HLA-B*15:04:04, a novel HLA allele identified during proficiency testing in Brazil.

HLA-B*15:04:04 differs from HLA-B*15:04:01 by one nucleotide at codon 238 (gat > gac).

Reduced Incidence of Cytomegalovirus Infection in Kidney Transplant Recipients Receiving Everolimus and Reduced Tacrolimus Doses.

This study compared the incidence of CMV infection/disease in de novo kidney transplant recipients receiving everolimus or mycophenolate and no CMV pharmacological prophylaxis. We randomized 288 patients to receive a single 3 mg/kg dose of antithymocyte globulin, tacrolimus, everolimus, and prednisone (r-ATG/EVR, n = 85); basiliximab, tacrolimus, everolimus, and prednisone (BAS/EVR, n = 102); or basiliximab, tacrolimus, mycophenolate, and prednisone (BAS/MPS, n = 101). The primary end-point was the incidence of first CMV infection/disease in the intention-to-treat population at 12 months. Patients treated with r-ATG/EVR showed a 90% proportional reduction (4.7% vs. 37.6%, HR 0.10, 95% CI 0.037-0.29; p < 0.001), while those treated with BAS/EVR showed a 75% proportional reduction (10.8% vs. 37.6%, HR 0.25, 95% CI 0.13-0.48; p < 0.001) in the incidence of CMV infection/disease compared to BAS/MPS. There were no differences in the incidence of acute rejection (9.4 vs. 18.6 vs. 15.8%, p = 0.403), wound-healing complications, delayed graft function, and proteinuria. Mean estimated glomerular filtration rate was lower in BAS/EVR (65.7 ± 21.8 vs. 60.6 ± 20.9 vs. 69.5 ± 21.5 ml/min, p = 0.021). In de novo kidney transplant recipients receiving no pharmacological CMV prophylaxis, reduced-dose tacrolimus and everolimus was associated with a significant reduction in the incidence of CMV infection/disease compared to standard tacrolimus dose and mycophenolate (ClinicalTrials.gov NCT01354301).

The HLA-A*02:481 allele was identified in unrelated Brazilians sharing HLA-B*15:17, C*07:01P, DRB1*13:02 and DQB1*06:04.

HLA-A*02:481 differs from HLA-A*02:01:01:01 by one nucleotide and was found in four unrelated Brazilians.

Minor H antigen matches and mismatches are equally distributed among recipients with or without complications after HLA identical sibling renal transplantation.

Studies of the effect of minor H antigen mismatching on the outcome of renal transplantation are scarce and concern mainly single center studies. The International Histocompatibility and Immunogenetics Workshops (IHIW) provide a collaborative platform to execute crucial large studies. In collaboration with 16 laboratories of the IHIW, the role of 15 autosomal, 10 Y-chromosome encoded minor H antigens and 3 CD31 polymorphisms, was investigated in relation to the incidence of renal graft rejection and graft loss in 444 human leukocyte antigens (HLA)-identical sibling renal transplantations. Recipient and donor DNA samples were genotyped for the minor H antigens HA-1, HA-2, HA-3, HA-8, HB-1, ACC-1, ACC-2, SP110, PANE1, UGT2B17, C19Orf48, LB-ECGF-1, CTSH, LRH-1, LB-ADIR and HY. The correlation between minor H antigen mismatch and the primary outcome graft rejection or graft loss was statistically analyzed. The incidence of rejection was very low and no correlation was observed between one or more minor H antigen mismatch(es) and a rejection episode (n = 36), of which only eight resulted in graft loss. In summary, in our study cohort of 444 renal transplants, mismatching for neither autosomal nor HY minor H antigens correlate with rejection episodes or with graft loss.

Algorithms for the determination of unacceptable HLA antigen mismatches in kidney transplant recipients.

One of the major tasks of human leukocyte antigen (HLA) laboratories is the pretransplant determination of unacceptable HLA antigen mismatches (UAM) in organ transplant recipients. HLA antigen specificities are determined against which the patient has circulating alloantibodies that are expected to harm the transplanted organ. Using the information on UAM, negative crossmatch (XM) prediction or 'virtual XM' is possible when a potential donor's complete HLA typing is available. Before the introduction of solid-phase antibody detection assays, UAM were determined using the complement-dependent cytotoxicity methodology. After the introduction of the single antigen bead technique, however, various UAM determination algorithms have emerged. In this report, six different laboratories worldwide present how they determine UAM in their collective of kidney transplant recipients in the pretransplant phase and proceed thereafter to transplantation.

