Anat R. Tambur

Cytokine gene polymorphisms in patients infected with hepatitis B virus.

OBJECTIVE:Cytokines play a key role in the regulation of the immune response. The maximal capacity of cytokine production varies among individuals and correlates with the polymorphism in the cytokine gene promoters. The aim of this study was to characterize gene polymorphism in patients with chronic hepatitis B virus (HBV) infection and to determine the different patterns in patient subgroups.METHODS:The study population consisted of 77 patients with chronic HBV infection (23 low-level HBV replicative carriers, 23 compensated high-level HBV replicative carriers, 21 decompensated liver transplant candidates, and 10 patients with documented hepatocellular carcinoma). The genetic profile of five cytokines was analyzed by polymerase chain reaction–sequence-specific primer (SSP), and subjects were genotyped as high or low producers of tumor necrosis factor-α and interleukin (IL)-6, and as high, intermediate, or low producers of transforming growth factor-β1, interferon (IFN)-γ, and IL-10 based on single nucleotide substitutions. The control group included 10 healthy individuals who recovered from HBV infection and 48 healthy controls.RESULTS:A highly statistically significant difference in the distribution of the IFN-γ gene polymorphism (at position +879) was observed between patients with chronic HBV infection and controls. The majority of the patients (65.2%) exhibited the potential to produce low levels of IFN-γ (A/A genotype) compared with 37.5% of the control group (p = 0.003). Healthy individuals who recovered from HBV infection had a similar distribution of IFN-γ gene polymorphism as the healthy controls. No statistically significant difference in IFN-γ production was found between patients with low- and high-level HBV replication and between compensated and decompensated patients. There was also no statistically significant difference in the genetic ability to produce tumor necrosis factor-α (at position −308), IL-6 (at position −174), IL-10 (at position −1082, −819, and −592), and transforming growth factor-β1 (at position +10 and +25).CONCLUSION:These findings suggest an association between the genetic ability to produce low levels of IFN-γ and the susceptibility to develop chronic HBV infection.

The presence of HLA-directed antibodies after heart transplantation is associated with poor allograft outcome.

Background. The clinical significance of HLA-directed antibodies newly detected after transplantation (HT) is controversial. Methods. Seventy-one HT recipients consented to enroll. Mean follow-up time was 28 months (range 6-48). Panel reactive antibody (PRA) analysis was performed on posttransplant sera (2 weeks, 1, 2, 3, 6, and 12 months and annually thereafter) using Flow-PRA. A mean of 6.9±1.2 serum samples per patient were obtained. Severity of cellular rejection was measured using the ISHLT grading system. Coronary angiography and intravascular ultrasound (IVUS) studies were performed annually to evaluate severity of allograft vasculopathy. Results. Twenty-five recipients had newly detected HLA-directed antibodies during the first year postHT. HLA class I antibodies were detected in 18 patients (25.4%), and class II in 11 patients (15.5%). The majority of donor recipient pairs were HLA mismatched (4.6±1.2 of the six major HLA antigens). Only mismatches at HLA-A locus had significant association with de novo posttransplant antibody formation. Length of ischemia time was correlated with early and sustained presence of de novo HLA-directed antibodies postheart transplant. Importantly, an association between de novo HLA-directed antibodies and cellular rejection was notes (P=0.0002). De novo HLA class II directed antibodies are also associated with IVUS documented vasculopathy (P<0.002). Finally, death due to allograft failure is associated with the presence of de novo formed HLA class II directed antibodies (P=0.008). Conclusions. Identifying the formation of de novo HLA-directed antibodies following heart transplantation may predict allograft outcome. This, in turn, may serve as a tool for individualization of immunosuppression protocols in heart transplant recipients.

