Marlene L. Rose

Antigenic Heterogeneity of Vascular Endothelium

The antigenic status of vascular endothelium from different sites of the normal adult and fetal human cardiovascular system was investigated. Tissues included aorta (n = 9), pulmonary artery (n = 8), coronary artery (n = 6), ventricle/atrium (n = greater than 10), lymph node (n = 2), fetal whole heart (n = 3), and umbilical cord (n = 7). Frozen sections were studied using monoclonal antibodies recognizing endothelial markers (EN4, vWf, Pal-E, and 44G4), vascular adhesion molecules (ICAM-1, ELAM, VCAM, and PECAM), the monocyte/endothelial marker (OKM5), and major histocompatibility complex (MHC) molecules (class I and class II). Results demonstrate that capillary endothelium is phenotypically different from endothelial cells (EC) lining large vessels. Capillary EC strongly express MHC classes I and II, ICAM, and OKM5, which are variably weak to undetectable on large vessels. In contrast, the large vessels strongly express vWf and appear to constitutively express ELAM-1. This suggests that the capillary EC may be more efficient at antigen presentation or more susceptible to immune attack in vivo. Interestingly, normal coronary arteries, unlike all other large vessels, express MHC class II and VCAM molecules. Future studies should concentrate on comparative functional studies between capillary, coronary, and large vessel EC.

Report from a consensus conference on antibody-mediated rejection in heart transplantation

BACKGROUND The problem of AMR remains unsolved because standardized schemes for diagnosis and treatment remains contentious. Therefore, a consensus conference was organized to discuss the current status of antibody-mediated rejection (AMR) in heart transplantation. METHODS The conference included 83 participants (transplant cardiologists, surgeons, immunologists and pathologists) representing 67 heart transplant centers from North America, Europe, and Asia who all participated in smaller break-out sessions to discuss the various topics of AMR and attempt to achieve consensus. RESULTS A tentative pathology diagnosis of AMR was established, however, the pathologist felt that further discussion was needed prior to a formal recommendation for AMR diagnosis. One of the most important outcomes of this conference was that a clinical definition for AMR (cardiac dysfunction and/or circulating donor-specific antibody) was no longer believed to be required due to recent publications demonstrating that asymptomatic (no cardiac dysfunction) biopsy-proven AMR is associated with subsequent greater mortality and greater development of cardiac allograft vasculopathy. It was also noted that donor-specific antibody is not always detected during AMR episodes as the antibody may be adhered to the donor heart. Finally, recommendations were made for the timing for specific staining of endomyocardial biopsy specimens and the frequency by which circulating antibodies should be assessed. Recommendations for management and future clinical trials were also provided. CONCLUSIONS The AMR Consensus Conference brought together clinicians, pathologists and immunologists to further the understanding of AMR. Progress was made toward a pathology AMR grading scale and consensus was accomplished regarding several clinical issues.

Antivimentin antibodies are an independent predictor of transplant-associated coronary artery disease after cardiac transplantation.

Background. Transplant-associated coronary artery disease (TxCAD) is the most serious long-term complication after cardiac transplantation. Anti-endothelial antibodies are associated with disease, and one of the major endothelial antigens recognized in the sera of patients has been shown to be the protein filament vimentin. In this study, we investigated whether antivimentin antibodies are associated with TxCAD and whether their presence can be used to identify patients at high risk of developing angiographically detectable TxCAD. Methods. Up to 5 years after transplantation, 880 sequential sera (7.07±1.8 samples/patient) were collected retrospectively from 109 patients; the majority were collected in the first 2 years. Sera were assessed for antivimentin antibodies using ELISA. TxCAD was assessed by annual angiography. Results. Mean titres of antivimentin antibodies, calculated up to 1, 2, and 5 years, were significantly higher in patients who developed TxCAD than those who remained disease free (P <0.0001, P <0.0038, and P <0.0001, respectively). A predictive test based on the first-year mean vimentin titre alone (≥120) produced a test with 63% sensitivity and 76% specificity. Inclusion of persistent rejection or high 1-year mean titre (≥270) as a risk factor produced a test with 66% sensitivity and 82% specificity. Multivariate analysis of time to occurrence of transplant vasculopathy showed that mean titre at 1 or 2 years was an independent predictor of time until disease in the presence of all other variables. Conclusions. Antivimentin antibodies are an independent predictor of TxCAD and can be used to identify some of the patients who are at high risk of developing this complication.

