CA2216090A1 - A process of prolonging organ allograft survival - Google Patents
A process of prolonging organ allograft survival Download PDFInfo
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- CA2216090A1 CA2216090A1 CA002216090A CA2216090A CA2216090A1 CA 2216090 A1 CA2216090 A1 CA 2216090A1 CA 002216090 A CA002216090 A CA 002216090A CA 2216090 A CA2216090 A CA 2216090A CA 2216090 A1 CA2216090 A1 CA 2216090A1
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- nematode
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- nippostrongylus
- brasiliensis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/56—Materials from animals other than mammals
- A61K35/62—Leeches; Worms, e.g. cestodes, tapeworms, nematodes, roundworms, earth worms, ascarids, filarias, hookworms, trichinella or taenia
Abstract
The present invention provides a process of prolonging organ allograft survival in an organism comprising down-regulating Th1 activity in the organism. The down-regulation of Th1 activity is accomplished either by infecting the organism with an effective Th2 up-regulating amount of a nematode or by administering to the organism an effective Th2 up-regulating amount of a soluble extract of a nematode. Exemplary and preferred nematodes for use in a process of the present invention are of the genus Nippostrongylus, Trichuris, Ascaris or Caenorhabditis. Most preferred is the nematode Nippostrongylus brasiliensis. A process of the present invention is particularly useful in prolonging kidney allograft survival.
Description
W 096/29082 PCT~B~. ~ C
A PROCESS OF PROLONGING ORGAN ALLOGRA~T SURVIVAL
Description Technical Field of the Invention The field of the present invention is organ allograft survival.
More specifically, the field of the present invention is prolongation of organ allograft survival. Organ allograft survival is prolonged by down-regulating Thl activity in an organism receiving an organ allograft.
Back~round of the Invention The development of novel immunosuppressive drugs has caused a dramatic increase in the short term survival of organ transplants in recent years. The use of such drugs, however, is associated with side effects such as opportunistic infections, tumors and ultimately, chronic rejection.
The recent description of two subsets of T helper cells (Th), with different cytokine secretion profiles and activities, may provide a new paradigm for immunoregulation of organ allograft rejection (Mocm~nn et al., Immuno]. Todav 8:223, 1987~. The two Th cell subsets, de~ignated Thl and Th2, can be characterized on the basis of their respective cytokine production profiles. Th1 cells produce and secrete interferon-gamma (IFN-y), lymphotoxin (LT) and interleukin-2 (IL-2). On the other hand, Th2 cells produce and secrete interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6) and interleukin-10 (IL-10).
Acute organ allograft rejection may result from responses associated with the pro-infl~mm~tory cytokines IL-2, IFN-y, and ~NF-~r, derived from Thl ce]ls. Down-regulation of Thl activity, therefore, may be a useful immunomodulatory therapy. It is also well known that inlEection by W O 96/29082 PCT~B96/00400 certain parasitic organisms can modulate the balance between Thl and Th2 activity. By way of example, Sher and coworkers have demonstrated that a depression of Thl-type responses can be achieved by helminth infection (Sher et al., J. Immunol., 147:2713, 1991). Helminth infection can also result in up-regulation or stimulation of Th activity. Nematode induced up-regulation can encc~mp~cs stimulation of IgE (and IgG1 in mice or IgG4 in humans), mast cell hyperplasia and eosinophilia in all species, including humans.
It is well known that the immune modulation associated with helminth infection is unrelated to the worm antigensper se. For example, greater than 80 percent of the IgE generated during Nippostrongylus brasiliensis (N. brasiliensis) infection was found not to be directed to N.
brasiliensis (Jarrett el al., Clin. Exp. Immunol., 24:326, 1976).
Such induced up-regulation may be due to the cross-regulatory actions of the Thl/Th2 system. The Thl and Th2 subsets have been found to be cross-regulatory with respect to differentiation and activity. By way of example, cytokines produced by Th2 cells (e.g., IL-4 and IL-10) inhibit Thl cell growth and cytokine production and ablate the effects of Thl cytokines (e.g., IFN-y) on their targets, while IFN-y inhibits Th2 function (Powrie et al., Immunol. Today. 14:270, 1993).
