There is substantial evidence that Kaposi sarcoma-associated herpesvirus (KSHV) plays an important role in the pathogenesis of all forms of Kaposi sarcoma (KS). It has been noted that KS commonly occurs in locations, such as the feet, where tissue may be poorly oxygenated. On the basis of this observation, the potential role of hypoxia in the reactivation of KSHV replication was explored by studying 2 KSHV-infected primary effusion lymphoma B-cell lines (BC-3 and BCBL-1) latently infected with KSHV. Acute and chronic exposure of these cells to hypoxia (1% O 2 ) induced KSHV lytic replication, as indicated by an increase in intracellular lytic protein expression and detection of virus in cell supernatants by Western immunoblotting. In addition, hypoxia increased the levels of secreted viral interleukin-6. Moreover, hypoxia enhanced the lytic replication initiated by the viral inducer 12-O-tetradecanoylphorbol-13-acetate. Desferoxamine and cobalt chloride, 2 compounds that increase the intracellular levels of hypoxia-inducible factor 1, were also able to induce KSHV lytic replication. These studies suggest that hypoxia is an inducer of KSHV replication. This process may play an important role in the pathogenesis of KS. ( IntroductionKaposi sarcoma (KS) is a multifocal proliferative disease of vascular origin. KS lesions are characterized by proliferation and growth of spindle-shaped neoplastic cells that appear to be of lymphatic endothelial cell origin. [1][2][3] KS is the most common neoplasm in human immunodeficiency virus type 1-infected individuals. 1 Even before the advent of acquired immunodeficiency syndrome (AIDS), KS was known to be more prevalent in certain geographic areas and groups of patients (classic and endemic KS). A classic form of KS is described as occurring in elderly men in southern Italy and other Mediterranean countries, 4 and an endemic form of KS has been observed to occur in sub-Saharan Africa. 5,6 In addition, transplant recipients and other immunosuppressed patients are at risk for developing KS. 4 Since the discovery of Kaposi sarcoma-associated herpesvirus (KSHV), also called human herpesvirus-8 (HHV-8), in 1994, 7 substantial evidence has been accumulated that implicates it as an essential factor in the pathogenesis of all forms of KS. 8,9 It is also involved in the pathogenesis of primary effusion lymphoma (PEL) and certain cases of multicentric Castleman disease. 10 KSHV DNA is detected in nearly all KS lesions. 7,11,12 As with other human herpesviruses, infection with KSHV can be latent or lytic. Activation of viral replication in latently infected B cells, endothelial cells, or other target cells may be responsible for viral spread and contribute to the development of KS. 4 KSHV encodes for several cellular gene mimics that are produced during lytic replication and that have proangiogenic activity. [13][14][15][16] The virus also encodes several cellular protein mimics with oncogenic potential, such as v-cyc, a D-type cyclin, and a member of the interferon regulatory factor ...

The skin is an attractive target for vaccine delivery. Topical application of adjuvants results in potent immune responses and good safety profiles. Adjuvants can be coadministered in a patch with vaccine antigens (transcutaneous immunization) or similar delivery format, or administered separately with an injection or IS patch (Iomai), leading to enhanced immune responses. These observations have moved into the clinic, highlighting the likelihood that skin delivery of vaccines will play an important future role in vaccine applications.

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Acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibitors are currently in clinical development as potential lipid-lowering and antiatherosclerotic agents. We investigated the effect of avasimibe (Cl-1011), a novel ACAT inhibitor, on bile acid synthesis and cholesterol 7␣-hydroxylase in cultured rat hepatocytes and rats fed different diets. Avasimibe dose-dependently decreased ACAT activity in rat hepatocytes in the presence and absence of ␤-migrating very low-density lipoproteins (␤VLDL) (by 93% and 75% at 10 mol/L) and reduced intracellular storage of cholesteryl esters. Avasimibe (3 mol/L) increased bile acid synthesis (2.9-fold) after preincubation with ␤VLDL and cholesterol 7␣-hydroxylase activity (1.7-and 2.6-fold, with or without ␤VLDL), the latter paralleled by a similar induction of its messenger RNA (mRNA). Hepatocytes treated with avasimibe showed a shift from storage and secretion of cholesteryl esters to conversion of cholesterol into bile acids. In rats fed diets containing different amounts of cholesterol and cholate, avasimibe reduced plasma cholesterol (by 52% to 71%) and triglyceride levels (by 28% to 62%). Avasimibe did not further increase cholesterol 7␣-hydroxylase activity and mRNA in cholesterol-fed rats, but prevented down-regulation by cholate. Avasimibe did not affect sterol 27-hydroxylase and oxysterol 7␣-hydroxylase, 2 enzymes in the alternative pathway in bile acid synthesis. No increase in the ratio of biliary excreted cholesterol to bile acids was found, indicating that ACAT inhibition does not result in a more lithogenic bile. Avasimibe increases bile acid synthesis in cultured hepatocytes by enhancing the supply of free cholesterol both as substrate and inducer of cholesterol 7␣-hydroxylase. These effects may partially explain the potent cholesterol-lowering effects of avasimibe in the rat. (HEPATOLOGY 1999;30:491-500.)

