Performance of a multiplexed amplicon-based next-generation sequencing assay for HLA typing.

Background: Next-generation sequencing (NGS) has enabled efficient high-resolution typing of human leukocyte antigen (HLA) genes with minimal ambiguity. Most commercially available assays amplify individual or subgroup of HLA genes by long-range PCR followed by library preparation and sequencing. The AllType assay simplifies the workflow by amplifying 11 transplant-relevant HLA genes in one PCR reaction.

Accelerated humoral renal allograft rejection due to HLA-C14 mediated allosensitization to HLA-Bw6.

Objectives: To investigate immunological mechanisms underlying accelerated antibody-mediated rejection (AMR) of a living-related renal allograft in a patient with no detectable antibodies to donor human leukocyte antigens (HLA) in pre-transplant sera.

Methods: Pre- and post-transplant HLA antibody specificities were determined by single-antigen bead assay, and crossmatching was performed by flow cytometry- and complement-dependent cytotoxicity-based methods. Intermediate- and high-resolution HLA typing were performed by molecular methods.

Quantitative Evaluation of the Impact of Ethylenediaminetetraacetic Acid Pretreatment on Single-Antigen Bead Assay.

Background: Ethylenediaminetetraacetic acid (EDTA) pretreatment has been shown to overcome complement interference in the single-antigen bead (SAB) assay. However, a quantitative evaluation of its impact on the assay for preemptive application to diverse clinical samples is still lacking.

Methods: Serum samples from 95 renal transplant candidates were tested with and without EDTA-pretreatment in parallel.

ABO incompatible renal transplants and decreased likelihood for developing immune responses to HLA and kidney self-antigens.

Immune responses to HLA and tissue-restricted self-antigens (SAgs) have been proposed to play a role in the pathogenesis of renal allograft (KTx) rejection. However, ABO incompatible (ABOi) KTx recipients (KTxR) following depletion of antibodies (Abs) to blood group antigens had fewer rejections. To determine the mechanisms, pre- and post-transplant sera from ABOi (n=18) and ABO-compatible (ABOc) (n=45) KTxR were analyzed for Abs against HLA class I and II by LABScreen single antigen assay.

Complement activation is not required for obliterative airway disease induced by antibodies to major histocompatibility complex class I: Implications for chronic lung rejection.

Background: The role of non-complement activating antibodies (ncAbs) to mismatched donor human leukocyte antigen (HLA) in the pathogenesis of chronic lung rejection is not known. We used a murine model of obliterative airway disease (OAD) induced by Abs to major histocompatibility major histocompatibility complex (MHC) class I and serum from donor-specific Abs developed in human lung transplant (LTx) recipients to test the role of ncAbs in the development of OAD and bronchiolitis obliterans syndrome (BOS).

Methods: Anti-MHC ncAbs were administered intrabronchially in B.

Cutoff values and data handling for solid-phase testing for antibodies to HLA: effects on listing unacceptable antigens for thoracic organ transplantation.

Application of single-antigen solid-phase immunoassay (SPI) in virtual crossmatch-based organ allocation has been hindered by continued debate over the biologic relevance of detected antibodies and the relationship between cutoff mean fluorescence intensity (MFI) values with crossmatch testing results. To define SPI parameters accurately predicting crossmatch testing, we analyzed a series of anti-HLA antibodies from highly-sensitized patients awaiting lung or heart transplantation. Serial dilution of serum for SPI and cytotoxic crossmatch (CXM) enabled comparison over a wide spectrum of antibody "strengths".

Alloimmunity-induced autoimmunity as a potential mechanism in the pathogenesis of chronic rejection of human lung allografts.

Background: Bronchiolitis obliterans syndrome (BOS) is a major cause of morbidity and mortality after lung transplantation (LTx). We sought to better understand the relationship between alloimmune responses and autoimmunity and, subsequently, how autoimmunity leads to chronic rejection.

Methods: We analyzed the development of donor-specific antibodies (Abs) in LTx by flow PRA and the development of Abs to K-α1 tubulin (K-α1T) and collagen V (ColV) by ELISA.

A role for antibodies to human leukocyte antigens, collagen-V, and K-α1-Tubulin in antibody-mediated rejection and cardiac allograft vasculopathy.

