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Antibody-mediated clearance of hepatitis B surface antigen (HBsAg) from the circulation of chronically infected patients (i.e., seroconversion) is usually associated with increased HBV-specific T cell responsiveness. However, a causative link between serum HBsAg levels and impairment of intrahepatic CD8+ T cells has not been established. Here we addressed this issue by using HBV replication-competent transgenic mice that are depleted of circulating HBsAg, via either spontaneous seroconversion or therapeutic monoclonal antibodies, as recipients of HBV-specific CD8+ T cells. Surprisingly, we found that serum HBsAg clearance has only a minimal effect on the expansion of HBV-specific naive CD8+ T cells undergoing intrahepatic priming. It does not alter their propensity to become dysfunctional, nor does it enhance the capacity of IL-2-based immunotherapeutic strategies to increase their antiviral function. In summary, our results reveal that circulating HBsAg clearance does not improve HBV-specific CD8+ T cell responses in vivo and may have important implications for the treatment of chronic HBV infection., Competing Interests: Disclosures: R. Jordan reported personal fees from Gilead Sciences during the conduct of the study and outside the submitted work, and is a shareholder of Gilead Sciences. C.R. Frey reported personal fees from Gilead Sciences during the conduct of the study. F.V. Chisari reported personal fees from Gilead Sciences, Vir Biotechnology, and Avalia Immunotherapies outside the submitted work. L.G. Guidotti reported grants and personal fees from Gilead Sciences during the conduct of the study. M. Iannacone reported grants from Gilead Sciences during the conduct of the study and personal fees from Gilead Sciences outside the submitted work. No other disclosures were reported., (© 2020 Fumagalli et al.)

Idelalisib is a highly selective oral inhibitor of PI3Kδ indicated for the treatment of patients with relapsed chronic lymphocytic leukemia in combination with rituximab. Despite additive clinical effects, previous studies have paradoxically demonstrated that targeted therapies potentially negatively affect anti-CD20 mAb effector mechanisms. To address these potential effects, we investigated the impact of PI3Kδ inhibition by idelalisib on the effector mechanisms of rituximab and obinutuzumab. At clinically relevant concentrations, idelalisib minimally influenced rituximab- and obinutuzumab-mediated Ab-dependent cellular cytotoxicity and phagocytosis on human lymphoma cell lines, while maintaining the superiority of obinutuzumab-mediated Ab-dependent cellular cytotoxicity. Consistent with this, idelalisib did not influence obinutuzumab-mediated B cell depletion in whole-blood B cell-depletion assays. Further, idelalisib significantly enhanced obinutuzumab-mediated direct cell death of chronic lymphocytic leukemia cells. In murine systems, in vivo inhibition of PI3Kδ minimally interfered with maximal rituximab- or obinutuzumab-mediated depletion of leukemic targets. In addition, the duration of rituximab- and obinutuzumab-mediated depletion of leukemia cells was extended by combination with PI3Kδ inhibition. Collectively, these data demonstrate that PI3Kδ inhibition does not significantly affect the effector mechanisms induced by rituximab or obinutuzumab and provides an effective in vivo therapeutic combination. Therefore, combinations of obinutuzumab and idelalisib are currently being assessed in clinical studies., (Copyright © 2018 The Authors.)