HLA class II alleles and chronic hepatitis C virus infection.

The aim of this study was to investigate association of human leucocyte antigens (HLA)-DRB1 and DQB1 polymorphisms with hepatitis C virus (HCV) infection and with the occurrence of severe liver fibrosis/cirrhosis in chronically infected patients. Ninety-nine white patients, from southeast Brazil, with confirmed HCV chronic infection were included in the study. Severe fibrosis/cirrhosis (METAVIR scores F3-F4) was present in 49 patients. HLA-DRB1 specificities and DRB1*11 and DQB1* alleles were determined by PCR-SSP, and their frequencies were compared between patients and a control group of 103 healthy white Brazilian individuals. The results confirmed previous reports of the association of DRB1*11 and DQB1*03 with protection from chronic HCV infection, but did not confirm their association with protection from severe fibrosis/cirrhosis. Furthermore, the results suggested that the polymorphic sites on HLA molecules responsible for protection from chronic HCV infection are encoded not only by the DRB1*1101 and DQB1*0301, as suggested in the literature, but also by other DRB1*11 and DQB1*03 alleles. Thus, we hypothesized that the common polymorphic residues shared by different DRB1*11 and/or DQB1*03 alleles might be responsible for selection of viral epitopes for presentation to CD4(+) T cells, leading to an efficient immune response against the virus.

Identification of a novel HLA-B*52 allele in a Brazilian individual: B*52:21.

HLA-B*52:21 differs from the closest, HLA-B*52:01:02, by two nucleotides (CTG → TGG), leading to an amino acid substitution from Leu to Trp at codon 156.

Post-transplant anti-HLA class II antibodies as risk factor for late kidney allograft failure.

The purpose of this study was to prospectively analyze the relationship between the post-transplant anti-HLA class I and/or class II panel reactive antibodies and graft failure due to chronic allograft nephropathy (CAN). We studied 512 first kidney recipients transplanted at a single center, with a graft functioning for at least 3 years. A single blood sample was collected from each patient for antibody evaluation. The median posttransplant time after blood collection was 4.4 years and did not differ between patients with (n = 91) or without anti-HLA antibodies (n = 421). Female gender, pregnancies and blood transfusions were associated with the presence of anti-HLA class I antibodies. Graft function deterioration was associated with anti-HLA class II antibodies. Multivariate analysis showed independent association for creatinine levels (RR = 7.5), acute rejection (RR = 2.6), recipient male gender (RR = 3.6) and anti-HLA class II antibodies (RR = 2.9) and CAN-associated graft loss. In conclusion, the presence of anti-HLA class II antibodies conferred a risk for graft loss before a decline in renal function and increased the risk of graft failure in patients who already had a decline in graft function. Thus, anti-HLA class II antibody monitoring is a useful tool for the management of long-term kidney recipients.

Expression of Fas, FasL, and soluble Fas mRNA in endomyocardial biopsies of human cardiac allografts.

Apoptosis mediated by the Fas/Fas ligand (FasL) has been implicated in rejection of solid organ allografts and it has been recently proposed that soluble forms of Fas could interfere with this interaction, blocking apoptosis. The purpose of this study was to analyze intragraft Fas, FasL, and soluble Fas mRNA levels in relation to acute rejection in cardiac allografts in humans. mRNA levels were determined by quantitative reverse transcriptase-polymerase chain reaction in 42 samples of endomyocardial biopsies obtained from 18 cardiac transplant recipients within the first 6 months after transplantation. FasL and Fas mRNA levels were higher in biopsies with rejection than in biopsies without rejection, and no difference was observed in soluble Fas mRNA. During rejection, there was a positive correlation between the mRNA levels of Fas-FasL, Fas-soluble Fas, and FasL-soluble Fas. During quiescent periods, however, the only correlation observed was between Fas and soluble Fas mRNA levels. In conclusion, our findings do not suggest a role for soluble Fas, confirm the heightened expression of FasL, and indicate, for the first time, an increased expression of Fas in acute rejection of cardiac allografts.

Characterization of CD28, CTLA4, and ICOS polymorphisms in three Brazilian ethnic groups.