Kaposi sarcoma-associated herpesvirus (KSHV) is associated with KS, primary effusion lymphoma (PEL), and multicentric Castleman disease. Reactivation of KSHV in latently infected cells and subsequent plasma viremia occur before the development of KS. Intracellular signaling pathways involved in KSHV reactivation were studied. In latently infected PEL cells (BCBL-1), KSHV reactivation in single cells was determined by quantitative flow cytometry. Viral particle production was determined by electron microscope analyses and detection of minor capsid protein in culture supernatants.Agents that mobilized intracellular calcium (ionomycin, thapsigargin) induced expression of KSHV lytic cycle-associated proteins and led to increased virus production. Calcium-mediated virus reactivation was blocked by specific inhibitors of calcineurin-dependent signal transduction (cyclosporine, FK506 IntroductionInfection with the ␥-herpesvirus Kaposi sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8), is causally involved in KS, primary effusion lymphoma (PEL), and some forms of multicentric Castleman disease. [1][2][3][4][5] The detection of KSHV in peripheral blood leukocytes is strongly predictive of the future development of KS. [6][7][8][9] Circulating B cells form the major virus reservoir in blood, 10-13 though KSHV has been found in monocytes, CD8 ϩ T cells, and CD34 ϩ cells. 11,[14][15][16] Active replication in blood and subsequent plasma viremia likely disseminate the virus throughout the body, leading to infection of dermal lymphatic endothelial cells, which are believed to be precursor cells for KS. 5 Furthermore, in KS lesions, tumor spindle cells are latently infected by KSHV; a small subpopulation of lytically infected cells produces virus and lytic viral gene products, which may contribute to the maintenance or progression of KS lesions. In general, understanding the pathways that activate virus replication (latent-tolytic switch), in blood as well as in KS lesions, is an important issue in fully understanding KS pathogenesis.In experimental systems to date, KSHV infection of primary B cells in vitro has been inefficient and unstable. 13,17 However, latently infected B cell lines, derived from patients with PEL, and infected immortalized endothelial cells are useful tools to study KSHV reactivation. [18][19][20][21][22][23] Expression of the KSHV lytic cycleassociated immediate-early ORF 50 protein (Rta) is sufficient to induce the entire lytic cycle of active viral replication. 24 Ionomycin, a Ca ϩϩ -ionophore, is known to induce expression of the ORF 50 protein. 25,26 This suggests that calcium-dependent signaling pathways may play a role in virus reactivation.In the immune system, calcium signaling is essential for the expression of many inducible genes encoding cytokines and cell surface receptors. 27,28 One of the enzymes activated by a sustained rise in [Ca ϩϩ ] i is calcineurin, a ubiquitously expressed serinethreonine phosphatase. Activation of the Ca ϩϩ -calcineurin signaling cas...

Intradermal (i.d.) immunization is a promising route of vaccine administration. Suitable i.d. adjuvants are important to increase vaccine efficacy in poorly responding populations such as the elderly or for dose-sparing strategies in the face of vaccine shortages. Bacterial exotoxins, such as Escherichia coli heat-labile enterotoxin (LT), exert strong immunostimulatory effects through binding to monosialoganglioside (GM1) cell surface receptors; however, injection is hampered by local inflammation. We demonstrate that the injection of LT formulations deficient in GM1 binding by mutation (LT(G33D)) or in vitro ligand coupling does not cause localized edema and inflammation in mice, yet these formulations retain potent adjuvant activity by enhancing functional Ab and cellular immune responses to coadministered Ags. Complete protection against in vivo lethal tetanus toxin challenge and the induction of Ag-specific CTL responses capable of killing target cells in vivo indicated in vivo efficacy of the induced immune responses. LT(G33D) proved superior to standard alum adjuvant regarding the magnitude and breadth of the induced immune responses. Immunizations in complex ganglioside knockout mice revealed a GM1-independent pathway of LT adjuvanticity. Immunostimulation by i.d. LT(G33D) is explained by its ability to induce migration of activated APCs to the proximal draining lymph nodes. LT(G33D) is a promising candidate adjuvant for human trials of parenteral vaccines in general and for current i.d. vaccine development in particular.

Isolated rat hepatocytes were incubated with extracellular ATP to induce a prolonged increase in intracellular Ca2l ([Ca2+]1) and a loss of viability within 2 h. By using video-intensified fluorescence microscopy, the effects of exposure to extracellular ATP on [Ca2-]i, mitochondrial membrane potential (MMP) and cell viability were determined simultaneously in individual living hepatocytes. The increase in [Ca2+]1 on exposure to ATP was followed by a decreasing MMP; there were big differences between individual cells. Complete loss of the MMP occurred before cell death was observed. Omission of K+ from the incubation medium decreased the cytotoxicity of ATP; under these conditions, intracellular K+ was decreased by more than 80 %. Treatment with nigericin also depleted intracellular K+ and decreased ATP-induced toxicity. Protection against loss of viability by means of a decrease in intracellular [K+] was reflected by maintenance of the MMP. These observations suggest that ATP-induced cell death may be caused by a mechanism that has been described for isolated mitochondria: after an increase in Ca2' levels, a K+ influx into mitochondria is induced, which finally disrupts the MMP and leads to cell death.

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