Maintaining detectable levels of antibodies to hepatitis B surface antigen (HBsAg) in serum after HBsAg sero-conversion is the key clinical endpoint indicative of recovery from infection with hepatitis B virus (HBV). As HBV-infected hepatocytes secrete HBsAg subviral particles in vast excess over HBV virions, detectable hepatitis B surface antibody (anti-HBs) titres imply complete elimination of HBV virions as well as HBsAg particles. Although intrahepatic phagocytes, for example Kupffer cells, are thought to mediate clearance of HBsAg via antibody (Ab)-dependent and Ab-independent mechanisms, the relative contributions of circulating phagocytic cell types to HBsAg elimination are poorly characterized. Understanding the role of various immune cell subsets in the clearance of HBsAg is important because Ab-dependent or Ab-independent phagocytic HBsAg uptake may modulate presentation of HBsAg-derived epitopes to antigen-specific T cells and hence plays a critical role in adaptive immunity against HBV. This study aims to characterize phagocytic leucocyte subsets capable of internalizing HBsAg immune complexes (HBsAg:IC) or un-complexed HBsAg particles in whole blood directly ex vivo. The data show that uptake of HBsAg:IC occurs most prominently in monocytes, B cells, dendritic cells and in neutrophils. In contrast, B cells, and to a lesser degree also monocytes, seem to be effective phagocytes for un-complexed HBsAg. Importantly, a similar pattern of phagocytic HBsAg uptake was observed in blood from chronic hepatitis B (CHB) patients compared to healthy controls, suggesting that phagocytosis-related cellular functions are not altered in the context of CHB., (© 2017 John Wiley & Sons Ltd.)

Cyclophilin inhibition has been a target for the treatment of hepatitis C and other diseases, but the generation of potent, drug-like molecules through chemical synthesis has been challenging. In this study, a set of macrocyclic cyclophilin inhibitors was synthesized based on the core structure of the natural product sanglifehrin A. Initial compound optimization identified the valine-m-tyrosine-piperazic acid tripeptide (Val-m-Tyr-Pip) in the sanglifehrin core, stereocenters at C14 and C15, and the hydroxyl group of the m-tyrosine (m-Tyr) residue as key contributors to compound potency. Replacing the C18-C21 diene unit of sanglifehrin with a styryl group led to potent compounds that displayed a novel binding mode in which the styrene moiety engaged in a π-stacking interaction with Arg55 of cyclophilin A (Cyp A), and the m-Tyr residue was displaced into solvent. This observation allowed further simplifications of the scaffold to generate new lead compounds in the search for orally bioavailable cyclophilin inhibitors.

GS-9620 is a potent and selective oral Toll-like receptor 7 (TLR7) agonist that directly activates plasmacytoid dendritic cells (pDCs). GS-9620 suppressed hepatitis B virus (HBV) in animal models of chronic infection and transiently activated HIV expression ex vivo in latently infected peripheral blood mononuclear cells (PBMCs) from virally suppressed patients. Currently, GS-9620 is under clinical evaluation for treating chronic HBV infection and for reducing latent reservoirs in virally suppressed HIV-infected patients. Here, we investigated the in vitro anti-HIV-1 activity of GS-9620. GS-9620 potently inhibited viral replication in PBMCs, particularly when it was added 24 to 48 h prior to HIV infection (50% effective concentration = 27 nM). Depletion of pDCs but not other immune cell subsets from PBMC cultures suppressed GS-9620 antiviral activity. Although GS-9620 was inactive against HIV in purified CD4 + T cells and macrophages, HIV replication was potently inhibited by conditioned medium derived from GS-9620-treated pDC cultures when added to CD4 + T cells prior to infection. This suggests that GS-9620-mediated stimulation of PBMCs induced the production of a soluble factor(s) inhibiting HIV replication in trans GS-9620-treated PBMCs primarily showed increased production of interferon alpha (IFN-α), and cotreatment with IFN-α-blocking antibodies reversed the HIV-1-inhibitory effect of GS-9620. Additional studies demonstrated that GS-9620 inhibited a postentry event in HIV replication at a step coincident with or prior to reverse transcription. The simultaneous activation of HIV-1 expression and inhibition of HIV-1 replication are important considerations for the clinical evaluation of GS-9620 since these antiviral effects may help restrict potential local HIV spread upon in vivo latency reversal., (Copyright © 2016 Bam et al.)