Costimulatory molecules CD28, CTLA-4 (cytotoxic T-lymphocyte-associated antigen-4), and ICOS (inducible costimulator) genes lie within the 300-kb chromosome region 2q33. CD28, CTLA-4, and ICOS have been described to be important regulators of T-cell activation. With the objective to study ethnic variations in allelic frequencies and linkage disequilibrium (LD) patterns, we genotyped CD28 intron 3 (+17 T>C), CTLA4 promoter (-319 C>T), CTLA4 exon 1 (+49 A>G), and ICOS 3' UTR (1564 T>C) polymorphisms in white (n = 103), mulatto (n = 97), and black (n = 79) Brazilian healthy individuals. No significant deviations from Hardy-Weinberg equilibrium were found in any of the population samples. A higher frequency of CD28 +17 C allele was detected in white (27%) in comparison with mulatto (15%) and black (13%) (p = 0.005) populations. LD between CD28 +17 C and CTLA4 -319 T alleles was observed in whites (p < 0.0001), mulattos (p = 0.0001), and blacks (p = 0.0002).

Maternal KIR repertoire is not associated with recurrent spontaneous abortion.

BACKGROUND: In view of evidence suggesting an immunological cause of recurrent spontaneous abortions (RSA) and the large number of maternal natural killer (NK) cells present in the pregnant uterus, we investigated the genetic polymorphism of the killer cell immunoglobulin-like receptors (KIR) in women with RSA. METHODS: KIR gene repertoire and KIR2DL4 (a receptor for HLA-G) genotyping were determined by SSP and SSCP respectively, in women experiencing RSA and controls. RESULTS: The KIR repertoire did not differ between RSA patients and controls in terms of: (i) the number of inhibitory receptors; (ii) the number of activating receptors; (iii) the ratio of inhibitory to activating receptors. KIR2DL4, a receptor for HLA-G, has different transmembrane alleles, which produce functionally different phenotypes. The frequency of KIR2DL4 transmembrane genotypes differed significantly between RSA patients and controls (P=0.03). However, although homozygosity for a membrane-bound receptor was more frequent in patients (25%) than controls (10%), other genotypes that would produce the same phenotype were not more frequent in patients than controls. CONCLUSIONS: The data provide little evidence that KIR polymorphism plays a role in predisposition to RSA.

Interleukin-2 gene polymorphism is associated with renal but not cardiac transplant outcome.

It was recently shown that IL-2 gene single nucleotide polymorphism (SNP) at position -330 (G-->T) is related to in vitro cytokine production levels, with the T/T and T/G genotypes being associated with low production and the G/G genotype associated with high production. The objective of this study was to investigate a possible influence of this polymorphism on renal and cardiac allograft outcomes. IL-2 SNP G-T (-330) was determined by PCR-RFLP in 67 recipients of heart allografts and in 63 recipients of renal grafts from HLA-haplo-identical, related donors. A higher frequency of the T/T genotype was observed in renal transplant patients who experienced at least one acute rejection episode during the first 3 months after transplantation than in those without rejection during this period (80% vs 49%, respectively, P <.05). Accordingly, the same genotype tended to be more frequent in renal recipients with a 6-month serum creatinine level above 1.5 mg/dL (median value for the whole group of kidney recipients) than in patients with lower creatinine levels (79% vs 45%, P <.08). Regarding cardiac transplant recipients, no associations were observed concerning acute rejection or graft survival. The finding of the association of T/T but not T/G genotype with acute kidney rejection was unexpected considering that both genotypes were shown to be associated with equal (low) IL-2 in vitro production. Further studies are necessary not only to dissect the nature of IL-2 T/T genotype association with kidney rejection, but also to explain why this genotype does not apparently influence cardiac allograft outcome.

Intragraft activation of genes encoding cytotoxic T lymphocyte effector molecules precedes the histological evidence of rejection in human cardiac transplantation.

BACKGROUND: The purpose of the present study was to investigate transcripts of perforin, granzyme B, and Fas ligand (FasL) in heart transplants undergoing rejection. METHODS: Quantitative reverse transcriptase-polymerase chain reaction was applied for mRNA detection in 29 endomyocardial biopsy specimens from 11 cardiac allograft recipients. RESULTS: The mRNA levels of granzyme B, perforin, and FasL were higher (P<0.05) in biopsy specimens with rejection than in biopsy specimens without rejection (granzyme B, 0.53 vs. 0.09; perforin, 0.34 vs. 0; FasL, 0.57 vs. 0.36). In prerejection biopsy specimens, granzyme B and FasL levels were significantly higher than in biopsy specimens without rejection. Any two of the three transcripts were increased in 100% of prerejection, in 92% of rejection, and in 36% of no rejection biopsy specimens (P<0.04). CONCLUSIONS: The assessment of intragraft levels of cytotoxic T lymphocyte effector molecule mRNA represents a valuable tool in the monitoring of cardiac allograft rejection, especially considering the predictive value for warning of impending acute rejection.

