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Aim The aim of this study was to investigate the expression of HLA genes in pre-implantation kidney biopsies (PIB) in relation to graft function after kidney transplantation (Tx) from deceased donors. Methods The analyses were performed using the expression data of PIB from our previous microarray study (paper in submission). Herein we analyzed the expression of HLA class I (A, B, C) and class II (DRA, DQA1, DQB1, DQA2, DQB2, DPA1, DPB1) genes in relation to delayed graft function (DGF) occurrence (27 cases with and 27 without) and graft dysfunction (estimated creatinine clearance ⩽ 45 mL/min) at 1 year (y) post-Tx (10 cases with and 41 cases without graft dysfunction). Finally, the genes were tested in an independent data set from the literature. Results No differences in expression levels of any HLA gene were observed in cases with or without DGF. HLA-B and HLA-C were not differentially expressed between kidneys that presented deteriorated or stable renal function at 1y. On the other hand, kidneys that presented deteriorated function at 1y post-Tx had significantly higher expression of HLA-A ( p = 0.02), -DRA ( p = 0.003), -DQA1 ( p = 0.002), -DQB1 ( p = 0.00002), HLA-DQA2 ( p = 0.0008), -DQB2 ( p = 0.00005), -DPA1 ( p = 0.02), -DPB1 ( p = 0.0006). All these genes, with the exception of HLA-DQA2, were represented in the microarray study performed by Park et al, 2010, in biopsies collected at 1y post-Tx and were also associated with poor outcome, represented by biopsies with interstitial fibrosis/tubular atrophy (IF/TA) and inflammation. Conclusion The association between increased expression of HLA genes in PIB and poor renal function at 1y post-Tx is consistent with the assumption that heightened expression of HLA genes contributes to increase allograft immunogenicity and consequently lowers long-term graft function and survival. Interestingly, among the genes with increased expression were the HLA-DQA2 and -DQB2 genes, whose normal expression pattern has not yet been determined. [ABSTRACT FROM AUTHOR]

Studies the individuals infected with human T-cell lymphotropic virus type I (HTLV-I) in Brazil. Variety of T-cell epitopes that are recognized in the context of specific classes I and II human leucocyte antigen (HLA) molecules expressed by HTLV-I infected-T-cells; Determination of HLA-A, -B and -DR specificities by polymerase chain reaction with sequence-specific primers; Association of HLA-DR8 with adult T-cell leukemia/lymphoma.

Aim False-low and false-negative reactions may occur in the Luminex single-antigen bead assay, particularly when the tested serum is fresh. The purpose of this study was to compare our current method for eliminating inhibitory factors, i.e., heat 56 °C, 1 min (H-1 min), with the methods that most of the laboratories worldwide appear to use, i.e., treatment of the serum with DTT or EDTA. Methods Two fresh sera with PRA I >80% (serum #1 and #2) and two fresh sera with PRA II >80% (serum #3 and #4), both with at least one reaction with >15,000 MFI, were tested with LABScreen® Single Antigen Class I and Class II (One Lambda), respectively. Serum treatments: H-1 min: the sera, already in Luminex plate, were heated, in a thermocycler, at 56 °C for 1 min; DTT: final concentration 5 mM, 37 °C, 30 min; EDTA: 10 mM. Differences ⩾50% in MFIs between treated and native sera were considered relevant. Results The different treatments did not significantly affect the reactions on negative nor positive control beads. Serum #1: 11 beads had higher MFIs in sera treated with H-1 min, DTT or EDTA and all these reactions could be explained by one antibody against an epitope shared by HLA-A2, -68, -69, -24, B-57 and B-58 molecules. Serum #2: no effect of any treatment was observed. Serum #3: six beads (five of them with DQB1 ∗ 03) had higher MFIs in H-1 min and EDTA treated sera (MFIs in native, H-1 min, DTT and EDTA treated sera: 13,854, 22,116, 16,494 and 23,744 respectively). Serum # 4: 18 beads had higher MFIs in sera treated with H-1 min, DTT or EDTA and, surprisingly, six reactions (all with beads with DQA1 ∗ 01/DQB1 ∗ 06) were practically abolished in EDTA-treated serum, being restored by the addition of calcium to the EDTA-treaded serum. Conclusions Heat-1 min, DTT and EDTA treatments were in general very similar regarding their ability to eliminate the inhibitory factors of the serum. We see the following advantages of H-1 min over DTT or EDTA treatments: (a) the same treatment can be used if we want to test for IgM antibodies, which would not be possible with the DTT treatment; and (b) there is no interference with calcium-dependent epitopes, which can happen with EDTA. To the best of our knowledge, this is the first report of a calcium-dependent anti-HLA antibody or, better yet, of an antibody directed against a Ca[2+]-dependent conformational epitope on an HLA molecule. [ABSTRACT FROM AUTHOR]

The study aimed to investigate the impact of HLA‐DPB1 allelic and molecular mismatches on the occurrence of acute rejection (AR) and low 5‐year graft function (5Y‐GF) in first kidney transplant (KT) recipients. This is a single center retrospective study of 130 deceased donor KT recipients transplanted between 2014 and 2016. HLA‐DPB1 allelic MM and the following molecular MM (mMM) were analyzed: expression MM with the high expression G allele in the donor; T cell epitope MM (TCE MM); epitope MM (EMM), considering all six hypervariable regions (EMM‐ABCDEF HVR), or only ABEF regions (EMM‐ABEF HVR); eplet MM (EpMM); antibody‐verified eplet MM (AbVer EpMM); and solvent accessible amino acid MM (SAMM). There was no association of allelic MM with AR or 5Y‐GF. The variables independently associated (Cox regression analyses) with AR were high donor final creatinine, nonpermissive TCE MM, ABCDEF EMM load ≥6, EpMM load ≥6; SAMM load ≥5, and AbVer EpMM load ≥3. No association between any HLA‐DPB1 mMM and 5Y‐GF was observed when all 130 transplant recipients were considered. However, when transplants from expanded criteria donors were excluded, independent associations were detected (logistic regression analyses) with AbVerEpMM load ≥2, SAMM load ≥7, cerebro‐vascular death, donor age, and AR. To our knowledge, this is the first study that shows that some HLA‐DPB1 mMM are associated with AR and low 5Y‐GF in a population of exclusively first kidney transplant recipients. [ABSTRACT FROM AUTHOR]