Tenofovir alafenamide fumarate (TAF) is an oral phosphonoamidate prodrug of the HIV reverse transcriptase nucleotide inhibitor tenofovir (TFV). Previous studies suggested a principal role for the lysosomal serine protease cathepsin A (CatA) in the intracellular activation of TAF. Here we further investigated the role of CatA and other human hydrolases in the metabolism of TAF. Overexpression of CatA or liver carboxylesterase 1 (Ces1) in HEK293T cells increased intracellular TAF hydrolysis 2- and 5-fold, respectively. Knockdown of CatA expression with RNA interference (RNAi) in HeLa cells reduced intracellular TAF metabolism 5-fold. Additionally, the anti-HIV activity and the rate of CatA hydrolysis showed good correlation within a large set of TFV phosphonoamidate prodrugs. The covalent hepatitis C virus (HCV) protease inhibitors (PIs) telaprevir and boceprevir potently inhibited CatA-mediated TAF activation (50% inhibitory concentration [IC50] = 0.27 and 0.16 μM, respectively) in vitro and also reduced its anti-HIV activity in primary human CD4(+) T lymphocytes (21- and 3-fold, respectively) at pharmacologically relevant concentrations. In contrast, there was no inhibition of CatA or any significant effect on anti-HIV activity of TAF observed with cobicistat, noncovalent HIV and HCV PIs, or various prescribed inhibitors of host serine proteases. Collectively, these studies confirm that CatA plays a pivotal role in the intracellular metabolism of TAF, whereas the liver esterase Ces1 likely contributes to the hepatic activation of TAF. Moreover, this work demonstrates that a wide range of viral and host PIs, with the exception of telaprevir and boceprevir, do not interfere with the antiretroviral activity of TAF., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

GS-9620 [8-(3-(pyrrolidin-1-ylmethyl)benzyl)-4-amino-2-butoxy-7,8-dihydropteridin-6(5H)-one] is a potent, orally bioavailable small-molecule agonist of Toll-like receptor 7 (TLR7) developed for finite treatment of chronic hepatitis B viral (HBV) infection, with the goal of inducing a liver-targeted antiviral effect without inducing the adverse effects associated with current systemic interferon-α (IFN-α) therapies. We characterized the pharmacodynamic response of GS-9620 in CD-1 mice and cynomolgus monkeys following intravenous or oral administration and showed that GS-9620 induces the production of select chemokines and cytokines, including IFN-α and interferon-stimulated genes (ISGs). It is noteworthy that we also demonstrated that, in animals and healthy human volunteers, oral administration of GS-9620 can induce a type I interferon-dependent antiviral innate immune response, as measured by whole-blood mRNA of the ISGs 2'5'-oligoadenylate synthetase 1 (OAS1) and myxovirus resistance 1 (MX1), without the induction of detectable systemic IFN-α, i.e., a presystemic response. Additionally, presystemic induction of hepatic OAS1 and MX1 mRNA was observed in CD-1 mice in the absence of detectable systemic IFN-α. We propose that the mechanism of this presystemic response is likely its high intestinal absorption, which facilitates localized activation of TLR7, probably in plasmacytoid dendritic cells at the level of gut-associated lymphoid tissue and/or the liver. This localized response is further supported by data that indicate only minimal contributions of systemic immune stimulation to the overall pharmacodynamic response to orally administered GS-9620. These data demonstrate that GS-9620 can induce an antiviral innate immune response without inducing a systemic IFN-α response and thus suggest the therapeutic potential of this approach in the treatment of chronic HBV infection.

