WO2009045104A1 - Method for diagnosing presymptomatic organ transplant rejection - Google Patents
Method for diagnosing presymptomatic organ transplant rejection Download PDFInfo
- Publication number
- WO2009045104A1 WO2009045104A1 PCT/NL2008/050632 NL2008050632W WO2009045104A1 WO 2009045104 A1 WO2009045104 A1 WO 2009045104A1 NL 2008050632 W NL2008050632 W NL 2008050632W WO 2009045104 A1 WO2009045104 A1 WO 2009045104A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- granzyme
- rejection
- subject
- expression level
- organ
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 54
- 210000000056 organ Anatomy 0.000 title claims abstract description 38
- 206010052779 Transplant rejections Diseases 0.000 title claims abstract description 23
- 108060005986 Granzyme Proteins 0.000 claims abstract description 125
- 102000001398 Granzyme Human genes 0.000 claims abstract description 124
- 230000014509 gene expression Effects 0.000 claims abstract description 50
- 239000002773 nucleotide Substances 0.000 claims description 19
- 125000003729 nucleotide group Chemical group 0.000 claims description 19
- 210000002700 urine Anatomy 0.000 claims description 17
- 238000003556 assay Methods 0.000 claims description 11
- 239000012530 fluid Substances 0.000 claims description 6
- 210000004369 blood Anatomy 0.000 claims description 2
- 239000008280 blood Substances 0.000 claims description 2
- 210000001185 bone marrow Anatomy 0.000 claims description 2
- 210000002216 heart Anatomy 0.000 claims description 2
- 210000000936 intestine Anatomy 0.000 claims description 2
- 210000003734 kidney Anatomy 0.000 claims description 2
- 238000012125 lateral flow test Methods 0.000 claims description 2
- 210000004185 liver Anatomy 0.000 claims description 2
- 210000004072 lung Anatomy 0.000 claims description 2
- 210000000496 pancreas Anatomy 0.000 claims description 2
- 210000003296 saliva Anatomy 0.000 claims description 2
- 210000000582 semen Anatomy 0.000 claims description 2
- 210000003491 skin Anatomy 0.000 claims description 2
- 210000000278 spinal cord Anatomy 0.000 claims description 2
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 62
- 230000001154 acute effect Effects 0.000 description 36
- 229940109239 creatinine Drugs 0.000 description 31
- 108090000765 processed proteins & peptides Proteins 0.000 description 26
- 210000002966 serum Anatomy 0.000 description 26
- 150000001413 amino acids Chemical group 0.000 description 23
- 102000004196 processed proteins & peptides Human genes 0.000 description 23
- 108020004999 messenger RNA Proteins 0.000 description 22
- 229920001184 polypeptide Polymers 0.000 description 21
- 208000003918 Acute Kidney Tubular Necrosis Diseases 0.000 description 17
- 206010038540 Renal tubular necrosis Diseases 0.000 description 17
- 235000001014 amino acid Nutrition 0.000 description 17
- 238000001514 detection method Methods 0.000 description 17
- 229940024606 amino acid Drugs 0.000 description 15
- 230000006870 function Effects 0.000 description 15
- 238000003745 diagnosis Methods 0.000 description 14
- 238000001574 biopsy Methods 0.000 description 13
- 108090000623 proteins and genes Proteins 0.000 description 10
- 238000002054 transplantation Methods 0.000 description 10
- 230000000694 effects Effects 0.000 description 9
- 239000003550 marker Substances 0.000 description 9
- 108020004463 18S ribosomal RNA Proteins 0.000 description 8
- 108020004414 DNA Proteins 0.000 description 8
- 230000004064 dysfunction Effects 0.000 description 8
- 150000007523 nucleic acids Chemical class 0.000 description 8
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 7
- 230000027455 binding Effects 0.000 description 7
- 238000005259 measurement Methods 0.000 description 7
- 206010063209 Chronic allograft nephropathy Diseases 0.000 description 6
- 108020004635 Complementary DNA Proteins 0.000 description 6
- 201000001200 Crouzon syndrome-acanthosis nigricans syndrome Diseases 0.000 description 6
- 101000836075 Homo sapiens Serpin B9 Proteins 0.000 description 6
- 102100025517 Serpin B9 Human genes 0.000 description 6
- 238000010804 cDNA synthesis Methods 0.000 description 6
- 239000002299 complementary DNA Substances 0.000 description 6
- 230000004054 inflammatory process Effects 0.000 description 6
- 230000035945 sensitivity Effects 0.000 description 6
- 238000011282 treatment Methods 0.000 description 6
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 5
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 5
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 5
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 230000007774 longterm Effects 0.000 description 5
- 108020004707 nucleic acids Proteins 0.000 description 5
- 102000039446 nucleic acids Human genes 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 230000002485 urinary effect Effects 0.000 description 5
- 208000021709 Delayed Graft Function Diseases 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 4
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 4
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 4
- 238000012300 Sequence Analysis Methods 0.000 description 4
- 210000001744 T-lymphocyte Anatomy 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 210000003810 lymphokine-activated killer cell Anatomy 0.000 description 4
- 230000001404 mediated effect Effects 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 238000011002 quantification Methods 0.000 description 4
- FSYKKLYZXJSNPZ-UHFFFAOYSA-N sarcosine Chemical compound C[NH2+]CC([O-])=O FSYKKLYZXJSNPZ-UHFFFAOYSA-N 0.000 description 4
- 230000009870 specific binding Effects 0.000 description 4
- 238000006467 substitution reaction Methods 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- 102000004631 Calcineurin Human genes 0.000 description 3
- 108010042955 Calcineurin Proteins 0.000 description 3
- 208000030453 Drug-Related Side Effects and Adverse reaction Diseases 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 208000034706 Graft dysfunction Diseases 0.000 description 3
- 206010062016 Immunosuppression Diseases 0.000 description 3
- 206010061218 Inflammation Diseases 0.000 description 3
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 3
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 3
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 3
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 3
- 206010070863 Toxicity to various agents Diseases 0.000 description 3
- 208000027418 Wounds and injury Diseases 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 239000000090 biomarker Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 230000001900 immune effect Effects 0.000 description 3
- 230000001506 immunosuppresive effect Effects 0.000 description 3
- 208000014674 injury Diseases 0.000 description 3
- 230000003907 kidney function Effects 0.000 description 3
- 239000000816 peptidomimetic Substances 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 230000002441 reversible effect Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 description 2
- 102100022997 Acidic leucine-rich nuclear phosphoprotein 32 family member A Human genes 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 2
- PMATZTZNYRCHOR-CGLBZJNRSA-N Cyclosporin A Chemical compound CC[C@@H]1NC(=O)[C@H]([C@H](O)[C@H](C)C\C=C\C)N(C)C(=O)[C@H](C(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)N(C)C(=O)CN(C)C1=O PMATZTZNYRCHOR-CGLBZJNRSA-N 0.000 description 2
- 108010036949 Cyclosporine Proteins 0.000 description 2
- 206010011831 Cytomegalovirus infection Diseases 0.000 description 2
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 2
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 206010048748 Graft loss Diseases 0.000 description 2
- 101000757200 Homo sapiens Acidic leucine-rich nuclear phosphoprotein 32 family member A Proteins 0.000 description 2
- 101000836351 Homo sapiens Protein SET Proteins 0.000 description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 2
- 108010006519 Molecular Chaperones Proteins 0.000 description 2
- 102100027171 Protein SET Human genes 0.000 description 2
- 102000006382 Ribonucleases Human genes 0.000 description 2
- 108010083644 Ribonucleases Proteins 0.000 description 2
- 108010077895 Sarcosine Proteins 0.000 description 2
- 108010060059 Sarcosine Oxidase Proteins 0.000 description 2
- 102000008118 Sarcosine oxidase Human genes 0.000 description 2
- QJJXYPPXXYFBGM-LFZNUXCKSA-N Tacrolimus Chemical compound C1C[C@@H](O)[C@H](OC)C[C@@H]1\C=C(/C)[C@@H]1[C@H](C)[C@@H](O)CC(=O)[C@H](CC=C)/C=C(C)/C[C@H](C)C[C@H](OC)[C@H]([C@H](C[C@H]2C)OC)O[C@@]2(O)C(=O)C(=O)N2CCCC[C@H]2C(=O)O1 QJJXYPPXXYFBGM-LFZNUXCKSA-N 0.000 description 2
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 230000000692 anti-sense effect Effects 0.000 description 2
- 229960001265 ciclosporin Drugs 0.000 description 2
- 238000004590 computer program Methods 0.000 description 2
- 230000021615 conjugation Effects 0.000 description 2
- 238000013211 curve analysis Methods 0.000 description 2
- 229930182912 cyclosporin Natural products 0.000 description 2
- 238000002405 diagnostic procedure Methods 0.000 description 2
- 238000010494 dissociation reaction Methods 0.000 description 2
- 230000005593 dissociations Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 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 2
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 229960000310 isoleucine Drugs 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 210000005087 mononuclear cell Anatomy 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 229940043230 sarcosine Drugs 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 150000003431 steroids Chemical class 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 229960001967 tacrolimus Drugs 0.000 description 2
- QJJXYPPXXYFBGM-SHYZHZOCSA-N tacrolimus Natural products CO[C@H]1C[C@H](CC[C@@H]1O)C=C(C)[C@H]2OC(=O)[C@H]3CCCCN3C(=O)C(=O)[C@@]4(O)O[C@@H]([C@H](C[C@H]4C)OC)[C@@H](C[C@H](C)CC(=C[C@@H](CC=C)C(=O)C[C@H](O)[C@H]2C)C)OC QJJXYPPXXYFBGM-SHYZHZOCSA-N 0.000 description 2
- 239000004474 valine Substances 0.000 description 2
- 235000014393 valine Nutrition 0.000 description 2
- 230000003442 weekly effect Effects 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- BEJKOYIMCGMNRB-GRHHLOCNSA-N (2s)-2-amino-3-(4-hydroxyphenyl)propanoic acid;(2s)-2-amino-3-phenylpropanoic acid Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1.OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 BEJKOYIMCGMNRB-GRHHLOCNSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 229940122739 Calcineurin inhibitor Drugs 0.000 description 1
- 101710192106 Calcineurin-binding protein cabin-1 Proteins 0.000 description 1
- 102100024123 Calcineurin-binding protein cabin-1 Human genes 0.000 description 1
- 108010077078 Creatinase Proteins 0.000 description 1
- 108010066906 Creatininase Proteins 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 102000002464 Galactosidases Human genes 0.000 description 1
- 108010093031 Galactosidases Proteins 0.000 description 1
- 108010044091 Globulins Proteins 0.000 description 1
- 102000006395 Globulins Human genes 0.000 description 1
- 206010056559 Graft infection Diseases 0.000 description 1
- 102000005623 HSP27 Heat-Shock Proteins Human genes 0.000 description 1
- 108010045100 HSP27 Heat-Shock Proteins Proteins 0.000 description 1
- 102000000588 Interleukin-2 Human genes 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 102000010789 Interleukin-2 Receptors Human genes 0.000 description 1
- 108010038453 Interleukin-2 Receptors Proteins 0.000 description 1
- 238000012313 Kruskal-Wallis test Methods 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 238000000585 Mann–Whitney U test Methods 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- FQISKWAFAHGMGT-SGJOWKDISA-M Methylprednisolone sodium succinate Chemical compound [Na+].C([C@@]12C)=CC(=O)C=C1[C@@H](C)C[C@@H]1[C@@H]2[C@@H](O)C[C@]2(C)[C@@](O)(C(=O)COC(=O)CCC([O-])=O)CC[C@H]21 FQISKWAFAHGMGT-SGJOWKDISA-M 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 206010053159 Organ failure Diseases 0.000 description 1
- KHGNFPUMBJSZSM-UHFFFAOYSA-N Perforine Natural products COC1=C2CCC(O)C(CCC(C)(C)O)(OC)C2=NC2=C1C=CO2 KHGNFPUMBJSZSM-UHFFFAOYSA-N 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 208000001647 Renal Insufficiency Diseases 0.000 description 1
- 206010063837 Reperfusion injury Diseases 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000005864 Sulphur Substances 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 206010060872 Transplant failure Diseases 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- WPVFJKSGQUFQAP-UHFFFAOYSA-N [2-[(2-amino-6-oxo-3h-purin-9-yl)methoxy]-3-hydroxypropyl] 2-amino-3-methylbutanoate Chemical compound N1C(N)=NC(=O)C2=C1N(COC(CO)COC(=O)C(N)C(C)C)C=N2 WPVFJKSGQUFQAP-UHFFFAOYSA-N 0.000 description 1
- LRIUKPUCKCECPT-UHFFFAOYSA-N [hydroxy(phenyl)-$l^{3}-iodanyl] 4-methylbenzenesulfonate Chemical compound C1=CC(C)=CC=C1S(=O)(=O)OI(O)C1=CC=CC=C1 LRIUKPUCKCECPT-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 230000003409 anti-rejection Effects 0.000 description 1
- 230000001494 anti-thymocyte effect Effects 0.000 description 1
- 229940124977 antiviral medication Drugs 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 238000003491 array Methods 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 229960004669 basiliximab Drugs 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 229940046731 calcineurin inhibitors Drugs 0.000 description 1
