Type III phosphatidylinositol-4-kinase beta (PI4KIII␤) was previously implicated in hepatitis C virus (HCV) replication by small interfering RNA (siRNA) depletion and was therefore proposed as a novel cellular target for the treatment of hepatitis C. Medicinal chemistry efforts identified highly selective PI4KIII␤ inhibitors that potently inhibited the replication of genotype 1a and 1b HCV replicons and genotype 2a virus in vitro. Replicon cells required more than 5 weeks to reach low levels of 3-to 5-fold resistance, suggesting a high resistance barrier to these cellular targets. Extensive in vitro profiling of the compounds revealed a role of PI4KIII␤ in lymphocyte proliferation. Previously proposed functions of PI4KIII␤ in insulin secretion and the regulation of several ion channels were not perturbed with these inhibitors. Moreover, PI4KIII␤ inhibitors were not generally cytotoxic as demonstrated across hundreds of cell lines and primary cells. However, an unexpected antiproliferative effect in lymphocytes precluded their further development for the treatment of hepatitis C. C hronic hepatitis C virus (HCV) infection, a major cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma, afflicts approximately 3% of the world's population (24). The current standard of care for treating hepatitis C is pegylated interferon and ribavirin, which shows poor tolerability and is capable of achieving a sustained viral response in only half of genotype 1 patients (7). Two NS3 protease inhibitors, telaprevir and boceprevir, have been approved recently, and additional direct-acting antivirals are in clinical development. While triple therapy with interferon, ribavirin, and a protease inhibitor increases the percentage of patients showing a sustained viral response to 75% and can shorten the treatment time, it still has limitations: only genotype 1 patients are responsive, side effects (such as anemia) prevent the use in transplant patients, and the inconvenient dosing schedule (three times a day) might cause noncompliance. Development of viruses resistant to direct antivirals occurs very rapidly and leads to relapse and viral breakthrough. A possible exception might be nucleoside inhibitors, since viruses with resistance mutations are not viable. We therefore executed high-throughput small interfering RNA (siRNA) screens in order to identify novel cellular targets for the treatment of HCV. Type III phosphatidylinositol-4-kinases (PI4KIIIs) were identified from these studies and in screens performed in other laboratories (3,4,(20)(21)(22).Mammalian cells express a large number of lipid kinases, including four enzymes that phosphorylate phosphatidylinositol at position four of the inositol ring, the phosphatidylinositol-4-kinases (PI4Ks). Lipid kinases are involved in multiple functions of the cell, of which phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) signaling is the most thoroughly investigated process. The four PI4Ks (type II ␣ and ␤ and type III ␣ and ␤) are localized to different sites in the cell by pro...

We describe the induction of autoimmune diabetes, insulitis, and thyroiditis in athymic rats following ujections of major histocompatibiity complex compatible spleen cells. Lymphocytes with these capabilities were found in normal rats ofthe YOS, WAG, PVG, and diabetes-resistant BB strains, and in diabetes-prone BB rats. Adoptive transfer was facilitated by prior in vivo depletion of RT6.1+ regulatory T cells and in vitro mitogen activation of donor spleen cells. By RT6 depleting diabetes-resistant donors and nsing nude recipients, transfer of diabetes and thyroiditis was accomplished by using fresh, unstimulated spleen cells. The data suggest that organ-specific autoreactive cells may be present to various degrees but suppressed to a variable extent in many rat strains. The equilibrium between autoreactive and regulatory cells appears to determine the expression of autoimmunity.Clonal deletion of autoreactive lymphocytes during repertoire development and active suppression of autoreactive cells in peripheral tissues contribute to the prevention of autoimmune diseases (1). In mice, T-lymphocyte populations are deleted in the thymus following interaction with a major histocompatibility complex (MHC) class II (I-E) molecule (2). Nonobese diabetic mice do not express I-E and develop autoimmune diabetes (3). These mice harbor T-cell populations normally deleted in I-E-expressing strains (4). In B6AF1 mice, neonatal thymectomy induces organ-specific autoimmunity (5). Thymectomy earlier or later is ineffective, suggesting that a sequence ofintrathymic events culminates in an immunological definition of self.Evidence for suppression of autoreactive T cells in peripheral tissues is also accumulating. Transplantation of Lyt-1-depleted spleen cells from nu/+ mice (6) or thymuses from cyclosporin-treated nu/+ mice (7) into nu/nu recipients results in autoimmune endocrinopathy. This can be prevented by cotransplantation of a nu/+ thymus or unfrictionated spleen cells together with the cyclosporin-treated thymus.An imbalance between autoreactive and regulatory (RT6+) cells may in part determine the expression of BB rat autoimmunity (8). Diabetes-prone (DP) BB rats are lymphopenic and deficient in T cells that express the RT6 alloantigen (9). They spontaneously develop insulitis, hyperglycemia, and thyroiditis (8,10 ITo whom reprint requests should be addressed. 7618The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.Proc. Natl. Acad. Sci. USA 87 (1990) 7619 isolated splenocytes were injected into recipients (108 cells in 0.5 ml per rat).Flow Microfluorimetry. To determine the number of native T cells present in athymic rats and to assay for chimerism in adoptive recipients, lymph node cell suspensions were labeled with 0X19 mouse anti-rat CD5 (pan-T cell) monoclonal antibody (mAb), with the rat anti-RT6.1 mAb DS4.23, or with the rat anti-RT6.2 mAb 6A5 (9). Cell...

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Information regarding the distribution and persistence of DNA encapsulated in poly-(lactide co-glycolide) microspheres was collected to provide additional information regarding the safety of DNA vaccines and to support the clinical testing of this new delivery system for DNA. Plasmid DNA was encapsulated in poly(lactide co-glycolide) microspheres and the distribution and persistence of plasmid in murine tissues resulting from parenteral administration were examined by a sensitive PCR assay. Encapsulated DNA delivered by intramuscular or subcutaneous injection can be detected for 100 days post-injection and is distributed primarily at the site of injection and the lymphoid organs. Intravenous administration results in more widespread dissemination with long term persistence limited to the lymphoid organs and those of the reticuloendothelial system. Specific cellular uptake of DNA by professional antigen presenting cells (APCs) following injection suggests the utility of microspheres as DNA delivery agents. Distribution and persistence studies support the safety of encapsulated DNA and the specific cellular uptake of DNA by professional APCs following injection suggests the utility of microspheres as DNA delivery agents.

Elderly patients presenting to St. Vincent's Health Emergency Department (ED) constitute approximately one third of presentations. A significant proportion of these involve preexisting conditions including depression that, within elderly patients, is associated with social isolation, physical and mental health problems, and barriers to accessing community services. It is also often overlooked as a clinical diagnosis among the elderly. This study aimed to assess the efficacy of a brief depression screening tool and examine the change over time in quality of life and social factors for elderly patients who present to ED. Patients aged 65 years and over were screened for depression using a short form of the Geriatric Depression Scale (GDS-15). Participants were randomized into control (usual care) and intervention (an assertive outreach community management program) groups and assessed in relation to depression, quality of life, and social support/functioning at recruitment and 6 weeks post discharge. Approximately one in four participants experienced mild to moderate depression that was related to medical factors and associated reduced mobility. This study suggests that an assertive outreach program, with the inclusion of community intervention and links to social supports and services, could improve the management of depression in the elderly and associated health outcomes.