C4d Fixing, Luminex Binding Antibodies—A New Tool for Prediction of Graft Failure After Heart Transplantation

The standard method to detect pretransplant antibodies has been the complement dependent cytotoxicity (CDC) test of donor leukocytes. Solid phase assays to detect HLA antibodies in pretransplant serum reveal a greater number of sensitized patients, but their clinical impact is less certain. Here we have developed a method of detecting C4d fixing HLA antibodies on Luminex beads. Pretransplant serum from 565 cardiac transplant patients was retrospectively tested for the presence of HLA antibodies using CDC, HLA coated Luminex beads and C4d deposition on Luminex beads, and the results correlated with graft survival. Whereas 5/565 patients had CDC positive donor specific antibodies (DSA) before their transplant, this number was increased by 19 using Luminex beads. The 1‐year survival of CDC –ve/Luminex +ve patients with DSA (n = 19) was 42% compared with 77% for CDC –ve/Luminex +ve without DSA (n = 39, p = 0.0039). Fixation of C4d (22/67 Luminex positive sera) had a negative effect on graft outcome; 1‐year graft survival was, C4d +ve/DSA +ve (n = 11) 20%, C4d +ve/DSA –ve (n = 11) 91%, C4d –ve DSA +ve (n = 13) 54%, C4d –ve DSA –ve (n = 32) 75%, compared with 75% for antibody‐negative patients (p = 0.0002). In conclusion, detection of Luminex +ve DSA in pretransplant serum provides a powerful negative predictor of graft survival, especially if they bind C4d.

De Novo Donor HLA-Specific Antibodies after Heart Transplantation Are an Independent Predictor of Poor Patient Survival

Preformed donor HLA‐specific antibodies are a known indicator for poor patient survival after cardiac transplantation. The role of de novo donor‐specific antibodies (DSA) formed after cardiac transplantation is less clear. Here we have retrospectively analyzed 243 cardiac transplant recipients, measuring HLA antibody production every year after transplantation up to 13 years post‐transplant. Production of de novo DSA was analyzed in patients who had been negative for DSA prior to their transplant. DSA including transient antibodies were associated with poor patient survival (p = 0.0018, HR = 3.198). However, de novo and persistent DSA was strongly associated with poor patient survival (p = 0.0001 HR = 4.351). Although complement fixing persistent DSA correlated with poor patient survival, this was not increased compared to noncomplement fixing persistent DSA. Multivariable analysis indicated de novo persistent DSA to be an independent predictor of poor patient survival along with HLA‐DR mismatch and donor age. Only increasing donor age was found to be an independent risk factor for earlier development of CAV. In conclusion, patients who are transplanted in the absence of pre‐existing DSA make de novo DSA after transplantation which are associated with poor survival. Early and regular monitoring of post‐transplant DSA is required to identify patients at risk of allograft failure.

Frequency and specificity of antiheart antibodies in patients with dilated cardiomyopathy detected using SDS-PAGE and western blotting

OBJECTIVES This study was designed to investigate the organ and disease specificity of antiheart antibodies in patients with dilated cardiomyopathy. BACKGROUND Autoimmune disease is characterized by the presence of circulating autoantibodies, and autoimmune mechanisms may play a role in the pathogenesis of dilated cardiomyopathy. METHODS An SDS-PAGE (sodium dodecylsulfate polyacrylamide gel electrophoresis) procedure followed by Western blotting was used to screen serum samples for antiheart antibodies of two immunoglobulin classes, IgM and IgG, from 52 patients with dilated cardiomyopathy and 48 patients with ischemic heart disease as control subjects. Use of two-dimensional gel electrophoresis followed by Western blotting and protein sequencing enabled us to identify the protein bands against which antiheart antibodies were produced in both groups of patients. RESULTS Strong IgG antiheart antibodies against myocardial proteins, cross-reacting with skeletal muscle proteins, were detected in significantly more patients with dilated cardiomyopathy (n = 24 [46%]) than with ischemic heart disease (n = 8 [17%]) (p = 0.001). Patients with dilated cardiomyopathy showed a significantly greater frequency and reactivity of IgG antiheart antibodies against six myocardial proteins (molecular weight 30, 35, 40, 60, 85 and 200 kD) than did patients with ischemic heart disease. These were identified as myosin light chain 1, tropomyosin, actin, heat shock protein (HSP)-60, an unidentified protein and myosin heavy chain, respectively. CONCLUSIONS We detected strong IgG antiheart antibodies in significantly more patients with dilated cardiomyopathy than with ischemic heart disease. The most immunogenic band was that corresponding to HSP-60. Antibodies against HSP-60 were found in 85% and 42% of patients with dilated cardiomyopathy and ischemic heart disease, respectively, confirming our hypothesis of an immune involvement in dilated cardiomyopathy.

