2. ~i~estion du ya~~~;r~as ~~. di~estiora est rea.lisee pendant 3C~ rr~in3~tes ~. ~?°~ ~,~~ivie dune agitation d~ l~ seoonde sons vortex.
Mlle est arr~t~e par une serge de lava.~.l dens du a'SS ~roid pule :°~..SS + IO °/a ~~tax ~ovi~ae S~r~~rr~
~~~5~ ~roid a 3~0 x g pendant 2 rr~ir~~e~,s. ~,e di~estat est e~s~ite ~l~zd ~
travers deux tarr~is de ~QO~.rr~ et %St~~m.. ~ a fltrat eontenant Ies ~lc~s est a~ors p~arif.~ ~~ tiss~.as aoir~e~ax payf ~r~ Uradient de F'-:eoll d' ~uroeollir~ (~. t~~, I.~95, 1.Q69, ~.~~~°~~. .tea co~.e~3e eontena~zt Ies dots est ens~;ite Iav~e du ~ieoll par du.
ASS +IQ ~/~ ~'~S event d'~tre rngse e~ ea~ltawre dares ~i~ Ci.~ 1~~~ +y~.fl ~/~ I:~~S ~'~~co~~npl~ment~ (3 t1 rr~irg~tes a -_~~°~~. ~,a suspension d'~~ots est eo~t°e e~s.
r~o~n~re d'~lots ~~ival~~nts ~~ ie~ = aS~J ~,rn de dia.etre). ~,a pLaretd peat-~tre ar~~rne~~t~e par seleetion ruar~oeile po~~r atte5.ndre plus de ~3~~/~ ca'llots.
2.2.2. ~~r~eranon de celi~~es ~ :
yes hats sort r~d~iLs en cellules par iner~l~ation pen8aant ~. rninvtes ~
~'~°~ a~reo ~r~e solution de ~_~S
~. ~ I~ eontenant ~.2~°/a (v/v~ de t~-~aps:~ne et 2?~ d'~I:~~'~..
t,'arr~~t de Ia ~°~aot~.c;n es'~ r~~lasde par I'ajout dune solutiorg froide de ~~S content ~.6 rngi~n~ de Soyl~ea~~ ~.r;rps~ne ~~al~i~ite~r ~S~'~I;~. ~a suspension eeilulaire est a.lors vortexes pendant ~ seoorades avan't iw lavabe et ~rriise en oulta.~re a 3'~°~':, 5~/~ C~~2 dons du ~'i~ tt365 eo.plet + ~~' ~/Q ~~S d~oo:~ple~~~:ent~.
2.2.x. ~ult~re de Ia Ii ;nee u°~ de eellvle sie Sertoti (~~ ~~'~~;~
sell lys-~e~
~,es eellules 'I'~~t de Sertoli sons ouit~v~es clans d~.~ r~~~Iieo ~-l:i~I~ ll +~ °~3 horse Serc~r~z + 2.5 ~/o _~5. ~,e rn~p.ieu est eha.rPbe tC3di5 lee ddux a, arro~s o~.=rs po~~
~err~e~Lre pan paSSa~e y~a.r Sea~ne. ~.,eS
oelluges sont tr~sir~~es et reensernene~es lorsciu7elles ~:.ttei~net~t ~~
~/° de oonfg~~enee. ~La dilution est de 1:25 entre Ia r'lasq$.~e rr~~re et Ies r'~as~ues ~Ines. ~,e surplus de o~:~I~~ies est oo~npt~ sun one eellule de ~ala.ssei~ au ~Ieu de trypan et eorP~eid dens use solut:ion de ~iliev~
os~n~~Iet eonter~ant ~ ~/~ de ~1~5~
dens I'a~ote. ~,a d~oon~~latior~ est rapide par 1'~,~out des ~,eFlutes dee.c,r~~elees ~. +:~7°~ dens un volurrle de r=~aliea~ ec~~nplet pr~al~blernent ?neub~ ~. :''~ ~ 5~/a ~~'2 p,u~darPt ~~
~ni~~utes.
l~
2.2.x. i°rotocole de tr~.r~s;~.~:.=.ioi: des spots ~a transfection est r~alise~ avec di.i I~a~ ' et l~, plasrnidc pCly~i~ po~:x~
la i~:rs~ a~ point. I~i~~ereratos C~~%r7,C~i~c''S~i2s d~:. trar&S~a°,C$iorl sOnw. ~.'SS~e)!C'.~."S:
'~'tdl~~C dE", la trailsl'~.'~t.tl4Jl~ ~J~, ~., ~~.; 'G~. 11~.L"si'..r-'S~, rr'3ti0 Z~1/s~~~' ~ ~i:3, ~:~, I:~, ~:~, 1::~ :2~, ~~~.ra~rt~ d'I~a ~:~, ~, ~, ~ 2 ~~i;, pry incubation ~:ies dots event Ia traiasfection ~fl, 3, 9, 2~- hearse). Iso~r ~flfl icy dens ~n nolo.rrie de 1.
~~i dry rr~ilif~ia de c~lt~are corriplet, tee conditions id~ales sort ~r~ ration 4: i '~~t : I~~ ~ ~~~y ~~bd' preparation I~~~~~'~a' F h~~,~r~ a~.%a~at la e:~ansl'cctioii ~l~i~s d~
tr~ili~v~ corr~pL~t sans s~n~ir tJrse p cV'.battor~ ~t~ ~ ~.~~r°s, d~~ ~~~<~ts ap-~Y~,s ~so~aLz~a~., ~~ar~t le ~~~~~t de la trai~s~'~c-tio~
J11C tr ai~s~C..Cti(7r1 i$~ G~ ~~lCka.~'.rCs C%l ', C.-'sCl'~;:a:, ~, SCr~~li ~ tau;~ de trar~s~ection est anal~~s~ par a~rr~pt~:~~c de l~~aocscoi~co d~
~~'~ a~ i~~roscop~ a.
~h~or~sc~nc~. i=,a ~ia~ilit~ doe dots tra~s~~ct~;~ gist oar~ti~~c par ~.i~
~t~st a 1'acridiiz~ ~ran~e ~r iodurc do ~ropidi'~,3?~.
2.2.x. ~'ransF~ction des c~ll~~ics ~'1~~~ de ~e~-~oli :
~,~s celluieu 'T1~~~. de Sertoli sons trarss~cct~es ~ j'fl ~i~ de eor~~i~ci~cc per d~~~ ~~~E~r~~6/pI~-~~-II. i~~~rcr~tes conditions oi1$ dte cssay~es : co~accr~tratior~s en ~~< ~'~ ratio l~/'~j~~onc&. I.,e protocolo do tr~.~s~cctiora a.~a.ret donne lee rneillc~rs r~s~lt~.ts ost : ~~:r~ ratio i:6 1~T/~'~~~rk~d pour 1 1.~~ d'r~~~t dens 2 gel de ~~ili~~.~ do c~lt~.rc a~ec du ser~~i~ p~r~da.rt 2~ ~~c~.tr~ . ~'~.nal~s~ d~
lctrans~'~ctic~:!~ so pit a~ microscope a ~l~orescence. pour 1'expression de ~~~~~. ~'~t~.de de la ~x_-ot~i.no I~~'.-II
est r~alis~e par I~ot ~3lot si-it 20~3~.1 de 2 i~l de rnilie~ de culture corr~p:et l~r~le~~ ~ u~ours a~:~~s la pransl~ctio~..
2. i .7. got plot ~,e rriilie~ de cu?t~re des c~ll~les tra.~°~.s~ec;$~es esi cei~tri~.z~~. ~. i 2 ~~~~flfl ~ ~. i=,e s~arria~ea.r~t ~sr recueilli et cc~nser~~ a, - ~fl ~. 2t3t~ ~,l de milie~~ es~~ d~pos~ s~,ar one r~~~rnlsranu d~ nitroceilulc~se. IJ~. i~,em~rane est traitee pendant ~ l~oure 30 r~zinutes a~ec. r~ anticorps ~~riinaire anti-I~~-I
o~ a~~ti-I~~'--II d~ iapin, dilu~
au z : iflOfl. ~~is, on ~~azt one it~c~x~atiox~ p~:ndan$ i ~uxv ~fl rr~inut~e.s a~rec ~r~ a.n.ticorps s~cor~daire (dil~.e x:iflOflfl~ couple ~. ~~r~e pert~~~%ds,se. ~a~tre c:da.p~e ir~c~l~atior~ p~zr .l~s anticorps pri~~iaires ~t l~
s°cor~dair~s, des series rte ia~a~e so~~ r~alis~°~~ ~~,r ci~.~
~~~~ ~.G:~'%., ~.C~~~ % '~~~ox~, ~.~ ~~o ~~f~. ~,~.
r~v~iatio~ se fait far o~ia~iiur~ines~,er~oe aveo to pit ~I'~~r~3ers~a~~~. ~ a si~~ai i~ir~eux est er~svite ~~tect~ far ia~~ressio~ sir zz~ ~il~~ ~~oto,~ra~i~i~,~v s~.iva~t ~iffs~~er~t s ~e~s pie ~or~taot. ~Jne a~aa~yse ~ensitornetzic~ue est faite poor a~~ro~<i~~~er fes ~,~a~tit~4~ ~e g~roteine,s.
~5 o~~ ~~tes~ d~s ~~v d~s s~~~~;~~ ~~d~~~~s df ~~~ ~~-:~"~:~
~.l.l ~xtractior~ de 1'1~~~L d;.~ ~oio dsouris ~'o~jcctif set 1'~.xtractior~ d'!=~.~'~'~ t co~tc~,~r~t lcs ~rt~ d~ 1' i:~~-~
Lt ~~~F'-~i ~srese~~ts au a~ivcaaa taepatidus. ~cux-ci r~ou.s scrvir~o~~s do ~asc ~ la sy~tla~;so do 1' I°~c ~,s s~q~c~ccs cod~.r~t dcs ~~F. ~,a fi~ur~ l ~-epr~sente lc r~s~ltat do #'~lcctropl~or~s~ ds dsux prod=nits d'cxtr:~.cti~r~s d'2~Tt fans sn parail~le. ~r~ cor~state la pr~so~cc d~. ;~ou~T prig-~cip~les l3ar~dos d'a~I~
~, ~ Pt 2 ~l~ ~corriparaiso~ avcv ~arq~~ur ~~ twills) qui c~rr~spor~d~r~t resp~~;~~iwor~crat ~~ux 1~-~lT
r:ic~c~s~ar~x ~fjs ct ~~3s. La pr~senc~ d~
css 'oandss proaa~c le b~~-~ stet d~; ~' ~~ _: ~~pr es cJxtr action. 1=a p~~°ct~ d~ws cxtraits ar~alysec par spectrophotorn~trc est sic ~.'~~ st ~.~~ ~~-atio ~~~°~~,/~,~6~) ~o~r dss ~~~.~a~°rtit~s d'At ds K.~ ~.b%~.l st 1.~7 ~~/#~l rsspsctivsrrasnt. l~,'~xtractfor~ d':~lTt set do:~c; bones sa~u d~bradatio~ do c~l~n-cl pendant is protocols d'sxtraction. ~'t set traaascrx= sn I~Tc =~otal par l~~'..p ~1~..
>~ s but cst ds s~lsctioransr et d'amplifs: iss r=i°~TC ds s~q~sr~ces ~~odan~s;s 1'~S.~F-~ s-' 1'~~-F-~~ obtsn~zss par 1~.~'-~'~~ sir 1' t. ~ a ~gurs ~ or~trs bus is protocols ds ~~;aplifis ~$ra ~~lc ayant sans tailje approxir~nat~vs ds ~S~ ~aircs do bases ~,pl~) pour 1'~~s~-~~ st ~0~ ply poaxr i'~~JF-~ sn co~parai,son au ~r~arqusur ds twills ~ ~0 pb ~~ssxs ~-~3. ass ta~ilss csr~respondsnt au.x s~q~zsrtcss ~~odantss ds l'~~F-I et de 1'~~~-~~ p~ubliess (~nsxs ~ st 2) et s~~scti~n:c~~ss par iss a~~o~-ces ds is r~actlo~~ ds ~~J~~. ~.Tr~e dibsstion par 1's~yz~e ds restriction ~~~des prod~i~s ds 1~~o~n~rs dss i~a~~~snts ds taillss ids-tiqucs a~a patron tl~eoriqa,~s ds di~sstiora ~d~rra~ss ~~cr~ p~r~ser~tess). ~l y ~ do~nc ds f~rtcs ~~roba'dilites pou~z- q~s tee ~I~c aplaf~~s par l~~~Z co~-espor~der~~ ac~x s~~iuences codar~tss d~I' ~~~-~~
et ~fe 1'~ ~h-:~.
