Many proteins of the CLC gene family are Cl ؊ channels, whereas others, like the bacterial ecClC-1 or mammalian ClC-4 and -5, mediate Cl ؊ /H ؉ exchange. Mutating a "gating glutamate" (Glu-224 in ClC-4 and Glu-211 in ClC-5) converted these exchangers into anion conductances, as did the neutralization of another, intracellular "proton glutamate" in ecClC-1. We show here that neutralizing the proton glutamate of ClC-4 (Glu-281) and ClC-5 (Glu-268), but not replacing it with aspartate, histidine, or tyrosine, rather abolished Cl ؊ and H ؉ transport. Surface expression was unchanged by these mutations. Uncoupled Cl ؊ transport could be restored in the ClC-4 E281A and ClC-5 E268A proton glutamate mutations by additionally neutralizing the gating glutamates, suggesting that wild type proteins transport anions only when protons are supplied through a cytoplasmic H ؉ donor. Each monomeric unit of the dimeric protein was found to be able to carry out Cl ؊ /H ؉ exchange independently from the transport activity of the neighboring subunit. CLC 6 transport proteins are encoded by a large gene family with members in all phyla (1, 2). Because the founding member of this gene family, ClC-0, from the electric organ of Torpedo (3), is a chloride channel, all CLC genes were believed to encode anion channels. This is undoubtedly true for mammalian ClC-1, -2, and -K, which belong to the same homology branch as ClC-0. However, the bacterial CLC protein ecClC-1 turned out to be an electrogenic Cl Ϫ /H ϩ exchanger (4). Our studies then revealed that endosomal ClC-4 and -5, which reach the plasma membrane to some degree, are Cl Ϫ /H ϩ exchangers as well (5, 6). The fact that several members of the gene family function as ion channels, whereas others carry out stoichiometrically coupled ion exchange, provides unprecedented opportunities to elucidate the structural basis for these different transport modes.The linear I/V relationship of ecClC-1 allowed the estimation of a 2:1 stoichiometry of transport from reversal potentials (4). Unlike ecClC-1, ClC-4 and -5 mediate strongly outwardly rectifying currents (7), precluding a precise determination of their coupling ratio from reversal potentials. Comparing Cl Ϫ and H ϩ transport rates yielded estimates for the Cl Ϫ /H ϩ stoichiometry between 1 and 5 (5, 6). The biological consequences of endosomal CLCs being Cl Ϫ /H ϩ antiporters rather than Cl Ϫ channels are intriguing (8). These proteins are thought to facilitate endosomal/lysosomal acidification by neutralizing proton pump currents, a process important for endocytotic trafficking and lysosomal function. Indeed, ClC-5 is crucial for renal endocytosis and is mutated in a human disorder associated with proteinuria and kidney stones (9).It remains unclear whether Cl Ϫ /H ϩ exchange depends on the dimeric structure of CLC proteins. It is known that both pores of the double-barreled ClC-0 Cl Ϫ channel can be shut closed simultaneously by a "common gate" that depends on both subunits (10 -13). Similarly, it may be that Cl Ϫ /H ϩ flux coupling ...

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The definition of the risk of hepatocellular carcinoma (HCC) recurrence after resection represents a central issue to improve the clinical management of patients. In this study we examined the prognostic relevance of infiltrating immune cell subsets in the tumor (TIL) and in nontumorous (NT) liver (LIL), and the expression of immune-related and lineage-specific mRNAs in HCC and NT liver derived from 42 patients. The phenotype of infiltrating cells was analyzed by flow cytometry, and mRNA expression in liver tissue was examined by real-time reverse transcription (RT)-PCR. The tumor immune microenvironment was enriched in inhibitory and dysfunctional cell subsets. Enrichment in CD4+ T-cells and in particular CD4 and CD8+ memory subsets within TIL was predictive of better overall survival (OS) and time to recurrence (TTR). Increased programmed death ligand 1 (PDL1) mRNA content and higher prevalence of invariant NKT (iNKT) cells were associated with shorter OS and TTR, respectively. By combined evaluation of infiltrating cell subsets along with mRNA profiling of immune and tumor related genes, we identified the intratumoral frequency of memory T-cells and iNKT-cells as well as PDL1 expression as the best predictors of clinical outcome. HCC infiltrate is characterized by the expression of molecules with negative regulatory function that may favor tumor recurrence and poor survival.

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Background: Quality of life (QoL) is becoming a major issue in the evaluation of any therapeutic intervention. Aims: To assess the QoL in patients with uncomplicated symptomatic diverticular disease (DD) and to elucidate the influence of two different treatments either on symptoms or QoL. Materials and Methods: 58 outpatients affected by uncomplicated symptomatic DD, admitted in our Gastroenterological Unit from October 2003 to March 2004, were enrolled. Patients were randomly assigned to two different treatments consisting of rifaximin or mesalazine for 10 days every month for a period of 6 months. QoL was evaluated by means of an SF-36 questionnaire and clinical evaluation was registered by means of a global symptomatic score (GSS) at baseline and after 6 months. Results: At baseline, lower values in all SF-36 domains were confirmed in patients with DD. Both rifaximin and mesalazine groups showed a significant reduction of their mean GSS (p < 0.01 and p < 0.001, respectively) and improvement of SF-36 mean scores after therapy, even though treatment with mesalazine showed better results. Conclusions: DD has a negative impact on QoL. Cyclic treatment with poorly absorbable antibiotics or anti-inflammatory drugs relieves symptoms and improves QoL.