Background & Aims: Direct-acting antiviral agents suppress hepatitis B virus (HBV) load, but they require life-long use. Stimulation of the innate immune system could increase its ability to control the virus and have long-lasting effects after a finite regimen. We investigated the effects of immune activation with GS-9620--a potent and selective orally active small molecule agonist of Toll-like receptor 7--in chimpanzees with chronic HBV infection., Methods: GS-9620 was administered to chimpanzees every other day (3 times each week) for 4 weeks at 1 mg/kg and, after a 1-week rest, for 4 weeks at 2 mg/kg. We measured viral load in plasma and liver samples, the pharmacokinetics of GS-9620, and the following pharmacodynamics parameters: interferon-stimulated gene expression, cytokine and chemokine levels, lymphocyte and natural killer cell activation, and viral antigen expression. Clinical pathology parameters were monitored to determine the safety and tolerability of GS-9620., Results: Short-term oral administration of GS-9620 provided long-term suppression of serum and liver HBV DNA. The mean maximum reduction of viral DNA was 2.2 logs, which occurred within 1 week of the end of GS-9620 administration; reductions of >1 log persisted for months. Serum levels of HBV surface antigen and HBV e antigen, and numbers of HBV antigen-positive hepatocytes, were reduced as hepatocyte apoptosis increased. GS-9620 administration induced production of interferon-α and other cytokines and chemokines, and activated interferon-stimulated genes, natural killer cells, and lymphocyte subsets., Conclusions: The small molecule GS-9620 activates Toll-like receptor 7 signaling in immune cells of chimpanzees to induce clearance of HBV-infected cells. This reagent might be developed for treatment of patients with chronic HBV infection., (Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background/aim: GS 9219 is a double prodrug of antiproliferative nucleotide analog 9-(2-Phosphonylmethoxyethyl)guanine (PMEG), with potent in vivo efficacy against various hematological malignancies. This study investigates the role of adenosine deaminase-like (ADAL) protein in the intracellular activation of GS-9219., Materials and Methods: A cell line resistant to 9-(2-Phosphonylmethoxyethyl)-N(6)-cyclopropyl-2,6-diaminopurine (cPrPMEDAP), an intermediate metabolite of GS-9219, was generated and characterized., Results: The resistant cell line was cross-resistant to cPrPMEDAP and GS-9219, due to a defect in the deamination of cPrPMEDAP to PMEG. Mutations in the ADAL gene (H286R and S180N) were identified in the resistant cells that adversely-affected its enzymatic activity. Introduction of the wild-type ADAL gene re-sensitized resistant cells to both cPrPMEDAP and GS-9219., Conclusion: The ADAL protein plays an essential role in the intracellular activation of GS-9219 by catalyzing the deamination of cPrPMEDAP metabolite to PMEG. Mutations affecting the activity of ADAL confer resistance to both GS-9219 and its metabolite cPrPMEDAP.

Background: GS-9620 is a novel oral agonist of Toll-like receptor 7 (TLR7) in development for the treatment of chronic viral hepatitis. TLR7 is a highly conserved innate immune receptor expressed primarily on plasmacytoid dendritic cells and B lymphocytes. The aim of this double-blind, placebo-controlled, single ascending-dose study was to investigate the safety, tolerability, pharmacokinetics and pharmacodynamics of GS-9620 in healthy volunteers., Methods: In total, 75 healthy volunteers (8 subjects in each of the 10 cohorts; 5 subjects participated in two cohorts) were randomized (6:2) to receive a single dose of GS-9620 (0.3, 1, 2, 4, 6, 8 or 12 mg) or placebo., Results: GS-9620 was well-absorbed and well-tolerated in oral doses up to 12 mg. Minimal treatment-related adverse events were seen at doses up to 8 mg. Serum interferon (IFN)-α was only detected in subjects who received 8 or 12 mg doses, and the adverse event profile at 8 and 12 mg doses was generally consistent with that associated with IFN-α exposure (flu-like symptoms), consistent with the mechanism of TLR7 agonism. All adverse events resolved within 72 h. Induction of chemokines/cytokines and IFN-stimulated genes were seen at GS-9620 doses ≥ 2 mg, well below doses that induced serum IFN-α or led to clinical adverse events., Conclusions: GS-9620 demonstrates safety and pharmacodynamic activity at doses up to 12 mg. Pharmacodynamic activity is seen before adverse events, suggesting the potential for induction of an antiviral response without systemic adverse events in subjects with chronic viral hepatitis.

