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M Storto, M Sallese, L Salvatore, R Poulet, DF Condorelli, P Dell'Albani, MF Marcello, R Romeo, P Piomboni, N Barone, F Nicoletti, and A De Blasi

The G protein-coupled receptor kinase type 4 mediates the homologous desensitisation of type-1 metabotropic glutamate (mGlu1) receptors and is predominantly expressed in the testis. Hence, we searched for the expression of mGlu1 or other mGlu receptor subtypes in rat and human testes. RT-PCR analysis showed the presence of mGlu1, -4 and -5 (but not -2 or -3) receptor mRNA in the rat testis. The presence of mGlu1 and -5 (but not mGlu2/3) receptor proteins was also demonstrated by Western blot analysis. In the rat testis, both mGlu1a and -5 receptors were highly expressed in cells of the germinal line. It is likely that these receptors are functional, because the agonist, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid, was able to stimulate inositol phospholipid hydrolysis in slices prepared from rat testes. Immunocytochemical analysis of bioptic samples from human testes showed a high expression of mGlu5 receptors inside the seminiferous tubuli, whereas mGlu1a immunoreactivity was restricted to intertubular spaces. mGlu5 receptors were also present in mature spermatozoa, where they were localised in the mid-piece and tail. This localisation coincided with that of beta-arrestin, a protein that is critically involved in the homologous desensitisation and internalisation of G protein-coupled receptors. Taken collectively, these results offer the first evidence for the expression of any glutamate receptor in testes, and suggest that at least mGlu5 receptors are present and functionally active in mature human sperm.

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L Monetini, F Barone, L Stefanini, A Petrone, T Walk, G Jung, R Thorpe, P Pozzilli, and MG Cavallo

Enhanced cellular immune response to bovine beta-casein has been reported in patients with type 1 diabetes. In this study we aimed to establish beta-casein-specific T cell lines from newly diagnosed type 1 diabetic patients and to characterise these cell lines in terms of phenotype and epitope specificity. Furthermore, since sequence homologies exist between beta-casein and putative beta-cell autoantigens, reactivity to the latter was also investigated. T cell lines were generated from the peripheral blood of nine recent onset type 1 diabetic patients with different HLA-DQ and -DR genotypes, after stimulation with antigen pulsed autologous irradiated antigen presenting cells (APCs) and recombinant human interleukin-2 (rhIL-2). T cell line reactivity was evaluated in response to bovine beta-casein, to 18 overlapping peptides encompassing the whole sequence of beta-casein and to beta-cell antigens, including the human insulinoma cell line, CM, and a peptide from the beta-cell glucose transporter, GLUT-2. T cell lines specific to beta-casein could not be isolated from HLA-matched and -unmatched control subjects. beta-Casein T cell lines reacted to different sequences of the protein, however a higher frequency of T cell reactivity was observed towards the C-terminal portion (peptides B05-14, and B05-17 in 5/9 and 4/9 T cell lines respectively). Furthermore, we found that 1 out of 9 beta-casein-specific T cell lines reacted also to the homologous peptide from GLUT-2, and that 3 out of 4 of tested cell lines reacted also to extracts of the human insulinoma cell line, CM. We conclude that T cell lines specific to bovine beta-casein can be isolated from the peripheral blood of patients with type 1 diabetes; these cell lines react with multiple and different sequences of the protein particularly towards the C-terminal portion. In addition, reactivity of beta-casein T cell lines to human insulinoma extracts and GLUT-2 peptide was detected, suggesting that the potential cross-reactivity with beta-cell antigens deserves further investigation.