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The interaction of Fas with FasL has been demonstrated to be implicated in the pathogenesis of several autoimmune and liver diseases. Recently, attention has been focused on the hypothesis that thyrocytes and beta cells undergo massive Fas/FasL-mediated apoptosis during autoimmune response. Similarly, hepatocyte cell death occurring following copper accumulation points towards the same mechanism.

Background: Many cases of idiopathic dilated cardiomyopathy (IDC) result from an inflammatory myocarditis. The specific immunological mechanisms are not yet defined. Various autoimmune diseases are associated with superantigen-triggered immune responses, resulting in massive T-cell activation and tissue damage. We studied 3 cases in a search for evidence that such a phenomenon is also implicated in IDC., Methods and Results: Myocardial, lymph node, and thymic tissue samples were obtained from IDC patients who were undergoing heart transplantation. Infiltrating immune-cell phenotypes and gene expression of T-cell receptor (TCR) alpha- and beta-chain variable (Valpha and Vbeta) regions were analyzed by immunostaining and polymerase chain reaction. Similar technical approaches were used to assay the tissues for the presence of coxsackievirus B (CVB). In all the specimens analyzed, an overexpression of the TCR Vbeta3, Vbeta7, and Vbeta13.1 gene families was detected among the infiltrating T cells. These tissues were also found to be CVB3-positive. In vitro exposure of peripheral blood mononuclear cells to lysates of cells infected with CVB3 was capable of stimulating expansion of the same TCR Vbeta families. The TCR Valpha repertoire was never found to be skewed., Conclusions: A superantigen-mediated immune response is involved in human heart disease. CVB3 may directly or indirectly trigger this response, suggesting a possible mechanistic link between CVB infection and myocarditis development progressing to IDC.

Fas is an apoptosis-inducing surface receptor involved in controlling tissue homeostasis and function at multiple sites. Here we show that beta cells from the pancreata of newly diagnosed insulin-dependent diabetes mellitus (IDDM) patients express Fas and show extensive apoptosis among those cells located in proximity to Fas ligand-expressing T lymphocytes infiltrating the IDDM islets. Normal human pancreatic beta cells that do not constitutively express Fas, become strongly Fas positive after interleuken (IL)-1beta exposure, and are then susceptible to Fas-mediated apoptosis. NG-monomethyl-L-arginine, an inhibitor of nitric oxide (NO) synthase, prevents IL-1beta-induced Fas expression, whereas the NO donors sodium nitroprusside and nitric oxide releasing compound (NOC)-18, induce functional Fas expression in normal pancreatic beta cells. These findings suggest that NO-mediated upregulation of Fas contributes to pancreatic beta cell damage in IDDM.

Activation-induced apoptosis is one of the primary control mechanisms for the negative selection of an immune response, leading to maintenance of immune homeostasis and selective T cell deletion. The interaction between the surface molecule Fas and its ligand (FasL) has been proposed as a primary mechanism initiating T cell apoptosis. The T cell receptor modulates the expression and function of these molecules. Defects in the Fas/FasL apoptosis pathway have been shown to result in autoimmune disease in humans and in murine models. Because subjects carrying the HLA-B8, DR3 haplotype show a number of immune dysfunctions, including membrano-proliferative glomerulonephritis, systemic lupus erythematosus, Graves' disease, and others, we investigated Fas expression on T and B cells, and sensitivity to Fas-mediated apoptosis of activated T cells, to determine whether abnormalities of the Fas pathway might be associated with the development of autoimmune diseases in this group of individuals. Our findings show that B cells and resting T cells from HLA-B8+, DR3+ subjects express markedly reduced levels of Fas compared with those isolated from HLA-B8-, DR3+ individuals. Reduced levels of Fas were also evident on the surface of T cells from HLA-B8+, DR3+ subjects activated in vitro by stimulation with OKT3 and phytohemoagglutinin. Cycling T cells from these subjects, evaluated for apoptotic nuclei by flow cytometry after incubation with a cytolytic anti-Fas mAb, showed a significantly lower percentage of Fas-mediated apoptosis than did those from HLA-B8-, DR3- individuals. Normal levels of apoptosis were restored after exposure to a synthetic ceramide analog (C2). Further elucidation of the interaction of these molecules in autoimmune diseases may lead to better understanding of the pathogenesis of these disorders.