- 239000011111 cardboard Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001493 electron microscopy Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000009093 first-line therapy Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 238000010820 immunofluorescence microscopy Methods 0.000 description 1
- 239000003018 immunosuppressive agent Substances 0.000 description 1
- 229940124589 immunosuppressive drug Drugs 0.000 description 1
- 238000002650 immunosuppressive therapy Methods 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 208000028867 ischemia Diseases 0.000 description 1
- 208000012947 ischemia reperfusion injury Diseases 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 235000014705 isoleucine Nutrition 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 201000006370 kidney failure Diseases 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 235000005772 leucine Nutrition 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 229940064748 medrol Drugs 0.000 description 1
- 229960001810 meprednisone Drugs 0.000 description 1
- PIDANAQULIKBQS-RNUIGHNZSA-N meprednisone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@@H]2[C@@H]1[C@@H]1C[C@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)CC2=O PIDANAQULIKBQS-RNUIGHNZSA-N 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- RTGDFNSFWBGLEC-SYZQJQIISA-N mycophenolate mofetil Chemical compound COC1=C(C)C=2COC(=O)C=2C(O)=C1C\C=C(/C)CCC(=O)OCCN1CCOCC1 RTGDFNSFWBGLEC-SYZQJQIISA-N 0.000 description 1
- 229960004866 mycophenolate mofetil Drugs 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 230000008816 organ damage Effects 0.000 description 1
- 230000005305 organ development Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 239000011087 paperboard Substances 0.000 description 1
- 230000007310 pathophysiology Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229930192851 perforin Natural products 0.000 description 1
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 238000002823 phage display Methods 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 238000002616 plasmapheresis Methods 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 230000023603 positive regulation of transcription initiation, DNA-dependent Effects 0.000 description 1
- 229960004618 prednisone Drugs 0.000 description 1
- XOFYZVNMUHMLCC-ZPOLXVRWSA-N prednisone Chemical compound O=C1C=C[C@]2(C)[C@H]3C(=O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 XOFYZVNMUHMLCC-ZPOLXVRWSA-N 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 238000003906 pulsed field gel electrophoresis Methods 0.000 description 1
- 150000004060 quinone imines Chemical class 0.000 description 1
- 238000000163 radioactive labelling Methods 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 210000005084 renal tissue Anatomy 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012502 risk assessment Methods 0.000 description 1
- 230000037390 scarring Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229940087854 solu-medrol Drugs 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000003146 transient transfection Methods 0.000 description 1
- 230000010024 tubular injury Effects 0.000 description 1
- 208000037978 tubular injury Diseases 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 230000003612 virological effect Effects 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
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
- G01N33/54388—Immunochromatographic test strips based on lateral flow
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
- G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
- G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
- G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
- G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
- G01N2333/96433—Serine endopeptidases (3.4.21)
- G01N2333/96436—Granzymes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/24—Immunology or allergic disorders
- G01N2800/245—Transplantation related diseases, e.g. graft versus host disease
Definitions
- the present invention relates to a method for diagnosing presymptomatic organ transplant rejection in a subject, using the determination of Granzyme A expression level as a marker of presymptomatic organ transplant graft rejection.
- organ transplantation is still the therapy of choice based on superior patient survival rates and cost effectiveness (1,2).
- progress has been made in the short-term success of renal transplantation which is mainly attributable to more efficient immunosuppressive therapies.
- long-term allograft survival has not improved similarly (3).
- the leading cause for allograft loss after the first year of transplantation is a chronic-progressive scaring process of renal tissue (4), the so called chronic allograft nephropathy (CAN) (5).
- CAN chronic allograft nephropathy
- CAN is the leading cause of graft loss in the long term.
- CAN is a complex multiplayer between immunological and non-immunological causes of graft injury.
- Acute rejection processes even when subclinical; i.e. without measurable concomitant allograft dysfunction (6,7), are an established, important factor for development of CAN.
- Other causes include acute tubular injury, toxic effects of drug treatments, especially from calcineurin inhibitors, viral graft infections and factors related to the graft quality, such as prolonged cold ischemia time, high donor age, graft origin from deceased donors (8,9).
- chronic rejection processes are implicated in the progressive scarring of renal allografts (5). Yet, the understanding of this in terms of definition and pathophysiology is less clear than for acute rejection. Because acute rejection is one of the key factors which determine long-term graft function and survival, timely detection and treatment are important goals in the post- transplant surveillance.
- Subclinical rejection is a common problem with a high occurrence rate, reported in studies performing planned protocol biopsies, with up to 33% at 3 months post-transplantation, when the first protocol biopsy is taken (10,11).
- a non-invasive method to detect also this kind of rejection is necessary and helpful in preserving graft function on long term. Treatment of these subclinical rejection episodes with steroids have been reported to be associated with better graft function and survival.
- Figure 1 shows the relative gene expression for granzyme A mRNA normalized to 18S rRNA. Measurements in a total of 17 patients with acute cellular rejection, 5 stable patients, 6 patients with subclinical acute rejection and 5 patients with acute tubular necrosis are shown. Levels were significantly higher in patients with rejection as compared to stable and acute tubular necrosis (p ⁇ 0.05).
- Figure 2 shows the relative gene expression for granzyme A mRNA normalized to 18S rRNA in a patient with subclinical acute rejection.
- the renal function was normal.
- mRNA for granzyme A can be detected in urine both before and after the protocol biopsy that shows subclinical acute rejection while kidney function was normal.
- Figure 3 shows the serial relative gene expression for granzyme A mRNA normalized to 18S rRNA in a patient with acute rejection.
- the granzyme A mRNA could be detected in urine even before graft dysfunction ensues as reflected by a significant rise in serum creatinine at which time a biopsy was performed.
- This figure is representative of 5 from 7 other experiments in patients with acute rejection.
- GrA mRNA is a sensitive and highly specific marker to distinguish both acute and subclinical rejection from stable graft function and ATN.
- ATN acute tubular necrosis
- CNI calcineurin drug toxicity
- SCR subclinical rejection
- TCMR T-cell mediated rejection
- Transcription levels of GrA, GrB, perform and SERPINB9 were analyzed by quantitative PCR and depicted as relative to the internal control 18s-rRNA. Horizontal lines represent medians and asterisks refer to a significant difference (p ⁇ 0.01) between two groups.
- a GrA mRNA is significantly elevated during both acute and subclinical rejection compared to stable graft function and ATN.
- b GrB is significantly elevated during acute rejection compared to stable and ATN.
- some patients with a stable graft or ATN have GrB expression levels comparable to ACR.
- c Perform transcripts are measurable in all groups tested. During ACR perform levels are significantly increased compared to stable and ATN d SERPINB9 can be detected in urine of patients in all groups. The mRNA levels of this gene are significantly increased during acute rejection compared to stable graft function.
- Granzyme A is a sensitive and highly specific marker to distinguish acute and subclinical rejection from stable graft function.
- the upper graphs represent receiver operating characteristic (ROC) curves visualizing sensitivity and specificity of GrA, GrB, perform and SERPINB9 to differentiate acute rejection (TCMR) or subclinical rejection (SCR) from stable graft function.
- the lower graphs represent ROC curves visualizing sensitivity and specificity of GrA, GrB, perform and SERPINB9 to differentiate acute rejection (TCMR) or subclinical rejection (SCR) from acute tubular necrosis (ATN).
- Detection of granzyme A in urine means 100% specificity in discriminating both TCMR and SCR from ATN and stable function with normal histological findings. Sensitivity of granzyme A in detection of both TCMR and SCR measures as high as 80%.
- FIG. 7 Urinary granzyme A is detectible prior to the rise in serum creatinine.
- a The first patient developed TCMR seven days after transplantation. The initial improvement of renal function is reversed by a 20% rise in serum creatinine at day zero. Both GrA and GrB mRNA levels were detectible at day minus one while no significant rise in creatinine was observed.
- granzyme A was a marker for rejection (both subclinical and acute rejection, identified as Acute Cellular Rejection or ACR and Subclinical Acute cellular Rejection identified as SAR). Surprisingly, granzyme A does not seem to be detectable in delayed graft function (or Acute Tubular Necrosis or delayed graft function, identified as ATN) or in calcineurin drug toxicity. The detection of granzyme A is therefore specific for mild or incipient rejection, i.e. subclinical rejection up to actual clinical acute rejection. However, it will not be detected in conditions with allograft infection and delayed graft function due to acute tubular necrosis caused by ischemia- reperfusion injury, which needs to be distinguished from inflammation caused by rejection.
- This invention has huge advantages: it allows the development of a non-invasive diagnosing method, which can be applied when no clinical signs of rejections are readily apparent. Therefore, the method might even be applied at home, as often as necessary, without the involvement of a medical doctor. Furthermore, the diagnosis is preferably made early enough in order to treat the subject to prevent rejection and/or graft dysfunction.
- the invention relates to a method for diagnosing presymptomatic organ transplant rejection in a subject, the method comprising the steps of:
- the diagnosis method of the invention is unique for granzyme A. It was already known that granzyme B is a marker of transplant rejection (Li B., et al, N. Engl. J. Med. (2001), 344: 947-954). However, the inventor surprisingly found that only granzyme A is a specific marker for diagnosing presymptomatic organ transplant rejection as demonstrated in the example (confere figures 1, 3).
- organ transplant is synonymous for “graft”.
- diagnosis presymptomatic organ transplant rejection preferably means that a diagnosis is reached before the actual (sub)clinical rejection of the transplanted organ is detectable via an increase in serum creatinine as explained below.
- the acute clinical rejection of the transplanted organ is usually detected by an increase in serum creatinine and confirmed by a subsequent biopsy revealing an acute inflammatory process showing mononuclear cells causing tubulitis and/or endothelialitis.
- Subclinical rejection preferably means that in this subject there is no other apparent clinical signs/symptoms of rejection (not even an increase in serum creatinine) or any organ transplant dysfunction has occurred.
- "before the actual (sub)clinical rejection” preferably means at least one day, at least two days, at least three days, at least four days, at least five days, at least six days, at least seven days, at least eight days, at least nine days, at least ten days at least 15 days, at least 20 days, at least 25 days, at least 30 days or more before the actual clinical rejection.
- rejection preferably means acute rejection, i.e.
- Organ transplant dysfunction is preferably assessed by measuring the creatinine serum level as described in the example. In a preferred embodiment, a detectable level of serum creatinine or an increase thereof at least 2% of its serum level indicates organ transplant dysfunction is occurring.
- an increase of the serum creatinine level of at least 4% indicates organ transplant dysfunction is occurring, even more preferably at least 5%, even more preferably at least 7%, even more preferably at least 10%, even more preferably at least 15%, even more preferably at least 20%, even more preferably at least 25%, even more preferably at least 30%, even more preferably at least 35%, or more.