The concept of resistance or susceptibility being related to Thl/Th2 balance has also been suggested in a variety of other models, including AIDS, and even successful pregnancy. The finding of pro-infl~mm~tory cytokines IL-2, IFN-y, TNF-~r and IL-6 in rejecting kidney allografts lends support to the hypothesis that Thl-type T cells orchestrate the immune response, which culminates in rejection. It likely that a WO 96/290g2 PCr/IB~C/C~ I~~
Thl/Th2 balance is established within the graft which changes over time, ~ differs among individuals, and is dependent on the treatment ~mini~tered but that the cellular mech~ni~mc behind graft rejection, such as infi~tration by macrophages and CI'L, are c~ e.~t with the activities of Thl cells.
s Brief Summary of the Invention In one aspect, the present invention provides a ~-ocess of prolonging organ allograft survival in an organism COlll~li',illg down-regulating Thl activity in the olg~i.~....
In a preferred embodiment, the down-regulation of Th1 activity is accomplished by infecting the organism with an effective Th2 up-regulating amount of a nematode.
In another preferred embodiment, the down-regulation of Thl activity is accomplished by ~-lmini~t~.ring to the org~ni~m an effective Th2 up-regulating amount of a soluble extract of a nematode.
Exemplary and preferred nematodes for use in a process of the present invention are of the genus N'ippostrongvlus, Tric~uns, Ascaris or Caenorhabditis. More preferred are Nippostrongylus brasiliensis, Trichuris muris, Trichuris suis, Ascaffs lum~ffcoides, Ascaris suum or Caenorha~ditis elegans. Most preferred is Nippostrongylus brasiliensis (N. brasiliensis). A
process of the present invention is particularly useful in prolonging hdney allograft survival.
Brief Description of the Drawin~s In the drawing, which forms a portion of the specification:
W 096/29082 PCT~B96100400 FIG. 1 shows IgG1 levels following N. brasiliensZs infection or treatment with N. br~siliens~s e~ctract.
., Detailed Descripbon of the Invention I. The Invenbon Infection with ~alasi~es such as nematodes induces strong IgE
responses not restricted to parasite antigens. In addition, certain he~minths have profound immunomodulatory effects on infected ~nim~l~, inducing strong Th2 responses while rliminiching Thl activity. The present invention provides that infection of organisms with n.om~todes or the ~(1mini~tration of soluble nematode extracts to organisms significantly prolonged organ allograft survival in those org~ni~n~
II. Process of Prolonging Allograft Survival In one aspect, the present invention provides a process of prolonging organ al~ograft survival in an organism culllpli~ing down-regulating Thl activity in the Ol~ iS~.
As used herein; the phrase "organ allograft" means a body organ graft from a donor organism of the same species but a different genotype than the recipient organism. As used herein, the term "organ"
means a vascularized internal organ such as heart, liver and kidney. An exemplary and preferred organ allograft is a hdney allograft. As used herein, an organ does not include skin. Skin differs from vascularized organs contemplated by the present invention because a substantial component of the graft vascular supply is derived from the donor rather than the recipient. Thus, shn allografts are much less susceptible to certain immune-mediated injuries (Bradley e~ aL, Immunol. Today. 13:434, 1992).
W 096129082 PCT/lbg~01 A
S
As used herein, the terms "up-regulation" and "down-~ regulation" and their gr~mm~t~c~l equivalents mean, respectively, shmulation and inhibition. Thus, down-regulahon of Thl activity means a depression or inhibition of the cascade of physio]ogical responses that acc~ pally a Thl cytokine production profile and up-regulahon of Th2 activity means a stimulahon or enhancement of the c~ de of physiological responses that acco.l.~.al,y a Th2 cytokine production profile.
In a preferred embodiment, the down-regulation of Thl activity is accomplished by infecting the o~ga~ - with an effective Th2 up-regulating amount of a nematode. An effective amount is that amount necessary to bring about the desired effect (e.g., down-regulation of Th1 activity). Means for determining an effective amount are well known in the art. By way of example, an organism is infected with various amounts of a particular nematode and the level of Thl activity monitored. An effective amount will depend inter alia, as is well known in the art, on the nature of the organism being injected and the nature of the particular nematode used.
Exemplary effective amounts using rodents and the nematode N. brasiliensis are set forth hereinafter in t~e Examples.
In another plefelled embodiment, the down-regulation of Thl activity is accomplished by ~mini~terjng to the organism an effective Th2 up-regulating amount of a soluble e~tract of a nem~tode. As set forth above, means for determining an effective amount of a soluble extract are well known and readily ascertainable by a skilled artisan. Means for making a soluble extract are also well known in the art (See. e.g., Lee e~ aL, Immunology, 55:721, 1985). By way of example, nematodes are homogenized in an aqueous medium such as saline (0.9% NaCl). The medium can further comprise buffers such as phosphate. Homogenization can be accomplished by mech~nir~l means or other disruptive means such as sonication. The homogenate is ~pically cleared of particulate matter using ultracentrifugation. The nematode is then filter sterilized to remove bacteria.
s A soluble nematode extract can be ~tlminictered to the organism by parenteral routes of ~tlminictration as is well known in the art.
