Altered Storage and Function of von Willebrand Factor in Human Cardiac Microvascular Endothelial Cells Isolated from Recipient Transplant Hearts.

The assembly of von Willebrand factor (VWF) into ordered helical tubules within endothelial Weibel-Palade bodies (WPBs) is required for the efficient deployment of the protein at sites of vascular injury. VWF trafficking and storage are sensitive to cellular and environmental stresses that are associated with heart disease and heart failure. Altered storage of VWF manifests as a change in WPB morphology from a rod shape to a rounded shape and is associated with impaired VWF deployment during secretion. In this study, we examined the morphology, ultrastructure, molecular composition and kinetics of exocytosis of WPBs in cardiac microvascular endothelial cells isolated from explanted hearts of patients with a common form of heart failure, dilated cardiomyopathy (DCM; HCMECD), or from nominally healthy donors (controls; HCMECC). Using fluorescence microscopy, WPBs in HCMECC (n = 3 donors) showed the typical rod-shaped morphology containing VWF, P-selectin and tPA. In contrast, WPBs in primary cultures of HCMECD (n = 6 donors) were predominantly rounded in shape and lacked tissue plasminogen activator (t-PA). Ultrastructural analysis of HCMECD revealed a disordered arrangement of VWF tubules in nascent WPBs emerging from the trans-Golgi network. HCMECD WPBs still recruited Rab27A, Rab3B, Myosin-Rab Interacting Protein (MyRIP) and Synaptotagmin-like protein 4a (Slp4-a) and underwent regulated exocytosis with kinetics similar to that seen in HCMECc. However, secreted extracellular VWF strings from HCMECD were significantly shorter than for endothelial cells with rod-shaped WPBs, although VWF platelet binding was similar. Our observations suggest that VWF trafficking, storage and haemostatic potential are perturbed in HCMEC from DCM hearts.

The interplay between the Rab27A effectors Slp4-a and MyRIP controls hormone-evoked Weibel-Palade body exocytosis.

Weibel-Palade body (WPB) exocytosis underlies hormone-evoked VWF secretion from endothelial cells (ECs). We identify new endogenous components of the WPB: Rab3B, Rab3D, and the Rab27A/Rab3 effector Slp4-a (granuphilin), and determine their role in WPB exocytosis. We show that Rab3B, Rab3D, and Rab27A contribute to Slp4-a localization to WPBs. siRNA knockdown of Slp4-a, MyRIP, Rab3B, Rab3D, Rab27A, or Rab3B/Rab27A, or overexpression of EGFP-Slp4-a or EGFP-MyRIP showed that Slp4-a is a positive and MyRIP a negative regulator of WPB exocytosis and that Rab27A alone mediates these effects. We found that ECs maintain a constant amount of cellular Rab27A irrespective of the WPB pool size and that Rab27A (and Rab3s) cycle between WPBs and a cytosolic pool. The dynamic redistribution of Rab proteins markedly decreased the Rab27A concentration on individual WPBs with increasing WPB number per cell. Despite this, the probability of WPB release was independent of WPB pool size showing that WPB exocytosis is not determined simply by the absolute amount of Rab27A and its effectors on WPBs. Instead, we propose that the probability of release is determined by the fractional occupancy of WPB-Rab27A by Slp4-a and MyRIP, with the balance favoring exocytosis.

Molecular analyses of human rabies virus associated with encephalitis in two children in Gabon.

Rabies is a zoonotic neurological life-threatening neglected tropical disease present worldwide, and Gabon is listed as an endemic country. However, despite strong clinical suspicion in humans and molecular confirmation of rabies virus (RABV) infections in dogs for several decades, no molecularly confirmed human case in Gabon has ever been reported. In this study, we describe two cases of human rabies and provide the first molecular diagnostic report on suspected human rabies cases in Gabon. Our results showed that the RABVs isolated from the patients are closely related to other RABV strains belonging to the African 1A subclade in the Cosmopolitan lineage isolated more than 20 years ago from Gabonese dog brains, suggesting that only this species circulates in the country. Because both patients had a history of dog bites a few weeks before symptom onset and the main causative agent of human rabies worldwide is dog-associated RABV, we conclude that dogs are likely to be the source of human infection in this study.

Protective effect of phosphorylated Hsp27 in coronary arteries through actin stabilization.