Background: We determined the role of donor-specific antibodies (DSA) and antibodies (Abs) to self-antigens, collagen-V (Col-V), and K-α1-Tubulin (KAT) in pathogenesis of acute antibody-mediated rejection (AMR) and cardiac allograft vasculopathy (CAV) after human heart transplantation (HTx).

Methods: One hundred thirty-seven HTx recipients, with 60 early period (≤ 12 months) and 77 late period (>12 months), were enrolled in this study. Circulating DSA was determined using LUMINEX.

Donor-specific antibodies to human leukocyte antigens are associated with and precede antibodies to major histocompatibility complex class I-related chain A in antibody-mediated rejection and cardiac allograft vasculopathy after human cardiac transplantation.

Humoral immune responses to mismatched donor human leukocyte antigen (HLA) and major histocompatibility complex (MHC) class I-related chain A (MICA) have been reported to contribute to immunopathogenesis of antibody-mediated rejection (AMR) in the early period and cardiac allograft vasculopathy (CAV) in the late period after cardiac transplantation (HTx). The goal of this study is to define the roles of donor-specific antibodies (DSA) and anti-MICA in AMR and CAV. A total of 95 post-HTx recipients were enrolled; 43 patients in the early period (≤ 12 months post-HTx) and 52 patients in the late period (>12 months post-HTx).

Characterization of immune responses to cardiac self-antigens myosin and vimentin in human cardiac allograft recipients with antibody-mediated rejection and cardiac allograft vasculopathy.

Background: Herein we study the role of donor-specific antibodies (DSA) to mismatched human leukocyte antigen (HLA) and antibodies (Abs) to the cardiac self-antigens myosin (MYO) and vimentin (VIM) in the pathogenesis of acute antibody-mediated rejection (AMR) in the early post-transplant period (EP, <12 months) and cardiac allograft vasculopathy (CAV) in the late post-transplant period (LP, >12 months) after heart transplantation (HTx).

Methods: One hundred forty-eight HTx recipients (65 in EP, 83 in LP) were enrolled in the study. Development of DSA was determined by Luminex.

CD4+ lymphocyte adenosine triphosphate determination in sepsis: a cohort study.

Introduction: Patients suffering from sepsis are currently classified on a clinical basis (i.e., sepsis, severe sepsis, septic shock); however, this clinical classification may not accurately reflect the overall immune status of an individual patient.

Development of antibodies to human leukocyte antigen precedes development of antibodies to major histocompatibility class I-related chain A and are significantly associated with development of chronic rejection after human lung transplantation.

The development of antibodies (Abs) to major histocompatibility (MHC) class I-related chain A (MICA) and human leukocyte antigen (HLA) and their role in the immunopathogenesis of chronic rejection (bronchiolitis obliterans syndrome [BOS]) after human lung transplantation (LTx) was analyzed. Sera from 80 LTx recipients were analyzed for anti-MICA and anti-HLA Abs using Luminex and flow PRA (panel reactive assay). Development of Abs either to MICA alone or MICA and HLA together significantly correlated (p < 0.

Virtual crossmatch by identification of donor-specific anti-human leukocyte antigen antibodies by solid-phase immunoassay: a 30-month analysis in living donor kidney transplantation.

Selection of donors for kidney transplantation depends on accurate prediction of risk factors for immunologic rejection. Historically, cytotoxicity crossmatch (CXM) examining lysis of donor cells by preformed anti-human leukocyte antigen (HLA) antibodies (Abs) has been considered the best predictor of immunologic rejection. However, there is much interest in defining anti-HLA Ab specificity in recipient sera by immunoassay to predict crossmatch results and aid in donor selection.

Increased erythrocyte C4D is associated with known alloantibody and autoantibody markers of antibody-mediated rejection in human lung transplant recipients.

Background: Immune responses to mismatched donor human leukocyte antigens (HLA) are important in the pathogenesis of chronic rejection. This study evaluated whether erythrocyte-bound C4d (E-C4d) is associated with known alloimmune and autoimmune markers of antibody-mediated rejection after human lung transplantation (LTx).

Methods: Flow cytometry was used to analyze 22 LTx recipients and 15 healthy individuals for E-C4d.

Living donor renal transplantation in the presence of donor-specific human leukocyte antigen antibody detected by solid-phase assay.