- diagnosis preferably means a predictive risk assessment of the subsequent development of an organ transplant rejection and/or organ transplant dysfunction in a subject.
- a subject may be an animal or a human being, said subject comprising a transplanted organ.
- any subject comprising a transplanted organ could be diagnosed using the method of the invention.
- the diagnosis method may be applied as often as necessary in a subject.
- a subject diagnosed is a subject suspected to have a high risk of rejecting a transplanted organ, due for example to potential genetic incompatibility between the transplanted organ and the subject transplanted and/or to previous unsuccessful transplantation attempts in this subject and/or to the age of the subject and/or to the risk of developing further complications leading to organ transplant dysfunction and/or rejection.
- a subject is a human being.
- a reference value for the expression level of granzyme A is preferably the average value for said expression level in a control subject or in control subjects. More preferably, a control subject is a subject, who has not been transplanted and for which no activation of the immune system has been detected (no infection). Alternatively according to an even more preferred embodiment, a control subject is a subject who has been successfully transplanted for the same type of organ as the subject to be diagnosed and for which no activation of the immune system has been detected (no infection, no rejection). Successfully transplanted means that no rejection has occurred when the subject is used as a control in this method within at least one month after measuring such subject's granzyme A level. According to another preferred embodiment, a reference value is a corresponding value for said subject before (one day or one week or one month before) or after (one day or one week or one month after) transplantation. Granzyme A may be not detectable in "a reference value”.
- the assessment of the expression level of granzyme A may be directly realised at the protein expression level (quantifying the amount of granzyme A), and/or indirectly by quantifying the amount of a nucleotide sequence encoding granzyme A (both the reference value from a control subject and the value from a subject wherein the method is being carried out).
- a nucleotide acid sequence encoding a granzyme A is given as
- a granzyme A to be quantified has:
- At least 60% or at least 65%, at least 70%, at least 75%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or more identity with SEQ ID NO:2 and/or - is encoded by a nucleotide acid sequence which has at least 60% (or at least 65%, at least 70%, at least 75%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or more) identity with SEQ ID NO:2 and/or - is encoded by a nucleotide acid sequence which has at least 60% (or at least
- a nucleotide acid sequence encoding granzyme A to be quantified has:
- At least 60% or at least 65%, at least 70%, at least 75%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or more identity with SEQ ID NO:1 and/or encodes an amino acid sequence of granzyme A that has at least 60% (or at least 65%, at least 70%, at least 75%, at least 75%, at least 80%, at least 85%, at least
- a nucleotide sequence encoding granzyme A means a messenger RNA (mRNA).
- mRNA messenger RNA
- the expression level of granzyme A is determined directly by quantifying the amount of granzyme A. Quantifying a polypeptide amount may be carried out by any known technique. Preferably, a polypeptide amount is quantified using a molecule which specifically binds to granzyme A.
- Preferred binding molecules are selected from: an antibody, which has been specifically raised for recognizing granzyme A, any other molecule which is known to specifically bind granzyme A.
- Such antibody could be used in any immunoassay known to the skilled person such as western blotting, or ELISA (Enzyme-Linked Immuno Sorbent Assay) or FACS (Fluorescence Activated Cell Sorting) using latex beads.
- the preparation of an antibody is known to those skilled in the art. A short explanation of methods that could be used to prepare antibodies is later herein given. Examples of suitable specific antibodies are described in FR-2673952 and in WO 99/54737.
- any other molecule known to bind granzyme A may be a nucleic acid, e.g. a DNA regulatory region, a polypeptide, a metabolite, a substrate, a regulatory element, a structural component, a chaperone (transport) molecule, a peptide mimetic, a non-pep tide mimetic, or any other type of ligand. Mimetic is later herein defined.
- Examples of molecules known to bind granzyme A include Putative HLA-DR Associated Protein I (PHAPI), PHAPII, a complex comprising PHAPII, or heat shock protein 27. These proteins are extensively described in WO 99/09206.
- Binding of granzyme A to a second binding molecule may be detected by any standard methods known to those skilled in the art. Suitable methods include affinity chromatography co-electrophoresis (ACE) assays and ELISA.
- ACE affinity chromatography co-electrophoresis
- ELISA ELISA
- transactivation of a target gene by granzyme A or a granzyme A binding molecule can be determined and quantified, e.g., in a transient transfection assay in which the promoter of the target gene is linked to a reporter gene, e.g., P- galactosidase or luciferase.
- a reporter gene e.g., P- galactosidase or luciferase.
- DNase activity of granzyme A, a granzyme A binding molecule, or a complex containing granzyme A binding molecule may be detected by in vitro degradation of soluble DNA visualized by radio labeling or EtBr staining after agarose electrophoresis, PFGE of genomic DNA, or SDS-DNA-PAGE analysis where DNA is incorporated into SDS PAGE gels, proteins are renatured in the presence of Ca 2+ and Mg 2+ and degraded DNA is visualized by absence of EtBr staining.
- the chaperone (transport) activity of granzyme A or a binding molecule may be detected by immunofluorescence microscopy, immuno electron microscopy or immunoblot.
- a sample from a subject is preferably used.
- the expression level (of a nucleotide sequence or polypeptide) is determined ex vivo in a sample obtained from a subject.
- a sample preferably comprises or consists of a fluid obtained from a subject. More preferably, a fluid comprises or consists of or is selected from: urine, blood, spinal cord fluid, saliva, semen, or bronchoalveolar lavage.
- nucleotide sequence encoding granzyme A and/or granzyme A are extracted and optionally purified using known methods to the skilled person.
- presymptomatic organ transplant rejection is diagnosed when the comparison leads to the finding of a detectable expression of granzyme A and/or an increase of the expression level of granzyme A.
- granzyme A is generally not detectable.
- Detection or an increase of the expression level of granzyme A and/or an increase or a detection of the expression level of a nucleotide sequence encoding granzyme A is preferably defined as being a detectable change of the expression level of granzyme A and/or of a nucleotide sequence encoding granzyme A (or steady state level of the encoded granzyme A or any detectable change in the biological activity of granzyme A) using a method as defined earlier on as compared to the expression level of granzyme A and/or of a corresponding nucleotide sequence (or steady state level of the corresponding encoded granzyme A) in a control subject.
- an increase of the expression level of a nucleotide sequence encoding granzyme A means an increase of at least 5% of the expression level of the nucleotide sequence using PCR.
- Preferred primers used for the PCR are identified as SEQ ID NO:3 5 '-AGGTGGAAGAGACTCGTGCAA-S ' and SEQ ID NO:4 5'- GGTCTCCGCATTTATTTTCAAG-3'.
- an increase of the expression level of a nucleotide sequence means an increase of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, at least 150%, or more.
- an increase of the expression level of granzyme A means an increase of at least 5% of the expression level of granzyme A using western blotting and/or using ELISA or a suitable assay. More preferably, an increase of the expression level of a polypeptide means an increase of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, at least 150%, or more.
- an increase of granzyme A activity means an increase of at least 5% of the polypeptide activity using a suitable assay. More preferably, an increase of the polypeptide activity means an increase of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, at least 150% or more.
- presymptomatic organ transplant rejection is diagnosed when the comparison leads to the finding of a detectable level or an increase of the level of expression of granzyme A or an increase or a detection of the expression level of a nucleotide sequence encoding granzyme A, said detection or increase being detected at the level of a nucleotide sequence encoding granzyme A (mRNA), more preferably an increase of at least 5% of the expression level of said nucleotide sequence using PCR as defined herein.
- mRNA nucleotide sequence encoding granzyme A
- the organ transplanted is selected from: kidney, heart, lung, liver, pancreas, skin, intestine or bone-marrow.
- Assay device
- an assay device for diagnosing presymptomatic organ transplant graft rejection in a subject, wherein said device comprises a molecule which specifically binds granzyme A.
- This device may be used in a diagnosis method of the invention. Any subject or physician could use this device at office/home, repeat the use of such device as often as necessary.
- a molecule which specifically binds granzyme A and which is present in the device is an antibody.
- an assay device is a lateral flow test strip also known as dipstick, preferably, though not necessarily, encased in a housing, designed to be read by the subject, and the assay is a sandwich immunoassay.
- Such devices are impregnated with reagents that specifically indicate the presence of a given molecule, here granzyme A by changing colour upon contact with a sample. Preferred subject's samples have already been defined herein.
- An antibody is preferably labeled by conjugation to a physically detectable label, and upon contacting with a sample containing granzyme A forms a complex.
- Said antibody-granzyme A complex is then contacted with a second antibody, which recognizes said first antibody and which is immobilized on a solid support within the device.
- a second antibody captures said antibody-granzyme A complex to form an antibody-granzyme A-antibody sandwich complex, and the resulting complex, which is immobilized on the solid support, is detectable by virtue of the label.
- a test strip may then be inserted into a reader, where a signal from said label in the complex is measured. Alternatively, a test strip could be inserted into the reader prior to addition of the sample.
- the presence of granzyme A is visualised by a subject as a change of color of at least part of a device. Dipsticks are usually made of paper or cardboard.
- a typical positive control could be an antibody recognizing a molecule which is known to be present in a sample to be tested.
- a typical negative control could be an antibody recognizing a molecule which is known to be absent in a sample to be tested.
- Sequence identity is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences.
- the identity between two amino acid or two nucleic acid sequences is preferably defined by assessing their identity within a whole SEQ ID NO as identified herein or part thereof. Part thereof may mean at least 50% of the length of the SEQ ID NO, or at least 60%, or at least 70%, or at least 80%, or at least 90%.
- identity also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences.
- similarity between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.
- Identity and similarity can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.
- Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include e.g. the GCG program package (Devereux, J., et al, Nucleic Acids Research 12 (1): 387 (1984)), BestFit, BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. MoI. Biol. 215:403-410 (1990).
- the BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al., J. MoI. Biol. 215:403-410 (1990).
- the well-known Smith Waterman algorithm may also be used to determine identity.
- Preferred parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch, J. MoI. Biol. 48:443-453 (1970); Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA.
- amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and iso leucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide- containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine.
- Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine- valine, and asparagine-glutamine.
- Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place.
- the amino acid change is conservative.
- Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to Ser; Arg to Lys; Asn to GIn or His; Asp to GIu; Cys to Ser or Ala; GIn to Asn; GIu to Asp; GIy to Pro; His to Asn or GIn; He to Leu or VaI; Leu to He or VaI; Lys to Arg, GIn or GIu; Met to Leu or He; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; and VaI to He or Leu.
- Antibodies are as follows: Ala to Ser; Arg to Lys; Asn to GIn or His; Asp to GIu; Cys to Ser or Ala; GIn to Asn; GIu to Asp; GIy to Pro; His to Asn or GIn; He to Leu or VaI; Leu to He or VaI; Lys to Arg, GIn or GIu
- Some aspects of the invention concern the use of an antibody or antibody- fragment that specifically binds to granzyme A.
- Methods for generating antibodies or antibody-fragments that specifically bind to a polypeptide are described in e.g. Harlow and Lane (1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) and WO 91/19818; WO 91/18989; WO 92/01047; WO 92/06204; WO 92/18619; and US 6,420,113 and references cited therein.
- the term "specific binding,” as used herein, includes both low and high affinity specific binding.
- Specific binding can be exhibited, e.g., by a low affinity antibody or antibody- fragment having a Kd of at least about 10 "4 M. Specific binding also can be exhibited by a high affinity antibody or antibody-fragment, for example, an antibody or antibody-fragment having a Kd of at least about of 10 "7 M, at least about 10 "8 M, at least about 10 "9 M, at least about 10 "10 M, or can have a Kd of at least about 10 "11 M or 10 "12 M or greater.
