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Biomedical subjects

E F Reed

Publications and source records attributed to E F Reed.

At least 19 recordsLinked to original sources
PubMed2006

Phosphorylated S6 ribosomal protein: a novel biomarker of antibody-mediated rejection in heart allografts.

We tested the hypothesis that phosphorylation of S6 ribosomal protein (S6RP), a downstream target of the PI3K/Akt/mTOR pathway, is a biomarker of antibody-mediated rejection (AMR) in heart allografts. Primary cultures of human aortic and microvascular endothelial cells (EC) were treated with anti-HLA class I and class II antibodies (Ab) and cell lysates were studied for phosphorylation of S6 ribosmal protein at Serine235/236 (p-S6RP). Treatment of cultured EC with anti-class I and class II Ab stimulated S6RP phosphorylation. Immunohistochemical techniques were used to detect the level of p-S6RP in endomyocardial biopsies (n = 131) from 46 heart transplant recipients and the results were correlated with histopathological diagnosis of rejection, C4d staining, production of posttransplant anti-HLA Ab and clinical outcome. Increased phosphorylation of S6RP in endomyocardial biopsies was significantly associated with the diagnosis of AMR (p < 0.0001). No significant association between acute cellular rejection (ACR) and p-S6RP was observed. C4d staining was positively associated with both AMR and p-S6RP. Posttransplant anti-HLA class II Ab production was also significantly associated with a positive p-S6RP status in cardiac biopsies. These results indicate that p-S6RP is a useful biomarker for the diagnosis of AMR.

Acute Disease↗
PubMed2004

Peptides from antibodies to DNA elicit cytokine release from peripheral blood mononuclear cells of patients with systemic lupus erythematosus: relation of cytokine pattern to disease duration.

Peptides from VH regions of antibodies to DNA drive immune responses in systemic lupus erythematosus (SLE). We studied peptide-induced cytokine release by peripheral blood mononuclear cells (PBMC) of patients, the influence of peptide concentration, disease characteristics and HLA-D haplotypes. Cells secreting cytokines (IFNgamma, IL-2, IL-4 and IL-10) were measured by ELISPOT in PBMC from 31 patients with SLE and 20 matched healthy controls in response to seven peptides (A-G) from the CDR1/FR2 to CDR2/FR3 VH regions of human anti-DNA MAbs. Disease activity was assessed by SELENA-SLEDAI. HLA-DR and -DQ alleles were determined by molecular typing techniques. PBMC from significantly higher proportions of SLE patients than controls responded to VH peptides by generating IFNgamma and IL-10. Type of cytokines released in response to at least one peptide (D) depended on antigen concentration. Cytokine release was not associated with clinical features of SLE except for disease duration. A shift occurred from IFNgamma, IL-4 and IL-10 production in early disease to IL-4 and IL-10 in late disease (suggesting increasing TH2-like responses over time). Three peptides (B, D, G) were more stimulatory in the SLE patients than controls. Although none of the peptides was restricted by any particular MHC class II allele, among responders there was increased prevalence of HLA- DQB1*0201 and/or DRB1*0301, alleles known to predispose to SLE. Thus, responses to some VH peptides are more frequent in SLE and vary with disease duration. Increased responses in individuals with HLA class II genotypes that predispose to SLE suggest that peptide presentation by those molecules permits brisker peripheral blood cell responses to autoantibody peptides, thus increasing risk for disease.

Amino Acid Sequence↗
PubMed1999

Alloantibody-mediated class I signal transduction in endothelial cells and smooth muscle cells: enhancement by IFN-gamma and TNF-alpha.

Chronic rejection is the major limiting factor to long term survival of solid organ allografts. The hallmark of chronic rejection is transplant atherosclerosis, which is characterized by the intimal proliferation of smooth muscle cells, endothelial cells, and fibroblasts, leading to vessel obstruction, fibrosis, and eventual graft loss. The mechanism of chronic rejection is poorly understood, but it is suspected that the associated vascular changes are a result of anti-HLA Ab-mediated injury to the endothelium and smooth muscle of the graft. In this study we have investigated whether anti-HLA Abs, developed by transplant recipients following transplantation, are capable of transducing signals via HLA class I molecules, which stimulate cell proliferation. In this report we show that ligation of class I molecules with Abs to distinct HLA-A locus and HLA-B locus molecules results in increased tyrosine phosphorylation of intracellular proteins and induction of fibroblast growth factor receptor expression on endothelial and smooth muscle cells. Treatment of cells with IFN-gamma and TNF-alpha up-regulated MHC class I expression and potentiated anti-HLA Ab-induced fibroblast growth factor receptor expression. Engagement of class I molecules also stimulated enhanced proliferative responses to basic fibroblast growth factor, which augmented endothelial cell proliferation. These findings support a role for anti-HLA Abs and cytokines in the transduction of proliferative signals, which stimulate the development of myointimal hyperplasia associated with chronic rejection of human allografts.