Presensitization of donor human leukocyte antigens (HLA) demonstrated through a positive crossmatch is detrimental to allograft function and best avoided through donor exclusion. The clinical significance of alloantibody detectable by sensitive solid-phase assay is not completely defined and is the focus of this study. Pretransplant sera from 64 consecutive living-donor renal transplant recipients were screened by enzyme-linked immunosorbent assay (ELISA) and Luminex assays.

Complement fragment C4d and C3d deposition in pediatric heart recipients with a positive crossmatch.

Background: Pediatric heart transplant recipients with a positive complement-dependent cytotoxic (CDC) donor-recipient crossmatch are at high risk for rejection. We sought to correlate the pattern of C3d and C4d myocardial capillary deposition and pericapillary macrophage infiltration, possible markers of antibody-mediated rejection, to clinical evidence of rejection in these patients.

Methods: Were studied 15 pre-sensitized pediatric patients who had 21 rejection episodes, as defined by International Society for Heart and Lung Transplantation (ISHLT) biopsy Grade >or=2R and/or the development of abnormal left ventricular (LV) function at >1 week after transplant.

Mortality and morbidity in pre-sensitized pediatric heart transplant recipients with a positive donor crossmatch utilizing peri-operative plasmapheresis and cytolytic therapy.

Background: The difficulty in obtaining a prospective negative donor/recipient crossmatch limits the ability to successfully transplant pediatric heart transplant candidates who show evidence of antibodies to multiple human leukocyte antigens (pre-sensitized patients).

Methods: We utilized a protocol that included peri-operative plasmapheresis, thymoglobulin and cyclophosphamide in 17 pre-sensitized (panel-reactive antibodies [PRA] >10%) pediatric patients to accept donors for these patients without a prospective crossmatch between 1995 and 2005. A retrospective review of survival, rejection and infection was performed, comparing the frequency of rejection and infection in our patients who were transplanted with a complement-dependent cytotoxic (CDC)-positive donor/recipient crossmatch to those patients transplanted with a negative crossmatch.

A significant role for histocompatibility in human islet transplantation.

Background: In recent years, transplantation of islets and pancreas has become a viable option for patients debilitated with type I diabetes. The success of islet transplantation has been attributed to the ability to isolate high quality islets for transplantation and capacity to maintain the recipient's immunosuppressive levels within a specific target range following transplantation. The purpose of this study was to determine the role of pretransplant sensitization to human leukocyte antigen (HLA) in islet transplantation.

HLA class I antibody mediated accommodation of endothelial cells via the activation of PI3K/cAMP dependent PKA pathway.

Allografts transplanted across ABO incompatibility or human leucocyte antigen (HLA)-sensitization undergoes antibody (Ab) mediated hyperacute rejection. Depleting anti-graft Ab from the recipient by plasmapheresis prior to transplantation can prevent this Ab-mediated rejection. Under these conditions, allografts have been shown to function even when the Ab rebound in the recipients.

Pre-exposure to sub-saturating concentrations of HLA class I antibodies confers resistance to endothelial cells against antibody complement-mediated lysis by regulating Bad through the phosphatidylinositol 3-kinase/Akt pathway.

Allografts transplanted across HLA-sensitization results in an antibody-mediated rejection known as hyperacute rejection. Depleting anti-graft antibodies from the recipient by plasmapheresis prior to transplantation can prevent this rejection. We developed an in vitro model using polyclonal HLA class I antibodies obtained from highly sensitized patients awaiting transplantation,and analyzed their ability to provide signals following binding to human aortic endothelial cells (EC).

Association of the HLA-DR15/HLA-DQ6 haplotype with development of choroidal neovascular lesions in presumed ocular histoplasmosis syndrome.

Associations of human leukocyte antigen DR2 (HLA-DR2) and HLA-B7 with presumed ocular histoplasmosis syndrome (POHS) in the United States has been previously described. However, these associations were determined by means of low-resolution, complement-dependent cytotoxicity assays for HLA-A, HLA-B, and HLA-DR molecules. To determine whether POHS is associated with other HLA alleles within the HLA-A, HLA-B, HLA-DR, and HLA-DQ loci, we performed a case control study of 34 patients diagnosed with macular choroidal neovascular membrane secondary to POHS and 45 healthy control individuals.