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Combined effects of cytokine genotypes, HLA class II alleles and CYP3A polymorphism and their expressions in renal transplantation
S68
Abstracts
6.1 #63
A ZERO A,B,DR MISMATCHED CADEVERIC KIDNEY DONOR LOW FLOW T AND B CROSSMATCH POSITIVE DUE TO A CW6 ANTIBODY Steven S. Geier,1 Jerry R. Niedzinski,1 Rui Pei,2 W. Ben Vernon,3 Melissa J. Yanover.3 1FLOW Laboratory, Laboratories At Bonfils, Denver, Colorado; 2 Research Department, One Lambda, Canoga Park, California; 3Porter Transplant Program, PorterCare Adventist Health System, Denver, Colorado HLA Cw alleles are polymorphic class I determinants like HLA A and B, and they can stimulate antibody and cytotoxic cell responses. HLA Cw mismatches between patients and donors can result in hyperacute or acute rejection of kidney and allogeneic bone marrow grafts, and GVHD in bone marrow recipients. In our case report, we describe the FLOW crossmatches with sera from a primary renal transplant high PRA patient (HLA A2,68 B39,61 DR4,8 possibly Cw3 or 7) and cells from a 0 HLA A, B, DR mismatched imported cadaveric donor HLA A2,68 B61,– DR4,8 DQ4,7 Cw6). The patient has a current PRA: I ⫽ 91%, II ⫽ 13% and peak 11/2000 PRA: I ⫽ 98, II ⫽ 99, with suggested specificities: anti A1,3,23,24,32,33 B8,35,49,57,75 Bw4, DR11,13,17,14. The sera for crossmatches were current, peak and 5/1999. The CDC B and T AHG crossmatches were negative as were most of the auto Flow crossmatches. However, in contrast to the CDC results, the FLOW crossmatches with the donor cells gave low positive results on donor T cells and B cells for all three serum dates. A decision was made not to transplant due to the high PRA of the patient and three low positive FLOW T cell crossmatches. The Flow reactivity could have been due to non HLA antibodies or HLA–Cw or DQ antibodies or an allele level response against the donor⬘s HLA A, B or DR. Auto IgG antibodies can have differential reactivity reacting more strongly with a donor⬘s cells then a patient⬘s own cells. DQ antibodies did not appear to be involved since the ELISA PRAs suggested that the patient did not have antibodies to the donor⬘s DQ4 or 7. Antibodies to Cw6 were suggested since the patient appeared to have B57 antibodies and Cw6 is often associated with B57. Samples of the three sera were sent to One Lambda to be analyzed with their Single Antigen (high definition) FLOW beads. The results identified fairly broad reactivity and strong Cw6 reactivity in all of the sera. In summary, our study demonstrates the importance of HLA typing for HLA Cw and screening for Cw antibodies, and the ability of the Single Antigen One Lambda FLOW beads to identify HLA antibody specificities in high PRA patients.
6.1 #64
COMBINED EFFECTS OF CYTOKINE GENOTYPES, HLA CLASS II ALLELES AND CYP3A POLYMORPHISM AND THEIR EXPRESSIONS IN RENAL TRANSPLANTATION D. Olga McDaniel,1 Donald E. Butkus,2 Kori Y. McDaniel,1 Maria E. Oropeza,1 William H. Barber.1 1Surgery, University of Mississippi Medical Center, Jackson, Mississippi; 2Medicine, University of Mississippi Medical Center, Jackson, Mississippi As a predictor of the outcome, effectiveness of cytokine, class II DRB1, DPB1 and cytochrome P450 CYP3A gene polymorphism and their expression levels were evaluated in a group of African–American (AA) patients who had undergone renal transplantation. It was hypothesized that possession of specific genotype might be influential in transplantation predisposing the recipient to allograft rejection. Cytokines (IL–2, TNF– ␣, TGF–1, IL–10, IL– 6, and IFN–␥); class II (DR1, and DP1); and CYP 3A (3A4 and 3A5) mRNA transcript levels were determined using peripheral blood cells. The mRNA transcript level was tested in pre and post transplantation, day 1 and day 4 using a RT–PCR assay. We assessed the combined effect of cytokine genotypes, class II and CYP3A alleles in relation to REs. Greater proportions of recipients (8 out of 13) with RE were TGF–1 high, IFN–␥ intermediate and IL–10 low producers. In addition the DR1 allele was found in REs with higher frequency as compared with those with RFG (0.38, 0.12 p ⬍ 0.04). The CYP 3A5 was found in a higher frequency but it was not statistically significant. High DR1 expression by recipient PBMCs before transplantation was associated with more RE rates (p ⬍ 0.001), and high DP1 expression before transplantation was associated with RFG (p ⬍ 0.05). There was an inverse relationship between the IFN–␥ and IL–10 expression associated with REs. Expression level of high IFN–␥, low IL–10 and high Cyp3A4 mRNA transcripts in recipients PBMCs was correlated with REs. Stimulation of PBMCs with PHA significantly increased the level of TGF–1 and IL–10 expression in individuals who carried high producer genotype, but it had little effect on IFN–␥ . In summary, testing peripheral blood cells for genetic markers diagnostic of rejection might allow predicting in advance some of the clinical outcome, and might reduce the need for tissue biopsy.
