PH26320A - Diagnosis and treatment of double-stranded RNA deficiency states - Google Patents
Diagnosis and treatment of double-stranded RNA deficiency states Download PDFInfo
- Publication number
- PH26320A PH26320A PH37488A PH37488A PH26320A PH 26320 A PH26320 A PH 26320A PH 37488 A PH37488 A PH 37488A PH 37488 A PH37488 A PH 37488A PH 26320 A PH26320 A PH 26320A
- Authority
- PH
- Philippines
- Prior art keywords
- dsrna
- mismatched
- cells
- rnase
- patients
- Prior art date
Links
- 108091032973 (ribonucleotides)n+m Proteins 0.000 title claims description 66
- 102000040650 (ribonucleotides)n+m Human genes 0.000 title claims description 56
- 230000007812 deficiency Effects 0.000 title claims description 35
- 238000011282 treatment Methods 0.000 title description 11
- 238000003745 diagnosis Methods 0.000 title description 6
- 210000004027 cell Anatomy 0.000 claims description 79
- 239000000284 extract Substances 0.000 claims description 46
- 238000000034 method Methods 0.000 claims description 41
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 claims description 35
- 238000002560 therapeutic procedure Methods 0.000 claims description 33
- 230000003834 intracellular effect Effects 0.000 claims description 28
- 241000700605 Viruses Species 0.000 claims description 25
- 230000037361 pathway Effects 0.000 claims description 22
- 230000009385 viral infection Effects 0.000 claims description 20
- 102000007445 2',5'-Oligoadenylate Synthetase Human genes 0.000 claims description 19
- 108010086241 2',5'-Oligoadenylate Synthetase Proteins 0.000 claims description 19
- 208000036142 Viral infection Diseases 0.000 claims description 18
- 102000003960 Ligases Human genes 0.000 claims description 13
- 108090000364 Ligases Proteins 0.000 claims description 13
- 230000002950 deficient Effects 0.000 claims description 12
- 238000009256 replacement therapy Methods 0.000 claims description 12
- 206010028980 Neoplasm Diseases 0.000 claims description 10
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 claims description 10
- 244000052769 pathogen Species 0.000 claims description 10
- 230000004069 differentiation Effects 0.000 claims description 9
- 230000001717 pathogenic effect Effects 0.000 claims description 9
- 230000007170 pathology Effects 0.000 claims description 8
- 210000000987 immune system Anatomy 0.000 claims description 7
- 238000001802 infusion Methods 0.000 claims description 6
- 230000001404 mediated effect Effects 0.000 claims description 6
- 230000008569 process Effects 0.000 claims description 6
- SIIZPVYVXNXXQG-KGXOGWRBSA-N [(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-4-[[(3s,4r)-5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-3-hydroxyoxolan-2-yl]methyl [(2r,4r,5r)-2-(6-aminopurin-9-yl)-4-hydroxy-5-(phosphonooxymethyl)oxolan-3-yl] hydrogen phosphate Polymers C1=NC2=C(N)N=CN=C2N1[C@@H]1O[C@H](COP(O)(=O)OC2[C@@H](O[C@H](COP(O)(O)=O)[C@H]2O)N2C3=NC=NC(N)=C3N=C2)[C@@H](O)[C@H]1OP(O)(=O)OCC([C@@H](O)[C@H]1O)OC1N1C(N=CN=C2N)=C2N=C1 SIIZPVYVXNXXQG-KGXOGWRBSA-N 0.000 claims description 5
- 201000011510 cancer Diseases 0.000 claims description 5
- 241000709661 Enterovirus Species 0.000 claims description 4
- 241001430294 unidentified retrovirus Species 0.000 claims description 4
- 229940035893 uracil Drugs 0.000 claims description 4
- XQFRJNBWHJMXHO-RRKCRQDMSA-N IDUR Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(I)=C1 XQFRJNBWHJMXHO-RRKCRQDMSA-N 0.000 claims description 3
- 239000013060 biological fluid Substances 0.000 claims description 3
- 230000002349 favourable effect Effects 0.000 claims description 3
- 230000035755 proliferation Effects 0.000 claims description 3
- 208000026278 immune system disease Diseases 0.000 claims description 2
- 241001529453 unidentified herpesvirus Species 0.000 claims description 2
- 231100001274 therapeutic index Toxicity 0.000 claims 2
- 230000009702 cancer cell proliferation Effects 0.000 claims 1
- 239000012530 fluid Substances 0.000 claims 1
- 239000000126 substance Substances 0.000 claims 1
- 244000052613 viral pathogen Species 0.000 claims 1
- 101000959820 Homo sapiens Interferon alpha-1/13 Proteins 0.000 description 50
- 102100040019 Interferon alpha-1/13 Human genes 0.000 description 50
- 102100027962 2-5A-dependent ribonuclease Human genes 0.000 description 46
- 108010000834 2-5A-dependent ribonuclease Proteins 0.000 description 46
- 230000000694 effects Effects 0.000 description 46
- 108090000623 proteins and genes Proteins 0.000 description 40
- 102000004169 proteins and genes Human genes 0.000 description 36
- 238000003556 assay Methods 0.000 description 31
- 208000030507 AIDS Diseases 0.000 description 29
- 210000004366 CD4-positive T-lymphocyte Anatomy 0.000 description 28
- 102000006382 Ribonucleases Human genes 0.000 description 23
- 108010083644 Ribonucleases Proteins 0.000 description 23
- 230000003612 virological effect Effects 0.000 description 23
- 230000001684 chronic effect Effects 0.000 description 19
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 19
- 208000015181 infectious disease Diseases 0.000 description 19
- 238000003776 cleavage reaction Methods 0.000 description 17
- 230000007017 scission Effects 0.000 description 17
- 201000010099 disease Diseases 0.000 description 16
- 102000000588 Interleukin-2 Human genes 0.000 description 13
- 108010002350 Interleukin-2 Proteins 0.000 description 13
- 230000001419 dependent effect Effects 0.000 description 13
- 108020002230 Pancreatic Ribonuclease Proteins 0.000 description 12
- 102000005891 Pancreatic ribonuclease Human genes 0.000 description 12
- 210000004698 lymphocyte Anatomy 0.000 description 12
- 201000008686 ARC syndrome Diseases 0.000 description 11
- 208000003685 Arthrogryposis-renal dysfunction-cholestasis syndrome Diseases 0.000 description 11
- 230000000840 anti-viral effect Effects 0.000 description 11
- 230000000975 bioactive effect Effects 0.000 description 11
- 239000003112 inhibitor Substances 0.000 description 11
- 206010008874 Chronic Fatigue Syndrome Diseases 0.000 description 10
- 108020004414 DNA Proteins 0.000 description 10
- 208000029766 myalgic encephalomeyelitis/chronic fatigue syndrome Diseases 0.000 description 10
- 210000001519 tissue Anatomy 0.000 description 10
- 102000004190 Enzymes Human genes 0.000 description 9
- 108090000790 Enzymes Proteins 0.000 description 9
- 230000007547 defect Effects 0.000 description 9
- 241000725643 Respiratory syncytial virus Species 0.000 description 8
- 239000002585 base Substances 0.000 description 8
- 239000001913 cellulose Substances 0.000 description 8
- 229920002678 cellulose Polymers 0.000 description 8
- 239000003795 chemical substances by application Substances 0.000 description 8
- 230000006870 function Effects 0.000 description 8
- 208000032791 BCR-ABL1 positive chronic myelogenous leukemia Diseases 0.000 description 7
- 241000282412 Homo Species 0.000 description 7
- 230000015572 biosynthetic process Effects 0.000 description 7
- 210000004369 blood Anatomy 0.000 description 7
- 239000008280 blood Substances 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 7
- 238000011084 recovery Methods 0.000 description 7
- 230000003442 weekly effect Effects 0.000 description 7
- 208000010833 Chronic myeloid leukaemia Diseases 0.000 description 6
- 102000008072 Lymphokines Human genes 0.000 description 6
- 108010074338 Lymphokines Proteins 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 6
- 208000033761 Myelogenous Chronic BCR-ABL Positive Leukemia Diseases 0.000 description 6
- 230000004913 activation Effects 0.000 description 6
- 238000001994 activation Methods 0.000 description 6
- 210000000601 blood cell Anatomy 0.000 description 6
- 230000007423 decrease Effects 0.000 description 6
- 230000007123 defense Effects 0.000 description 6
- 230000006735 deficit Effects 0.000 description 6
- 230000002458 infectious effect Effects 0.000 description 6
- 230000009467 reduction Effects 0.000 description 6
- 238000006722 reduction reaction Methods 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- 208000024891 symptom Diseases 0.000 description 6
- 230000001225 therapeutic effect Effects 0.000 description 6
- 208000031886 HIV Infections Diseases 0.000 description 5
- 208000037357 HIV infectious disease Diseases 0.000 description 5
- 241000725303 Human immunodeficiency virus Species 0.000 description 5
- 210000001744 T-lymphocyte Anatomy 0.000 description 5
- 238000011888 autopsy Methods 0.000 description 5
- 230000000593 degrading effect Effects 0.000 description 5
- 238000005755 formation reaction Methods 0.000 description 5
- 208000033519 human immunodeficiency virus infectious disease Diseases 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 230000006698 induction Effects 0.000 description 5
- 230000002401 inhibitory effect Effects 0.000 description 5
- 230000003902 lesion Effects 0.000 description 5
- 238000012423 maintenance Methods 0.000 description 5
- 230000002829 reductive effect Effects 0.000 description 5
- 230000004044 response Effects 0.000 description 5
- 230000035945 sensitivity Effects 0.000 description 5
- 210000004881 tumor cell Anatomy 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 241000701022 Cytomegalovirus Species 0.000 description 4
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 4
- 108091034117 Oligonucleotide Proteins 0.000 description 4
- 230000007022 RNA scission Effects 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 238000001574 biopsy Methods 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 230000036737 immune function Effects 0.000 description 4
- 230000006872 improvement Effects 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 210000005259 peripheral blood Anatomy 0.000 description 4
- 239000011886 peripheral blood Substances 0.000 description 4
- 208000011580 syndromic disease Diseases 0.000 description 4
- HBOMLICNUCNMMY-XLPZGREQSA-N zidovudine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](N=[N+]=[N-])C1 HBOMLICNUCNMMY-XLPZGREQSA-N 0.000 description 4
- 206010061818 Disease progression Diseases 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 108010050904 Interferons Proteins 0.000 description 3
- 102000014150 Interferons Human genes 0.000 description 3
- 208000007766 Kaposi sarcoma Diseases 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 3
- 206010058874 Viraemia Diseases 0.000 description 3
- 230000002159 abnormal effect Effects 0.000 description 3
- 230000035508 accumulation Effects 0.000 description 3
- 238000009825 accumulation Methods 0.000 description 3
- 230000001028 anti-proliverative effect Effects 0.000 description 3
- 230000000259 anti-tumor effect Effects 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 210000003719 b-lymphocyte Anatomy 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 210000001124 body fluid Anatomy 0.000 description 3
- 239000010839 body fluid Substances 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 230000024245 cell differentiation Effects 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 230000034994 death Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 230000005750 disease progression Effects 0.000 description 3
- 208000035475 disorder Diseases 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 206010016256 fatigue Diseases 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 201000009277 hairy cell leukemia Diseases 0.000 description 3
- 208000006454 hepatitis Diseases 0.000 description 3
- 231100000283 hepatitis Toxicity 0.000 description 3
- 238000009396 hybridization Methods 0.000 description 3
- 210000002865 immune cell Anatomy 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 230000000977 initiatory effect Effects 0.000 description 3
- 238000012544 monitoring process Methods 0.000 description 3
- 239000013610 patient sample Substances 0.000 description 3
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 108020004418 ribosomal RNA Proteins 0.000 description 3
- 210000003705 ribosome Anatomy 0.000 description 3
- 231100000419 toxicity Toxicity 0.000 description 3
- 230000001988 toxicity Effects 0.000 description 3
- 230000001052 transient effect Effects 0.000 description 3
- 102000003390 tumor necrosis factor Human genes 0.000 description 3
- 230000004580 weight loss Effects 0.000 description 3
- 229960002555 zidovudine Drugs 0.000 description 3
- 229920000936 Agarose Polymers 0.000 description 2
- 206010056375 Bile duct obstruction Diseases 0.000 description 2
- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 206010016654 Fibrosis Diseases 0.000 description 2
- 241000700721 Hepatitis B virus Species 0.000 description 2
- 208000008771 Lymphadenopathy Diseases 0.000 description 2
- 241000699660 Mus musculus Species 0.000 description 2
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 2
- 208000007027 Oral Candidiasis Diseases 0.000 description 2
- 102000001253 Protein Kinase Human genes 0.000 description 2
- 101710149951 Protein Tat Proteins 0.000 description 2
- 206010037660 Pyrexia Diseases 0.000 description 2
- 108091034057 RNA (poly(A)) Proteins 0.000 description 2
- 108020005202 Viral DNA Proteins 0.000 description 2
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 2
- 230000001594 aberrant effect Effects 0.000 description 2
- 230000005856 abnormality Effects 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 230000001668 ameliorated effect Effects 0.000 description 2
- 230000008827 biological function Effects 0.000 description 2
- 230000031018 biological processes and functions Effects 0.000 description 2
- 230000008499 blood brain barrier function Effects 0.000 description 2
- 210000001218 blood-brain barrier Anatomy 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000011088 calibration curve Methods 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 230000007969 cellular immunity Effects 0.000 description 2
- 230000033077 cellular process Effects 0.000 description 2
- 238000003759 clinical diagnosis Methods 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 230000002596 correlated effect Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 2
- 230000001747 exhibiting effect Effects 0.000 description 2
- 102000034356 gene-regulatory proteins Human genes 0.000 description 2
- 108091006104 gene-regulatory proteins Proteins 0.000 description 2
- 239000003365 glass fiber Substances 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 230000002962 histologic effect Effects 0.000 description 2
- 230000009215 host defense mechanism Effects 0.000 description 2
- 239000000411 inducer Substances 0.000 description 2
- 229940079322 interferon Drugs 0.000 description 2
- 239000000543 intermediate Substances 0.000 description 2
- 230000037041 intracellular level Effects 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 208000019423 liver disease Diseases 0.000 description 2