~m ~~~ d~s ~~~~~~° p~y~~ .~ ~~~~~o~~d~ ~a~. ~d~s ~ ~~ ~t 1'~~~°~p~
~.a synt~~ss d'~n ~rectezar procar;~ots p~.~ _aoss~dar~1'~ ~'-~ st 1'~~~-~~
r~ou.s psrettr~a d'it~s~rsr pus facilsgnsnt st rapidsr~nent lee I~~ da.ra ~~~ ~cctcux~ e~car;ots er~'~tillsar~t ces sites poly i~:r~lcsr. f,a selection dss clor~ss b~.ct~rlsns positlfs set felts par cul~:urs dares ~~ra ~r°sii~s~ s~;secti~des l~ac,'~~racs transfsct~ss st par I'analyse den produits d'exs~ractio~ ~.es l~~:ir~i Preps par ~a~e c~iges~ioa~
e~~~~natiq~e aver I~v~~~ et ~ccWl.
1_,'arnplificatio~ ales clones positifs par ~~~.~i :~r4ps :~~o~s perrr~e~~
I'ob$:e~tior~ de plas~~ide:~ de ptz:ete ele~,~re et erx q~~ar~tite s~~~'ISaa~te po~.~r is pos~rs~i~.e ~~~ ~:~-ot~scole. yes prod~its de ~~axi Preps so~1-~ ~, r~o~veau sournis a ~r~e aerie de digestion par ~~v~~l~ et ~~coqaai co~~'zr~e Ies r~s~Itats den l~fi~~~i Preps. ~,e sequeriqage de p~~.1--1~F-1 r~o~s r~v~Ie I'1-~~Jrr~ologie ~. I~a~/fl aver la s~q~aence publiee de I'I~F-I rnais sors ir~ser~ior~ dean Ie rna~vais serfs ~donr~ee r~on rreserztee}.
~~rc~;~~ p~
~.~.1. ~~r~tl~~se de p~I~~l'~~h I~~~-I_ ~p~~~'-1}; p~=~-I~s~l~-II: ~pl~~~-1I;
et p~:i~~!-~Z~~-I~~-I1 ~p~-I~~'-II}:
~,'objecti~ eat la ~;r~atio:~ d'~.n plasr-ode d'e.xpressio~~ e~car~rote exprir~~a~t a'IGF-I o~ <'I~F-II o~ Ies deazx ~ partir ales plasrr~ides d'expression~ pr~,cai~ote. ~~o~ar ce faiwb, no~.~s ~.vor:s ~,xtilis~ I':~oologie den ext~-~~~it~s cohesives ties I~ apres Iei~r dig,estio~ far certaizxes e~.~~rnes de r~;strictio~ ~scla~r:~a 3}. yes c~~igestions vont orienter et azag~~enter- Ie re:~~err~er~t rIe I'ir!ser~:ion den s~qa~~,rdces codar~tes des l~~' dens le vec~eur en elir~ir~ar~t lea rr~a~vaises ins~r~ior~s sans steps de depl!~~ospl~orylati~~~s den extrerr~it~s ~'. ~,a i<gure ~ rr~c~r~tre I'electrophorese den prod~its ~e digestions avar~t ~;t apr~;s prelcwezr~e~t. bra ~.'~oit gets lea digestions opt biers e~ lier.~, car Ies tailles den prod~its de digestion correspor~denr~ a~zx ~~-agnrer~ts t:~eoriq~es attendees ~anr~exe 2). I,es ~~-~~~s d'i~~~:~.r~t se:~or~t pr~lev~;s:, soi~ : 57f~ pb po;~r ~'I~~'-I ; 7I3 pb poaxr I'I~P -II; 5~'~~ pb poa~r ie p r;~ P~r~pa~°~ pour i'vrasertior~
d'I~~~'--1; ~~-3C~ pb po~~r Ie p~ prepare po~~r d'I~F-II saran Ia s~que~~ce :~ ~r ~~7~:~ pava~.~r Ie ply pr~~;ar~ po:.ar I'ir~ser~iorø d'i~F'--II aver; Ia s~que~ce I~. I=.a selection den clones positi~s pea digestion den prod~.~its de l~~lirci Preps est ,T~alisee par Ies e~,~~es de ~-estricti.on ~coRl, arr:ydo~~:~~ees n~~n presentec;~s}.
1_,'arnplil°ication den ces clo~~es eat realises par ~,~Q rnl de Maxi Prep.
~,'ar~alyse den prod~zits d'extractior°z de;s a:~i P;-eps acs spect~'ophotor~evre ~ ~~Ci r~rn Ft 2~ nr~~ d.~z pl~'r~'-1, pIC~P-~I et p ~,~ LAP -II. or~tre ~~m c~JLgcea~~rati~r~ de 1.7~
rr°~g/~~~I, l.~~f gI~~~I et x.93 g/~nl respecciver_~erat. ~,e ratio de p,~arer~e .~.'~b~~m,'' '~'~,~C3nrr~ donrte respective~r~~~t~t 1.:~2, 1.~9 et 1.~:~
respective~-ne~t poor 1es train plasrrrda:,s, ce qua cor~-esp~~d ~. ~r~e excellertte p~rere. L,a digestion den prod~ats de Maxi Preps co~~rrr~e I'ir~se~-tior~ den irbse~~s provenant ~~e Ia digesaiors de p~;,.1. I_,e res~gtat d~ se~~en~age r~o~s apporte to pre~ave de I'ic~er~tite des inserts corxme et~s~t IC'-~1?-?~ et i~~'_II dens pS
avec Brie born~IOgie proche de 10~°,% den sequences co~a~tes p~blaees ~er~ reco~zpar~t ie sequer~~age den i~riris seas et anti-sans). I=,es s~cluen~a,~es novas ~;orl~r~~~ent ~~ssi li'inse~ion duns le ~o~~ sans des ~1~T
c~.ns le vecteur ~~'i~-~res 4 et ~).
3.4.2. ~yntlaese de p~~ I~~~-I +I~I~'-I
ve ~recte~~- ply a I'~jrantage de po~vc~ir exi::rier deux ~~ries di~'~'~rents ~~~~.oe ~ sa sdduenee I~~~
sons oor~firdle d'ura rn~~~e prorno'ceur : ~'~I. ~~ela noun perrnettra ti'~c~dier I'i~~ter~otion des deux I~~ et de eomp~.rer leer ef~°~ca~.it~ dens i~. protec-tior~ dcs ;ilc~ts. ~Jn p~rotooole sirnil~ire est r~alis~ pour I~, s~rnt~~se du pl~srnide ply I~~'-I + ~~"~~_II. ~..a digestion dpi pl~~~'-a se I'ait pa.r I~tl~e~/ gall pour Iibdrer ~~~'-I et ma digestion du p ~ I~~-~ par :~~I~eI/~aho~ pour prelparer le ~eote~ur a I'insertion d'IG~-I.
~~nnexe 3). ~pres oon~irrnati~n des ::;i~~nes posixil='s par digesfiion d~~s prodazits de ~~ini prep par ~co~ et ~arr~, ceux-ei sont an~plii~es par ~fl~xi r re,p et .na~~s~s par le speetrop~otornetre et des dibestions ~fa,gure n). ~Jes produits de digestions so-nt ide~~ti~ues a~~ patrons t~~~ori~ues ~r~,~~raexe 3), ee tlui ~;on:~yr~~e la tonne orienfiation des inserts dens a ~recreur. ~e sdsluen~a~e c~.es inserfis r~'dst pas g~~~.lis~, oar noes s,vor~s utilises les ~n~~nes 1°~ cque pour Ies plasr~-~ides pI~-~'-I et ply-II ~ui onfi ate s~~eenc~s.
~e resv~ltat noes ~:~ont~e due les pr~-~fi~~;oles c~e ~iolc>gie ~raol~eulai<v rfi,=alisds sort efldx:,°aces et ont per~uis la erdation de plasides d'expressio~~ cr~ea~-yote poss~dant les s~~raer~ees eodar~t de i'~~'~~-I, de I'I~~-IlL
ec de I'I~F'-~ + I~~'-II.
mn s~~~r~ d~~~~~ ~ p~~~:~°~~~ d~ ~~t~~~s ~d~ ~~~s ~,e protoeoie d'isolation d'~I~ts de rats et so~.~ris est ~tnw tape i~npc~r'~~.r~te d~.r~s 1'.~ tr~,r~splantation d'ilc~ts.
~ette tape izaer~ee directerne~~t Iw~~ ~rial~ilyt~ et le~f:~°
~'~~°efiion~~~.lit~. L,'o~je~~ti~' est d'o~ten.ir a ~?us d'llots possi~le a~eo la ~~eilleure via~ilit~ possil~ie. i°~o~s ol~te~~ons des Aei~deents de I'c~rdre de 300 ~ec~.
par par°~oreas de souris et 1200 ~ec~ per p~ncr~~s de raga. ~~~
~r~,hilit~, r~est~e p~.r I~ efi ~.~, des dots est tcsu~ours sul~erieure ~ ~~ ~/o dens les eontrcsies n~~,~.til"s des firar~m~eotions, soft de 2 ~ 5 fours apx~s I'isolation.
l~
~u I~~~ ~~~~~~ ~°~~~~~ ~ ~~°~s~:~~~ p~~ ~~~~~ ~s ~~~~t~ ~
~~~~°~s ~ ~°~~~~
~,e but est d'obtenir un taux d'exr~ressior~ du tr~,vs~.ene suftlsa~n~~°~er~~: di~vc~ poor acciu~~-ir u~ e~fet protecteur le pl~zs important possible s~.~r les dots de I,s,~a~erh~.ns. f~-r~°~~re;~~:es condftior~s. de miss au point soot testees : eluaratite d'II'~, radio liposon~es/~~~'.%-~~, terr~ps d'g~~cubatgon, terr~ps de culture apr~s l'isol~taor~ et event is trans~'ectio:~a. ~,es r~sutt~.ts de cette noise au poi~~t pd~ur les dots de rats sont repr~s~r~t~s ~ i'a~~nexe 6. retie f~ise ~.u rorat a p°r~~is po~:r 1.
~~milleure cor~~'ition ~7r~ tau~ de transfectidn cue ~~%. ~.:es ~~~urES 7 et ~ r~ao:ntfen$w des dots d°
rats ~:::~ans~e~,t~s; o~:~ .constate la presence a celluies et/ou d'arnas cellulairo expri~~ant Ie :~e. ~n peer voir c~~se po~~r ce protoco:~e_ la ~r~ajorate des Mots soa~t tr~nsfPct~s au rnoir~s anti fois ct ~u~ cert~.ins dots sort vra.nsfect~;~ ~lzas de cin~ ~'ois. t~es r~,sultats ~ta'~leau ~~ des trar~sf~ctior~s corresponda~-~t ~~~a~ p~oto~~-;:~pl~ies don~zt~nt 6~a °~~ Pt ~~°'° d'ilots transfectes respectivernent a. cinci et sip ~o~ars. ~a ~~ure ~ .rw~Ie la pr~se~~ce de poirns l~.~mineu~ de plus iaible intensite pouvar~t titre des ceil~~ies tra~sfect~es. ~,'~tude de xa via;bili_t~ par pp et ~~ r~alisee en parallels a i'~t~de du ~~~.a~ de r~-c;.r~s~'e~,tio~z r~.ontrc ~~.~e le protocols d~~ tra~.;~fectgo;c~ n'affecte pas la viabglit~ des zlots ~tab~eau 4). n ez~ cor~~;Iut c~~ae le protocols de tra~~s~ecti~r~ r~'ai~ecte pas la vzabilite des dots et pernaet i'e~g~ression day tr~.nsg~ne dens plus de 6a~ ~r~ % ~le.s dots aver anti ~~aa~or=°.t~ de ce~~-ci transfect~s plus dune foil.
i=,~ transfection des cellules ~I4 est =~mporeante co~~~me outil pour i'~t~dc de la production des prot~ines ~~~~mI et I~~~-~ par dot blot, ~r~ais ~.ussi po:.~r des ap~l3catior~s iztL~resS comrne la cr~~:~ion de lign~;es stables e~prrr~tant ~~--~ ou ~~~-i~ ou l~~s deu.~. i~es prearniers r~sultats rnr~i~trent anti t~~~icite irtaporta~~te du ~~'~'1~P ~ la difference du ~'ubene6 ~~i a2e s~;mble pas aecter ies cellul~es ~I~~~. ~,a toxicity du ~~~'~~ se traduit par anti inort inapox-~~.nte des eelx3.~les vasibles so~.~s ~~r~ic~ oscop~e et par ~a co~.oration au bleu de ~ou~~assie. ~.,es r~s~itats pr~~irrir~aires donnena: ,des ta~~:~ de transfection de l'ordre de 30 % pour le ~~~avec ura ratio de ~ :6 pour ~ ~,~ iii et ~~3 ~ifl a~rec un ratio de ~d :6 pour ~ p,~ d'2~ po~.~r le ~u~er~e6 trois ~oa~rs apres la fin de la tra:~s~'ection.