Aim: To assess the impact of HLA compatibility and of pre-transplant anti-HLA antibodies (Ab) on deceased donor (DD) kidney graft survival (GS). Methods: This study analyzed 2,441 consecutive DD kidney transplants (Tx) performed from Jan 1, 2002 to June 30, 2011. The only exclusion criterium was lack of pre-TX PRA data which occurred in 24 recipients (R). More than 90% of the 2.417 Tx included were performed in a single institution and all histocompatibility tests, except for HLA typing of some donors, were performed in a single laboratory: HLA, by PCR-SSP or PCR-SSO; PRA: by CDC until 2005, thereafter by ELISA and, from 2008 on, by Luminex-based methodology (One Lambda). All Tx were performed with negative T and B CDC crossmatches (XM). Since August 2009 the presence of anti-HLA I or II donor specific antibodies (DSA) detected with Luminex-single antigen beads with MFI >1.500 has been considered a contra-indication to the Tx, in spite of negative T and B XM. Kaplan-Meier GS curves, considering patient death or return to dialysis, were compared with the log-rank test. All the calculations were performed using the database and software package of the Regional Registry of Transplants of the State Secretary for Health. Results: The 8-y GS was 82.4% in Tx with 0 MM HLA-A,B,DR, 73.3%, in Tx with 0-6 MM in R with PRA 0-5%, and 67.3% in Tx with 0-6 MM in R with PRA 6-100% (p< 0.001 in all comparisons). Considering only Tx 1-6 MM performed after August 2009, when Luminex-detected DSA >1.500 MFI contra-indicated the Tx, no difference was observed in GS in R with PRA 0-5% and 6 to 100%. Comparison of 1-y GS of 1-6 MM Tx performed between Aug 2009 and June 2011 (N=1,042) with Tx performed from 2002 to 2005 (N=400) showed no difference in R with PRA 0-5% (90.7 vs 93.4) and higher GS in R with PRA 6-100% (92.3 vs 84.9%, p<0.05). Conclusions: The data support the value of HLA matching in DD kidney Tx and shows the beneficial effect of avoiding Tx with Luminex detected DSA in cases with negative T and B CDC XMs. [Copyright &y& Elsevier]

HLA‐DQB2 is a gene of limited polymorphism, with unknown function that presents at least two transcript variants: v1, which encodes the full‐length beta‐chain, and v2, which lacks exon 4 and could give rise to a soluble protein. We previously showed a strong correlation between high v2 expression in preimplantation biopsies (PIB) of kidneys from young (18‐ to 49‐year olds) but not from old, deceased donors and 1‐year posttransplant low (estimated glomerular filtration rate < 45 ml/min/1.73 m2) graft function (GF). In this study, we aimed to investigate the impact of posttransplant soluble HLA‐DQB2 (sDQB2) serum levels, v1 expression in PIB, and recipient HLA‐DQB2 rs7453920 A/G polymorphism on GF. sDQB2 was evaluated by enzyme‐linked immunosorbent assay in sera from 114 recipients, collected at least 1 year (median 2.1 years) after transplantation. Higher sDQB2 levels were observed in recipients of kidneys from young, but not from old, donors that had a ≥30% decline in GF within 1 year after blood collection for sDQB2 determination. Among the 15 recipients of kidneys from young donors with sDQB2 ≥ 1.52 ng/ml, 40% presented a ≥30% decline in GF, whereas this occurred in none of the 43 recipients with lower sDQB2 levels (p = 0.007; OR: 36.5). Expression of HLA‐DQB2 variant 1, measured by reverse transcription‐polymerase chain reaction (RT‐PCR) in 92 PIB from young or old donors, did not significantly differ between transplants with high or low 4‐year GF. HLA‐DQB2 rs7453920 single nucleotide polymorphism (SNP) frequencies did not significantly differ between recipients with low or high 4‐year GF. We conclude that HLA‐DQB2 variant 1 expression in PIB and recipient rs7453920 SNP polymorphism are not associated with graft outcome. On the other hand, the association, in transplants of kidneys from young donors, between high posttransplant serum sDQB2 levels and decline in GF is a very interesting finding that deserves a validation study in a larger cohort. [ABSTRACT FROM AUTHOR]

Abstract: The TNF superfamily ligands BAFF and APRIL and receptors BCMA, TACI and BAFF-R play an important role in the regulation of B cell immunity. A number of functionally important splice isoforms have already been characterized for these molecules, stimulating the search for new transcript variants (TVs). Here we report two new BAFF TVs and three BCMA TVs, all potentially codifying new proteins. BAFF TVs were expressed in peripheral blood mononuclear cells (PBMC) of nearly all the individuals studied, decreasing in level when PBMC were activated by PMA and ionomycin. They were also detected in PBMC cytoplasmic RNA. Low levels of the BAFF TVs in all lymphocyte subpopulations analyzed suggest that their main source in PBMC are monocytes. BCMA TVs were observed only in some CD19+ cell samples. Functional studies concerning interaction between isoforms of BAFF, APRIL and their receptors are needed for elucidation of their significance in the immune response. [Copyright &y& Elsevier]

