We recently identified CD8+CD122+ regulatory T cells that directly control CD8+ and CD4+ cells without intervention of APCs. In this study, we investigated the effector mechanism of CD8+CD122+ regulatory T cells by using an in vitro regulation system. The profile of cytokine expression revealed that IL-10 was predominantly produced by CD8+CD122+ cells, whereas other cytokines were similarly expressed in CD8+CD122+ cells and CD8+CD122− cells. Suppression of both proliferation and IFN-γ production by CD8+CD122− cells by CD8+CD122+ cells was blocked by adding anti-IL-10 Ab to the culture but not by adding anti-TGF-β Ab. When IL-10 was removed from the conditioned medium from CD8+CD122+ cells, the conditioned medium no longer showed regulatory activity. Finally, CD8+CD122+ cells from IL-10-deficient mice had no regulatory activity in vitro and reduced regulatory activity in vivo. Our results clearly indicate that IL-10 is produced by CD8+CD122+ cells and mediates the regulatory activity of these cells.

ABSTRACT. Apoptosis signal-regulating kinase 1 (ASK1) is a ubiquitously expressed mitogen-activated protein (MAP) kinase kinase kinase that activates the c-Jun N-terminal kinase (JNK) and p38 MAP kinase signaling cascades. Recent findings from analyses of ASK1-deficient mice have revealed that ASK1 is required for apoptosis induced by oxidative stress, TNF and endoplasmic reticulum (ER) stress. In addition, several lines of evidence have suggested that ASK1 has diverse functions in the decision of cell fate beyond its pro-apoptotic activity. Thus, ASK1 appears to be a pivotal component not only in stress-induced cell death but also in a broad range of biological activities in order for cells to adapt to or oppose various stresses. Key words: MAPKKK/ASK1/JNK/p38/apoptosisThe mitogen-activated protein (MAP) kinase cascades are multifunctional signaling pathways that are evolutionally well conserved in all eukaryotic cells (Ichijo, 1999;Widmann, 1999;Kyriakis and Avruch, 2001). Three MAP kinase cascades that converge on ERKs, c-Jun N-terminal kinases (JNKs), and p38 MAP kinases have been extensively characterized, and each consists of three classes of serine/threonine kinases, MAP kinase, MAP kinase kinase (MAPKK, also referred to as MEK) and MAPKK kinase (MAPKKK). MAPKKK phosphorylates and thereby activates MAPKK, and activated MAPKK in turn phosphorylates and activates MAP kinase. Among the three MAP kinase cascades, two of them that converge on JNKs and p38 MAP kinases are preferentially activated by cytotoxic stresses such as UV radiation, X-ray, heat shock and osmotic shock, and by proinflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-1 (Tibbles and Woodgett, 1999). One of the important biological responses mediated through these stress-activated MAP kinase pathways appears to be the decision of cell fate by regulating apoptosis. The possible roles of the JNK pathway in proapoptosis signaling have been demonstrated by knockout mouse studies. Mice lacking the JNK3 gene were reported to exhibit marked reduction in excitotoxicity-induced apoptosis of hippocampal neurons . JNK2 was shown to be required for apoptosis of immature thymocytes induced by anti-CD3 antibody but not for activationinduced cell death of mature T cells (Sabapathy et al., 1999). Compound mutant mice lacking the JNK1 and JNK2 genes suggested that JNK1 and JNK2 regulate region-specific apoptosis during early brain development (Kuan et al., 1999). Several lines of evidence have also suggested the pro-apoptotic roles of the p38 pathway (Xia et al., 1995;Kawasaki et al., 1997;Harper and LoGrasso et al., 2001), although they have not yet been supported by data of mice deficient for the p38 genes.Apoptosis signal-regulating kinase 1 (ASK1)/MAPKKK5 is a ubiquitously expressed MAPKKK that activates the JNK and p38 pathways by directly phosphorylating and thereby activating their respective MAPKKs, MKK4 (SEK1)/MKK7 and MKK3/MKK6 Ichijo et al., 1997) (Fig. 1). Overexpression of wild-type or constitutively active ASK1 induces apop...

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Hepatitis C virus (HCV) infection is a major cause of liver disease and hepatocellular carcinoma. Glycan shielding has been proposed to be a mechanism by which HCV masks broadly neutralizing epitopes on its viral glycoproteins. However, the role of altered glycosylation in HCV resistance to broadly neutralizing antibodies is not fully understood. Here, we have generated potent HCV neutralizing antibodies hu5B3.v3 and MRCT10.v362 that, similar to the previously described AP33 and HCV1, bind to a highly conserved linear epitope on E2. We utilize a combination of in vitro resistance selections using the cell culture infectious HCV and structural analyses to identify mechanisms of HCV resistance to hu5B3.v3 and MRCT10.v362. Ultra deep sequencing from in vitro HCV resistance selection studies identified resistance mutations at asparagine N417 (N417S, N417T and N417G) as early as 5days post treatment. Comparison of the glycosylation status of soluble versions of the E2 glycoprotein containing the respective resistance mutations revealed a glycosylation shift from N417 to N415 in the N417S and N417T E2 proteins. The N417G E2 variant was glycosylated neither at residue 415 nor at residue 417 and remained sensitive to MRCT10.v362. Structural analyses of the E2 epitope bound to hu5B3.v3 Fab and MRCT10.v362 Fab using X-ray crystallography confirmed that residue N415 is buried within the antibody-peptide interface. Thus, in addition to previously described mutations at N415 that abrogate the β-hairpin structure of this E2 linear epitope, we identify a second escape mechanism, termed glycan shifting, that decreases the efficacy of broadly neutralizing HCV antibodies.

