Toxoplasma gondii infects astrocytes, neurons and microglia cells in the CNS and, after acute encephalitis, persists within neurons. Robust astrocyte activation is a hallmark of Toxoplasma encephalitis (TE); however, the in vivo function of astrocytes is largely unknown. To study their role in TE we generated C57BL/6 GFAP-Cre gp130^fl/fl mice (where GFAP is glial fibrillary acid protein), which lack gp130, the signal-transducing receptor for IL-6 family cytokines, in their astrocytes. In the TE of wild-type mice, the gp130 ligands IL-6, IL-11, IL-27, LIF, oncostatin M, ciliary neurotrophic factor, B cell stimulating factor, and cardiotrophin-1 were up-regulated. In addition, GFAP⁺ astrocytes of gp130^fl/fl control mice were activated, increased in number, and efficiently restricted inflammatory lesions and parasites, thereby contributing to survival from TE. In contrast, T. gondii- infected GFAP-Cre gp130^fl/fl mice lost GFAP⁺ astrocytes in inflammatory lesions resulting in an inefficient containment of inflammatory lesions, impaired parasite control, and, ultimately, a lethal necrotizing TE. Production of IFN-γ and the IFN-γ-induced GTPase (IGTP), which mediate parasite control in astrocytes, was even increased in GFAP-Cre gp130^fl/fl mice, indicating that instead of the direct antiparasitic effect the immunoregulatory function of GFAP-Cre gp130^fl/fl astrocytes was disturbed. Correspondingly, in vitro infected GFAP-Cre gp130^fl/fl astrocytes inhibited the growth of T. gondii efficiently after stimulation with IFN-γ, whereas neighboring noninfected and TNF-stimulated GFAP-Cre gp130^fl/fl astrocytes became apoptotic. Collectively, these are the first experiments demonstrating a crucial function of astrocytes in CNS infection.

The purpose of this study was to evaluate the expression of proinflammatory and immunoregulatory cytokines in chronic otitis media. The expression levels of TNF-α, IL-1α, IL-6 and IL-10 were determined by Western blot analysis of tissue samples obtained during ear surgery. The expression levels of TNF-α, IL-1α and IL-6 in cholesteatoma tissues were substantially higher compared to those determined in the granulation tissue. The highest levels of TNF-α, IL-1 and IL-6 proteins were observed in patients with bone destruction. There were no significant differences in the expression of IL-10 levels in cholesteatoma and normal skin, but in the granulation tissue its level was substantially higher. The level of IL-10 in cholesteatoma tissues inversely correlated with the expression of proinflammatory cytokines, the degree of bone destruction and cholesteatoma invasion. Increased expressions of TNF-α, IL-1α and IL-6 in chronic otitis media and a strong positive correlation between these cytokine levels and the degree of bone destruction indicate the destructive behavior of cholesteatoma or granulation tissue.

Evidence that the time course of insulin-induced changes in adenosine level in diabetic rats is different from that observed for expression of adenosine kinase prompted us to study the insulin effect on expression of nucleoside transporters in tissues of diabetic rats. RNase protection assay demonstrated that mRNA levels of equilibrative (rENT) and Na⁺-dependent nucleoside transporters (rCNT) were altered in diabetic tissues. The rENT1 mRNA level with respect to values obtained in age- and sex-matched nondiabetic rats was decreased by 45, 32, and 10% in diabetic heart, liver, and kidney, respectively. The level of rENT2 mRNA was lowered by 40% in diabetic kidney and heart, and by 24% in diabetic liver. Changes in the expression pattern of rCNT1 and rCNT2 in diabetic tissues differed significantly from that observed for rENT. The levels of rCNT1 and rCNT2 mRNA did not change significantly in diabetic kidney. In diabetic heart, the mRNA levels of rCNT1 and rCNT2 increased 1.7- and 2-fold, respectively. Changes in expression of nucleoside transporters were accompanied by alterations in adenosine content. Administration of insulin to diabetic rats resulted in a drop in adenosine concentration in examined tissues and return of the rCNT1, rCNT2, and rENT2 but not rENT1 mRNA levels to values observed in nondiabetic rats. In summary, these data demonstrate that insulin affects expression of nucleoside transporters in a cell-specific manner. We conclude that change in the expression level of the nucleoside transporters occurring in tissues of diabetic rat is an important factor influencing adenosine levels in the cell.

Summary: The proliferative response of T lymphocytes is a crucial step in cell‐mediated immunity. This study was undertaken to investigate the mechanisms leading to the impaired proliferative response of diabetic T lymphocytes. T cells that had been isolated from the spleen of normal rats and cultured in medium containing 20 mmglucose and no insulin displayed the same degree of proliferative impairment as cells isolated from diabetic rats. The rate of T‐cell proliferation, when induced with concanavalin A or anti‐CD3 and anti‐CD28 antibodies, was not affected by the inhibition of nucleoside transporters. T cells cultured at high glucose concentrations in the absence of insulin displayed decreased expression of adenosine kinase, and released measurable extracellular quantities of adenosine. Under resting conditions, the level of cAMP was 5·9‐fold higher in these cells compared to cells grown in low glucose and in the presence of insulin. Experiments with specific adenosine receptor agonists and antagonists showed that adenosine‐induced suppression of diabetic T cell proliferation was mediated by the A_2Aadenosine receptor, but not by the A_2Breceptor. Treatment of diabetic T cells with 10 μmH‐89, a specific protein kinase A inhibitor, restored T‐cell proliferation. These results show that suppressed proliferation of diabetic T lymphocytes is evoked by the decreased expression of adenosine kinase, leading to the outflow of adenosine from the cell. Extracellular adenosine then stimulates the A_2Areceptor and induces cAMP production, leading to the activation of protein kinase A, and suppression of T‐cell proliferation.