~~ ~~s~ ~ ~~~~~~~~~ ~: ~a p~~~:~~ ~'-~~r L~ ~~t gist ~~ v~rifi~r s> les cell-~l~s tr~nsfoctees paY~ pT~~-II s~cr~t~a~t i~, tr~.ns,°~~~ne ~t si ool~i-of est dens one oonfarrnation r~can~~~ao par ~.a~~ ant-ioarps ~a~~~~or~;iaa anti-I~IF'-Ih.
T~Ta~s ~~rons ~aur vela realise pan pat riot aw~c le rr~ilie~ de oultur~ o~ ~oll~.~~~s '~T~I~ ~rar~sfoot~~;> par ~a,~ rn~l<~n~e pF~~-Ilf~~ageneE et plop'-~IW~'f. I~~ f~b~re ~ montr~ i~, roa~altas dv I~o~ ~Ios:. I)n ,b~z~t cansta~~c.r ~~o los ~~~za canrrales positifs rdalises av~c TO~r~n d'ICIf-II a~p~raiss~r~3t ~.~~sss iaion ~~n~s 3~
~ ~~ ~~o aans io rnilie~ de ~,ult~r~
~a~plot. F,a lirr~ito ~e deteoti~a~ vst a L~into ar~ro~ 1~ oantr~lo pasitif r~aiis~ a~m~, 1J nsi'I~F-II dens du ~~3~ ~i~i n'apparait pas. Las oontr~l.~s ~~~~atil~ ~~~eevt~nt: en ~vv~on~;~;
l~. prdsonoe: ~'I~-f~-II dens le mil~ou de o-ulture ocsanpl~t des ~,eli~ies 'fT~I~- ~v~c ~~~ s~~ ~~n rnais pas ~t~.ras to rniii~~_a ~l~ o~~alt~re s~r~s s~rurn (~r~sonc~ ~a'I~f-II ~a~as le sdra~rn~. ~.-~ c;onstat~ ~~:a.~;s~ ~an~
pracl~~;tiar~ ~~as~.le ~~'I~~-II par l~s o~ll~l~s ''~T~~ s~rva~°~t clo ~antrs~l~ r~d~atif a is t~-~.nsf~~=iar~.
L_'~t~.~lo donsita~-n~tri~~e rdvelo o~~; 1'imtar~site est d~
d~~x fois s~~perie~~r~ ps~~ar l~s ~oll~i~,s '~'~'~:4 rra.nsf~ct~;os par pI~~T'-:~.i elve pour les c~~l'~~~les 'fI4 non transf~ct~~s (-~aialea~ 5~. I~o plays, 1'x~2to~sitd r~ fat I~lot pair les ~,oll~los transfoot~os ost s~,~p~rie~ur~ ~
1'int~nsit~ d~ oontr~le positif (lOt~ n~ ~'I~~~'-'-l dens d~ iii~~ a~ro ~~~
se:~~a, ~e p~zi signi~e d~.ie los coll~l~s ~'I~ transr'°~otdos pra~l~aisor~t pies ~~° TC~~~ n~
~i'I~,~~~-II. u. p~~Lt dano c,oncl~r~ ~aae los o~ll~les 'fc+ do ~o_~ali trar~sfeoteos par pl~~'.-I~k. prc~d~:zs~nt plr~s d'I~~-II
cIE~~ l~~s ooll~~l~s nova transfe~t~os ~ (yes co~contrat:ons approximativos s~zp~r~~~,s.res ~. 5~~ n~l~l ~~~ barr~p~~~'aison a~.~ oe:ntrt~lo pasitif (I~at blot realise a'm~; 2~i3 ~.l d~ ~. ml tatal do ~~aiii~La. cl~ c~lt~ro c~c~rnpar~ ~, Tp~ ng d'Ii~I~~II). ~;e r~sazltat gist or~co~:~ag~ant of ~~orstre ~a~e io plasan:~~ pI~-p'-I ~ p ~rFc~~E
1'o~:pr~ssia~'< d~~ trnns~~rke I~~I'-II.
'?.0 s ~~t~to etude se sitrie dais to cadre des r~c~°~~ro~iio;~, a~aor~~~s sri r la prop octiorF des ''°iiots do f,a.rager bais corers I'apoptose of is necrose porir 1'ain~iior~.tior~ des pi°otocoles de trarr.spiar~t~aioi o~e~ ie diabetiqa:~~ do tape i=. ~oilri~e ions I'a~ora v~~ dens i~. ~eev~~e nee iitt~rar:~re, differs fits ia'ooratoir; s ~ tr~~rers ie rriorde, ~tiiisait differente.s approc'ies, ~ta~~~~e~~t ie. ~=~o~~ei de diriinrier i~=
r~~oi~bre d'ilots yar transplantation, i'objectif ~tai~t d'obtenir rin pancreas po3~z ririe transplantation.
f~e~~eo~ap de pietas sont ir~~,~estigu~es dens rafts res;~iero~ie : differeites prot~i~es a~~x capacia:es proteotris;e, diffe,rents r,~o;~eizs de traisfection, is i~ioroe~oaps~iatioi, ies trai~splar~tatiori d'~øovs aiio~;diiq~es on ;c~nog~r~iq~~~,s. l~o~.~s a~or~s cboisi de trawaiiier dens Sri prerr~ier temps a~~ee ies proteines de is fa~~iaie des ~~~p" et ~n ~rotooole de transpiantation par liposoi3es por r pi~.~.se~rs ~-~isoras.
m I6 ~~°~~ ~~~
~orrie ions i'avons dens 1'ii~trod~otio:~, ies I~~ ont rii bait ~o~:vc:~ir de i~roteoti.o~ pour dimin~aer i'apopto,se et is necrose s~.zr ies dots oo~r~nae srzr d'aritres t~pvs oeil~iaires. f.'aaantage de cette stride par r~.ppoa~ ~. ri'arirres tztilisant des proteiries aiti-apoptotiqries coiri~~e ~~ci-2, p35, ~'~,0, ~~~s3 est i~otai~ii~erat ie rriode d'a~,tior~. ~i effet, s.'aoti~~ation d'I~'-~~ indriit is rx~obiiis~:~ioi de differents senders celia~laires, a~ssi b~er~ a~ ii~ea~ oytoi~ias~~iqa~e q~i'a~~ iivea~
rr~~i.~og~naq~,ae. p a fonctionnerrient d'riie ceilrale est coa~iple~e et 1'ii~bibition on F'aeoengz~atio~i d'~.an sei~itier s~~uli~iaire coridriit dens is i~ajorite des ~aS ~ ~'aCtd~%ati012 ~1'i..~llC', joie Cse 53gia~isatlo~.'i N~aia~I~l.~' de i~o~tlpeu'is~tloi~i '.old d, i'l~a %bitloYi d'riii effet v~i~eessaire art fonctionierg°aent de is ~,eiric~ie ~art~eie~. g'ar exeizpie, b~oqa:er ies effete des e~to~ines aia.
' ~~i~; sari de ses effecterirs e~coplasrniq3ues pe~~ s~~~~ria~~;r ~i effet '~;~,ri~f~q ve dø c.e?lemc~i - ~ r , ;~: ~a . ie~ei~pie oaf'-.
,J;se ~~. i_,es ~C~~' sont secretes et or~t ~i~e aot=~rit° ~.~ssi bie.n paraerin~; q~~°a~~:~orir~~; du i~ is ~~atior~ sur des '.~' r~,cepte~rs extraceiirilaires. ~,e fonctior.rierxeit ~~e is c.e Ii~ie i~'est do~~~c; has r~°~odifi~, ii s'agit j~~iste de i'activation d'rine joie bioiogiqrae iri~i~cei~te ~, is celli~ie az~ ni~ea~
cy~topias~~iiq~ze er ivcieaire. I~Tous pori~ao~is faire 1'~oi~~ologie eitre i'acti~fit~ proieccrice i:;~diaite par yes vr~nsg~nas exprirnait ies ~~-~ et i'activite protectrice indriite par lee ~_~' ari coi~rs dye ~,~veioppPe~it ei~br~oinaire, ~~oc~s n~iriois rive activite protoctrice endogene sacs rnodi~ne~° ie foi~ctionnei~er~t iori~mi des celi~tes Vii.
~,a deu~ieir~e raison est i'utilisation d~~Es g.e laboatoir~; de is i~icr~~yeicapsriiatioi. ~'n eft=et, is poursuite de cette stride a tree co~i~ terrine porte:~~-~ sr$r i~. tarispla:r~-~:ation d'~lo~tv r~iicroericapsraies transfectes par ies plasi~ides d'expressioi ci'x~~. ~3r, dew. et-aades oi~t r~~o~~tre qrie la ~:~~icroei~icapsiilation geiere rin rnic;ro ~i er~viror~rler~er~t autour des hots. ~J'ir~t~rvt de rravaiiier ,~,vec des z~~>i~~cu:a~s protectrices secretes Pst donc i'ohtention d'ur~ ~radie~t de cor~ce~~traticr~ au~c~~~r de i'~iot. ~.d~rsi;
~~~r~~e si i.e norrihre de ceiiuies tra.~sfectees, dens un giot est faihie, <'activite prtctectri~:e pour~-a ~~re pari:a~~e par toutes les ceiiuies de i'~iot. ~e qui noun a~nene a discuter d~~ c~oi~ de is trar~~sfectiora.
~v ~u~°~~ u~~ ~-r ~~~~~~~~~ ~r ~~~~~
~.or~~~e nous venous de is voir, l~ r~icroeracaps~viatio~ perr~~et is b~~~ratao~~ d'ur~ ~radier~t de corcerntration des ~~~' secr~t~es pa:r ies ceiiuies tr~~sfectdes. ~~~ous :,~e so~nt~es done pas obii~~s d'obterzir url taux de transfectsoi~ de ~~0% ~u sein des ceiiules de i'iiot.
yes hots soa~t r~aturelie~ner~t diffciies ~ trarisfecter, ~ sont des ~~as~ ceiiuiair~ er~tour~ d'u~~e capsule clui f~~~j~ obstacle a. is tra~~si"ectior~. ~a~~s cette etude, noun ~~~orrtror~s due :~oa.~s sornrnes capahic;s de transfecter des hots anec plus de is rr~ajorit~ des dots trar~sfect~s ~u ~t~ir~s une fois. yes r~sultats sprat proette~~~-s e~~ cor~paraxsor~
avec d'autres ~t~des utilisargts des !iposves ~so~~~~ne ~thodes de t~wr~sfectiorz cles dots. ~i_n_si une etude utiiis~r~t du ~,ipofecta~ine et p~~'-~:~'~~ ou p~'~~f obtient d~~:~ tau~ de taarisfectior~ faiblc sur des hots i3urnain tout erg ohse~var~t une au~fr~eratatio~~ de is production .de ~~:~~ de; 6 fois par ceux-ci {30~.
~r~e autre ~~~ude ~vec ie ~ ~ ~..~ svr d~~s dots de pore ~btie~~t un -~au:~~
de rra~sf~ctiorA des hots de 25 %
{3 ~). dais ce protocols Boat pouvc~rr ~tre ar~eEio~~c~, des tau~ de ~~-~0 ~/~
d'~lots tra~sfectes aver ie ~~~'1~..~ et is ~-naiactosidase corn~~e ~e~~e rappor~eur out d~j~. ~~t~ obtenu {32~. ~i est ir~tEressant de cc~~~stater clue l'on sous-estirne ie rende~er t de is transfection. ~r~
~ffe~;; certain:3 ps~i~ts lurr~arse~~ molds i~ter~ses ~or~ prig' en vorr~pte dens le ta~:~x de ~:rarafecti~r~~ peL~venr ~tre de s ceii~xies trar~sfect~es dons ie sibriai est a~~a~u~ par la faille de i'~loi. ~T~a~ai~oit~s, avec ce bia;is positif, ~oo_gs ohter~or~s u.r~ -~au~ de traa~sfectiorl de ~~ ~ 7C3 % des dots tr~~~sfectvs avec ~=ius de is ~~oiti~ de ceux-ci au .pins une fois cor~r~e dit pr~c~derr~rr~ent, ~e slui Esc sup~rAeur ~ d'~r.atres ~tude;s fa5tc~s aver des ii~osoa~~es. l~Tous anor~trc~ns aussi, ~r~e sl ii s'abit d'~tvde pr~iir~ir~air~;, sue is t~-ansfectior~ de ce:iiute de ~ertoii ~'1~4 par p~:~f-~~ conduit a is production d~, plus de ~ ~lrc~I d'~Cl~ -~~ {caicul~sur i'~tude demirog~~~triciue). ~r, ie ta~~ de trar~sfectior~ des ceiiuies de hex loll ~,st de '~(~ ~% ~.°t ies yravau;~ de ~oberi .obitaxiie prat o~tr~ que i'ajout de SQ~ri~/rni d'~~~'-~~ dares ie _r~ilieu de c~.iture perr~zet de b<~isser de r~oi~:ie is ~uantit~ d'~iots par trarlspiantation n~cessaire a. i'obtention dune ~~~rr~~ogl~rc~rr~ie. Sri peut donc s'atter~ci.ro ~ observer un effet protecteur des dots g~icroer~capsui~s transfectes par p~~~"-~~ ~.