- Peptide-like molecules referred to as peptidomimetics
- non-peptide molecules that specifically bind to granzyme A or to its receptor polypeptide and that may be applied in any of the methods of the invention as defined herein (for altering the activity or steady state level of a polypeptide of the invention) may be identified using methods known in the art per se, as e.g. described in detail in US 6,180,084 which is incorporated herein by reference. Such methods include e.g. screening libraries of peptidomimetics, peptides, DNA or cDNA expression libraries, combinatorial chemistry and, particularly useful, phage display libraries. These libraries may be screened for agonists and antagonists of polypeptides by contacting the libraries with substantially purified polypeptides of the invention, fragments thereof or structural analogues thereof.
- the verb "to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.
- the verb "to consist” may be replaced by "to consist essentially of meaning that a peptide or a composition as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention.
- reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements.
- the indefinite article “a” or “an” thus usually means “at least one”.
- Urine from patients was collected and total RNA was isolated from the cell pellet with the GenEluteTM Mammalian Total RNA kit (Sigma) according to the instructions of the manufacturer.
- Creatinine serum level was determined using the following enzymatic method: CREA Plus (11775685 216) from Roche/Hitachi, Roche Diagnostics and following the manufacturer's instructions.
- this method determines the concentration of sarcosine after conversion of creatinine with the aid of creatininase, creatinase, and sarcosine oxidase.
- Sarcosine is converted to glycine, formaldehyde and hydrogen peroxide in presence of oxygen by the action of sarcosine oxidase.
- the liberated hydrogen peroxide reacts with 4-aminophenazone and HTIB to form a quinone imine chromogen.
- the reaction is catalized by peroxidase.
- the color intensity is directly proportional to the concentration of creatinine (31, 32, 33, 34).
- Renal transplant recipients may suffer from a CMV infection derived from the transplant.
- the present study reveals that in CMV infected patients, granzyme A is always detected, i.e. even when the transplant is not rejected.
- Prophylactic treatment with antiviral medication, i.e. valgancyclovir to prevent CMV infection in the early post transplant period alleviates this problem.
- detection of granzyme A mRNA can be used as a reliable non-invasive diagnostic tool to monitor graft function in urine specimens of the majority of transplant recipients. In case of a positive test, one should be alert of acute rejection or subclinical rejection.
- Creatinine serum level was determined using the following enzymatic method: CREA Plus (11775685 216) from Roche/Hitachi, Roche Diagnostics and following the manufacturer's instructions.
- RNA was quantified and reverse transcribed to complementary DNA (cDNA) using random hexamers (pd(N)6, Amersham Biosciences, Piscataway, NJ, USA) and Superscript II, RNase H-reverse transcriptase kit (Invitrogen, Breda, The Netherlands).
- the following primer sets were developed for the following genes: Granzyme A: (5'-AGGTGGAAGAGACTCGTGCAA-S', 5'- GGTCTCCGC ATTT ATTTTCAAG-3'), Granzyme B: (5'-
- Primers were selected to span exon-intron junctions to prevent amplification of genomic DNA. Primers were validated on cDNA of lymphokine activated killer (LAK) cells, as LAK cells express both granzyme A (GrA) (35), granzyme B (GrB), perforin(36) and SerpinB9 (37). AK cells were generated by stimulating isolated PBMCs of healthy volunteers with 6000 U/ml IL-2 for 7 days at 37°C in a 5% CO 2 - incubator. Product specificity of each primer set was verified by agarose gel electrophoresis and sequence analysis of the amplified PCR product.
- LAK lymphokine activated killer
- Gene expression levels were measured in the ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA) using the SYBR green method (Applied Biosystems). Serial dilutions of control samples of LAK cells were assayed to determine the range of linear amplification for reliable mRNA measurements of each gene. Thresholds of detection were set at the lowest C t values that were still within the linear amplification range. Product specificity of each primer set in each reaction was verified by dissociation curve analysis. Transcript levels and relative gene expression of mRNA were determined as described by Pfaffl (30). All results were normalized to the internal control 18S rRNA, and are expressed relative to the expression levels found in LAK cells.
- ROC receiver-operating-characteristic
- urinary granzyme A is a novel, reliable and simple diagnostic molecular biomarker for both acute T-cell mediated rejection and subclinical rejection.
- Urinary granzyme A can distinguish between rejection; both acute T-cell mediated rejection and subclinical rejection on the one hand, and acute tubular necrosis, calcineurin drug toxicity or stable graft function with normal histological findings on the other hand, i.e. those conditions that should be discriminated from rejection in case of graft dysfunction. Sensitivity and specificity measure as high as 80% and 100%, respectively. In a limited number of patients with serially obtained urine samples, detection of urinary granzyme A precedes the clinical signs of rejection as defined by a significant rise in serum creatinine up to 20% of the lowest baseline value which is compatible with characteristics of an early biomarker for rejection.
- Nankivell BJ Borrows RJ, Fung CL, O'Connell PJ, Allen RD, Chapman JR. Natural history, risk factors, and impact of subclinical rejection in kidney transplantation. Transplantation 2004;78: 242-249.
- Nankivell BJ Borrows RJ, Fung CL, O'connell PJ, Allen RD, Chapman JR. Natural history, risk factors, and impact of subclinical rejection in kidney transplantation. Transplantation 2004;78: 242-249. 17.
- Bohmig GA Cikivell BJ, Borrows RJ, Fung CL, O'connell PJ, Allen RD, Chapman JR. Natural history, risk factors, and impact of subclinical rejection in kidney transplantation. Transplantation 2004;78: 242-249. 17.
- Bohmig GA Cikivell BJ, Borrows RJ, Fung CL, O'connell PJ, Allen RD, Chapman JR. Natural history, risk factors, and impact of subclinical rejection in kidney transplantation. Transplantation 2004;78: 242-249. 17.
- Bohmig GA Cikivell BJ, Borrows RJ, Fung CL, O'connell PJ, Allen RD, Chapman JR. Natural history, risk factors, and impact of subclin
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Analytical Chemistry (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to amethod for diagnosing presymptomaticorgan transplant rejection in a subject, the method comprisingthe steps of: (a) determining the expressionlevel of granzyme A in a subject which comprises a transplanted organ; and, (b) comparing the expression level of granzyme A as defined in (a) witha reference value for saidexpressionlevel, the reference value preferablybeing the average value for saidexpressionlevel in a control subject.
Description
Method for diagnosing presymptomatic organ transplant rejection
Field of the invention The present invention relates to a method for diagnosing presymptomatic organ transplant rejection in a subject, using the determination of Granzyme A expression level as a marker of presymptomatic organ transplant graft rejection.
Background of the invention
In case of organ failure such as renal failure, organ transplantation is still the therapy of choice based on superior patient survival rates and cost effectiveness (1,2). In the past decades, progress has been made in the short-term success of renal transplantation which is mainly attributable to more efficient immunosuppressive therapies. However, long-term allograft survival has not improved similarly (3). The leading cause for allograft loss after the first year of transplantation is a chronic-progressive scaring process of renal tissue (4), the so called chronic allograft nephropathy (CAN) (5). Next to death with a functioning graft, CAN is the leading cause of graft loss in the long term. CAN is a complex multiplayer between immunological and non-immunological causes of graft injury. Acute rejection processes, even when subclinical; i.e. without measurable concomitant allograft dysfunction (6,7), are an established, important factor for development of CAN. Other causes include acute tubular injury, toxic effects of drug treatments, especially from calcineurin inhibitors, viral graft infections and factors related to the graft quality, such as prolonged cold ischemia time, high donor age, graft origin from deceased donors (8,9). Also, chronic rejection processes are implicated in the progressive scarring of renal allografts (5). Yet, the understanding of this in terms of definition and pathophysiology is less clear than for acute rejection. Because acute rejection is one of the key factors which determine long-term graft function and survival, timely detection and treatment are important goals in the post- transplant surveillance. The standard care with serum creatinine measurements followed by biopsy in case of allograft dysfunction, as reflected by a rise in serum creatinine; implies that acute rejection is detected in an advanced stage. Moreover, subclinical acute rejection cannot even be detected or is not obvious to clinicians at the
time of occurrence by serial measurements of creatinine as it does not lead to an instant rise in serum creatinine. In other words, subclinical rejection which is shown to lead to late graft failure in large scale studies performing planned protocol biopsies, is actually a hidden silent form of an acute inflammatory process like acute rejection, but not accompanied by an acute rise in creatinine at the time of occurrence. Subclinical rejection is a common problem with a high occurrence rate, reported in studies performing planned protocol biopsies, with up to 33% at 3 months post-transplantation, when the first protocol biopsy is taken (10,11). A non-invasive method to detect also this kind of rejection is necessary and helpful in preserving graft function on long term. Treatment of these subclinical rejection episodes with steroids have been reported to be associated with better graft function and survival.
Up to now, a robust, non-invasive diagnostic method which would allow easier and more frequent monitoring of the patient and the graft is lacking. Reliable and early detection of rejection (presymptomatic stage of rejection, i.e. at the stage that rejection cannot be detected using the current standard methods for detection, or even suspicion of rejection, e.g. by serial measurements of serum creatinine and/or other methods for calculating creatinine clearance) is particularly important in efforts to spare patients from over-immunosuppression by using minimized and tailored immunosuppressive protocols, and to combat the graft injury and inflammation in an early stage. Surveillance of patients using repetitive measurements of serum creatinine implies recognition of the rejection at a relatively late stage, i.e., when immunological injury has reached a degree that causes impaired graft function. Increased serum creatinine can only be detected when inflammation in the graft, i.e. rejection process, has reached an advanced stage. In theory, protocol biopsy could be used as surveillance strategy for organ transplant rejection (presymptomatic as defined above). In practice, however, a protocol comprising taking weekly biopsies will never be implemented due to the associated risk of losing the transplanted organ and infection, the costs and the heavy burden such procedure would impose on the patient (12, 13).
In summary, there is still a need for a non-invasive method for diagnosing a presymptomatic organ transplant rejection. Such method is attractive since the diagnosis is reached early enough in order to treat a diagnosed subject to prevent organ
damage. Furthermore, this method is non-invasive, simple, reproducible, sensitive, specific, and time and cost efficient.
Description of the figures
Figure 1 shows the relative gene expression for granzyme A mRNA normalized to 18S rRNA. Measurements in a total of 17 patients with acute cellular rejection, 5 stable patients, 6 patients with subclinical acute rejection and 5 patients with acute tubular necrosis are shown. Levels were significantly higher in patients with rejection as compared to stable and acute tubular necrosis (p< 0.05).
Figure 2 shows the relative gene expression for granzyme A mRNA normalized to 18S rRNA in a patient with subclinical acute rejection. In agreement with the definition of subclinical acute rejection, the renal function was normal. As depicted, mRNA for granzyme A can be detected in urine both before and after the protocol biopsy that shows subclinical acute rejection while kidney function was normal.
Figure 3 shows the serial relative gene expression for granzyme A mRNA normalized to 18S rRNA in a patient with acute rejection. The granzyme A mRNA could be detected in urine even before graft dysfunction ensues as reflected by a significant rise in serum creatinine at which time a biopsy was performed. This figure is representative of 5 from 7 other experiments in patients with acute rejection.
Figure 4. GrA mRNA is a sensitive and highly specific marker to distinguish both acute and subclinical rejection from stable graft function and ATN.
Urine samples were collected from either recipients with a stable graft function and normal histological findings (stable, n=10), acute tubular necrosis (ATN, n=9), calcineurin drug toxicity (CNI, n=4), subclinical rejection (SCR, n=10) or T-cell mediated rejection (TCMR; acute rejection type I, n=20). Transcription levels of GrA, GrB, perform and SERPINB9 were analyzed by quantitative PCR and depicted as relative to the internal control 18s-rRNA. Horizontal lines represent medians and asterisks refer to a significant difference (p<0.01) between two groups.
a GrA mRNA is significantly elevated during both acute and subclinical rejection compared to stable graft function and ATN. b GrB is significantly elevated during acute rejection compared to stable and ATN. Yet, some patients with a stable graft or ATN have GrB expression levels comparable to ACR. c Perform transcripts are measurable in all groups tested. During ACR perform levels are significantly increased compared to stable and ATN d SERPINB9 can be detected in urine of patients in all groups. The mRNA levels of this gene are significantly increased during acute rejection compared to stable graft function.