The e7~tract can be dispersed or suspended in a physiologically acceptable diluent prior to ~f~minictratiom A preferred route of a-lminictration is subcutaneous. An extract can be ~tlminictered prior to, simultaneously with or after organ transplantation. In a ~lefelled embodiment, an extract is minictered prior to, simultaneously with and after transplantation. One of ordinary skill in the art can readily dete~nine the optimum ~minictratjon schedule for a given transplant and treatment regimen.
Any nematode that down-regulates Thl activity can be used in a process of the present invention. Exemplary and preferred nematodes for use in a process of the present invention are of the genus Nippostrongylus, Trichuris, Ascaris or Caenorhabditis. More preferred are Nippostrongylus brasiliensis, Trichuris muris, Trichuris suis, Ascaris lumbricoides, Ascaris suum or Caenorhabditis elegans. Most preferred is Nippostrongylus brasiliensis (~.
brasiliensis).
A process of the present invention prolongs allograft survival when compared to allograft survival performed in the absence of nematode treatment. In other words, a process of the present invention improves or 4 increases survival time of the allograft.
W O 96/29082 PCTAB~ OIC~
A detailed description of the use of a process of the present invention to prolong kidney allograft survival is set forth hereinafter in the FY~mples. Briefly, rats were infected with N. brasilienszs (Nb) or treated with a soluble extract of N. brasiliensis (Nb extract) and received a kidney S allograft. The surviva] time of the kidney al]ograft was increased from 9.7 +
1.2 days (no treatment) to 32.0 + 10.0 days (Nb infection) and 21.5 + 4.6 days (Nb extract treatment). By day S post tr~n~pl~nt, untreated allografts contained a substantial mononuclear cell infiltrate and kidney destruction was advanced. In contrast, hdneys transplanted into Nb treated animals had only a mild cellu]ar infiltrate and retained normal architecture. FACS
analysis revealed a significant decrease in the number of CD8+ cells infiltrating Nb treated grafts. Prelimin~ry RT-PCR analysis of mRNA
expression by graft-infiltrating ce]ls indicated that the production of IL-4 wasup-regulated in Nb-treated kidneys as opposed to untreated allografts.
In additional studies, Nb treatrnent was associated with substantial and significant increases in IgG1 levels (See Example 2).
The following Examples illustrate preferred embodiments of the present invention and are not limiting of the claims or specification in any way.
EX~MPLE 1: Kidnev Allo~raft Survival Lewis (LEW,RT1l) and Brown Norway (BN, RT1n) rats were chosen as recipients and donors, respectively. Male rats were purchased from Harlan Sprague Dawley (Indianapolis, IN) and provided with water and rat chow ad libitum. ~nim~lc receiving a ~.brasiliensis infection were injected with 3~00 third stage infective larvae subcutaneously four days prior to transplantation. ~nim~l~ receiving the nematode products were injected CA 022l6090 l997-09-22 W 096/29082 PCTAB9~'001 subcutaneously, at day -4, 0 and +4 (with day 0 being the day of transplant) with 200 worm equivalents of adult worm homogenate. All procedures were performed under sodium pentobarbital ~n~çst~esia (65 mglkg).
S The left kidney of the donor (BN) rat was perfused in situ withcold (4~C) saline, mobilized, and removed. It remained in cold saline during the pl~a~ation of the recipient. Following left nephrectomy of the Lewis recipient, the BN kidney was transplanted in a heterotopic position.
Ureteric reconstruction in these experiments was achieved by end-to-end anastomosis. Four days after the transplants, the recipients were re-anaesthetized and a right native nephrectomy was perforrned. ~nim~lc were sacrificed when they showed signs of morbidity due to failure of the transplanted kidney.
Graft survival data are shown below in Table 1.
Kidney Allograft Survival Following Nematode Infection Treatment Survival (days) Mean + SD
No treatment 10, 8, 11, 11 9,8,11,9,10 9.7+1.2 Nb infection 34, 27, 34, 23 25, 34, 25, 54 32.0 + 10.0 Nb extract 30, 21, 22, 20 16, 20 21.5 + 4.6 These results clearly show a significant prolongation of kidney allograft survival in anirnals g*en either a N.brasiliensis infection or soluble W 096/29082 PCT/Lb,'~
_ 9 _ worm extract. In untreated ~nim71~ kidneys survived a mean of only 9.7 +
1.2 (mean + SD, n=9) days, whereas kidneys in nematode infected rats retained functional capacity much longer, resulting in a mean survival time of 32.0 + 10.0 (mean + DS, n-8) days, with one animal retaining grafted S kidney function up to 54 days post tr~nq-l~nt. In ~nim~l~ treated with the worm extract the survival was also significantly longer (mean 21.5 + 4.6).