There is evidence for an inverse association between cellular expression of Hsp27 and vascular disease with carotid plaques, endarterectomy specimens, and cardiac biopsies investigated to date. Here we compare non-diseased coronary arteries from human heart transplant donors and patients with dilated cardiomyopathy (DCM) with no evidence of coronary artery disease, to coronary arteries from patients with ischemic heart disease (IHD) in order to determine abundance of phosphorylated Hsp27 (phospho-Hsp27) in plaque-free diseased vessels and elucidate how this protective effect is brought about through protein regulation. Western blotting identified phospho-Hsp27, phosphorylated on Ser82, Ser78, and Ser15, to be specifically decreased in IHD, but not DCM, compared to non-diseased vessels. Immunohistochemistry confirmed these results and revealed phospho-Hsp27 was located within both smooth muscle and endothelial cells. Disease-free coronary arteries and from patients with IHD were then subjected to 2-Dimensional Difference Gel Electrophoresis (2D-DIGE) analysis to detect proteins with altered abundance, which were subsequently identified by mass spectrometry. Hsp27 showed decreased abundance in ischemic vessels as expected. The expression of cytoskeletal proteins, namely vimentin was significantly reduced, while transgelin and tropomyosin showed significantly increased abundance in vessels with IHD. Immunohistochemistry studies suggested an increase in G-actin abundance to be present within IHD vessels. The results are consistent with the hypothesis that phospho-Hsp27 protects against vascular disease possibly by stabilizing the actin cytoskeleton within endothelial and/or smooth muscle cells.

Effect of phosphorylated hsp27 on proliferation of human endothelial and smooth muscle cells.

Recent studies have suggested a protective role of hsp27 against atherosclerosis and transplant graft vasculopathy. Here we have investigated the effects of over-expression of wild-type hsp27 and its phosphorylation mimics on proliferation of human endothelial cells (ECs) and smooth muscle cells (SMCs). ECs and SMCs cultured from human blood vessels or cells lines (human microvascular endothelial cell line and human telomerase reverse transcriptase subunit SMC) were infected with adenovirus containing DNA from wild-type hsp27, hyper-phosphorylated hsp27 mimic (3D hsp27), hypo-phosphorylated hsp27 mimic (3A hsp27) or anti-sense hsp27, and proliferation measured over the next 5 days. Protein extracts from infected cells were subjected to proteomic analysis using 2-D DIGE. Over-expression of 3D hsp27 and anti-sense hsp27 but not 3A hsp27 mimic caused significant inhibition of proliferation of ECs and SMCs. Proteomic analysis focussed on proteins that were significantly down-regulated by the 3D hsp27 mutant. The cell cycling proteins stathmin, cofilin and ubiquitination enzymes fullfilled these criteria. 1-D Western blots of infected human microvascular endothelial cell line and human telomerase reverse transcriptase subunit SMC confirmed down-regulation of stathmin, cofilin and ubiquitination enzymes by 3D hsp27. The phosphorylation status of hsp27 is an important regulator of proliferation of human vascular ECs and SMCs; possibly contributing to cardiovascular protection.

Expression of endothelial intercellular adhesion molecule-1 is determined by genotype: effects on efficiency of leukocyte adhesion to human endothelial cells.

Two biallelic polymorphisms, previously described in the human intercellular adhesion molecule (ICAM)-1 gene at codon 241 (glycine [G] to arginine [R] substitution) and codon 469 (glutamic acid [E] to lysine [K] substitution) have been associated with a number of diseases including myocardial infarction, transplant rejection, and diabetes. However, the functional significance of these polymorphisms has not been determined. ICAM-1 cell surface expression and ICAM-1-mediated leukocyte adhesion were investigated using Cos7 transfected with ICAM-1 polymorphic variants or human umbilical vein endothelial cells (HUVEC) of different ICAM-1 genotypes. There was significantly higher expression of surface ICAM-1 on Cos7 transfected with a plasmid encoding the GE (G241/E469) ICAM-1 variant or untreated HUVEC of GEGE (G241/E469 homozygous genotype). ICAM-1-mediated adhesion of peripheral blood mononuclear cells (PBMC) to GE-Cos7 cells or TNF-treated GEGE HUVEC was significantly increased. However, there was no significant difference in adhesion of PBMC to recombinant ICAM-1 of each polymorphic variant plated onto plastic wells. We conclude that the GE genotype of ICAM-1 is associated with greater cell surface expression of ICAM-1, which in turn leads to greater adhesion of leukocytes. This may explain the previously described associations of ICAM-1 polymorphisms with chronic inflammatory disease.