Figures 5, 6: Granzyme A is a sensitive and highly specific marker to distinguish acute and subclinical rejection from stable graft function. The upper graphs represent receiver operating characteristic (ROC) curves visualizing sensitivity and specificity of GrA, GrB, perform and SERPINB9 to differentiate acute rejection (TCMR) or subclinical rejection (SCR) from stable graft function. The lower graphs represent ROC curves visualizing sensitivity and specificity of GrA, GrB, perform and SERPINB9 to differentiate acute rejection (TCMR) or subclinical rejection (SCR) from acute tubular necrosis (ATN). Detection of granzyme A in urine means 100% specificity in discriminating both TCMR and SCR from ATN and stable function with normal histological findings. Sensitivity of granzyme A in detection of both TCMR and SCR measures as high as 80%.
Figure 7: Urinary granzyme A is detectible prior to the rise in serum creatinine. We collected urine samples at different time points from 8 patients with an acute rejection. Relative gene expression levels of GrA (blue) and GrB (red) of two representative patients are depicted in these graphs; the grey line represents serum creatinine values. Biopsy (BX) confirmed acute rejection, denominated day 0, is indicated by the dotted line. a The first patient developed TCMR seven days after transplantation. The initial improvement of renal function is reversed by a 20% rise in serum creatinine at day zero. Both GrA and GrB mRNA levels were detectible at day minus one while no significant rise in creatinine was observed.
b In the second patient GrA and GrB were detectible three days prior to diagnosis of TCMR, preceding the rise in serum creatinine.
Description of the invention
To compensate for the limitations of allograft biopsy, many attempts have been made to establish surrogate markers of rejection. Yet, none of these markers have made their way into today's clinical practice as a universally accepted diagnostic tool suggesting that the ideal marker(s) is still to be discovered. Surprisingly, the inventors discovered that granzyme A is an ideal marker for diagnosing presymptomatic organ transplant rejection and subclinical rejection. Granzyme A is detectable in urine. Therefore, sample collection is easy and repeatedly possible. Analysis of granzyme A is simple, reproducible, and time and cost efficient. Sensitivity and specificity of the detection of granzyme A relate to its reliability to detect rejection and/or subclinical rejection. The inventor found that granzyme A was a marker for rejection (both subclinical and acute rejection, identified as Acute Cellular Rejection or ACR and Subclinical Acute cellular Rejection identified as SAR). Surprisingly, granzyme A does not seem to be detectable in delayed graft function (or Acute Tubular Necrosis or delayed graft function, identified as ATN) or in calcineurin drug toxicity. The detection of granzyme A is therefore specific for mild or incipient rejection, i.e. subclinical rejection up to actual clinical acute rejection. However, it will not be detected in conditions with allograft infection and delayed graft function due to acute tubular necrosis caused by ischemia- reperfusion injury, which needs to be distinguished from inflammation caused by rejection.
This invention has huge advantages: it allows the development of a non-invasive diagnosing method, which can be applied when no clinical signs of rejections are readily apparent. Therefore, the method might even be applied at home, as often as necessary, without the involvement of a medical doctor. Furthermore, the diagnosis is preferably made early enough in order to treat the subject to prevent rejection and/or graft dysfunction.
Diagnosis method
In a first aspect, the invention relates to a method for diagnosing presymptomatic organ transplant rejection in a subject, the method comprising the steps of:
(a) determining the expression level of granzyme A in a subject which comprises a transplanted organ and, (b) comparing the expression level of granzyme A as defined in (a) with a reference value for said expression level, the reference value preferably being the average value for said expression level in a control subject.
The diagnosis method of the invention is unique for granzyme A. It was already known that granzyme B is a marker of transplant rejection (Li B., et al, N. Engl. J. Med. (2001), 344: 947-954). However, the inventor surprisingly found that only granzyme A is a specific marker for diagnosing presymptomatic organ transplant rejection as demonstrated in the example (confere figures 1, 3).
In the context of the invention, "organ transplant" is synonymous for "graft".
In the context of the invention, "diagnosing presymptomatic organ transplant rejection" preferably means that a diagnosis is reached before the actual (sub)clinical rejection of the transplanted organ is detectable via an increase in serum creatinine as explained below. The acute clinical rejection of the transplanted organ is usually detected by an increase in serum creatinine and confirmed by a subsequent biopsy revealing an acute inflammatory process showing mononuclear cells causing tubulitis and/or endothelialitis. Alternatively, according to another preferred embodiment, it means that a diagnosis is reached of a subclinical rejection of the transplanted organ.
Subclinical rejection preferably means that in this subject there is no other apparent clinical signs/symptoms of rejection (not even an increase in serum creatinine) or any organ transplant dysfunction has occurred. In this context, "before the actual (sub)clinical rejection" preferably means at least one day, at least two days, at least three days, at least four days, at least five days, at least six days, at least seven days, at least eight days, at least nine days, at least ten days at least 15 days, at least 20 days, at least 25 days, at least 30 days or more before the actual clinical rejection. In this context, "rejection" preferably means acute rejection, i.e. the histopathological inflammatory process in the graft with mononuclear cells infiltrating the graft causing tubulitis and/or endothelialitis. "Before" has preferably the same meaning as earlier defined herein. Organ transplant dysfunction is preferably assessed by measuring the creatinine serum level as described in the example. In a preferred embodiment, a
detectable level of serum creatinine or an increase thereof at least 2% of its serum level indicates organ transplant dysfunction is occurring. More preferably, an increase of the serum creatinine level of at least 4% indicates organ transplant dysfunction is occurring, even more preferably at least 5%, even more preferably at least 7%, even more preferably at least 10%, even more preferably at least 15%, even more preferably at least 20%, even more preferably at least 25%, even more preferably at least 30%, even more preferably at least 35%, or more.
In the context of the invention, diagnosis preferably means a predictive risk assessment of the subsequent development of an organ transplant rejection and/or organ transplant dysfunction in a subject.
In the context of the invention, a subject may be an animal or a human being, said subject comprising a transplanted organ. In principle, any subject comprising a transplanted organ could be diagnosed using the method of the invention. The diagnosis method may be applied as often as necessary in a subject. Preferably, a subject diagnosed is a subject suspected to have a high risk of rejecting a transplanted organ, due for example to potential genetic incompatibility between the transplanted organ and the subject transplanted and/or to previous unsuccessful transplantation attempts in this subject and/or to the age of the subject and/or to the risk of developing further complications leading to organ transplant dysfunction and/or rejection. Preferably, a subject is a human being.
In the context of the invention, "a reference value" for the expression level of granzyme A is preferably the average value for said expression level in a control subject or in control subjects. More preferably, a control subject is a subject, who has not been transplanted and for which no activation of the immune system has been detected (no infection). Alternatively according to an even more preferred embodiment, a control subject is a subject who has been successfully transplanted for the same type of organ as the subject to be diagnosed and for which no activation of the immune system has been detected (no infection, no rejection). Successfully transplanted means that no rejection has occurred when the subject is used as a control in this method within at least one month after measuring such subject's granzyme A level. According to another preferred embodiment, a reference value is a corresponding value for said subject before (one day or one week or one month before) or after (one day or one
week or one month after) transplantation. Granzyme A may be not detectable in "a reference value".
The assessment of the expression level of granzyme A may be directly realised at the protein expression level (quantifying the amount of granzyme A), and/or indirectly by quantifying the amount of a nucleotide sequence encoding granzyme A (both the reference value from a control subject and the value from a subject wherein the method is being carried out). A nucleotide acid sequence encoding a granzyme A is given as
SEQ ID NO:1. A corresponding granzyme A amino acid sequence is given as SEQ ID
NO:2. The skilled person will understand that it is possible to isolate multiple iso forms of granzyme A depending on the subject to be tested.
In a preferred embodiment, a granzyme A to be quantified has:
- at least 60% (or at least 65%, at least 70%, at least 75%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or more) identity with SEQ ID NO:2 and/or - is encoded by a nucleotide acid sequence which has at least 60% (or at least
65%, at least 70%, at least 75%, at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, at least 97%, at least 99% or more) identity with SEQ ID
NO:1.
In another preferred embodiment, a nucleotide acid sequence encoding granzyme A to be quantified has:
- at least 60% (or at least 65%, at least 70%, at least 75%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or more) identity with SEQ ID NO:1 and/or encodes an amino acid sequence of granzyme A that has at least 60% (or at least 65%, at least 70%, at least 75%, at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, at least 97%, at least 99% or more) identity with an amino acid sequence encoded by SEQ ID NO:1.
Identity is later herein defined. The quantification of the amount of a nucleotide sequence encoding granzyme A is preferably performed using classical molecular biology techniques such as (real time) PCR, arrays or northern analysis. In this embodiment, a nucleotide sequence encoding granzyme A means a messenger RNA (mRNA). Alternatively, according to another preferred embodiment, in the diagnosis method the expression level of granzyme A is determined directly by
quantifying the amount of granzyme A. Quantifying a polypeptide amount may be carried out by any known technique. Preferably, a polypeptide amount is quantified using a molecule which specifically binds to granzyme A. Preferred binding molecules are selected from: an antibody, which has been specifically raised for recognizing granzyme A, any other molecule which is known to specifically bind granzyme A. Such antibody could be used in any immunoassay known to the skilled person such as western blotting, or ELISA (Enzyme-Linked Immuno Sorbent Assay) or FACS (Fluorescence Activated Cell Sorting) using latex beads. The preparation of an antibody is known to those skilled in the art. A short explanation of methods that could be used to prepare antibodies is later herein given. Examples of suitable specific antibodies are described in FR-2673952 and in WO 99/54737. In the context of the invention, any other molecule known to bind granzyme A may be a nucleic acid, e.g. a DNA regulatory region, a polypeptide, a metabolite, a substrate, a regulatory element, a structural component, a chaperone (transport) molecule, a peptide mimetic, a non-pep tide mimetic, or any other type of ligand. Mimetic is later herein defined. Examples of molecules known to bind granzyme A include Putative HLA-DR Associated Protein I (PHAPI), PHAPII, a complex comprising PHAPII, or heat shock protein 27. These proteins are extensively described in WO 99/09206. Binding of granzyme A to a second binding molecule may be detected by any standard methods known to those skilled in the art. Suitable methods include affinity chromatography co-electrophoresis (ACE) assays and ELISA. The skilled person will understand that alternatively or in combination with the quantification of a nucleic acid sequence encoding granzyme A and/or a corresponding polypeptide, the quantification of a substrate of a corresponding polypeptide or of any compound known to be associated with a function or activity of a corresponding polypeptide or the quantification of a function or activity of a corresponding polypeptide using a specific assay is encompassed within the scope of the diagnosis method of the invention. For example, transactivation of a target gene by granzyme A or a granzyme A binding molecule can be determined and quantified, e.g., in a transient transfection assay in which the promoter of the target gene is linked to a reporter gene, e.g., P- galactosidase or luciferase.
Such evaluations can be done in vitro or in vivo or ex vivo.
As another example, DNase activity of granzyme A, a granzyme A binding molecule, or a complex containing granzyme A binding molecule, may be detected by in vitro degradation of soluble DNA visualized by radio labeling or EtBr staining after agarose electrophoresis, PFGE of genomic DNA, or SDS-DNA-PAGE analysis where DNA is incorporated into SDS PAGE gels, proteins are renatured in the presence of Ca2+ and Mg2+ and degraded DNA is visualized by absence of EtBr staining.
As an additional example, the chaperone (transport) activity of granzyme A or a binding molecule may be detected by immunofluorescence microscopy, immuno electron microscopy or immunoblot.