In addition to graft survival the histological appearance of the transplanted kidneys was ~ccecse~ in the control versus ~. brasiliensis treated recipients 5 days after transplant. Transplanted kidneys were flushed with cold saline and cut into 2-3 mm thick slices before fixation in formalin.
Sections were taken and stained with hem~toxylin and eosin using standard protocols.
The results demonstrate clear differences between the kidneys taken from the N. brasiliensis infected and the control groups. The intensity of the infiltration was, in a blinded evaluation, found to be much more extensive in the control group when compared to the 1~. brasiiiensis infected group. In the control group,'there was extensive infiltration of the interstitiaby mononuclear cells and evidence of damage to the tubular endothelium in some tubules. In the kidneys isolated from N. brasiliensis infected recipients, in contrast, there was only a small amount of mononuclear infiltrate around major vessels and almost no interstitial infiltrate. Kidney architecture in the N. brasiliensis infected recipients was virtually normal.
These data show that infection of rats by the nematode 1~.
brasiliensis or treatment with N. brasiliensis extracts significantly prolonged kidney allograft survival across a known strong histocompatibility barrier.
CA 022l6090 l997-09-22 W 096129082 PCTAB~6 This prolongation likely results from the ability of N. ~rasiliensis to stronglyactivate Th2 responses with a resultant inhibition of Thl activity.
EX~MPLE 2: ~e,eulation Of I~G1 Response Bv Nematodes Increases in the serum levels of total (non-specific) IgE and IgG1 or IgG4 are associated with nematode infections in mice and humans respectively. There are several points where this response can be regulated.
One of such, is during the process of Immunoglobulin class switch.
A group of five BALB/c mice was infected with 500 infective larvae of the nematode Nippostrongylus brasiliensis and a second group of five BALB/c mice (age matched) was injected subcutaneously with an extract from N. brasiliensis (Adult Worm Homogenate, AWH) at a concentration of 200 worm equivalent emulsified in Freund's Incomplete Adjuvant. The levels of total IgG1 in the serum obtained from the blood taken from these mice, were measured weekly for ten weeks, by IgG1 caplure ELISA.
The data showed clearly that, the nematode extract, AWH
induces signiScant increases in the production of total serum IgG1. The result agrees with the massive increase in total IgE aDd IgG1 levels associated with infection with the larvae. As shown in FIG. 1, a ten fold increase in the IgG1 levels was observed. Prior to the injection of AWH, the total serum IgG1 level in the mice was 0.25mg/ml. This level increased continuously from week 1 (0.30mg/ml) through week 4 (1.15 mglml).
Further increases in the serum IgG1 level upon re-injection with the nematode extract at week 4, were also observed. It reached its maximum (2.15 mg/ml) at week 7, before it started to decline. AT. ~rasiliensis extract AWH, also induced significant increase iD total IgG1 levels in vivo.
A PROCESS OF PROLONGING ORGAN ALLOGRA~T SURVIVAL
Description Technical Field of the Invention The field of the present invention is organ allograft survival.
More specifically, the field of the present invention is prolongation of organ allograft survival. Organ allograft survival is prolonged by down-regulating Thl activity in an organism receiving an organ allograft.
Back~round of the Invention The development of novel immunosuppressive drugs has caused a dramatic increase in the short term survival of organ transplants in recent years. The use of such drugs, however, is associated with side effects such as opportunistic infections, tumors and ultimately, chronic rejection.
The recent description of two subsets of T helper cells (Th), with different cytokine secretion profiles and activities, may provide a new paradigm for immunoregulation of organ allograft rejection (Mocm~nn et al., Immuno]. Todav 8:223, 1987~. The two Th cell subsets, de~ignated Thl and Th2, can be characterized on the basis of their respective cytokine production profiles. Th1 cells produce and secrete interferon-gamma (IFN-y), lymphotoxin (LT) and interleukin-2 (IL-2). On the other hand, Th2 cells produce and secrete interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6) and interleukin-10 (IL-10).