Collagen and fibrin are important extracellular matrix (ECM) components in the body, providing structural integrity to various tissues. These biopolymers are also common scaffolds used in tissue engineering. This study investigated how co-gelation of collagen and fibrin affected the properties of each individual protein network. Collagen-fibrin co-gels were cast and subsequently digested using either plasmin or collagenase; the microstructure and mechanical behavior of the resulting networks were then compared with the respective pure collagen or fibrin gels of the same protein concentration. The morphologies of the collagen networks were further analyzed via three-dimensional network reconstruction from confocal image z-stacks. Both collagen and fibrin exhibited a decrease in mean fiber diameter when formed in co-gels compared with the pure gels. This microstructural change was accompanied by an increased failure strain and decreased tangent modulus for both collagen and fibrin following selective digestion of the co-gels. In addition, analysis of the reconstructed collagen networks indicated the presence of very long fibers and the clustering of fibrils, resulting in very high connectivities for collagen networks formed in co-gels., (Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Fibrin and collagen, biopolymers occurring naturally in the body, are biomaterials commonly-used as scaffolds for tissue engineering. How collagen and fibrin interact to confer macroscopic mechanical properties in collagen-fibrin composite systems remains poorly understood. In this study, we formulated collagen-fibrin co-gels at different collagen-to-fibrin ratios to observe changes in the overall mechanical behavior and microstructure. A modeling framework of a two-network system was developed by modifying our micro-scale model, considering two forms of interaction between the networks: (a) two interpenetrating but noninteracting networks ("parallel"), and (b) a single network consisting of randomly alternating collagen and fibrin fibrils ("series"). Mechanical testing of our gels show that collagen-fibrin co-gels exhibit intermediate properties (UTS, strain at failure, tangent modulus) compared to those of pure collagen and fibrin. The comparison with model predictions show that the parallel and series model cases provide upper and lower bounds, respectively, for the experimental data, suggesting that a combination of such interactions exists between the collagen and fibrin in co-gels. A transition from the series model to the parallel model occurs with increasing collagen content, with the series model best describing predominantly fibrin co-gels, and the parallel model best describing predominantly collagen co-gels.

GS-9191, a bis-amidate prodrug of the nucleotide analog 9-(2-phosphonylmethoxyethyl)-N6-cyclopropyl-2,6-diaminopurine (cPrPMEDAP), was designed as a topical agent for the treatment of papillomavirus-associated proliferative disorders, such as genital warts. In this study, we investigated the mechanism of conversion of GS-9191 to cPrPMEDAP. We observed that GS-9191 is hydrolyzed in the presence of the lysosomal carboxypeptidase cathepsin A (CatA) in vitro and is less efficiently metabolized in CatA-deficient fibroblasts than in control cells. In addition, knockdown of CatA by small interfering RNA (siRNA) reduced the intracellular accumulation of GS-9191 metabolites. However, intracellular CatA levels did not correlate with the susceptibility of tested cell lines to GS-9191, indicating that the CatA step is unlikely to be rate limiting for the activation of GS-9191. Further analysis showed that upon the hydrolysis of the carboxylester bond in one of the GS-9191 amidate moieties, the unmasked carboxyl group displaces L-phenylalanine 2-methylpropyl ester from the other amidate moiety. The cPrPMEDAP-L-phenylalanine conjugate (cPrPMEDAP-Phe) formed is not metabolized by Hint1 (histidine triad nucleotide binding protein 1) phosphoramidase but undergoes spontaneous degradation to cPrPMEDAP in acidic pH that can be significantly enhanced by the addition of SiHa cell extract. Pretreatment of SiHa cells with bafilomycin A or chloroquine resulted in an 8-fold increase in the intracellular concentration of cPrPMEDAP-Phe metabolite and the accumulation of GS-9191 metabolites in the lysosomal/endosomal fraction. Together, these observations indicate that the conversion of GS-9191 to cPrPMEDAP occurs in lysosomes via CatA-mediated ester cleavage, followed by the release of cPrPMEDAP, most likely through the combination of enzyme-driven and spontaneous pH-driven hydrolysis of a cPrPMEDAP-Phe intermediate.