Since the expression level of a nucleotide sequence encoding granzyme A and/or amounts of a corresponding polypeptide may be difficult to detect in a subject, a sample from a subject is preferably used. According to another preferred embodiment, the expression level (of a nucleotide sequence or polypeptide) is determined ex vivo in a sample obtained from a subject. A sample preferably comprises or consists of a fluid obtained from a subject. More preferably, a fluid comprises or consists of or is selected from: urine, blood, spinal cord fluid, saliva, semen, or bronchoalveolar lavage.
Subsequently, a nucleotide sequence encoding granzyme A and/or granzyme A are extracted and optionally purified using known methods to the skilled person.
In a more preferred diagnosis method, presymptomatic organ transplant rejection is diagnosed when the comparison leads to the finding of a detectable expression of granzyme A and/or an increase of the expression level of granzyme A. In control subjects as defined before, granzyme A is generally not detectable.
Detection or an increase of the expression level of granzyme A and/or an increase or a detection of the expression level of a nucleotide sequence encoding granzyme A (or steady state level of granzyme A) is preferably defined as being a detectable change of the expression level of granzyme A and/or of a nucleotide sequence encoding granzyme A (or steady state level of the encoded granzyme A or any detectable change in the biological activity of granzyme A) using a method as defined earlier on as
compared to the expression level of granzyme A and/or of a corresponding nucleotide sequence (or steady state level of the corresponding encoded granzyme A) in a control subject. According to a preferred embodiment, detection or an increase of granzyme A activity is quantified using a specific mRNA assay for the Granzyme A gene as earlier defined herein. Preferably, an increase of the expression level of a nucleotide sequence encoding granzyme A means an increase of at least 5% of the expression level of the nucleotide sequence using PCR. Preferred primers used for the PCR are identified as SEQ ID NO:3 5 '-AGGTGGAAGAGACTCGTGCAA-S ' and SEQ ID NO:4 5'- GGTCTCCGCATTTATTTTCAAG-3'. More preferably, an increase of the expression level of a nucleotide sequence means an increase of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, at least 150%, or more.
Preferably, an increase of the expression level of granzyme A means an increase of at least 5% of the expression level of granzyme A using western blotting and/or using ELISA or a suitable assay. More preferably, an increase of the expression level of a polypeptide means an increase of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, at least 150%, or more.
Preferably, an increase of granzyme A activity means an increase of at least 5% of the polypeptide activity using a suitable assay. More preferably, an increase of the polypeptide activity means an increase of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, at least 150% or more.
In a most preferred diagnostic method, presymptomatic organ transplant rejection is diagnosed when the comparison leads to the finding of a detectable level or an increase of the level of expression of granzyme A or an increase or a detection of the expression level of a nucleotide sequence encoding granzyme A, said detection or increase being detected at the level of a nucleotide sequence encoding granzyme A (mRNA), more preferably an increase of at least 5% of the expression level of said nucleotide sequence using PCR as defined herein.
In another preferred embodiment, the organ transplanted is selected from: kidney, heart, lung, liver, pancreas, skin, intestine or bone-marrow.
Assay device
In a second aspect, an assay device is provided for diagnosing presymptomatic organ transplant graft rejection in a subject, wherein said device comprises a molecule which specifically binds granzyme A. This device may be used in a diagnosis method of the invention. Any subject or physician could use this device at office/home, repeat the use of such device as often as necessary.
The type of molecules that are known to specifically bind granzyme A have already been earlier described herein. In a preferred embodiment, a molecule which specifically binds granzyme A and which is present in the device is an antibody.
In a preferred embodiment, an assay device is a lateral flow test strip also known as dipstick, preferably, though not necessarily, encased in a housing, designed to be read by the subject, and the assay is a sandwich immunoassay. Such devices are impregnated with reagents that specifically indicate the presence of a given molecule, here granzyme A by changing colour upon contact with a sample. Preferred subject's samples have already been defined herein. An antibody is preferably labeled by conjugation to a physically detectable label, and upon contacting with a sample containing granzyme A forms a complex. Said antibody-granzyme A complex is then contacted with a second antibody, which recognizes said first antibody and which is immobilized on a solid support within the device. A second antibody captures said antibody-granzyme A complex to form an antibody-granzyme A-antibody sandwich complex, and the resulting complex, which is immobilized on the solid support, is detectable by virtue of the label. A test strip may then be inserted into a reader, where a signal from said label in the complex is measured. Alternatively, a test strip could be inserted into the reader prior to addition of the sample. Alternatively and according to a preferred embodiment, the presence of granzyme A is visualised by a subject as a change of color of at least part of a device. Dipsticks are usually made of paper or cardboard. Usually additional molecules are present in a device as a positive or negative control. A typical positive control could be an antibody recognizing a molecule which is known to be present in a sample to be tested. A typical negative control could be an antibody recognizing a molecule which is known to be absent in a sample to be tested.
Sequence identity
"Sequence identity" is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. The identity between two amino acid or two nucleic acid sequences is preferably defined by assessing their identity within a whole SEQ ID NO as identified herein or part thereof. Part thereof may mean at least 50% of the length of the SEQ ID NO, or at least 60%, or at least 70%, or at least 80%, or at least 90%.
In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988).
Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include e.g. the GCG program package (Devereux, J., et al, Nucleic Acids Research 12 (1): 387 (1984)), BestFit, BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. MoI. Biol. 215:403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al., J. MoI. Biol. 215:403-410 (1990). The well-known Smith Waterman algorithm may also be used to determine identity.
Preferred parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch, J. MoI. Biol. 48:443-453 (1970); Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992); Gap Penalty: 12; and Gap Length Penalty: 4. A program useful with these parameters is publicly available as the "Ogap" program from Genetics Computer Group, located in Madison, WI. The aforementioned parameters are the default parameters for amino acid comparisons (along with no penalty for end gaps).
Preferred parameters for nucleic acid comparison include the following: Algorithm: Needleman and Wunsch, J. MoI. Biol. 48:443-453 (1970); Comparison matrix: matches=+10, mismatch=O; Gap Penalty: 50; Gap Length Penalty: 3. Available as the Gap program from Genetics Computer Group, located in Madison, Wis. Given above are the default parameters for nucleic acid comparisons.
Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called "conservative" amino acid substitutions, as will be clear to the skilled person. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and iso leucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide- containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine- valine, and asparagine-glutamine. Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid change is conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to Ser; Arg to Lys; Asn to GIn or His; Asp to GIu; Cys to Ser or Ala; GIn to Asn; GIu to Asp; GIy to Pro; His to Asn or GIn; He to Leu or VaI; Leu to He or VaI; Lys to Arg, GIn or GIu; Met to Leu or He; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; and VaI to He or Leu.
Antibodies
Some aspects of the invention concern the use of an antibody or antibody- fragment that specifically binds to granzyme A. Methods for generating antibodies or antibody-fragments that specifically bind to a polypeptide are described in e.g. Harlow and Lane (1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) and WO 91/19818; WO 91/18989; WO 92/01047; WO 92/06204; WO 92/18619; and US 6,420,113 and references cited therein. The term "specific binding," as used herein, includes both low and high affinity specific binding. Specific binding can be exhibited, e.g., by a low affinity antibody or antibody- fragment having a Kd of at least about 10"4 M. Specific binding also can be exhibited by a high affinity antibody or antibody-fragment, for example, an antibody or antibody-fragment having a Kd of at least about of 10"7 M, at least about 10"8 M, at least about 10"9 M, at least about 10"10 M, or can have a Kd of at least about 10"11 M or 10"12 M or greater.
Peptidomimetics
Peptide-like molecules (referred to as peptidomimetics) or non-peptide molecules that specifically bind to granzyme A or to its receptor polypeptide and that may be applied in any of the methods of the invention as defined herein (for altering the activity or steady state level of a polypeptide of the invention) may be identified using methods known in the art per se, as e.g. described in detail in US 6,180,084 which is incorporated herein by reference. Such methods include e.g. screening libraries of peptidomimetics, peptides, DNA or cDNA expression libraries, combinatorial chemistry and, particularly useful, phage display libraries. These libraries may be screened for agonists and antagonists of polypeptides by contacting the libraries with substantially purified polypeptides of the invention, fragments thereof or structural analogues thereof.
General
In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb "to consist" may be replaced by "to consist essentially of meaning that a peptide or a composition as defined herein may comprise additional component(s) than the ones specifically
identified, said additional component(s) not altering the unique characteristic of the invention. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".
All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.
Examples
Example 1 (figures 1-3) Material and Methods
Urine from patients was collected and total RNA was isolated from the cell pellet with the GenElute™ Mammalian Total RNA kit (Sigma) according to the instructions of the manufacturer.
The isolated RNA was quantified and reverse transcribed to complementary DNA (cDNA) using random hexamers (pd(N)6, Amersham Biosciences, Piscataway, NJ, USA) and Superscript II, RNase H-reverse transcriptase kit (Invitrogen, Breda, The Netherlands).
Gene expression levels was measured in the ABI PRISM 7000 Sequence Detection System (Applied Bio systems, Foster City, CA) using the SYBR green method (Applied Biosystems). Primers were developed to prevent amplification of genomic DNA. Product specificity of each primer set was verified by agarose gel electrophoresis and by dissociation curve analysis. PCR products were further confirmed by sequence analysis.
Primer sets (sense sequence, antisense sequence) for the following genes were used: GrA (5'-AGGTGGAAGAGACTCGTGCAA-S', 5'-
GGTCTCCGCATTTATTTTCAAG-3'); GrB (5'-TGCGAATCTGACTTACGCCAT- 3', 5'-GGAGGCATGCCATTGTTTCG-S'); Perform (5'- CCGCTTCTACAGTTTCCATG-3', 5'-GTTGTCCGTGAGCCCTTCC-S'); PI9 (5'- TGTC AAGATAACCCTTCGC AC-3', 5'-AGCTCAGCATGGTAGAATTGAA-S'). AS an internal control, primers specific for human 18S rRNA were used (5'- CGGCTACCAC ATCC AAGGAA-3', 5'-GCTGGAATTACCGCGGCT-S'). Transcript levels and relative gene expression of mRNA were determined as described in (30). All results were normalized to the internal control 18S rRNA, and are expressed relative to the expression levels found in LAK cells.
Creatinine serum level determination
Creatinine serum level was determined using the following enzymatic method: CREA Plus (11775685 216) from Roche/Hitachi, Roche Diagnostics and following the manufacturer's instructions.
The principle of this method is briefly outlined hereafter: this method determines the concentration of sarcosine after conversion of creatinine with the aid of creatininase, creatinase, and sarcosine oxidase. Sarcosine is converted to glycine, formaldehyde and hydrogen peroxide in presence of oxygen by the action of sarcosine oxidase. The liberated hydrogen peroxide reacts with 4-aminophenazone and HTIB to form a quinone imine chromogen. The reaction is catalized by peroxidase. The color intensity is directly proportional to the concentration of creatinine (31, 32, 33, 34).
Results
We investigated the role of granzyme A as an early biomarker for rejection in renal transplant recipients. We found that granzyme A is detectable in the early phase of rejection, preceding the clinical symptoms and the rise in serum creatinine (confer figure 3). We show that this marker can distinguish between acute tubular necrosis (delayed graft function) and rejection which is clinically important and crucial (confer figures 1, 2).
We showed that also subclinical rejection is accompanied by detectable granzyme A mRNA levels in urine which enables us to diagnose this important clinically silent state of rejection, because subclinical rejection is considered to be an important risk factor for development of chronic allograft nephropathy, the leading cause of graft loss on the long term (confer figures 1, 2). The values of figure 1 are also presented in table 1 below.
Table 1 : Relative gene expression for granzvme A mRNA
Renal transplant recipients may suffer from a CMV infection derived from the transplant. The present study reveals that in CMV infected patients, granzyme A is always detected, i.e. even when the transplant is not rejected. Prophylactic treatment with antiviral medication, i.e. valgancyclovir to prevent CMV infection in the early post transplant period, alleviates this problem. As the new guidelines recommend renal transplant recipients to be treated prophylactically, detection of granzyme A mRNA can be used as a reliable non-invasive diagnostic tool to monitor graft function in urine specimens of the majority of transplant recipients. In case of a positive test, one should be alert of acute rejection or subclinical rejection.