- 208000018555 lymphatic system disease Diseases 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 210000001616 monocyte Anatomy 0.000 description 2
- 210000005087 mononuclear cell Anatomy 0.000 description 2
- 231100000252 nontoxic Toxicity 0.000 description 2
- 230000003000 nontoxic effect Effects 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 238000011580 nude mouse model Methods 0.000 description 2
- 238000005222 photoaffinity labeling Methods 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 229920002401 polyacrylamide Polymers 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 108060006633 protein kinase Proteins 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 239000003419 rna directed dna polymerase inhibitor Substances 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 241000712461 unidentified influenza virus Species 0.000 description 2
- 108020004463 18S ribosomal RNA Proteins 0.000 description 1
- 229940124321 AIDS medicine Drugs 0.000 description 1
- 206010001513 AIDS related complex Diseases 0.000 description 1
- 102100030088 ATP-dependent RNA helicase A Human genes 0.000 description 1
- 208000004998 Abdominal Pain Diseases 0.000 description 1
- 206010000087 Abdominal pain upper Diseases 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 208000000044 Amnesia Diseases 0.000 description 1
- 108020005544 Antisense RNA Proteins 0.000 description 1
- 206010005003 Bladder cancer Diseases 0.000 description 1
- 206010006448 Bronchiolitis Diseases 0.000 description 1
- 206010007134 Candida infections Diseases 0.000 description 1
- 206010008609 Cholangitis sclerosing Diseases 0.000 description 1
- 208000017667 Chronic Disease Diseases 0.000 description 1
- 102000007644 Colony-Stimulating Factors Human genes 0.000 description 1
- 108010071942 Colony-Stimulating Factors Proteins 0.000 description 1
- 125000000824 D-ribofuranosyl group Chemical group [H]OC([H])([H])[C@@]1([H])OC([H])(*)[C@]([H])(O[H])[C@]1([H])O[H] 0.000 description 1
- 206010012289 Dementia Diseases 0.000 description 1
- 206010012434 Dermatitis allergic Diseases 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 208000010201 Exanthema Diseases 0.000 description 1
- 201000008808 Fibrosarcoma Diseases 0.000 description 1
- 206010019842 Hepatomegaly Diseases 0.000 description 1
- 206010019973 Herpes virus infection Diseases 0.000 description 1
- 208000007514 Herpes zoster Diseases 0.000 description 1
- 108020004996 Heterogeneous Nuclear RNA Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102100037850 Interferon gamma Human genes 0.000 description 1
- 108010047761 Interferon-alpha Proteins 0.000 description 1
- 102000006992 Interferon-alpha Human genes 0.000 description 1
- 108090000467 Interferon-beta Proteins 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 102000015696 Interleukins Human genes 0.000 description 1
- 108010063738 Interleukins Proteins 0.000 description 1
- 208000008839 Kidney Neoplasms Diseases 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 206010028851 Necrosis Diseases 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 208000025966 Neurological disease Diseases 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 206010061876 Obstruction Diseases 0.000 description 1
- 102000043276 Oncogene Human genes 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 208000001388 Opportunistic Infections Diseases 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 208000037581 Persistent Infection Diseases 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 208000005384 Pneumocystis Pneumonia Diseases 0.000 description 1
- 206010073755 Pneumocystis jirovecii pneumonia Diseases 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 229920012196 Polyoxymethylene Copolymer Polymers 0.000 description 1
- 108010069820 Pro-Opiomelanocortin Proteins 0.000 description 1
- 102100027467 Pro-opiomelanocortin Human genes 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 206010038997 Retroviral infections Diseases 0.000 description 1
- 208000005074 Retroviridae Infections Diseases 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 206010039491 Sarcoma Diseases 0.000 description 1
- 208000032023 Signs and Symptoms Diseases 0.000 description 1
- 241000287411 Turdidae Species 0.000 description 1
- 206010053613 Type IV hypersensitivity reaction Diseases 0.000 description 1
- 241000812633 Varicus Species 0.000 description 1
- 108010067390 Viral Proteins Proteins 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 208000021240 acute bronchiolitis Diseases 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 210000001691 amnion Anatomy 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000036436 anti-hiv Effects 0.000 description 1
- 239000000729 antidote Substances 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- 230000004596 appetite loss Effects 0.000 description 1
- 230000003416 augmentation Effects 0.000 description 1
- 230000003190 augmentative effect Effects 0.000 description 1
- 230000008952 bacterial invasion Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 210000000941 bile Anatomy 0.000 description 1
- 210000000013 bile duct Anatomy 0.000 description 1
- 238000011325 biochemical measurement Methods 0.000 description 1
- 238000006065 biodegradation reaction Methods 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000008512 biological response Effects 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 201000001531 bladder carcinoma Diseases 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000004820 blood count Methods 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 210000003123 bronchiole Anatomy 0.000 description 1
- 201000003984 candidiasis Diseases 0.000 description 1
- 230000003306 cell dissemination Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000036755 cellular response Effects 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 230000008131 children development Effects 0.000 description 1
- 208000003167 cholangitis Diseases 0.000 description 1
- 230000007882 cirrhosis Effects 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 230000005757 colony formation Effects 0.000 description 1
- 229940047120 colony stimulating factors Drugs 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 239000003184 complementary RNA Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000000120 cytopathologic effect Effects 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 230000000254 damaging effect Effects 0.000 description 1
- 230000005860 defense response to virus Effects 0.000 description 1
- 238000011257 definitive treatment Methods 0.000 description 1
- 230000003413 degradative effect Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 230000003292 diminished effect Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000000239 endoribonucleolytic effect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000008029 eradication Effects 0.000 description 1
- 238000002481 ethanol extraction Methods 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 201000005884 exanthem Diseases 0.000 description 1
- 208000028327 extreme fatigue Diseases 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 230000004761 fibrosis Effects 0.000 description 1
- 239000000727 fraction Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 210000002443 helper t lymphocyte Anatomy 0.000 description 1
- 230000002489 hematologic effect Effects 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 208000002672 hepatitis B Diseases 0.000 description 1
- 206010019847 hepatosplenomegaly Diseases 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000003118 histopathologic effect Effects 0.000 description 1
- 230000000652 homosexual effect Effects 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 229940047124 interferons Drugs 0.000 description 1
- 229940047122 interleukins Drugs 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 210000001865 kupffer cell Anatomy 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 238000007449 liver function test Methods 0.000 description 1
- 231100001252 long-term toxicity Toxicity 0.000 description 1
- 235000021266 loss of appetite Nutrition 0.000 description 1
- 208000019017 loss of appetite Diseases 0.000 description 1
- 231100000863 loss of memory Toxicity 0.000 description 1
- 201000005296 lung carcinoma Diseases 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 210000003810 lymphokine-activated killer cell Anatomy 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 238000001471 micro-filtration Methods 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000004264 monolayer culture Methods 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 229910052754 neon Inorganic materials 0.000 description 1
- 210000004498 neuroglial cell Anatomy 0.000 description 1
- 230000009251 neurologic dysfunction Effects 0.000 description 1
- 208000015015 neurological dysfunction Diseases 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 206010029410 night sweats Diseases 0.000 description 1
- 230000036565 night sweats Effects 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 210000004738 parenchymal cell Anatomy 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 210000005105 peripheral blood lymphocyte Anatomy 0.000 description 1
- 150000004713 phosphodiesters Chemical class 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 230000004983 pleiotropic effect Effects 0.000 description 1
- 201000000317 pneumocystosis Diseases 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229910052573 porcelain Inorganic materials 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 208000037821 progressive disease Diseases 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 208000030279 prolonged fever Diseases 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 238000000159 protein binding assay Methods 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 206010037844 rash Diseases 0.000 description 1
- 230000000306 recurrent effect Effects 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 230000000284 resting effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 208000010157 sclerosing cholangitis Diseases 0.000 description 1
- MSTPNDZQVGSTET-UHFFFAOYSA-M sodium;2-anilino-6-sulfanylidene-1h-1,3,5-triazine-4-thiolate Chemical compound [Na+].N1C(=S)N=C([S-])N=C1NC1=CC=CC=C1 MSTPNDZQVGSTET-UHFFFAOYSA-M 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000007863 steatosis Effects 0.000 description 1
- 231100000240 steatosis hepatitis Toxicity 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 230000008093 supporting effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 230000008337 systemic blood flow Effects 0.000 description 1
- 230000002992 thymic effect Effects 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 230000005740 tumor formation Effects 0.000 description 1
- 231100000588 tumorigenic Toxicity 0.000 description 1
- 230000000381 tumorigenic effect Effects 0.000 description 1
- 230000005951 type IV hypersensitivity Effects 0.000 description 1
- 208000027930 type IV hypersensitivity disease Diseases 0.000 description 1
- 208000010570 urinary bladder carcinoma Diseases 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 230000009723 vascular congestion Effects 0.000 description 1
- 231100000925 very toxic Toxicity 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 208000016261 weight loss Diseases 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Virology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Toxicology (AREA)
- Epidemiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Description
: A
BACKGROUND OF THE INVENTICHN
New methods in diagnosis and therapy of viral - diseases , chronic pathogen infections generally and cancer emerge when the diverse roles in host defense played by natural dsRNA are uncovered. For example, : development of AIDS (a retroviral infection/cancer) is associated with progressively severe dysfunction in biological processes which I have found to be catalyzed by dsRNA including not only interferon biosynthesis, but also bioactive 2-54 production, RNase L activity and various cell-mediated immune functions. I show that specific reduction in bioactive dsRNA, or enzymes which depend on dsRNA, within specific cells contri- oo butes to disease progression. :
A deficiency state in pathways involving bioactive dsRNA also lies at the core of various cellular lesions . in host defense systems leading to death. Earlier, I detected an inhibitor of RNase LI in lymphocytes of
EIV-infected ARC and AIDS patients which is just one result (from many) of inadequate intracellular dsRNA levels. Therapy with appropriately configured synthetic " , dsRNA eliminates. this inhibitors), which can explain part of the clinical responses I observed with respect to reduction in virus concentration and resto- ration of immune function in patients with a variety of chronic viremias which at present continue to largely defy definitive medical intervention. * Accordingly, I have now developed an integrative in- vention which can explain, quantitate, and correct a variety of dsRNA deficiency states in varied disease settings. I have also discovered that certain dsRNAs, e.g., mismatched dsENA, can actually substitute for a naturally occuring dsRNA which help maintain functions essential for normal cell processes. The absence of ’ . such dsRNA regulation, if not corrected with a suitable CL exogenous dsRNA, can cause varicus abnormal cellular processes to develop. Abnormal cellular processes then lead to chronic viral or other intracellular "pathogen infections, reduced immune cell functions and, ultimately, chronic morbidity and possibly death itself.
Clinical Strategies. Viral infections can often follow partial disturbance of the immune system : through agents as diverse as stress and viruses which oo ~3-
directly attack immune function. For example, AIDS (Acquired Immune Deficiency Syndrome) follows the progressive deterioration of the immune system caused EE by infection of T lymphocytes by human immunodeficiency virus, HIV (Coffin et al., Science vol. 232, p. 697, 1986).
For completeness, it should be noted that different designators for the HIV virus exist; LAV is the desig- nator for the AID3 virus isolated at the Pasteur
Institute, Paris, France and HTLV-III is the designator for the AIDS virus isolated at the National Institute of Health, Bethesda, Maryland, U. S. A. Currently, HIV ’ is used as a generic term for the virus. Frequently in this text, the HIV virus will be referred to generically or designated HIV or HTLV-III or LAV with- out intending to differentiate between them. Further- more, the term HIV in the specification and claims includes all other viruses which may be associated with ‘producing AIDS, whether yet isolated or not.