Flow cytometric detection of HLA-specific antibodies as a predictor of heart allograft rejection

BACKGROUND Historically, panel reactive antibody (PRA) analysis to detect HLA antibodies has been performed using cell-based complement-dependent cytotoxicity (CDC) techniques. Recently, a flow cytometric procedure (FlowPRA) was introduced as an alternative approach to detect HLA antibodies. The flow methodology, using a solid phase matrix to which soluble HLA class I or class II antigens are attached is significantly more sensitive than CDC assays. However, the clinical relevance of antibodies detected exclusively by FlowPRAhas not been established. In this study of cardiac allograft recipients, FlowPRA was performed on pretransplant sera with no detectable PRA activity as assessed by CDC assays. FlowPRA antibody activity was then correlated with clinical outcome. METHODS PRA analysis by anti-human globulin enhanced (AHG) CDC and FlowPRA was performed on sera corresponding to final cross-match specimens from 219 cardiac allograft recipients. In addition, sera collected 3-6 months posttransplant from 91 patients were evaluated. The presence or absence of antibodies was correlated with episodes of rejection and patient survival. A rejection episode was considered to have occurred based on treatment with antirejection medication and/or histology. RESULTS By CDC, 12 patients (5.5%) had pretransplant PRA >10%. In contrast, 72 patients (32.9%) had pretransplant anti-HLA antibodies detectable by FlowPRA (34 patients with only class I antibodies; 7 patients with only class II antibodies; 31 patients with both class I and class II antibodies). A highly significant association (P<0.001) was observed between pretransplant HLA antibodies detected by FlowPRA and episodes of rejection that occurred during the first posttransplant year. Fifteen patients died within the first year posttransplant. Of nine retrospective flow cytometric cross-matches that were performed, two were in recipients who had no pretransplant antibodies detectable by FlowPRA. Both of these cross-matches were negative. In contrast, five of seven cross-matches were positive among recipients who had FlowPRA detectable pretransplant antibodies. Posttransplant serum specimens from 91 patients were also assessed for antibodies by FlowPRA. Among this group, 58 patients had FlowPRA antibodies and there was a trend (although not statistically significant) for a biopsy documented episode of rejection to have occurred among patients with these antibodies. CONCLUSIONS Collectively, our data suggest that pre- and posttransplant HLA antibodies detectable by FlowPRA and not AHG-CDC identify cardiac allograft recipients at risk for rejection. Furthermore, a positive donor reactive flow cytometric cross-match is significantly associated with graft loss. Thus, we believe that detection and identification of HLA-specific antibodies can be used to stratify patients into high and low risk categories. An important observation of this study is that in the majority of donor:recipient pairs, pretransplant HLA antibodies were not directed against donor antigens. We speculate that these non-donor-directed antibodies are surrogate markers that correspond to previous T cell activation. Thus, the rejection episodes that occur in these patients are in response to donor-derived MHC peptides that share cryptic determinants with the HLA antigens that initially sensitized the patient.

Assessing Antibody Strength: Comparison of MFI, C1q, and Titer Information

The presence of donor‐specific HLA antibodies before or after transplantation may have different implications based on the antibody strength. Yet, current approaches do not provide information regarding the true antibody strength as defined by antigen–antibody dissociation rate. To assess currently available methods, we compared between neat mean fluorescence intensity (MFI) values, C1q MFI values, ethylenediaminetetraacetic acid (EDTA)‐treated samples, as well as titration studies and peak MFI values of over 7000 Luminex‐based single‐antigen HLA antibody data points. Our results indicate that neat MFI values do not always accurately depict antibody strength. We further showed that EDTA treatment (6%) does not always remove all inhibitory factors compared with C1q or titration studies. In this study of patients presenting with multiple antibody specificities, a prozone effect was observed in 71% of the cohort (usually not affecting all antibody specificities within a single serum sample, though). Similar to titration studies, the C1q assay was able to address the issue of potential inhibition; however, its limitation is its low sensitivity and inability to detect the presence of weak antibodies. Titration studies are the only method among the approaches used in this study to provide information suggesting antigen–antibody dissociation rates and are, therefore, likely to provide better indication of true antibody strength.