Acute organ allograft rejection may result from responses associated with the pro-infl~mm~tory cytokines IL-2, IFN-y, and ~NF-~r, derived from Thl ce]ls. Down-regulation of Thl activity, therefore, may be a useful immunomodulatory therapy. It is also well known that inlEection by W O 96/29082 PCT~B96/00400 certain parasitic organisms can modulate the balance between Thl and Th2 activity. By way of example, Sher and coworkers have demonstrated that a depression of Thl-type responses can be achieved by helminth infection (Sher et al., J. Immunol., 147:2713, 1991). Helminth infection can also result in up-regulation or stimulation of Th activity. Nematode induced up-regulation can encc~mp~cs stimulation of IgE (and IgG1 in mice or IgG4 in humans), mast cell hyperplasia and eosinophilia in all species, including humans.
It is well known that the immune modulation associated with helminth infection is unrelated to the worm antigensper se. For example, greater than 80 percent of the IgE generated during Nippostrongylus brasiliensis (N. brasiliensis) infection was found not to be directed to N.
brasiliensis (Jarrett el al., Clin. Exp. Immunol., 24:326, 1976).
Such induced up-regulation may be due to the cross-regulatory actions of the Thl/Th2 system. The Thl and Th2 subsets have been found to be cross-regulatory with respect to differentiation and activity. By way of example, cytokines produced by Th2 cells (e.g., IL-4 and IL-10) inhibit Thl cell growth and cytokine production and ablate the effects of Thl cytokines (e.g., IFN-y) on their targets, while IFN-y inhibits Th2 function (Powrie et al., Immunol. Today. 14:270, 1993).
The concept of resistance or susceptibility being related to Thl/Th2 balance has also been suggested in a variety of other models, including AIDS, and even successful pregnancy. The finding of pro-infl~mm~tory cytokines IL-2, IFN-y, TNF-~r and IL-6 in rejecting kidney allografts lends support to the hypothesis that Thl-type T cells orchestrate the immune response, which culminates in rejection. It likely that a WO 96/290g2 PCr/IB~C/C~ I~~
Thl/Th2 balance is established within the graft which changes over time, ~ differs among individuals, and is dependent on the treatment ~mini~tered but that the cellular mech~ni~mc behind graft rejection, such as infi~tration by macrophages and CI'L, are c~ e.~t with the activities of Thl cells.
s Brief Summary of the Invention In one aspect, the present invention provides a ~-ocess of prolonging organ allograft survival in an organism COlll~li',illg down-regulating Thl activity in the olg~i.~....
In a preferred embodiment, the down-regulation of Th1 activity is accomplished by infecting the organism with an effective Th2 up-regulating amount of a nematode.
In another preferred embodiment, the down-regulation of Thl activity is accomplished by ~-lmini~t~.ring to the org~ni~m an effective Th2 up-regulating amount of a soluble extract of a nematode.
Exemplary and preferred nematodes for use in a process of the present invention are of the genus N'ippostrongvlus, Tric~uns, Ascaris or Caenorhabditis. More preferred are Nippostrongylus brasiliensis, Trichuris muris, Trichuris suis, Ascaffs lum~ffcoides, Ascaris suum or Caenorha~ditis elegans. Most preferred is Nippostrongylus brasiliensis (N. brasiliensis). A
process of the present invention is particularly useful in prolonging hdney allograft survival.
Brief Description of the Drawin~s In the drawing, which forms a portion of the specification:
W 096/29082 PCT~B96100400 FIG. 1 shows IgG1 levels following N. brasiliensZs infection or treatment with N. br~siliens~s e~ctract.
., Detailed Descripbon of the Invention I. The Invenbon Infection with ~alasi~es such as nematodes induces strong IgE
responses not restricted to parasite antigens. In addition, certain he~minths have profound immunomodulatory effects on infected ~nim~l~, inducing strong Th2 responses while rliminiching Thl activity. The present invention provides that infection of organisms with n.om~todes or the ~(1mini~tration of soluble nematode extracts to organisms significantly prolonged organ allograft survival in those org~ni~n~
II. Process of Prolonging Allograft Survival In one aspect, the present invention provides a process of prolonging organ al~ograft survival in an organism culllpli~ing down-regulating Thl activity in the Ol~ iS~.
As used herein; the phrase "organ allograft" means a body organ graft from a donor organism of the same species but a different genotype than the recipient organism. As used herein, the term "organ"
means a vascularized internal organ such as heart, liver and kidney. An exemplary and preferred organ allograft is a hdney allograft. As used herein, an organ does not include skin. Skin differs from vascularized organs contemplated by the present invention because a substantial component of the graft vascular supply is derived from the donor rather than the recipient. Thus, shn allografts are much less susceptible to certain immune-mediated injuries (Bradley e~ aL, Immunol. Today. 13:434, 1992).