EIV infection is a progressive disease although : the rate of transition from one phace to another is variable. Asymtomatic individuals infected by HIV =f can develop a condition (LAS or pre-ARC) characterized by lymphadenopathy. Paitents progress to AIDS-related complex or ARC by exhibiting the T4 cell deficit and later show reduced ability of lymphocytes to undergo antigen-stimulated proliferation and IFN-gamma produc- tion. ® These patients lose various cell mediated immune functions such as delayed cutaneous hypersensitivity, eventually becoming completely anergic. They exhibit evidence of breakdown in host defense mechanisms including Herpes Zoster infections, oral candidiasis (thrush), and symptoms such as prolonged fevers, night sweats diarrhea and weight loss. Finally, these patients experience certain severe opportunistic infec- tions (e.g., Pneumocystis carinii pneumonia) and are then defined as having full-blown ATDS, with an approximately 50% mortality within 12 months. The
HIV virus infection is chosen as an illustrative case simply because the level of dsRNA deficiency is so pronounced. Many other diseases, including indolent viral infections, will be associated with similar deficiencies; thus, my invention has unexpected wide applicability. po | -5=-
Importantly, the typical progression outlined : above does not describe the remarkable disparity in clinical symptomatology among HIV-infected patients or many other patients with chronic disease such as
Ipstein Barr or EB infection, hepatitis virus infec- tion, cytomegalovirus, herpes infections, etc.
Components of the immune system decay with vastly different rates in various patients and the lack of bioactive dsRNA is a cause or contributing factor in this process. HIV infection may develop in a variety ; of different types of cells (e.g., blood cells, glial cells). Some patients develop neurological dysfunction as their first symptom. Thus, various individuals may have vastly different therapeutic requirements even though all will probably require HIV eradication from
T4 cells.
Two general pathological features need to be addressed in many different viral infections inclu- ding those of ARC patients: a collection of immune deficits and ongoing viral infection (reviewed in
Fauci, Proc. Nat'l. Acad. Sci. USA, vol. 83, p. 9278, 1986). ‘Therapeutic intervention which attacks only So.
the immune deficits but fajls to control virus repli- cation appears to be counterproductive especially when such treatment activates T4 cells. For example,
T-helper cells with Tac receptors are responsive to the lymphokine IL-2. In AIDS victims, IL-2 causes expansion of HIV-sensitive cells and apparent worsen- ing of the disease.
Attacking viral infections directly, e.g., the \ HIV infection, has met with some success especially evidenced by the viral reverse transcriptase inhibitor designated azidothymidine (AZT) which can reduce HIV or "]oad" in some patients and prolong life. Such therapy is not without undesirable results. In a minority of patients, AZT also causes temporary recovery of T-cell meditated immunity as measured by a transient rise in
T4 cells and by appearance of delayed type hypersen- sitivity (Fischi et al. New Eng. J. Med., July, 1987)
However, reverse transcriptase inhibitors are toxic : at doses needed to produce significant antiviral effects and are thus poorly tolerated over time.
Antiviral agents may need to be administered for the life of the patient. , i.
IFN is both ineffective as a general antiviral compound and as an anti-AIDS drug, probably because : it is not an antidote for the dsRNA-related defects vhich I have now uncovered. Certainly, ARC/AIDS victims have various "defects" in IFN pathways, such as the production of a defective, acid-latile IFN and inability of ™ cells to produce appropriate amounts of IFN gamma which are well described in the medical literature. ZFarlier, I concluded that these various . 10 IFN-related defects in ARC and AIDS patients may prove to be related in whole or part to a lack of normal
RNase I. (a terminal pathway mediator) activity in ’ lymphocytes which I described in my copending applica- tion Serial No. 021,372 filed March 3, 1987, which is hereby incorporated by reference. Although dsRNA- dependent 2-5A synthetase is elevated in blood lymphocytes of individuals with ARC/AIDS as well as Cr other chronic viremias, I have found that the same : blood cells show diminished levels of authentic 2-5A : 20 and low RNase IL (see below). I correlated the low : RNase IL activity with low Quantities of M_80,000 protein capable of binding a photoaffinity labeled analogue of 2-5A. liy findings are consistent with a , a dsRNA pathway(s) which has (have) been shut down and the relative absence and/or inhibitor of RNase L.
Vaccination against HIV and other viruses, and the use of passive anti-HIV antibodies, are also under congideration for viral prevention and treatment, respectively,, but may have significant limitations; for example, HIV (as well as influenza virus in particular) readily can infect cells covertly. I also described a role for dsRNA to enhance antibody forma- tion against viruses in general and retroviruses in particular in the above-mentioned patent application. ,
I describe herein a new phenomenon that argues for double-stranded RHAs (dsRNA) to constitute the first group of molecules which are effective against both the viral and immune lesions of various subacute/chronic viral infections such as HIV and others. These defects can be corrected with lacer- like precision without adversely affecting other bodily functions which is a common limitation of presently availavle therapies for chronic viral in- fections. I conclude that patients with unresolved ’ ~9- . ve and indolent viral infecticns often have specific defects in vital intracellular dsiliA-dependent path- , ways which can be raversed or ameliorated at least in part by an exogenously supplied dsRNA (this is explained graphically in the flow chart of Figure 1), and that monitoring these pathwaysy before and during treatment, provides a novel efficacious manner to gauge the degree of dsRNA replacement therapy required an individual patient or specific disease basis. +
Critical enzymes associated with host defense require dsRNAs for expressing bioactivity. These ’ enzymes can be activated by exogenous dsRNA if the level of natural intracellular dsRNA is too low. }
Part of the pleiotropic action of dsRNA derives from its ability to induce the synthesis of the entire range of IFNs - alpha, beta and gamma - which operate through a group of intracellular mediators including a dsRNA- dependent protein kinase, dsRNA-dependent 2-5A synthetase and 2-5A-dependent RNase L. These dsRNA- : 20 dependent enzymes may carry out many biological activities attributable to IFN as reviewed by Lengyel (Ann. Rev. Biochem., vol. 51, p. 251, 1982). For -10- —
LAD QRiGiNg, | 2 example, production of an antisense RNA which blocks expression of 2-5A-synthetase causes profound sensiti- vity of animal cells to infection by different viruses (Bendetti et al. Prac. Natl. Acad. Sci USA, vol. 84, p. 658, 1987). The dsRNA-activated protein kinase phosphorylates eIF2 which inhibit protein syntheses; it also has proteolytic activity to degrade viral proteins. DsRNA-activated 2-5A synthetase synthesizes 2-5A which activate RNase L, a pathway which I suggest or plays a crucial role both in the inhibition of various animal viruses and in cell growth control (escape from which may lead to tumor formation). 2'-5' oligo- ’ adenylic acids are unusual in that the normal 32',5! phosphodiester linkage characteristics of most dsRNA. has been replaced by a novel phosphodiester bond with acquisition of new biological properties.
Antiviral Activity of dsRNA. dsRNA is a well- known inducer of an antiviral state (Marcus et al,
Nature, vol. 66, p. 815, 1977). Similarly, poly(A) :poly(U) generated an antiviral state without inducing IFN. But, prior to my current invention, it was not known that natural dsRNA regulators exist and, therefore, that actual dsRNA deficiency states are common in humans, particularly associated with pathogenic episodes (viral, fungal, protozoan, bacte- rial invasions, etc., especially those which have intracellular (human) presence as a valuable or necessary components of their life cycle).
Antiproliferative Activity of dsRNA. Earlier,
I found (J. IFN. Res., Vol. 6, p. 373, 1986) that 42% of more than 100 fresh human tumor specimens, when CL studied in soft agar, showed a 50% or greater reduc- tion in tumor cell colony formation after only one ’ exposure to dsRNA. These cells are dsRNA deficient.
I also described independent IFN and dsRNA sensitivity in various human tumor cell lines and found that human tumors propagated in nude mice were sensitive to dsRNA; dsRNA therapy was curative of some tumors (e.g. , renal) and the animals lived to a normal life expectancy (see published European patent application 0,113,162).
Herein, I report a strong correlation between clinical : 20 ~ responses and dsRNA-dependent enzyme activation as opposed to detectable IFN or IL-2 induction secondary to dsRNA administration.
Immune Enhancement Activity of dsRNA. Earlier
I reported (J. Immunol., vol. 124, p. 1852, 1980) ' that dsRNA increased human natural killer NK cytlytic activity against human leukemia cells. The dsRNA structural requirements for NK augmentation paralleled those needed for 2-5A synthetase activation and for
Co IFN induction.
Gene Control Mediated by dsRNA. Sullo et al (Cell, vol. 43, p. 793, 1985) demonstrated that dsRNA induces competence genes in resting fibroblasts - including the oncogenes designated fos and myc. As used herein, "competence genes" are those genes in a cell whose actions are required in order to modulate vital actions such as multiplication, growth, etc.
The IFN-beta gene induction by dsRNA may utilize a regulatory protein which is removed upon exposure of cells to dsRNA (Zinn et al. Cell, vol. 45, p. 611, 1986). I have determined that, to biological activity, oo “exogenously supplied dsRNAs mimic a natural, nonviral, intracellular dsRNA such as heterogeneous nuclear RNA or mRNA complexed with poly(U). For example, I recently discovered an IFN-inducible dsRNA in Hela cell nuclei capable of activating 2-5A synthetase in ) vitro. The advent of cellular immunity far back in the evolutionary cycle in animals apparently set the stage for the evolution of IFN/dsRNA molecules toward implementing cellular differentiation, particularly differentiation promoted by IFN, I was able to achieve the more general insight that dsRNA can implement differentiation promoted by other regulatory proteins which set the stage for my definition of dsRNA deficiency states.
BRIEF DESCRIPTION OF THE DRAWINGS ot
Figure 1 is a flow chart illustrating the dual : pathways to host morbidity and the pathway to host recovery;
Figure 2A 4s a graph plotting the number of days prior to and after initiation of IFN therapy in the amount indicated against synthetase activity;
Figure 2B is a graph comparing white blood cell count and quantity of mismatched dsRNA after 120 days of treatment;
Figure 3 is a photograph of polyacrylamide gel electrophoresis plates showing various zones and bands along each track; and oo
Figure 4 is a graph comparing absolute number of
T4 lymphocyte levels per cubic millimeter of blood, expressed as % change from baseline, to months of therapy. :
I describe herein a new phenomenon that argues for double-stranded RNAs (dsRNA) to constitute the first group of molecules which are effective against both the viral and immune lesions of various subacute/chronic viral infections such as HIV and : others. These defects can be rorrected with laser- like precision without adversely affecting other bodily functions which is a common limitation of presently available therapies for chronic viral infec- tions.
I conclude that patients with unresolved and indolent viral infections often have cpecific defects in vital intracellular dsRNA-dependent pathways which can be reversed or ameliorated at least in part by an exogenously supplied dsRNA (this is explained graphi- cally in the flow chart of Figure 1), and that monitoring these pathways, before and during treatment, =~ °° provides a novel efficacious manner to gauge the degree of dsRNA replacement therapy required an indi- vidual patient or specific disease basis. , this invention provides a diagnostic procedure for determining a dsRNA deficiency state in a patient's sample, quantifying this deficiency (if any), and providing a therapeutic procedure for restoring any : dsRNA deficiency identified by the administration of. } exogenous dsRNA optionally in combination with a : lymphokine.
Typically, a dsRNA deficiency state is evidenced within a component cell of the immune . system whether or not the component cell is in the process of multiplication or differentiation.
DsRNA co -16-
deficiency is evidenced, for example, by the inability of a component cell of the immune system to maintain a sufficient level of bioactive RNase IL.
Monitoring of the intracellular dsRNA-dependent pathways before, during and after therapy enables the clinician to gauge the degree of dsRNA replacement therapy required : on an individual basis.
Other means to asess dsRNA deficiencies are given in the detailed description that follows. "Mediators" are herein operationally defined as any intracellular moieties (e.g., specific oligonucleotides, proteins, dsRNAs - alone or in combination) which effect specific biochemical functions initiated by presence of dsRNA.
Such biochemical functions will contribute to a strengthening of host defense mechanism(s) at either the single cell or whole (organism) level. : Vonditions susceptible to the therapeutic proce- dures of this invention are generally thosein which a deficiency of the intracellular dsRNA level is below normal limits, as compared with healthy or individuals, dsENA deficiency causing tissue patho- logy and/or in the presence of an abnormally low dsRNA
: level coupled with or coexisting with the presence of an intracellular pathogen. More specific conditions or tissue pathologies and constitutional symptoms in- clude viral infections such as retrovirus infections including HIV, herpes family virus, paranyxovirus, rhinovirus, hepatitis and chronic fatigue syndrome.
Others include uncontrolled proliferation of cancer cells and pathologies of the immune system whether or i i» not the component cell is in the process of multipli- cation or differentiation. :
By "mismatched dsRNAs" are meant those in which hydrogen bonding (base stacking) between the counter- part strand is relatively intact, i.e., is interrupted on average less than one base pair in every 29 conse- cutive base residues. Mismatching is an interruption of the normal geometry of the RNA double helix by in~ pouching (or out-pouching) of the strands which represent points of vulnerability of the dsRNA to digestion by ribonucleases. The term "mismatched dsRNA" should be understood accordingly. »
The dsRNA may be a complex of polyinosinate and © | _18-
‘a polycytidylate containing a proportion of uracil bases or guanidine bases, e.g., from 1 in 5 to 1 in 30 such bases (poly 1. poly (Cy_pg x>U or G).
The dsRNA may be of the general formula : 5 rl .r(Cq,U), - Other suitable examples of dsRNA are discussed below.