Life and death of peripheral lymphocytes is strictly controlled to maintain physiologic levels of T and B cells. Activation-induced cell death (AICD) is one mechanism to delete superfluous lymphocytes by restimulation of their immunoreceptors and it depends partially on the CD95/CD95L system. Recently, we have shown that hematopoietic progenitor kinase 1 (HPK1) determines T-cell fate. While full-length HPK1 is essential for NF-kappaB activation in T cells, the C-terminal fragment of HPK1, HPK1-C, suppresses NF-kappaB and sensitizes toward AICD by a yet undefined cell death pathway. Here we show that upon IL-2-driven expansion of primary T cells, HPK1 is converted to HPK1-C by a caspase-3 activity below the threshold of apoptosis induction. HPK1-C selectively blocks induction of NF-kappaB-dependent antiapoptotic Bcl-2 family members but not of the proapoptotic Bcl-2 family member Bim. Interestingly, T and B lymphocytes from HPK1-C transgenic mice undergo AICD independently of the CD95/CD95L system but involving caspase-9. Knock down of HPK1/HPK1-C or Bim by small interfering RNA shows that CD95L-dependent and HPK1/HPK1-C-dependent cell death pathways complement each other in AICD of primary T cells. Our results define HPK1-C as a suppressor of antiapoptotic Bcl-2 proteins and provide a molecular basis for our understanding of CD95L-independent AICD of lymphocytes.

We studied monocytic differentiation of primary mouse progenitor cells to understand molecular mechanisms of differentiation. We found a tightly controlled non-apoptotic activation of caspase-3 that correlated with differentiation. Although caspase activity was already detected during monocytic differentiation, a caspase-3 target has not been identified yet. We show that hematopoietic progenitor kinase 1 (HPK1) is processed towards its N- and C-terminal fragments during monocytic differentiation. While HPK1 is an immunoreceptor-proximal kinase in T and B cells, its role in myeloid cells is elusive. Here, we show that the N-terminal cleavage product, HPK1-N, comprising the kinase domain, confers progenitor cell survival independent of the growth factor IL-3. Furthermore, HPK1-N causes differentiation of progenitor cells towards the monocytic lineage. In contrast to full-length kinase, HPK1-N is constitutively active causing sustained JNK activation, Bad phosphorylation and survival. Blocking of caspase activity during differentiation of primary mouse progenitor cells leads to reduced HPK1-N levels, suppressed JNK activity and attenuated monocytic differentiation. Our work explains growth factor-independent survival during monocytic differentiation by caspase-mediated processing of HPK1 towards HPK1-N.

An adaptive immune response implies expansion of activated T cells and subsequent elimination to maintain homeostasis in a process called activation-induced cell death. Some cells, however, differentiate into memory cells and ensure a strong secondary immune response. To analyze the apoptosis phenotype of memory T cells on a cellular and molecular level, we have established an in vitro model of T cell activation and generation of cells phenotypically and functionally similar to memory cells. These long-term cultured T cells show a CD95-resistant phenotype, although they are still sensitive towards TCR/CD3-mediated apoptosis. Biochemical analysis revealed that these cells shift from CD95 type I (direct signaling from the receptor) during the effector phase to CD95 type II cells (dependent on the mitochondrial amplification loop). Moreover, their mitochondria are protected, probably due to high expression levels of Bcl-x(L) and Bcl-2. Thus, our data suggest a mechanism how memory T cells acquire resistance towards bystander cell death via the CD95 system.

While insufficient cell death of activated T cells can result in autoimmune disorders, elimination of too many T cells can lead to immunodeficiency. Therefore, T lymphocyte fate is highly regulated and requires that cells can switch from an apoptosis-resistant towards an apoptosis-sensitive state. This switch is tightly controlled by various effector molecules. Basically, two separate pathways control the fate of antigen-activated T cells: activation-induced cell death (AICD) and activated T cell autonomous death (ACAD). Autoreactive T lymphocytes are eliminated by restimulation via their T cell receptor (TCR) and undergo AICD involving death receptors (extrinsic pathway). In contrast, ACAD can lead to T cell deletion without TCR restimulation, and is determined by the ratio between anti- and pro-apoptotic Bcl-2 family members at the mitochondria (intrinsic pathway). While the extrinsic and the intrinsic pathway lead to caspase activation, non-caspase proteases (e.g., cathepsins) can be released by the lysosomes and might contribute to AICD as well as to ACAD. Activated T cells poses cell death escape mechanisms which are needed for survival of (memory) T cells, but are deleterious for autoimmune disorders or progression of T cell lymphomas.