The purpose of this single center retrospective study was to investigate the relationship between HLA and ABO polymorphisms and COVID‐19 susceptibility and severity in kidney transplant recipients. It included 720 recipients who had COVID‐19 and 1680 controls composed by recipients in follow‐up who did not contact the transplantation center for COVID‐19 symptoms, up to the moment of their inclusion in the study. HLA‐A, ‐B, and ‐DRB1 allele groups and ABO frequencies were compared between recipients with COVID‐19 (all cases, or separately mild/moderate and severe disease) and controls. The HLA association study was conducted in two case–control series and only associations that showed a p‐value <0.05 in both series were considered. No HLA association regarding COVID‐19 occurrence or severity met this criterion. Homozygosity at HLA‐A locus was associated with COVID‐19 susceptibility (odds ratio 1.4) but not severity. Blood groups A and O were associated with susceptibility and resistance to COVID‐19, respectively. COVID‐19 severity was associated only with older age and cardiac disease, in a multivariate analysis. We conclude that an influence of HLA on COVID‐19 susceptibility is supported by the association with homozygosity at HLA‐A locus but that there is no evidence for a role of any particular HLA‐A, ‐B, or ‐DRB1 polymorphism. Thus, we suggest that what matters is the overall capability of an individual's HLA molecules to present SARS‐CoV‐2 peptides to T cells, a factor that might have a great influence on the breadth of the immune response. [ABSTRACT FROM AUTHOR]

Optimizing antithymocyte globulin (rATG) dosage is critical for high immunological risk patients undergoing a repeat kidney transplant. This natural retrospective cohort study compared clinical outcomes of two successive cohorts of consecutive recipients of retransplants receiving 5 x 1 mg/kg (rATG-5, n = 100) or a single 3 mg/kg (rATG-3, n = 110) dose of rATG induction therapy. All patients had negative complement-dependent cytotoxicity crossmatch and no anti-HLA A, B, DR donor-specific antibodies (DSA). The primary endpoint was efficacy failure (first biopsy-proven acute rejection, graft loss, or death) at 12 months. There was no difference in the cumulative incidence of efficacy failure (18.0% vs. 21.8%, HR = 1.22, 95% CI 0.66–2.25), respectively. There were no differences in 3-years freedom from biopsy proven acute rejection, and patient, graft, and death-censored graft survivals. There were no differences in the incidence of surgical complications (25.0% vs. 18.2%; p 0.151), early hospital readmission (27.8% vs. 29.5%; p = 0.877) and CMV infections (49% vs. 40%; p = 0.190). There were also no differences in the incidence (59.6% vs. 58.7%, p = 0.897) and duration of delayed graft function but a stable difference in estimate glomerular filtration rate was observed from month 1 (54.7±28.8 vs. 44.1±25.3 ml/min/1.73 m2, p = 0.005) to month 36 (51.1±27.7 vs. 42.5±24.5, p = 0.019). Mean urinary protein concentration (month 36: 0.38±0.81 vs. 0.70±2.40 g/ml, p = 0.008) and mean chronic glomerular Banff score in for cause biopsies (months 4–36: 0.0±0.0 vs. 0.04±0.26, p = 0.044) were higher in the rATG-3 group. This cohort analysis did not detect differences in the incidence of efficacy failure and in safety outcomes at 12 months among recipients of kidney retransplants without A, B, and DR DSA, receiving induction therapy with a single 3 mg/kg rATG dose or the traditional 5 mg/kg rATG. [ABSTRACT FROM AUTHOR]

Highlights from the article: The presence of the monoclonal antibody (mAb) rituximab (RIT) in the serum causes false-positive B cell crossmatch results, due to binding of the antibody to CD20 molecules on the B cell surface. The addition of anti-RIT to the serum completely abolished the false-positive B cell XM results and did not interfere with T cell XM or with HLA antibody detection.

Summary: The optimal immunosuppressive regimen for recipients of expanded criteria donor (ECD) kidneys has not been identified. In this single‐center study, 171 recipients of ECD kidney transplants were randomized to receive antithymocyte globulin induction, and delayed introduction of reduced dose tacrolimus, prednisone and everolimus (r‐ATG/EVR, n = 88), or mycophenolate (r‐ATG/MPS, n = 83). No cytomegalovirus (CMV) pharmacological prophylaxis was used. The primary endpoint was the incidence of CMV infection/disease at 12 months. Secondary endpoints included treatment failure [first biopsy‐proven acute rejection (BPAR), graft loss, or death] and safety. Patients treated with EVR showed a 89% risk reduction (13.6 vs. 71.6%; HR 0.11, 95% CI 0.06–0.220, P < 0.001) in the incidence of first CMV infection/disease. Incidences of BPAR (16% vs. 5%, P = 0.021), graft loss (11% vs. 1%, P = 0.008), death (10% vs. 1%, P = 0.013), and treatment discontinuation (40% vs. 28%, P = 0.12) were higher in the r‐ATG/EVR, leading to premature study termination. Mean glomerular filtration rate was lower in r‐ATG/EVR (31.8 ± 18.8 vs. 42.6 ± 14.9, P < 0.001). In recipients of ECD kidney transplants receiving no CMV pharmacological prophylaxis, the use of everolimus was associated with higher treatment failure compared with mycophenolate despite the significant reduction in the incidence of CMV infection/disease (ClinicalTrials.gov.NCT01895049). [ABSTRACT FROM AUTHOR]