Adjuvants, Immunologic↗
PubMed1999

HLA class I-mediated induction of cell proliferation involves cyclin E-mediated inactivation of Rb function and induction of E2F activity.

Chronic rejection of transplanted organs is manifested as atherosclerosis of the blood vessels of the allograft. HLA class I Ags have been implicated to play a major role in this process, since signaling via HLA class I molecules can induce the proliferation of aortic endothelial as well as smooth muscle cells. In this study, we show that HLA class I-mediated induction of cell proliferation correlates with inactivation of the Rb protein in the T cell line Jurkat as well as human aortic endothelial cells. HLA class I-mediated inactivation of Rb can be inhibited specifically by neutralizing Abs to basic fibroblast growth factor (bFGF), suggesting a role for FGF receptors in the signaling process. Signaling through HLA class I molecules induced cyclin E-associated kinase activity within 4 h in quiescent endothelial cells, and appeared to mediate the inactivation of Rb. A cdk2 inhibitor, Olomoucine, as well as a dominant-negative cdk2 construct prevented HLA class I-mediated inactivation of Rb; in contrast, dominant-negative cdk4 and cdk6 constructs had no effect. Furthermore, there was no increase in cyclin D-associated kinase activity upon HLA class I ligation, suggesting that cyclin E-dependent kinase activity mediates Rb inactivation, leading to E2F activation and cell proliferation.

Carrier Proteins↗
PubMed1998

Ligation of HLA class I molecules on smooth muscle cells with anti-HLA antibodies induces tyrosine phosphorylation, fibroblast growth factor receptor expression and cell proliferation.

The development of transplant atherosclerosis, a manifestation of chronic rejection, is the major obstacle to long-term survival of cardiac and renal allografts. The incidence of transplant atherosclerosis is increased in transplant recipients producing antidonor HLA antibodies following transplantation, suggesting that anti-HLA antibodies play a role in the pathogenesis of the disease. We have postulated that anti-HLA antibodies mediate the development of transplant atherosclerosis by binding to class I molecules on the endothelium and smooth muscle of the graft and transducing signals which stimulate cell proliferation. In this report we demonstrate that anti-HLA class I antibodies transduce signals in smooth muscle cells stimulating increased tyrosine phosphorylation of intracellular proteins and up-regulation of fibroblast growth factor (FGF) receptors. Antibody binding to class I molecules on smooth muscle cells is also accompanied by increased responsiveness to basic FGF and augmented cell proliferation. These findings may explain the increased occurrence of transplant atherosclerosis in recipients producing anti-donor HLA antibodies.

Antibodies↗
PubMed1997

Induction of high affinity fibroblast growth factor receptor expression and proliferation in human endothelial cells by anti-HLA antibodies: a possible mechanism for transplant atherosclerosis.

The major limitation to long term survival of organ allografts is chronic rejection, which is manifested as atherosclerosis of the vessels of the transplanted organ. There is a significant association between transplant atherosclerosis and the development of Abs to the disparate HLA Ags present on the graft vasculature. We have investigated the effect of anti-HLA Abs on endothelial cells (EC) and smooth muscle cells cultured in vitro. Ab ligation of class I molecules on ECs results in increased high affinity fibroblast growth factor receptor mRNA expression, and enhanced basic fibroblast growth factor ligand binding. Ab binding to class I molecules on EC and smooth muscle cells is also accompanied by augmented cell proliferation. These results suggest that the intimal thickening observed in transplant atherosclerosis is the result of the proliferative effects of anti-HLA Abs.

Antibodies↗
PubMed1997

Polymorphism in the 5' terminal region of the mRNA of HLA-DQA1 gene: identification of four groups of transcripts and their association with polymorphism in the alpha 1 domain.

Relative to other loci in the MHC, the HLA-DQ locus exhibits an exceptional degree of polymorphism of both A1 and B1 genes, particularly in the region coding for alpha and beta chains. Diversification of the association between different alpha and beta molecules either in cis or in trans contributes to the structural diversity of the repertoire of cell-surface class II protein's in the population. In addition, structural allelic polymorphisms in the 5' regulatory region of both DQB1 and DQA1 shows several linkage groups with respect to the allelic coding sequence of the respective genes. We describe here the allelic polymorphism in the DQA1 mRNA structure located at the 5' untranslated terminal region. This portion of the mRNA molecule represents, in many genes, a cis-acting regulatory sequence playing a role in the posttranscriptional mechanisms by which gene expression can be modulated. Based on detailed transcriptional analysis, we have been able to define at least four groups of transcripts in DQA1. The mRNA variability was associated with the polymorphism of the second exon of the DQA1 gene, coding for the alpha 1 domain and not with the DNA polymorphism in the 5' regulatory region.