Example 2 (figures 4-7) Materials and Methods
Renal allograft recipients
We included 72 renal transplant candidates who were transplanted between November 2004 and April 2008. All patients received standard immunosuppression consisting of
induction therapy by monoclonal antibodies against IL-2 receptor (Basiliximab, Roche: 20 mg op day 1 and day 4, wherein day 1 is the day of the transplantation) and triple maintenance immunosuppression with Prednisone (Roche, 50 mg first three days, i.e. day 1 , day 2 and day 3 wherein day 1 is the day of the transplantation, subsequently lOmg per day) calcineurin inhibitor (either Tacrolimus from Astellas (0.1 mg/kg twice a day) or Cyclosporine from Novartis (4mg/kg twice a day)) and Mycophenolate mofetil (Roche Nederland BV, twice a day 500mg when used with tacrolimus or twice 1000mg/day when used with cyclosporine) . These doses are standard doses. When acute rejection was suspected, a percutaneous graft biopsy was done, which was histological graded by a local pathologist according to the Banff '05 classification Patients experiencing acute rejection were treated with methylprednisone (SoIu- Medrol) 1000 mg for three consecutive days. Anti Thymocyte Globulin (rATG, Genzyme, 4mg/kg to start with and subsequently adjusted depending on the number of lymphocytes detected) was given for steroid-resistant rejection episodes and for humoral rejection combined with plasmapheresis. Rejection was considered steroid- resistant if no stabilization or improvement to 20% of baseline serum creatinine occurred within 7 days after treatment with Solu-Medrol (Pfizer BV, 1 g/day during three days). An episode of T-cell mediated rejection was classified as reversible if the serum creatinine level returned to within 15 percent of the prerejection level within four weeks after the initiation of antirejection treatment.
Urine sample collection
Fresh urine samples were collected weekly, up to ten weeks after transplantation. In case of a biopsy, an extra urine sample was obtained at the same day prior to adjustments in immunosuppressive drug treatment.
Creatinine serum level determination.
Creatinine serum level was determined using the following enzymatic method: CREA Plus (11775685 216) from Roche/Hitachi, Roche Diagnostics and following the manufacturer's instructions.
Isolation of total RNA.
At the day of collection, 100 ml of urine was centrifuged for 10 minutes, 1700rpm. The supernatant was discarded and cells were washed with PBS prior to addition of 700 μl of lysis solution (GenElute™ Mammalian Total RNA kit, Sigma). After thorough mixing, samples were stored until further use at -800C. RNA was isolated from the urinary cell-lysates according to the instructions of the manufacturer. Purity and concentration of the samples were determined with a Nanodrop spectrophotometer NDlOOO (Nanodrop technologies, Wilmington,USA).
Semi-quantitative real-time mRNA measurements. The isolated RNA was quantified and reverse transcribed to complementary DNA (cDNA) using random hexamers (pd(N)6, Amersham Biosciences, Piscataway, NJ, USA) and Superscript II, RNase H-reverse transcriptase kit (Invitrogen, Breda, The Netherlands). The following primer sets (sense sequence, antisense sequence) were developed for the following genes: Granzyme A: (5'-AGGTGGAAGAGACTCGTGCAA-S', 5'- GGTCTCCGC ATTT ATTTTCAAG-3'), Granzyme B: (5'-
TGCGAATCTGACTTACGCCAT-3', 5'-GGAGGCATGCCATTGTTTCG-S'),
Perform: (5'-CCGCTTCTACAGTTTCCATg-3', 5'-GTTGTCCGTGAGCCCTTCC-S'), SerpinB9 (also named PI9 in previous example) : (5'- TGTC AAGATAACCCTTCGC AC-3', 5'-AGCTCAGCATGGTAGAATTGAA-S'), 18S rRNA: (5'-CGGCTACCACATCCAAGGAA-S', 5'-GCTGGAATTACCGCGGCT- 3').
Primers were selected to span exon-intron junctions to prevent amplification of genomic DNA. Primers were validated on cDNA of lymphokine activated killer (LAK) cells, as LAK cells express both granzyme A (GrA) (35), granzyme B (GrB), perforin(36) and SerpinB9 (37). AK cells were generated by stimulating isolated PBMCs of healthy volunteers with 6000 U/ml IL-2 for 7 days at 37°C in a 5% CO2- incubator. Product specificity of each primer set was verified by agarose gel electrophoresis and sequence analysis of the amplified PCR product. Gene expression levels were measured in the ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA) using the SYBR green method (Applied Biosystems). Serial dilutions of control samples of LAK cells were assayed to determine the range of
linear amplification for reliable mRNA measurements of each gene. Thresholds of detection were set at the lowest Ct values that were still within the linear amplification range. Product specificity of each primer set in each reaction was verified by dissociation curve analysis. Transcript levels and relative gene expression of mRNA were determined as described by Pfaffl (30). All results were normalized to the internal control 18S rRNA, and are expressed relative to the expression levels found in LAK cells.
Statistical analyses. Patient characteristics are presented as respectively mean ± SD, median ± quartiles, percentages or proportions. Comparisons between the groups were performed in SPSS (version 16.0), a one-way ANOVA or Kruskal-Wallis test was used when appropriate. Experimental data are presented as relative gene expression, median and quartiles. Multiple two tailed Mann- Whitney U tests were used to compare relative mRNA levels between groups. To control the false discovery rate we performed a Benjamini- Hochberg correction, p<0.016 was considered statistical significant. Receiver-operating characteristics curves were generated to find the best cutoff points for the diagnosis of acute cellular and subclinical rejection. Analyses were performed in GraphPad Prism (version 5) and SPSS (versionlό.O).We used receiver-operating-characteristic (ROC) curves to analyze mRNA levels in order to determine the cutoff points that yielded the highest combined sensitivity and specificity for predicting the outcome of an episode of rejection.
Here we show that urinary granzyme A is a novel, reliable and simple diagnostic molecular biomarker for both acute T-cell mediated rejection and subclinical rejection.
Urinary granzyme A can distinguish between rejection; both acute T-cell mediated rejection and subclinical rejection on the one hand, and acute tubular necrosis, calcineurin drug toxicity or stable graft function with normal histological findings on the other hand, i.e. those conditions that should be discriminated from rejection in case of graft dysfunction. Sensitivity and specificity measure as high as 80% and 100%, respectively. In a limited number of patients with serially obtained urine samples, detection of urinary granzyme A precedes the clinical signs of rejection as defined by a
significant rise in serum creatinine up to 20% of the lowest baseline value which is compatible with characteristics of an early biomarker for rejection.
Reference lists
1. van Dijk PC, Jager KJ, de CF et al. Renal replacement therapy in Europe: the results of a collaborative effort by the ERA-EDTA registry and six national or regional registries. Nephrol Dial Transplant 2001;16: 1120-1129.
2. de Wit GA, Ramsteijn PG, de Charro FT. Economic evaluation of end stage renal disease treatment. Health Policy 1998;44: 215-232.
3. Meier-Rriesche HU, Schold JD, Kaplan B. Long-term renal allograft survival: have we made significant progress or is it time to rethink our analytic and therapeutic strategies? Am J Transplant 2004;4: 1289-1295.
4. Kreis HA, Ponticelli C. Causes of late renal allograft loss: chronic allograft dysfunction, death, and other factors. Transplantation 2001;71: SS5-SS9.
5. Solez K, Colvin RB, Racusen LC et al. Banff '05 Meeting Report: differential diagnosis of chronic allograft injury and elimination of chronic allograft nephropathy ('CAN'). Am J Transplant 2007;7: 518-526.
6. Rush D, Nickerson P, Gough J et al. Beneficial effects of treatment of early subclinical rejection: a randomized study. J Am Soc Nephrol 1998;9: 2129- 2134.
7. Moreso F, Ibernon M, Goma M et al. Subclinical rejection associated with chronic allograft nephropathy in protocol biopsies as a risk factor for late graft loss. Am J Transplant 2006;6: 747-752.
8. Massy ZA, Guijarro C, Kasiske BL. Clinical predictors of chronic renal allograft rejection. Kidney Int Suppl 1995;52: S85-S88.
9. Sebekova K, Feber J, Carpenter B et al. Tissue viral DNA is associated with chronic allograft nephropathy. Pediatr Transplant 2005;9: 598-603.
10. Nankivell BJ, Borrows RJ, Fung CL, O'Connell PJ, Allen RD, Chapman JR. The natural history of chronic allograft nephropathy. N Engl J Med 2003;349: 2326-2333.
11. Nankivell BJ, Borrows RJ, Fung CL, O'Connell PJ, Allen RD, Chapman JR. Natural history, risk factors, and impact of subclinical rejection in kidney transplantation. Transplantation 2004;78: 242-249.
12. Huraib S, Goldberg H, Katz A et al. Percutaneous needle biopsy of the transplanted kidney: technique and complications. Am J Kidney Dis 1989;14:
13-17.
13. Schwarz A, Gwinner W, Hiss M, Radermacher J, Mengel M, Haller H. Safety and adequacy of renal transplant protocol biopsies. Am J Transplant 2005;5: 1992-1996.
14. Choi BS, Shin MJ, Shin SJ et al. Clinical significance of an early protocol biopsy in living-donor renal transplantation: ten-year experience at a single center. Am J Transplant 2005;5: 1354-1360.
15. Kirk AD, Jacobson LM, Heisey DM, Radke NF, Pirsch JD, Sollinger HW. Clinically stable human renal allografts contain histological and RNA-based findings that correlate with deteriorating graft function. Transplantation
1999;68: 1578-1582.
16. Nankivell BJ, Borrows RJ, Fung CL, O'connell PJ, Allen RD, Chapman JR. Natural history, risk factors, and impact of subclinical rejection in kidney transplantation. Transplantation 2004;78: 242-249.
17. Bohmig GA, Regele H, Horl WH. Protocol biopsies after kidney transplantation. Transpl Int 2005; 18: 131-139.
18. Lipman ML, Shen Y, Jeffery JR et al. Immune-activation gene expression in clinically stable renal allograft biopsies: molecular evidence for subclinical rejection. Transplantation 1998;66: 1673-1681.
19. Rush D, Somorjai R, Deslauriers R, Shaw A, Jeffery J, Nickerson P. Subclinical rejection—a potential surrogate marker for chronic rejection—may be diagnosed by protocol biopsy or urine spectroscopy. Ann Transplant 2000;5: 44-49.
20. Rush D. Protocol biopsies should be part of the routine management of kidney transplant recipients. Pro. Am J Kidney Dis 2002;40: 671 -673.
21. Takemoto SK, Zeevi A, Feng S et al. National conference to assess antibody- mediated rejection in solid organ transplantation. Am J Transplant 2004;4: 1033-1041.
22. Pelletier RP, Hennessy PK, Adams PW, VanBuskirk AM, Ferguson RM, Orosz CG. Clinical significance of MHC-reactive alloantibodies that develop after kidney or kidney-pancreas transplantation. Am J Transplant 2002;2: 134-141.
23. Worthington JE, Martin S, Al Husseini DM, Dyer PA, Johnson RW. Posttransplantation production of donor HLA-specific antibodies as a predictor of renal transplant outcome. Transplantation 2003;75: 1034-1040.
24. Piazza A, Poggi E, Borrelli L et al. Impact of donor-specific antibodies on chronic rejection occurrence and graft loss in renal transplantation: posttransplant analysis using flow cytometric techniques. Transplantation 2001;71: 1106-1112.