Background: Accurate pre-transplant prediction of late graft function remains an unmet need in kidney transplantation. The aim of this study was to evaluate HLA genes expression levels in pre-implantation biopsies (PIB) of deceased donor kidneys as markers for long-term graft outcome. Methods: HLA genes expression analysis was initially performed using microarray data of 53 PIB, previously generated by our laboratory. The validation analysis was performed by real-time PCR in 116 PIB from an independent cohort. Results: The microarray data showed association between high expression levels of HLA class II genes, especially HLA-DQB1 and -DQB2, in kidneys from young (18 to 49-year-old) donors and poor (eGFR < 45 mL/min/1.73 m 2 ) 1- and 5-year graft function. A subsequent study in an independent cohort, in which only HLA-DQB2 expression was evaluated, validated the association between increased HLA-DQB2 expression in PIB of kidneys from young donors and poor 1-year graft function: expression levels ≥0.0025 relative units conferred an odds ratio of 22.5, with positive and negative predictive values of 71.4% and 90.0%, respectively. Conclusion: Heightened expression of HLA-DQB1 and -DQB2 in PIB are promising tools for pre-transplant risk assessment of poor late graft function in transplants with kidneys from 18 to 49-year-old donors. [ABSTRACT FROM AUTHOR]

Background Soluble CD30 (sCD30) is a suggested marker for kidney transplantation outcomes. We investigated whether sCD30 serum levels are influenced by immunosuppression and whether they correlate with findings in protocol biopsies and with CD30 gene expression in peripheral blood mononuclear cells (PBMC). Methods We studied 118 kidney transplant recipients that initially received tacrolimus (TAC) and, at month-3, were converted or not to sirolimus (SRL). Results sCD30 serum levels gradually declined after transplantation, being the decline more pronounced in the SRL group. CD30 gene expression in PBMC was higher in the SRL group than in the TAC group. Patients with IF/TA ≥ I in the month-24 protocol biopsy had higher sCD30 levels than patients without IF/TA, in the SRL group (P = .03) and in the TAC group (P = .07). CD30 + cells were observed in three out of 10 biopsies with inflammatory infiltrate from the SRL group. In mixed lymphocyte cultures, SRL and TAC diminished the number of CD30 + T cells and the sCD30 levels in the supernatant, but the effect of SRL was stronger. Conclusions Overall, sCD30 levels are lower in SRL-treated patients, but the association between increased sCD30 levels and IF/TA at month-24 post-transplantation is stronger in SRL than in TAC-treated patients. [ABSTRACT FROM AUTHOR]

The purpose of this study was to investigate possible markers for predicting delayed graft function (DGF). To this end we analyzed, in pre-implantation biopsies (PIB) and in first-day post-Tx peripheral blood mononuclear cells (PBMC), the expression of five genes ( ACSL4 , CUBN , DEFB1 , FABP3 , GK ) through real-time TaqMan PCR assays. These genes were selected from a large scale gene expression study in PIB. DEFB1 , FABP3 and GK expression levels in PIB were lower in cases with DGF and, in a multivariate analysis which included these genes and clinical variables, only FABP3 expression remained independently associated with DGF. FABP3 expression lower than −1.32 units of relative expression conferred an odds ratio for DGF of 41.1. Compared to the PBMC of recipients without DGF, recipients with prolonged DGF (pDGF) had lower ACSL4 and higher DEFB1 expression levels. In a multivariate analysis, including PBMC gene expression levels of ACSL4 , DEFB1 and TLR4 (data from a previous study with the same patients) and clinical variables, only TLR4 remained independently associated with pDGF. In summary, this study revealed FABP3 expression in PIB as a marker for DGF and disclosed new genes possibly involved in the pathogenesis of DGF. [ABSTRACT FROM AUTHOR]

Several studies have shown association of high pre- or post-transplant levels of soluble CD30 (sCD30) with acute rejection and poor late kidney transplant outcome. Our goal was to investigate whether sCD30 levels at month-3 post-transplant are associated with subclinical rejection, presence of CD30 + cells within the graft, and expression of immune response genes in peripheral blood mononuclear cells. The study comprised 118 adult first kidney graft recipients, transplanted at a single center, receiving tacrolimus in low concentration. All were submitted to a protocol biopsy at month-3. Subclinical rejection was identified in 10 biopsies and sCD30 levels ≥ 61.88 ng/mL (P = 0.004), younger recipient age (P = 0.030) and non-Caucasian ethnicity (P = 0.011) were independently associated with this outcome. Rare CD30 + cells were present in only two biopsies. There was a correlation between sCD30 levels and CD30 gene expression in peripheral blood mononuclear cells (r = 0.385, P = 0.043). These results show that high sCD30 levels are independent predictors of graft dysfunction and may contribute to patient selection protocols by indicating those who could benefit from a more thorough evaluation. [ABSTRACT FROM AUTHOR]

The purpose of this study was to evaluate the association of post-transplant soluble CD30 (sCD30) levels, isolated or in combination with of anti-HLA class II antibodies and of serum creatinine levels, with kidney graft loss due to chronic allograft nephropathy (CAN), and type of lesions in graft biopsies for cause. The study comprised 511 first kidney graft recipients, transplanted at a single center, with a graft functioning for at least 2.8 years. A single blood sample was collected from each patient sCD30 levels were determined by ELISA and HLA antibodies by Luminex assay. The minimum follow-up after testing was 9.3 years. High sCD30 levels, set at sCD30 >34.15 ng/mL the presence of HLA class II antibodies, and serum creatinine >1.9 mg/dL were independently associated with CAN-graft loss (P values <0.0001, 0.05, <0.0001, respectively), and the combined hazard ratio for CAN-graft loss was 20.2. Analyses of 166 biopsies for cause showed that high sCD30 levels and creatinine were independently asso-ciated with interstitial lesions. Post-transplant sCD30 serum levels, especially in conjunction with information regard-ing HLA class II antibodies and serum creatinine levels, provide valuable information regarding graft outcome and could be useful for the management of kidney transplant recipients. [ABSTRACT FROM AUTHOR]