B-Lymphocytes↗
PubMed1997

Differential expression of insulin-dependent diabetes mellitus-associated HLA-DQA1 alleles in vivo.

The strong association of HLA-DQ genes with insulin-dependent diabetes mellitus (IDDM) susceptibility is persuasive evidence of their central role in the etiology of this autoimmune disease. Among other possibilities, it has been proposed that an unbalanced expression of IDDM-associated DQA, and/or DQB alleles may lead to alterations in the composition of alpha beta heterodimers and preferential expression of a particular heterodimer on the antigen-presenting cell surface, leading to self-recognition. In this report, we demonstrate the differential expression of DQA1 alleles in vivo, in particular of the two diabetogenic alleles DQA1*0301 and DQA1*0501. Family studies suggest that unequal HLA-DQA1 allele expression in heterozygous individuals is not associated in cis with the HLA-DQA1 gene, but may be affected by trans-acting determinant(s). We also discuss the segregation of this phenotype in IDDM-affected members. Furthermore, we examined historical samples of PBL from an IDDM-affected individual and an HLA-identical unaffected sibling acting in a kidney transplant program as donor and recipient, respectively. This analysis allowed us to establish that unbalanced expression of DQA1*0301 and DQA1*0501 can be induced by microenvironmental conditions. Inducible differential expression of HLA-DQA1 alleles may account for the discordance in the outcome of autoimmune disease in monozygotic twins and HLA-identical siblings.

Alleles↗
PubMed1997

Anti-HLA antibody ligation to HLA class I molecules expressed by endothelial cells stimulates tyrosine phosphorylation, inositol phosphate generation, and proliferation.

The major threat to long-term survival of solid organ allografts is chronic rejection. Progressive narrowing and ultimate luminal occlusion of the arteries and arterioles of the transplanted organ are the hallmarks of the disease. The mechanism of chronic rejection is poorly understood, but it is suspected that the associated vascular changes are a result of anti-HLA antibody-mediated injury to the endothelium. We have postulated that anti-HLA antibodies initiate chronic rejection by binding to class I molecules on the endothelium and transducing signals that result in endothelial cell activation and proliferation. Our data demonstrate that anti-HLA class I antibodies transduce signals in endothelial cells stimulating increased tyrosine phosphorylation of intracellular proteins. Antibody binding to class I antigens also leads to the generation of inositol phosphate and endothelial cell proliferation. These results indicate that anti-HLA antibodies can deliver functionally important signals to endothelial cells, a finding that may be fundamental to an understanding of the mechanisms of chronic rejection.

Antibodies, Monoclonal↗
PubMed1996

Indirect recognition of donor HLA-DR peptides in organ allograft rejection.

To determine whether indirect allorecognition is involved in heart allograft rejection T cells obtained from peripheral blood and graft biopsy tissues were expanded in the presence of IL-2 and tested in limiting dilution analysis (LDA) for reactivity to synthetic peptides corresponding to the hypervariable regions of the mismatched HLA-DR antigen(s) of the donor. Serial studies of 32 patients showed that T cell reactivity to donor allopeptides was strongly associated with episodes of acute rejection. The frequency of allopeptide reactive T cells was 10-50-fold higher in the graft than in the periphery indicating that T cells activated via the indirect allorecognition pathway participate actively in acute allograft rejection. In recipients carrying a graft differing by two HLA-DR alleles the response appeared to target only one of the mismatched antigens of the donor. Indirect allorecognition was restricted by a single HLA-DR antigen of the host and directed against one immunodominant peptide of donor HLA-DR protein. However, intermolecular spreading was demonstrated in patients with multiple rejection episodes by showing that they develop allopeptide reactivity against the second HLA-DR antigen. These data imply that early treatment to suppress T cell responses through the indirect pathway of allorecognition, such as tolerance induction to the dominant donor determinant, may be required to prevent amplification and perpetuation of the rejection process.

Cells, Cultured↗
PubMed1996

Monitoring of soluble HLA alloantigens and anti-HLA antibodies identifies heart allograft recipients at risk of transplant-associated coronary artery disease.

The development of accelerated transplant-related coronary artery disease (T-CAD) is the major obstacle to long-term survival of cardiac allografts. We have investigated the role of various demographic and immunologic parameters as prognostic indicators of T-CAD in a population of 274 heart allograft recipients. Our data demonstrate that patients who experience more than 1 episode of acute rejection per year and/or develop antidonor HLA antibodies are at increased risk of developing T-CAD. Using HLA-A2 as a marker for the release of soluble HLA antigens from the donor, we established that recipients displaying circulating donor alloantigens for more than 26 weeks following transplantation are at increased risk of developing T-CAD (P=0.008). This association suggests that the release of alloantigens from the allograft is indicative of chronic injury and/or that it stimulates chronic rejection via the indirect allorecognition pathway. Our findings indicate that patients at risk of developing T-CAD can be identified by monitoring the release of donor alloantigens and production of antidonor HLA antibodies following transplantation.