In the preferred mismatched dsRNA, rI .1(Cy5 0) a region consisting of an uninterrupted stretch of 6 to 12 base pairs, i.e., one-half to one full turn of an RNA helix, serves both as biotrigger causing , release of lymphokines and as an obligate intracellular co-factor for enzymes comprising the natural antiviral pathways. The mismatched regions consisting of uracil: residues is periodically inserted in the polypyrimidine strand to accelerate dsRNA hydrolysis and thus prevent toxicity.
The mismatched dsRNAs preferred for use in the present invention are based on copolynucleotides selected from poly (C16) in which n is an integer : having a value of from #4 to 29, and are mismatched co -19-
analogs of complexes of polyriboinosinic and poly- ribocytydilic acids, formed by modifying rl .rC, to incorporate unpaired bases (uracil or guanidine) along the polyribocytidylate (rc) strand. Alterna- tively, the dsRNA may be derived from poly (I). poly (C) dsRNA by modifying the ribosyl backbone of poly- riboinosinic acid (v1) e.g., by including 2' -0O-methyl ribosyl residues. These mismatched analogs of rl .rC., preferred ones of which are of the general formula rI .v(Cyq_ 1420), and rl .t(Chq 6) ps are described by Carter and Ts'o in U. S. Patents 4,120,641 and 4,024,222 the disclosures of which are , hereby incorporated by reference. The dsRNAs des- cribed therein generally are suitable for use according : 15 to the present invention. In certain instances, com- plementary oligonucleotide duplexes (helices) will also suffice as replacement therapy.
Other examples of mismatched dsRNA for use in : the invention include: poly (I).poly (Cy, UD poly (I).poly (Cp\U)
poly (I).poly (C121) poly (I).poly (C,5,U) poly (I).poly (C5016) poly (I).poly (Cg @) and poly (I).poly (cy) 23 G>p dsRNA-Tymphokine Uynergy. Since dsRNA exerts a significant component of its biological action through a specific lymphonine system mediated in part by IFN,
I reasoned that it must (a) induce IFN in IFN-deficient cells as well as (b) activate relevant dsRNA-dependent enzyme mediators, the cellular dsRNA being non-induci- ° ble in cells failing to respond to exogenously applied
IFN.
If dsRNA operate® solely thought the IFN system and if dsRNA is in excess in cells, then an excess of
IFN alone would exert as much bioactivity as the 7 ) combinations of IFN and dsRNA. However, this must be a relatively rare condition since I have now found many examples of dsRNA-IFN synergy and a paucity of examples of no synergy. For example, I found that dsRNA was synergistic with human IFN alpha, beta or gamma in inhibiting the growth of cells form human , bladder carcinoma, lung carcinoma, and fibrosarcoma.
I extended these results to 15 other human tumor cell lines with only one line failing to show antiproli- ferative synergy. 1 observed similar antitumor synergism was seen in some human tumor engrafted in nude mice and, clinically, I consistently observed : | that the combination of IFN and dsRNA was superior to either agent alone as an anticancer regimen in both kidney tumors and CML. dsRNA Deficiency States. As a prototype pathway, : the 2-5A synthetase/RNase L pathway includes one or more dsRIAs, 2-54, linked enzymes and inhibitors. Cr
There exists a direct biochemical connection between dsRNA and RNase IL in that 2-5A, the product of dsRNA- dependent 2-5A synthetase, is a necessary cofactor for RNase LI activity. The examples below are intended - to exemplify a few of many possible kinds of defects ' in dsRNA-mediated pathways which can be reversed by exogeneously supplied dsRNA.
The flow chart of Figure 1 further illustrates the relationship between dsRNA sufficiency, leading SE to host recovery, and dsRNA deficiency, leading to host morbidity. Bioactive dsRNA is produced intra- cellularly or is introduced by certain events which lead to an antiviral state as well as immune cell differentiation and host recovery.
The in vitro measurement of 2-54 synthetase is of limited value, since this assay does not reflect the in vivo activity of the 2-54 synthetase. There- fore, I have developed a method to extract and quantitate the 2-54 from the peripheral blood mono- : oo nuclear cells (PBMC) of healthy individuals and from pathogen-infected patients before and after mismatched dsRNA therapy. The concentration of 2-54 can be determined by the ability of the 2-54 to activity affinity-purified RNase I to degrade poly(U)/ 2°B7pCp.
CPE EON = . " pe oS . Fe . - TABLE 1 - Intracellular 2-5A Concentration and In Vitro Activity of 2-BA j Synthetase and Biase L in POMC Extracts of Viral-Infected Individuals
E PBMC Weeks on 2-BA Intracellular RNase L : b } 5 source mismatched synthetase 2-BA p a d d ~ dsRNA activity latent" ’ activated x i } } column: ' 1 2 3 4 5 8 £1 AIDS 0 49 <0.2 260 - - + [3 105 ND - 2 “10 ks ? 4 © «0.2 - N
Z 15 10 1.8 +4 ’ i 19 21 0.8 - ce. 3 : 24 Cc - 26 © >10.0 : : +444 ~. i 28 20 >10.0 sree $2 AIDS © -k 205 <0.2 240 - - : : * 0 180 oD - - 3 xs 4 117 1.4 + * + 8 19 2.4 +ee
PCP 21 ND 6.8 +" 3 . 26 9 B.& +444 ) ’ 23 ARC’ -6 11 <0.2 270 - - . t 2 13 ND - 4 19 <0.2 - 17 3 1.8 +444 ’ , #6 ARC 0 130 <0.2 270 - - oR . P- neon 0 : Smad , ‘ . — pens - ~24~
T ET Fy yr ry r——— rea Pret tem ry mire mg ery ee. reaper
A
. 7 / 2 185 ND - oo i 4 18 0.4 + 8 ND <0.2 - 22 ND >10.0 +44 #5 LAS 0 30 0.6 280 - - : a 36 <0.2 - 9 12 1.0 + 17 21 3.0 ees ‘ . Healthy 5 0.3-1.1 1350 +434
Volunteers 2 nmole ATP incorporated into 2-5A/mg protein as determined in poly(I):poly(C)-agarose assays; standard deviation 9%: ND, not determined. b nmole/g protein as determined in core-cellulose assays (in duplicate); standard deviation 20% : ¢ dpm/50 ug protein as determined in radiobinding assays (in duplicate) (column 3); standard deviation 20%. d determined in rRNA cleavage assays (columns 4 and 5) in two independent assays. Specific cleavage product (SCP) formation was quantitated by densitometric tracings of photographs of gels and is expressed as a ratio of SCP formation divided by 18S and 28S rRNA x 100; -, O; +, 10-30; ++, 31-63; 64-85; ++++, 86-100.
Methods. Heparinized peripheral blood was obtained from ten healthy individuals and from five homosexual male patients with LAS, ARC and AIDS (as defined by the Centers for Disease
Control). Clinical, virological and immunological features of patients involved in this study (patients #1-5) were 4 ee et etree yp ee - reer errr >. .m2o= characterized previously as patients 10, 8, 1, 7 and 2, respectively in my Lancet June 6, 1987 article.
PBMC were isolated on Ficoll-Hypaque. L929 and HL929 (an RNase L-deficient subclone of 1.929) cells were maintained in monolayer culture. Cytoplasmic extracts from 1929 and T.929 cells were prepared in glycerol buffer and PBMC extracts were prepared in NP40O lysis buffer. Protein concentration of the extracts ranged from 10 - 15 pg/pl. KS, Kaposi's sarcoma; PCP,
Ineumocystis carnii pneumonia. :
Activity of 2-5A synthetase (column 1) was ’ . determined in FBMC extracts (50 1g protein/assay) using poly(I):poly(C)-agarose. 2-5A was isolated from the ethanol-soluble fraction of FBMC extracts (100 pg protein) (70% ethanol, v/v). The ethanol-extracted 2-5A was then lyophilized and redissolved in water (20 pL). Concentrations of intracellular 2-5A present in PBMC extracts (column 2) were determined in core- : cellulose assays with 1929 cell extracts as determined 20’ in core-cellulose assays with 1929 cell extracts as to the source of RNase IL from calibration curves obtained
Lo oe
\ . with authentic 2-5A. Activation of RNase L by 2-5A in this assay is based on the conversion of poly (U)/ >2p7pCp to acid-soluble fragments. In : control experiments (in the absence of 2-51), 6500 dpm was retained on glass fiber filters from a total of 19700 dpm of poly(U)/ 7°P7pCp added (1.3 pCi/nmole). /
In the presence of <1 x 10710 1 authentic 2-54, poly(U)/ 2%p7pCp was degraded 0%; in the presence of >1 x 10-7 authentic 2-54, poly(U)/ 2“P7pCp was degraded 100%. Latent RNase L levels were measured by two methods: (i) in radiobinding assays after the addition of 20,000 dpm of pyhy/ > E7PCD to PBMC ~ extracts (50 pg protein/assay) (column 3) and (ii) in ribosomal ENA cleavage assays in the presence of 2-5A : (column 4). Extracts (150 pg protein/assay) prepared ” from L929 cells were incubated in the presence of 5 pl of ethanol-soluble fractions of PBMC extracts and 1 x 10°21 Pzhz in a final volume of 20 ul for 60 minutes at %0°C. The total RNA was extracted, denatured and analyzed by electrophoresis on 1.8% agarose gels. The ethidium bromide stained RNA bands were visualized under UV light. ‘Activated RNase L levels (column 5) were determined by ribosomal cleavage assays in the absence of added 2-5A.
The concentration of functional 2-5A in PBMC extracts varied from 0.3 to 1.1 nmole/g protein for healthy humans and from below detectable levels to 0.6 nmole/g protein in PBMC extracts from virus- : infected patients before mismatched dsRNA treatment (Table 1, column 2). However, 2-58 accumulated to greater than 10.0 nmole/g protein as mismatched dsRNA therapy progressed. 2-5A activated, partially- CL purified RNase L has been previously reported to pre- ferentially aleave polu(U), but not poly (GC). The isolated human 2-5A, like authentic 2-5A,:-could . activate affinity-purified RNase I from either 1929 cells or from PBMC of healthy humans to specifically degrade poly(U); see table #., It is important to note that when TCA is used to extract 2-5A from PBMC, a poly(C) degrading activity was extracted in addition to the poly(U) degrading (RNase L) activity. This poly(C) degrading TCi-extractable activity could be eliminated by protease digestion or further ethanol extraction. The poly(C) degradative activity was found in TCA-soluble fractions of PBMC from all humans, 5 , but in none of the permanent cell lines (i.e., Hela
1,929) tested thus far. The poly(C) degrading activity was not detectable in any of the ethanol-soluble frac- tions of PBMC.
TABLE B
| Effect of dsRNA on Infectious Center Formation by Respiratory Syncitial Virus-Infected CCL 25 Cells
Concentration of Infectious Centers? Percent mismatched dskNA® Plate Counts Mean Counts Reduction . ——— ee v 0 pg/ml 49, 44, 15%, 33 42.0 0.5 pg/ml 38, %6, 26, 16* 33,3 21.7 1.0 pg/ml 54, 44, 42, 53 48.5% 15.5 2.5 pg/ml 27, 22, 24, 30 20.8 26.7 5.0 pg/ml 27, 14, 17, 18 19.0 54.8 10.0 pg/ml 1,1, 7, 5 3.5 91.7 © 15 25.0 pg/ml 0, 2, 0, O 0.5 98.8
J i ’ . ' * . } Data not used to calculate mean counts.
The highest concentration of mismatched dsRNA (T1.7(Cy 5,
U),, 25 pg/ml7 was non-toxic to OCL 25 cells. or
Bh (2) muadruplicate wells of confluent CCL 25 cells in oh well plates were re-fed with 1.5 ml maintenance medium containing dsRNA at concentrations indicated above 1 hour prior to plating of R3V-infected cells.
Mock-treated cells were re-fed dsRNA-free maintenance medium. : (v) 4 x 10% CCL 25 cells in suspension were infected in a total volume of 1 ml containing 5 x 10° PFU of ’
RSV (101 = 12). Viral adsorption occured during a 2 hour incubation at 37°C with intermittent resuspension of the cells. At the end of this period, the suspen- aion was diluted 1/100 in maintenance medium. 0.5 ml aliquots of the infected cell suspension were added directly to each experimental well, and tc each infectious center-control well. 0.5 ml volumes of maintenance medium were added to cell control wells. £11 cultures were incubated at 290 in 5% C0,~95% air for 72 hours, fixed with methanol and stained with
Giemsa. Flaques (Infectious Centers) were counted microscopically. : moi refers to multiplicity of ‘infection; that is, the : approximate number of viral particles avaialtle to 5. infect each target cell. TFU refers to plaque forming units; that is, the number of viral units as determined by counting the cytopathic (dead cells). . Although there have been reports on the presence of 2-5A in tissues isolated from untreated animals as well as the accumulation of 2-54 in tissues from mice ’ treated with dsRNA, mine is the first report of the accumulation of 2-5A in a human tissue. It is well established that authentic 2-5A binds to and activates
RNase L to degrade rRNA to highly characteristic ‘specific cleavage products (SCP). Therefore, I charac- terized the activity of the 2-5A isolated from FBMNC extracts in rRNA cleavage assays. The specific : cleavage pattern generated by RNase I, with ethanol- extracted 2-5A was the same as that obtained in the presence of authentic 2-5A. These results show that i.v. administration ’of mismatched dsRNA to virus-'
infected patients caused dramatic increases of 2-5A in blood cells beginning from concentrations which were below detection.