Abstract: False-positive anti-HLA reactions may occur in Luminex-single antigen (SA) beads assays, and it is important to recognize them to correctly interpret the test. The purpose of this report is to describe a peculiar pattern of reactivity, characterized by positivity with beads coated with HLA-DRB1∗09:01, DRB3∗01:01, DRB3∗02:02, DRB3∗03:01, DPB1∗02:01, DPB1∗20:01 and DPB1∗28:01, that was observed in 141 of 8121 serum samples tested in our laboratory with three different lots of the same kit (LABScreen® SA, One Lambda). These 141 serum samples came from 56 different patients on the kidney transplant waiting list, corresponding to 1% of the patients. Of these, 10 males had never been transfused or transplanted. About 66% of the patients had positive reactions against self-antigen HLA-DRB3 alleles. No reactions against native HLA-DRB1∗09:01 were observed in flow cytometry crossmatch and in absorption/elution experiments, leading to the conclusion that the reactivity was due to antibodies against epitopes present in denatured forms of HLA-class II antigens. The occurrence of this reactivity pattern was associated with female gender and systemic lupus erythematosus (SLE). [Copyright &y& Elsevier]

The present study assessed, in cervical carcinoma, expression levels of seven immune response genes and sought correlation to response to treatment. The expression levels of CD28, CTLA4, ICOS, ICOSL, CD80 and CD86 and granzyme B genes were assessed by real-time RT-PCR in pre-treatment tumor fragments. During the six-month follow-up after treatment, 8 patients presented tumor and 10 survived free of tumor. The only gene whose expression levels were higher in patients with poor outcome (p = 0.03) was granzyme B. Further evaluation, in adequately powered prospective studies is warranted to confirm the data and to translate this observation to the clinical setting. [ABSTRACT FROM AUTHOR]

Abstract: Lymphotoxin alpha (LTA) is a member of the TNF cytokine superfamily, produced principally by lymphocytes. It plays an important role in immune and inflammatory responses. Many TNF superfamily members have functionally important isoforms generated by alternative splicing but alternative splicing of LTA has never been studied. The known LTA protein is encoded by a transcript containing four exons. Here we report seven new LTA splice variants, three of them evolutionary conserved. We demonstrate their presence in cytoplasmic RNA suggesting that they could be translated into new LTA isoforms. We observed that their expression is differentially regulated upon activation of peripheral blood mononuclear cells and lymphocyte subpopulations (CD4+, CD8+, and CD19+). Our data suggest that the new LTA splice variants might play a role in the regulation of the immune response. [Copyright &y& Elsevier]

Various single nucleotide polymorphisms (SNPs) have been investigated regarding association with gene expression levels or human diseases. Although different SNPs within one gene are frequently analyzed individually, it is highly probable that in the majority of the cases, a precise combination of SNP alleles, i.e., haplotype, determines a functional trait. Methods commonly used for haplotype determination, involving studies in families, cloning, or somatic cell hybrids, are expensive and time-consuming. We herein suggest a novel and simple strategy for haplotype determination, involving selective haplotype depletion with a restriction enzyme, followed by sequencing. We studied 11 LTA gene polymorphisms in 102 Brazilian individuals, and we applied this novel methodology for haplotyping 67 out of 70 LTA heterozygous individuals. We concluded that the method is rapid and efficient, and, as it includes only simple and widespread-used techniques, it could be used in most of the laboratories without further investment in equipments. The wider usage of haplotyping could be important to clarify contradictory results frequently observed among studies that focus on a single SNP. [ABSTRACT FROM AUTHOR]

Abstract: Use of internal reference gene(s) is necessary for adequate quantification of target gene expression by RT-PCR. Herein, we elaborated a strategy of control gene selection based on microarray data and illustrated it by analyzing endomyocardial biopsies with acute cardiac rejection and infection. Using order statistics and binomial distribution we evaluated the probability of finding low-varying genes by chance. For analysis, the microarray data were divided into two sample subsets. Among the first 10% of genes with the lowest standard deviations, we found 14 genes common to both subsets. After normalization using two selected genes, high correlation was observed between expression of target genes evaluated by microarray and RT-PCR, and in independent dataset by RT-PCR (r =0.9, p <0.001). In conclusion, we showed a simple and reliable strategy of selection and validation of control genes for RT-PCR from microarray data that can be easily applied for different experimental designs and tissues. [Copyright &y& Elsevier]

Abstract: Two transcript variants (TV) of the T cell immune regulator gene 1 (TCIRG1) have already been characterized. TV1 encodes a subunit of the osteoclast vacuolar proton pump and TV2 encodes a T cell inhibitory receptor. Based on the search in dbEST, we validated by RT-PCR six new alternative splice events in TCIRG1 in most of the 28 human tissues studied. In addition, we observed that transcripts using the TV1 transcription start site and two splice forms previously described in a patient with infantile malignant osteopetrosis are also expressed in various tissues of healthy individuals. Studies of these nine splice forms in cytoplasmic RNA of peripheral blood mononuclear cells showed that at least six of them could be efficiently exported from the nucleus. Since various products with nearly ubiquitous tissue distribution are generated from TCIRG1, this gene may be involved in other processes besides immune response and bone resorption. [Copyright &y& Elsevier]

: BackgroundThe purpose this study was to investigate the relationship of anti-myosin and anti–heat shock protein immunoglobulin G (IgG) serum antibodies to the original heart disease of cardiac transplant recipients, and also to rejection and patient survival after cardiac transplantation.: MethodsAnti-myosin and anti–heat shock protein (anti-hsp) IgG antibodies were evaluated in pre-transplant sera from 41 adult cardiac allograft recipients and in sequential post-transplant serum samples from 11 recipients, collected at the time of routine endomyocardial biopsies during the first 6 months after transplantation. In addition, the levels of these antibodies were determined from the sera of 28 healthy blood donors.: ResultsHigher anti-myosin antibody levels were observed in pre-transplant sera than in sera from normal controls. Moreover, patients with chronic Chagas heart disease showed higher anti-myosin levels than patients with ischemic heart disease, and also higher levels, although not statistically significant, than patients with dilated cardiomyopathy. Higher anti-hsp levels were also observed in patients compared with healthy controls, but no significant differences were detected among the different types of heart diseases. Higher pre-transplant anti-myosin, but not anti-hsp, levels were associated with lower 2-year post-transplant survival. In the post-transplant period, higher anti-myosin IgG levels were detected in sera collected during acute rejection than in sera collected during the rejection-free period, whereas anti-hsp IgG levels showed no difference between these periods.: ConclusionsThe present findings are of interest for post-transplant management and, in addition, suggest a pathogenic role for anti-myosin antibodies in cardiac transplant rejection, as has been proposed in experimental models of cardiac transplantation. [Copyright &y& Elsevier]