The mechanisms responsible for thyrocyte destruction in Hashimoto's thyroiditis (HT) are poorly understood. Thyrocytes from HT glands, but not from nonautoimmune thyroids, expressed Fas. Interleukin-1beta (IL-1beta), abundantly produced in HT glands, induced Fas expression in normal thyrocytes, and cross-linking of Fas resulted in massive thyrocyte apoptosis. The ligand for Fas (FasL) was shown to be constitutively expressed both in normal and HT thyrocytes and was able to kill Fas-sensitive targets. Exposure to IL-1beta induced thyrocyte apoptosis, which was prevented by antibodies that block Fas, suggesting that IL-1beta-induced Fas expression serves as a limiting factor for thyrocyte destruction. Thus, Fas-FasL interactions among HT thyrocytes may contribute to clinical hypothyroidism.

Interferon (IFN)-gamma, interleukin (IL)-10, IL-6, and tumor necrosis factor (TNF)-alpha were significantly increased in sera from Sicilian patients with acute boutonneuse fever (BF) compared with those of healthy controls. IFN-gamma levels dropped sharply within the second week after infection. IL-6, IL-10, and TNF-alpha levels gradually declined; in convalescent patients only were they in the normal range. In contrast, peripheral blood mononuclear cells (PBMC) stimulated in vitro with phytohemagglutinin (PHA) produced low levels of IL-10 and IFN-gamma in acute BF that were compatible with the reduction in the levels of CD4+, CD4+/CD45RO+, and CD4+/CD45RA+ cells. In vitro production of TNF-alpha and IL-6 from PBMC stimulated with PHA was not significantly modified during the various phases of the infection compared with control PBMC, which could be due to the persistence of high levels of CD14+ monocytes compensating for the decrease in CD20+ B cells.

The result of human islet isolation procedures to transplant in type I insulin-dependent diabetic patients is significantly conditioned by the technique used for pancreas procurement from multiorgan donor. In fact, during multiorgan procurement, an improper handling of the gland could result in edema or degranulation of the acinar tissue, detrimental for the islet purification step. The surgical technique used by our surgical team, includes some important refinements to obtain the largest quantity and the best quality of purified endocrine material.

Triggering of CD95 (Fas/APO-1) cell surface receptors regulates the elimination of autoreactive T and B lymphocytes through a mechanism of cell suicide called apoptosis. Three different mutations involving CD95 or its ligand are responsible for induction of autoimmunity in susceptible mouse strains. To determine whether a defect involving the CD95 receptor is associated with human insulin-dependent diabetes mellitus (IDDM), we have studied the expression of CD95 on peripheral blood mononuclear cells from IDDM patients at different stages of the disease. Three-colour flow cytometry and mean fluorescence analysis showed that T and B lymphocytes from newly diagnosed IDDM and patients with long-standing disease, and subjects at high risk of developing the disease were highly defective in CD95 expression (p < 0.001), whereas monocytes from all the groups studied expressed normal amounts of CD95 molecules on their cell surface. T-cell subset analysis showed that the impairment of CD95 expression in IDDM patients and high-risk subjects involved both CD3+ CD4+ (p < 0.001) and CD3+ CD8+ cells (p range: < 0.01-0.001), suggesting that this alteration concerns both helper and cytotoxic T cells. Moreover, after activation in vitro with anti-CD3 monoclonal antibody, T cells from newly diagnosed IDDM patients maintained a reduced CD95 expression during the entire cell culture period (24-72 h) in comparison to the control population (p < 0.001). In conclusion, we found a reduced expression of the apoptosis-inducing CD95 receptor on T and B lymphocytes of individuals with clinical and preclinical IDDM. We hypothesize that this defective expression may impair the capacity of autoreactive lymphocytes to undergo CD95-mediated apoptosis, contributing to the lack of control on beta-cell specific B- and T-cell clones.