Mycophenolate mofetil may allow cyclosporine and steroid sparing in de novo heart transplant patients.

BACKGROUND: Mycophenolate mofetil (MMF) provides superior prophylaxis against acute rejection when compared with azathioprine (AZA) in heart and renal transplantation. However, it remains unclear whether this results in improved survival or reduced morbidity after heart transplantation. METHOD: In a sequential study, 240 cardiac transplant patients were treated with either MMF (n=119) or AZA (n=121) both in combination with cyclosporine and corticosteroids after rabbit antithymocyte globulin induction. RESULTS: By protocol lower cyclosporine levels were targeted in the MMF group during the first year (e.g. 203+/-52 ng/mL MMF vs. 236+/-59 ng/mL AZA, P=0.0006 at 6 months). Patient survival at 1 year (82% MMF vs. 79% AZA, P=0.55) and at 3 years was similar in both groups. The cumulative probability of receiving antirejection treatment within 1 year was lower in the MMF group, as was biopsy-proven acute rejection with International Society of Heart and Lung Transplantation grade > or =3A (24% vs. 35%, P=0.03). The MMF group also had fewer episodes requiring cytolytic therapy (6% vs. 13%, P=0.04) and more patients had steroids withdrawn by 1 year (66% vs. 32%, P<0.001). Renal function was better in the MMF group with lower creatinine levels at 1 year (133+/-45 vs. 155+/-46 micromol/L, P=0.0004). Calculated creatinine clearance (Cockcroft and Gault formula) at 1 year was also better (MMF 74+/-32 mL/min vs. AZA 62+/-24 mL/min, P=0.004). CONCLUSION: Our results suggest that immunosuppression with MMF rather than AZA may allow lower cyclosporine levels, better renal function, and increased steroid weaning at 1 year while also achieving better control of acute rejection.

Leukocyte Adhesion Under Hemodynamic Flow Conditions.

Vascular endothelial cells (ECs) line the luminal side of all blood vessels and act as a selective barrier between blood and tissue. ECs are constantly exposed to biochemical and biomechanical stimuli from the blood and underlying tissue. Fluid shear stress acts in parallel to the vessel wall, resulting from friction of blood against EC. Despite the importance of flow on normal EC function, much of the information regarding EC function and dysfunction has been derived from cells harvested, grown, and studied in static culture.In order to study the effects of shear stress on EC function a number of in vitro models have been developed. This manuscript provides methodology for use of a system which enables recirculation of leukocytes and cell culture medium over the endothelium for a period of several minutes to days and enables investigation of the effects of prolonged leukocyte coculture on both the endothelial and leukocyte populations.

Chronic rejection in the heart.

The dramatic improvements in 1-yr survival following cardiac transplantation have not been matched by similar improvements in long-term graft survival. Long-term survival of allografted hearts is limited by a progressive fibroproliferative disease, resulting in intimal thickening and occlusion of the grafted coronary vessels. This disease, variously known as accelerated transplant coronary artery disease or cardiac graft vasculopathy, is also known as chronic rejection. The histology and clinical sequelae are briefly described. The disease can be thought of as a model for non-transplant atherosclerosis, postangioplasty restenosis, and vein graft atherosclerosis. There is compelling evidence that it is driven by alloantigen-dependent mechanisms. The evolution of the disease consists of three phases, an antibody-mediated phase, a cell-mediated phase, and a phase of tissue remodeling that is dependent on cytokines and growth factors. Experimental studies show that adoptive transfer of immunoglobulin can transfer features of intimal hyperplasia to transplanted arteries in immunodeficient recipients. Damage to donor endothelium is likely to be an important initiating factor in this disease because it exposes a thrombogenic subendothelial matrix. Whether T cells of antibody are most important in damaging the endothelium is currently the subject of much research. Although T cells are sometimes present in atherosclerotic lesions, an association with acute rejection has never been consistently shown.

Detection and clinical relevance of antibodies after transplantation.