Expression of CD40 ligand, interferon-gamma and Fas ligand genes in endomyocardial biopsies of human cardiac allografts: correlation with acute rejection

The purpose of the present study was to investigate the expression (mRNA) of CD40 ligand (CD40L), interferon-gamma (IFN-gamma) and Fas ligand (FasL) genes in human cardiac allografts in relation to the occurrence of acute cardiac allograft rejection as well as its possible value in predicting acute rejection. The mRNA levels were determined by a semiquantitative reverse transcriptase-polymerase chain reaction method in 39 samples of endomyocardial biopsies obtained from 10 adult cardiac transplant recipients within the first six months after transplantation. Biopsies with ongoing acute rejection showed significantly higher CD40L, IFN-gamma and FasL mRNA expression than biopsies without rejection. The median values of mRNA expression in biopsies with and without rejection were 0.116 and zero for CD40L (P<0.003), 0.080 and zero for IFN-gamma (P<0.0009), and 0.156 and zero for FasL (P<0.002), respectively. In addition, the levels of IFN-gamma mRNA were significantly increased 7 to 15 days before the appearance of histological evidence of rejection (median of 0.086 in pre-rejection biopsies), i.e., they presented a predictive value. This study provides further evidence of heightened expression of immune activation genes during rejection and shows that some of these markers may present predictive value for the occurrence of acute rejection.

Expression of CD40 ligand, interferon-gamma and Fas ligand genes in endomyocardial biopsies of human cardiac allografts: correlation with acute rejection.

The purpose of the present study was to investigate the expression (mRNA) of CD40 ligand (CD40L), interferon-gamma (IFN-gamma) and Fas ligand (FasL) genes in human cardiac allografts in relation to the occurrence of acute cardiac allograft rejection as well as its possible value in predicting acute rejection. The mRNA levels were determined by a semiquantitative reverse transcriptase-polymerase chain reaction method in 39 samples of endomyocardial biopsies obtained from 10 adult cardiac transplant recipients within the first six months after transplantation. Biopsies with ongoing acute rejection showed significantly higher CD40L, IFN-gamma and FasL mRNA expression than biopsies without rejection. The median values of mRNA expression in biopsies with and without rejection were 0.116 and zero for CD40L (P<0.003), 0.080 and zero for IFN-gamma (P<0.0009), and 0.156 and zero for FasL (P<0.002), respectively. In addition, the levels of IFN-gamma mRNA were significantly increased 7 to 15 days before the appearance of histological evidence of rejection (median of 0.086 in pre-rejection biopsies), i.e., they presented a predictive value. This study provides further evidence of heightened expression of immune activation genes during rejection and shows that some of these markers may present predictive value for the occurrence of acute rejection.

Monitoring of intragraft and peripheral blood TIRC7 expression as a diagnostic tool for acute cardiac rejection in humans.

T-cell immune response cDNA 7 (TIRC7) is a recently described T-cell costimulatory molecule that exhibits a central role in T-cell activation in vitro and in vivo. The present study was undertaken to investigate association between intragraft and peripheral blood mononuclear cell (PBMC) TIRC7 mRNA levels and cardiac allograft rejection in humans. TIRC7 gene expression levels were determined by a quantitative-competitive reverse transcriptase-polymerase chain reaction (QC-RT-PCR) in endomyocardial biopsies and in PBMC from cardiac transplant recipients. Biopsies collected during rejection or up to 15 days before rejection showed heightened TIRC7 mRNA expression in comparison with biopsies without rejection. All prerejection and rejection biopsies showed TIRC7 mRNA upregulation, while this was present in only 30% of the biopsies without rejection. Regarding TIRC7 mRNA in PBMC, transplant recipients showed lower levels than healthy individuals and, in contrast to the results obtained in biopsies, the levels were lower during rejection than in rejection-free periods. In summary, TIRC7 mRNA expression levels increase in biopsies and decrease in peripheral blood during acute cardiac rejection. We conclude that intragraft detection of TIRC7 transcripts is a useful tool not only for the diagnosis but also for the prediction of acute heart allograft rejection episodes.