RNase L Activity
The absence of RNase L activity in HIV-infected individuals was confirmed and extended (Table 1, columns 3, 4, 5). A sensitive technique based on the cleavage specificity of 2-5A activated RNase LI on rRNA (Wreschner et al, Nucleic Acids Res., vol. 9, pp. 1571-1581) permitted the measurement of activated
RNase I in extracts of PBMC isolated from as little as one ml of peripheral blood. Ixtracts of an RNase
L-deficient subclone of the 1929 cell line (HL929) were utilized as the source of ribosomes. PBMC |, extracts of HIV-infected patients had 5-7 times lower levels of latent RNase IL than those of healthy individuals as determined in radiobinding assays (Table 1, column 3; 240-280 dpm vs. 1350 dom).
Similar observations have been reported by %Wu et al ’ (AIDS Research, vol. 2, pp. 127-131, 1986). The application of the radiobinding assay 1s limited with a -32-
respect to the determination of latent RNase IL level in patients treated with mismatched dsRNA because accumulated 2-54 present in PRMC samples from these patients (Tabld 1, column 2) can compete for binding to RNase L. Therefore, determination of the level of the 2-5A-activated RNase L was essential.
First, levels of activated RNase L were deter- mined by measuring the specific cleavage of rRNA by
PBMC extracts in the absence of exogenously added 2-5A (Table 1, column 5). Using this assay, I demonstrated that RNase I, was not activated in PBMC extracts of Co,
HIV-infected patients before therapy (polyacrylamide gels shown in Figure 2). Knowing that the levels of : intracellular 2-5A were low in these IBINC samples (Table 3, column 2), this result was not unexpected.
However, extracts of PBMC from all healthy individuals tested showed the presence of activated RNase L which produced specific cleavage products in TRNA cleavage assays, even though 2-5A in some healthy individuals was as low as 0.3% nmole/g protein (Table %, column 5).
It is significant that the absence of RNase L activity in samples from HIV-infected patients before therapy cannot be due to accumulation of a competitive inhibi- tor of 2-5A as reported (Caylet et al European J. Bioch. , vol. 143, pp. 165-177, 1984) RNase L activity could not _ be restored by the addition of authentic 2-5A (Table 1, column 4).
Next, levels of latent RNase L were determined by two independent measures. Latent RNase IL activity was determined in the rRNA specific cleavage assay, : but in the presence of added 2-54 (Table 3, column 4). | In samples taken before mismatched dsRNA therapy, no latent RNase IL activity could be detected in any of the 5 patients examined.
The technique of photoaffinity labeling with
Dh, / 22E700D was used to determine further if RNase L was missing or altered in PBMC of HIV-infected patients.
I'revious photoaffinity labeling studies have identified a protein of 1,,80,000 with binding affinity for psd, > E700D and specific poly(U) endoribonucleolytic activity as Rllase L. Affinity labeling of RNase L present in FBMC extracts was determined using p3h/ "> “E7p0D. RNase IT, from TBMC extracts (50pe : 5 protein) from-a normal individual was compared with : | | | a an ARC patient (patient before mismatched dsRNA therapy) or L929 cell extracts (50pg protein) was photolabeled after the addition of pA, /"270Cp (20,000 dpm, 3000 ci/mmole) in the absence or the presence of authentic Phy (1 x 1078 1). RNase L from
PBMC extracts from a normal individual (100pg protein) was purified on 2-54 core-cellulose and photolabeled after the addition of Phyl > F7pCp (30,000 dpm; 3000
Ci/mmole). After incubation at 0°C for 90 minutes, the samples were transferred to ice-cold porcelain spot plates and photolysed for 3 minutes, using 254 nm UVG-11 Mineralight lamp (Ultraviolet Products) at a distance of 2 cm (1.0 I/m°). The positions of the protein markers and the 1.,80,000 ENase L were deter- + mined.
My photolabel ing studies. revealed that ph, / >F7pCp was covalently linked to a protein with zn M, of 80,000 in extracts of PBMC from healthy humans (polyacrylamide gel data not shown). However, no protein was labeled in extracts of FBMC from a chronically virally- infected" h patient. Under idenfical experimentsl conditions, a protein of M., 80,000 was specifically photolabeled in 1929 cell extracts. Addition of Pgh, (1 x 1078 1) to incubation mixtures containing the pahy/ E7RCD prevented photolabeling thus providing me additional evidence that the photolabeling was highly specific for RNase I... Fhotolabeling studies with the protein purified from FBMC extracts from a healthy human by the core-cellulose method revealed covalent linkage to a protein of M_.80,000, which was capable of degra- ding poly(U), but not poly(A), poly(C) nor poly (G), in the presence of authentic 2-4A (Table 4). Taken together, these results confirm that the protein purified from PBMC of helathy individuals and photo- labeled by ph/22E700p.
After 4 to 17 weeks on mismatched dsRNA therapy, activated Rllase I was first detected in PBIMC extracts from all five chronically infected patients; by 17 to CL 28 weeks of mismatched dsRNA therapy, activated RNase
L activity for all five patients was equivalent to
RNase I levels observed ir healthy individuals (Table 1, column 5). <The level of activated RNase L showed very close correlation with the concentration of functional 2-5A isolated from the same samples (Table . 1, as compared with columns 2 and 5). Of utmost interest, patient #1 maintained an elevated intra- cellular 2-5A level and fully activated RNase L at 28 weeks, 3 weeks after mismatched dsRNA therapy was Cee discontinued (Table 1, columns 2 and 5). oe
The experiments described here show that five
MIV-infected patients possess a common molecular phenctype in blood mononuclear cells: reduced levels of 2-5A and absence of detectable RNase IL activity.
Blood mononuclear levels for healthy individuals exhibited a different phenotype in that 2-54 levels were higher, on average, and RNase L activity was readily detectable. The latter phenotype is more consistent with that expected of a functional 2-5A synthetase/RNase L pathway. In normal individuals, the steady state pool of 2-5A is detectable; an appreciable fraction of this intracellular 2-5A may become tightly associated with RNase L, thereby © 20 activating the RNase I.. In the experimental proce- dures elucidated herein, it is clearly established that in blood mononuclear cells of chronic virally-
infected persons, the intracellular levels of 2-5A are below detection and there is no activated RNase ’ L as determined by presently available technology.
In summary, an important discovery here was that defects in the 2-5A synthetase/RNase L pathway were reversed by therapy with mismatched dsRNA. My results are not explained simply by normal levels of RNase L protein without 2-5A, since RNase L activity in vitro was not restored by adding 2-5A to PBMC extracts.
RNase I protein must be absent or inhibited, a . conclusion .confirmed by lack of binding by a ’ photoaffinity-labeled analog of 2~5A. In the treated : patients described here, HIV RNA levels in PBMC were reduced within 10-20 days and the number of infections centers (co-cultivation) of HIV-infected PBMC declined more slowly. The increase in intracellular 2-5A and
RNase L activity determined here more closely parallel . the loss of infectious centers in these patients. My : results clearly show that the 2-5A synthetase/RNase L " pathway became more active in treated HIV-infected patients than in healthy humans after several weeks of Sherepy with mismatched dsBNA. My results are thus fully consistent with the hypothesis that certain chronic viral infections represent a dsRNA deficiency state which can be reversed by supplying exogenous source of bioactive dsRNA.
The invention is further described with reference to the following illustrative examples in which all parts and percentages are by weight unless otherwise indicated.
EXAMPLE 1 - Coordinated IFN and dsRNA Treatments
A dsRNA-deficiency state is one in which IFN-treated tumor cells show little or no IFN-induced growth arrest.
This is a functional or operational test of dsRNA deficiency which can be confirmed rigorously if need be by various biochemical measurements described below. . : 15 In many instances, a clinical diagnosis of a "dsRNA deficiency" will become sufficient as my invention Co . becomes more widely practiced. IFN" tumor cells are . known to be rarely deficient in IFN receptors and may contain high or low levels of mediators, but in either case I find that they respond to exogenously supplied oo ~39-
dsRNA.
These examples are of clinical relevance since . ’ they indicate that dsRNA will consistently expand the ; therapeutic range of IFN in particular and also other lymphokines as well, e.g., IL-2, various colony stimulating factors and TNF.
Chronic myelogenous leukemia (CML) is a case in point.
About 60% of CML patients respond to IFN alone, while 40jc do not.
The latter fail to induce 2-5A synthetase in blood mono- Co nuclear cells (IINC) as described by Rosenblum et al. oo in Cancer Res., vol. 46, p. 4848, (1986). Tigure 2-4 presents an IlFN-resistant case which I treated with nismatched dsRNA plus IFN following a short course ’ (7 days) of IFN alone then with mismatched RNA in } combination with IFN.
This patient failed to induce 2-54 synthetase activity while receiving IFN alone but did show a substantial increase in activity of this mediator with the combination of IFN and : mismatched dsRNA.
Induction of 2-5A synthetase was accompanied by complete hematologic remission which has lasted for several months, after a transient increase in blood cell number caused by interrupting prior chemotherapy.
The activity of RNase L was also Co measured and found to be 10 to 100-fold higher than _10-
normal (Figure 3A, lane 1) yielding RNA cleavage products in vitro which represented further degradation beyond the usual rRNA cleavage products of normal RNase
L activity returned to normal levels and cleavage ot : 5 profiles after 30 days of therapy with the IFN/d SRA combination (Figure 3A, lane 2). I note here that Bh this kind of response was observed regularly.
EXAMPLE 2 ~ Viral Infection of T4 Cells
Another dsRNA-deficiency state is one where the = animal virus itself (or its replicative intermediates) supply an inhibiting dsRNA or a partly bioactive dsRNA for mediator activations, even though IFN and its mediators are induced by virus infeectioms. Many viruses associated with chronic symptomatology in the host will fall in this category. Infection of T# cells by HIV : can also constitute such a situation as suggested by
Figure 1, HIV replication in T4 cells was effectively blocked by mismatched dsRNA in tissue culture (see my published Buropean patent application No. 0,213,921) despite the fact that HIV meplication in these same oo ~41-
cells was only moderately suppresed by IFN alpha, - beta or gamma, alone or in physiological mixtures. | ) “ince HIV RNA itself displays considerable secondary structure which does not block its own expression, different dsRNAs probably elicit different biological responses. One region of extensive dsRNA structure at the 5' end of HIV mRNA binds an Mr 15000 polypeptide suspected to be the HIV transacting (tat) protein : (Muesing et al., Cell, vol. 48, p. 691, 1987) and I suggest that mismatched dsRNA may compete with HIV RNA for tat protein. The property of supporting efficient .
HIV infection, as well as other chronic viremias, may ’ he a second dsRUA deficiency state in ARC and AIDS.
BXAMFLE %2 - HIV infected Cells
A third kind of dsRNA deficiency state is observed in blood lymphocytes of individuals with chronic viral . infections such as HIV in ARC and AIDS. Preble et al. (J. Infect. Dis., vol. 152, 1985) reported that these cells possess elevated levels of dsRNA-dependent 2-5A synthetase, a finding I confirmed. This result was typical of a series of consecutive ARC and AIDS patients which I studied and helped form the basis for my invention in respect of the central role of dskNA in : host recovery and the necessity to replace dsRNA deficiency conditions by an appropriate source of exogenous dsRNA.
I reported RNase L to be often depleted in ARC and AIDS patients (June 6, 1987, The Lancet); however,
I reported that RNase L can return to more normal activity after several weeks of therapy with mismatched dsRNA in certain cases. As Figure 3B, lane 4 suggests,
ARC lymphocyte extracts can inhibit the activity of normal ENase I under certain conditions. Since tri- choloacetic acid (TCA) and ethanol-soluble extracts lacked inhibiting activity, the transferable inhibitor observed in ARC patient's MNC cannot be that described by Williams et al in herpes-infected cells (Virology, vol. 151, p. 23%, 1986). Also, I consistently saw or no inhibitor in MNC extracts from healthy individuals (Lane 5) studied to date.
Since mismatched dsRNA acts directly on these cells, they must also be dskNA-deficient. Thus, I have shown that different dsRNAs have different bio- activities (the HIV dsRNA being inhibitory whereas nismatched dsRNA is not). It appears that lymphocytes from ARC and AIDS patients ere dsRii-deficient in the . sense that only some of the neccessary dsRNA-dependent functions are carried out. One function at least, the maintenance of sufficient levels of bioactive RRase L, is not carried out unless exogenous dshlif is added.
EXAMPLE 4 - Chronic Fatigue Syndrome
An example of dsRNA deficiency requiring appropriate replacement therapy is Chronic Fatigue Syndrome (CFS), a condition involving some 10 to 12 million Americans, a difficult to diagnose, ubiquitous disorder characterized by extreme fatigue, lymph gland enlargement and cons- titutional symptoms ‘such as weight loss, loss of appetite and the like. <The condition occurs primarily in young, active people. ‘hile some CF3 patients manifest neuro- psychiatric changes such as depression, loss of memory and similar derangements, chronic fatigue syndrome is sometimes difficult to distinguish from entirely lye , : | Ap ORIGINAL d neurological disorders, particularly depression. CL.
