US20060127383A1 - Pre-conditioning cells against death - Google Patents
Pre-conditioning cells against death Download PDFInfo
- Publication number
- US20060127383A1 US20060127383A1 US11/347,059 US34705906A US2006127383A1 US 20060127383 A1 US20060127383 A1 US 20060127383A1 US 34705906 A US34705906 A US 34705906A US 2006127383 A1 US2006127383 A1 US 2006127383A1
- Authority
- US
- United States
- Prior art keywords
- disorders
- use according
- apoptosis
- aliquot
- blood
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 230000034994 death Effects 0.000 title description 3
- 231100000517 death Toxicity 0.000 title description 3
- 230000006907 apoptotic process Effects 0.000 claims abstract description 68
- 210000004369 blood Anatomy 0.000 claims abstract description 65
- 239000008280 blood Substances 0.000 claims abstract description 65
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 44
- 230000017074 necrotic cell death Effects 0.000 claims abstract description 32
- CBENFWSGALASAD-UHFFFAOYSA-N Ozone Chemical compound [O-][O+]=O CBENFWSGALASAD-UHFFFAOYSA-N 0.000 claims abstract description 19
- 230000000694 effects Effects 0.000 claims abstract description 18
- 230000037406 food intake Effects 0.000 claims abstract description 10
- 230000001590 oxidative effect Effects 0.000 claims abstract description 10
- 230000036760 body temperature Effects 0.000 claims abstract description 8
- 208000012902 Nervous system disease Diseases 0.000 claims abstract description 6
- 208000014674 injury Diseases 0.000 claims abstract description 6
- 208000025966 Neurological disease Diseases 0.000 claims abstract description 5
- 230000008733 trauma Effects 0.000 claims abstract description 4
- 210000000056 organ Anatomy 0.000 claims description 27
- 210000001519 tissue Anatomy 0.000 claims description 14
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 10
- 229910052760 oxygen Inorganic materials 0.000 claims description 10
- 239000001301 oxygen Substances 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 9
- 239000003814 drug Substances 0.000 claims description 8
- 208000024891 symptom Diseases 0.000 claims description 8
- 239000000126 substance Substances 0.000 claims description 7
- 229940079593 drug Drugs 0.000 claims description 6
- PMATZTZNYRCHOR-CGLBZJNRSA-N Cyclosporin A Chemical compound CC[C@@H]1NC(=O)[C@H]([C@H](O)[C@H](C)C\C=C\C)N(C)C(=O)[C@H](C(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)N(C)C(=O)CN(C)C1=O PMATZTZNYRCHOR-CGLBZJNRSA-N 0.000 claims description 5
- 229930105110 Cyclosporin A Natural products 0.000 claims description 5
- 108010036949 Cyclosporine Proteins 0.000 claims description 5
- 231100000699 Bacterial toxin Toxicity 0.000 claims description 4
- 239000000688 bacterial toxin Substances 0.000 claims description 4
- QKSKPIVNLNLAAV-UHFFFAOYSA-N bis(2-chloroethyl) sulfide Chemical compound ClCCSCCCl QKSKPIVNLNLAAV-UHFFFAOYSA-N 0.000 claims description 4
- 229960001265 ciclosporin Drugs 0.000 claims description 4
- 229930182912 cyclosporin Natural products 0.000 claims description 4
- 239000003053 toxin Substances 0.000 claims description 4
- 231100000765 toxin Toxicity 0.000 claims description 4
- 108700012359 toxins Proteins 0.000 claims description 4
- 229960003444 immunosuppressant agent Drugs 0.000 claims description 3
- 239000003018 immunosuppressive agent Substances 0.000 claims description 3
- 230000002633 protecting effect Effects 0.000 claims description 3
- 231100000331 toxic Toxicity 0.000 claims description 3
- 230000002588 toxic effect Effects 0.000 claims description 3
- 206010008428 Chemical poisoning Diseases 0.000 claims description 2
- 206010016952 Food poisoning Diseases 0.000 claims description 2
- 208000019331 Foodborne disease Diseases 0.000 claims description 2
- 208000029462 Immunodeficiency disease Diseases 0.000 claims description 2
- 206010029155 Nephropathy toxic Diseases 0.000 claims description 2
- 239000002647 aminoglycoside antibiotic agent Substances 0.000 claims description 2
- 210000001185 bone marrow Anatomy 0.000 claims description 2
- 239000000994 contrast dye Substances 0.000 claims description 2
- 239000008246 gaseous mixture Substances 0.000 claims description 2
- 208000018706 hematopoietic system disease Diseases 0.000 claims description 2
- 208000015181 infectious disease Diseases 0.000 claims description 2
- 230000005865 ionizing radiation Effects 0.000 claims description 2
- 208000017169 kidney disease Diseases 0.000 claims description 2
- 201000006370 kidney failure Diseases 0.000 claims description 2
- 208000019423 liver disease Diseases 0.000 claims description 2
- 230000007694 nephrotoxicity Effects 0.000 claims description 2
- 231100000417 nephrotoxicity Toxicity 0.000 claims description 2
- 239000003958 nerve gas Substances 0.000 claims description 2
- 208000017520 skin disease Diseases 0.000 claims description 2
- 239000002904 solvent Substances 0.000 claims description 2
- 230000001629 suppression Effects 0.000 claims description 2
- 206010073306 Exposure to radiation Diseases 0.000 claims 3
- 208000035475 disorder Diseases 0.000 abstract description 26
- 238000011282 treatment Methods 0.000 abstract description 20
- 230000003750 conditioning effect Effects 0.000 abstract description 9
- 230000001939 inductive effect Effects 0.000 abstract description 5
- 230000003247 decreasing effect Effects 0.000 abstract 1
- 238000000034 method Methods 0.000 description 56
- 230000008569 process Effects 0.000 description 41
- 241001465754 Metazoa Species 0.000 description 34
- 210000004027 cell Anatomy 0.000 description 32
- 210000003734 kidney Anatomy 0.000 description 27
- 239000007924 injection Substances 0.000 description 16
- 238000002347 injection Methods 0.000 description 16
- 230000000302 ischemic effect Effects 0.000 description 16
- 230000001413 cellular effect Effects 0.000 description 14
- 208000028867 ischemia Diseases 0.000 description 13
- 210000003470 mitochondria Anatomy 0.000 description 12
- 230000005855 radiation Effects 0.000 description 12
- 239000007789 gas Substances 0.000 description 11
- 210000001700 mitochondrial membrane Anatomy 0.000 description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 9
- 239000012528 membrane Substances 0.000 description 9
- 241000700159 Rattus Species 0.000 description 8
- 210000004379 membrane Anatomy 0.000 description 8
- 230000010410 reperfusion Effects 0.000 description 8
- 239000011780 sodium chloride Substances 0.000 description 8
- 238000013467 fragmentation Methods 0.000 description 7
- 238000006062 fragmentation reaction Methods 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 241000282472 Canis lupus familiaris Species 0.000 description 6
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 6
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 6
- 230000030833 cell death Effects 0.000 description 6
- 238000005259 measurement Methods 0.000 description 6
- 230000007423 decrease Effects 0.000 description 5
- 230000002438 mitochondrial effect Effects 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 230000035882 stress Effects 0.000 description 5
- 208000024827 Alzheimer disease Diseases 0.000 description 4
- 230000001133 acceleration Effects 0.000 description 4
- 230000001965 increasing effect Effects 0.000 description 4
- 210000000231 kidney cortex Anatomy 0.000 description 4
- 230000007246 mechanism Effects 0.000 description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- 206010063837 Reperfusion injury Diseases 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- 208000027418 Wounds and injury Diseases 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 230000009931 harmful effect Effects 0.000 description 3
- 230000036542 oxidative stress Effects 0.000 description 3
- 238000012552 review Methods 0.000 description 3
- 239000001509 sodium citrate Substances 0.000 description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- 238000001356 surgical procedure Methods 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- GKWLIQDHWRWNRS-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC(N)(CO)CO.OCCN1CCN(CCS(O)(=O)=O)CC1 GKWLIQDHWRWNRS-UHFFFAOYSA-N 0.000 description 2
- 208000023275 Autoimmune disease Diseases 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- BMZRVOVNUMQTIN-UHFFFAOYSA-N Carbonyl Cyanide para-Trifluoromethoxyphenylhydrazone Chemical compound FC(F)(F)OC1=CC=C(NN=C(C#N)C#N)C=C1 BMZRVOVNUMQTIN-UHFFFAOYSA-N 0.000 description 2
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 2
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 206010020772 Hypertension Diseases 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 2
- 208000018737 Parkinson disease Diseases 0.000 description 2
- 206010039966 Senile dementia Diseases 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 239000003146 anticoagulant agent Substances 0.000 description 2
- 229940127219 anticoagulant drug Drugs 0.000 description 2
- 238000003782 apoptosis assay Methods 0.000 description 2
- LMEKQMALGUDUQG-UHFFFAOYSA-N azathioprine Chemical compound CN1C=NC([N+]([O-])=O)=C1SC1=NC=NC2=C1NC=N2 LMEKQMALGUDUQG-UHFFFAOYSA-N 0.000 description 2
- 229960002170 azathioprine Drugs 0.000 description 2
- 210000001772 blood platelet Anatomy 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 229960004397 cyclophosphamide Drugs 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 229910001882 dioxygen Inorganic materials 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000007824 enzymatic assay Methods 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 238000010255 intramuscular injection Methods 0.000 description 2
- 239000007927 intramuscular injection Substances 0.000 description 2
- 230000002530 ischemic preconditioning effect Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004770 neurodegeneration Effects 0.000 description 2
- 210000002569 neuron Anatomy 0.000 description 2
- 239000007800 oxidant agent Substances 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 230000005522 programmed cell death Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 210000002254 renal artery Anatomy 0.000 description 2
- 210000002796 renal vein Anatomy 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 230000001052 transient effect Effects 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- 208000012260 Accidental injury Diseases 0.000 description 1
- 208000031261 Acute myeloid leukaemia Diseases 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 108010063104 Apoptosis Regulatory Proteins Proteins 0.000 description 1
- 102000010565 Apoptosis Regulatory Proteins Human genes 0.000 description 1
- 208000006029 Cardiomegaly Diseases 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 108010008286 DNA nucleotidylexotransferase Proteins 0.000 description 1
- 102100033215 DNA nucleotidylexotransferase Human genes 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 108010083687 Ion Pumps Proteins 0.000 description 1
- 102000006391 Ion Pumps Human genes 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 239000012839 Krebs-Henseleit buffer Substances 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 208000033776 Myeloid Acute Leukemia Diseases 0.000 description 1
- 108010025020 Nerve Growth Factor Proteins 0.000 description 1
- 102000007072 Nerve Growth Factors Human genes 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 208000018262 Peripheral vascular disease Diseases 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- 208000005374 Poisoning Diseases 0.000 description 1
- 108010090931 Proto-Oncogene Proteins c-bcl-2 Proteins 0.000 description 1
- 102000013535 Proto-Oncogene Proteins c-bcl-2 Human genes 0.000 description 1
- 102000006270 Proton Pumps Human genes 0.000 description 1
- 108010083204 Proton Pumps Proteins 0.000 description 1
- 208000003782 Raynaud disease Diseases 0.000 description 1
- 208000012322 Raynaud phenomenon Diseases 0.000 description 1
- 206010063897 Renal ischaemia Diseases 0.000 description 1
- 241000219061 Rheum Species 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 206010042496 Sunburn Diseases 0.000 description 1
- 101710119418 Superoxide dismutase [Mn] Proteins 0.000 description 1
- 101710202572 Superoxide dismutase [Mn], mitochondrial Proteins 0.000 description 1
- 102100032891 Superoxide dismutase [Mn], mitochondrial Human genes 0.000 description 1