:i'aade d'ur~ lipos~~e.
~J~p autre raison est i'absence d'itrzur~c~~~~~c~it~, de is transfectio~r par iiposor~~e. ~r~ eff°t, i'e~p~rience ~~nor~tre que i'utiiisation de vecteurs viA~au~, bleu ~~.e ~:~.pab;e d'iruduire urn ta~~:;~ de ~~ ansr'°ection ~iev~, induit aussi uz~e fore r~ponse irnrr~u~~it~~rgre, tableau :~. fir, ii a ciairerr~~r~t ate d~~montre due ie s~ster:e irn~nitaire per I'ir~ter~~~diaire des ~v~tol~ines ii~flar~ar~~~toires esfi, er~'~re autre, respor~sable d~. ~ornl:~re ~lev~ d'ilots par crar~splanfiafiion. "'e pl~.s, I'~fiztisation de prornotc~~~rs vir~~a~ coi~~e Ie ~l pest voir leer efflcacit~ di~ni~.uer par E'IF'~T-~ se~:r~fi~ ate ,ours d3~ la r~porse;
ir~rn~nitaire anti-virale. ~lotre etude fait abstracfiion de ce probl~me par Irailisatio n de Iiposon°xe. ~I
est vrai s.i~e la capsa.~.Ie poss~de ~n mile protec;teur contre la, r~acLion I~6te ~rers~s ~ref~~sn, a~ssb biers qLze d~~s l~rrr~phoc~ces c-~fiot~xiq~~°s qve des anfiicorps, rna.is poor I'instant auc~r~e ~~.~de n~o~~fi~-e I a r~Ic pro~t~cfie~.~r des r~~icroc~ps~,~les contra Ies particles v hales, a:~ssi bier ~~te vwr3~S brel ~>n q~e ~reffor~ v~:i-~~s I~i~te. ~e iaoi~~fi est d'a.i3le~rs en ~-°'~'"'- ir~vest:i~~tion dens le I~,borafioire d~r f'r ~ea~~-~iWrre ~~Ile. oar cefi~e ~visor~, noes n'~cartons pas. la possil~ilafie de firansf~rer les bones des Iw~~ par des vectr~vrs vir~~a~. ~-~
contraire, Ies ~;t~des faites sir Ies plasrrlides pIS I~~'-I efi I~~-II efi l.'expressior des pro~dir~xes no~ss facilifiero~fi la ~~n~r~.fiion de plas~~ides vira~x exprin~anfi I~~-I, I~"~_II o~z I~~s dc~.~.
~ma ~~ ~-~~~~~~~su~~ ~~
3e tiers ~ disc~fier de I'~tilis~,;~ior~x de is r~icz-oenca.ps~~latic~~, car na~:~x~e si ~e re I'ai has encore realise dens le cadre de r:~on ~t~de dv ~~~icroerrcaps~i~,t3or~, ~e devr~.i, ~.~~ v~~e des r~sa~afi;~cs, c-ornrnencer les protoccles d':ici peg. ~,a rnicroencaps~l,~fiio~ cor~siste ~, isoler les ~Ic~~s d; I_,an~er~~.ns d e~ ap~ticorps et des I~~~pi'~oc~tes c~fio'~oxiqnes to~~: en p=~~L:er~rar~t l.e pass~~e des n~firi~~e~~fis et de I'irs~.~I_~.ne. Le bit est de s~pprie.r la r~ponse irnn~~znit~.ire do I'I~c~ve verses ire-I'fon pr~ser~~:wt dens is firarzsplan'cati~r:. et I~ r~ponse ~~.fio-i~n~~une responsable de is desfi~action des ~=ots de ~.,ange:r~ars ~Iaras le diab~fie de type ~. f,e ~aboratoire d~.a I~r I~all~ fait pai~ie des I~~ior~aiers d~.~.s l~;
d~veiopper=~er~t de ces fiechniques. II travaille sir g~. ~~ise a~ poinfi de rnic~-ocaps~Ies ~~~;~, s~~r la biocorr~patibilite ~~4~~, s~.~r I~, protecfiion apport~e par les ~~~icrocaps~Ies cor~~tre Is; s~sfi~me i~r~-~~r3taire ~3~e;> spar I~, ir~.r,~sp>antation de ces rricrocapsvles a~
nivea.n h~patig~e ~~6).
L,es firava~~ r~alisds depuis ~oiobre ;~af~2 s'ir~scrivenfi d~~:ls r. pro~et de doctorafi e~~ co-fi~ztelle entre Ie l~.b~r~.toirv d~z ~r alle et le ~I:orafioire d~.~ ~'r ~svr~t de I'-~Jr3t~ :i~te de ~'h~r~.pie ~ell~laire et ~°ass~,Iaire de ~reroble, ~°rxertar~t ainsi ~: prof~~ Ies ~:onr~~3issances d~es de~a;~ laboratoires d~,r~s la recherche sir I<z transplantation d'ilots. yes r~s~stats obfie~a~s d~r~s ce firava~.i or~fi ~~era~~is la raise a~z point d'ouatils c;ssuntie's a l~. r~alisafiior°x de la d~.~xi~~~e ~:fiape de cefifie ~~~:a~de, snit Ia transplantation d'~lots atl2cg'oE,'Ylc~,pSL$iL'S ~r~3nSf~Tt2l~~",s ~eT'xeiT.t'~~.41'T''aa.°.,:~'3t. "a~.~.°li:a°', ~$3."sdE.' 'a'%a'~''~-~ c°3i.A6'sS! :~s~rlMlt~.$°'~S'~e a'STC:~;
I'~'.Z'I:~r13~1EJ11$afilOr.
2' sp..~r les ani~na~x et is ca~lt~xre cel~~laire des dots ~.e -~,an~g rhan.s °t de la ligzree ~'~. de cellules de er~~li.
I~io~s po~ve~ns aar~tenant comrnence~- ;'~t~de ~. proprernent pane cbe proi:ecta~c~ des dots de ~,an~er~ar~s par les proteines I~~'. ~o~.r cela, des prt~tocole9 de ~.ra~splantati~~n d'i~.ots rnicrc~er~caps~,al~s (ou non, transfectes par les plasrnides I~~ se~-o-l~t realises c~~e~ la so~:~-is e~
:;1~~,~, le rat en cornparaison aver des clots non transfectes. IJe model ani~nc:i ~~tilise ;~er4~ ron~e~zr (s~~ris ~~
et rats ~~''i tart'; ies ar~irr~.a~~ seront rend~.rs diab~ticl~es par le strepto~ot~ci-r~, ~~~ ~y~e~~t o~dan-t capable ~'ir~tiuire ie diabete par destruction des il,~ts de ~,angerl~ans. yes pr otoco~~es z~o~as ypb~or~eror;s des in3aor3:~a~io~as s~.nr la pro~~ection apportee par les IC~~, naais aussi la comparaison ~'activit~ entre des de~~x I~~i. ?~o~as devrions e~~ ool~plea~ent des etazdes ~z~ -~x~~~ etudier ~~ va~~ la protection, Ia fiabilite et la forktio~;~.lit~~ des dots icroer~capsi~les transfectes par ies plasrnides. rmJn a~ ~tr~~ axe f~'~tude s~;r a la raise ~r~
poinr d' urge li~ne° stable de cell~ale de ~ertoii ~~~~ exprirna~at de z~ani~.r~; cE~nstit~ti~~e 1'~_~~-I, I~~'-I~ et I~~~-I-~-II. IJna felts 1-ibnee gurus perm~ttra de realiser des co--c~tt7~res et des c~~--er9caps~s~,tions entry:
les Il~sts et cette li~n~e. I~'objectif est d'evaluer les benefices d'~ne tells app,.-oche par rappor~ a~ ~~ransf~;; ~ des ~~nes dens ies hots. ~e rnir~i-projet a 1'a~,~a.ntage d'~tre rapidement rnis en r lace en par~ant des ~~reiE~rs resultats de transfectiorg des celiui.es '~i~~4 et de I'expression des ~~o~.~$s~enes dens c~.s cell~les. ~Lrn int~;~~~t secondai.re, non fe :noindre, est la anise au point d'un protocols ds~ rr~icroer~capsu~ation de c~::~:i~les adlaere~~tes ~~~orpi~oic~giduerraent tres dif'ferer~te des dots. ~n tel protoc;.~~e de rr~icroenca~;sulat~r~n poa.~rrait ~~tre transpose ~ d'autres iig~ees cell'~laires et tro~ver d'avtres applicati~~ns, notarnr~ent en car~cerolc~gie.
2~
~.e b~~t pre>~ier de cet~e ~t~de est a~,ar?t ts~~~t ~.e ~-e~,lierc:~e.r des ~
°~s~~e~s c~r~cret pc~~ar a~~~li~rer ae preto~.ole de la tra~splantataorl d'~lc~ts. ~~ es de~.~;~ stapes li~~itar~tes de la trarasplantati~~ d'ilots sat la via~ilite des slots q~i se trad~it per ~~~ ~~r~~~~re d'-elots r~~cessaire ~le~re ~ t,'c~hter~tierz d'F~r~e ~~rrn~~lrc~.ie et la ~aladie auto-i~:~~~n~r~e irzl~~re~te ~ la ~~aladie.
~.'~~rp~tl~~se de d~~ar~t de cette et~rde est la capacity de 1'~C~~'-~~ aj~~tee ~a~~s le r~ile~~ de c~lt~a~:e a c~irr~i~~r par de~x le r~e~~'cre d'alots r~~cessaire ~ 1'c~~ter~ti~r~ d'~~e r~c~r~r~~~l~c~~na° via la p3.~tecti~r~ ~.~;,~ ~x~ts w~r~tre 1'ap~ptose. ~.,'ir~t~ret de cette ~t~de est la ~~r~eratic~~ d'c~~tils c~.pa~~e de rras~s~rrer 1'~~~-l:l, ~
ais a~ssi 1'~~~~-:~ s~~ lee de~tx dais yes ilC~tS. ~..e 1?Yit a°,st d'&5'~te31~.3' d~S r~s'~~~t='zt5 ~3r~7~~,1r'~~.5 ~. ~c~~C3~"t ~'~'~~.~;V-~~ a~? r~lll~lt, 112aas V~51A"e peLEt°vtl'e de ~a~eiilea~rs r~s~ltats d~ Fait de la d~r~e s~;ipe~~e~re a 1'ea~pressis~n de tr;~r~s~~~e. ~,rr~ a~tre ~.tc~~at est la p~ssi~ilite de ct~parer lee e~°~'ets des d~~x ~~_~~' sir ~a pr~tecti~,~,r~ des dots. ~r~~r~; lee ~a~tils ~en~r~s ~~vre~t le chaps d'u~~ larbe ~~er~tail d'~t~des sir is pr~tectic~r~ d~e.s dots via la tra~s~ecti~p~ des dots r~~
la co--e~caps~lati~~°~ a~ec ~tr~e lib~ee ~,erl~aaaire a x~ra~~a~~t des pr~t~i~es prcstectrices ~~~ la c~o-c~zlt~re avec one li~r~ee exprir~ant des pr~t~i~es p'~otectrices. ~'ei a per~~~ d'~t~dier dit°fere~tes appr~cl~es dares la protection des dots. ~ette dt~.~de ~~vre ~~~ lar~~, c~a~~p ~~'applicati~r~ par l~;s d:u~~rerates tec~~r~i~iaaes mise~t erg ~e~a, ~~e ce snit erg biotogie mcj~~~Iair~;; e~ c~lt~~re cell~~lairy, la arlar~ap~latio~ d'ar~i~~~a.nx o~~ la I:ficroencaps~latzo~.. vela reed cette ~~:~de tr~s znorivr~~jte et tree v~t~ressar~te, ~~.ai pivs est, cette ~t~.z~e s'i~scrit dares Ie ca.dre d'~r~e co-t~te.Ie er~tre de~x lai~c~rat~ires, ~. ~a co~~pi~~~e~~tarit~ certai~ze9 ~a~e colia=~oratio~a no~~relle et pro~~ette~se.