W 096129082 PCT/lbg~01 A
S
As used herein, the terms "up-regulation" and "down-~ regulation" and their gr~mm~t~c~l equivalents mean, respectively, shmulation and inhibition. Thus, down-regulahon of Thl activity means a depression or inhibition of the cascade of physio]ogical responses that acc~ pally a Thl cytokine production profile and up-regulahon of Th2 activity means a stimulahon or enhancement of the c~ de of physiological responses that acco.l.~.al,y a Th2 cytokine production profile.
In a preferred embodiment, the down-regulation of Thl activity is accomplished by infecting the o~ga~ - with an effective Th2 up-regulating amount of a nematode. An effective amount is that amount necessary to bring about the desired effect (e.g., down-regulation of Th1 activity). Means for determining an effective amount are well known in the art. By way of example, an organism is infected with various amounts of a particular nematode and the level of Thl activity monitored. An effective amount will depend inter alia, as is well known in the art, on the nature of the organism being injected and the nature of the particular nematode used.
Exemplary effective amounts using rodents and the nematode N. brasiliensis are set forth hereinafter in t~e Examples.
In another plefelled embodiment, the down-regulation of Thl activity is accomplished by ~mini~terjng to the organism an effective Th2 up-regulating amount of a soluble e~tract of a nem~tode. As set forth above, means for determining an effective amount of a soluble extract are well known and readily ascertainable by a skilled artisan. Means for making a soluble extract are also well known in the art (See. e.g., Lee e~ aL, Immunology, 55:721, 1985). By way of example, nematodes are homogenized in an aqueous medium such as saline (0.9% NaCl). The medium can further comprise buffers such as phosphate. Homogenization can be accomplished by mech~nir~l means or other disruptive means such as sonication. The homogenate is ~pically cleared of particulate matter using ultracentrifugation. The nematode is then filter sterilized to remove bacteria.
s A soluble nematode extract can be ~tlminictered to the organism by parenteral routes of ~tlminictration as is well known in the art.
The e7~tract can be dispersed or suspended in a physiologically acceptable diluent prior to ~f~minictratiom A preferred route of a-lminictration is subcutaneous. An extract can be ~tlminictered prior to, simultaneously with or after organ transplantation. In a ~lefelled embodiment, an extract is minictered prior to, simultaneously with and after transplantation. One of ordinary skill in the art can readily dete~nine the optimum ~minictratjon schedule for a given transplant and treatment regimen.
Any nematode that down-regulates Thl activity can be used in a process of the present invention. Exemplary and preferred nematodes for use in a process of the present invention are of the genus Nippostrongylus, Trichuris, Ascaris or Caenorhabditis. More preferred are Nippostrongylus brasiliensis, Trichuris muris, Trichuris suis, Ascaris lumbricoides, Ascaris suum or Caenorhabditis elegans. Most preferred is Nippostrongylus brasiliensis (~.
brasiliensis).
A process of the present invention prolongs allograft survival when compared to allograft survival performed in the absence of nematode treatment. In other words, a process of the present invention improves or 4 increases survival time of the allograft.
W O 96/29082 PCTAB~ OIC~
A detailed description of the use of a process of the present invention to prolong kidney allograft survival is set forth hereinafter in the FY~mples. Briefly, rats were infected with N. brasilienszs (Nb) or treated with a soluble extract of N. brasiliensis (Nb extract) and received a kidney S allograft. The surviva] time of the kidney al]ograft was increased from 9.7 +
1.2 days (no treatment) to 32.0 + 10.0 days (Nb infection) and 21.5 + 4.6 days (Nb extract treatment). By day S post tr~n~pl~nt, untreated allografts contained a substantial mononuclear cell infiltrate and kidney destruction was advanced. In contrast, hdneys transplanted into Nb treated animals had only a mild cellu]ar infiltrate and retained normal architecture. FACS
analysis revealed a significant decrease in the number of CD8+ cells infiltrating Nb treated grafts. Prelimin~ry RT-PCR analysis of mRNA
expression by graft-infiltrating ce]ls indicated that the production of IL-4 wasup-regulated in Nb-treated kidneys as opposed to untreated allografts.
In additional studies, Nb treatrnent was associated with substantial and significant increases in IgG1 levels (See Example 2).
The following Examples illustrate preferred embodiments of the present invention and are not limiting of the claims or specification in any way.