Sera from Sicilian patients with confirmed visceral leishmaniasis (Leishmania donovani infantum) were analysed at the moment of the diagnosis, during the course of the disease and after clinical recovery, for the concentration of IL-10, IFN-gamma, IL-4 and IL-2. The results show high concentrations of IL-10 and IFN-gamma in the sera at the beginning of infection that return to the normal range following successful chemotherapy. By contrast, PBMC stimulated in vitro with Ag and mitogen produced low levels of IL-10 and IFN-gamma when collected at the time of the diagnosis and normal levels when assayed after recovery. IL-2 was undetected in the sera and was significantly reduced in the supernatants of actively infected patients, returning to the normal level after recovery. IL-4 was absent in the sera and in high concentrations in the supernatants in all the phases of the disease. The levels of CD4+ and CD8+ T cells were within the normal range, but acute VL patients had markedly reduced levels of memory T cells (CD3+/CD45RO+) compared with healthy controls. These cells returned to the normal levels following successful chemotherapy. T cells are strongly activated in acute VL patients as indicated by the elevated number of CD3+ HLA-DR+ and by the increase in HLA-DR antigen on these cells. There was a significant reduction in the cell membrane DR antigen of the monocytes (CD 14+) during the acute phase of the disease, but it returned to the normal range after clinical recovery. These findings therefore suggest that in Sicilian patients with active VL the cytokine profile is not clearly characterized by Th2 phenotype as in mice, and both Th1-like and Th2-like cells appear to proliferate and to be activated. Furthermore, IL-10, rather than IL-4, could play an important part in the inhibition of IFN-gamma-induced macrophage activation and could reflect the levels of HLA-DR antigen expressed by the monocytes.

Gram-negative bacterial infections of the central nervous system are generally associated with high morbidity and mortality rates. In patients with ventriculitis induced by gram-negative liquoral sepsis, a reduction in cerebrospinal fluid formation has been reported, suggesting that gram-negative ventriculitis is able per se to alter the normal functioning of the choroid plexus. The aim of the present study was to analyse, for the first time in the rat, the effects of acute ventriculitis on the ultrastructure of the choroid plexus. A simple and inexpensive experimental model of acute ventriculitis was developed: we injected into the cisterna magna of rats 10(3) CFU of live Escherichia coli, inducing septic ventriculitis without major neurological deficits. Histological examinations of rodent choroid plexus 24 h after the injection revealed patches of altered epithelium, with swollen and vacuolated ependymal cells associated with leukocyte infiltration. Electron microscopy demonstrated a reduced number of microvilli and flattening of the epithelial surface. These results (a) indicate that gram-negative septic ventriculitis is able to induce visible ultrastructural alterations of the choroid plexus which (b) are consistent with a picture of marked reduction of the functioning epithelial choroid plexus surface, and (c) highlight the potential usefulness of our rodent acute ventriculitis model for developing treatment modalities.

The bcl-2 gene product has been shown to regulate apoptotic cell death, and its dysregulation has been shown to induce several abnormalities in the immune system. No data exist regarding bcl-2 expression in autoimmune diseases, such as human insulin-dependent diabetes mellitus (IDDM). We investigated bcl-2 protein expression by testing T lymphocytes from 15 newly-diagnosed (< 3 weeks) IDDM patients in comparison to 10 age-matched control subjects. The expression of bcl-2 on CD3+ lymphocyte subsets was investigated after membrane permeabilization by two- or three-colour immunofluorescence. When the percentage and mean fluorescence intensity (MFI) of bcl-2+/CD3+ cells from normal individuals and patients were compared, we found that bcl-2 expression within the CD3+ and CD4+ CD45R0+ T-cell populations was reduced significantly in IDDM patients (46.8 +/- 15.4 vs 79.6 +/- 11.7; 25.7 +/- 3.8 vs 47.15 +/- 5.7, respectively; p < 0.001). To establish whether low bcl-2 expression in T cells from newly-diagnosed patients reflects their susceptibility to death by an apoptotic process, we also evaluated DNA staining with propidium iodide in CD3+ lymphocyte suspension after a (24-72 h) culture period (spontaneous apoptosis). We found that IDDM patients have higher levels of spontaneous apoptosis (mean +/- SEM: 24 h = 4.6 +/- 0.8; 48 h = 9.9 +/- 1; 72 h = 12.8 +/- 1.1) than control subjects (24 h = 1.8 +/- 0.4; 48 h = 4.6 +/- 0.4; 72 h = 5.7 +/- 0.3; p < 0.02-0.001). Our study suggests that recent onset IDDM is characterised by reduced bcl-2 expression, which in turn may be associated with the increased spontaneous apoptosis we observed.