Significant Frequencies of T Cells With Indirect Anti-Donor Specificity in Heart Graft Recipients With Chronic Rejection

BACKGROUND The purpose of this study was to determine whether T cells with indirect allospecificity could be detected in heart transplant recipients with chronic rejection. METHOD AND RESULTS Human T-cell clones were used to determine the most effective way to deliver major histocompatibility complex alloantigens for indirect presentation. Seven allograft recipients with evidence of progressive, chronic rejection were selected. Four heart graft recipients with no evidence of chronic rejection were used as controls. Peripheral blood T cells and antigen-presenting cells from the recipients were cultured with frozen/thawed stored donor cells or major histocompatibility complex class I-derived synthetic peptides in limiting dilution cultures and then compared with controls using tetanus toxoid and frozen/thawed third-party cells with no human leukocyte antigens in common with the donor. In 5 of 7 patients analyzed who had chronic rejection, elevated frequencies of T cells with indirect, anti-donor specificity (iHTLf) were detected. No such elevated iHTLf were detected in recipients without chronic rejection. DISCUSSION iHTLf can be obtained from human transplant recipients, which supports the contention that the indirect pathway is involved in chronic transplant rejection.

A comparison of primary endothelial cells and endothelial cell lines for studies of immune interactions

The purpose of this study was to assess the suitability of using endothelial cell (EC) lines for studies of endothelial/immune interactions. The immortal human EC lines HMEC-1, ECV304 and EaHy926 were compared to human umbilical vein endothelial cells (HUVEC) for constitutive and induced expression of surface antigens known to be involved in interactions with T cells. These cell lines were also compared to HUVEC in transendothelial migration assays. Flow cytometry was used to measure cell surface expression of platelet/endothelial cell adhesion molecule-1 (PECAM-1), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, major histocompatibility complex (MHC) class I and MHC class II, CD40, CD95 (fas) and lymphocyte function associated antigen-3 (LFA-3) before and after treatment with the cytokines tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). Polymerase chain reaction (PCR) was used to detect expression of the MHC class II transactivator. Significant differences were found in the ability to respond to cytokines between HUVEC and the cell lines, the greatest differences being induction of VCAM-1 and E-selectin in response to TNF-alpha and induction of MHC class II antigens in response to IFN-gamma. Thus unlike HUVEC, induction of VCAM-1 and E-selectin was not detectable on EaHy926 and ECV304 and barely detectable on HMEC-1. MHC class II antigens were not induced on ECV304 in response to IFN-gamma and nor was the class II transactivator (CIITA). Unlike HUVEC and the other cell lines, ECV304 were constitutively negative for PECAM-1. Constitutive and induced expression of MHC class I, ICAM-1, LFA/3, CD40 and fas were most conserved between the cell lines and showed little difference to HUVEC. The migration of peripheral blood mononuclear cells (PBMC) through all cell lines was significantly reduced compared to through HUVEC, suggesting that there is a functional difference between the cell lines with regard to interactions with lymphocytes. In conclusion this study has demonstrated significant differences in the ability of endothelial cell lines to respond to cytokines compared to primary HUVEC cultures. In particular ECV304 compares very poorly with HUVEC. Whether these differences are caused by immortalization procedures or reflect heterogeneity of EC arising from different vascular beds is discussed.

CYCLOSPORIN IN JUVENILE DERMATOMYOSITIS

Juvenile dermatomyositis in fourteen children who had not responded fully to steroids and other immunosuppressants and who had had chronic active disease for an average of 3 years was successfully treated with cyclosporin. Twelve patients had serious complications of the disease or of previous treatment. The response to cyclosporin included recovery of muscle strength and function and resolution of complications. It was possible to stop steroids or to reduce the steroid dose, which had previously been difficult, in all fourteen patients. In general, a low dose of cyclosporin (2.5-7.5 mg/kg daily) was sufficient and no serious side-effects were seen.

Report from a consensus conference on the sensitized patient awaiting heart transplantation.

A consensus conference took place on April 8, 2008 to assess the current status of sensitization in the pre–heart transplant patient, the use and efficacy of desensitization therapies, and the outcome of desensitized patients after heart transplantation. The conference had 71 participants (transplant cardiologists, surgeons, immunologists and pathologists; see Appendix) representing 51 heart transplant centers from North America, Europe, Asia and Australia. Prior to the conference, survey data (regarding the sensitized patient) were submitted by 23 of the 51 centers participating in the conference (Table 1).