- 102100031988 Tumor necrosis factor ligand superfamily member 6 Human genes 0.000 description 1
- 108050002568 Tumor necrosis factor ligand superfamily member 6 Proteins 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 238000001949 anaesthesia Methods 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 229940045799 anthracyclines and related substance Drugs 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 210000002469 basement membrane Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 210000004958 brain cell Anatomy 0.000 description 1
- 230000005587 bubbling Effects 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000002575 chemical warfare agent Substances 0.000 description 1
- YTRQFSDWAXHJCC-UHFFFAOYSA-N chloroform;phenol Chemical compound ClC(Cl)Cl.OC1=CC=CC=C1 YTRQFSDWAXHJCC-UHFFFAOYSA-N 0.000 description 1
- 230000019771 cognition Effects 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 230000001143 conditioned effect Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000001120 cytoprotective effect Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000001085 differential centrifugation Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- XVLXYDXJEKLXHN-UHFFFAOYSA-M dioc6 Chemical compound [I-].O1C2=CC=CC=C2[N+](CCCCCC)=C1C=CC=C1N(CCCCCC)C2=CC=CC=C2O1 XVLXYDXJEKLXHN-UHFFFAOYSA-M 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 230000000547 effect on apoptosis Effects 0.000 description 1
- 210000001163 endosome Anatomy 0.000 description 1
- 230000003511 endothelial effect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 231100001261 hazardous Toxicity 0.000 description 1
- 230000002489 hematologic effect Effects 0.000 description 1
- 210000000777 hematopoietic system Anatomy 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 231100000234 hepatic damage Toxicity 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 150000002432 hydroperoxides Chemical class 0.000 description 1
- 230000007954 hypoxia Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000002055 immunohistochemical effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000006882 induction of apoptosis Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000009434 installation Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000001361 intraarterial administration Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000001678 irradiating effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000008818 liver damage Effects 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 210000005060 membrane bound organelle Anatomy 0.000 description 1
- 238000000329 molecular dynamics simulation Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 210000004498 neuroglial cell Anatomy 0.000 description 1
- 239000003900 neurotrophic factor Substances 0.000 description 1
- 230000007959 normoxia Effects 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000001706 oxygenating effect Effects 0.000 description 1
- LCCNCVORNKJIRZ-UHFFFAOYSA-N parathion Chemical compound CCOP(=S)(OCC)OC1=CC=C([N+]([O-])=O)C=C1 LCCNCVORNKJIRZ-UHFFFAOYSA-N 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 210000000512 proximal kidney tubule Anatomy 0.000 description 1
- 239000013014 purified material Substances 0.000 description 1
- 230000009396 radiation induced apoptosis Effects 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 238000000163 radioactive labelling Methods 0.000 description 1
- 230000003134 recirculating effect Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- TUFFYSFVSYUHPA-UHFFFAOYSA-M rhodamine 123 Chemical compound [Cl-].COC(=O)C1=CC=CC=C1C1=C(C=CC(N)=C2)C2=[O+]C2=C1C=CC(N)=C2 TUFFYSFVSYUHPA-UHFFFAOYSA-M 0.000 description 1
- 230000033764 rhythmic process Effects 0.000 description 1
- 210000005245 right atrium Anatomy 0.000 description 1
- 229940080817 rotenone Drugs 0.000 description 1
- JUVIOZPCNVVQFO-UHFFFAOYSA-N rotenone Natural products O1C2=C3CC(C(C)=C)OC3=CC=C2C(=O)C2C1COC1=C2C=C(OC)C(OC)=C1 JUVIOZPCNVVQFO-UHFFFAOYSA-N 0.000 description 1
- 230000026799 smooth muscle cell apoptotic process Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 238000011699 spontaneously hypertensive rat Methods 0.000 description 1
- 238000013222 sprague-dawley male rat Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 231100000167 toxic agent Toxicity 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 230000036642 wellbeing Effects 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
- A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
- A61M1/3681—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits by irradiation
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
- A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
- A61M1/3621—Extra-corporeal blood circuits
- A61M1/3623—Means for actively controlling temperature of blood
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
- A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
- A61M1/3681—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits by irradiation
- A61M1/3683—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits by irradiation using photoactive agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
- A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
- A61M1/3687—Chemical treatment
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
- A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
- A61M1/369—Temperature treatment
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
- A61M1/14—Dialysis systems; Artificial kidneys; Blood oxygenators ; Reciprocating systems for treatment of body fluids, e.g. single needle systems for hemofiltration or pheresis
- A61M1/32—Oxygenators without membranes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M2202/00—Special media to be introduced, removed or treated
- A61M2202/02—Gases
- A61M2202/0216—Ozone
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M2205/00—General characteristics of the apparatus
- A61M2205/05—General characteristics of the apparatus combined with other kinds of therapy
- A61M2205/051—General characteristics of the apparatus combined with other kinds of therapy with radiation therapy
- A61M2205/053—General characteristics of the apparatus combined with other kinds of therapy with radiation therapy ultraviolet
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
- A61N5/00—Radiation therapy
- A61N5/06—Radiation therapy using light
- A61N2005/0658—Radiation therapy using light characterised by the wavelength of light used
- A61N2005/0661—Radiation therapy using light characterised by the wavelength of light used ultraviolet
Definitions
- This invention relates to the field of medicine. More specifically, the invention relates to means for preconditioning the mammalian body, including the human body, so as to enable cellular organs thereof better to resist subsequently encountered cell death as induced by apoptosis-inducing events, or by necrosis-inducing events, including events inducing both apoptosis and necrosis.
- Apoptosis is the biological process of controlled, programmed cell death, by means of which cells die by a process of condensation without the release of cell contents into the surrounding milieu.
- Cells of most organs and tissues divide and multiply over time, a process that is normally in equilibrium with cell death by apoptosis, resulting in optimal cell numbers in the healthy body.
- Apoptosis therefore, can be considered to act as a control on the total number of cells in organs and tissues. Residues of apoptosed cells are largely consumed by other cells, by a process of phagocytosis.
- necrosis the natural, well-regulated process by which the body undertakes removal of unwanted cells
- necrosis the process of necrosis by which the cells die largely in an uncontrolled manner, as a result of membrane rupture.
- necrosed cells The intracellular components of necrosed cells are released into the organism in an uncontrolled manner, commonly resulting in inflammatory reactions as the body attempts to deal with these suddenly encountered components. Apoptosed cells cause virtually no harmful inflammatory reactions.
- Some medical disorders in a living body, or an individual organ of a living body can be attributed at least in part to an undue acceleration in the rate of apoptosis. This can occur, for example, when a body ingests chemical poisons or encounters excessive amounts of harmful radiation (radioactivity, UV exposure, etc.). Other disorders involve both apoptosis and necrosis. Still other disorders involve an accelerated rate of cell death due primarily to necrosis.
- Apoptosis of the cell is understood to be initiated by an alteration in the functioning of the mitochondria of the cell.
- Mitochondria as is well known, are membrane-bounded organelles, located within the cell, and occupying a major fraction of the total cell volume. They contain large amounts of internal membrane.
- the main function of mitochondria is to convert energy from foodstuffs to forms that can be used to drive cellular reactions. This is accomplished by a process of chemiosmotic coupling, by which membrane-bound ion pumps transfer ions from one side of the mitochondrial membrane to the other.
- the proton pumps generate an electrochemical proton gradient across the membrane, which is used to drive various energy-requiring reactions when the protons flow through membrane embedded proteins such as the enzyme ATP synthase.
- mitochondrial membrane potential As an ionic process, the potential across the mitochondrial membrane is important in the efficient operation of this energy-providing mechanism. Mitochondria participate directly in the induction of apoptosis by releasing pro-apoptotic proteins Decreases in mitochondrial membrane potential are known to be indicative of the commencement of apoptosis.
- Organs undergoing apoptosis exhibit oligonucleosomal DNA fragmentation into 180-200 base pairs, in a specific pattern which appears as a ladder after gel electrophoresis.
- the degree of DNA fragmentation correlates with the progression of apoptosis in the organ, and can be measured by extracting the DNA, radiolabelling it, subjecting it to electrophoresis and quantifying the radioactivity associated with various DNA fragments.
- Such techniques can be used to determine the numbers of cells undergoing apoptosis or exhibiting an apoptotic condition or predisposition, so as to determine an extent or degree of apoptosis in a body organ or tissue.
- LDH lactate dehydrogenase
- U.S. Pat. No. 4,968,483 Mueller et al. describes an apparatus for oxygenating blood, by treating an aliquot of a patient's blood extracorporeally, with an oxygen/ozone mixture and ultraviolet light, at a controlled temperature.
- the apparatus is proposed for use in hematological oxidation therapy.
- U.S. Pat. No. 5,591,457 Bolton discloses a method of inhibiting the aggregation of blood platelets in a human, a method of stimulating the immune system and a method of treating peripheral vascular diseases such as Raynaud's disease, by extracting an aliquot of blood from a patient, subjecting it to ozone/oxygen gas mixture, and ultraviolet radiation at a temperature in the range of about 37-43° C., and then reinjecting the treated blood in the human patient.
- U.S. Pat. No. 5,834,030 Bolton describes a similar process for increasing the content of nitric oxide in the blood of a mammalian patient, potentially useful in treating conditions such as high blood pressure in mammalian patients.
- WO98/07436 described an autoimmune vaccine for administration to human patients to alleviate the symptoms of autoimmune diseases such as rheumatoid arthritis, the vaccine comprises an aliquot of the patient's blood which has been subjected extracorporeally to an oxidizing environment, UV radiation and elevated temperature.
- the present invention provides a process whereby a mammalian body may be preconditioned so that the cells of organs and tissues can better resist subsequently encountered apoptosis- and/or necrosis-inducing events.
- the process involves in vitro treatment of an aliquot of the blood from the mammalian body, with certain stressors to effect modification of the blood aliquot. Then the treated blood aliquot is reintroduced into the mammalian body.