25. Meyers CM, Kirk AD. Workshop on late renal allograft dysfunction. Am J Transplant 2005 ;5: 1600-1605.
26. Terasaki PI, Cai J. Humoral theory of transplantation: further evidence. Curr Opin Immunol 2005;17: 541-545.
27. Lipman ML, Shen Y, Jeffery JR et al. Immune-activation gene expression in clinically stable renal allograft biopsies: molecular evidence for subclinical rejection. Transplantation 1998;66: 1673-1681.
28. Kirk AD, Jacobson LM, Heisey DM, Radke NF, Pirsch JD, Sollinger HW. Clinically stable human renal allografts contain histological and RNA-based findings that correlate with deteriorating graft function. Transplantation
1999;68: 1578-1582.
29. Wittke S, Haubitz M, Walden M et al. Detection of acute tubulo interstitial rejection by proteomic analysis of urinary samples in renal transplant recipients. Am J Transplant 2005;5: 2479-2488.
30. Pfaffl MW.:A new mathematical model for relative quantification in real-time
RT-PCR. Nucleic Acids Res. (2001), May I;29(9):e45.
31. Junge W, Wilke B, Halabi A, Klein G. Determination of reference intervals for serum creatinine, creatinine excretion and creatinine clearance with an enzymatic and a modified Jaffe method.Clin Chim Acta. 2004 Jun;344(l- 2):137-48.
32. Fuentes-Arderiu X, Alvarez-Funes V, Coca-Fabregas L, Cruz-Placer M, Diaz-Fernandez J, Herrero-Bernal P, Garcia-Caballero F, del Mar Larrea-Ortiz-Quintan M, La-Torre-Marcellan P, Mar-Medina C, Victoria Rodriguez-Hernandez M, Juve-Cuxart S. Multicentre physiological reference values for the concentration of creatininium in plasma and diagnostic specificity
of glomerular filtration rate estimated with the MDRD equation.Clin Chem Lab Med. 2007;45(4):531-4.
33. Owen LJ, Keevil BG. Does bilirubin cause interference in Roche creatinine methods? Clin Chem. 2007 Feb;53(2):370-l.
34. McKillop DJ, Cairns B, Duly E, Van Drimmelen M, Ryan M.
The effect of serum creatinine method choice on estimated glomerular filtration rate determined by the abbreviated MDRD formula. Ann Clin Biochem. 2006 May;43(Pt 3):220-2.
35. Spaeny-Dekking EH, Kamp AM, Froelich CJ, Hack CE. Extracellular granzyme A, complexed to proteoglycans, is protected against inactivation by protease inhibitors. Blood. 2000;95: 1465-72.
36. Ozdemir O, Ravindranath Y, Savasan S. Mechanisms of superior anti-tumor cytotoxic response of interleukin 15-induced lymphokine-activated killer cells. J Immunother. 2005;28: 44-52.
37. Barrie MB, Stout HW, Abougergi MS, Miller BC, Thiele DL. Antiviral cytokines induce hepatic expression of the granzyme B inhibitors, proteinase inhibitor 9 and serine proteinase inhibitor 6. J Immunol. 2004; 172: 6453-9
Claims
1. A method for diagnosing presymptomatic organ transplant rejection in a subject, the method comprising the steps of: (a) determining the expression level of granzyme A in a subject which comprises a transplanted organ; and,
(b) comparing the expression level of granzyme A as defined in (a) with a reference value for said expression level, the reference value preferably being the average value for said expression level in a control subject.
2. A method according to claim 1, wherein a presymptomatic organ transplant graft rejection is diagnosed when the comparison leads to the finding of a detectable expression level or an increase of the expression level of granzyme A.
3. A method according to claim 1 or 2, wherein the expression level of granzyme A is determined by directly quantifying the amount of granzyme A and/or indirectly by quantifying the amount of a nucleotide sequence encoding granzyme A.
4. A method according to any one of claims 1 to 3, wherein the expression level is determined ex vivo in a sample obtained from the subject.
5. A method according to claim 4, wherein the sample is a fluid obtained from the subject.
6. A method according to claim 5, wherein the fluid is selected from: urine, blood, spinal cord fluid, saliva, semen or bronchoalveolar lavage.
7. A method according to any one of claims 1 to 6, wherein the organ is selected from: kidney, heart, lung, liver, pancreas, skin, intestine or bone-marrow.
8. An assay device for diagnosing presymptomatic organ transplant graft rejection in a subject, wherein the device comprises a molecule which specifically binds granzyme A.
9. A device according to claim 8, wherein the molecule which specifically binds granzyme A is an antibody.
10. A device according to claim 8 or 9, wherein the device is a lateral flow test strip.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP07117971.7 | 2007-10-05 | ||
EP07117971 | 2007-10-05 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2009045104A1 true WO2009045104A1 (en) | 2009-04-09 |
Family
ID=38657879
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/NL2008/050632 WO2009045104A1 (en) | 2007-10-05 | 2008-10-02 | Method for diagnosing presymptomatic organ transplant rejection |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2009045104A1 (en) |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2010112934A1 (en) * | 2009-03-31 | 2010-10-07 | Biofortuna Ltd | Assay method and device |
EP2999482A4 (en) * | 2013-05-21 | 2017-08-09 | Biogen MA Inc. | Prodrugs and drugs |
EP3146076A4 (en) * | 2014-05-22 | 2018-05-09 | The Scripps Research Institute | Gene expression profiles associated with sub-clinical kidney transplant rejection |
US10451636B2 (en) | 2014-04-09 | 2019-10-22 | The Regents Of The University Of California | Protein biomarkers for immune assessment and prediction of transplant rejection |
US11104951B2 (en) | 2014-05-22 | 2021-08-31 | The Scripps Research Institute | Molecular signatures for distinguishing liver transplant rejections or injuries |
US12060611B2 (en) | 2014-05-22 | 2024-08-13 | The Scripps Research Institute | Gene expression profiles associated with sub-clinical kidney transplant rejection |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999054737A1 (en) * | 1998-04-17 | 1999-10-28 | Stichting Sanquin Bloedvoorziening | Immunodiagnostic method for granzymes |
EP1170589A1 (en) * | 2000-06-05 | 2002-01-09 | Stichting Sanquin Bloedvoorziening | Method for histocompatibility testing |
WO2003099780A2 (en) * | 2002-05-24 | 2003-12-04 | Molecular Devices Corporation | Luminogenic protease substrates |
WO2004074815A2 (en) * | 2003-02-14 | 2004-09-02 | Beth Israel Deaconess Medical Center, Inc. | Predicting graft rejection |
-
2008
- 2008-10-02 WO PCT/NL2008/050632 patent/WO2009045104A1/en active Application Filing
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999054737A1 (en) * | 1998-04-17 | 1999-10-28 | Stichting Sanquin Bloedvoorziening | Immunodiagnostic method for granzymes |
EP1170589A1 (en) * | 2000-06-05 | 2002-01-09 | Stichting Sanquin Bloedvoorziening | Method for histocompatibility testing |
WO2003099780A2 (en) * | 2002-05-24 | 2003-12-04 | Molecular Devices Corporation | Luminogenic protease substrates |
WO2004074815A2 (en) * | 2003-02-14 | 2004-09-02 | Beth Israel Deaconess Medical Center, Inc. | Predicting graft rejection |
Non-Patent Citations (3)
Title |
---|
ALPERT S ET AL: "THE RELATIONSHIP OF GRANZYME A AND PERFORIN EXPRESSION TO CARDIAC ALLOGRAFT REJECTOIN AND DYSFUNCTION", TRANSPLANTATION, WILLIAMS AND WILKINS, BALTIMORE, MD, US, vol. 60, no. 12, 27 December 1995 (1995-12-27), pages 1478 - 1485, XP001080216, ISSN: 0041-1337 * |
BEYER JANA ET AL: "The induction of cytotoxic molecules in urine is associated with acute rejection: a non-invasive diagnostic approach in kidney transplantation", GENES AND IMMUNITY, vol. 5, no. Suppl. 1, May 2004 (2004-05-01), & 18TH EUROPEAN HISTOCOMPATIBILITY CONFERENCE; SOFIA, BULGARIA; MAY 08-11, 2004, pages S16, XP002459999, ISSN: 1466-4879 * |
GRIFFITHS G M ET AL: "Granzyme A and perforin as markers for rejection in cardiac transplantation", EUROPEAN JOURNAL OF IMMUNOLOGY, WILEY - V C H VERLAG GMBH & CO. KGAA, DE, vol. 21, 1 January 1991 (1991-01-01), pages 687 - 692, XP002092298, ISSN: 0014-2980 * |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2010112934A1 (en) * | 2009-03-31 | 2010-10-07 | Biofortuna Ltd | Assay method and device |
EP2999482A4 (en) * | 2013-05-21 | 2017-08-09 | Biogen MA Inc. | Prodrugs and drugs |
US10451636B2 (en) | 2014-04-09 | 2019-10-22 | The Regents Of The University Of California | Protein biomarkers for immune assessment and prediction of transplant rejection |
EP3146076A4 (en) * | 2014-05-22 | 2018-05-09 | The Scripps Research Institute | Gene expression profiles associated with sub-clinical kidney transplant rejection |
US11104951B2 (en) | 2014-05-22 | 2021-08-31 | The Scripps Research Institute | Molecular signatures for distinguishing liver transplant rejections or injuries |
EP3825416A3 (en) * | 2014-05-22 | 2021-09-15 | The Scripps Research Institute | Gene expression profiles associated with sub-clinical kidney transplant rejection |
US12060611B2 (en) | 2014-05-22 | 2024-08-13 | The Scripps Research Institute | Gene expression profiles associated with sub-clinical kidney transplant rejection |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Zollner et al. | Faecal biomarkers in inflammatory bowel diseases: calprotectin versus lipocalin-2—a comparative study | |
Beunders et al. | Proenkephalin (PENK) as a novel biomarker for kidney function | |
AU2004213839B2 (en) | Predicting graft rejection | |
Gwinner | Renal transplant rejection markers | |
WO2009045104A1 (en) | Method for diagnosing presymptomatic organ transplant rejection | |
AU2009253696A1 (en) | Methods of diagnosing rejection of a kidney allograft using genomic or proteomic expression profiling | |
Oetting et al. | Urinary β2-microglobulin is associated with acute renal allograft rejection | |
KR20110005708A (en) | Methods of diagnosing acute cardiac allograft rejection | |
US20090053195A1 (en) | Biomarkers for acute graft rejection | |
Raza et al. | The association of urinary interferon-gamma inducible protein-10 (IP10/CXCL10) levels with kidney allograft rejection | |
CA2983542A1 (en) | A method for predicting the risk of incidence of chronic kidney disease | |
TING et al. | Urinary tubular biomarkers as potential early predictors of renal allograft rejection | |
EP1850130B1 (en) | Gene markers and utilzation of the same | |
US20120165207A1 (en) | Methods for Monitoring Allograft Rejection | |
DK2585089T3 (en) | CCL18 blockade of signaling through CCR6 as a therapeutic option in fibrotic diseases and cancer | |
EP1861718B1 (en) | Use of xcri for diagnosing or monitoring of immune tolerance | |
Nickerson | Post-transplant monitoring of renal allografts: are we there yet? | |
EP3653724A1 (en) | Methods to determine risk of chronic allograft lung dysfunction | |
WO2020077267A1 (en) | Biomarker of chronic kidney disease in liver transplant recipients | |
Bayram et al. | High prolidase levels in patients with Familial Mediterranean Fever (FMF) | |
Sumiyoshi et al. | Exploring the role of insulin-like growth factor binding protein-1 in identifying idiopathic multicentric Castleman's disease types: Implications for the mTOR signaling pathway | |
Hribova et al. | Intrarenal cytokine and chemokine gene expression and kidney graft outcome | |
Michon et al. | Investigation of new biomarkers of kidney injury in renal transplant recipients undergoing graft biopsy | |
WO2015129324A1 (en) | Mental illness marker and use therefor | |
JP5010220B2 (en) | Understanding the pathology of patients undergoing hematopoietic stem cell transplantation |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 08835539 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 08835539 Country of ref document: EP Kind code of ref document: A1 |