The HLA-B8, DR3 haplotype is overrepresented in several autoimmune diseases, implying that genes predisposing to these disorders are linked to this haplotype. In the patients affected by these diseases, as well as in healthy HLA-B8, DR3 individuals, various dysfunctions reflecting an impairment of T-cell activation have been found. To better characterize T-cell impairment of HLA-B8, DR3-positive healthy individuals, we analyzed the surface expression of early (CD69) and late (CD71) activation phenotypes. MNC cultures were stimulated with PHA and used for T-cell phenotyping by flow cytometry analysis. The results showed that the percentage of CD69+ T cells was significantly decreased in MNC from HLA-B8, DR3+ subjects. This defect was detected in cell cultures from all subjects studied, but it attained significance only in females in the early hours after stimulation. The difference in CD69 expression between HLA-B8, DR3-positive individuals and -negative ones was not due to differences in CD4 and CD8 ratios in the HLA-B8, DR3 cells that underwent activation, as following activation the pattern of CD4 and CD8 antigen expression was the same in both groups of subjects. Concerning the late antigen CD71, no significant difference in percentage was observed between T lymphocytes from HLA-B8, DR3+ and HLA-B8, DR3- subjects at all the times studied. The analysis of the requirements for CD69 expression has suggested that sustained PKC activation and an increase of intracellular CA2+ could be responsible for TCR/CD3-mediated CD69 induction. Thus, present data suggest a defect in the signal transduction pathway of the TCR/CD3 complex.(ABSTRACT TRUNCATED AT 250 WORDS)

A case of thyroiditis due to Brucella Melitensis is reported. Brucellosis anticipated by about two months the onset of the characteristic symptoms of acute thyroiditis. Cultures of specimens obtained by fine needle aspiration biopsy and microbiological investigations allowed isolation and identification of the germ. This observation allowed the recognition that thyroid gland might harbored secondary localization of a prolonged brucellosis. A microbiological study (the protocol of which is proposed) of specimen obtained by fine needle aspiration biopsy should be performed in the presence of symptoms and signs of an inflammatory process associated to an acute swelling of the thyroid gland.

The levels of soluble CD4 (sCD4), sCD8 and beta 2-microglobulin (beta 2-M) were measured in sera from patients with visceral leishmaniasis during the course of infection. Levels of sCD4, sCD8 and beta 2-M were raised significantly above levels in normal sera and returned to the normal range after recovery. The decrease in the levels of sCD8 was related to a reduction of anaemia, leukopenia and thrombocytopenia. In contrast, sCD4 levels fluctuated during the period of infection. beta 2-M returned within normal range more rapidly than sCD8 secretion. Our results suggest that T cells are activated during infection, and that it is also possible that the raised levels of these soluble molecules play a role in the impairment of protective immunity.

The autoimmune process leading to the destruction of pancreatic beta-cells is mediated by T lymphocytes. Peripheral T cells from subjects with preclinical and clinical type I diabetes respond weakly in vitro to lectin stimulation. We, therefore, investigated in a group of newly diagnosed diabetic patients the presence of a defect in the signal transduction pathway of the T cell receptor (TcR)/CD3 complex. Following stimulation with anti-CD3-coupled beads, the proliferative response in diabetic T cells was significantly decreased in comparison with that from normal T cells. Interestingly, addition of either recombinant interleukin (IL)-2 or phorbol 12-myristate 13-acetate to the cell culture was able to completely restore impaired anti-CD3-induced proliferation in diabetic T cells, suggesting the presence of a defect through the TcR/CD3 pathway, located upstream of protein kinase C (PKC) activation and resulting in low IL-2 production and proliferation. Intracellular Ca2+ measurements by Fluo-3 labeling and flow cytometry analysis on diabetic and control T cells after anti-CD3 stimulation gave comparable results, indicating that this defect does not involve events leading to intracellular Ca2+ mobilization. In contrast, anti-CD3 stimulation of diabetic T cells resulted in a marked impairment of PKC translocation and CD69 antigen expression, as assessed by peptide substrate phosphorylation and by flow cytometry analysis, respectively. Taken together, our data clearly show the presence in individuals at the onset of the disease of an in vitro defect in the signal transduction pathway of the TcR/CD3 complex, resulting in ineffective PKC activation which is not able to induce normal IL-2 production and proliferation of diabetic T cells.