CD95 (APO-1/Fas)-mediated apoptosis of hepatocytes plays a central role in the pathophysiology of various human liver diseases. Hepatocyte growth factor (HGF) was shown to exert antiapoptotic functions in rodent hepatocytes. We previously showed that primary human hepatocytes (PHH) are a valuable tool for the investigation of apoptotic processes in liver cells. In this study, we analyzed the influence of HGF on CD95-mediated apoptosis of PHH and its molecular determinants. HGF significantly inhibited CD95-mediated apoptosis of PHH as well as cleavage of caspase-8 and poly (ADP-ribose)polymerase. HGF transcriptionally induced the expression of the anti-apoptotic Bcl-2 family member myeloid cell leukemia-1 (Mcl-1). In contrary, HGF did not alter the expression levels of Bcl-2 or Bcl-x(L). HGF activated survival pathways such as the phosphatidylinositol-3 kinase (PI3K)/Akt pathway, the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase/ERK and the signal transducer and activator of transcription 3 (STAT3) pathway. Notably, HGF triggered serine(727)--but not tyrosine(705)--phosphorylation of STAT3. Pretreatment of PHH with the PI3K inhibitor LY294002 as well as adenoviral transduction of dominant negative Akt1 prevented HGF-mediated Mcl-1 induction and reversed the antiapoptotic effects of HGF. In conclusion, HGF confers survival of PHH by activation of the PI3K/Akt pathway. PI3K/Akt activation by HGF results in the induction of antiapoptotic proteins such as Mcl-1. Thus, application of HGF may be a therapeutic approach to prevent CD95-mediated hepatocellular damage in human liver diseases.

Varicella-zoster virus (VZV) causes varicella, establishes neuronal latency, and can reactivate, resulting in herpes zoster. VZV-specific T cells are important for controlling infection. VZV immediate early protein 62 (IE62) is recognized by cytotoxic T cells from immune individuals, but no CD8(+) T cell epitopes have been defined for any VZV protein. CD8(+) T cell frequencies were assessed by cytokine flow cytometry (CFC), by use of synthetic-peptide pools corresponding to the IE62 sequence. IE62 peptide-specific CD8(+) T cells were below the threshold of detection, by direct CFC of either whole blood or peripheral blood mononuclear cells (PBMCs). Activated CD8(+)CD69(+) T cells that produced interferon-gamma were detectable after in vitro restimulation of PBMCs, and restricted epitopes were identified for HLA-A*0201-positive subjects. Varicella vaccination of 3 VZV-immune subjects was associated with increases in IE62 peptide-specific CD8(+) T cells, a finding indicating that in vivo re-exposure boosts memory immunity to this important viral protein.

JNK proteins are ubiquitously expressed, evolutionarily conserved MAP kinases that are involved in stress responses. Recently, it was shown that the JNK cascade in Xenopus oocytes exhibits sustained, all-or-none responses to graded, transient stimuli. Here, we have examined the character of the JNK cascade's response in mammalian cells. The steady-state responses of JNK to sorbitol and anisomycin were found to be highly ultrasensitive in HeLa cells, HEK 293 cells, and Jurkat T cells. The JNK responses were also reversible, not sustained, as was the case in oocytes. Jurkat cells activated their JNK in response to phorbol myristate acetate (PMA), and the response of the entire population of Jurkat cells was graded. However, analysis of subpopulations of the PMA-treated Jurkat cells revealed that the steady-state responses of both JNK and CD69, a T cell surface activation marker, were essentially all-or-none in character. These studies show that the JNK cascade commonly exhibits switch-like responses to a variety of stimuli.