Various laboratory studies indicate that many different viruses may replicate in individuals having Chronic
Fatigue Syndrome, and that these individuals become, in effect, "virus sewers". Viruses such as Epstein-
Barr, cytomegalovirus, retroviruses, herpes viruses, etc., are often present in such individuals.
The in vivo concentration of 2'-5'A molecules, a biochemically related, accurate measurement of intra- cellular dsRNA levels, in normal individuals and subjects with Chronic Fatigue Syndrome, is assessed as follows: Ethanol-soluble fractions of patient gamples (Ficoll-Hypaque-purified peripheral blood lymphocytes) were analyzed for their 2'-5'A content jn 2'-5'A core-cellulose assays (affinity chromato- graphy (with poly u-13°p} -pcp. In this assay, the ability of 2'-5'A-activated RNase L to hydrolyze poly(U) is used to determine the concentration of functional 2'-5'A.
Reference values were established by testing 15 normal subjects with no recent history of viral infections as evidenced by lack of fever, absence of constitutional symptoms, rashes, etc. Concentrations of their lymphocyte 2'-5'A levels were determined using calibration curves obtained with authentic 2'-5'A molecules. Normal individual reference values, expresses as ndnamoles of 2'-5'A within a range of 0.3 . to 1.1 nanamole per gram of PBMC cells.
Using this assay method, ten patients exhibiting the usual symptoms of Chronic Fatigue Syndrome were tested and the results obtained were as follows: ’ . TABLE 2
Subject n moles 2'-5'A
Number per gram lymphocyte protein 1 <0.08 2 <0.05 3 <0.05 4 | ~~ <0.05 5 nd* ==
6 "nd* 7 <0.01 8 < 0.01 9 <0.01 <0.08 *not detectable
Patients with Chronic Fatigue Syndrome have generally below 0.1 and always below about 0.2 n moles of 2'-5'A per gram of lymphocyte protein. 10 Definitive treatment of such individuals with Chronic
Fatigue Syndrome is provided by supplying exogenous dsRNAs, as required, until the intracellular level of 2'-5'A oligonucleotides reaches normal indicating the return of the intracellular dsRNA level to normalcy CT and/or the patient's clinical symptomology sbates.
The patient's resistance to Chronic Fatigue Syndrome and opportunistic viruses is maintained by continuing to measure the patient's intracellular 2'=-5'A oligo- nucleotide levels and supplying exogenous dsRNA, as required, to maintain the 2'-5'A level in the normal range, usually in exéess of 0:2 nanamoles of 2'-5'A , ur .
per gram of lymphocyte protein. he natural dsRNAs play a role in host defense when challenged with a viral disease such as Chronic
Fatigue Syndrome. Specific reduction in bioactive dsRNA, or enzymes which depend on dsRNA, notably a viral-associated inhibitor of RNase LI coupled with Ce abnormally low levels of 2'-5'A in peripheral blood: lymphocytes, within specific cells contributes to disease progression. dsRNA, notably mismatched dsRNAs, : 10 reverse disease progression.
Patients having Chronic Fatigue Syndrome are teeated with intravenous infusions of 200 to 600 mg (depending upon disease severity and viral burden, etc.) of rI.r(Cyq_ 14,0) twice weekly and 1-'S'A levels increase in association with clinical improvement.
The amount of dsRNA administered and the frequency of administration will be guided by the 2'-5'A levels measured in conjunction with the patient's clinical improvement. Amounts of dsRNA administered will provide a level of from 0.01 to 1,000 micrograms of dsRNA per milliliter of the patient's systemic blood circulation immediately following administration measured at a point distal from the point of infusion.
EXAMPLE 5 - Antiviral Effect on T4 Cell Populations dsRNA, particularly mismatched dsRNAs, exogenously administered restore a viral-infected patient's ability to respond to viral challenge. As an illustration of a viral condition, HIV was selected as one of the most damaging. The primary target of an HIV infection are the T4 lymphocytes and their population is dramatically reduced as the viral infection progresses. Further, ’
T4 cell population decline is an unwelcomed, quanti- fiable disease parameter whose decline reflects a worsening of the disease. Conversely, an increase in the T4 cell population is a favorable diagnostic indi- cation showing an improvement in therapeutic interven- tion. the normel T4 cell population for a healthy individual is 400 cells per cubic millimeter of blood.
Thirty-nine patients (see Figure 4) diagnosed as being in the pre-ARC or ARC’ category were measured and divided into three categories based upon the absolute number of T4 cells per mm? of blood: (a) greater than 300 (18 patients, data not shown); (b) 150 to 200 (13 patients); and (c) less than 150 (8 patients, mean = 92). The patients in groups (b) and (c) were treated with 100-200 mg rI .(Cy5,V) (Aopligen ® twice weekly by IV infusion for 3 to 16 months (mean = 8.4 months). For purposes of comparison, T4 cell populs- tion data from untreated HIV-infected patients (181) are included (connected deltas) to show the otherwise “ inevitable decline in the T4 population in such - individuals. ’
The baseline is the initial absolute T4 lympho- cyte concentration before initiating therapy.
Improvement is expressed in terms of percent change, positive or negative, from this baseline as shown - - graphicallly in Figure 4. Patients were initially observed and T4 cell concentration measured. The (bv) group of patients, shown in Figure 4 by which the open circle connected line, had T4 cell concentrations in the range of 150 to 300 cells/mm’. These patients received initially 100 mg Ampligen (IV twice weekly) , | co RE as a precautionary measure and observed for adverse reactions, then the dose was increased to 200 mg. given at the same frequency. A second group of patients : (¢) whose T4 cell coneentration was less than 150 . 5 cells/mm’ were given 300 mg Ampligen (IV, twice weekly).
The percent change from baseline is shown in the line connecting the solid circles. Untreated patients (181), with absolute T4 lymphocyte continued with a sharp decline as shown in the line connecting the deltas.
Mean T4 cells increased 4.3% after 4 months in 30 patients, 8.0% after 8 months in 10 patients and , 17% after 12 months in 6 patients. Only 2 patients oo (mean baseline T4 = 82 cells) advanced to AIDS while receiving continuous rl .(C;,,U) therapy and another
ARC patient who discontinued therapy for 2 months developed Kaposi's sarcoma.
These data demonstrate that infected individuals whose absolute T4 lymphocyte levels are less than 150 cells/mm require significant quantities of the drug 2C to Siow the rate of T4 cell reduction; however, patients in the 150-300 range are effectively maintained at lower dosage levels. These data also underscore the importance of initiating therapy before drastic decline in the HIV patient's condition.
These data further demonstrate the direct rela- tionship between the magnitude of the dsRNA deficit : (assessed indirectly bw accurately by absolute T4 cell populations) and the quantity of exogenous dsRNA required to correct the deficiency and restore the patient towards full health (as a restoration of the
T4 cell population to values approaching that in less severely infected and uninfected individuals) -- the ’ larger the T4 deficit, the greater the quantity of dsRNA required to restore the deficit. ) -
EXAMPLE 6 - dsRNA Replacement Therapy in Paramyxovirus
Infection
Another example of a transient dsRNA deficiency requiring appropriate replacement therapy may be various phases in neonatal life and infant development wherein the body's immune system is not fully developed.
In these instances, potentially life-threatening infections may emerge from what would otherwise be self-limiting infections of adolescence or adulthood.
A specific example follows: members of the paramyxo- virus family such as respiratory syncytial virus (RSV) can cause acute bronchiolitis in infants (typically 6 to 18 months of age) with *heir yet-to- be fully developed immune intriasic antiviral defense ’ systems. In such cases, I show below that replacement therapy with dsRNA [FI .x(Cy5,U),7 can be crucial and abort what otherwise may be a lethal event (death).
To develop this novel insight, I grew a human ’ amnion-derived cell line designated CCl 25 under standard laboratory conditions. CCL 25 cells (obtained from the American Type Culture Collection, Rockville,
Maryland, USA) support the growth and replication of
RSV in a manner analogous to human neonatal bronchiole tissues. The attached table shows conclusively that
I was able to reduce the multiplication of RSV by : approximately 99% using doses of dsRNA which I have independently shown are well-tolerated in humans. :
. 0 ! o od . . Ka . L 0
Q . : ~ 0 8 0 2 LR | g
NN ot 0 NN NJ ~~ al
Po i =
Cl — Oo © 0
Q © o | od ol AN P 0 0 o «©
A —~ rf or . 0 —- +» [ joe] © Lo Ry oO od °F 2 : - N 0 an #4 PON < a 60 a "1 H 0 oN oO bh NN tn 9 = a ~~ NN lo) + oJ o | dq g xR aN : a $9 m 0 “ ! Qo | in 0 je! - 0 ~~ [oN oO = ON or , ~~ ul + o bh [LE o ~~ NY Oo Oo s)
H Oo oO 1 A o A mI = . 4 + i H — o ® . ce re] $ o o jo) : v 3 eo 5 | 2 o ra) © Qa 0 PON + * —~ A ~~ Wl. I % go 0 bh ve) Ny © . 0 RN - Nn . o . o = a 2 3 H ° [We | Q se . hol . nq « = . o
Ee . } o Rg
J 5 i ! Q “ g oJ s o
Lo) Oo HA © -® —] © a ° = bo d 3 5 oe § gS «<« # > : ~ H PP nN © os 0 0 = ! 4 125] . a ON os
P a > od : » . © < s
Methods. RNase I, purified from extracts (100 pg protein) of FBMC from a healthy individual or from L929 cells, were incubated under core-cellulose assay condi- tions in the presence of poly (U)/ >2P7pCp (11000 dpm, 0.10 pCi/mmole) or poly(C)/ 2°P7pCp (12400 spm, 0.33 pCi/nmole) after the addition of authentic 2-5A (PAs, 5 x 1077 1) or 2-5A isolated from an extract (12.5 pg protein) of PBMC from patient #5 (at 9 weeks of mismatched dsRNA therapy). The poly(U)/ >2E7pCp and poly(C)/ 22p7pCp were synthesized from poly(U) or poly(C) (Sigma) and / "3270p (Pmersham-) by T4 ligase : (Bethesda Research laboratories). In control experi- 7 ments (in the absence of hs) 32300 dpm of poly(U) /~32p7pCp and 11000 dpm of poly(C)/ >2p7pCp were retained on glass fiber filters and are referred to as no (0%) : degradation.
Since it is established that infectious center formation induced by RSV involves direct cell to cell gpread of the virus, I have now established that simply by raising the intracellular concentration of dsRNA effectively I can reduce the cell to cell dissemination of RSV without significant untoward effects on other bodily tissues.
Thus, disease pathology caused by other potentially self-limited pathogens such as RSV (paramyxovirus family) as well as other RNA producing viruses which affect the nasgal/tracheal/bronchial tree, such as 7 rhinoviruses, or influenza viruses, may be effectively contained by a dsRNA replacement therapy mechanism. :
Local portals of pathogen entry, such as (but not limited to) the airways, may have intrimsically or transiently lower dsRNA content (dsRNA being herein , operationally defined as any of those molecules which trigger the relevent immune/entiviral defense cascades "to thwart pathogen reproduction/tissue-induced pathology).
EXAMPLE 7 - Treatment of Hepatitis
Numerous reviews of liver disease in patients with AIDS have appeared in the literature over the - last 2-3 years. The recurrent theme is the wide array of histologic findings in both needle directed , : se vo r,s va biopsy specimens and biopsy at time of autopsy. In all studies below mentioned the U. S. Center for ; Disease Control, Atlanta, Georgia, USA, criteria for a diagnosis of AIDS was satisfied. Typical present- ing symptoms and signs of patients found to have extensive hepatic disease are entirely nonspecific and include weight loss, fever, lymphadenopathy, | : hepatosplenomegaly, and abdominal pain. Serum trans- sminasges typically are elevated in most AIDS patients. | There is a propensity for elevated alkaline phospha- tases to be disproportionately high; it has been : suggested that such elevations of alkaline phosphatase are correlated with a histologic diagnosis of granu-
A lomates disease. Autopsy studies done in unselected patients (i.e., unbiased patient selection as opposed to biopsy studies in patients with hepatomegaly and elevated liver function tests) universally demonstrate a wide array of intrahepatic pathology. Approximately 80-90% of all autopsy cases in AIDS-patients demons- trate abnormal liver anatomy. Hepatic derangements include vascular congestion, portal inflammation, steatosis, focal necrosis, granulomata, bile stasis, , 59. : Ce cirrhosis and Kupffer cell hyperplasia (in approximate order of frequency). Of interest, the intraphepatic diagnosis of Kapos Sarcoma at autopsy was the most common specific intrahepatic pathogen (18%) in one study (Schneiderman et al, Annals Internal Med., Vol. 105, pg. 207, 1987).