The present study investigated gene expression of costimulatory molecule CD27 in relation to the occurrence of acute cardiac rejection. CD27 transcripts were measured by means of quantitative competitive reverse transcriptase–polymerase chain reaction in 120 endomyocardial biopsies and in 89 samples of blood mononuclear cells from 31 recipients. Higher levels of CD27 transcripts were observed in biopsies with rejection than in samples without rejection (medians, 7.1 and 1.9; P = 0.06). In contrast, blood mononuclear cells collected during rejection showed lower levels than blood mononuclear cells from rejection-free periods (medians, 3.3 vs. 7.9; P = 0.03). Considering only endomyocardial biopsies without rejection, the values were lower in samples from recipients who did not present any rejection during the first 6 months after transplantation than in those from recipients who had at least one rejection during the same period (medians, 0 vs. 3.5, P < 0.001; percentage of biopsies expressing CD27, 44% vs. 77%). In conclusion, the presence of intragraft CD27 mRNA may identify recipients at risk for developing acute rejection. [Copyright &y& Elsevier]

In the present study we determined the HLA-A, B and DR antigenic and haplotypic frequencies in unrelated Brazilian Caucasian paediatric patients with IgA deficiency (IgA-D). Out of 17 IgA-D subjects typed for HLA A and B specificities, 12 (71%) presented B8 and/or B14; of 15 patients also typed for HLA-DR specificities, 14 (93%) were positive for at least one of the HLA markers previously reported to be associated with IgA-D, i.e. B8, B14, DR1, DR3 or DR7. The haplotypes B8, DR3, B14, DR1 and B13, DR7 were present in 43, 21 and 14% of the cases, respectively, while they have a frequency in the general population of 2, 2 and 1%, respectively. The concomitant association with the three IgA-associated haplotypes found in our study probably reflects the admixture of European genetic influences present in the Brazilian Caucasian population of São Paulo. [ABSTRACT FROM AUTHOR]

Highlights from the article: We have previously showed that decreased FABP3 expression, in preimplantation kidney biopsies (PIB), was associated with delayed graft function (DGF) (Mourão et al., 2016). In a multivariate analysis, including clinical variables related to DGF, as well as FABP3 expression, only TMEM52B expression (p = 0.010) and donor last creatinine (DCr) (p = 0.001) remained independently associated with DGF.

The best available unrelated donor (URD) for a patient with acute lymphoblastic leukemia in need of hematopoietic stem cell transplant was HLA 11/12 with a DPB1*04:01 mismatch. Search for donor-specific HLA antibodies (DSA) by solid phase assay with isolated HLA molecules (SAB) on a first panel (manufacturer 1) identified a DSA against DPB1*04:01 (MFI = 3090). Epitope analysis using HLA Fusion and HLAMatchmaker was not conclusive due to an atypical 85GPM epitope pattern, showing positive reactions with DPB1*04:01 (MFI = 3090), DP2 (MFI = 1655), DPB1*04:02 (MFI = 4571), DP23 (MFI = 1549) and DP28 (MFI = 1409, 1324, 1256), but lack of reactivity with DP15 and DP18, which also express 85GPM. The DP α 31 M + β 85GPM + β 96 R configuration, obtained with the HLA Epitopia Map, justified the observed pattern because it is present in the DP4, DP2, DP23 and DP28 heterodimers that reacted positively, but absent in DP15 ( α 31 Q + β 85GPM + β 96 K ) and DP18 ( α 31 Q + β 85GPM + β 96 K and α 31 M + β 85GPM + β 96 K ) whose reactions were negative. To validate this α / β epitope predicted in silico , the serum was tested with an SAB panel which included five distinct DP4 heterodimers (manufacturer 2) composed of DPB1*04:01 combined to different alpha chains. The result showed that only heterodimers with α 31 M + β 85GPM + β 96 R configuration were reactive: DPA1*01:03/DPB1*04:01 (MFI = 3609), DPA1*03:01/DPB1*04:01 (MFI = 3778) and DPA1*04:01/DPB1*04:01 (MFI = 3750). The α 31 Q + β 85GPM + β 96 R heterodimers showed negative reactions (DPA1*02:01/DPB1*04:01, MFI = 298; DPA1*02:02/DPB1*04:01, MFI = 94) (Table). Patient was transplanted with this URD without previous desensitization due to the weak reactivity of this antibody. This DP αβ DSA did not show clinical relevance in the rejection vector, since there was hematological engraftment on day +26 and chimerism with 100% donor cells on day +30. This report suggests the presence of a new conformational DP αβ epitope and shows that HLA Epitopia Map tool contributes to the elucidation of atypical reactivity patterns in SAB solid phase assays. [ABSTRACT FROM AUTHOR]