S~q~enee ~ocJar~t ~ø~F-~ rnuri~
~~~.g~~t~~ gtt~~~~t~~_~~=t ~z:.~,:~~~~v~::~g ~~~~gk~:t~tg ~~~.~~g~~~t~
gg~~~.~~.rc~
~odor~ st~.:~~:
g~t~;~~~.~~~ ~~~ag~~~~~. ~~~~~g~:~gg ~~:e~~g~c;~~~ ~~~~~~-wgggt~~g~g~~E gg~
gg~-~g~~~.t~
~~g~~~~gtg~ gtgg~.~.~g~g g9g~';:~:t~.~~~~.~~~.g~ ~:~~.c~.gg~~~ ~gg:~~~~~gc:
at~gg~ggg c~~~tc~g~~ ~.gg~~t~gtg g~~g~g.gt~ g~t~:~~g~~~g ~~gt~~~~.tg ~gg~g~.~tgg ~g~~gt~~tg ~g~~~v~~~tg ~.~g~e~~~~~ ~.~.g~.gt~~~:g ~~~:v~~~~.g~ g~~~~~g~g~z~ ~~~.~rg~~~t g~~~~ag~~~
~~g~.~gg~~g ~~~~~t~g~.~ g~~~~~~.g~ ~.g~gg~~gtg ~~gg;~~~~~ g~.~~~~~~g~
~~g~~~g~.g9 ~.g~.~~~~~~.~ ~g~ ~~.g~.a~ ~tg~~~.~~~~
~o~on stop __ ~~~L~ellCe ~7es a~T3~ra~es ~etl~ : gtgtt~a~i,$~g~~t~ata.~~.
~haaager~er~t ci';~r~e g~a~it~e p~~~° :,wre a~~r~i~e poor ~~irrar~~er ie:> st~~act~.~res seeondaires ~'apparie~ent ~e l'arnoree.
~r~ti-seas : ~.gg~gcagaaaa~gc~,~~atc.
~,(~ailgei~lel3i; ~s~Lr9e tyZtll~Pe pCS:.~r =v?~ ~ ~yto~~~'z~, Seq~~;r~ee eo~a~te ~e Ia ~ase~~3 ~, ~~6 so t 3~~ ps, ar~~~ia~~atio~ far :3~~~~
~e i'a~o~ ~e seas a, anti-seas snit ~ a is base 59 ~, j4fl : 4~ ~ pb fl ~e~°
a ,~Qq~~e~cc,~ ~,~ Ztv~i° c~'~~~s ~~c~~'t~~~ ~~a ~~tf~'c~ca~eF ~~~~
t~~a~liF~-l:~k~ ~~~a~th ~'~cto~° 1 ~pwc~.c~.~~~~o'~ei1 ~~, St<~~~ien ig.'~, ~ o:vg ~il~~~., a.i~,~, l~I~eleic ~_eic~s yes ~~~6 bet 2~-; ~4~20~:?'~'Z~--~2 o ~ns~.iin-like growth fa~,tc~r ~e~~iates ,~e~.ibone ar~u~rai ~ensivy ~r~
~~i~.e by ~ot~ grovath ~s~r~~or~e-deper~~e~t any -i~~depe~ent ~~ec~anisz~s4 I~~oi~a~. S, ohrna~a ~;
~.~uo ~, !~rnaar , ~onah~e ~..~, ~~~e.~;eci~.i ~, ~~a~~iir~: ~~~ ~,r~~~oerxrfotogy 2~G3 ar;~.44~~~:!~29.~36 S°quu~ce codar~~t ~~ ~'~~:F~_iC'~: ~~:~~:~~~.:
~~~:~ge~.~c: ~tt~~~c.~~.e t~~~~gt~~~: ~.~'ggg~a~t~ ~~~gtggg~~~ ~g~~g~'~g~v gg~g~tt~~~
~.t:'3G~'~8 Se~S ,'_~C3E~~Ii St~ti ~.~~~~_~~tgg ~~~~~g~~a~ gtg~~g~:~':~w g~~g~tt~.~~ g~~.~gg~.g~ g:~.~~~.tgtg~
~g~ggg~9~
ttgttg~~~~: g~tt~~.g~~~ g~~~g~~:~g~~ :~.~~g~gg~tt ~~a~~~~~g~~gc~.
~~t~~~.~g~~c~'tg~~~~~~g~~gc:r~g~~gtgc>~~t~~~~gg~ag~.gtg~tg~ttc~gc~
~.g~~g~g~~~.~g~c~~~~c~g~g~.~~_~~~..~g~.g~e~..a~.~.~.~~gc~.ia~.~g~~u~.~g~.g~ggg~
,'r~~g~~~~~~t ~~6~~S~g~~J~~~'L~.~~gg~.~g~.~lwt~~.~.~~~~~~~~~.a..g~ggg~.~~gt~~t~~~~.~~~tg~~~~~
.~g~d~g ~~~g~c~g~g~g~~9~~~g~g~~ggg~~t g~~tg~~~~t~~~g~g~g~~~::g~c.g9~g~~g~~tg~~tg~~
~~~.~~~g~t~.~~.g~~~t~~g~g~gg,.~~.~~.~~g~~~t~.~g~~~~~~~~.t~gf~g~~~~~~.~.~~:~.~:~
g~.cc~:~g ~6..~a~gggg~~-~~~~~~~~gg~g~,~3~~~~~.g~~.~~~r~~~:g~g~~$.~.f~.~.~~~~g~~~~ ~-F~t~iSg~~~~
~~~~a3 ,~t~p giag~~.~~~.~ ~~~~
~~.~.~i.tg~g~ ~
~~~~~..~~~~.~
~. ~~~
~ '~<
~~~~
~.
~~ ~,~tli~~~
~~C!~t~~'tc~ ~~S ~.iTlQiccS
~~uS : bc~~cctc~Lt~.cc~LC~a~cttc~
~~~,~;~err~er~t d'~~c g~~~inc ~c~~r ~~~ ~.~~~r~~~~m ~~~~r ~it~~r~~a~r ~~S
St~~~ct~rea scc~r~da.~r~s ~'~~rp~r~er~cr~t de ~'~~sorcc ~°~t~-Ser~S : ctct~ga;c~.g~~~c~~,~a~;,c~.tc ~ec°~~"r~c~ c~cla.r~tc d~ ccad~~ Step ~. St~~, ~c ~~. ~~5~ ~'~ ~. ~7~
Sc~~t ~~~2 ~~. r~c~ ~'!-~I~i~yf ~.a~~~a~ per :w~
e~c a~'~~SC 3 ~ ~4~ spit ~~5 p Re~°
~'~c;a~~;~cc~~ ~e~l~c~~~~c~~ c.~l~T~z ~.°~~cr~r~~~~ ~.' e~ ~cer.~w ~~~~'~.~~z~-~apk~: ~°r)s~r~~a~°c~c~r~a ?~~~E~c~c°~~~~a ~~'~~
~tc~~~3i~~ i~~., ~o~~, ~~~ ~,~, ~~ ~xo ~~~~ ~ct;~j~;5~:~~°~-6t ,~n,s~~i~~-t~~Ce ~a-~w~~ae,~e~~~~~ Y~~~~~~z~~.~~ ~~~k ~~~~~ ~~~~~~~~~~
de~.s~Ey ~aa c~i~~ ~~ ~~~,~ ~°~~v~~ ~~~~r~~a~-d~~e~~~~~ ~~~~' -~~ac~'e~a~~~~~~ ~r~~~c~~~;;~~:~~ ~~~~3~~~.r: ~, c:~b~~,n ~;', a:~~c. ~~., ~~.r~~~,r ~', ~oel~.h~e t,, ~~r~~c~al ~, a~~~r~~~~~a ~~~~cr~~~~=~g;~ 2G~~~3 ~r;~~~~;~~:~~9-3~
~7 ~,~r~~ ~i~~~riciu~ ~.~,s di~e~ii~l3s ~r~~~~.~~ v /i~zs~;~ ~~ T~~~~i ~reia:~~
~~~-~ + ~~~-~~
spel, l~hes :. I~--! (5no ~asj I~l~~: ~."°e. ":, ,J ~, 'e ~;
~Cl~.'! a~~-! ~: ~vo ~ 8~P=-a Y~~~
~. :3a ~$3 ACS ~~~~ ~~. ~(..~~
'~ ...a ~~c:.S
r~ -._.ysho!
''~V~aP
~..,-'~~.~°'""'.
igesiicn lOigestion Ciges:ion i bands 3 5?U pb (IGF-I) + 1 bands a 71U pb {iGF-i.) + 1 bands: a 810 pb (GFh) +
1 bands a 3830 pb 1 bands a 3840 pb (~CR2.1) (pCR2.1) 1 bands: a 3900 p!> (vecteur) 1CS r.Xho! F~C5;,~l~lee! A
~/~
!P''a!e a ~z I$s"S ra I~~,S
i ~l~~S
~~9G ~b ~u1~4 ~~ q~ ~ '~E9~~! ~b ~.~ba8 CS B vICS S ~~
'''~Xlaa! P'i5of~ ~' '~CS 8 °~..'..-_....~ ~~~_,_..... "~o~&
Cigestior:. ~ digestion Cigesfion 1 bands a 650 pb (II~FS) + 1 bands a e,'6U pb ((F:rS) - 1 oande a ?0 pi; +
1 5ande a 3250 pb (vecteur) 1 bands a : 24U pb (vecteur) 1 bands a 3880 pb (~,recfeur) ~h I ~~'~~ CS B CS..t~31t2! !RE ~c:~~~!
EcoPi! -E\d?le~,~'~Sln '~ Ses~,~6 PdlCS
!CF-!1 ...-._ , ,MCS .~ ~~CF"i1 a ,~t:a~S !~',,~-i! ° ~ 1n &CF-!
&8003 ~b ~WF-j I~F- ~l~ir~~S !~~h_! .=-!CF-!!
t ' 658PJ ~h ~~ ~ r4~61 ~t~
IVlCS S ~~''~CS S ~:W .~"' y JlCS A ;~C& .~ ~
...-,~ ~..-..'~Saii -.~.~Rd SercB~!
---~-~ ~ __ i7igestion iUigesfion 'Digestion t t~ande a 515 pb (IGF=-i)+ i bands ~ 650 pb (IFtES) ,~ ~i bands a 6fiU (IGF-II) +
1 bands a 10 pb + 1 bandP i 59U pb (tCF-i) j- 1 bands ~ 5490 pb (ve:cfeur) + 1 hands is 30 pb 1 bands d 6780 pb {vecteur) 1 bands ~; 5520 pb (vec".ur) i~~S i~~~ 8~d~
Sarn~ll ~, I=ar I
hACS iviCS '° ~C~;
Ear llfiCS A g,-CvICS A ~ CF-!9 ~~lC F-!! r~CS A ~" ~ wF-f!
!CF-~ PIF~~S ~~F-! ~IGF-!! g~p.~' plf~~S fi~"~F-9 +l~fi'-!! ~ &~F-~9~t~S iGE-I
+l~f~-I
~s~00 pb a ~~UO )3tz p Ear'r~' 7400 !s!c CS >~. I~ICS CS A iViCS =a:r ! ?CS A !tJ! ~,S , 5sp! ~~ ~'~ p~ ,. Ssp! ~--. Ear !
i ~ ~~anai-!! (~a30~) '~Sspl ar 1 °° .