- the result is a significant increase in resistance to apoptosis and apoptosis/necrosis of the cells of the body, as indicated by changes in mitochrondrial membrane potential, decrease of DNA laddering, and decrease of release of LDH, when the cells are subsequently exposed to stressing or toxic agents.
- the aliquot of blood is treated by being subjected to one or more stressors which have been found to modify the blood.
- the blood aliquot can be modified by subjecting the blood, or separated cellular or non-cellular fractions of the blood, or mixtures of the separated cells and/or non-cellular fractions of the blood, to stressors selected from heat, ultraviolet light and oxidizing environments.
- the stressors may be applied individually, or in any combination of two or more of such stressors, simultaneously or sequentially.
- the process of the invention may be used for pre-conditioning the mammalian body against the effects of a wide range of subsequently encountered factors known to cause pathological conditions which are associated with excessive degrees of apoptosis of necrosis of cells of various body organs.
- Medical disorders associated with excessive degreess of apoptosis and/or necrosis in various organs or tissues, and for which, accordingly, the process of the present invention is indicated for use, either as a treatment thereof or as preconditioning against the effects thereof, can be classified into four general categories. These are:
- radiation exposure disorders which include exposure to excessive amounts of ionizing radiation such as nuclear radiation, therapeutic radiation or X-rays; or ultraviolet light (resulting in skin disorders such as sunburn, for example).
- ionizing radiation such as nuclear radiation, therapeutic radiation or X-rays; or ultraviolet light (resulting in skin disorders such as sunburn, for example).
- the fact that such radiation exposure disorders are associated with increases in apoptosis is known, for example from Blankenberg et.al. “Dying a thousand deaths. Radionuclide imaging of apoptosis”, O. J. Nucl. Med. 1999 June; 43(2): 170-6 and various references cited therein; from Wong, G. H. “Protective roles of cytokines against radiation: induction of mitochondrial MnSOD”, Biochim. Biophys.
- chemical exposure and ingestion disorders which include chemical poisoning; food poisoning from bacterial toxins; toxic drug ingestion overdoses and side effects; disorders from exposure to chemical warfare agents such as nerve gases and mustard gas; liver disorders from chemicals and toxins (including alcohol); kidney disorders e.g. resulting from ingestion of aminoglycoside antibiotics, radiographic contrast dyes or cyclosporin nephrotoxicity; hematopoietic disorders and immunodeficiency disorders derived from drug or toxin induced bone marrow suppression; infections from bacterial toxins; ozone exposure; solvent exposure; and the effects of immunosuppressants such as cyclosporin, cyclophosphamide or azathioprine.
- immunosuppressants such as cyclosporin, cyclophosphamide or azathioprine.
- neurological disorders such as Parkinson's disease (which involves apoptosis of specific brain cells), senile dementia, and Alzheimer's disease and like diseases.
- the fact that such neurological disorders are associated with increases in apoptosis is known, for example from Desjardins P, Ledoux S “The role of apoptosis in neurodegenerative diseases,” Metab. Brain Dis. 1998 June; 13(2):79-96; from Dragunow M, McGibbon G. A. et.al. “Apoptosis, neurotrophic factors and neurodegeneration”, Rev. Neurosci.
- the determination of whether or not a particular process or procedure has an effect on apoptosis in tissues or organs of the living mammalian body is best determined at the cellular level, e.g. by determination of mitochondrial membrane potential or by determination of the degree of DNA fragmentation. These measurements are described in more detail in the specific examples which follow. A determination by such measurements that a process or procedure leads to a decrease in apoptosis is an indication that such a process or procedure is effective in treating or preconditioning against any of the apoptosis related disorders listed in the four categories above.
- the process of the invention is primarily indicated for use by people who are likely to encounter conditions where they are exposed to such factors, such as workers in chemical manufacturing facilities, nuclear installations and the like, or physically hazardous situations such as emergency response teams. Potential military applications whereby troops may be pre-conditioned against a wide variety of hazards, will be apparent. More specific indications for use of the process are in connection with patients undergoing medical treatments, including administration of toxic drugs, which are accompanied by undesired side effects. For example, the administration of immunosuppressants such as cyclosporin, cyclophosphamide and azathioprine to assist in organ transplants and for other purposes leads commonly to apoptosis and/or necrosis acceleration associated disorders.
- the use of the process of the present invention on patients involved in such treatments can be beneficial, particularly since the treatment regimen for such patients, both with drugs or radiation and with the process of the invention, can be carefully planned in advance and conducted according to a carefully controlled schedule.
- the process of the invention can also be used to control or to alleviate the symptoms of a medical disorder involving increased apoptosis and/or necrosis.
- the term “alleviating or protecting against the symptoms” as used herein refers to both pre-conditioning to afford protection, and treatment of manifested symptoms.
- use of the process of the invention by patients suffering from the disorder, in order to control it or to alleviate its symptoms is the most practical use of it. Indeed, clinical tests have provided evidence of improvement in cognition and general well-being of elderly patients.
- the present invention provides a process of alleviating or protecting against the symptoms of a medical disorder involving accelerated rates of apoptosis or necrosis in a mammalian body, said disorder being selected from radiation exposure disorders; chemical exposure and ingestion disorders; neurological disorders; and physical trauma disorders; which comprises reducing the rate of or susceptibility to apoptosis or necrosis of tissues and organs of the mammalian body by (a) reacting an aliquot of blood from the mammalian body ex vivo with at least one stressor selected from the group consisting of a temperature above or below body temperature, ultraviolet light, and an oxidative environment; and (b) administering the aliquot of blood treated in step (a) to the mammalian body.
- FIG. 1 of the accompanying drawings is a graphical presentation of the results obtained from Example 1 described below;
- FIGS. 2 and 3 of the accompanying drawings are graphical presentations of the results obtained from Example 2 described below;
- FIG. 4 of the accompanying drawings is a graphical presentation of the results obtained according to Example 3 below.
- an aliquot of blood is extracted from a mammalian subject, preferably a human, and the aliquot of blood is treated ex vivo with certain stressors, described in more detail below.
- the terms “aliquot”, “aliquot of blood” or similar terms used herein include whole blood, separated cellular fractions of the blood including platelets, separated non-cellular fractions of the blood including plasma, and combinations thereof.
- the effect of the stressors is to modify the blood, and/or the cellular or non-cellular fractions thereof, contained in the aliquot.
- the modified aliquot is then re-introduced into the subject's body by any suitable method, most preferably intramuscular injection, but also including subcutaneous injection, intraperitoneal injection, and oral, nasal or rectal administration. intra-arterial injection or intravenous injection.
- the stressors to which the aliquot of blood is subjected ex vivo according to the method of the present invention are selected from temperature stress (blood temperature above or below body temperature), an oxidative environment and ultraviolet light, individually or in any combination, simultaneously or sequentially.
- the aliquot has a volume sufficient that, when re-introduced into the subject's body, a pre-conditioning against apoptosis level is achieved in the subject.
- the volume of the aliquot is up to about 400 ml, preferably from about 0.1 to about 100 ml, more preferably from about 5 to about 15 ml, even more preferably from about 8 to about 12 ml, and most preferably about 10 ml.
- the temperature stressor warms the aliquot being treated to a temperature above normal body temperature or cools the aliquot below normal body temperature.
- the temperature is selected so that the temperature stressor does not cause excessive hemolysis in the blood contained in the aliquot and so that, when the treated aliquot is injected into a subject, an effective pre-conditioning against apoptosis and/or necrosis will be achieved.
- the temperature stressor is applied so that the temperature of all or a part of the aliquot is up to about 55° C., and more preferably in the range of from about ⁇ 5° C. to about
- the temperature of the aliquot is raised above normal body temperature, such that the mean temperature of the aliquot does not exceed a temperature of about 55° C., more preferably from about 40° C. to about 50° C., even more preferably from about 40° C. to about 44° C., and most preferably about 42.5 ⁇ 1° C.
- the aliquot is cooled below normal body temperature such that the mean temperature of the aliquot is within the range of from about 4° C. to about 36.5° C., even more preferably from about 10° C. to about 30° C., and even more preferably from about 15° C. to about 25° C.
- the oxidative environment stressor can be the application to the aliquot of solid, liquid or gaseous oxidizing agents.
- it involves exposing the aliquot to a mixture of medical grade oxygen and ozone gas, most preferably by bubbling through the aliquot, at the aforementioned temperature range, a stream of medical grade oxygen gas having ozone as a minor component therein.
- the ozone content of the gas stream and the flow rate of the gas stream are preferably selected such that the amount of ozone introduced to the blood aliquot, either on its own or in combination with other stressors, does not give rise to excessive levels of cell damage.
- the gas stream has an ozone content of up to about 300 ⁇ g/ml, preferably from about 10 to about 100 ⁇ g/ml, more preferably about 30 ⁇ g/ml, even more preferably up to about 20 ⁇ g/ml, particularly preferably from about 10 ⁇ g/ml to about 20 ⁇ g/ml, and most preferably about 14.5 ⁇ 1.0 ⁇ g/ml.
- the gas stream is suitably supplied to the aliquot at a rate of up to about 2.0 litres/min, preferably up to about 0.5 litres/min, more preferably up to about 0.4 litres/min, even more preferably up to about 0.33 litres/min, and most preferably about 0.24 ⁇ 0.024 litres/min.
- the lower limit of the flow rate of the gas stream is preferably not lower than 0.01 litres/min, more preferably not lower than 0.1 litres/min, and even more preferably not lower than 0.2 litres/min.
- the ultraviolet light stressor is suitably applied by irradiating the aliquot under treatment from a source of UV light while the aliquot is maintained at the aforementioned temperature and while the oxygen/ozone gaseous mixture is being bubbled through the aliquot.
- Preferred UV sources are UV lamps emitting UV-C band wavelengths, i.e. at wavelengths shorter than about 280 nm.
- Ultraviolet light corresponding to standard UV-A (wavelengths from about 315 to about 400 nm) and UV-B (wavelengths from about 280 to about 315) sources can also be used.
- an appropriate dosage of such UV light can be obtained from lamps with a power consumption of from about 15 to about 30 watts and useful UV output of about 5-10 watts, arranged to surround the sample container holding the aliquot.
- lamps with a power consumption of from about 15 to about 30 watts and useful UV output of about 5-10 watts arranged to surround the sample container holding the aliquot.
- Up to eight such lamps surrounding the sample bottle operated at an intensity to deliver a total UV light energy at 253.7 nm at the surface of the blood of from about 0.025 to about 10 joules/cm 2 , preferably from about 0.1 to about 3.0 joules/cm 2 , may advantageously be used.
- Such a treatment provides a modified blood aliquot which is ready for injection into the subject.
- the time for which the aliquot is subjected to the stressors is normally within the time range from about 0.5 up to about 60 minutes. The time depends to some extent upon the chosen intensity of the UV light, the temperature, the concentration of the oxidizing agent and the rate at which it is supplied to the aliquot. Some experimentation to establish optimum times may be necessary on the part of the operator, once the other stressor levels have been set. Under most stressor conditions, preferred times will be in the approximate range of from about 2 to about 5 minutes, more preferably about 3 minutes.
