Leo Kei Iwai

Radical production from free and peptide-bound methionine sulfoxide oxidation by peroxynitrite and hydrogen peroxide/iron(II).

Methionine sulfoxide is a post‐translational protein modification that has been receiving increasing attention in the literature. Here we used electron paramagnetic resonance spin trapping techniques to show that free and peptide‐bound methionine sulfoxide is oxidized by hydrogen peroxide/iron(II)‐EDTA and peroxynitrite through the intermediacy of the hydroxyl radical to produce both CH3 and CH2CH2CH radicals. The results indicate that methionine sulfoxide residues are important targets of reactive oxygen‐ and nitrogen‐derived species in proteins. Since the produced protein‐derived radicals can propagate oxidative damage, the results add a new antioxidant route for the action of the enzyme peptide methionine sulfoxide reductase.

The gp43 from Paracoccidioides brasiliensis: A Major Diagnostic Antigen and Vaccine Candidate

The gp43, a 43,000 dalton glycoprotein from Paracoccidioides brasiliensis, is one of those rare examples of a major diagnostic antigen in serological assays (e.g., Mendes-Giannini et al. 1990) that also contains epitopes eliciting an immunoprotective cellular immune response. Its role in fungal virulence is not clearly defined which raises the ques-tion as to the selective pressure that keeps its expression constant in both yeast and mycelium phase of this dimorphic pathogenic species.

Systematic Analysis of the Combinatorial Nature of Epitopes Recognized by TCR Leads to Identification of Mimicry Epitopes for Glutamic Acid Decarboxylase 65-Specific TCRs

Accumulating evidence indicates that recognition by TCRs is far more degenerate than formerly presumed. Cross-recognition of microbial Ags by autoreactive T cells is implicated in the development of autoimmunity, and elucidating the recognition nature of TCRs has great significance for revelation of the disease process. A major drawback of currently used means, including positional scanning synthetic combinatorial peptide libraries, to analyze diversity of epitopes recognized by certain TCRs is that the systematic detection of cross-recognized epitopes considering the combinatorial effect of amino acids within the epitope is difficult. We devised a novel method to resolve this issue and used it to analyze cross-recognition profiles of two glutamic acid decarboxylase 65-autoreactive CD4+ T cell clones, established from type I diabetes patients. We generated a DNA-based randomized epitope library based on the original glutamic acid decarboxylase epitope using class II-associated invariant chain peptide-substituted invariant chains. The epitope library was composed of seven sublibraries, in which three successive residues within the epitope were randomized simultaneously. Analysis of agonistic epitopes indicates that recognition by both TCRs was significantly affected by combinations of amino acids in the antigenic peptide, although the degree of combinatorial effect differed between the two TCRs. Protein database searching based on the TCR recognition profile proved successful in identifying several microbial and self-protein-derived mimicry epitopes. Some of the identified mimicry epitopes were actually produced from recombinant microbial proteins by APCs to stimulate T cell clones. Our data demonstrate the importance of the combinatorial nature of amino acid residues of epitopes in molecular mimicry.

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.

T-Cell Recognition of Paracoccidioides brasiliensis gp43-Derived Peptides in Patients with Paracoccidioidomycosis and Healthy Individuals

ABSTRACT Vaccines with synthetic peptides induce the immune response to epitopes that bind to several HLA alleles. By using a TEPITOPE algorithm, we selected and analyzed the T-cell responses of peripheral blood mononuclear cells from 29 paracoccidioidomycosis (PCM) patients to peptides of the immunodominant gp43 antigen of Paracoccidioides brasiliensis, the causative agent of PCM.

Diversity of physiological cell reactivity to heat shock protein 60 in different mouse strains

Abstract Heat shock proteins (Hsp) are families of highly conserved molecules and immunodominant antigens in some infections and in autoimmune diseases. Some reports suggest that different regions of the Hsp60 molecule induce distinct immune responses. However, there are no reports comparing physiological T-cell reactivity to Hsp60 in mice. In this study, we have analyzed T-cell proliferation and cytokine production induced by Hsp60, under physiological conditions, in three mouse strains bearing distinct major histocompatibility complex (MHC) backgrounds. Proliferative response predominantly was found in C57BL/6 mice, mostly induced by N-terminal and intermediate Hsp60 peptides (P < 0.0001). Interferon-γ (IFNγ) production was broadly induced by different regions of Hsp60 in all three mouse strains, although response was focused in different peptide groups in each strain. We did not observe an exclusive Th1 or Th2 cytokine profile induced by any particular region of Hsp60. However, we identified a strain hierarchy in IL-10 production induced by Hsp60 peptides from different regions, mostly detected in C3H/HePas, and in BALB/c, but not in C57BL/6 mice. In contrast, IL-4 production only was induced by the intermediate and C-terminal region peptides in both C3H/HePas and BALB/c mice. Our data give original information on physiological cellular reactivity to Hsp60. We also have identified peptides with the capacity to induce the production of anti-inflammatory cytokines, bringing perspectives for their use in immunotherapy of chronic inflammatory diseases and allograft rejection.