i7igestion Cigest'son' Digesfi ~ ar , far I
1 bands a 2970 pb {iGF-I!) + 1 bands a 373U ;~'L (1C=s, IRES) +
i hands a 4380 pb (vecteur) 1 bands a 185U pc + 1 bands d 1?15 pb+ 2850 pb +
1520 plo ;- 1260 p'o +
1 bands a 50 et 2°~ pb T00 pb + 630 pb +. 'Z10 pb + 500 pb :
~dqtaez~rage p~ar~-~
~E~'Cil~C2lCp CflC'ari$C ~,'-~,7LtCe'~,CCtC ~~CC$Ca$~.~~, aCC~;aC~Ci,g aCC$gCtg$g' ~~aaaCgaCCC ~pc'la:'Cl,at;~~.a aaa$gaCCgC as.CtgCusat~
grin ~erss ~ppariemen$ ar~s~rce ~~~s aacgaccc F3;gav,cta~~;ca aa'-~~:gaccgc acctgcaata a~rIi1 i~l~$=-~CI15 gtgttCaCCtC agCCtCa$Lagt a~'Ca~~,r',aCtCtgaCC$gCGgt~~øa'r~aCgr.'~.CCC apg~,CCt~!~Ca aa~$gaCC~~,=L' ~'..;C?CgCsx.a$a aaga'~tc~tCaC~i $C~~;~ Cg~C $tCaCc,~.CC$C ttC$aCC$gg C~C$4;$gC~L gC$Cs~aCC$x~C
~ia~CagCtCCa CCGS.Cc,gCi.ff;g aCCagagaCC
a~lt~'~tt931'~C$ $C~~~~CT~~T$ ~2$CaCc~.CCtC $tCtaCCtgg '~:,gC'.'.CtgCt$
~>CtvaCC'LLC ~a.CCc"2gC$CCa Cd,FaCagCt~g aCCimgagaCC
aaga$acaca $ca~~ cgtc tt~cacacctc $tctacctgg cgcs:c~atgc$t ,~~~cacc$~~;
accagctcca c;cacaget,~g ac.°°cagag~.cc ctt~gcgggg ctgagctggt gga$gctct$ cad tcgtgE g:~ggaccgag gggct~.~ta!;
ttcaa~caagc c,cacaggwta c$ttgcgggg ctgagc$ggt gga~~gctctt cagttcgtgt gtggaccgag gggc$t$tac $tcaacaagc c,cacagg to c$ttgcgggg c$gagc$gb ggatgctctt cag~tcgtg~ g$ggaccgag gggc~.~$a~; ttc;aacaagc c .: acagg;~ta $ggc$ccagc: ai~tcggaggg cacctcaga., aggcat~tg~g g~:.$gagtgt~;, gcttccgg~,g ctgtgatc$g .ggagactgg $ggctccagcv a$tcggaggg caccteagac aggcat$g~tg gr tgagtgtE gct$ccggag crgtga$c$g ~:ggagactgg $ggctccagc a$tcggaggg cacc$cagac aggcattgtg g~,tgagtg~~ gct$ccgg.~ag ctgtgatctg rxggagactgg agatgtactg tgccccactg aagcctacaa aagcagcccg c~c:$a$ceg-v gcc.°cagcgtc acactgacw gccca;~gact aga$gtactg tgccccactg aagcc$acaa aagcagcccg c$ctavcc~~ gcccagc~;cc acadtgaca'.
gccc~;~gact agatg~:ac$g tgccccactg aagcctacaa aagcagcccg c'.:ctatcggaaccagc' ~~c acac$gaca ~ g~~ccate.~t Cagaaggaag $aCc'3$~$ga~i, gaaCc'3Caa~'~ a~aggaag~~ C:3ggaaaCa~. gaCC$aCaga a$g$d8~g~~.,~~,~ ;gCCtCCCa~
Cagaagga2g $aCa$~'$gaa ge:aCc'3.Cac'1~~~'e agaggaagtg Ca2g,~~.'lac'~C~7s~
gsaCCtaCaga a$g~;'c$~g~g~ ~gCa:.$CCCaC
c~i~aaggaag ta-~aaariaa gaacacaagt agaggaag~tg caggaaac~.a gacctacag~a, a ~.~. .aacaaaaa a$~ccaca~tc gga~agcag~.aa acgcca ~pp~rie~er~$ a~~or~e as~$i-sera ~~as$ seq~ei~ee a~d:r~g i~~-~ ~~~c~c~r~ s~;~~°t-s$~*~) i seci~:ea~eir~~; r~~ s~~a~s:
~eore = ~ i7 bi$s ~~7~), ~~pec$ _ ~.0 ~der~3:i~~c;s = ~'~4/3'7~ ~99~/~) ~tran~. _ ~i3~s / ~I~s~
~iasv seci~e~ee ec~dircg I~~-~ ~ec~~~r1 s~ar$...s$~p) ,' seq~etrein'; ~rir~
a=~$i-seas:
~cere = ~C~G bits ~3 ~ 2), ~.xpec$ = e-~ ~9 i~er~$iries = ~6213~ i ~9~°/m), raps = jal~~~ i ~ i~'i~) ~$ra~d = ~'i=.~s / Ii~~s r ~~~7~.er~rage ~~~~-I~
~CL$ilsrlCC CC5da~2tC ~'~C,~~CCtC CttaCCCaaC ttC~'$~i,CC, aQ$~g~~a$C
CC~,~~'~.'gggga agtCg~~gtt ;~~gCttCtC r~.-~CvCtt~,~'~~.g CCttCgCCtt ~5'~~rl~&1'?~Ilt ~113OTC~ ~øk'~s gtgctgcatc gctgcttacg gccecggaga gactctgtgc gg~,~~ggagc $tgtvgacac g~.,tLcagt'~ g~ctg~.'tc.gg accgcggct~~
tacg gcc.ceggaga gactctgti~..r, $~.',c,t~gggagc ttg? ~ga~.a~zi~~cag~i.~i:
gvctgttEagg acegcgge~'~
tgctgcate gctg~7tttaeg gccec~ggagagactcttgcg~,~aggggagc$~gt$g_=~c cacgcttca~;~'~tg~~ctgL~cgg accgcggctt etacttcagc aggcet~bcaa gcegtgceaa ccgtcgcagc c~tggcatcg tggaag~gtg ctgcttccgc;
agetg<gacc tggccctcct etacttcagc aggccttcaa gccgtgceaa ccgtcgcagc cgtggcateg $ggaagagtg ctgcttcegc;
agetgc:gaec tggccctce$
ctacttcagc aggecttcaa gccgtgccaa ccgtcgcage cvtggcatcg $ggaagagi:g ctgcttccgc ~.gc$gc,gacc tggecctcct ggagacatas; tgtgceaccc ccgceaagtc cgagagggac ~'.gte~aect cteaggccgt ~:~~,'tc;cggae gacrødccca gataccccgt ggagacatac~ tgtgccaeec ccgccaagte egagagggac ~gtc.t~acct cteaggocg$
aettceggiac ga'cv~dceea ~atacc.cegt ggagacatac tgtgccaece ecgccaagtc egagagggac s;tgtctace$ ctcaggccgt ae$tccggae gact$~~ccc~ gataccccgt gggcaagttc; i-tccaatatg acacctggag ac.agtrcgcg ggaegcctgc gcagaggect gcctgcccS9: c~Wge~;cgccc gccggggtcg gggeaagttc ttceaatatg acaectggag aeagteegcggg~~e.gccctgegeagags;ec$g~ee$gccc;$eet:gcg,~ gcccgceggggtcg gggcaag~$c; viceaa atg ~cacctggc~gacagtcegcg ggaegcctgc gcagaggect gectgccce:c ctgc~ ~gccc gccggg~,~tcg C~ztgC$~:gCC aaagagCtCa aagag$;~Cag agaggCCaaC. ,ri~CC~l,$e~~:, C~.C~gc'~.tegt g$$aCCaCCC; ~a~gaCCCCg CCCaCggggg Ca$gCttgCC aaas3gagetCaaagag$tc~yl~~gaggCCsl~~ ~,~tCf~tC~:CC~°Ctg~tC;;~
gt~3.CCaccC awaa4~aC.Ce,Cg C~~'~~,ggg~g CatgCttgCC aaagagCtCa adgagttCag ~gaggCCai"~~ C~7 Cc'~t~glC d~el~$Y~at2~~gt gi~aCCgllg,:.Ela~ags~.eCCCgeCCc~Cggggg agcctcttcg gagatgccca geaaccatca gtgaatca~a =~~tgtggta attctgcaat ,gtagtaccat ca~atctgtg~ cctcc$c$~
agcctct$cggagatg~cc ~ri~~ sc~~s agcnt~itc ~r$rs ar~~~ s;~s a~ca~~~aca_ ceatc ~,~~ar~er~ea~~t a~c~rce a~c~-sins east ~e~~e~~~ ~.~da~at~ ~~~d~r~ Mart-~c~~~~ st;~~u se~~~r:~ag~; saris ~;~r~-~s~ssrl~~.~,t ~c~re = ?3 3 tits ~3 ~ ~ ~, ~~~e~t = ~. Q
3si~~$iti~;s = 4~J7,~~~~ X95%~9 has = ~/~~~~~%~
~tra~~ -_ ~'l~s / ~l~s Mast sec~,~a~r~ce ~~dar~t (sari-st~~~/ s~~~~r~c,ag~ a~~ti-s~~.s ~~~~ - 7~4 bats ~~~7;9 ~X~~~$ -- k~.~
~~~~3$itze;s = ~r~~/46~ ~~~°/'m~; ~'~~3s = ~1~~5~'~ ~~%) ~trarc~ = Plus / i~~s 3~
~su~~~~s d~s rnis~s au ~c~i~t du ~r~tcsv~~~ d~ ~r~~s.~~~tic~~~ pay ~~'~~~Ip~F~' suø d~s a~ws d~ r~~.s.
~ ~ ~ I ~ I - I
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~~~ ~o~l ~ ~ + ' ~ ~- ; ~ ~ ~ ~ ~~ aa~ S~
i~_ i~ ~_ i ~ -~ ~ ~- ~ ~ ~a~ ~ ~~ ~~~ ~~~
s ~~~/~ ~ ~~ ~~~ ~~~
~ ~l~ j I i, ~ ~U~~°~ ~ ~~ ~~~ ~~~
t~ca ~ o~ ;~~J ~ ' ~ __~I ~U~/~a ~ ~.~ ~~~ ~S~
=~U~, ~3 ~ ,3,~ ~~~ 5S~
- j S~~f~ a~ ~,~ a~~ ~s _ ' ~_~ _ j ~g~ ~ n~ ,~ , ~U'~r~. ~~ ~~ aye ~U~ ~
j_ _ ~~~ ~ el ~- ~ ~- I ~ .~~'~/~ ~ :~~ ale 5~~
I
6 ~ 1~
tip ~ ~~ ! , ~ _~. _. ~ - ~~~~~ ~ S~ aa~ ~7 i ~~,~ t' I ~ ~ ~~
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I 1 -~ -' SU~f~ ~ ~~ axis ~0~
i ~a~~e~ -~ I ( ~r _ ~~ ~/~~4~ai<~4~c I
II ~~~ ~ ~ __~ -G l~ I
I I
6 ~ 1~ j ~ , ~ ' ~-~~~ 5 e~ I ~ ~ ~ ~ ~_v. ~' ~ ~~°l~ ~ S,~ eke ~~~
I ~ .a- ~ ',,- ~ -a- 4 6~f%~ ~ S,~ ~~e ~2~
~ 6ys,~ i'~T i ~ i i i ~ __ j S~~/~ :~ ~a~ ale ~US~ _ __ ~f~ Sri I I _~. -_~~ ~ ~~ ~ h ~. S~ Q~e ~U~a ~, 1 ~
?~~a~ 4 ~~ ~ ~ ~.- ~ ~ ~~/~ ~. S~ a~a; X33 ~ ~ IbT~d ~ i _~_ f 3~
.c'~, T~~~r~u.e~ar ~~ ~~,ilg~ F ~~ ~~ ~=v!u ~~ ~e'~r~vi~rc~b~~. a ~tiEis~s d~.a~~ ~e5 ~~e~tr~phor~ses ~2 ~i~re~ra.tior~s utiiis~~s dens ~o ~~~rr:oy ~~~'~'~ ~ : ~r~it~ ~~f~o de ~'h~ra~i~ ~'.°xaTai~,ir~ ~t '~iss~z'c:ire ~~.ci : ~ ie~ est ~n hots dont i° da~r~Wtrv os~r~~ ~~~
-cc : ~u~no~~ ~i~cr~c~sis ~~oto~--a ~'~.1-~~: Inter~°orc~r~ ~~.mr~n~~, ~i~I~: ~nreri~gakir~o-~.~
~~F: ~ndot~~ii~r~~ ~~-ow-th z~~,ot;~r ~~~-~ et ~: -ons~iir~ ~ro~~~ia ~~,ctor~ ~ ~
~~F~~: ~ns~aiin (~ro~th ~'aoto~- ~ir~din~ ~rotoi~~
~~~-7 ~~~-: ~~~' ~oo~~tor typo ~ ~~: ~
l°~'~'->~: l~T~oi~~,r ~'~otor-~~~~x: ~ro~wth ~orone l~t~: rnor3oxido dy~.~oto i~~s: 1~r~ s~~nthdt~,so :~: ~ratra ~-i~oso~e ~ntor se~a.~~r~oe ~~u/~.n,-'r~ ~~~: ~~:por tyxyd~ ~is:~Y~,.so ~~ ~~ai~~rw o~.~ ~in~, ov T~~y~r~.~sia~~
~~: : ~etl~zl~ d~ So~to~i ~~~ : ~ree~ ~lao~-oscent Protein ~~1~~ : ~~~ol~~~n~.~~ ~~2'L~s E~~Tc: ~ oornrlerr~~ntaar~
~Tt: ~d total fir: 1 ri~oso~nio~~~
3~
?. Shapirca ~,1~. :~~ax>ey ~;. ~~yar~ ~~g., ~~b~.~t t4iS, '~~th ~,, ~~~lar~m~~
~a-~,; r~~te~~an Iii, ~ajott~ : '~I. 2000 s~i. Is~°t transp~a~aatic~~ in sv~~r! patents ~~it~~type 1 ~.iab~t~s ~~eliitus ,sing a ~i~~nc~3-tic~id-free i<~~~~n~°s~~~pr~ssiv~ regirner4. 1~1 sngl ~ ~d. ~%;3~r3~~-~:23~-2. ~r°~ac~a~, ~.~~'., ~. ~b~rb~i~~r, ~.. ~'~s~, I=,. ~ssi~r, ~.