Until recently, the role of antibodies in graft failure has been hampered by poor methods of defining specificity. Development of solid phase assays using purified major histocompatibility complex (MHC) molecules has greatly advanced our ability to monitor anti-human leukocyte antigen (HLA) antibodies in patients and to distinguish between HLA and non-HLA antibodies. The purpose of this chapter is to describe the methods for detecting antibodies and what we have learned in recent years regarding the role of well-defined antibodies to HLA and non-HLA antigens. Use of the complement-dependent lymphocytotoxic test was instrumental in defining patients who are sensitized to donor HLA antigens, and it still plays a major role in avoiding transplantation of organs into sensitized patients. However, solid phase assays are more useful for following patients posttransplant. A major advance has been the demonstration that anti-MHC class II antibodies are made late after transplantation and contribute to late graft failure. This has been demonstrated for renal and lung transplantation, but has not yet been confirmed for other organs. Clearer definition of non-HLA antibodies has been achieved, such as the autoantigen vimentin and MHC I-related chain A. Experimental studies using minor mismatched strain combinations confirm that non-HLA antibodies bind to donor endothelial cells; these antibodies seem to cause apoptosis but not complement-mediated lysis.

Species differences of endothelial extracellular nucleotide metabolism and its implications for xenotransplantation.

There is a severe shortage of human organs available for transplantation and xenotransplantation - use of animal organs has long been suggested to overcome this problem. Recent advances in understanding rejection in xenotranplantation and development of genetically engineered pigs that reduced hyperacute rejection were fundamental steps forward but other unresolved mechanisms remain an obstacle. Endothelium is a major target for all rejection mechanisms in xenotransplantation. This is caused not only by location of these cells at the first line of contact but also because endothelium is a very variable cell type across different species. This variability affects not only its immune characteristics but also physiology and metabolism. Nucleotide metabolism is particularly variable in endothelial cells of different species. We attributed particular importance to one such difference - much lower activity of ecto-5'-nucleotidase (E5'N) in pig endothelial cells as compared to human. To study its significance our group developed pig endothelial cell line stably expressing human E5'N. This allowed us to determine that E5'N controls the rate of adenosine formation from extracellular nucleotides even with ATP as the substrate. Expression of human E5'N in pig cells attenuated several mechanisms involved in xenotransplant rejection such as cytotoxicity induced by human NK cells, human platelet aggregation or human platelet adherence to endothelium. We conclude that species differences of endothelial nucleotide metabolism could contribute to rejection following xenotransplantation. These studies suggests that expression of human ecto-5'-nucleotidase in pigs genetically engineered for xenotransplantation could help to prolong graft survival.

Activation of autoimmune B cells and chronic rejection.

Allotransplantation into immunosuppressed individuals results in long-term survival of grafts. However, the grafts are damaged, probably at many stages before, during and after implantation. The hypothesis to be presented is that release of antigens and autoantigens from the chronically damaged graft results in breaking tolerance to self-antigens and an autoimmune response. There is experimental evidence that autoimmune responses following allotransplantation are damaging and cause accelerated graft rejection.

Anti-intercellular adhesion molecule-1 antibodies in sera of heart transplant recipients: a role in endothelial cell activation.

BACKGROUND: Antiendothelial antibodies to non-human leukocyte antigens are made by a subset of heart transplant recipients, but the specificity of such antibodies is undefined. Intercellular adhesion molecule (ICAM)-1 is an abundantly expressed adhesion molecule with polymorphic residues, expressed on the surface of endothelial cells. The hypothesis that ICAM-1 acts as a minor histocompatibility antigen and that anti-ICAM-1 antibodies, directed against polymorphic residues, could be one component of the antiendothelial antibodies found after heart transplantation has been tested. METHODS: Chinese hamster ovary cells were transfected with full-length polymorphic variants of human ICAM-1. The binding of antibodies (immunoglobulin [Ig] G or IgM) to these cells was measured using sera from 50 heart transplant recipients (pretransplant and 1 and 2 years posttransplant) and sera from 20 normal volunteers by flow cytometry. The recipients and donors were genotyped for ICAM-1 polymorphisms. RESULTS: Sixty-eight percent (n=34) of patients made IgM antibodies that bound to ICAM-1. However, it seems unlikely that ICAM-1 is a minor transplantation antigen, because there were no differences in antibody production from recipients matched or mismatched for ICAM-1 alleles. The antibodies bound to mouse endothelial cells that were engineered to overexpress human ICAM-1, and induced a robust activation of the Erk-2 mitogen-activated protein kinase pathway. CONCLUSIONS: Anti-ICAM-1 antibodies are produced after cardiac transplantation, but not to polymorphic residues. Such antibodies may contribute to the endothelial activation by binding to the endothelium, causing activation of proinflammatory signaling pathways.

The autoimmune response to vimentin after renal transplantation in nonhuman primates is immunosuppression dependent.

