Hepatobiliary long-term consequences of COVID-19: dramatically increased rate of secondary sclerosing cholangitis in critically ill COVID-19 patients.

BACKGROUND: Increasing evidence suggests that secondary sclerosing cholangitis (SSC), which can lead to cirrhosis or liver failure, may be a hepatobiliary long-term complication of COVID-19. The aim of this study was to estimate the frequency and outcome of this COVID-19 sequela and to identify possible risk factors. METHODS: This observational study, conducted at University Hospital Charité Berlin and Unfallkrankenhaus Berlin, Germany, involved hospitalized patients with COVID-19 pneumonia, including 1082 ventilated COVID-19 patients. We compared COVID-19 patients who developed SSC with a COVID-19 control group by univariate and multivariate analyses. RESULTS: SSC occurrence after COVID-19 was observed exclusively in critically ill patients with invasive ventilation, albeit with extreme clustering among them. One in every 43 invasively ventilated COVID-19 patients developed this complication. Risk factors preceding the development of secondary sclerosing cholangitis in critically ill COVID-19 patients (SSC-CIP) were signs of systemic reduced blood oxygen supply (e.g., low PaO2/FiO2, ischemic organ infarctions), multi-organ failure (high SOFA score) at admission, high fibrinogen levels and intravenous ketamine use. Multivariate analysis confirmed fibrinogen and increased plasma lactate dehydrogenase as independent risk factors associated with cholangiopathy onset. The 1-year transplant-free survival rate of COVID-19-associated SSC-CIP was 40%. CONCLUSIONS: COVID-19 causes SSC-CIP in a substantial proportion of critically ill patients. SSC-CIP most likely develops due to severe tissue hypoxia and fibrinogen-associated circulatory disturbances. A significant increase of patients with SSC-CIP is to be expected in the post-COVID era.

Therapeutic Potential of TLR8 Agonist GS-9688 (Selgantolimod) in Chronic Hepatitis B: Remodeling of Antiviral and Regulatory Mediators.

BACKGROUND AND AIMS: GS-9688 (selgantolimod) is a toll-like receptor 8 agonist in clinical development for the treatment of chronic hepatitis B (CHB). Antiviral activity of GS-9688 has previously been evaluated in vitro in HBV-infected hepatocytes and in vivo in the woodchuck model of CHB. Here we evaluated the potential of GS-9688 to boost responses contributing to viral control and to modulate regulatory mediators. APPROACH AND RESULTS: We characterized the effect of GS-9688 on immune cell subsets in vitro in peripheral blood mononuclear cells of healthy controls and patients with CHB. GS-9688 activated dendritic cells and mononuclear phagocytes to produce IL-12 and other immunomodulatory mediators, inducing a comparable cytokine profile in healthy controls and patients with CHB. GS-9688 increased the frequency of activated natural killer (NK) cells, mucosal-associated invariant T cells, CD4+ follicular helper T cells, and, in about 50% of patients, HBV-specific CD8+ T cells expressing interferon-γ. Moreover, in vitro stimulation with GS-9688 induced NK-cell expression of interferon-γ and TNF-α, and promoted hepatocyte lysis. We also assessed whether GS-9688 inhibited immunosuppressive cell subsets that might enhance antiviral efficacy. Stimulation with GS-9688 reduced the frequency of CD4+ regulatory T cells and monocytic myeloid-derived suppressor cells (MDSCs). Residual MDSCs expressed higher levels of negative immune regulators, galectin-9 and programmed death-ligand 1. Conversely, GS-9688 induced an expansion of immunoregulatory TNF-related apoptosis-inducing ligand+ NK cells and degranulation of arginase-I+ polymorphonuclear MDSCs. CONCLUSIONS: GS-9688 induces cytokines in human peripheral blood mononuclear cells that are able to activate antiviral effector function by multiple immune mediators (HBV-specific CD8+ T cells, CD4+ follicular helper T cells, NK cells, and mucosal-associated invariant T cells). Although reducing the frequency of some immunoregulatory subsets, it enhances the immunosuppressive potential of others, highlighting potential biomarkers and immunotherapeutic targets to optimize the antiviral efficacy of GS-9688.

Serum HBsAg clearance has minimal impact on CD8+ T cell responses in mouse models of HBV infection.

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.

PD-1 blockade partially recovers dysfunctional virus-specific B cells in chronic hepatitis B infection.