Biliary disease has recently been described in a small number of AIDS patients. Isclated case reports have described sclerosing cholangitis, intrahepatic destructive bile duct lesions, and benign extrahepatic biliary obstruction in patients with AIDS. No specific etiologic agent has been documented in these cases; cytomegalovirus has been implicated as a possible cause i secondary to histopathologic findings on biopsy. In
AIDS patients with benign extrahepatic biliary obstruc- tion, common presenting complaints have included liver, right upper quadrant pain and modest bilirubin eleva- tions. At autopsy or leparatomy a diagnosis of ampulai of vater structure secondary to fibrosis was made. In at least two cases of extrahepatic obstruc- tion, cryptosporidia were isolated from the biliary aE tree. Other cases were notable for cytomegalovirus inclusive bodies in the vicinity of the ampulae of vater. As with the above cases of intrahepatic cholangitis, specific etiology is unknown and the specific pathogens may have only a casual role in the pathogenesis of these disorders.
I determined that dsRNA infusion [FI.v(C 5,0), 200 mg, twice weekly, IV/ also corrects or significantly ameliorates the underlying cellular abnormality asso- ciated with hepatitis B virus (HVB) multiplication.
To accomplish this insight, the following method was used:
Evaluation of Sera or Plasma for HBV-Specific DNA:
The presence of serum (or plasma)-associated HBV DNA, an indicator of the presence of hepatitis B virus, was determined by slot-blot hybridization. Serum samples were diluted in NH, OAc to a final concentration of 1 M salt. Using a 72-wall microfiltration apparatus Ce (Schleicher & Schuell, Keene, N.H.), the samples were applied idirectly to a nitrocellulose filter that had been presosked for 10 minutes in 1 M NH, OAc. The DNA was denatured in situ with alkali and then neutralized. i
Dilutions of purified HBV DNA in normal human serum were assayed in an identical manner to quantitate each slot-blot hybridization. The filters were baked, prehybridized, hybridized, and washed under conditions : of high stringency, and autoradiographed using standard conditions (Maniatis, T., Fritsch, E.F. and Sambrook, ~J., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory, Cold Sprig Harbor, 1982).
The HBV probe DNA used in the hybridization was obtained from a recombinant plasmid containing a single, unit length copy of HBV DNA (HBsAG serotype adw2) cloned at the EcoRl site. The plasmid was digested with EcoRl and the HBV DNA was purified by 2 cycles of preparative agarose gel elecrophoresis and electroelution. The purified HBV DNA was labeled with 32p_gcTP to a specific activity of 1-2 x 107 cpm/ yg using the random-prime labeling technique (Feinberg, A. P. and Vogelstein, B.,
Analyt. Biochem. 132:6-13, 1983). The level of sensi- tivity obtained with this assay is 1 pg of HBV DNA.
At this level of sensitivity, I am detecting 2.9 x 10° molecules of HBV DNA.
The autoradiographs produced were evaluated for degree of relative intensity and numerically ranked according to observed intensity and are reported in the following Table. Days of therapy are given parenthetically. [
Ce E | -61-
. o . © 3 = . ED 0 rl 0 NAN NN NAN MY bd ~ ~~ ~~ InN OO © Oo Wu F OO do] ~ 4 oN OW A & NN x OO NN @© . gel OO «©» o- 0 A ~ NANNY ND wl ~~ vv A ’ 4
NY
Pp we ’ & A A & . * . . gm & A & & & & & ~~ — — -
Pp o
A Me CL 4y ~~ o o
Q
8 | z
A dd 85k $d Ba! ~ os o nm ~~ ~ ~~ OO OO
CHA gl ~ 4 NN vw fF 4 © :
Py ol oo oN nn 0 ~~ dH . fe] dl 'v vv © J 9 = rH 0 ° aque & A oo o ol + + «+ + . m Sl ld HA . 0 +m : aq 4 d er o
S « ©
TB 2 od : n Coe ce. % gq wu H : oO = ~ QO he g dO © | ° 0 o b> ~~ H HH o Bal jot NAN AN NN SIN ON + eH + Mm gs ~ 9 a on 9 Oo Bn 09 OO
[2] Bl ~ © OO HH OVO +4 nn OO Wn
H n u oO oN WOW A H oN a KK Nn FF .
FL] al ul Tv NT ~~’ ~ n + > br el ord o & =" q + uP o al] rer Ek + & + + + + + + + In wu MN KN a NN AH +A oO 85 8 oA 2 «< 9 Io Pp <4 28 d = oH fH
QO 1°] wn HP ~~ od Po
El © 4 mm|m H
Oo Q . w » . Q 13 } ‘ " bv 2 x
A) g Et ' =
Z| oe : ; . BH <A oN Mog Nn 0 ~~ © an O oo o ~ od r | 5 oo | ¥ cp ’
relative intensity of x-ray film exposed for five days to >°p containing viral DNA. Larger pluses (e.g. z* yg. 1*) refer to more intense and larger slot-blots , on x-ray film indicating presence of more viral DNA.
In order to increase sensitivity of viral measurements, various dilutions of patient sera were used, typically dilutions of 1:40, 1:200, 1:40 and 1:1,500. Confirma- tory results were established with densitometric tracings of slot blots. Methodology was based on reagents provided by Schleicher and Schnell (materials designated
No. 370 2/284). Complementary results were obtained with analysis of viral specific antigen (immunoassay for the detection of hepatitis B surface antigen in human serum or plasma) kit available from Abbott Labora- tories (designated technical exhibit 83-0804/R12 dated
July 1985)
As the results of my invention become known publicly and further developed and applied to additional conditions/infections/disorders, it may well become | possible to make a clinical diagnosis of dsRNA deficiency without relying on extensive laboratory date and perhaps even the scientific insight and novelty
I put forth in this document. Many less obvious dsRNA- Nn deficiency states can also exist. For example, since
IFN operates naturally as a feback inhibitor of proliferative pathways (Aullo et al, Cell, Vol. 43 pp. 79%, 1985), and since dsRNA-dependent mediators implement thede events, then cells undergoing differen- tiations or with tumorigenic/metastaic potential may be dsRNA-deficient.
From the examples cited above, it is apparent that by interrelating biologic function (e.g., cell response to exogenous IFN) or biochemical function (e.g., elevated 2-5A synthetase) or presence of 2'-5' oligo adenylate pathway intermediates, I can readily diagnose dsRNA deficiency states and quantitate the degree of derangement by various means including: (a) Determination of endogenous 2',5' oligoadeny- late concentrations. (bb) Quantitative analysis of 2-5A concentration and molecular size of 2-5A from patient samples via high pressure liquid chromatography.
(¢) Proof of biological functionality of the 2-5) synthesized in vivo in patients utili- zing ribosomal RNA cleavage assays. (4d) Binding assays with 2', 5'-pgh, | *7P1-3'-p0p to determine the level of free (unbound)
RNase IL in patients' samples. (e) Core-cellulose assays (affinity chromatography) with poly u-{>2p)}-pCp to characterize the specificity of activation of RNase L by 2-5A synthesized in patient samples. (f) Ribosomal RNA cleavage assays to determine the level of activate RNase L by 2-5A synthesized : in patient samples. (g) Ribosomal RNA cleavage assays to determine the level of activated RNase IL in patients samples.
Some of these methods are described in detail in my patent applications entitled "Elaboration of Host
Defense Mediators into Biological Fluid", Serial No. oo 20 028,823 filed August 12, 1987, and "Double Stranded
RNA Correction of Aberrant Metabolic Pathways",
‘Serial No. 074,649 filed July 17, 1987, which are herewith incorporated by reference.
DsRNA sufficiency states can also exit. For ’ : example, CML and hairy cell leukemia (HCL) are often responsive to IFN given as sole therapy and reflect intracellular situations of natural dsRNA sufficiency.
To support this notion, I found similar levels of 2-5A synthetase-activating dsRNA in nuclei of mononuclear blood cells form untreated patients with HCL as in = nuclei of IFN-treated Hela cells.
Mismatched dsRNA. While many dsRNAs can induce various lymphokines, including IFN, and activate 2-54 synthetase, not all dsRNAs have these properties. Of : synthetic dsRNAs, poly(I):poly(C) is an excellent inducer of IFN and activator of 2-5A synthetase but is also very toxic. Poly(I):poly(Cy,5,U), also referred to as "mismatched dsRNA" or Ampligen ® (HEM Research,
Inc., Rockville, Maryland, USA), contains periodic uracil residues in the polypyrimidine strand. Mis- matching induces rapid biodegradation without destroying oe -66-
biological function. For example, poly(I):poly(Cy,,U) i exhibits antiviral, antitumor and immune-enhancing activity, and is nontoxic.
DsRNA As A Replacement Therapy in Broad Spectrum
Viral and Cancer Therapy. Mismatched dsRNA may be particularly well-suited for chronic viral infections including ARC treatment because it is both 8 broad- spectrum antiviral snd an immune enhancer; even at high doses it is essentially without long term toxicity.
My data on 45 consecutive patients treated intravenously with 200-250 mg of poly(I):poly(Cy 5, U) twice weekly Se showed rapid decline in virus concentration (HIV, CMV and herpes) and durable enhancement of both T and B cell immunity accompanied by improved performance with no significant side effects. In many patients, the clinical responses continue over approximately 18 months, or as long as they continued dsRNA therapy (100-400 mg twice weekly). In some cases larger doses, more frequently applied, nay be necessary. Viral infections and immune lesions in AIDS patients may differ from one individual to another and no single agent can be expected to reverse all the potential clinical problems. However, dsRNA may play a pivotal role in various treatment regimens. For example, since mismatched dsRNA displayed synergy with AZT in blocking
HIV infection of T4 cells (see my co-pending application serial no. 028,823), mismatched dsRNA and very low dose
AZT may be an important combination in early phases of infection including ARC. Both dsRNA and IL-2 enhance
T cell, NK cell and LAK cell activity and thus far, where combined, I have shown that the two biologicals have displayed therapeutic synergism without added toxicity. Further, I anticipate that, insofar as IFNs enhance differentiation of B cells and monocytes, certain lymphokines may increase the ability of dsRNA to promote specific neutralizing antibodies against
HIV and increase monocyte killing activity. Similarly, since retinoids increase the number of T4 cells and promote differentiation of both T and B cells with IFN, the combination of dsRNA and retinoids may provide additional benefits in ARC and AIDS. The combinations of dsRNA and thymic peptides may also have T4 cell : restorative effects, especially in patients with very 7 low T4 cell numbers. Finally, the fact that dsRNA exhibits direct antiproliferative activity against
Kaposi's sarcoma cells which can be augmented by adding either IFN alpha or IL-2, suggests a new anti- tumor regimen in advanced disease.
Control of blood cell parameters in AIDS will reveal increased CNS problems.
To that end, mismatched dsRNA crosses the murine blood brain barrier in sufficient quantities to activate oligo 2'S' adenylate synthetase in brain parenchymal cells; thus, combinations of dsRNA and other agents including lymphokines which cross the 10 . blood-brain barrier will be useful in viral-related dementias.
Lymphokines will be understood to include the interferons, preferably interferon alpha, the ’ interleukins, specifically interleukin-2 (IL-2) and recombinant interleukin-2 (rIL-2), and tumor necrosis factor (TNF). Also included are lymphokine activated : killer (LAK) cells formed in animals in response to exposure to a lymphokine. : When interferon (alpha) is used as the lymphokine, an amount of from 0.01 to 100,000 IRU per milliliter of the patient's body fluid is provided.
When IL-2, . preferably rIL-2, is the lymphokine, the amount administered lies wi thin a range of about 10° IL-2 ~69- oo units per kg of the patient's body weight up to a value approaching unacceptable levels of toxicity in the patient, which may be as high as 108 IL-2 units. How- ever, most effective, toxic-reaction manageable values are in the range of from about 10° to about 10" IL-2 per kg of body weight.
The usual amounts of dsRNA administered provide a level of from 0.1 to 1,000 micrograms dsRNA per milliliter of the patient's body fluid. The term body fluid is intended to refer to that solution of serum, salts, vitamins, etc., which circulates within the organism and bathes the tissues. When both agents (a dsRNA and a lymphokine) are administered they may be administered as a mixture, administered separately but simultaneously, or sequentially.
Administration of a dsRNA and a lymphokine "in a. combination" includes presentations in which both agents are administered together as a therapeutic mix~ ture, and also procedures in which the two agents are administered separately but simultaneously.
FIGURE 1 is a flow chart illustrating a typical. - ~ dsRNA deficiency which leads to host morbidity and dsRNA sufficiency which leads to host recovery. Bioactive dsRNA is produced intracellularly or introduced by cer- tain viruses (e.g., EMC) and triggers a series of enzymatic events, including lymphokine production and mediator activation, which lead to an antiviral state as well as immune cell differentiation and host recovery.
DsRNA introduced by various virusex (e.g., HIV) or tumor 7 cells can subvert this pathway by acting as an inhibitor.
Physiological events can limit dsRNA production or cause . production of aberrant dsRNA. Such abnormalities lead to chronic infections and hose morbidity. In HIV and other chronic viral infections, immune dysfunction can also result from an inhibitor of RNase I (shown as an alternate pathway) which is overcome by a supply of appropriately configured exogenous dsRNA. :
FIGURE 2A and 2B illustrate induction of 2'S5' . oligoadenylate synthetase by mismatched dsRNA in MNC or a CMI patient. A CML patient was treated with IFN alone for seven days (Fig. 2A), then with mismatched oo 71-
dsRNA and IFN (Fig. 2B). At times before and during treatment, synthetase activity was measured in Ficoll- purified MNC.