Adolescent↗
PubMed1996

Immunologic monitoring in lung allograft recipients.

To identify patients with increased risk of chronic lung allograft rejection, we assessed the utility of an in vitro biopsy-derived lymphocyte growth assay and serum anti-HLA antibody screening as a complement to currently available methods of monitoring lung allograft recipients. Lymphocyte growth assay was performed on bronchoscopic fragments of tissue cultured in medium with rIL-2. Seventy-nine biopsies from 31 lung transplant recipients were tested by lymphocyte growth assay, and results were correlated with histopathology findings. Positive lymphocyte growth was found in 12/26 (46%) episodes of acute rejection, 5/44 biopsies without rejection (11%), and 0/9 episodes of bronchitis. Positive lymphocyte growth was seen in 7/16 (44%) grade A1 rejections and in 5/10 (50%) grade A2 rejections, as opposed to only 5/44 (11%) grade A0 (no rejection) biopsies (P < 0.01 for both A1 and A2 with respect to A0). Actuarial probability of remaining free from obliterative bronchiolitis (OB)* tended to be higher in patients who did not exhibit lymphocyte growth in biopsies. Sequential samples of sera obtained at the time of the biopsy were screened for lymphocytotoxic anti-HLA antibodies. Twenty-two of 44 recipients (50%) developed anti-HLA antibodies during the first postoperative year, exhibiting greater than 10% reactivity to an HLA reference panel of lymphocytes in four or more consecutive serum samples. Actuarial survival of lung allograft recipients with anti-HLA antibodies (n = 22) was lower than in those without anti-HLA antibodies (n = 22; P = 0.03). Of the 22 antibody producers, 7/12 died as a consequence of OB. Of the 22 non-antibody-producers, 1/2 deaths occurred as a consequence of OB. Anti-HLA antibodies were present in 9/11 instances of OB (82% sensitivity) and in 13/33 patients without OB (61% specificity; P = 0.03). These data indicate that lung transplant recipients with positive lymphocyte growth and anti-HLA antibodies are at an increased risk of chronic allograft rejection.

Antibodies↗
PubMed1996

Indirect recognition of donor MHC Class II antigens in human transplantation.

To investigate the role of the indirect pathway of recognition in human allograft rejection, we have mapped the dominant T cell determinant of the HLA-DRbeta1*0101 molecule presented by the DRbeta1*1101 antigen. A synthetic peptide (pp 22-35) corresponding to the sequence of the dominant peptide determinant was used for testing the frequency of in vivo activated T cells in the graft and in the periphery. DRbeta1*1101-positive patients carrying a heart allograft mismatched for the HLA-DR1 antigen showed no reactivity to pp 22-35 during quiescence. However, interleukin-2-responsive T cells, which were pp 22-35 specific, were found in the circulation prior to and at the time of acute and chronic rejection. The response of in vivo and in vitro activated T cells was inhibited at high concentrations of peptide 22-35. This data suggests that indirect recognition plays an important role in allograft rejection and that it can be abolished by high zone tolerance induction.

Acute Disease↗
PubMed1995

Suppression of the indirect pathway of T cell reactivity by high doses of allopeptide.

T helper cells, which recognize allopeptides processed and presented by self APC, contribute to the generation of both cellular and humoral immune responses against allogeneic transplants. We have explored the hypothesis that the indirect T cell recognition pathway is initiated by soluble MHC antigens and that it can be suppressed by high doses of synthetic peptides corresponding to the dominant alloepitope. T cells from a DR11/7 responder were immunized in vitro with recombinant HLA-DR4 (rDR4). Experiments using partially overlapping synthetic peptides showed that the resulting T cell line (TCL) recognized a single dominant epitope mapping within residues 69-88 of the first domain of the DR4 molecule. In vitro immunization with synthetic allopeptides corresponding to other polymorphic regions, were unable to elicit T cell reactivity against rDR4, although at least one of these peptides (corresponding to residues 13-27) was immunogenic, behaving like a cryptic epitope. The rDR4-specific TCL expressed a limited TCR repertoire and provided help to autologous B cells for the production of specific antibodies. The T cell blastogenic response as well as the transcription and secretion of IL-4 (but not IL-2) was efficiently suppressed by high doses of the dominant allopeptide. These findings support the concept that selective immunointervention of indirect allorecognition can be achieved by use of high doses of antigen or TCR vaccination, as proposed for autoimmune diseases.

Dose-Response Relationship, Immunologic↗