~~~f~rr~is, F'. ~ayi~, ~.
'~biv~I~t, ~~. t~~artin, f. ~~is, L-.. ~ad~t, ~. ~~ii~P, and L~. i?~~~r~~.
2C~OI . I~~?an isles: trar~sp?antatir~ra r~~t~~c~rk for the treat~~er~t c~3_'°'Typ~ 1 diabetes: ~~r st data ~-crr~ the Spas-Fr~r~~;i~ ~~I~, ~:~~s~r~:i~rr~ (1999-~~0~~.
l~~c~~~~o~og~~. 4~: X59-~4.
R.esenberg, ~., b~.. $Jarxg, S. aras~nas, arid ~. ~:aysin~~r. 1999.
Strri~t~rai and fian~ti~nai ehanbes rest~itir~g fr~~xa islet Isoiati~n lead t~ islet sell d~:at~~. ~~~~~r~. 126::~9~-~.
~iiwa, ~., S. ~a~~t, .~~. ~~~ang, . ~gapit;L~s, ~~..1. ill, ~r~d ~.
R~ser~berg. 1999.
~ar~~r~ati~ islet cell s~r~i~aai ~~il~~ir~b asset isc~iati~n: tb~r~i~ ~f ~~li~iar ir~t~ra~ti~ns in the pan~r~;as. .~~r~r~oori~aod. 16l :~~"~-6~.
~~obr~aeier ~-, ~'~gigperz ~~, '~'rar~. ~J'~, a~ri;~ ~; ~I~~garc~ ~. 199 I~uay. Stabh e:~p~~essic~~ c~f rnar~ga~ese s~.~p~.rq~~id~ ~ist~tas~ (l~~S~~~~ in ir~s~.l~in~t~a cetis ;~re~rer~ts ~~.-lbeta-in~~=ced eytot~~sieity arfd red~~~s r~itria; ~~~i~e pr~~~t,ti~r~. a ~~"'i~:~
Lr~aest. ~;101~;9):~.~11-2~.
~,i~iri~, f~.L,., a~~:c1 ~. ~~~rdr-~p-f~e~isen. ~~I~. ~~ eb~i~,e ~f death-~~~e sig~~al-rransd~cti~n c~fir~~r~e-a~ediatecl beta-c;eøl apc~ptc~sis. ~')i~a~»~o~o~~~~.
~~.:2zI~-~3.
9. fhQmas I~ar~dr~p~~~~.;ise~~. 2C~~1. ~-eeli I-~p~p':~sis StirraF~ai and Signa1png.
~ia'~etes 5C :~~~-S6~
1~3. ~bd~l~cadir ~~~s:r~, i~iu~:shell ~~.eegaz~, ~-ra ~~is:a~adia, ~~aazl ~e ~'~s, I~l~ce 2~ar ~~~i~er~, S~asar~ ~r~r~e~=~eir arid ~'~rd~n C. ~~eir. ~~0?. l~aere~~~a~age ~epietis~~ ipr~~es s~rviwal ~1°
pc~rcirce r~e~r~atai par~ereatie sail cla~st,c~rs c~ntair~ed i~~ alginate ~~~aer~~,aps'~i~~s %ra~spianted irit~ rats.
~~er~c~trartsplantatic~r~; 1° ~; Z~f~-251.
11. grey, S~~'., ~. ~ ~ek, ~.~-~. ~ae~, arid ~. ~err.~ar~. ~~c~s.
,~_den~~Erir~s-mediated ~e~e transfer ~f ~2C~ ire a~rine islets i~bibirs gas-irl~~vee~ ap~pt~:~sis.
~~~~~,~~~~~~c~ ~r°o~. ~3:5'~7-~.
1~. d~abiz~~~itel°g 1~~ S.-~.L, to ~r~a~.ica , ~~ a~, ~~elsrei~~ f.~.;
brc~~~anl~:~ , ~~rb~tiv ~, aj~tte . 1999. tra~sfeeti~r~ ~f l~t~rfi~an. panc::~°eatie isleta>
~ait~~ are a~~ti-ap~ptc~tie gene (gel-~) ~r~tects beta eelis ~r~a~ ~ytel~~r~e-~nd~eed I~estr~etic~r~. t~~c~b~~~,~. 4S:
I~~2~-1'~~9.
1~. ~laa~ E. arise~~. Sif ~y. .e~~, ~~arer~ ~~,indbe,rg., ;lesper ;ic~l~an~~ese~~, Elisabth D;
~alsgaard, ~lernrr~ir~g l~oe~~:, yens ~I. i'~ielsea~, ~lZ~mas ?~a~~c.'~r~.~p-I~cs~ls~;~, 3c~rr~ I'~erc~~p, _'i°~ils iiiestr~~. 2~0 i . ~~~~ressor of ~y'~olci~e si~L~alik~L~ ~ ~~~C~,-~~ ~rc~teets ~~-cells a'ains-e inter~e~a~in-~~ arLd Interferon-~,_r~ediated to:~icity. I~tf~.~; vo~9~; '~I;
~2~9a-12i9G.
date, 'f., ~..~ s=~eia~;~er, ~. _'~a;e~i.~iai4~, ~.~. ~;axiiiva~; ~.... ~,iaz, ~. ;~earia, 5.~.
~~e3z~, ~...~. ~re~ory, and ~;. ~~a._a~. '~~(~~. ~de~ovir~as-~nedia~rod ex~ressior~ of~~~ prevents ypoxialreo~y~;er~ation ira~~ry bar red~x~;~rL~ reaoti~e o~y~~L~ se~;ies and caspase activity.
C~c~;~~a~vc~sc Re.~. 55:09-19.
~ 5. ~eL~i-~al~o~, .~., ~. i~~o:iscot, ~.~~.~. x~~ieixard, ~. ~eatri~_ ~,.
cadet, ~'. ~~a&~to~, 1. ~err-~'on-re; ~. ~iLroboe~ek, ~ . ~,err°aarehar~d, ~Y~d ~. ~~aairgai.
~.~39~. ~de~~~~~°ir~gs-r~ediat~~d c~ta~ase gene transfer rPd~ees o~idar~t stress in ~~arL, porcine ~L~d L~at par~ere~ti~, islets.
~~~~abeto~.o~ia. Vii.: ~~93-~~0.
Mann~~c~a~is, i~., ~. ~~i, °i.~. ~.v~der~, l~_. ~a~ottc~; ~~. ~fv~eco, arLd ~. ~.obbira.s. X000.
re~lerLtion ofbeta cell dysf~r~ction a~~d apoptcbsis ~.o~i~mtion irL
da~~r~~~.r~ asie°a by aderLt~nirai ;~er~e transfer of tie ia~suls~a-liife ~ro~t~L factor 4. ~<:~~~; I~~°. '~::~~~~-~~'.
17. ~~drea Ice, z~iir5ce~~r arid ~.~:~ ~~,. ~'cidmarg. 2~~~:. control o~'ceil s~rvi~al ~y ~~F
si~~aallir~~ pat~~aays. ~ro~%vtf~ ~orrrLO:~e and ~~~-~ r~,sear~;~~. $ 5.~~-19°?
~alCae ~, ~ido ~I, ~~cciii ~. ~~~~ sec . istir~ct aL~d o~rerlappinn ~.~~nctio~~s of insrair~ and ~a~p'-~ receptor so ~,v~ciocr .c~v.;~2~~):~?I~-~~.
19. ~!ioan S, f~ic,~~~a~~.r~ ~ , ~7-~0 ., ~a-~:~aar ~, or~4~~~~e ~,_~, w,r~ecal :~, aylin~ ~.~.
~~~~ ~~ai. ~a~Sdz~LLi-~1~CG' ~r~~~~ i~.~ti~r re:.~t"rla~~"s ~~;a~ ~~Ile rLILFL~za~ C~etISL$y l~L ri~i~e by ~3Gt$k ~rO~Jv~i1 horz~one-deperLdent and -indepe ndent ~nea,~La_~isn~s.
~,r~docriL<olo~y.;~~4~:~):9i9-3~i 2~;. ~a:~ter ~. 2~~ ~ ~~~;~t ~~~s~Ik~a-ii~e ~rov~~t~ factor ,;~~~~'~-bin~iiL~~
proteins:
z~ateractions ~~aitl~ ~Gfs arLd intrinsic bioactivities. ~L~ 3 ~I~ys~ol ~L~docr,not Ietab.;27~~6~:96 i-7~.
Zi. ~'etri~ ~, ~elj ~'~~, ~~-any ~., i~~c~o~~aid'f~,, f~ear~ a,%~,; ~~;ii~ , ~:.i11 .~. 1999 ~a» .
erexpressiorL o~°ir~s~iirL-ii~e ~~w~~factor-%~ ire transbenis; :i~.;e i.s associated ~.~it~ pa~mreatic islet cell ~y;~Prplasia. ~~~docriroso~y.;l~~m;~):~~~~~-~3.
'~2. obitaiile F~, ..1., .eL~le~ 1~, .~ser~berb ~, 1-~afz~ J~ . ~~~~. i~f'-il allo~%~s pro~o~°~ged blood ~l~cose norL~alization. ~~it~ a red~,~ced islet cell ~~ass trarsspia~nati;~r~. ~~~c~~~~aa~~~~~%.
2.j. ~rrLit~, f.~., ~.. oseL~, ~,. Vila-:oa~-off, ~.~. ~~F.eir, any ~.
~~:~raner-~.~leir. 1991.
vla~i~.FIGeC~ L3~sLFl~ii1-illCe ~roaL? factor ~ ~~I~e e34~3ressloll L~2 rC~,4~Tt,Izt,rat:~?'L~ ~c"~~ ~=ai'L;Lrz;s'~.S. ~v'ad~ l~,ICL'~~'.~~C'lt~~'CZ
2~. ~vorge, 1~., ~,. :'~r~~.LSO, ~~:. ~ aseilau, ~. ~:osta, .;a.~::.
~e~ed~iaa~, arLd ~'. ~osc~a. 2~G2.
:~e~da veil e.xpressi~~z s~f~~f-~ leads t~ re~,o~er~~ fr~~~ type 1 diabetes. ~
~'1~~~ I~vc,~t. 109:1 ~ ~~-~4 .
~5 '~5. ~.i~, ~~L, ~. ~~air-~'~a~ce, ~. ~. ~.,ee; and ~~%~~.~,. l~o~~re, Jr.
:~~~0~,. ~ctivatior~ of phosph.atidylinositol ~-lCir~ase cor~~t=h.utes t:~; 3~s~li_:~-like grow=el~
fac~.tor ~-media~ed ir~hibitior~ of pancreatic beta-cell deatl~..~'~~'~cr°i~~i~,~%. 1~.~:~~(~~-1>.
~ir~ ~', f~~~otte ~ ~~, ~~~~ c~~r ~iV~, orb~att ~a~. 2J~2. e~r{~lcc~~er~t of are irrgrr~~noprivileged site tc prolong islet allograft s~ar~rivaa. ;ell 'frargsplant.;i ~~~~:~~.'~_~;~_ 27. I~~ca C~, ~alvitti I'~, I~Ie~ i ~,~~i, eccl~etti ~, ~~,pitarn ~, ~,~sta ice, ~rageletti ~, fanelli ~, ~ru~etti ~, ~alaf~ore ~.0 2f~01? ~Vov. Sertoli cell--ir~d~ced reversal of ~.d~~ia rat pancreatic islet hate-cells into fetal-li4ce status: potential i~plic:~~or~s foi° islet tra:
spl~.r~tatior~. ~:~ type ~ diahetes r:riellitus. 3 ~nvestig 1'~~ed;~~~~~;:~~1~-~.
2~. ~ ar~g I~. ~lrigl~t fir. -~ X99. ~ome~caps~~lated of ~~~~oii enriched ~estic~ziar cell fractions f~r~-~°~c;r prolongs fis3~~-to-mouse islet ~erograft s~~~-vival.
'~'rar~splai~trrLior~ ~',~; 6'~~r~): ~~~-~Zi~.
29. ~ngeliqne ~~cray, Iic~,~le ~3locl~.el, etsia ~:~ess, ~~~~offi~e~r ~.
~~.arr~~or~d, ~~ubert ~erard, ~e ~~rard_ ~~9~. ~stablisl~ent of ~~ o~ase Sertoli well line prodvcin;g rat arsdrogeta-hir~ding po~:ein ~~~. Steroids ~~: ~~~-2~'~.