- the starting blood temperature, and the rate at which it can be warmed or cooled to a predetermined temperature tends to vary from subject to subject. Preferably four such lamps are used.
- the blood aliquot may be treated with the stressors using an apparatus of the type described in aforementioned U.S. Pat. No. 4,968,483 to Mueller.
- the aliquot is placed in a suitable, sterile, UV light-transmissive container, which is fitted into the machine.
- the UV lamps are switched on for a fixed period before the gas flow is applied to the aliquot providing the oxidative stress, to allow the output of the UV lamps to stabilize.
- the UV lamps are typically on while the temperature of the aliquot is adjusted to the predetermined value, e.g. 42.5 ⁇ 1° C.
- the oxygen/ozone gas mixture of known composition and controlled flow rate, is applied to the aliquot, for the predetermined duration of up to about 60 minutes, preferably 2 to 5 minutes and most preferably about 3 minutes as discussed above, so that the aliquot experiences all three stressors simultaneously.
- blood is appropriately modified according to the present invention to achieve the desired effects.
- a patient In operating the process of the invention, it is preferred to give a patient a course of treatments, comprising a daily or alternate day treatment, over a period of one or two weeks. Each treatment is substantially identical, with the same volume aliquot being extracted, stressed and re-injected. The course of treatments is scheduled to be completed shortly before the patient is to be exposed to an apoptosis-accelerating factor as described above, for most effective pre-conditioning against the effects thereof.
- An 8-ml aliquot of blood was extracted from the animal, treated with sodium citrate (2 ml) and placed in a sterile container. It was subjected simultaneously to the UV radiation, oxygen/ozone gas oxidative environment and elevated temperature stressors, in an apparatus as generally described in the aforementioned Mueller U.S. Pat. No. 4,969,483. More specifically, the blood sample in the sterile, UV-transparent container was heated using infra-red lamps to 42.5° C., and whilst being maintained at that temperature, it was subjected to UV radiation of wavelength 253.7 nm under the preferred conditions previously described.
- a mixture of medical grade oxygen and ozone, of ozone content 13.5-15.5 ug/ml was bubbled through the blood sample at a flow rate within the range from 60-240 mls/min.
- the time of simultaneous UV exposure and gas mixture feed was 3 minutes.
- a 5 ml portion of the treated blood aliquot was reinjected intramuscularly into each test animal.
- Groups B and D were the control groups, given two 10-day courses of daily injections of 5 ml of physiological saline, with a three-week rest period between the 10-day courses.
- the animals were observed for 6 days after the ischemia procedure, and then sacrificed.
- the ischemic kidney of each animal was surgically removed and divided into two parts. One part was kept frozen at ⁇ 80° C., and the other part was fixed in 10% formalin for immuno- and routine histopathology studies.
- Mitochondrial membrane potential was measured in proximal tubular cells isolated from the ischemic and control kidneys, both at the time of removal of the control kidney and following sacrifice.
- dog kidney proximal tubes were purified from normal or ischemic kidney cortexes by the collagenase treatment procedure described by Marshansky et. al., “Isolation of heavy endosomes from dog proximal tubes in suspension”, J. Membr. Biol 153(1), 59-73, 1996. Renal mitochondria were isolated in suspension by differential centrifugation (see Marshansky, “Organic hydroperoxides at high concentrations cause energization and activation of AATP synthesis in mitochondria”, J. Biol. Chem. 264(7), 3670-3673. 1989.
- Mitochondrial membrane potential was measured as described by Kroemer, G., Zamzam, N. and Susin, S. A., “Mitochondrial control of apoptosis”, (Review) Immunology Today (1997) v.18, p 44-51; with JC-1 dye—see Salvioli et.al., “JC-1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess delta psi changes in intact cells: implications for studies on mitochondrial functionality during apoptosis”, FEBS Letters 411 (1), 77-82. 1987.
- JC-1 fluorescence in the suspension of purified mitochondria from normal and ischemic kidneys was monitored continuously on a Deltascan Model RFM-2001 spectrofluorimeter (Photon Technology International, South Brunswick, N.J.).
- the excitation wavelength was 490 nm (slit width 2 nm) and the emission wavelength was 590 nm (slit width 4 nm).
- the signals were recorded using Felix® (Version 1.1) software. All measurements were performed with continuous stirring at 37° C.
- the incubation buffer for measurement of mitochondrial membrane potential contained 200 mM sucrose, 5 mM MgCl 2 , 5 mM KH 2 PO 4 , 0.1 ⁇ M of JC-1 and 30 Mm HEPES-Tris (pH 7.5).
- the concentrations of the substrate and inhibitors were 10 mM succinate, 0.1 ⁇ M rotenone with or without 0.1 ⁇ M FCCP.
- Proximal tubule mitochondrial membrane potential was estimated in the right (control) kidney prior to ischemia and in the left (ischemic) kidney after sacrifice of the dogs on day 6 following ischemia and was estimated as difference of JC-1 fluorescence after uncoupling of mitochondria with FCCP as shown in the accompanying FIG. 1A .
- 50 ⁇ g protein of purified material was used for each measurement.
- JC-1 fluorescence is proportional to the mitochondrial membrane potential.
- the contralateral nephrectomized kidney served as control.
- the ischemic kidney of the saline-injected animals showed significantly lower (p ⁇ 0.05) fluorescence compared to the control kidney.
- the preservation of mitochondrial membrane potential evidences the capacity of the therapy to protect mitochondria, and thereby to precondition cells against apoptosis.
- a group of 12 male SHR rats was treated with either injections of pooled blood stressed as described in Example 1 above, or, in control animals, with injections of saline. Since the blood from all of the animals of this genetic strain is identical, blood from one animal of this same strain was treated by the process of the invention for administration to the test animal. The blood was treated with sodium citrate as anti-coagulant, and placed in a sterile container They received either injections of 150 ⁇ l of stressed blood on days-14 and -13 followed by a rest period of 11 days and a third injection the day before ischemic surgery, or injections in parallel with saline.
- the rats were anaesthetized with light flurane, and the right kidney was removed through a mid-abdominal incision.
- the left kidney was then subjected to transient ischemia by occlusion of the left renal artery and vein using a micro-clip.
- the skin was then temporarily closed. After 60 minutes of occlusion, the clip was removed and the wound was closed with a suture.
- the animals were sacrificed 12 hours after reperfusion.
- ischemic and non-ischemic kidneys of the test animals were removed and subjected to DNA laddering tests. Oligonucleosomal DNA fragmentation into 180 to 200 base pairs is a specific pattern which appears as a ladder after agarose gel electrophoresis in various organs undergoing apoptosis.
- To estimate the degree of DNA fragmentation in the kidney cortex an aliquot of pulverized kidney cortex was weighed and total tissue DNA was extracted by the phenol-chloroform procedure after tissue digestion with proteinase K and RnaseA in the presence of EDTA.
- a regression line for each sample was drawn for the radioactivity as a function of DNA loaded on the gel (see deBlois et.al., ‘Smooth muscle cell apoptosis during vascular regression in spontaneously hypertensive rats.’ Hypertension 29, 340-349, 1997).
- the slope of the linear regression line served as a DNA fragmentation index (cpm/pixel per ⁇ g DNA).
- FIG. 2 The results from ischemic-reperfused (I/R) kidneys and from normal, non-I/R kidneys, all from animals which did not receive injections of stressed blood, are shown graphically on FIG. 2 , a plot of the slope of the regression lines for the various samples (vertical axis) against time after initiation of reperfusion.
- FIG. 3A of the accompanying drawings is a picture of the electophoresis gel of the fragmented DNA, in the 150-1500 bp range, radio-labeled as described to attach radioactivity labels to the DNA fragments.
- Trace S derives from DNA of kidneys from animals which received saline injections prior to kidney ischemia-reperfusion
- trace V derives from DNA of kidneys of animals which received injections of the stressed blood prior to kidney ischemia-reperfusion.
- the Figure shows that 60 minutes renal ischemia induced a clear accumulation of fragmented DNA in both groups of rats at 12 h but the level of this parameter was significantly lower (p ⁇ 0.05) in animals receiving the treated blood.
- FIG. 1 shows that 60 minutes renal ischemia induced a clear accumulation of fragmented DNA in both groups of rats at 12 h but the level of this parameter was significantly lower (p ⁇ 0.05) in animals receiving the treated blood.
- 3B quantifies the amount of irradiation from the samples, in arbitrary units, and shows that DNA fragmentation-laddering occurs in both S and V samples as a result of ischemia/reperfusion, but that the extent is markedly reduced in V samples as compared with S samples.
- the results presented on FIG. 3B are the means of six animals in each case.
- One group of rats (n 4) received a saline injection and served as controls.
- the blood was treated with sodium citrate as anticoagulant, and placed in a sterile container. It was heated and subjected simultaneously to the UV stressor and oxygen/ozone stressor in the amounts and under the conditions set out in Example 1 above.
- test animal received on day 1 an injection of 150 ⁇ l of the treated blood, followed by a 10-day rest period. Then each animal received a 150 ⁇ l injection of treated blood on both day 12 and on day 13. Each control animal received similar injections, on the same schedule, of physiological saline. The animals were then sacrificed on day 14.
- the heart was removed and perfused ex vivo according to the Langendorf mode with non-recirculating Krebs Henseleit buffer gassed with 95% oxygen/5% carbon dioxide pH 7.4, containing glucose as energy substrate.
- the heart was submitted to an ischemia-reperfusion insult as typically is used in studies of cardiac ischemic preconditioning (see for example R. T. Rowland et.al., Am. J. Physiol. 272, H2708-H2715; E. O. Weselcouch et.al., Cardiovasc. Res. 29; 126-132, 197).
- the heart was submitted to a 25 minute global ischemia at 37° C.
- FIG. 4 indicates data measuring the perfusion protocol for the lactate dehydrogenase released into the effluent perfusate, an index of cellular necrosis, as evaluated by a standard enzymatic assay.
- the curve based on the triangular-form points is derived from organs of animals which received blood treated with stressors as described.
- the curve based on square-form points is derived from the organs of the control animals which received saline solution.
- FIG. 4 indicates a significant reduction in LDH release (cumulative LDH release during the 45 minute reperfusion period; p ⁇ 0.05; treated vs saline), indicative of significantly reduced cell necrosis in organs treated with stressor as described.
Landscapes
- Health & Medical Sciences (AREA)
- Heart & Thoracic Surgery (AREA)
- Vascular Medicine (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Anesthesiology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Cardiology (AREA)
- Animal Behavior & Ethology (AREA)
- Engineering & Computer Science (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Description
- This invention relates to the field of medicine. More specifically, the invention relates to means for preconditioning the mammalian body, including the human body, so as to enable cellular organs thereof better to resist subsequently encountered cell death as induced by apoptosis-inducing events, or by necrosis-inducing events, including events inducing both apoptosis and necrosis.