In Type I diabetes the observation of a decreased release of interleukin-2 (IL-2) and soluble IL-2 receptors by means of stimulated lymphocytes in vitro indicates that a primary immunoregulatory defect may be involved. To confirm this hypothesis we investigated the T-cell activation trend, evaluating the surface expression of IL-2 receptor (CD25), transferrin (CD71), HLA class II (DR), and CD69 phenotypes after in vitro stimulation with phytohemagglutinin (PHA; 1 and 10 micrograms/ml) and concanavalin A (12.5 micrograms/ml) in six newly diagnosed Type I diabetics and six islet cell- and insulin autoantibody-positive first-degree relatives. As controls were studied six long-standing Type I diabetics and six healthy subjects. T-cell cultures from the four groups were performed on the same day and examined at 0, 24, 48, 96, 120, and 144 hr. Cytometric analysis was performed, keeping PBMC gating constant on the basis of physical parameters (scatter and volume). Using both PHA concentrations, a lower level of CD25, CD71, CD69, and DR antigen expression was found in newly diagnosed patients at all observation times with respect to control cultures (P < 0.001). Unexpectedly, pre-Type I diabetic subjects, after 1 microgram/ml of PHA, showed a significantly reduced expression of CD69 (P < 0.001) and CD71 (P < 0.001). The levels remained low, also with high PHA, at the different observation periods, while CD25 expression was found to be reduced in prediabetics only after 1 micrograms/ml of PHA (P < 0.001). The long-standing patients showed a T cell activation trend very close to the latter. Our data show that in Type I diabetes and in the early phases of the disease, the initial activation signal(s) appears to be affected, particularly with one or more subsequent events necessary to initiate the appearance of "activation antigens." This study suggests that the natural history of immunoregulation in pre-Type I and Type I diabetes is characterized by a primary defect in this system, which also persists in patients with long-standing disease.

Problem: Our aim was to investigate the immunological status of diabetic pregnancy, which is an overlap of diabetic immunity abnormalities and the immunological modifications normally occurring during pregnancy., Method: We studied lymphocyte subpopulations and lymphokine production, after 96 h of phytohemagglutinin (PHA) stimulation, from normal and Type I diabetic pregnant women at delivery time and from the respective cord blood., Results: Peripheral blood mononuclear cells (PBMC) from both normal and Type I diabetic mothers showed an increase in CD8+ and a decrease in CD4+ cells compared to the respective cord blood mononuclear cells (CBMC). Moreover, Type I PBMC showed a lower number of "activated" CD3+ DR+ cells and a higher number of CD8+ CD25+ cells with respect to normal women, which may reflect the dysregulatory pattern due to the autoimmune condition. Type I CBMC showed a big increase in the number of CD4+ Leu8+ cells, a cell subpopulation characterized by inhibitory activity. Finally, as regards lymphokine release in culture supernatants, type I diabetes seemed to be associated with an over-production of IL1 and IL6, although the latter increase is less evident in CBMC cultures., Conclusions: The present study shows that diabetic pregnancy is associated with major alterations of cell-mediated immunity leading to a state of immunodepression. Moreover, our study suggests that the maternal immunological status influences fetal immunity, as demonstrated by the increase in the number of regulatory cells and by the altered pattern of lymphokine production (IL1 and IL6) by lymphocytes derived from diabetic CBMC. The latter phenomenon perfectly mirrors maternal PBMC characteristics.

91 strains of Staphylococci belonging to different species were investigated for their adhesive capacity to urinary and buccal human epithelial cells. Furthermore the adherence of the same bacterial strains was evaluated in relation with their methicillin-sensitive and methicillin-resistant properties. S. aureus showed an high adhesion to buccal and urinary cells; S. saprophyticus and S. epidermidis attached mainly to uroepithelial cells and to buccal cells, respectively. Bacterial strains, either sensitive or resistant to methicillin antibiotic, did not exhibit significant differences of adhesive capacity.

The Staphylococcal strains, identified by "The Simplified Lyogroups System" were tested for their susceptibility to methicillin and some aminoglycosides. The results, besides showing a higher ratio of susceptibility against aminoglycosides in methicillin-susceptible (MS) strains, show a different trend within each lyogroup. A total of 1616 wild Staphylococcus strains were isolated in microbiological units in Catania, Messina, Rome and Genoa. The results show a high susceptibility to aminoglycosides, both in MS and methicillin-resistant (MR) groups but with different trends among lyogroups.