The Banff 2015 Kidney Meeting Report: Current Challenges in Rejection Classification and Prospects for Adopting Molecular Pathology

The XIII Banff meeting, held in conjunction the Canadian Society of Transplantation in Vancouver, Canada, reviewed the clinical impact of updates of C4d‐negative antibody‐mediated rejection (ABMR) from the 2013 meeting, reports from active Banff Working Groups, the relationships of donor‐specific antibody tests (anti‐HLA and non‐HLA) with transplant histopathology, and questions of molecular transplant diagnostics. The use of transcriptome gene sets, their resultant diagnostic classifiers, or common key genes to supplement the diagnosis and classification of rejection requires further consensus agreement and validation in biopsies. Newly introduced concepts include the i‐IFTA score, comprising inflammation within areas of fibrosis and atrophy and acceptance of transplant arteriolopathy within the descriptions of chronic active T cell–mediated rejection (TCMR) or chronic ABMR. The pattern of mixed TCMR and ABMR was increasingly recognized. This report also includes improved definitions of TCMR and ABMR in pancreas transplants with specification of vascular lesions and prospects for defining a vascularized composite allograft rejection classification. The goal of the Banff process is ongoing integration of advances in histologic, serologic, and molecular diagnostic techniques to produce a consensus‐based reporting system that offers precise composite scores, accurate routine diagnostics, and applicability to next‐generation clinical trials.

Comprehensive assessment and standardization of solid phase multiplex-bead arrays for the detection of antibodies to HLA.

Solid phase multiplex‐bead arrays for the detection and characterization of HLA antibodies provide increased sensitivity and specificity compared to conventional lymphocyte‐based assays. Assay variability due to inconsistencies in commercial kits and differences in standard operating procedures (SOP) hamper comparison of results between laboratories. The Clinical Trials in Organ Transplantation Antibody Core Laboratories investigated sources of assay variation and determined if reproducibility improved through utilization of SOP, common reagents and normalization algorithms. Ten commercial kits from two manufacturers were assessed in each of seven laboratories using 20 HLA reference sera. Implementation of a standardized (vs. a nonstandardized) operating procedure greatly reduced MFI variation from 62% to 25%. Although laboratory agreements exceeded 90% (R2), small systematic differences were observed suggesting center specific factors still contribute to variation. MFI varied according to manufacturer, kit, bead type and lot. ROC analyses showed excellent consistency in antibody assignments between manufacturers (AUC > 0.9) and suggested optimal cutoffs from 1000 to 1500 MFI. Global normalization further reduced MFI variation to levels near 20%. Standardization and normalization of solid phase HLA antibody tests will enable comparison of data across laboratories for clinical trials and diagnostic testing.

Role of cytokine gene polymorphism in hepatitis C recurrence and allograft rejection among liver transplant recipients.

Background. Cytokines play a key role in the regulation of immuneresponses. The maximal capacity of cytokine production varies betweenindividuals and was shown to correlate with polymorphism in cytokine genepromoters. The objective of this study was to analyze the role of cytokineallelic variations in susceptibility to early graft rejection episodes andrecurrence of hepatitis C infection in liver transplant (LTx)recipients. Methods. The genetic profile of five cytokines was studied in 68 LTxrecipients and 49 controls using polymerase chain reaction sequence specificprimers. All individuals were genotyped as high or low producers of TNF-&agr;and IL-6 and high, intermediate, or low producers of transforming growthfactor &bgr; (TGF-&bgr;), interferon &ggr; (IFN-&ggr;), and interleukin 10(IL-10) based on single nucleotidesubstitutions. Results. No statistically significant differences were observedbetween patients with or without early rejection episodes. A significantproportion of patients more prone to rejection were genotyped as having a lowproduction profile of IL-10 compared with the control population(P =0.04). These data are inaccordance with reports regarding other solid-organ transplant recipients.Patients with no recurrence of hepatitis C had the inherent ability to producehigher TGF-&bgr; levels than did patients with recurrent disease(P =0.042). Among nonrecurrentpatients, the percentage of genetically low IL-10 producers was higher thanamong recurrent patients(P =0.07). Furthermore, agenetic tendency to produce higher levels of IFN-&ggr; was noted among LTxrecipients with nonrecurrent hepatitis C than among those with recurrenthepatitis C. Conclusions. While no significant correlation was detected betweenparticular cytokine profile and early rejection episodes, our data stronglysuggest an association between cytokine gene polymorphism of TGF-&bgr;, IL-10,and INF-&ggr; and recurrence of hepatitis C in LTxrecipients.