- The two known forms of cellular death are necrosis and apoptosis. Apoptosis is the biological process of controlled, programmed cell death, by means of which cells die by a process of condensation without the release of cell contents into the surrounding milieu. Cells of most organs and tissues divide and multiply over time, a process that is normally in equilibrium with cell death by apoptosis, resulting in optimal cell numbers in the healthy body. Apoptosis, therefore, can be considered to act as a control on the total number of cells in organs and tissues. Residues of apoptosed cells are largely consumed by other cells, by a process of phagocytosis. The process of apoptosis, the natural, well-regulated process by which the body undertakes removal of unwanted cells is to be contrasted with the process of necrosis by which the cells die largely in an uncontrolled manner, as a result of membrane rupture. Importantly, however, in many instances apoptosis and necrosis behave as a continuum. The intracellular components of necrosed cells are released into the organism in an uncontrolled manner, commonly resulting in inflammatory reactions as the body attempts to deal with these suddenly encountered components. Apoptosed cells cause virtually no harmful inflammatory reactions.
- Some medical disorders in a living body, or an individual organ of a living body, can be attributed at least in part to an undue acceleration in the rate of apoptosis. This can occur, for example, when a body ingests chemical poisons or encounters excessive amounts of harmful radiation (radioactivity, UV exposure, etc.). Other disorders involve both apoptosis and necrosis. Still other disorders involve an accelerated rate of cell death due primarily to necrosis.
- Apoptosis of the cell is understood to be initiated by an alteration in the functioning of the mitochondria of the cell. Mitochondria, as is well known, are membrane-bounded organelles, located within the cell, and occupying a major fraction of the total cell volume. They contain large amounts of internal membrane. The main function of mitochondria is to convert energy from foodstuffs to forms that can be used to drive cellular reactions. This is accomplished by a process of chemiosmotic coupling, by which membrane-bound ion pumps transfer ions from one side of the mitochondrial membrane to the other. The proton pumps generate an electrochemical proton gradient across the membrane, which is used to drive various energy-requiring reactions when the protons flow through membrane embedded proteins such as the enzyme ATP synthase. As an ionic process, the potential across the mitochondrial membrane is important in the efficient operation of this energy-providing mechanism. Mitochondria participate directly in the induction of apoptosis by releasing pro-apoptotic proteins Decreases in mitochondrial membrane potential are known to be indicative of the commencement of apoptosis.
- Organs undergoing apoptosis exhibit oligonucleosomal DNA fragmentation into 180-200 base pairs, in a specific pattern which appears as a ladder after gel electrophoresis. The degree of DNA fragmentation correlates with the progression of apoptosis in the organ, and can be measured by extracting the DNA, radiolabelling it, subjecting it to electrophoresis and quantifying the radioactivity associated with various DNA fragments. Such techniques can be used to determine the numbers of cells undergoing apoptosis or exhibiting an apoptotic condition or predisposition, so as to determine an extent or degree of apoptosis in a body organ or tissue.
- In the course of necrosis, enzymes and other cell contents normally contained in the cytoplasm are released, as a result of disintegration of cell membranes, a hallmark of necrosis. One of these is the enzyme lactate dehydrogenase (LDH), the levels of which are commonly used to determine the degree of necrosis.
- U.S. Pat. No. 4,968,483 Mueller et al. describes an apparatus for oxygenating blood, by treating an aliquot of a patient's blood extracorporeally, with an oxygen/ozone mixture and ultraviolet light, at a controlled temperature. The apparatus is proposed for use in hematological oxidation therapy.
- U.S. Pat. No. 5,591,457 Bolton, discloses a method of inhibiting the aggregation of blood platelets in a human, a method of stimulating the immune system and a method of treating peripheral vascular diseases such as Raynaud's disease, by extracting an aliquot of blood from a patient, subjecting it to ozone/oxygen gas mixture, and ultraviolet radiation at a temperature in the range of about 37-43° C., and then reinjecting the treated blood in the human patient.
- U.S. Pat. No. 5,834,030 Bolton, describes a similar process for increasing the content of nitric oxide in the blood of a mammalian patient, potentially useful in treating conditions such as high blood pressure in mammalian patients.
- International Patent Application PCT/CA97/00564 Vasogen Inc. (WO98/07436) described an autoimmune vaccine for administration to human patients to alleviate the symptoms of autoimmune diseases such as rheumatoid arthritis, the vaccine comprises an aliquot of the patient's blood which has been subjected extracorporeally to an oxidizing environment, UV radiation and elevated temperature.
- It is an object of the present invention to provide means for pre-conditioning a mammalian patient to better withstand external cellular insults likely to effect acceleration of or to increase the degree of apoptosis in tissues or organs of the mammalian patient.
- It is a further object of the invention to provide means for pre-conditioning a mammalian patient to better withstand external cellular insults likely to effect acceleration of or increase the degree of necrosis in tissues or organs of the patient.
- It is a further object of the invention to provide a pre-conditioning process for mammalian patients against the harmful effects of chemical and radiation poisoning.
- It is a further object of the invention to provide a process for alleviating or decelerating the progression of the symptoms of apoptosis-related or necrosis-related medical disorders.
- The present invention provides a process whereby a mammalian body may be preconditioned so that the cells of organs and tissues can better resist subsequently encountered apoptosis- and/or necrosis-inducing events. The process involves in vitro treatment of an aliquot of the blood from the mammalian body, with certain stressors to effect modification of the blood aliquot. Then the treated blood aliquot is reintroduced into the mammalian body. The result is a significant increase in resistance to apoptosis and apoptosis/necrosis of the cells of the body, as indicated by changes in mitochrondrial membrane potential, decrease of DNA laddering, and decrease of release of LDH, when the cells are subsequently exposed to stressing or toxic agents.
- The aliquot of blood is treated by being subjected to one or more stressors which have been found to modify the blood. According to the present invention, the blood aliquot can be modified by subjecting the blood, or separated cellular or non-cellular fractions of the blood, or mixtures of the separated cells and/or non-cellular fractions of the blood, to stressors selected from heat, ultraviolet light and oxidizing environments. The stressors may be applied individually, or in any combination of two or more of such stressors, simultaneously or sequentially.
- Accordingly, the process of the invention may be used for pre-conditioning the mammalian body against the effects of a wide range of subsequently encountered factors known to cause pathological conditions which are associated with excessive degrees of apoptosis of necrosis of cells of various body organs.
- Medical disorders associated with excessive degreess of apoptosis and/or necrosis in various organs or tissues, and for which, accordingly, the process of the present invention is indicated for use, either as a treatment thereof or as preconditioning against the effects thereof, can be classified into four general categories. These are:
- (1) radiation exposure disorders, which include exposure to excessive amounts of ionizing radiation such as nuclear radiation, therapeutic radiation or X-rays; or ultraviolet light (resulting in skin disorders such as sunburn, for example). The fact that such radiation exposure disorders are associated with increases in apoptosis is known, for example from Blankenberg et.al. “Dying a thousand deaths. Radionuclide imaging of apoptosis”, O. J. Nucl. Med. 1999 June; 43(2): 170-6 and various references cited therein; from Wong, G. H. “Protective roles of cytokines against radiation: induction of mitochondrial MnSOD”, Biochim. Biophys. Acta 1995 May 24; 1271(1): 205-209 and various references cited therein, from Zhao et.al. “Mitochondrial and intracellular free-calcium regulation of radiation-induced apoptosis in human leukemic cells”, Int J Radiat Biol 1999 April; 75(4): 493-504; and from Reap E. A. et.al., “Radiation and stress-induced apoptosis: a role for Fas/Fas ligand interactions”, Proc Natl Acad Sci USA. 1997 May 27; 94(11):5750-5
- (2) chemical exposure and ingestion disorders, which include chemical poisoning; food poisoning from bacterial toxins; toxic drug ingestion overdoses and side effects; disorders from exposure to chemical warfare agents such as nerve gases and mustard gas; liver disorders from chemicals and toxins (including alcohol); kidney disorders e.g. resulting from ingestion of aminoglycoside antibiotics, radiographic contrast dyes or cyclosporin nephrotoxicity; hematopoietic disorders and immunodeficiency disorders derived from drug or toxin induced bone marrow suppression; infections from bacterial toxins; ozone exposure; solvent exposure; and the effects of immunosuppressants such as cyclosporin, cyclophosphamide or azathioprine. The fact that such chemical ingestion and exposure disorders are associated with increases in apoptosis is known, for example from Losser M R and Payen D, “Mechanisms of liver damage”, Semin Liver Dis, 1996 November; 16(4): 357-67; from Smith K. J. et.al., Immunohistochemical studies of basement membrane proteins and proliferation and apoptosis markers in sulfur mustard induced cutaneous lesions in weanling pigs”, J. Dermaol. Sci. 1997 September; 15(3): 173-82; from Dabrowska M. I. et. al., Sulfur mustard induces apoptosis and necrosis in endothelial, cells”, Toxicol Appl Pharmacol 1966 December; 141(2>: 569-83; from Muller et.al., “Anthracycline-derived chemotherapeutics in apoptosis and free radical cytotoxicity (Review)”, Int J Mol Med 1998 Febuary: 1(2): 4914, and various references cited therein; from Healey et.al., “Apoptosis and necrosis: mechanisms of cell death induced by cyclosporine A in a renal proximal tubular cell line”, Kidney Int, 1998 December; 54(6): 1955-66; from Hatake K et.al., “Apoptosis-gene expression in hematopoietic system: normal and pathologicAl conditions (Review)”, Int J Mol Med 1998 January; 1(1): 121-9 and various references cited therein; from Banker D. E. et.al., “Measurement of spontaneous and therapeutic agent-induced apoptosis with BCL-2 protein expression in acute myeloid leukemia”, Blood, 1997
Jan 1;(1):243-55; from Voetberg B. J. et.al., “Apoptosis accompanies a change in the phenotypic . . . ”, Clin Immunol Immunopathol 1994 May; 71(2): 190-8; and from Mountz J. D. et.al. “Autoimmune disease. A problem of defective apoptosis”, Arthritis Rheum 1994 October; 37(10): 1415-20; - (3) neurological disorders such as Parkinson's disease (which involves apoptosis of specific brain cells), senile dementia, and Alzheimer's disease and like diseases. The fact that such neurological disorders are associated with increases in apoptosis is known, for example from Desjardins P, Ledoux S “The role of apoptosis in neurodegenerative diseases,” Metab. Brain Dis. 1998 June; 13(2):79-96; from Dragunow M, McGibbon G. A. et.al. “Apoptosis, neurotrophic factors and neurodegeneration”, Rev. Neurosci. 1997 July-December;8(3-4): 223-265; from Kitamura Y, Taniguchi T, Shimohama S, “Apoptotic cell death in neurons and glial cells: implications for Alzheimer's disease”, Jpn J. Pharmacol. 1999 January; 79(1): 1-5; and from Budd S. L. and Nicholls D. G. “Mitochondria in the life and death of neurons”, Essays Biochem 1998;33;43-52; and other publications both preceding and following those detailed above;
- (4) physical trauma disorders such as physical accident injuries, wounding, thermal injuries (burns), and losses of blood such as occur during surgery. The fact that such disorders are associated with increases in apoptosis is known, for example from Wilson S. E, “Molecular cell biology for the refractive corneal surgeon: programmed cell death and wound healing”, J Refract Surg, 1997 March-April; 13(2): 171-5.