EX~MPLE 1: Kidnev Allo~raft Survival Lewis (LEW,RT1l) and Brown Norway (BN, RT1n) rats were chosen as recipients and donors, respectively. Male rats were purchased from Harlan Sprague Dawley (Indianapolis, IN) and provided with water and rat chow ad libitum. ~nim~lc receiving a ~.brasiliensis infection were injected with 3~00 third stage infective larvae subcutaneously four days prior to transplantation. ~nim~l~ receiving the nematode products were injected CA 022l6090 l997-09-22 W 096/29082 PCTAB9~'001 subcutaneously, at day -4, 0 and +4 (with day 0 being the day of transplant) with 200 worm equivalents of adult worm homogenate. All procedures were performed under sodium pentobarbital ~n~çst~esia (65 mglkg).
S The left kidney of the donor (BN) rat was perfused in situ withcold (4~C) saline, mobilized, and removed. It remained in cold saline during the pl~a~ation of the recipient. Following left nephrectomy of the Lewis recipient, the BN kidney was transplanted in a heterotopic position.
Ureteric reconstruction in these experiments was achieved by end-to-end anastomosis. Four days after the transplants, the recipients were re-anaesthetized and a right native nephrectomy was perforrned. ~nim~lc were sacrificed when they showed signs of morbidity due to failure of the transplanted kidney.
Graft survival data are shown below in Table 1.
Kidney Allograft Survival Following Nematode Infection Treatment Survival (days) Mean + SD
No treatment 10, 8, 11, 11 9,8,11,9,10 9.7+1.2 Nb infection 34, 27, 34, 23 25, 34, 25, 54 32.0 + 10.0 Nb extract 30, 21, 22, 20 16, 20 21.5 + 4.6 These results clearly show a significant prolongation of kidney allograft survival in anirnals g*en either a N.brasiliensis infection or soluble W 096/29082 PCT/Lb,'~
_ 9 _ worm extract. In untreated ~nim71~ kidneys survived a mean of only 9.7 +
1.2 (mean + SD, n=9) days, whereas kidneys in nematode infected rats retained functional capacity much longer, resulting in a mean survival time of 32.0 + 10.0 (mean + DS, n-8) days, with one animal retaining grafted S kidney function up to 54 days post tr~nq-l~nt. In ~nim~l~ treated with the worm extract the survival was also significantly longer (mean 21.5 + 4.6).
In addition to graft survival the histological appearance of the transplanted kidneys was ~ccecse~ in the control versus ~. brasiliensis treated recipients 5 days after transplant. Transplanted kidneys were flushed with cold saline and cut into 2-3 mm thick slices before fixation in formalin.
Sections were taken and stained with hem~toxylin and eosin using standard protocols.
The results demonstrate clear differences between the kidneys taken from the N. brasiliensis infected and the control groups. The intensity of the infiltration was, in a blinded evaluation, found to be much more extensive in the control group when compared to the 1~. brasiiiensis infected group. In the control group,'there was extensive infiltration of the interstitiaby mononuclear cells and evidence of damage to the tubular endothelium in some tubules. In the kidneys isolated from N. brasiliensis infected recipients, in contrast, there was only a small amount of mononuclear infiltrate around major vessels and almost no interstitial infiltrate. Kidney architecture in the N. brasiliensis infected recipients was virtually normal.
These data show that infection of rats by the nematode 1~.
brasiliensis or treatment with N. brasiliensis extracts significantly prolonged kidney allograft survival across a known strong histocompatibility barrier.
CA 022l6090 l997-09-22 W 096129082 PCTAB~6 This prolongation likely results from the ability of N. ~rasiliensis to stronglyactivate Th2 responses with a resultant inhibition of Thl activity.
EX~MPLE 2: ~e,eulation Of I~G1 Response Bv Nematodes Increases in the serum levels of total (non-specific) IgE and IgG1 or IgG4 are associated with nematode infections in mice and humans respectively. There are several points where this response can be regulated.
One of such, is during the process of Immunoglobulin class switch.
A group of five BALB/c mice was infected with 500 infective larvae of the nematode Nippostrongylus brasiliensis and a second group of five BALB/c mice (age matched) was injected subcutaneously with an extract from N. brasiliensis (Adult Worm Homogenate, AWH) at a concentration of 200 worm equivalent emulsified in Freund's Incomplete Adjuvant. The levels of total IgG1 in the serum obtained from the blood taken from these mice, were measured weekly for ten weeks, by IgG1 caplure ELISA.
The data showed clearly that, the nematode extract, AWH
induces signiScant increases in the production of total serum IgG1. The result agrees with the massive increase in total IgE aDd IgG1 levels associated with infection with the larvae. As shown in FIG. 1, a ten fold increase in the IgG1 levels was observed. Prior to the injection of AWH, the total serum IgG1 level in the mice was 0.25mg/ml. This level increased continuously from week 1 (0.30mg/ml) through week 4 (1.15 mglml).