3~. F~arr~ ~. ah~.to, saes ~~enry, r~~it S. i~~rar~g, ~~ai;rr~a Sa~~e,k, ~~.a~iel f raga, ~al~.l~ I~ctb, ~sa~na gaher. 20G~. cationic ~,ipid and ~s~iy~~r~e~~-used gene ~~~li~re~~r to ~-~urnar~ ~~.r~cre~tic islets.
P~xoieccalar ~'herapy ~?~; ~9- l ~G.
Pierre ~. ~erL~ao~, ~'wisLit~e I~:ao~~isc~i~, f'hilippe l-~re~aost, ~;rac olla~ad, Serge ali~~.i, ~ad~wiga ~~rol~ocsek. ~9~7. ,Star~dardi~atiov of pr ocedur° for er'~aci~::r~t ex viva gene tr~.nsfer into porcine pancreatic islets with catio~iv liposo3~~e,~~ ranspl;~~tati~~a~
(~3~7~'<~~~1f~3.
32. ~--~ '~ ~,akey. '~0~ ~ . ~o~x ~iiraa tr ar~sfec'~io.r~ of intact lF~~,ncr eratic islets. ~ r~.nspl~.r~tation cell;
i(~:697-7~3~
3~. Le~ic~nd, ~'..A., ~. ~'essie~, a~~d ~.t~. :alle. 1~~~. ~ar~titacive rc~ethod for tl~e eval~atioa~ of bioicrocapsvlo resistance to rnecl~a~.ic~.l stress. ~~~c~tw~~~c~i'~.
t7:~;~'~'~~-1(a~.
3~. t~obitaillc, ., 3,f. ~a~~a~~~~:~, F'.~. ~ ehlc~nd, lh~w. ~,ayr~o~.~re~x, "~~. ~,epage, ~~d :~.. I-~alle.
19~~. Studies ors small ~<~~~ ~icrorr~,~ alginate-poly-~~,-lysine rrai~:,roc,ap~~ales. ~~~. ~ioc;oa~apati'oility of smaller versus standard g~nicrocapsuleN. ~~~'~~~r~!~l~~e~~ ~~.~. ~~:1 l~-~~_ 35. ~alle, J.~., S. ~o~zrassa., ~.~. ~ebls~nd, S. ~h.evalier, . e~.udry, ~..
~~apdelair~e, S.
~oa~si~eau, w. Sai~tor~ge, and s. f : a'ah=.. 1 ~~~. .~rot.ectior~ of islets of iJ~.ngeri~a.r~s from antibodies by ~~acroencapsulatic~r~ wit~°~ alginate-poly-~ mlysia~e ~er~brd~anes.
~'~~,x~soc~~~~c~~~~~a~. 5~:35(~-~.
3C~. ~.ebi~~ad, ~.~., ~. Si~~ard, ~~1. ~enavy , t~. f~ochele~.~~~, ~.T~i.
~.et, ar d ~.~. ~~;lle. 199.
Studies on s~aaller (approxirr~ately ~ ~ ~ ~ic~-oa~~ r~.icr oc~.psoles: iV.
~e~.sit~ility and safety of ir~tra~~epatic irr~plant~.tior°~s of srr~all alginate poly-~~ ,-l.~sine t~~icr.o aps,~~les. ~~~1 ~~~,~~.~,~a~~~s~~. ~:3~ f-3 ~.
~~c~~e~aca~~ p~~~' i .~~tt~a~~t~ ~~~t~atagt a~~~aw~~:.tg a~~tg~tgtg taaa~ga~:~~ gga~~t°a~~.
aaatga~~g~
a~~tg~aata aa~c~~a~~~ gga~~ta~~a aa-tga~~:g~
a~<=tg~aata gtgtt~~.~~t~ ag~~t~attagt ~.~~~~w:.~t~tg a~~tg~tgtg taaa~a~a~~~. gga~~ta~~~~
aaatga~~g~
~i~c~tg~~.ata ~.ac~ata~~~~a t~~gt~gt~ vt~a~a~~.~~ tt~ta~~agg ~ug~t~tg~tt g~:~a~~tt~ a~~ag~-~~~.a ~~z~~agctgg a~~agaga~~
~~a~~ttttr~&~t t~tgt~~ar~ ~'t~~~~~~~ ~t~.~~itr~~g~~ ~g~a~.tg~i.,s ~~~~.~4ma~~tt.~ a~~ag'.t~~~
~~~~~~.g~tgg a~~~gaga~~
aa~~at~c~.~a t~~~gt~:gt~ ttt~a~a~.~~t~ tt~~~~:~w~~tgg ~.g~t~g~~wt g~tca~~.tt~
a~r:ag~t~~~.
~~~L~ag~tgg a~~agaga~~
~~.t~:g~ggg9 ~tgag~tggt ggatg~.t~~tt ~:agtt~gtgt gtgga~~gs~~g gg~tttta~
tt~a~.~aag~
~ca.~agg~ta ~tttg~gggg ~tg~.g~tggt ggatg~t~ tt ~~gtt~.gvgt gtgga~~g~g gg~tttta~
tt~a~.~aag~
~~~.~agg~ta ~tt~:gcgggg ~tgag~tggt ggatg~tfwtt ~agtw~:gtg~ gtgga~cg~.g ggg~tttta~
tt~aa~aag~
~~~.~agg~ta tge~~t~~~.r~~ att~gg~.gg~a~~t~aga~ a~ng~ attgv:g g~.tgag~:,:tt gcat~~ggag ~tgtg~at~tg aggaga~mgg ~~gg~t~~ag~ att~ggagg~~.~~t~:a<~a~ agg~artg~~:~g~.tgagt:gtt g~at~~ggag ~tgtgat~.tg agc~~:ga~tgg tgg~t~~ag~ att~ggaggg ~a~~t~a~~a~ agg~attg~:g gatgagtgtt g~:tt~~ggag ~vgtga~c~tg aggag~.~tgg ag~agta~tg tg~~,c~a~tg aag~~t~.~~a a~.g~ag~~~g ~t~t~.t~~gt g~~:~ag~g~~
~~a~°tg~.~at g~~~aaga~t ag~.tgta~tg tg~~c~actg aag~~t~~~.a aag~ag~~~g ~t~tatc~~~t g~~~~ag~g~~
~~:a~tg~.~.at g~~~aaga~t aga.tgta~ttg~~~~a~tg aagc~t.~.~ ~ag~~g~~~g ~t~tat~~aa~:~ag~gc~ a~:a~tg~.~at g~~~-!tit ~~g~.~gg~~g ~~~~~~~g~.~ c~~,~.~~.~~~.g~ ~g~gg.~,g~c~. ~~gg~,~~.~~ g~~~~~~~g~.
~.~gt~gg~9g ~g~W~~~~~~~
~.~c~~~.gga~g ~~~~.~~tg~~ g~.~~~.~~~s~~ ~.gg,g~~.~~6~~~.gg~.;~~~~~ g~::~~~~~g~
~.~g~gag9 ~g~~~~~~:.~~
c~~~~~ ~~g~~~c~~~
ggc~g~~ga~~ ~~g~.~~
~~,si s~c~~~r~~~ c~darice I~~~~~ ~d~~ sv~, ~-s~~~) r s~c~~e~~~;~~~ ~~ir~
s~r~s:
~co~~ _ '~~'~ bits ~3'~~), ~xpe~~ = f~.~'~
~d~r~~a~ta~s = 374r~75 (9~%) ~~r~r~.d = ~~~s / ~l~~s~
~ ~~t se~~.~err~~ ~~dar~~e ICS-~<-li ~~~d~~ s~~dr~~s~~~.~) r s~q~~~~c~~~ ~rvr~
~~~~4s~~s:
~c;or~ = 500 bits (3I2), ~xp~c~ _ ~- ~ ~~
~de~~i~i~s = 3~~r3~ i X95%), ~a~s - ~r3~ i '~ ~'~) ~~~~.r~d = ~~~~ r ~r~v~
,~~~~e~a~~ ~od~~:~ I~~-I:
t~~c~.~g~t't ~~~a~.z'~~~~~ ~~~~~.~~a.s~.~cs. ~a~~~~.~tgt n~.~t~s~~g~~:
t~~g'~t~t ~~ I c~ttcac~t~g ~~~t~at~.~t ~.~~:a~t~tg a~t~tg~ttg b ~4I tga;~tgg~ ga.t~t~t~ .~~'~~e~tt tc~~.c~a~ c~.~vtt~ta~
~tc°~~r~~~~c~~~~~c~~s ~~I
~,~1 ~o~~~ s~or~
~eI .gc~t~~~ac'~ac~agaa ~~~.~:~~~:. ~.~~~~aggat ~:~y~~tttg~tg~ ttgag~a~.~.~:
a ~~~I tg~~.a.a~at ~gaaa~a~~t ~c~~aata~~ ~~.t~.taagt ~~s~.ataa~:at t~.~a~~gatg ~~~ I ggeat'~t~~~. ~~aatgaaat a~~~~.~gv~ a~ ato:y:.
~~q~~~~~ ~~~~t~ dI ~~-~
~,ef o ,~~~~~ea~~es ~~'l~we~ ~~3~~~',s ~~e~~z~a~ ~"~~r~ ~a're~~e~f ~~~~s~v i~asa~l~a~-~z~~ ~~~~th f~~~'oa°
~~r~e~a~~sn~se'~oii ~~, ~torra~~r~ I~~.~, ~~oi~g ~-, I~~ii ~,~~, l~v~i~i~
~a.hds ~.os 1~~6 ~~~ ~~;~~.t~o~: ~~~~-~s~
o ~~s~iir~-Iiic~ ~ro~rt~b I'~~c~or r~~~i~~~s ~~~is ~or~~ ~~i~~~r~i d~~sr~Jr i~
r~~~~ by both bro~v'ch hor~o~e-.d~pvr~d~r~~~ ~~d -i~gd~;~~r~d~~~ rr~~;i~h~.r~iss~ I~,~ohar ~'t, oha~~~~ ~, ~o ~, l~.z~~ar ~, I~or~~h~ao ~,~, l~y~~~dai ~, ~ayi~nic ~:~p ~r~do~rir~oio~~
~0~3~
~~/Ia.x; I ~.~.(~ ):92~-3 DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVETS
COMPRI~:ND PLUS D'UN TOME.
CECI EST L,E TOME 1 DE 2 NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadien des Brevets.
JUMBO APPLICATIONS / PATENTS
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Claims
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| Application Number | Priority Date | Filing Date | Title |
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| CA 2437250 CA2437250A1 (en) | 2003-08-14 | 2003-08-14 | Microencapsulation of living cells in covalently crosslinked alginate-poly-l-lysine-alginate membranes |
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| CA 2437250 CA2437250A1 (en) | 2003-08-14 | 2003-08-14 | Microencapsulation of living cells in covalently crosslinked alginate-poly-l-lysine-alginate membranes |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011011962A1 (en) * | 2009-07-31 | 2011-02-03 | 清华大学 | Method for preparing complex multi-layer tissue organ precursor |
| US8202701B2 (en) | 2004-10-08 | 2012-06-19 | Georgia Tech Research Corporation | Microencapsulation of cells in hydrogels using electrostatic potentials |
| US11077289B2 (en) | 2013-03-07 | 2021-08-03 | Viacyte, Inc. | 3-dimensional large capacity cell encapsulation device assembly |
| CN113730372A (en) * | 2021-10-15 | 2021-12-03 | 中国人民解放军陆军军医大学第二附属医院 | Phospholipid membrane microcapsule of neural stem cell and preparation method thereof |
| CN115737600A (en) * | 2022-11-25 | 2023-03-07 | 苏州大学 | Continuous hydrogen production biological microsphere and preparation method and application thereof |
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2003
- 2003-08-14 CA CA 2437250 patent/CA2437250A1/en not_active Abandoned
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8202701B2 (en) | 2004-10-08 | 2012-06-19 | Georgia Tech Research Corporation | Microencapsulation of cells in hydrogels using electrostatic potentials |
| WO2011011962A1 (en) * | 2009-07-31 | 2011-02-03 | 清华大学 | Method for preparing complex multi-layer tissue organ precursor |
| US11077289B2 (en) | 2013-03-07 | 2021-08-03 | Viacyte, Inc. | 3-dimensional large capacity cell encapsulation device assembly |
| CN113730372A (en) * | 2021-10-15 | 2021-12-03 | 中国人民解放军陆军军医大学第二附属医院 | Phospholipid membrane microcapsule of neural stem cell and preparation method thereof |
| CN115737600A (en) * | 2022-11-25 | 2023-03-07 | 苏州大学 | Continuous hydrogen production biological microsphere and preparation method and application thereof |
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