- The determination of whether or not a particular process or procedure has an effect on apoptosis in tissues or organs of the living mammalian body is best determined at the cellular level, e.g. by determination of mitochondrial membrane potential or by determination of the degree of DNA fragmentation. These measurements are described in more detail in the specific examples which follow. A determination by such measurements that a process or procedure leads to a decrease in apoptosis is an indication that such a process or procedure is effective in treating or preconditioning against any of the apoptosis related disorders listed in the four categories above. Such a determination of apoptosis inhibiting effects of a process or procedure, at the cellular level, in conjunction with a demonstrated efficacy of that process in alleviating or preconditioning against a disorder in one of the above categories is strong evidence of potential clinical success of that process or procedure in alleviating or preconditioning against other disorders in the same category.
- Thus the process of the invention is primarily indicated for use by people who are likely to encounter conditions where they are exposed to such factors, such as workers in chemical manufacturing facilities, nuclear installations and the like, or physically hazardous situations such as emergency response teams. Potential military applications whereby troops may be pre-conditioned against a wide variety of hazards, will be apparent. More specific indications for use of the process are in connection with patients undergoing medical treatments, including administration of toxic drugs, which are accompanied by undesired side effects. For example, the administration of immunosuppressants such as cyclosporin, cyclophosphamide and azathioprine to assist in organ transplants and for other purposes leads commonly to apoptosis and/or necrosis acceleration associated disorders. The use of the process of the present invention on patients involved in such treatments can be beneficial, particularly since the treatment regimen for such patients, both with drugs or radiation and with the process of the invention, can be carefully planned in advance and conducted according to a carefully controlled schedule.
- In addition to the use of the process of the invention to pre-condition a body or body organ against subsequently encountered factors, the process can also be used to control or to alleviate the symptoms of a medical disorder involving increased apoptosis and/or necrosis. The term “alleviating or protecting against the symptoms” as used herein refers to both pre-conditioning to afford protection, and treatment of manifested symptoms. In situations where the causative factor of the medical disorder is associated with ageing (Parkinson's disease or senile dementia, for example), use of the process of the invention by patients suffering from the disorder, in order to control it or to alleviate its symptoms, is the most practical use of it. Indeed, clinical tests have provided evidence of improvement in cognition and general well-being of elderly patients.
- Accordingly, in one aspect the present invention provides a process of alleviating or protecting against the symptoms of a medical disorder involving accelerated rates of apoptosis or necrosis in a mammalian body, said disorder being selected from radiation exposure disorders; chemical exposure and ingestion disorders; neurological disorders; and physical trauma disorders; which comprises reducing the rate of or susceptibility to apoptosis or necrosis of tissues and organs of the mammalian body by (a) reacting an aliquot of blood from the mammalian body ex vivo with at least one stressor selected from the group consisting of a temperature above or below body temperature, ultraviolet light, and an oxidative environment; and (b) administering the aliquot of blood treated in step (a) to the mammalian body.
-
FIG. 1 of the accompanying drawings is a graphical presentation of the results obtained from Example 1 described below; -
FIGS. 2 and 3 of the accompanying drawings are graphical presentations of the results obtained from Example 2 described below; -
FIG. 4 of the accompanying drawings is a graphical presentation of the results obtained according to Example 3 below. - According to a preferred process of the present invention, an aliquot of blood is extracted from a mammalian subject, preferably a human, and the aliquot of blood is treated ex vivo with certain stressors, described in more detail below. The terms “aliquot”, “aliquot of blood” or similar terms used herein include whole blood, separated cellular fractions of the blood including platelets, separated non-cellular fractions of the blood including plasma, and combinations thereof. The effect of the stressors is to modify the blood, and/or the cellular or non-cellular fractions thereof, contained in the aliquot. The modified aliquot is then re-introduced into the subject's body by any suitable method, most preferably intramuscular injection, but also including subcutaneous injection, intraperitoneal injection, and oral, nasal or rectal administration. intra-arterial injection or intravenous injection.
- The stressors to which the aliquot of blood is subjected ex vivo according to the method of the present invention are selected from temperature stress (blood temperature above or below body temperature), an oxidative environment and ultraviolet light, individually or in any combination, simultaneously or sequentially. The aliquot has a volume sufficient that, when re-introduced into the subject's body, a pre-conditioning against apoptosis level is achieved in the subject. Preferably, in human patients, the volume of the aliquot is up to about 400 ml, preferably from about 0.1 to about 100 ml, more preferably from about 5 to about 15 ml, even more preferably from about 8 to about 12 ml, and most preferably about 10 ml.
- It is preferred, according to the invention, to apply all three of the aforementioned stressors simultaneously to the aliquot under treatment, in order to ensure the appropriate modification to the blood. It may also be preferred in some embodiments of the invention to apply any two of the above stressors, for example to apply temperature stress and oxidative stress, temperature stress and ultraviolet light, or ultraviolet light and oxidative stress. Care must be taken to utilize an appropriate level of the stressors to thereby effectively modify the blood to achieve the desired effect.
- The temperature stressor warms the aliquot being treated to a temperature above normal body temperature or cools the aliquot below normal body temperature. The temperature is selected so that the temperature stressor does not cause excessive hemolysis in the blood contained in the aliquot and so that, when the treated aliquot is injected into a subject, an effective pre-conditioning against apoptosis and/or necrosis will be achieved. Preferably, the temperature stressor is applied so that the temperature of all or a part of the aliquot is up to about 55° C., and more preferably in the range of from about −5° C. to about
- In some preferred embodiments of the invention, the temperature of the aliquot is raised above normal body temperature, such that the mean temperature of the aliquot does not exceed a temperature of about 55° C., more preferably from about 40° C. to about 50° C., even more preferably from about 40° C. to about 44° C., and most preferably about 42.5±1° C.
- In other preferred embodiments, the aliquot is cooled below normal body temperature such that the mean temperature of the aliquot is within the range of from about 4° C. to about 36.5° C., even more preferably from about 10° C. to about 30° C., and even more preferably from about 15° C. to about 25° C.
- The oxidative environment stressor can be the application to the aliquot of solid, liquid or gaseous oxidizing agents. Preferably, it involves exposing the aliquot to a mixture of medical grade oxygen and ozone gas, most preferably by bubbling through the aliquot, at the aforementioned temperature range, a stream of medical grade oxygen gas having ozone as a minor component therein. The ozone content of the gas stream and the flow rate of the gas stream are preferably selected such that the amount of ozone introduced to the blood aliquot, either on its own or in combination with other stressors, does not give rise to excessive levels of cell damage. Suitably, the gas stream has an ozone content of up to about 300 μg/ml, preferably from about 10 to about 100 μg/ml, more preferably about 30 μg/ml, even more preferably up to about 20 μg/ml, particularly preferably from about 10 μg/ml to about 20 μg/ml, and most preferably about 14.5±1.0 μg/ml. The gas stream is suitably supplied to the aliquot at a rate of up to about 2.0 litres/min, preferably up to about 0.5 litres/min, more preferably up to about 0.4 litres/min, even more preferably up to about 0.33 litres/min, and most preferably about 0.24±0.024 litres/min. The lower limit of the flow rate of the gas stream is preferably not lower than 0.01 litres/min, more preferably not lower than 0.1 litres/min, and even more preferably not lower than 0.2 litres/min.
- The ultraviolet light stressor is suitably applied by irradiating the aliquot under treatment from a source of UV light while the aliquot is maintained at the aforementioned temperature and while the oxygen/ozone gaseous mixture is being bubbled through the aliquot. Preferred UV sources are UV lamps emitting UV-C band wavelengths, i.e. at wavelengths shorter than about 280 nm. Ultraviolet light corresponding to standard UV-A (wavelengths from about 315 to about 400 nm) and UV-B (wavelengths from about 280 to about 315) sources can also be used. For example, an appropriate dosage of such UV light, applied simultaneously with the aforementioned temperature and oxidative environment stressors, can be obtained from lamps with a power consumption of from about 15 to about 30 watts and useful UV output of about 5-10 watts, arranged to surround the sample container holding the aliquot. Up to eight such lamps surrounding the sample bottle, operated at an intensity to deliver a total UV light energy at 253.7 nm at the surface of the blood of from about 0.025 to about 10 joules/cm2, preferably from about 0.1 to about 3.0 joules/cm2, may advantageously be used. Such a treatment provides a modified blood aliquot which is ready for injection into the subject.
- The time for which the aliquot is subjected to the stressors is normally within the time range from about 0.5 up to about 60 minutes. The time depends to some extent upon the chosen intensity of the UV light, the temperature, the concentration of the oxidizing agent and the rate at which it is supplied to the aliquot. Some experimentation to establish optimum times may be necessary on the part of the operator, once the other stressor levels have been set. Under most stressor conditions, preferred times will be in the approximate range of from about 2 to about 5 minutes, more preferably about 3 minutes. The starting blood temperature, and the rate at which it can be warmed or cooled to a predetermined temperature, tends to vary from subject to subject. Preferably four such lamps are used.
- In the practice of the preferred process of the present invention, the blood aliquot may be treated with the stressors using an apparatus of the type described in aforementioned U.S. Pat. No. 4,968,483 to Mueller. The aliquot is placed in a suitable, sterile, UV light-transmissive container, which is fitted into the machine. The UV lamps are switched on for a fixed period before the gas flow is applied to the aliquot providing the oxidative stress, to allow the output of the UV lamps to stabilize. The UV lamps are typically on while the temperature of the aliquot is adjusted to the predetermined value, e.g. 42.5±1° C. Then the oxygen/ozone gas mixture, of known composition and controlled flow rate, is applied to the aliquot, for the predetermined duration of up to about 60 minutes, preferably 2 to 5 minutes and most preferably about 3 minutes as discussed above, so that the aliquot experiences all three stressors simultaneously. In this way, blood is appropriately modified according to the present invention to achieve the desired effects.
- In operating the process of the invention, it is preferred to give a patient a course of treatments, comprising a daily or alternate day treatment, over a period of one or two weeks. Each treatment is substantially identical, with the same volume aliquot being extracted, stressed and re-injected. The course of treatments is scheduled to be completed shortly before the patient is to be exposed to an apoptosis-accelerating factor as described above, for most effective pre-conditioning against the effects thereof.
- The invention is further illustrated and described with reference to the following specific examples, namely animal studies conducted in approved manner.