Further increases in the serum IgG1 level upon re-injection with the nematode extract at week 4, were also observed. It reached its maximum (2.15 mg/ml) at week 7, before it started to decline. AT. ~rasiliensis extract AWH, also induced significant increase iD total IgG1 levels in vivo.
Claims (18)
1. A process of prolonging organ allograft survival in an organism comprising down-regulating Th1 activity in the organism by administering to the organism an effective Th2 up-regulating amount of a nematode.
2. The process according to claim 1 wherein down-regulating Th1 activity is accomplished by administering to the organism an effective Th2 up-regulating amount of a soluble extract of a nematode.
3. The process according to claim 1 wherein the nematode is of the genus Nippostrongylus, Trichuris, Ascaris or Caenorhabditis.
4. The process according to claim 2 wherein the nematode is of the genus Nippostrongylus, Trichuris, Ascaris or Caenorhabditis.
5. The process according to claim 3 wherein the nematode is of the genus Nippostrongylus.
6. The process according to claim 4 wherein the nematode is of the genus Nippostrongylus.
7. The process according to claim 5 wherein the nematode is Nippostrongylus brasiliensis.
8. The process according to claim 6 wherein the nematode is Nippostrongylus brasiliensis.
9. The process according to claim 1 wherein the organ is kidney.
10. A use of an effective Th2 up-regulating amount of nematode to prepare a medicament to prolong organ allograft survival in an organism by down-regulating Th1 activity in the organism.
11. The use according to claim 10 wherein the medicament is prepared with a soluble extract of a nematode.
12. The use according to claim 10 wherein the nematode is of the genus Nippostrongylus, Trichuris, Ascaris or Caenorhabditis.
13. The use according to claim 11 wherein the nematode is of the genus Nippostrongylus, Trichuris, Ascaris or Caenorhabditis.
14. The use according to claim 12 wherein the nematode is of the genus Nippostrongylus.
15. The use according to claim 13 wherein the nematode is of the genus Nippostrongylus.
16. The use according to claim 14 wherein the nematode is Nippostrongylus brasiliensis.
17. The use according to claim 15 wherein the nematode is Nippostrongylus brasiliensis.
18. The use according to any one of claims 10 to 17 wherein the medicament prolongs survival of a kidney.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US40921195A | 1995-03-23 | 1995-03-23 | |
| US08/409,211 | 1995-03-23 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2216090A1 true CA2216090A1 (en) | 1996-09-26 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002216090A Abandoned CA2216090A1 (en) | 1995-03-23 | 1996-03-25 | A process of prolonging organ allograft survival |
Country Status (3)
| Country | Link |
|---|---|
| AU (1) | AU5344996A (en) |
| CA (1) | CA2216090A1 (en) |
| WO (1) | WO1996029082A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1041994A4 (en) * | 1997-12-31 | 2003-02-19 | Univ Iowa Res Found | Use of parasitic biological agents for prevention and control of autoimmune diseases |
| US6521232B1 (en) * | 1998-03-31 | 2003-02-18 | Nisshin Flour Milling Co., Ltd. | Proteins having immunomodulatory activity and remedies for immunological diseases |
| GB9824034D0 (en) | 1998-11-03 | 1998-12-30 | Univ Nottingham | Immunomodulatory factors forimmunosuppressant and antiallergic treatment |
| JP5935200B2 (en) * | 2010-10-27 | 2016-06-15 | 国立大学法人山口大学 | Immunostimulants from helminth parasites |
| GB201402909D0 (en) * | 2014-02-19 | 2014-04-02 | Univ Southampton | Treating infection |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| FR2487198A1 (en) * | 1980-07-28 | 1982-01-29 | Berri Balzac Sa | NOVEL IMMUNO-SUPPRESSIVE SUBSTANCE, ITS METHOD OF ISOLATION AND ITS THERAPEUTIC APPLICATION |
| EP0629130A1 (en) * | 1992-03-04 | 1994-12-21 | Schering Corporation | Use of interleukin-10 to suppress graft-vs.-host disease |
| AU1987695A (en) * | 1994-03-11 | 1995-09-25 | Board Of Regents, The University Of Texas System | Immunomodulatory trichinella substances |
-
1996
- 1996-03-25 CA CA002216090A patent/CA2216090A1/en not_active Abandoned
- 1996-03-25 WO PCT/IB1996/000400 patent/WO1996029082A1/en active Application Filing
- 1996-03-25 AU AU53449/96A patent/AU5344996A/en not_active Abandoned
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| Publication number | Publication date |
|---|---|
| WO1996029082A1 (en) | 1996-09-26 |
| AU5344996A (en) | 1996-10-08 |
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