The purpose of the present communication is to report a new epitope that explains the reaction against HLA-B∗18:01 of an antibody produced by an individual carrying HLA-B∗18:05. This reactivity was detected in the Luminex-single antigen assay (LSA) and could not be explained by the HLA MatchMaker algorithm. Comparison between amino acid sequences in alfa-1, −2 and −3 domains of the two B∗18 alleles revealed that the sole difference was in position 127: 127 K, in B∗18:05, and 127 N, in B∗18:01. Interestingly, the residue 127 N was also present in the self-alleles A∗03:01, B∗44:02, C∗05:01 and C∗12:03. We then proceeded to search for combinations between 127 N and residues within a 15 angstrom radius and we found that the combination of the self-residues 127 N and 113H was present in B∗18:01, as well as in other antigens that reacted with the antibody: B∗07:02, B∗08:01, B∗13:01/02, B∗15:01/03/10/11/12/16, B∗35:01, B∗38:01, B∗39:01, B∗40:01/02/06, B∗41:01, B∗42:01, B∗46:01, B∗48:01, B∗51:01/02, B∗52:01, B∗53:01, B∗54:01, B∗55:01, B∗56:01, B∗57:01/03, B∗58:01, B∗59:01, B∗67:01, B∗78:01, B∗81:01, B∗82:01, C∗15:02. On the other hand, this combination was not present in any of the self-antigens. The serum with the B∗18:01 antibody produced by the individual typed as HLA-A∗02,03; B∗18:05,44:02; C∗05,12 was absorbed with a cell typed as HLA-A∗02,03; B∗18:01,-; C∗05,12 and the antibodies recovered in the eluate reacted only with the beads that contained HLA molecules with the presently described epitope (127 N + 113H). We conclude that the amino acid sequences comparison that led us to describe this interesting new epitope, formed by two self-amino acids, represents an additional useful tool for understanding epitopes recognized by the antibodies. [ABSTRACT FROM AUTHOR]

The purpose of this report is to describe a new epitope present on denatured HLA class I molecules. Serum from a kidney transplant candidate, male, and with only one previous blood transfusion was tested with the IgG-binding assay with single-antigen beads on a Luminex platform (LSA) (One Lambda - OL). The following positive reactions were observed: HLA-B- 13, 18, 37, 41, 44, 45, 47, 48, 49, 50, 52, 60, 61, 62, 72, 75, 76, 77. This reaction pattern could be explained by the HLA-Matchmaker eplet 66IS (65Q66I67S69T) which is considered an exposed eplet ( http://www.epregistry.com.br ). The serum was tested with flow-cytometry using three different cells carrying one of these specificities: HLA-B37 (Ac with MFI of 5250), B60 (Ac with MFI of 3661) or B50 (Ac com MFI of 4752) and the results were negative. In addition, no positive reactions were observed when the serum was tested with a LSA kit from another vendor. In order to investigate whether the OL Luminex reactions were false-positive, the serum was tested with OL beads bearing HLA molecules denatured by acid treatment and the same reactions were observed, although with higher MFI values. The reaction pattern observed on the beads with denatured HLA could not be explained by any cryptic epitope described by El-Awar, 2009. In order to search for the targeted epitope on the denatured HLA molecules, we analyzed the amino acid sequences and we found that the position 67S, cryptic in the HLA molecule, was present only in the molecules with which the serum reacted. In fact, this position is included in the polymorphic residues of HLAMatchMaker eplet 66IS. This finding underlines that not all reactions that can be explained by the HLA-Matchmaker are true-positive reactions. [ABSTRACT FROM AUTHOR]

Aim This study sought to investigate the association between single nucleotide polymorphisms (SNPs) in genes coding for molecules involved in ischemia–reperfusion injury and the occurrence of delayed graft function (DGF). Methods Using a prioritization system (SNPLogic - www.snplogic.org ) based on SNP’s potential to influence gene expression and their allelic frequencies in Caucasian populations, we selected SNPs in the following genes: ATF3, BAX, BCL2, CLCN6, CX3CL1, CXCL1, CXCL10, EDN1, EPAS1, FABP1, GSTP1, HAVCR1, HMOX1, ICAM1, IL8, JAG1, LGALS3, MCPH1, NGAL, NOS2, PARP1, SELP, SHROOM3, SPATA5L1, STC1, TLR4, UMOD and VEGFA. The study included 579 transplants performed in a single center with kidneys from deceased donors. SNPs were determined in 312 donors and 466 recipients of predominantly Caucasian ancestry. DGF was defined as the need for dialysis in the first week after transplantation, and occurred in 303 transplants. TaqMan® OpenArray™ System was used to genotype 29 SNPs in 28 genes. Results Only the SNP rs2146323, +7149 A > C intron 2 in VEGFA of donors (AA/AC vs CC genotypes: p = 0.0004, OR = 2.310, 95% CI = 1.465–3.643) and the SNP rs1871042, +2778 T > C intron 6 in GSTP1 of recipients (TT/TC vs CC genotypes: p = 0.0066, OR = 1.680, 95% CI = 1.159–2.435) were associated with DGF. These two SNPs are located at predicted transcription factors binding sites. A multiple regression analysis including only clinical data showed that only donor serum creatinine (Cr) > 1.5 mg/dL before kidney removal remained associated with DGF (p = 0.0005, OR = 1.544, 95% CI = 1.304–1.829). A posterior multiple regression analysis, including the final Cr of the donor and the VEGFA and GSTP1 SNPs, revealed that all three variables were independently associated with DGF. Conclusions Out of 29 SNPs investigated, only one SNP in the gene VEGFA (vascular endothelial growth factor A) of the donor, and one SNP in the gene GSTP1 (glutathione S-transferases P1) of the recipient were found to be significantly associated with DGF. [ABSTRACT FROM AUTHOR]

