Detection of GAD65 autoreactive T-cells by HLA class I tetramers in type 1 diabetic patients.

Type 1 diabetes (T1D) is an autoimmune disease, in which pancreatic beta cells are destroyed in genetically predisposed individuals. While the direct contribution of autoantibodies to the disease pathogenesis is controversial, it is generally recognised that the mechanism of beta cell destruction is mediated by autoreactive T cells that had escaped the thymic selection. We aimed to design a method to detect circulating CD8+ T cells autoreactive against an epitope of the glutamic acid decarboxylase autoantigen, isoform 65 (GAD65) ex vivo in T1D patients by using HLA class I tetramers. Low frequencies of GAD65 peptide-specific CD8+ cytotoxic T lymphocytes were detected in peripheral blood lymphocytes (PBMC) of normal controls after GAD65 peptide-specific stimulation. Conversely, their frequencies were significantly higher than in controls in PBMC of T1D patients after GAD65 peptide stimulation. These preliminary data are encouraging in order to develop a reliable assay to be employed in large-scale screening studies.

Cryptosporidium parvum at different developmental stages modulates host cell apoptosis in vitro.

We studied apoptosis in a human ileocecal adenocarcinoma tumor cell line (HCT-8) infected with Cryptosporidium parvum, from 2 to 72 h postinfection (h.p.i.). At 2 h.p.i., the percentage of annexin V-positive cells in the cell culture had increased to 10% compared to 2.5% in noninfected control culture; sorted infected cells expressed mRNA of FasL, the active form of caspase 3, and high caspase 3 activity, whereas the noninfected neighboring cells sorted from the same culture showed no signs of apoptosis. At 24 h.p.i., the percentages of early (annexin V positive) and late (DNA fragment) apoptotic cells were 13 and 2%, respectively, in the entire cell culture, and these percentages were not statistically significant in comparison with those from noninfected control cultures. At this time, sorted infected cells expressed the inactive form of caspase 3, a low caspase 3 activity, and the antiapoptotic protein Bcl-2. Noninfected cells sorted from the same culture showed expression of the active form of caspase 3, a moderate caspase 3 activity, and no Bcl-2 expression. At 48 h.p.i., the percentages of early and late apoptotic cells and caspase 3 activity had increased in the total cell culture, and both sorted infected and noninfected cells showed the active form of caspase 3. These results show that C. parvum, depending on its developmental stage, can inhibit (at the trophozoite stage) or promote (at the sporozoite and merozoite stages) host cell apoptosis, suggesting that it is able to interact with and regulate the host-cell gene expression.

Cryptosporidium parvum-specific CD4 Th1 cells from sensitized donors responding to both fractionated and recombinant antigenic proteins.

T-cell-mediated immunity plays a central role in the host response to Cryptosporidium parvum. Human T-cell clones (TCC) were isolated from peripheral blood mononuclear cells of five healthy donors with prior cryptosporidiosis by use of a C. parvum crude extract, two antigen fractions obtained by ion-exchange chromatography (IEC1 and IEC2), and two recombinant peptides (SA35 and SA40) from C. parvum sporozoites. The T-cell lines derived from the one recently infected donor had a higher proportion (26 to 38%) of T cells exhibiting the gamma/delta T-cell receptor (gamma/delta-TCR) than those from donors who had recovered from cryptosporidiosis several years earlier, suggesting that the gamma/delta T-cell population is involved in the early stage of the infection. The specific TCC had the alpha/beta-TCR, had the phenotype CD45RO(+) CD4(+) CD8(-), and were characterized by either hyperproduction of gamma interferon (IFN-gamma) alone, with a Th1 profile, or IFN-gamma hyperproduction together with interleukin-4 (IL-4) or IL-5 production, with a Th0 profile. SA35, SA40, IEC1, and IEC2 may be considered good targets of the cellular response against C. parvum and may play a role in maintaining the T-cell-mediated memory response to this parasite. Furthermore, the SA35 and SA40 peptides may be regarded as immunodominant antigens involved in the maintenance of the T-cell response in healthy C. parvum-sensitized persons.

A new modular protein of Cryptosporidium parvum, with ricin B and LCCL domains, expressed in the sporozoite invasive stage.

The recombinant SA35 peptide has been described as an antigenic portion of a larger Cryptosporidium parvum protein. We identified and characterized the encoding Cpa135 gene and the entire protein, Cpa135. The Cpa135 gene was found to consist of a single exon of 4671 bp, and the mRNA transcribed in the sporozoites was identified. The predicted 1556 amino-acid protein showed the presence of domains which are widely conserved also in other unrelated phylogenetic groups (i.e. a ricin B and a LCCL motif). Comparison of Cpa135 sequence with genomic and protein databases revealed many related genes in other apicomplexan species and high homology with CCP2 protein from Plasmodium yoelii and Plasmodium berghei. The Cpa135 protein was identified and localized by using a monoclonal antibody (Mab) directed against the SA35 antigen (anti-SA35). In oocyst-sporozoite lysate, the anti-SA35 MAb recognized a 135 kDa protein that forms a protein complex larger than 200 kDa, which is mediated by disulfide bridges. Cpa135 synthesis was up-regulated during the excystation process. After host-cell invasion, Cpa135 gene expression was undetectable up to 48 h, whereas mRNA synthesis was newly observed at 72 h post-infection. The Cpa135 protein was localized in the apical complex, and it was found to be secreted by sporozoites during their gliding. Cpa135 persisted during the intracellular stages of the parasite, and it defined the boundaries of the parasitophorous vacuole in the infected cells. The unique array of domains and the homology with other apicomplexan proteins indicate that the Cpa135 protein is representative of a new family of proteins.

Increased CD8(+)-T-cell expression and a type 2 cytokine pattern during the muscular phase of Trichinella infection in humans.

Cell-mediated immunity during the muscular phase of Trichinella infection in humans was studied. Cell proliferation, the phenotypic changes in the T-cell population, and expression and production of cytokines were examined by using peripheral blood mononuclear cells (PBMC) collected at different times postinfection from 10 individuals who had acquired Trichinella spiralis and five individuals who had acquired Trichinella britovi in two distinct outbreaks. T. spiralis and T. britovi crude worm extracts induced proliferation of PBMC from T. spiralis- and T. britovi-infected donors. Cytokine gene expression showed a predominant type 2 pattern for the entire period of infection studied, although gamma interferon (IFN-gamma) was expressed. Interleukin-2 (IL-2), IL-5, IL-10, and IFN-gamma production was found in PBMC of all donors. There was a good correspondence between the cytokine expression and production patterns. Changes in PBMC composition, with a trend toward an increase in CD8(+) lymphocyte counts, were observed.