Abstracts
6.1 #63
A ZERO A,B,DR MISMATCHED CADEVERIC KIDNEY DONOR LOW FLOW T AND B CROSSMATCH POSITIVE DUE TO A CW6 ANTIBODY Steven S. Geier,1 Jerry R. Niedzinski,1 Rui Pei,2 W. Ben Vernon,3 Melissa J. Yanover.3 1FLOW Laboratory, Laboratories At Bonfils, Denver, Colorado; 2 Research Department, One Lambda, Canoga Park, California; 3Porter Transplant Program, PorterCare Adventist Health System, Denver, Colorado HLA Cw alleles are polymorphic class I determinants like HLA A and B, and they can stimulate antibody and cytotoxic cell responses. HLA Cw mismatches between patients and donors can result in hyperacute or acute rejection of kidney and allogeneic bone marrow grafts, and GVHD in bone marrow recipients. In our case report, we describe the FLOW crossmatches with sera from a primary renal transplant high PRA patient (HLA A2,68 B39,61 DR4,8 possibly Cw3 or 7) and cells from a 0 HLA A, B, DR mismatched imported cadaveric donor HLA A2,68 B61,– DR4,8 DQ4,7 Cw6). The patient has a current PRA: I ⫽ 91%, II ⫽ 13% and peak 11/2000 PRA: I ⫽ 98, II ⫽ 99, with suggested specificities: anti A1,3,23,24,32,33 B8,35,49,57,75 Bw4, DR11,13,17,14. The sera for crossmatches were current, peak and 5/1999. The CDC B and T AHG crossmatches were negative as were most of the auto Flow crossmatches. However, in contrast to the CDC results, the FLOW crossmatches with the donor cells gave low positive results on donor T cells and B cells for all three serum dates. A decision was made not to transplant due to the high PRA of the patient and three low positive FLOW T cell crossmatches. The Flow reactivity could have been due to non HLA antibodies or HLA–Cw or DQ antibodies or an allele level response against the donor⬘s HLA A, B or DR. Auto IgG antibodies can have differential reactivity reacting more strongly with a donor⬘s cells then a patient⬘s own cells. DQ antibodies did not appear to be involved since the ELISA PRAs suggested that the patient did not have antibodies to the donor⬘s DQ4 or 7. Antibodies to Cw6 were suggested since the patient appeared to have B57 antibodies and Cw6 is often associated with B57. Samples of the three sera were sent to One Lambda to be analyzed with their Single Antigen (high definition) FLOW beads. The results identified fairly broad reactivity and strong Cw6 reactivity in all of the sera. In summary, our study demonstrates the importance of HLA typing for HLA Cw and screening for Cw antibodies, and the ability of the Single Antigen One Lambda FLOW beads to identify HLA antibody specificities in high PRA patients.
6.1 #64
COMBINED EFFECTS OF CYTOKINE GENOTYPES, HLA CLASS II ALLELES AND CYP3A POLYMORPHISM AND THEIR EXPRESSIONS IN RENAL TRANSPLANTATION D. Olga McDaniel,1 Donald E. Butkus,2 Kori Y. McDaniel,1 Maria E. Oropeza,1 William H. Barber.1 1Surgery, University of Mississippi Medical Center, Jackson, Mississippi; 2Medicine, University of Mississippi Medical Center, Jackson, Mississippi As a predictor of the outcome, effectiveness of cytokine, class II DRB1, DPB1 and cytochrome P450 CYP3A gene polymorphism and their expression levels were evaluated in a group of African–American (AA) patients who had undergone renal transplantation. It was hypothesized that possession of specific genotype might be influential in transplantation predisposing the recipient to allograft rejection. Cytokines (IL–2, TNF– ␣, TGF–1, IL–10, IL– 6, and IFN–␥); class II (DR1, and DP1); and CYP 3A (3A4 and 3A5) mRNA transcript levels were determined using peripheral blood cells. The mRNA transcript level was tested in pre and post transplantation, day 1 and day 4 using a RT–PCR assay. We assessed the combined effect of cytokine genotypes, class II and CYP3A alleles in relation to REs. Greater proportions of recipients (8 out of 13) with RE were TGF–1 high, IFN–␥ intermediate and IL–10 low producers. In addition the DR1 allele was found in REs with higher frequency as compared with those with RFG (0.38, 0.12 p ⬍ 0.04). The CYP 3A5 was found in a higher frequency but it was not statistically significant. High DR1 expression by recipient PBMCs before transplantation was associated with more RE rates (p ⬍ 0.001), and high DP1 expression before transplantation was associated with RFG (p ⬍ 0.05). There was an inverse relationship between the IFN–␥ and IL–10 expression associated with REs. Expression level of high IFN–␥, low IL–10 and high Cyp3A4 mRNA transcripts in recipients PBMCs was correlated with REs. Stimulation of PBMCs with PHA significantly increased the level of TGF–1 and IL–10 expression in individuals who carried high producer genotype, but it had little effect on IFN–␥ . In summary, testing peripheral blood cells for genetic markers diagnostic of rejection might allow predicting in advance some of the clinical outcome, and might reduce the need for tissue biopsy.