BACKGROUND: Chronic allograft nephropathy (CAN) is a common late complication of kidney transplantation. Antibodies to both human leukocyte antigen and nonhuman leukocyte antigen antigens have been implicated in the development of this condition. Here we investigated the presence of antivimentin antibodies in nonhuman primate recipients of kidney allografts as a possible predictor of CAN and the effects of immunosuppression. METHODS: Thirty seven rhesus monkeys received a kidney allograft to study the potency of several different immunosuppressive regimens (conventional immunosuppression, n=19, vs. costimulatory blockade, n=18). Monkeys were tested for antivimentin antibody by enzyme-linked immunosorbent assay and for anti-donor antibody by staining donor spleen cells with recipient serum. The appearance of antibodies was correlated with the graft pathology in biopsy and necropsy material. RESULTS: Antivimentin antibodies were found in 31 of 37 animals, whereas only 15 of 32 animals made anti-donor antibodies. Conventional immunosuppression did not prevent antivimentin antibody formation. Costimulation blockade, in particular blocking CD40 and CD86, significantly delayed or prevented antivimentin antibody formation, but did not prevent CAN. Antivimentin antibodies were not significantly associated with development of CAN. CONCLUSIONS: We postulate that vimentin acts as an autoantigen after renal transplantation; it elicits an autoimmune response that is not regulated by cyclosporine. This autoimmune response may be part of the complex immunologic events occurring posttransplantation and may contribute to the development of CAN, but cannot be considered as a major cause of CAN because this condition also develops without antivimentin antibodies.

Sequential expression of three known protective genes in cardiac biopsies after transplantation.

BACKGROUND: The expression of the "protective" genes A20, heme oxygenase (HO)-1, and Bcl-xl in rodent allografts and xenografts correlates with long-term survival of transplanted hearts. We investigated the expression of HO-1, Bcl-2, and A20 in sequential biopsies from nine cardiac transplant recipients by using quantitative real-time reverse-transcriptase polymerase chain reaction and immunohistochemistry. METHODS: Five to 16 endomyocardial biopsies were analyzed from each patient 7 to 365 days after transplantation. Biopsies were classified as acute rejection (AR) by International Society of Heart and Lung Transplantation criteria. mRNA values were normalized against an endogenous control gene (18S), and protein expression was analyzed by immunohistochemistry. RESULTS: All genes were expressed at every time point. HO-1 was significantly higher in the first 2 months (2 months vs. 10+ months, P<0.05) and was associated with AR (0.30+/-0.07) versus nonrejection (0.16+/-0.02, P=0.026). In contrast, expression of Bcl-2 and A20 was low at 2 months, but both increased with time (P<0.05, 2 months vs. 10+ months for Bcl-2 and A20). There was no significant association of Bcl-2 or A20 with AR. Immunocytochemistry revealed that HO-1 localizes to infiltrating cells and not parenchymal cells in cardiac biopsies. In contrast, Bcl-2 and A20 were found to localize to endothelial, smooth muscle, and infiltrating cells. CONCLUSIONS: HO-1 is induced early after transplantation, whereas Bcl-2 and A20 seem to be induced as part of the chronic response. These differences together with different localization sites in vivo suggest they have different roles in protection from injury after cardiac transplantation.

Blood transfusion, serum ferritin, and iron in hemodialysis patients in Africa.

Background and Objectives. There is no data analyzing the outcome of blood transfusions and oral iron therapy in patients with kidneys failure in sub-Saharan Africa. The present study aimed to fill that gap and assess the value of ferritin in the diagnosis of iron overload and deficiency. Design. From January to February 2012, we prospectively studied 85 hemodialysis patients (78% of males and 22% of females aged 20 to 79 years) attending the Gabonese National Hemodialysis Centre. Results. Correlation studies showed (a) a strong positive linear relationship between the number of blood transfusions and high serum ferritin in hemodialysis patient (Spearman r : 0.74; P value: 0.0001); (b) a weak association between the number of blood transfusions and serum iron concentrations (Spearman r : 0.32; P value: 0.04); (c) a weak association between serum ferritin and serum iron (Spearman r : 0.32; P value: 0.003). Also, the strength of agreement beyond chance between the levels of ferritin and iron in the serum was poor (κ = 0.14). The prevalence of iron overload was 10.6%, whereas the prevalence of iron deficiency was 2.3%, comparing (1) patients with a maximum of one transfusion not on iron therapy; (2) patients with a maximum of one transfusion on iron therapy; (3) polytransfused patients not on iron therapy; and (4) polytransfused patients on oral iron therapy. The "Kruskal-Wallis test" showed that ferritin levels varied significantly between the groups (P value: 0.0001). Conclusion. Serum ferritin is not reliable as a marker of iron overload. For patients undergoing regular transfusion we recommend routine serum ferritin measurement and yearly measurement of LIC.