FIGURE 3A is a photograph of polyacrylamide gel electrophoresis plates showing the indicate number of a tracks and bands or zones along each track. Elevated activity of RNase L associated with novel cleavage products in MNC or a CML patient are illustrated on this plate. Hungarian L929 cells which supply rRNA but no RNase IL, are deposited under the terms of the
Budapest Convention at the American Type Culture
Collection as ATCC No. CRL 9659; 1929 cells as deposited as CCL 1 and MNC from the CML patient were prepared according to Kariko and Ludwig and Silverman et al, then
RNase L from the MNC was measured as described in my . co-pending application Serial No. 074,649 filed July 17, 1987, the disclosure of which is hereby incorporated by reference.
FIGURE 3B is also a photograph of a polyacrylamide gel electrophoresis plates showing the presence of an
RNase L inhibitory factor in PBMC extracts of an ARC patient as determined in the rRNA cleavage assay.
Extracts of 1929 cells (which supply both RNase L and rRNA) and MNC were prepared according to Kariko and ’ Ludwig and Silverman et al. respectively.
Lane 1: 18 nl 1929 cell extract (150 pg total protein). . was incubated in the presence of 4 pl NP40 buffer used for MNC lysis plus 5.5 ul water.
Lane 2: identical to Lane 1, except that in lieu of water, 5.5 pl of 5 x 1078 M authentic P3A3% was added to incubations.
Lane %: identical to Lane 1 except that 5.5 pl of TCA- soluble extract from MNC extract (100 pg total protein) from an ARC patient was added prior to incubation.
Lane #4: 18 pl L929 cell extract was incubated with’ 4 pl MNC extract from an ARC patient (100 ve total protein) and 5.5 pl water.
Lafle 5: 18 pl 1929 cell extract was incubated with 4 pl MNC extract from a healthy individual (100 pg -7 Fm
\ total protein) and 5.5 pl water. 28S and 18S rRNA as well as normal levels of 2'5'A activated RNase L : specific cleavage products are indicated.
Claims (22)
1. A method of diagnosing a dsRNA deficiency state in a patient comprising the steps of:
a. obtaining a sample of the patient's peripheral blood mononuclear cells (PBMC); 7 b. preparing an extract of same and determining the quantity of 2'-5' oligoadenylate (2-5 A) synthetase in the PBMC;
c. comparing the amount of 2-5 A synthetase determined in step b. to the amount of same in a normal ) : individual and assessing the difference, if any, and, if deficient;
d. administering by infusion an amount of exoge- nous dsRNA sufficient to replace the deficient dsRNA and return the patient's dsRNA level to a normal amount.
2. A method of claim 1 wherein the dsRNA deficiency state is due to an immune disorder, a viral infection, or uncontrolled cancer cell proliferation. 3, A method of claim 2 wherein the causative virus is a retrovirus, a paramyxovirus, rhinovirus, or a
N . member of the herpes virus family.
4, The method of claim 1 wherein the dsRNA deficiency state is assessed in PBMC whether or not these cells are in the process of multiplication or differentiation.
5. A method of claim 1 of disgnosing dsRNA : deficiencies consisting of first infusing an amount of synthetic dsRNA sufficient to simulate the expected concentration of natural dsRNA in a healthy person. and, thereafter, determining whether any positive changes are observed in any dsRNA mediated pathways or parameter of clinical status as a result of said infusion.
6. A method of claim 1 wherein the dsRNA replace- : ment therapy is carried out with a matched dsRNA.
" 7. A method of claim 1 where the dsRNA replacement therapy is carried out with a mismatched dsRNA.
8. The method of claim 7 in which the mismatched ‘dsRNA is a complex of polyinosinate and a polycytidylate containing from 1 4n 5 to; 1 in 30 uracil or guanadine bases. .
:
9. The method of claim 7 in which the mismatched . I dsRNA is r n.x(Cyq _y4)p- :
10. The method of claim 7 in which the dsRNA contains regions of bond breakage and exhibits the . . I favorable therapeutic ratio property of r nT 1470p"
11. The method of claim 7 in which the mismatched dsRNA is administered in an amount which will result ’ in a level of from 1 to 1,000 micrograms of dsRNA per milliliter of the person's primary biological fluid and proportional amounts of dsRNA-induced mediator substances in the person's secondary or compartmenta- lized fluids.
12. A method of ameliorating tissue pathology in a person caused by a deficiency of dsRNA coexisting oo with the presence of an intracellular pathogen : comprising the steps of:
a. obtaining a sample of the patient's peripheral blood mononuclear cells (PBMC); -
~ b. preparing an extract of same and determining the quantity of 2'-5' oligoadenylate (2-5 A) synthetase in the PBMC; 0
¢. comparing the amount of 2-5 A synthetase determined in step b. to the amount of same in a normal individual and assessing the difference, if any, and, if deficient;
d. supplying exogenous dsRNA to the person's affected tissue and restoring the intracellular dsRNA level.
13. The method of claim 12 in which the intra- cellular pathogen is a virus or uncontrolled prolifera- tion of cancer cells. ,
14. The method of claim 13, in which the viral pathogen is a retrovirus, a paramyxovirus, & rhinovirus or a herpes family virus.
15. The method of claim 12 in which the tissue’ pathology is in a component of all the immune system or whether or not the component cell is in the process of multiplication or differentiation.
16. The method of claim 1 in which a lymphokine is administered in conjunction with the exogenous dsRNA.
17. A method of claim 1 wherein the dsRNA replace- ment therapy is carried out with a matched dsRNA.
18. A method of claim 1 where the dsRNA replace- ] ment therapy is carried out with a mismatched dsRNA.
19. The method of claim 7 in which the mismatched dsRNA is a complex of polyinosinate and a polycytidylate containing from 1 in 5 to; 1 in 30 uracil or guanadine bases.
20. The method of claim 7 in which the mismatched dsRNA is rl n.r(Cyy_34)p
21. The method of claim 7 in which the dsRNA - N contains regions of bond breakage and exhibits the favorable therapeutic ratio property rI .v(Cy1_14)p°
22. The method of claim 7 in which the mismatched dsRNA is administered in an amount which will result in a level of from 1 to 1,000 micrograms of dsRNA per milliliter of the person's primary biological fluid. WILLIAM A. CARTER Inventor v | .
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US9352387A | 1987-09-04 | 1987-09-04 |
Publications (1)
Publication Number | Publication Date |
---|---|
PH26320A true PH26320A (en) | 1992-04-29 |
Family
ID=22239406
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PH37488A PH26320A (en) | 1987-09-04 | 1988-09-02 | Diagnosis and treatment of double-stranded RNA deficiency states |
Country Status (15)
Country | Link |
---|---|
JP (1) | JPH0825884B2 (en) |
KR (1) | KR890005277A (en) |
CN (1) | CN1031651A (en) |
AU (3) | AU2186488A (en) |
CA (1) | CA1336683C (en) |
DK (1) | DK491088A (en) |
FI (1) | FI884069A (en) |
IL (1) | IL87664A (en) |
NO (1) | NO883868L (en) |
NZ (1) | NZ226033A (en) |
OA (1) | OA08911A (en) |
PH (1) | PH26320A (en) |
PT (1) | PT88415B (en) |
RU (1) | RU2001917C1 (en) |
ZA (1) | ZA886581B (en) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1088344C (en) * | 1997-10-17 | 2002-07-31 | 青岛市卫生防疫站 | Process for preparing selenium or zinc enriched earthworm dry powder or extract |
US20060035859A1 (en) * | 2003-05-16 | 2006-02-16 | Hemispherx Biopharma | Treating severe and acute viral infections |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4024222A (en) * | 1973-10-30 | 1977-05-17 | The Johns Hopkins University | Nucleic acid complexes |
JPS57200310A (en) * | 1981-05-26 | 1982-12-08 | Merck & Co Inc | Local application of interferon inducer |
AU1820388A (en) * | 1987-07-17 | 1989-01-19 | Hem Research, Inc. | Double stranded rna correction of aberrant metabolic pathways associated with uncontrolled tumor cell and virus growth cycles |
-
1988
- 1988-08-30 NO NO88883868A patent/NO883868L/en unknown
- 1988-09-01 CA CA000576351A patent/CA1336683C/en not_active Expired - Fee Related
- 1988-09-02 RU SU884356574A patent/RU2001917C1/en not_active IP Right Cessation
- 1988-09-02 PH PH37488A patent/PH26320A/en unknown
- 1988-09-02 NZ NZ226033A patent/NZ226033A/en unknown
- 1988-09-02 FI FI884069A patent/FI884069A/en not_active IP Right Cessation
- 1988-09-02 DK DK491088A patent/DK491088A/en not_active Application Discontinuation
- 1988-09-02 PT PT88415A patent/PT88415B/en not_active IP Right Cessation
- 1988-09-02 IL IL8766488A patent/IL87664A/en not_active IP Right Cessation
- 1988-09-02 AU AU21864/88A patent/AU2186488A/en not_active Abandoned
- 1988-09-03 CN CN88106411A patent/CN1031651A/en not_active Withdrawn
- 1988-09-03 JP JP63219654A patent/JPH0825884B2/en not_active Expired - Fee Related
- 1988-09-03 KR KR1019880011423A patent/KR890005277A/en not_active Application Discontinuation
- 1988-09-05 ZA ZA886581A patent/ZA886581B/en unknown
- 1988-09-05 OA OA59419A patent/OA08911A/en unknown
-
1992
- 1992-06-03 AU AU17366/92A patent/AU1736692A/en not_active Abandoned
-
1995
- 1995-01-04 AU AU10014/95A patent/AU1001495A/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
JPH01131118A (en) | 1989-05-24 |
IL87664A (en) | 1995-03-15 |
CA1336683C (en) | 1995-08-15 |
KR890005277A (en) | 1989-05-13 |
DK491088A (en) | 1989-03-05 |
PT88415A (en) | 1989-07-31 |
AU1736692A (en) | 1992-07-30 |
DK491088D0 (en) | 1988-09-02 |
NO883868D0 (en) | 1988-08-30 |
FI884069A0 (en) | 1988-09-02 |
OA08911A (en) | 1989-10-31 |
AU1001495A (en) | 1995-03-30 |
IL87664A0 (en) | 1989-02-28 |
JPH0825884B2 (en) | 1996-03-13 |
PT88415B (en) | 1992-10-30 |
NZ226033A (en) | 1999-08-30 |
AU2186488A (en) | 1989-03-09 |
RU2001917C1 (en) | 1993-10-30 |
NO883868L (en) | 1989-03-06 |
ZA886581B (en) | 1989-04-26 |
FI884069A (en) | 1989-03-05 |
CN1031651A (en) | 1989-03-15 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP0306347B1 (en) | Diagnosis of double-stranded RNA deficiency states | |
US5593973A (en) | Treatment of viral hepatitis with mismatched dsRNA | |
Fazely et al. | Pentoxifylline (Trental) decreases the replication of the human immunodeficiency virus type 1 in human peripheral blood mononuclear cells and in cultured T cells [see comments] | |
US20040138167A1 (en) | Method of treating HIV infection and related secondary infections thereof | |
JP3208138B2 (en) | Diagnosis and treatment of viral hepatitis | |
EP0347501A1 (en) | Modulation of lymphokine-resistant cellular states by dsRNAs | |
AU660973B2 (en) | RNase L inhibitor as a marker for virus infections | |
AU754242B2 (en) | Method of treating HIV infection and related secondary infections thereof | |
PH26320A (en) | Diagnosis and treatment of double-stranded RNA deficiency states | |
AU692433B2 (en) | Method for using polynucleotides, oligonucleotides and derivatives thereof to treat various disease states | |
RU2049336C1 (en) | Method for diagnosing hiv | |
AU724056B2 (en) | Diagnosis and treatment of double stranded deficiency states | |
US5194245A (en) | Diagnosis of viral hepatitis | |
WO2021198535A1 (en) | Use of an arsenic compound for treating a short or long cytokine storm in various autoimmune/inflammatory diseases in humans or animals | |
Bahr et al. | Selective regulation of human immunodeficiency virus-infected CD4+ lymphocytes by a synthetic immunomodulator leads to potent virus suppression in vitro and in hu-PBL-SCID mice | |
RU2021810C1 (en) | Method of action on disease development caused by hiv-virus or virus causing similar biochemical or clinical picture | |
Sarin et al. | Treatment of AIDS with drugs targeted to inhibit different stages of the HIV life cycle | |
El-Reshaid et al. | Rituximab Treatment for Recurrent Skin Vasculitis Due to Cryoglobulinemia Associated with Hepatitis C Despite Viral Clearance with Sofosbuvir | |
IE922145A1 (en) | Modulation and diagnosis of cytokine dysfunctions | |
Carnicer et al. | Tratamiento con PEG interferón alfa 2b y ribavirina en enfermos previamente tratados con interferón estándar en monoterapia o combinado con ribavirina |