Mônica Spadafora-Ferreira

Cellular autoreactivity against heat shock protein 60 in renal transplant patients: peripheral and graft-infiltrating responses

Autoreactivity to heat shock protein 60 (Hsp60) has been implicated in the pathogenesis and regulation of chronic inflammation, especially in autoimmune diseases. In transplantation, there is a lack of information regarding the cytokine profile and specificity of cells that recognize self‐Hsp60 as well as the kinetics of autoreactivity following transplantation. We studied the cellular reactivity of peripheral and graft‐infiltrating lymphocytes against Hsp60 in renal transplant patients. Cytokine production induced by this protein in peripheral blood mononuclear cells indicated a predominance of interleukin (IL)‐10 during the late post‐transplantation period, mainly in response to intermediate and C‐terminal peptides. Patients with chronic rejection presented reactivity to Hsp60 with a higher IL‐10/interferon (IFN)‐γ ratio compared to long‐term clinically stable patients. Graft‐infiltrating T cell lines, cocultured with antigen‐presenting cells, preferentially produced IL‐10 after Hsp60 stimulation. These results suggest that, besides its proinflammatory activity, autoreactivity to Hsp60 in transplantation may also have a regulatory role.

CD4+CD25+Foxp3+ Indirect Alloreactive T cells from Renal Transplant Patients Suppress Both the Direct and Indirect Pathways of Allorecognition

Alloreactive T cells recognize donor antigens by two routes: direct and indirect pathways of allorecognition. Although the direct pathway is reported to be dominant in allograft rejection, indirect allorecognition also plays an important role. Indirect alloreactivity is also observed in renal transplant patients irrespective of rejection. Previously we showed a predominance of interleukin (IL)‐10 induced by indirect allorecognition of donor human leucocyte antigen (HLA)‐DR peptides, suggesting the existence of indirect alloreactive T cells displaying regulatory activity. In the present work, our objective was to characterize these regulatory T cells. We detected indirect alloproliferation of peripheral blood mononuclear cells (PBMC) from renal transplant patients, induced by donor HLA‐DR peptides, dependent on IL‐4 or IL‐10, suggesting regulatory activity as part of the alloreactive T‐cell repertoire. PBMC‐derived indirect alloreactive T‐cell lines were established and produced both inflammatory and regulatory cytokines. We showed that two of these T‐cell lines which were able to inhibit both direct and indirect alloproliferation of another T‐cell line from the same patient presented a CD4+CD25+Foxp3+ T‐cell population. These data support the idea that indirect alloreactive T cells may also have regulatory activity and may contribute to the maintenance of the human renal allograft.

Two novel anti-von Willebrand factor monoclonal antibodies.

Von Willebrand Factor is a multimer produced by endothelial cells and megakaryocytes, being stored in intracellular organelles, such as the Weibel-Palade bodies and alpha-granules in endothelial cells and platelets, respectively. This molecule acts as a carrier protein for factor VIIIc, involved in the intrinsic pathway of blood coagulation maintaining its stability in circulation. Von Willebrand Factor also plays an important role in platelet aggregation and adhesion to injured vessel wall. It interacts with platelets through two distinct glycoproteins, GPIb and GPIIb/IIIa. We raised two monoclonal antibodies, ECA-3 and ECA-4, against human umbilical vascular endothelial cells that recognize and immunoprecipitate von Willebrand Factor. Interestingly, ECA-4 monoclonal antibody is able to completely inhibit platelet agglutination induced by ristocetin, suggesting that it binds to von Willebrand Factor close to platelet GPIb binding site. The use of monoclonal antibodies to identify von Willebrand Factor binding regions to factor VIII or platelets has been reported by others. In pulmonary hypertension, abnormalities have been detected on the multimeric structure of the molecule as well as on its proteolytic fragments, by using monoclonal antibodies. Moreover, monoclonal antibodies raised against specific regions of von Willebrand Factor molecule may allow studies of functional abnormalities of this protein in inherited and acquired disorders like subtypes of von Willebrands disease.

Indirect alloreactivity and cytokine production to HLA-DR peptides in human renal transplantation

T HAS BEEN proposed that the indirect pathway of allorecognition to donor major histocompatibility antigens (MHC) plays a significant role in allograft rejection in different animal models and in humans. In human studies, an association between positive proliferative response to donor HLA-DR peptides has been described with both acute and chronic rejection in cardiac transplantation 1,2 as well as in renal 3 and liver 4 chronic rejection. On the other hand, in our previous work we detected in the peripheral blood the indirect alloresponse to a pool of naturally processed alloantigens in a group of long-term renal transplanted patients, irrespective of rejection. 5 In the present study, we evaluated prospectively the indirect alloresponse against HLA-DR allopeptides in renal transplanted patients pretransplantation and in different periods posttransplantation (post-Tx) as well as in normal individuals. To better characterize the T cell repertoires involved in allorecognition, we studied the indirect alloresponse by proliferation and cytokine production.

Contrasting roles of donor and recipient TGFB1 and IFNG gene polymorphic variants in chronic kidney transplant rejection.

OBJECTIVE To assess the long-term impact (minimum of 3 years follow-up) of polymorphisms in cytokine genes in donor:recipient pairs on the results of the transplant. METHODS We compared genetic cytokine polymorphisms and the primary factors of risk for the development of chronic rejection in paired groups of renal transplant patients with and without chronic allograft nephropathy [CAN]. RESULTS Multivariate analysis indicated that the presence of the high-production TT genotype (codon 10) of the transforming growth factor beta-1 (TGFB1) was protective in receptors (p=0.017), contrasting with the increased risk when present in donor samples (p=0.049). On the other hand, in the case of the gamma interferon studied, the greater frequency of the high production allele was protective in the analysis of the donor group (p=0.013), increasing the risk of chronic nephropathy of the allograft when present in the recipients (p=0.036). CONCLUSION Our results highlight the importance of TGFB1 genotyping in donors, and indicate that polymorphisms in the gene of this cytokine in donor cells might contribute to the development of chronic allograft nephropathy.

Reactivity of renal transplant sera against a 17 kD mononuclear cell antigen.

In recent years, studies have shown that non-HLA antigens can be involved in renal graft rejection. The so called minor antigens have been found to be expressed on a variety of cells, including endothelial cells. With the aim of understanding better the role of minor antigens in graft rejection, we undertook a Western blot screening of sera from patients waiting for or having received grafts from living-related or cadaveric donors. In this study we described the reactivity of these sera against a 17 kD antigen with expression in many different cell types.