Transition from proliferation to quiescence brings about extensive changes in cellular behavior and structure. However, the genes that are crucial for establishing and/or maintaining quiescence are largely unknown. The fission yeast Schizosaccharomyces pombe is an excellent model in which to study this problem, because it becomes quiescent under nitrogen starvation. Here, we characterize 610 temperature-sensitive mutants, and identify 33 genes that are required for entry into and maintenance of quiescence. These genes cover a broad range of cellular functions in the cytoplasm, membrane and nucleus. They encode proteins for stress-responsive and cell-cycle kinase signaling pathways, for actin-bound and osmo-controlling endosome formation, for RNA transcription, splicing and ribosome biogenesis, for chromatin silencing, for biosynthesis of lipids and ATP, for cell-wall and membrane morphogenesis, and for protein trafficking and vesicle fusion. We specifically highlight Fcp1, a CTD phosphatase of RNA polymerase II, which differentially affects the transcription of genes that are involved in quiescence and proliferation. We propose that the transcriptional role of Fcp1 is central in differentiating quiescence from proliferation.

Little correlation was observed between imaging response of colorectal cancer liver metastases to chemotherapy and pathologic response. Liver surgery should be undertaken even after a complete response by imaging. Outcome after hepatectomy was favorable in patients showing complete pathologic response of least one metastasis.

While proteasome is central to the degradation of cellular ubiquitinated proteins, the control of its nuclear function is barely understood. Here we show that the fission yeast ubiquitin-conjugating Rhp6/Ubc2/Rad6 and ligating enzymes Ubr1 are responsible for nuclear enrichment of proteasome through the function of Cut8, a nuclear envelope protein. Cut8 is an Rhp6 substrate that physically interacts with and tethers proteasome. Nonubiquitinatable K-all-R Cut8 weakly interacts with proteasome and fails to enrich nuclear proteasome. Consistently, the nuclear enrichment of proteasome also fails in rhp6 and ubr1 null mutants. Further, cut8 null and cut8 K-all-R mutants are hypersensitive to DNA damage, probably due to the paucity of nuclear proteasome. Thus, Rhp6 enhances the retention of nuclear proteasome through regulating Cut8. The short-lived nature of Cut8 is crucial for feedback enrichment of the proteasome within the nucleus. This is likely to be a conserved mechanism as we describe a Cut8 homolog in flies.

Massive arterial hemorrhage is, although unusual, a life-threatening complication of major pancreatobiliary surgery. Records of 351 patients who underwent major surgery for malignant pancreatobiliary disease were reviewed in this series. Thirteen patients (3.7%) experienced massive hemorrhage after surgery. Complete hemostasis by transcatheter arterial embolization (TAE) or re-laparotomy was achieved in five patients and one patient, respectively. However, 7 of 13 cases ended in fatality, which is a 54% mortality rate. Among six survivors, one underwent selective TAE for a pseudoaneurysm of the right hepatic artery (RHA). Three patients underwent TAE proximal to the proper hepatic artery (PHA): hepatic inflow was maintained by successful TAE of the gastroduodenal artery in two and via a well-developed subphrenic artery in one. One patient had TAE of the celiac axis for a pseudoaneurysm of the splenic artery (SPA), and hepatic inflow was maintained by the arcades around the pancreatic head. One patient who experienced a pseudoaneurysm of the RHA after left hemihepatectomy successfully underwent re-laparotomy, ligation of RHA, and creation of an ileocolic arterioportal shunt. In contrast, four of seven patients with fatal outcomes experienced hepatic infarction following TAE proximal to the PHA or injury of the common hepatic artery during angiography. One patient who underwent a major hepatectomy for hilar bile duct cancer had a recurrent hemorrhage after TAE of the gastroduodenal artery and experienced hepatic failure. In the two patients with a pseudoaneurysm of the SPA or the superior mesenteric artery, an emergency relaparotomy was required to obtain hemostasis because of worsening clinical status. Selective TAE distal to PHA or in the SPA is usually successful. TAE proximal to PHA must be restricted to cases where collateral hepatic blood flow exists. Otherwise or for a pseudoaneurysm of the superior mesenteric artery, endovascular stenting, temporary creation of an ileocolic arterioportal shunt, or vascular reconstruction by re-laparotomy is an alternative.