Aim: The aim of this study was to investigate the relationship between soluble CD30 (sCD30) serum levels and lesions in protocol graft biopsies (PBx), in recipients (R) of living or deceased donor kidney transplant (Tx), under triple immunosuppressive therapy including Tacrolimus (TAC) or Sirolimus (SRL). Methods: All Tx were performed with negative T/B CDC crossmatches, and without donor-specific antibodies (DSA) detected with Luminex-single antigen assay. Up to the third month post-Tx, all R (n=169) received TAC/PRED/MPS. At this point, a PBx was performed and R with evidence of acute rejection in PBx or in previous biopsies for cause, proteinuria/creatinine (P/Cr) >0.05, estimated glomerular filtration rate (eGFR) <40 mL/min or any active post-operative complication were excluded. Among the R that persisted in the study, 58 continued to be treated with TAC/PRED/MPS and 60 were converted to SRL/PRED/MPS. Results: At 2 years (y) post-Tx, 53/58 TAC-R and 52/60 SRL-R had a functioning graft. In both groups post-Tx sCD30 levels (ELISA) gradually decreased. The decrease was more pronounced in the SRL group, resulting in lower 2 y post-Tx levels than in TAC group (15.3 x 24.6 U/mL, p<0.0001). The proportion of R with P/Cr ⩾0.15 was higher in the SRL group (59.3% x 23.6%, p=0.001), and no significant differences were observed concerning eGFR, serum Cr or DSA appearance. PBx were performed in 100/105 R at 2 y post-Tx, and graded according to Banff’07. The SRL group had a higher proportion of biopsies with total inflammation (ti) score ⩾1% (61.7% x 35.4%, p<0.05) and IF/TA ⩾I (70.2% x 45.8%, p<0.02). Furthermore, in the SRL group sCD30 levels differ among R without IF/TA and ti-score 0%, with IF/TA I, and with IF/TA II/III (median sCD30 levels of 10.5, 15.8 and 25.6 U/mL, respectively, p=0.02). Conclusions: In sum, the most interesting observations of our study were the lower sCD30 levels in SRL-treated than in TAC-R and the association of sCD30 levels with fibrosis and inflammation in PBx in SRL-treated R. [Copyright &y& Elsevier]

Aim: Delayed graft function (DGF) remains a major problem in solid organ transplantation, as it adversely impacts both short and long term outcomes. There is evidence that activation of the innate immune system triggered by “danger signals” associated with brain death and/or ischemia-reperfusion injury (IRI) plays a significant role in the pathogenesis of DGF. The objective of this work was to investigate association between expression of the innate immunity genes TLR4, TLR2, MYD88, TRIF, SARM1, NOD1 and NOD2 in deceased donor (DD) grafts and occurrence of DGF. Methods: Expression levels were evaluated in pre-implantation biopsies (PIB) from kidneys of 72 DD and 18 living donors (LD). DGF was defined as dialysis requirement within the first week after transplantation (Tx). Total RNA was extracted from PIB with RNeasy Mini Kit (Qiagen). Relative expression (2-ΔΔCt method) of the innate immunity genes and TATA-binding protein (TBP, used as internal control) were obtained by Taqman assays (Applied Biosystems). Statistics: Mann-Whitney and Fisher’s exact tests; significance was set at p<0.05. Results: SARM1 and TRIF expression did not differ between DD and LD, neither between cases with and without DGF. Expression levels of TLR4, TLR2, MYD88, NOD1 and NOD2 were higher in DD than in LD kidneys, but only NOD1 expression was higher in DD transplants with than without DGF (1.7 vs 1.4, p=0.03). Conclusions: Overall, these data emphasize the involvement of innate immunity in the physiopathology of kidneys from DD; the fact that TRIF was not differentially expressed in DD kidneys suggests that TLR4 signaling in this setting occurs preferentially through MyD88-dependent pathway; among all genes tested, only the expression of NOD1 showed association with the occurrence of DGF. [Copyright &y& Elsevier]

Aim: To determine the clinical relevance in kidney transplantation (Tx) of preformed HLA donor specific antibodies (DSA) revealed by Luminex single antigen beads assay (SAB). Methods: We analyzed 298 recipients (R) transplanted in a single center, between 2001 and 2006, with negative T and B CDC crossmatches (XM) and without any information regarding Luminex-detected antibodies. Stored pre-Tx sera of 165 R with ELISA PRA 0% and of 133 R with ELISA PRA 50% were screened with Labscreen Mixed and the positive ones were tested with the SAB assay (One Lambda). In the SAB assay, reactions with MFI >300 were recorded as positive; DSAs were defined as Abs against HLA-A, B or DR mismatches. Graft survival (GS) curves (Kaplan-Meier) were compared with the log-rank test; Cox regression analysis was used to calculate risk of graft loss. Results: No difference in GS was detected among R with PRA ELISA and Luminex 0% (N=165), PRA ELISA 50% without DSA (N=44), PRA ELISA 50% with DSA 300-1,500 MFI (N=19): overall 1-y and 8-y GS of 92.1% and 76.3%, respectively; R with DSA >1,500 MFI against HLA class I only (29) or class I and class II (N=33) presented significanly (p<0.001) lower 1-y and 8-y GS: 75.8% and 51.6%, respectively. We could not analyze the impact of isolated HLA class II DSA because only 8 R felt into this category. HLA class I DSA >1500 MFI conferred a relative risk of 4.4 for graft loss at one year and of 3.0 for late graft loss in R with functional kidneys at one year. In addition, R with HLA class I DSA >1500 MFI presented lower estimated glomerular filtration rates (Cockroft-Gault formula), at the first month (43.0±9.9 vs. 56.0±20.3ml/min, p<0.001) and at first year (56.6±17.2 vs. 65.0±21.2ml/min, p<0.01) post-Tx. Conclusions: HLA class I DSAs with MFIs up to 1,500 do not affect Tx outcome, whereas DSAs with MFIs >1,500, although not associated with immediate graft loss, confer risk for graft dysfunction already at the end of the first month, and a risk for long-term graft loss. [Copyright &y& Elsevier]