- The experiments reported in the examples demonstrate, by use of an animal model system involving ischemia and subsequent reperfusion of various body organs, that the process of the present invention has the effect of reducing apoptosis and necrosis. Ischemia-reperfusion injuries are known to involve increase of apoptosis and necrosis in the affected organs and tissues—see for example Saikumar p, et. al. “Mechanisms of cell death in hypoxia/reoxygenation injury”, Oncogene 1998 Dec. 24; 17(25):3341-9; and Burns A. T. et.al., “Apoptosis in ischemia/reperfusion injury of human renal allografts”, Transplantation, 1998 Oct. 15; 66(7): 872-6, and other publications both preceding and following those. Known techniques of determination of apoptosis at the cellular level are employed in the examples. The finding that the process of the invention decreases apoptosis and necrosis in this model is indicative of its utility in the various categories of apoptosis-associated disorders discussed above.
- Pure-bred normal beagle dogs, aged 1-2 years, equal numbers of males and females, were used as the experimental animals. The animals were separated into four groups, A, B, C and D, each group consisting of six animals, three males and three females. Animals of groups A and C were subjected to the process of the invention, by being subjected to two 10-day courses of daily removal of an 8 ml aliquot of blood, extracorporeal treatment of the aliquot with oxygen/ozone, UV radiation and heat, and re-administration of 5 ml of the treated aliquot to the same animal, by intramuscular injection.
- Each such treatment was conducted as follows.
- An 8-ml aliquot of blood was extracted from the animal, treated with sodium citrate (2 ml) and placed in a sterile container. It was subjected simultaneously to the UV radiation, oxygen/ozone gas oxidative environment and elevated temperature stressors, in an apparatus as generally described in the aforementioned Mueller U.S. Pat. No. 4,969,483. More specifically, the blood sample in the sterile, UV-transparent container was heated using infra-red lamps to 42.5° C., and whilst being maintained at that temperature, it was subjected to UV radiation of wavelength 253.7 nm under the preferred conditions previously described. Simultaneously, a mixture of medical grade oxygen and ozone, of ozone content 13.5-15.5 ug/ml was bubbled through the blood sample at a flow rate within the range from 60-240 mls/min. The time of simultaneous UV exposure and gas mixture feed was 3 minutes. A 5 ml portion of the treated blood aliquot was reinjected intramuscularly into each test animal.
- Each animal of groups A and C, receiving the courses of treatment according to the invention, experienced a three week rest period between the 10-day courses of treatment. Groups B and D were the control groups, given two 10-day courses of daily injections of 5 ml of physiological saline, with a three-week rest period between the 10-day courses.
- One day following the second course of injections, the animals were anaesthetized under light gas anaesthesia, and the right kidney of each animal was removed through a back incision. An occlusive clip was placed on the remaining renal artery and vein, to expose the left kidney to transient ischemia, for 60 minutes; Then the clip was removed to allow reperfusion of the kidney by normal blood flow.
- The animals were observed for 6 days after the ischemia procedure, and then sacrificed. The ischemic kidney of each animal was surgically removed and divided into two parts. One part was kept frozen at −80° C., and the other part was fixed in 10% formalin for immuno- and routine histopathology studies.
- Mitochondrial membrane potential was measured in proximal tubular cells isolated from the ischemic and control kidneys, both at the time of removal of the control kidney and following sacrifice. For this purpose, dog kidney proximal tubes were purified from normal or ischemic kidney cortexes by the collagenase treatment procedure described by Marshansky et. al., “Isolation of heavy endosomes from dog proximal tubes in suspension”, J. Membr. Biol 153(1), 59-73, 1996. Renal mitochondria were isolated in suspension by differential centrifugation (see Marshansky, “Organic hydroperoxides at high concentrations cause energization and activation of AATP synthesis in mitochondria”, J. Biol. Chem. 264(7), 3670-3673. 1989. after tissue homogenization in a buffer containing 250 mM sucrose, 10 mM HEPES-Tris (pH 7.5), and 250 μM EDTA. Cell debris was removed by centrifugation at 10,000 g for 30 minutes. The mitochondria were washed with the sucrose/HEPES buffer without EDTA.
- Mitochondrial membrane potential was measured as described by Kroemer, G., Zamzam, N. and Susin, S. A., “Mitochondrial control of apoptosis”, (Review) Immunology Today (1997) v.18, p 44-51; with JC-1 dye—see Salvioli et.al., “JC-1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess delta psi changes in intact cells: implications for studies on mitochondrial functionality during apoptosis”, FEBS Letters 411 (1), 77-82. 1987. JC-1 fluorescence in the suspension of purified mitochondria from normal and ischemic kidneys was monitored continuously on a Deltascan Model RFM-2001 spectrofluorimeter (Photon Technology International, South Brunswick, N.J.). The excitation wavelength was 490 nm (
slit width 2 nm) and the emission wavelength was 590 nm (slit width 4 nm). The signals were recorded using Felix® (Version 1.1) software. All measurements were performed with continuous stirring at 37° C. The incubation buffer for measurement of mitochondrial membrane potential contained 200 mM sucrose, 5 mM MgCl2, 5 mM KH2PO4, 0.1 μM of JC-1 and 30 Mm HEPES-Tris (pH 7.5). The concentrations of the substrate and inhibitors were 10 mM succinate, 0.1 μM rotenone with or without 0.1 μM FCCP. Proximal tubule mitochondrial membrane potential was estimated in the right (control) kidney prior to ischemia and in the left (ischemic) kidney after sacrifice of the dogs onday 6 following ischemia and was estimated as difference of JC-1 fluorescence after uncoupling of mitochondria with FCCP as shown in the accompanyingFIG. 1A . For each measurement, 50 μg protein of purified material was used. - JC-1 fluorescence is proportional to the mitochondrial membrane potential. The contralateral nephrectomized kidney served as control. As is clear from the
FIG. 1B , the treatment process of the invention did not modify the membrane potential of the non-ischemic control right kidney (p=0.445 for treated vs saline). However, the ischemic kidney of the saline-injected animals showed significantly lower (p<0.05) fluorescence compared to the control kidney. The stress treatment according to the invention prevented the uncoupling of mitochondria during ischemic/reperfusion, and membrane potential showed no significant difference (p=0.244) between ischemic and control kidneys. This parameter remained significantly higher (p=0.0006) vs saline-injected dogs) in the ischemic kidneys of dogs pretreated according to the process of the invention for at least 6 days post-reperfusion. - These results indicate that the process of the invention effects protection of the kidney against apoptosis and/or accelerates recovery at the mitochondrial level. Accordingly the process of the invention is indicated for preconditioning of the cells, tissues and organs of a mammalian body against subsequently encountered factors which will normally accelerate apoptosis.
- Specifically, the preservation of mitochondrial membrane potential evidences the capacity of the therapy to protect mitochondria, and thereby to precondition cells against apoptosis.
- A group of 12 male SHR rats was treated with either injections of pooled blood stressed as described in Example 1 above, or, in control animals, with injections of saline. Since the blood from all of the animals of this genetic strain is identical, blood from one animal of this same strain was treated by the process of the invention for administration to the test animal. The blood was treated with sodium citrate as anti-coagulant, and placed in a sterile container They received either injections of 150 μl of stressed blood on days-14 and -13 followed by a rest period of 11 days and a third injection the day before ischemic surgery, or injections in parallel with saline. On the day of surgery, the rats were anaesthetized with light flurane, and the right kidney was removed through a mid-abdominal incision. The left kidney was then subjected to transient ischemia by occlusion of the left renal artery and vein using a micro-clip. The skin was then temporarily closed. After 60 minutes of occlusion, the clip was removed and the wound was closed with a suture. The animals were sacrificed 12 hours after reperfusion.
- The ischemic and non-ischemic kidneys of the test animals were removed and subjected to DNA laddering tests. Oligonucleosomal DNA fragmentation into 180 to 200 base pairs is a specific pattern which appears as a ladder after agarose gel electrophoresis in various organs undergoing apoptosis. To estimate the degree of DNA fragmentation in the kidney cortex, an aliquot of pulverized kidney cortex was weighed and total tissue DNA was extracted by the phenol-chloroform procedure after tissue digestion with proteinase K and RnaseA in the presence of EDTA. One μg of extracted DNA was labeled by enzymatic assay using terminal deoxynucleotidyl transferase with P32-dCTP (see Teiger et.al., ‘Apoptosis in pressure overload-induced heart hypertrophy in the rat’, J. Clin. Invest. 97, 2891-2897, 1996). Increasing quantities of radio-labelled DNA were loaded onto 1.5% agarose gels. After electrophoresis, DNA was transferred onto nylon membranes (Hybond) and the radioactivity associated with 150 to 1500 bp DNA fragments was quantified in a PhosphorImager (Molecular Dynamics). A regression line for each sample was drawn for the radioactivity as a function of DNA loaded on the gel (see deBlois et.al., ‘Smooth muscle cell apoptosis during vascular regression in spontaneously hypertensive rats.’ Hypertension 29, 340-349, 1997). The slope of the linear regression line served as a DNA fragmentation index (cpm/pixel per μg DNA).
- The results from ischemic-reperfused (I/R) kidneys and from normal, non-I/R kidneys, all from animals which did not receive injections of stressed blood, are shown graphically on
FIG. 2 , a plot of the slope of the regression lines for the various samples (vertical axis) against time after initiation of reperfusion. The DNA laddering, indicative of DNA fragmentation, was clearly increased in the ischemic kidney cortex compared to the contralateral non-ischemic organ and the maximal attained at twelve hours returned to near basal values by 48 hours. Twelve hours was thus selected as the time point for study of the effect of the stressed blood of the invention on early ischemia-induced renal apoptosis. -
FIG. 3A of the accompanying drawings is a picture of the electophoresis gel of the fragmented DNA, in the 150-1500 bp range, radio-labeled as described to attach radioactivity labels to the DNA fragments. Trace S derives from DNA of kidneys from animals which received saline injections prior to kidney ischemia-reperfusion, and trace V derives from DNA of kidneys of animals which received injections of the stressed blood prior to kidney ischemia-reperfusion. The Figure shows that 60 minutes renal ischemia induced a clear accumulation of fragmented DNA in both groups of rats at 12 h but the level of this parameter was significantly lower (p<0.05) in animals receiving the treated blood.FIG. 3B quantifies the amount of irradiation from the samples, in arbitrary units, and shows that DNA fragmentation-laddering occurs in both S and V samples as a result of ischemia/reperfusion, but that the extent is markedly reduced in V samples as compared with S samples. The results presented onFIG. 3B are the means of six animals in each case. - These results confirm that the cytoprotective effect of the administration of stressed blood according to the invention on renal reperfusion injury involves the inhibition of early or late apoptosis.