Effect of cognate human CD4+ T cell and endothelial cell interactions upon chemokine production.

BACKGROUND: In vitro studies have shown that cognate recognition of antigen presented by endothelial cells (EC) causes T cell activation, proliferation, and cytokine release and alters the transmigration of T cells. Here we have investigated chemokine induction caused by cognate interactions between human CD4+ T cells and MHC class II-expressing EC. METHODS: HLA-DR-restricted CD4+ T cells were cocultured with HLA-DR-expressing allogeneic Eahy.926, aortic, or heart microvascular EC. Chemokine mRNA expression was measured by RTPCR, and chemokine protein secreted was measured by a cytokine array system and ELISA. Molecules involved in chemokine secretion were identified using blocking monoclonal antibodies, and cellular sources of chemokines determined by intracellular chemokine staining. Coculture supernatants were also used in chemotaxis assays. RESULTS: Nine different chemokine mRNA and proteins were expressed because of noncognate interactions between T cells and EC. Cognate interactions induced de novo expression of four chemokines and upregulation of seven chemokines. Levels of CCL3, CCL8, and CXCL10 secreted into supernatants were in the nanomolar range and were chemotactic for T cells and monocytes. Blocking antibodies to HLA-DR and LFA-3 abrogated production of CCL3, CCL8, and CXCL10. Blocking antibodies to interferon-gamma and tumor necrosis factor-alpha inhibited CCL8 and CXCL10 but not CCL3 production. CCL3 and CXCL10 were produced by both T cells and EC. CONCLUSIONS: Cognate interactions between alloreactive CD4+ T cells and MHC class II-expressing EC results in a specific pattern of chemokine production. These chemokines could play important roles in recruitment of leukocytes into vascularised allografts.

High diversity of non-human leukocyte antigens in transplant-associated coronary artery disease.

BACKGROUND: Antibodies to endothelial derived non-human leukocyte antigens (HLA) have been associated with transplant (Tx)-associated coronary artery disease (CAD) after cardiac transplantation; however, few have been identified. The aim of this study was to screen a human coronary artery endothelial cell cDNA library with patient sera to establish the diversity and nature of the target antigens. METHODS: A human coronary artery endothelial cell cDNA library was screened with sera from seven long-term cardiac transplant patients with angiographically diagnosed TxCAD and sera from five healthy volunteers. RESULTS: Of the seven patients' sera, five showed reactivity, as did sera from two of the five normal subjects. Eighteen positive cDNA clones were isolated by TxCAD sera; DNA sequence analysis and DNA database searching identified all but one clone; 16 were nuclear or cytoplasmic proteins and 1 of them was the cell surface protein neuropilin 2. Five clones were targeted by normal sera. A different spectrum of reactive clones was identified by the sera of each patient where reactive clones were evident. CONCLUSIONS: A high diversity of non-HLA antigens, probably autoantigens, are involved in the pathogenesis of TxCAD.

Detection of vimentin-specific autoreactive CD8+ T cells in cardiac transplant patients.

BACKGROUND: Evidence is emerging that autoimmunity can play a role in allograft rejection. Reports have described the presence of autoantibodies in transplant patients and CD4+ autoreactive T cells in rodent models of allograft rejection. The objective of this study was to seek evidence of CD8+ T-cell-mediated autoimmunity in the transplant setting. The author have previously observed autoimmunity to the non-polymorphic cytoskeletal protein vimentin in cardia transplant patients. In this study, vimentin antibody positive patients were screened for the presence of vimentin-specific self-major histocompatibility complex class I-restricted CD8+ T cells. METHODS: Two peptide sequences from vimentin that bound HLA-A*0201 were identified and fluorochrome-labeled A*0201 tetramers with each peptide were constructed to screen for vimentin-specific T cells. RESULTS: Tetramer-binding CD8+ T cells were detected in peripheral blood lymphocytes from two of six patients after expansion by in vitro stimulation with peptide. Tetramer-binding T cells produced interferon-gamma in an antigen-specific fashion. No autoreactive T cells specific for vimentin were detected after peptide stimulation of T cells from eight healthy A*0201-positive volunteers. CONCLUSIONS: This finding is the first evidence of CD8+ T-cell-mediated autoimmunity in human transplant patients.