The complexity of human leukocyte antigen (HLA)-DQ antibodies and its effect on virtual crossmatching.

Background. We previously reported that in patients possessing human leukocyte antigen (HLA)-DQ-directed antibodies, the target molecule may include the patient’s own DQ&bgr; chain if it is paired with non–self-DQ&agr; chain, thus forming a different DQ target. Herein, we sought to assess the breadth of this phenomenon. Methods. Serum samples from 104 patients awaiting kidney transplantation, known to have DQ antibodies, were studied. Antibody identification was performed using luminex-based HLA class II single-antigen bead assays from two vendors; DQA1/DQB1 typing was performed using luminex polymerase chain reaction – sequence specific oligo prob hybridization (PCR-SSO) technology. Results. A total of 71% of the 104 serum samples studied contained antibodies reactive against test beads coated with the patient’s own DQ&agr;- or &bgr;-chain components. Of those, 35 patients (34%) exhibited antibodies to their own DQ&bgr; chain when in combination with non–self-DQ&agr; chains; and 64 patients (62%) had antibodies to their own DQ&agr; chain when in combination with non–self-DQ&bgr; chains. This is a striking observation. Conclusions. To the best of our knowledge, this is the first systematic, high-resolution evaluation of DQ antibody repertoire. With the expansion of virtual crossmatching, particularly in the context of a national registry, the need for more detailed DQ antibody or antigen evaluation is critical to improve operational efficiency and patient outcomes.

Perception versus reality ?: Virtual crossmatch - How to overcome some of the technical and logistic limitations

The goal of this work was to evaluate concordance between (a) actual flow cytometric crossmatch (FCXM) that is performed by the OPO laboratory servicing our transplant center and (b) virtual XM (vXM) prediction based on antibody identification by solid‐phase methods performed in our laboratory.

Genomic Biomarkers Correlate with HLA-Identical Renal Transplant Tolerance

The ability to achieve immunologic tolerance after transplantation is a therapeutic goal. Here, we report interim results from an ongoing trial of tolerance in HLA-identical sibling renal transplantation. The immunosuppressive regimen included alemtuzumab induction, donor hematopoietic stem cells, tacrolimus/mycophenolate immunosuppression converted to sirolimus, and complete drug withdrawal by 24 months post-transplantation. Recipients were considered tolerant if they had normal biopsies and renal function after an additional 12 months without immunosuppression. Of the 20 recipients enrolled, 10 had at least 36 months of follow-up after transplantation. Five of these 10 recipients had immunosuppression successfully withdrawn for 16-36 months (tolerant), 2 had disease recurrence, and 3 had subclinical rejection in protocol biopsies (nontolerant). Microchimerism disappeared after 1 year, and CD4(+)CD25(high)CD127(-)FOXP3(+) regulatory T cells and CD19(+)IgD/M(+)CD27(-) B cells were increased through 5 years post-transplantation in both tolerant and nontolerant recipients. Immune/inflammatory gene expression pathways in the peripheral blood and urine, however, were differentially downregulated between tolerant and nontolerant recipients. In summary, interim results from this trial of tolerance in HLA-identical renal transplantation suggest that predictive genomic biomarkers, but not immunoregulatory phenotyping, may be able to discriminate tolerant from nontolerant patients.