- Experiments were carried out in rats, more specifically in male Sprague-Dawley rats to demonstrate the protection of removed organs, deprived of the donor's blood, against a sustained ischemic insult as typically observed with classical ischemic preconditioning protocol (K. Przylenk and R. A. Kloner, Progress in Cardiovasc. Dis. vol 40: 517-547, 1998).
- Two groups of four rats, 270-285 g body weight, were used. One group of rats (n=4) received a saline injection and served as controls. The other group of rats received blood treated by a protocol in accordance with the invention. Since the blood from all of the animals of this genetic strain is identical, blood from one animal of this same strain was treated by the process of the invention for administration to the test animal. The blood was treated with sodium citrate as anticoagulant, and placed in a sterile container. It was heated and subjected simultaneously to the UV stressor and oxygen/ozone stressor in the amounts and under the conditions set out in Example 1 above.
- Each test animal received on
day 1 an injection of 150 μl of the treated blood, followed by a 10-day rest period. Then each animal received a 150 μl injection of treated blood on bothday 12 and on day 13. Each control animal received similar injections, on the same schedule, of physiological saline. The animals were then sacrificed on day 14. - From each animal, the heart was removed and perfused ex vivo according to the Langendorf mode with non-recirculating Krebs Henseleit buffer gassed with 95% oxygen/5% carbon dioxide pH 7.4, containing glucose as energy substrate. The heart was submitted to an ischemia-reperfusion insult as typically is used in studies of cardiac ischemic preconditioning (see for example R. T. Rowland et.al., Am. J. Physiol. 272, H2708-H2715; E. O. Weselcouch et.al., Cardiovasc. Res. 29; 126-132, 197). Briefly, after a 20 minute equilibration period under normoxia, the heart was submitted to a 25 minute global ischemia at 37° C. Then, it was reperfused for 45 minutes as follows: (i) for the initial 25 min of reperfusion, the heart was allowed to beat spontaneously, the (ii) it was paced using pacing wires fixed to the right atrium to achieve a rhythm of 300 beats/min.
-
FIG. 4 indicates data measuring the perfusion protocol for the lactate dehydrogenase released into the effluent perfusate, an index of cellular necrosis, as evaluated by a standard enzymatic assay. InFIG. 4 of the drawings, the curve based on the triangular-form points is derived from organs of animals which received blood treated with stressors as described. The curve based on square-form points is derived from the organs of the control animals which received saline solution.FIG. 4 indicates a significant reduction in LDH release (cumulative LDH release during the 45 minute reperfusion period; p<0.05; treated vs saline), indicative of significantly reduced cell necrosis in organs treated with stressor as described.
Claims (16)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US11/347,059 US20060127383A1 (en) | 1999-01-12 | 2006-02-02 | Pre-conditioning cells against death |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US11563699P | 1999-01-12 | 1999-01-12 | |
US09/480,260 US7045124B1 (en) | 1999-01-12 | 2000-01-11 | Pre-conditioning against cell death |
US11/347,059 US20060127383A1 (en) | 1999-01-12 | 2006-02-02 | Pre-conditioning cells against death |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/480,260 Division US7045124B1 (en) | 1999-01-12 | 2000-01-11 | Pre-conditioning against cell death |
Publications (1)
Publication Number | Publication Date |
---|---|
US20060127383A1 true US20060127383A1 (en) | 2006-06-15 |
Family
ID=36318066
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/480,260 Expired - Fee Related US7045124B1 (en) | 1999-01-12 | 2000-01-11 | Pre-conditioning against cell death |
US11/347,059 Abandoned US20060127383A1 (en) | 1999-01-12 | 2006-02-02 | Pre-conditioning cells against death |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/480,260 Expired - Fee Related US7045124B1 (en) | 1999-01-12 | 2000-01-11 | Pre-conditioning against cell death |
Country Status (1)
Country | Link |
---|---|
US (2) | US7045124B1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016022972A1 (en) * | 2014-08-08 | 2016-02-11 | Alfred E. Mann Institute For Biomedical Engineering At The University Of Southern California | Apoptotic bodies |
Families Citing this family (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7045124B1 (en) * | 1999-01-12 | 2006-05-16 | Vasogen Irelend Limited | Pre-conditioning against cell death |
US9358292B2 (en) * | 2007-04-08 | 2016-06-07 | Immunolight, Llc | Methods and systems for treating cell proliferation disorders |
US20090104212A1 (en) * | 2007-08-06 | 2009-04-23 | Immunolight | Methods and systems for treating cell proliferation disorders using two-photon simultaneous absorption |
TWI458512B (en) | 2008-02-21 | 2014-11-01 | Immunolight Llc | Compositions for treating cell proliferation disorders and systems for producing auto-vaccines using plasmonics enhanced photospectral therapy (pepst) and exciton-plasmon enhanced phototherapy (epep) |
WO2009124189A1 (en) | 2008-04-04 | 2009-10-08 | Immunolight, Llc | Non-invasive systems and methods for in-situ photobiomodulation |
EP2729175B1 (en) | 2011-07-08 | 2021-12-01 | Duke University | System for light stimulation within a medium |
ITMI20131667A1 (en) * | 2013-10-09 | 2015-04-10 | Ind Paolo Gobbi Frattini | METHOD TO PRE-TREAT THE HEMATIC CELLS BEFORE SEPARATION. |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5116312A (en) * | 1989-11-03 | 1992-05-26 | The Uab Research Foundation | Method and apparatus for automatic autotransfusion |
US5215519A (en) * | 1990-03-07 | 1993-06-01 | Shettigar U Ramakrishna | Autotransfusion membrane system with means for providing reverse filtration |
US5344393A (en) * | 1992-02-28 | 1994-09-06 | Alliance Pharmaceutical Corp. | Use of synthetic oxygen carriers to facilitate oxygen delivery |
US5591457A (en) * | 1992-02-07 | 1997-01-07 | Vasogen Inc | Method of inhibiting the aggregation of blood platelets and stimulating the immune systems of a human |
US5658271A (en) * | 1996-02-08 | 1997-08-19 | Loubser; Paul G. | Closed circuit autologous sequestration reservoir system |
US5834030A (en) * | 1992-02-07 | 1998-11-10 | Vasogen, Inc. | Method of increasing the concentration of nitric oxide in human blood |
US6136308A (en) * | 1997-09-12 | 2000-10-24 | Vasogen Ireland Limited | Treatment of stress and preconditioning against stress |
US7045124B1 (en) * | 1999-01-12 | 2006-05-16 | Vasogen Irelend Limited | Pre-conditioning against cell death |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP4141636B2 (en) | 1997-09-12 | 2008-08-27 | ヴァソゲン アイルランド リミテッド | Treatment of stress and preliminary adjustment to stress |
CA2271190A1 (en) | 1999-05-06 | 2000-11-06 | Vasogen Ireland Limited | Improved method for treating mammals with modified mammalian blood |
-
2000
- 2000-01-11 US US09/480,260 patent/US7045124B1/en not_active Expired - Fee Related
-
2006
- 2006-02-02 US US11/347,059 patent/US20060127383A1/en not_active Abandoned
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5116312A (en) * | 1989-11-03 | 1992-05-26 | The Uab Research Foundation | Method and apparatus for automatic autotransfusion |
US5215519A (en) * | 1990-03-07 | 1993-06-01 | Shettigar U Ramakrishna | Autotransfusion membrane system with means for providing reverse filtration |
US5591457A (en) * | 1992-02-07 | 1997-01-07 | Vasogen Inc | Method of inhibiting the aggregation of blood platelets and stimulating the immune systems of a human |
US5834030A (en) * | 1992-02-07 | 1998-11-10 | Vasogen, Inc. | Method of increasing the concentration of nitric oxide in human blood |
US5344393A (en) * | 1992-02-28 | 1994-09-06 | Alliance Pharmaceutical Corp. | Use of synthetic oxygen carriers to facilitate oxygen delivery |
US5658271A (en) * | 1996-02-08 | 1997-08-19 | Loubser; Paul G. | Closed circuit autologous sequestration reservoir system |
US6136308A (en) * | 1997-09-12 | 2000-10-24 | Vasogen Ireland Limited | Treatment of stress and preconditioning against stress |
US7045124B1 (en) * | 1999-01-12 | 2006-05-16 | Vasogen Irelend Limited | Pre-conditioning against cell death |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016022972A1 (en) * | 2014-08-08 | 2016-02-11 | Alfred E. Mann Institute For Biomedical Engineering At The University Of Southern California | Apoptotic bodies |
CN106659740A (en) * | 2014-08-08 | 2017-05-10 | 南加州大学阿尔弗雷德·E·曼生物医学工程研究所 | Apoptotic bodies |
US10646518B2 (en) * | 2014-08-08 | 2020-05-12 | Alfred E. Mann Institute For Biomedical Engineering At The University Of Southern California | Apoptotic bodies |
Also Published As
Publication number | Publication date |
---|---|
US7045124B1 (en) | 2006-05-16 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20060127383A1 (en) | Pre-conditioning cells against death | |
EP1480658B1 (en) | Methods of treating vascular disease | |
US7223391B2 (en) | Method for treating mammals with modified mammalian blood | |
US6136308A (en) | Treatment of stress and preconditioning against stress | |
EP1146885B1 (en) | Pre-conditioning against cell death | |
US6733748B2 (en) | Chronic lymphocytic leukemia treatment | |
AU741181B2 (en) | Treatment of stress and preconditioning against stress | |
Sugahara et al. | Studies on a sulfhydryl radioprotector of low toxicity | |
EP1171140B1 (en) | Treatment of hypersensitivity reaction disorders | |
US6432399B1 (en) | Treatment of stress and preconditioning against stress | |
Benke et al. | Scavengers of free radical oxygen affect the generation of low molecular weight DNA in stimulated lymphocytes from patients with systemic lupus erythematosus | |
CA2372624A1 (en) | Improved method for treating mammals with modified mammalian blood | |
Naseem et al. | Morphological Perturbation of Human Erythrocytes Exposed to Photoilluminated Riboflavin | |
Ali et al. | Morphological Perturbation of Human Erythrocytes Exposed to Photoilluminated Riboflavin | |
CA2230836A1 (en) | Inducing resistance to ischemic stresses | |
Ali | Morphology perturbation of human erythrocytes exposed to photoilluminated riboflavin | |
CA2436515A1 (en) | Ex vivo oxidatively stressed cll cells in chronic lymphocytic leukemia (cll) treatment |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: VASOGEN IRELAND LIMITED, IRELAND Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:HAMET, PAVEL;TREMBLAY, JOHANNE;DESROSIERS, CHRISTINE;AND OTHERS;REEL/FRAME:017392/0497;SIGNING DATES FROM 20020819 TO 20020903 Owner name: CENTRE DE RECHERCHE DU CENTRE HOPSITALIER DE L'UNI Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:HAMET, PAVEL;TREMBLAY, JOHANNE;DESROSIERS, CHRISTINE;AND OTHERS;REEL/FRAME:017392/0497;SIGNING DATES FROM 20020819 TO 20020903 |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |