Anti-N and anti-Doa immunoglobulin G alloantibody-mediated delayed hemolytic transfusion reaction with hyperhemolysis in sickle cell disease treated with eculizumab and HBOC-201: case report and review of the literature.

BACKGROUND: Delayed hemolytic transfusion reaction (DHTR) with hyperhemolysis is a potentially fatal complication resulting from alloimmunization that can cause severe hemolysis of both transfused and intrinsic red blood cells (RBCs). Patients with sickle cell disease often receive multiple RBC units during their lifetime and thus are likely to develop alloantibodies that increase the risk for DHTR. Treatment to decrease hemolysis includes intravenous immunoglobulin (IVIG), steroids, eculizumab, rituximab, and plasmapheresis in addition to erythropoietin (EPO), intravenous (IV) iron, vitamin B12, and folate to support erythropoiesis. RBC transfusion is preferably avoided in DHTR due to an increased risk of exacerbating the hemolysis. CASE REPORT: We report a rare case of anti-N and anti-Doa immunoglobulin (Ig)G alloantibody-mediated life-threatening DHTR with hyperhemolysis in a patient with hemoglobin SS after RBC transfusion for acute chest syndrome who was successfully treated with eculizumab and HBOC-201 (Hemopure) in addition to steroids, IVIG, EPO, IV iron, and vitamin B12. HBOC-201 (Hemopure) was successfully used as a RBC alternative in this patient. CONCLUSION: Anti-N and anti-Doa IgG alloantibodies can rarely cause severe life-threatening DHTR with hyperhemolysis. HBOC-201 (Hemopure) can be a lifesaving alternative in this scenario. Our report also supports the use of eculizumab in DHTR; however, prospective studies are needed to determine the appropriate dose and sequence of eculizumab administration.

Late manifestation of alloantibody-associated injury and clinical pulmonary antibody-mediated rejection: Evidence from cell-free DNA analysis.

BACKGROUND: Antibody-mediated rejection (AMR) often progresses to poor health outcomes in lung transplant recipients (LTRs). This, combined with the relatively insensitive clinical tools used for its diagnosis (spirometry, histopathology) led us to determine whether clinical AMR is diagnosed significantly later than its pathologic onset. In this study, we leveraged the high sensitivity of donor-derived cell-free DNA (ddcfDNA), a novel genomic tool, to detect early graft injury after lung transplantation. METHODS: We adjudicated AMR and acute cellular rejection (ACR) in 157 LTRs using the consensus criteria of the International Society for Heart and Lung Transplantation (ISHLT). We assessed the kinetics of allograft injury in relation to ACR or AMR using both clinical criteria (decline in spirometry from baseline) and molecular criteria (ddcfDNA); percent ddcfDNA was quantitated via shotgun sequencing. We used a mixed-linear model to assess the relationship between and ddcfDNA levels and donor-specific antibodies (DSA) in AMR+ LTRs. RESULTS: Compared with ACR, AMR episodes (n = 42) were associated with significantly greater allograft injury when assessed by both spirometric (0.1 liter vs -0.6 liter, p < 0.01) and molecular (ddcfDNA) analysis (1.1% vs 5.4%, p < 0.001). Allograft injury detected by ddcfDNA preceded clinical AMR diagnosis by a median of 2.8 months. Within the same interval, spirometry or histopathology did not reveal findings of allograft injury or dysfunction. Elevated levels of ddcfDNA before clinical diagnosis of AMR were associated with a concurrent rise in DSA levels. CONCLUSION: Diagnosis of clinical AMR in LTRs lags behind DSA-associated molecular allograft injury as assessed by ddcfDNA.

Human neutrophil antigen-3a antibodies induce neutrophil stiffening and conformational activation of CD11b without shedding of L-selectin.

BACKGROUND: HNA-3a antibodies induce severe transfusion-related acute lung injury (TRALI) in which neutrophils play a major role. As neutrophil passage through the pulmonary microvasculature is a critical step in the pathogenesis of TRALI, we investigated the impact of HNA-3a antibodies on two important factors that could impair granulocyte passage through lung capillaries: the elasticity of neutrophils and the expression and activation of adhesion molecules. STUDY DESIGN AND METHODS: The impact of HNA-3a antibodies on the elasticity of neutrophils was investigated using atomic force microscopy (AFM). Neutrophils were settled on poly-2-hydroxyethyl-methacrylate-coated glass slides before treatment with anti-HNA-3a plasma samples, control plasma, or control plasma containing formyl-methionyl-leucyl-phenylalanine (fMLP). Elasticity measurements were carried out in a temperature-controlled perfusion chamber using an atomic force microscopy (AFM) device. The impact of HNA-3a antibodies on the surface expression of total CD11b, activation of CD11b, and L-selectin (CD62L) shedding was investigated by flow cytometry. The functional impact of HNA-3a antibodies on neutrophil adhesion was assessed using fibrinogen-coated plates. RESULTS: HNA-3a antibodies induced stiffening of neutrophils (+24%-40%; p < 0.05) to a similar extent as fMLP. This effect was blocked by treatment of neutrophils with cytochalasin D. While total surface expression of CD11b and L-selectin on neutrophils was largely unaffected, HNA-3a antibodies induced alloantigen-specific activation of CD11b (+72%-107%; p < 0.05) and increased adhesion of neutrophils to fibrinogen. CONCLUSION: Accumulation of neutrophils in the pulmonary microvasculature during severe TRALI is likely mediated by increased rigidity and CD11b-mediated adhesion of neutrophils leading to retention of neutrophils.

Monitoring B cell subsets and alloreactivity in kidney transplantation.

B cells are the precursors of antibody producing plasma cells that can give rise to the formation of donor-specific antibodies. However, recent data suggest that besides their role in antibody production, B cells participate in antibody-independent responses, potentially leading to allograft rejection or allograft tolerance. The presence of CD20(+) B cells in kidney graft biopsies has been shown during severe acute rejection episodes and during chronic rejection. Furthermore, operationally tolerant kidney transplant recipients showed a clear B cell dominated fingerprint of tolerance. Several techniques exist to study B cells on different levels. Numerous classification schemes allow for the distinction of many different B cell subsets using flow cytometry. Regardless, data on B cell subsets during stable graft function, rejection or tolerance remain scarce. To obtain a complete picture of the role of B cells during transplantation, antigen specific B cell assays may be required. Therefore, techniques have now been developed that allow for studying the specificity and frequency of HLA specific B cells. Here, we present an overview of the existent assays, panels and techniques intended to characterize peripheral B cells, and the currently available HLA specific B cell functional assays that may allow for monitoring the humoral alloimmune response in transplant recipients.

Platelets in early antibody-mediated rejection of renal transplants.

Antibody-mediated rejection is a major complication in renal transplantation. The pathologic manifestations of acute antibody-mediated rejection that has progressed to functional impairment of a renal transplant have been defined in clinical biopsy specimens. However, the initial stages of the process are difficult to resolve with the unavoidable variables of clinical studies. We devised a model of renal transplantation to elucidate the initial stages of humoral rejection. Kidneys were orthotopically allografted to immunodeficient mice. After perioperative inflammation subsided, donor-specific alloantibodies were passively transferred to the recipient. Within 1 hour after a single transfer of antibodies, C4d was deposited diffusely on capillaries, and von Willebrand factor released from endothelial cells coated intravascular platelet aggregates. Platelet-transported inflammatory mediators platelet factor 4 and serotonin accumulated in the graft at 100- to 1000-fold higher concentrations compared with other platelet-transported chemokines. Activated platelets that expressed P-selectin attached to vascular endothelium and macrophages. These intragraft inflammatory changes were accompanied by evidence of acute endothelial injury. Repeated transfers of alloantibodies over 1 week sustained high levels of platelet factor 4 and serotonin. Platelet depletion decreased platelet mediators and altered the accumulation of macrophages. These data indicate that platelets augment early inflammation in response to donor-specific antibodies and that platelet-derived mediators may be markers of evolving alloantibody responses.

The clinical relevance of alloantibody in liver transplantation.

The transplanted liver appears resistant to antibody-mediated injury compared to other transplanted organs such as kidney or heart. However, a growing number of reports suggest that alloantibody to the liver is associated with poorer outcomes. The data surrounding this field are unclear, and their interpretation remains controversial. Mechanistically, there is not a clear explanation for the liver's resistance to antibody-mediated injury, and the pathological criteria for antibody-mediated rejection (AMR) remain ill-defined. Furthermore, treatment of AMR is non-uniform. The field would benefit from better outcome data based on measurement of antibody at the time of transplantation and at the time of rejection. Consensus opinion regarding antibody and the liver might emerge with better standardization of antibody measurement and pathological definition of AMR.

Impact of IgM and IgG3 anti-HLA alloantibodies in primary renal allograft recipients.

BACKGROUND: With standard IgG donor-specific anti-HLA antibody (DSA) testing, it is unclear which immunoglobulin-G (IgG) DSA positive patients will fail. We looked further into the immune response by studying immunoglobulin-M (IgM) and IgG subclass 3 (IgG3) DSA to determine if these identify the IgG DSA patients at highest risk for allograft loss. METHODS: In 189 consecutively transplanted primary renal allograft recipients, sera were collected sequentially pre- and posttransplant. Of the 189, 179 patients had sera available to retrospectively test for anti-HLA IgG, IgM, and IgG3 antibodies via LABScreen single-antigen bead assay and were included in the study. All patients had a negative crossmatch. Per patient, all DSA (IgM, IgG3, and IgG) refers to the same serologic specificity. RESULTS: Overall, 100 (56%) patients developed an alloimmune response (IgM or IgG DSA positive, or both). Ninety-five patients developed IgM DSA and 47 patients developed IgG DSA. IgM DSA was detected in 42 of 47 patients with IgG DSA. IgM DSA alone did not increase the allograft loss risk, whereas IgG DSA did (P=0.002). Once IgG DSA appeared, IgM DSA persisted in 33 patients and an isotype switch to IgG3 positive DSA occurred in 25 patients. Patients with IgM persistent IgG3 positive DSA (n=19) were more likely to have allograft failure than those without (P=0.02). CONCLUSION: This study shows the evolution of the humoral immune response from IgM to IgG DSA posttransplant. We found that development of IgM persistent IgG3 positive DSA identifies the most dangerous IgG DSA subpopulation.

Desensitization for prevention of chronic antibody-mediated rejection after kidney transplantation.

Chronic antibody-mediated rejection (C-AMR) is the most important and leading cause of graft loss after kidney transplantation. Although it is well known that chronic renal allograft dysfunction or failure is caused by various immunological or non-immunological factors, donor-specific anti-human leukocyte antigen antibodies (DSAs) are considered to be the most detrimental to graft survival and could cause C-AMR. Despite the use of intensive treatment for C-AMR, outcomes have not always been promising. Recently, prevention, rather than treatment, of C-AMR has been attempted, and this approach appears to be a more effective option for reducing the incidence of C-AMR and, ultimately, improving long-term survival. To prevent C-AMR, removal of antibodies, inactivation of antibodies, and prevention of antibody production after kidney transplantation are essential. Preconditioning treatment including plasmapheresis, intravenous immunoglobulin, and rituximab injection seems the most effective of current desensitization protocols. In this minireview, we will focus on the prevention of C-AMR through desensitization and improving long-term graft survival.

Alloantibody and complement promote T cell-mediated cardiac allograft vasculopathy through noncanonical nuclear factor-κB signaling in endothelial cells.

BACKGROUND: Cardiac allograft vasculopathy is the major cause of late allograft loss after heart transplantation. Cardiac allograft vasculopathy lesions contain alloreactive T cells that secrete interferon-γ, a vasculopathic cytokine, and occur more frequently in patients with donor-specific antibody. Pathological interactions between these immune effectors, representing cellular and humoral immunity, respectively, remain largely unexplored. METHODS AND RESULTS: We used human panel reactive antibody to form membrane attack complexes on allogeneic endothelial cells in vitro and in vivo. Rather than inducing cytolysis, membrane attack complexes upregulated inflammatory genes, enhancing the capacity of endothelial cells to recruit and activate allogeneic interferon-γ--producing CD4(+) T cells in a manner dependent on the activation of noncanonical nuclear factor-κB signaling. Noncanonical nuclear factor-κB signaling was detected in situ within endothelial cells both in renal biopsies from transplantation patients with chronic antibody-mediated rejection and in panel-reactive antibody--treated human coronary artery xenografts in immunodeficient mice. On retransplantation into immunodeficient hosts engrafted with human T cells, panel-reactive antibody--treated grafts recruited more interferon-γ--producing T cells and enhanced cardiac allograft vasculopathy lesion formation. CONCLUSIONS: Alloantibody and complement deposition on graft endothelial cells activates noncanonical nuclear factor-κB signaling, initiating a proinflammatory gene program that enhances alloreactive T cell activation and development of cardiac allograft vasculopathy. Noncanonical nuclear factor-κB signaling in endothelial cells, observed in human allograft specimens and implicated in lesion pathogenesis, may represent a target for new pharmacotherapies to halt the progression of cardiac allograft vasculopathy.

Anti-donor HLA class I antibodies: pathways to endothelial cell activation and cell-mediated allograft rejection.

BACKGROUND: The development of donor-specific human leukocyte antigen (HLA) class I antibodies after organ transplantation is associated with subsequent acute and chronic rejection. The aim of this study was to examine the role of anti-HLA class I antibody in modulating endothelium-leukocyte interaction. METHODS: Human microvascular endothelial cells (HMEC-1) stimulated with HLA class I antibody (W6/32) or allospecific antibodies from sensitized patients (n=6) were examined for activation of transcription factor CREB by Western blotting. Up-regulation of endothelial adhesion molecules and chemokines was measured by flow cytometry and quantitative polymerase chain reaction, respectively. Leukocyte adhesion was evaluated by chemotaxis and in vitro flow-based assays. RESULTS: Treatment of HMEC-1 cells with HLA class I antibody resulted in the phosphorylation of CREB in protein kinase A-dependent pathway. Furthermore, there was a significant increase in the expression of cell surface VCAM-1 (Akt-dependent) and ICAM-1 in Akt-dependent and extracellular signal-regulated kinase-dependent manner (P<0.001). Additionally, exposure to W6/32 antibody induced significant expression of interleukin-6, CXCL8, CXCL10, and CCL5. Knockdown of CREB produced a reduction in W6/32-induced CXCL8 expression (P<0.001). Media from W6/32-treated endothelial cells induced a significant monocyte chemotaxis (P<0.001) and flow-based adhesion assay demonstrated an increase in monocyte adhesion to endothelial cells compared with the control group (P<0.001). Importantly, allospecific antibodies from sensitized patients also activated endothelial CREB and significantly up-regulated VCAM-1, ICAM-1, and CXCL8. CONCLUSION: These findings suggest that donor-specific HLA class I antibodies directly activate endothelial cells leading to an increase in their potential to recruit and bind recipient leukocytes, thereby increasing the potential for allograft inflammation.

Antibody alone is not a stimulator of exocytosis of Weibel-Palade bodies from human endothelial cells.

BACKGROUND: The mechanisms of antibody-mediated damage to allografts are not well understood. We have examined the effect of antibodies to human leukocyte antigens on secretion of von Willebrand factor (vWF) from endothelial cells (ECs). METHODS: The effect of monoclonal antibodies (W6/32, L2, and L243), in the presence and absence of sublytic concentrations of complement, on the release of vWF from Weibel-Palade bodies (WPBs) in human umbilical vein ECs (HUVECs), human aortic ECs (HAECs), and human heart microvascular ECs (HHMECs) was investigated using biochemical and live-cell imaging. Fura-2-loaded ECs expressing the WPB marker proregion-enhanced green fluorescence protein were imaged simultaneously for intracellular Ca(2+) changes ([Ca(2+)](i)) and WPB exocytosis. RESULTS: Stimulation of ECs with 1- or 10-µg/mL W6/32, L2, or L243 did not evoke significant vWF release above control IgG. In live-cell imaging studies, exposure of proregion-enhanced green fluorescence protein-expressing HAECs to physiologic saline, 10-µg/mL U9F4, or W6/32 alone for 5 to 10 min induced irregular (Ca(2+))(i)\ spiking but no WPB exocytosis. Histamine-evoked WPB exocytosis was not changed by preexposure of HAECs to physiologic saline, U9F4, or W6/32. Stimulation of HUVECs with sublytic complement concentrations evoked WPB exocytosis; however, the addition of W6/32 did not change the amount of vWF released. CONCLUSION: Antibodies to human leukocyte antigen class I or II do not elicit significant WPB exocytosis or vWF secretion from ECs in the absence of exogenous complement.

Role of plasma exchange in ABO-incompatible kidney transplantation.

BACKGROUND: In the past, ABO incompatibility was an absolute contraindication for solid organ transplantation. However, multiple recent trials have suggested strategies for overcoming the reactions between graft antigens and recipient antibodies that cause graft rejection. In this study, we determined the usefulness of plasma exchange (PE) for removing anti-A/B antibodies that cause hyperacute/acute humoral graft rejection in patients undergoing ABO-incompatible kidney transplantation. METHODS: In our study, 12 patients underwent ABO-incompatible kidney transplantation. All recipients received pre-transplantation conditioning by PE or intravenous immunoglobulin (IVIG) administration. After pre-transplantation conditioning, anti-A/B antibody titers were evaluated, and transplantation was performed when the titer was below 1:8. To assess the transplantation outcome, anti-A/B antibody titers, creatinine level, estimated glomerular filtration rate (eGFR), and proteinuria levels were measured. RESULTS: Anti-A/B antibody titers were below 1:8 in all patients at the time of transplantation. eGFR measured on post-transplant day 14 showed that 10 patients had immediate recovery of graft function, while 2 patients had slow recovery of graft function. Short-term outcomes of ABO-incompatible kidney transplantation (measured as creatinine levels) after reducing anti-A/B antibody titers were similar to those of ABO-compatible kidney transplantation. After transplantation, the anti-A/B antibody titers were below 1:8 in 7 patients, but the remaining 5 patients required post-transplantation PE and IVIG treatment to prevent antigen-antibody reactions. CONCLUSIONS: With the increasing demand for kidney donations, interest in overcoming the ABO incompatibility barrier has increased. PE may be an important breakthrough in increasing the availability of kidneys for transplantation.

[Delayed haemolytic transfusion reaction due to anti-JK1 antibody]. / Accident hémolytique retardé post-transfusionnel par allo-immunisation anti-JK1.

INTRODUCTION: The sensitivity of the detection of irregular antibodies (DIA) is one of the fundamental basis of transfusion safety. The production of alloantibodies is the first cause of adverse events following transfusion. CASE REPORT: We report a 77-year-old woman who was transfused and presented with a delayed haemolytic anemia due to anti-JK1 alloimmunization. This event highlights the limits of DIA performed before a transfusion, the hazard of this specific type of antibody and the difficulties of the diagnosis of haemolytic anaemia. The preventive measures necessary to avoid this undesirable effect are reminded. CONCLUSION: Despite the sensitive routine test method, the anti-JK1 antibodies could be missed. We should keep in mind the possibility of an anaemia due to alloantibodies we confronted to an unexplained haemolytic episode.

IL-13 antibodies influence IL-13 clearance in humans by modulating scavenger activity of IL-13Rα2.

Human studies using Abs to two different, nonoverlapping epitopes of IL-13 suggested that epitope specificity can have a clinically significant impact on clearance of IL-13. We propose that Ab modulation of IL-13 interaction with IL-13Rα2 underlies this effect. Two Abs were administered to healthy subjects and mild asthmatics in separate dose-ranging studies and allergen-challenge studies. IMA-638 allows IL-13 interaction with IL-13Rα1 or IL-13Rα2 but blocks recruitment of IL-4Rα to the IL-13/IL-13Rα1 complex, whereas IMA-026 competes with IL-13 interaction with IL-13Rα1 and IL-13Rα2. We found ∼10-fold higher circulating titer of captured IL-13 in subjects treated with IMA-026 compared with those administered IMA-638. To understand how this difference could be related to epitope, we asked whether either Ab affects IL-13 internalization through cell surface IL-13Rα2. Humans inducibly express cell surface IL-13Rα2 but lack the soluble form that regulates IL-13 responses in mice. Cells with high IL-13Rα2 expression rapidly and efficiently depleted extracellular IL-13, and this activity persisted in the presence of IMA-638 but not IMA-026. The potency and efficiency of this clearance pathway suggest that cell surface IL-13Rα2 acts as a scavenger for IL-13. These findings could have important implications for the design and characterization of IL-13 antagonists.

The diagnostic value of the anti-PF4/heparin immunoassay high-dose heparin confirmatory test in cardiac surgery patients.

There are limited and conflicting data on how a confirmatory step using high-dose heparin can improve diagnostic specificity of the antiplatelet factor 4/heparin enzyme immunoassay for heparin-induced thrombocytopenia (HIT). We investigated sera from a recently published study on cardiac surgery patients and found that only half of the sera that were heparin-induced platelet activation assay positive could be inhibited (optical density <40%) by high-dose heparin (100 IU/mL) in the enzyme immunoassay. More importantly, only 2 of the 3 patients with definite HIT were confirmatory test positive. Therefore, the high-dose heparin confirmatory test should be used with caution to exclude platelet-activating antiplatelet factor 4/heparin antibodies or clinical HIT.

Alloantibodies prevent the induction of transplantation tolerance by enhancing alloreactive T cell priming.

Circulating alloantibodies in transplant recipients are often associated with increased Ab-mediated as well as cellular rejection. We tested the hypothesis that alloantibodies facilitate cellular rejection by functioning as opsonins to enhance T cell activation using a BALB/c to C57BL/6 heart or skin transplant model. Long-term heart and skin survival induced with anti-CD154 alone or in combination with donor-specific transfusion (DST), respectively, was abrogated by the presence of anti-K(d) mAbs, and alloreactive T cell activation as well as acute rejection was observed. The prevention of graft acceptance in the skin model was dependent on anti-K(d) binding to and converting DST from tolerigenic to immunogenic. Adoptive transfer of CFSE-labeled TCR-transgenic T cells into B6 recipients treated with anti-CD154/DST revealed the ability of anti-K(d) to enhance the proliferation of anti-K(d)-specific T cells via the indirect pathway as well as of non-K(d)-reactive, recipient MHC-restricted CD4(+) and CD8(+) T cells. Thus, alloantibodies with restricted specificity are able to facilitate the indirect presentation as well as the cross-presentation of a larger repertoire of "linked" donor-derived Ags. These observations highlight the ability of alloantibodies to function not only in classical humoral rejection but also as opsonins that facilitate the CD40-CD154-independent activation of alloreactive T cells.

Molecular determination of RHD zygosity: predicting risk of hemolytic disease of the fetus and newborn related to anti-D.

OBJECTIVE: Development of an accurate molecular method for paternal RHD zygosity to predict risk to a fetus for hemolytic disease of the fetus and newborn (HDFN) related to anti-D. METHODS: Quantitative fluorescence polymerase chain reaction (QF-PCR) was used to detect RHD exons 5 and 7, using RHCE exon 7 as an internal control. The genotype and zygosity were determined from the peak area ratios of RHD exon 5 or 7 to RHCE exon 7. We tested 25 Caucasian and 25 African American (AA) samples whose zygosity was predicted from the Rh phenotype and an alternate molecular method. In addition, we tested 71 paternal samples from prenatal cases where fetal testing was performed. RESULTS: RHD/RHCE ratios clearly distinguished the RHD/D and RHD/d genotypes. RHD variants were recognized when RHD exon 5 copy number was discordant with exon 7. The molecular assay identified eight cases where the phenotype incorrectly assigned zygosity and we observed three false-negatives in the hybrid Rhesus box assay. The prenatal results were consistent with the zygosity determined for the paternal samples in our study. CONCLUSIONS: This QF-PCR method accurately determines RHD zygosity in Caucasians and AAs and will help predict the risk that a fetus will inherit RHD.

Alport alloantibodies but not Goodpasture autoantibodies induce murine glomerulonephritis: protection by quinary crosslinks locking cryptic α3(IV) collagen autoepitopes in vivo.

The noncollagenous (NC1) domains of alpha3alpha4alpha5(IV) collagen in the glomerular basement membrane (GBM) are targets of Goodpasture autoantibodies or Alport posttransplant nephritis alloantibodies mediating rapidly progressive glomerulonephritis. Because the autoepitopes but not the alloepitopes become cryptic upon assembly of alpha3alpha4alpha5NC1 hexamers, we investigated how the accessibility of B cell epitopes in vivo influences the development of glomerulonephritis in mice passively immunized with human anti-GBM Abs. Alport alloantibodies, which bound to native murine alpha3alpha4alpha5NC1 hexamers in vitro, deposited linearly along the mouse GBM in vivo, eliciting crescentic glomerulonephritis in Fcgr2b(-/-) mice susceptible to Ab-mediated inflammation. Goodpasture autoantibodies, which bound to murine alpha3NC1 monomer and dimer subunits but not to native alpha3alpha4alpha5NC1 hexamers in vitro, neither bound to the mouse GBM in vivo nor induced experimental glomerulonephritis. This was due to quinary NC1 crosslinks, recently identified as sulfilimine bonds, which comprehensively locked the cryptic Goodpasture autoepitopes in the mouse GBM. In contrast, non-crosslinked alpha3NC1 subunits were identified as a native target of Goodpasture autoantibodies in the GBM of squirrel monkeys, a species susceptible to Goodpasture autoantibody-mediated nephritis. Thus, crypticity of B cell autoepitopes in tissues uncouples potentially pathogenic autoantibodies from autoimmune disease. Crosslinking of alpha3alpha4alpha5NC1 hexamers represents a novel mechanism averting autoantibody binding and subsequent tissue injury by posttranslational modifications of an autoantigen.

Two cases of platelet transfusion refractoriness associated with anti-CD36.

BACKGROUND: Antibodies to platelet (PLT) glycoprotein (GP) IV (CD36) have been implicated in rare cases of PLT refractoriness, particularly in non-Caucasians. We report two cases of PLT transfusion refractoriness linked to anti-CD36. STUDY DESIGN AND METHODS: A 5-year-old female of Lebanese descent and a 70-year-old male of Chinese descent both failed to respond to HLA-matched PLT transfusions during acute myelogenous leukemia induction therapy. Antibody screening was performed using a PLT antibody solid-phase kit (PAKPLUS, GTI Diagnostics), followed by the monoclonal antibody-specific immobilization of PLT antigen (MAIPA) test and, for the second case, the modified antigen capture enzyme-linked immunosorbent assay (MACE). RESULTS: Both patients demonstrated antibody to GP IV (CD36) on the PAKPLUS assay. On MAIPA testing, both phenotyped as CD36 negative. Anti-CD36 was demonstrated by MAIPA in the first case. In the second case, antibodies were not detected by MAIPA and variably detectable by MACE, depending on the mouse monoclonal antibody (MoAb) used. Because no Canadian CD36-negative donors were available, antigen-negative plateletpheresis units from the BloodCenter of Wisconsin were successfully transfused. CONCLUSION: Two cases of clinically significant CD36 antibodies are reported. Investigation of one case was complicated by steric inhibition of binding in the MAIPA and MACE assays with certain MoAbs. The cases demonstrate the importance of maintaining an ethnically diverse pool of rare donors and the value of international cooperation in the management of these patients.