Chronic HBV (CHB) infection suppresses virus-specific T cells, but its impact on humoral immunity has been poorly analyzed. Here, we developed a dual-staining method that utilizes hepatitis B virus (HBV) surface antigens (HBsAg) labeled with fluorochromes as "baits" for specific ex vivo detection of HBsAg-specific B cells and analysis of their quantity, function, and phenotype. We studied healthy vaccinated subjects (n = 18) and patients with resolved (n = 21), acute (n = 11), or chronic (n = 96) HBV infection and observed that frequencies of circulating HBsAg-specific B cells were independent of HBV infection status. In contrast, the presence of serum HBsAg affected function and phenotype of HBsAg-specific B cells that were unable to mature in vitro into Ab-secreting cells and displayed an increased expression of markers linked to hyperactivation (CD21lo) and exhaustion (PD-1). Importantly, B cell alterations were not limited to HBsAg-specific B cells, but affected the global B cell population. HBsAg-specific B cell maturation could be partially restored by a method involving the combination of the cytokines IL-2 and IL-21 and CD40L-expressing feeder cells and was further boosted by the addition of anti-PD-1 Abs. In conclusion, HBV infection has a marked impact on global and HBV-specific humoral immunity, yet HBsAg-specific B cells are amenable to a partial rescue by B cell-maturing cytokines and PD-1 blockade.

The PI3Kδ-Selective Inhibitor Idelalisib Minimally Interferes with Immune Effector Function Mediated by Rituximab or Obinutuzumab and Significantly Augments B Cell Depletion In Vivo.

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.

Discovery of Potent Cyclophilin Inhibitors Based on the Structural Simplification of Sanglifehrin A.

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.

TLR7 Agonist GS-9620 Is a Potent Inhibitor of Acute HIV-1 Infection in Human Peripheral Blood Mononuclear Cells.

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.

Intracellular Activation of Tenofovir Alafenamide and the Effect of Viral and Host Protease Inhibitors.

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.

Pharmacokinetic and pharmacodynamic properties of GS-9620, a novel Toll-like receptor 7 agonist, demonstrate interferon-stimulated gene induction without detectable serum interferon at low oral doses.

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.

Mutations in adenosine deaminase-like (ADAL) protein confer resistance to the antiproliferative agents N6-cyclopropyl-PMEDAP and GS-9219.

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.

Safety, pharmacokinetics and pharmacodynamics of GS-9620, an oral Toll-like receptor 7 agonist.

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.

GS-9620, an oral agonist of Toll-like receptor-7, induces prolonged suppression of hepatitis B virus in chronically infected chimpanzees.

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.

[How up-to-date are metal-based resin-bonded fixed partial dentures in the era of full-ceramics and dental implants? A case report]. / Wie aktuell sind metallkeramische Adhäsivbrücken im Zeitalter der Vollkeramik und der Implantologie? Ein Fallbericht.

Regardless of the success of preventive measures the replace¬ment of missing teeth in young patients with caries-free dentitions is a daily challenge for the clinician. The decision-making process concerning the different treatment options is difficult, based on several equivalent solutions. Thereby the significance of minimal invasive approaches is steadily increasing. In indicated cases, resin-bonded fixed partial dentures which were introduced in the last century still represent a well documented and minimally invasive procedure, even in the era of dental implants. In this article, clinical aspects of a conventional, metal-based resin-bonded fixed partial dentures are discussed according to the current literature and the clinical steps are presented in a clinical case.

Role of cathepsin A and lysosomes in the intracellular activation of novel antipapillomavirus agent GS-9191.

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.

Psychometric properties of an International Classification of Functioning, Disability and Health (ICF)-oriented, adaptive questionnaire for the assessment of mobility, self-care and domestic life.

OBJECTIVE: Development of an adaptive, International Classification of Functioning, Disability and Health (ICF)-oriented patient questionnaire on mobility and self-care based on an item response theory model (MOSES questionnaire). METHODS: Using item reconstruction rules, items were developed for the ICF chapters "mobility", "self-care" and "domestic life". The resulting instrument, together with other instruments (SF-36, Short Musculoskeletal Function Assessment Questionnaire (SMFA), MacNew, Functional Independence Measure (FIM), Barthel) was presented to 549 patients with musculoskeletal disease, 212 patients with cardiac disease and 258 neurological rehabilitation patients in rehabilitation clinics in Germany. RESULTS: The MOSES questionnaire includes 58 items on 12 scales and fulfills the requirements of the 1-parameter item response theory model (Rasch model). The results indicate good reliability and high construct validity and sensitivity to change of the instrument. In the construction and selection of items, ICF contents that include complex processes of evaluation, and which presuppose skills that are not acquired prior to the individual learning process, were omitted due to a lack of unidimensionality. CONCLUSION: The successful implementation of the concept of applying rules to ICF categories in formulating the items of a patient questionnaire showed that the goal of a theory-driven measurement of activities using the ICF is feasible. The results of the project also raise questions as to the homogeneity of the contents of some ICF categories.

Caspase-cleaved HPK1 induces CD95L-independent activation-induced cell death in T and B lymphocytes.

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.

Goals in cardiac rehabilitation: influencing factors, relation to outcome, and relevance of physicians' illness perception.

PURPOSE: The study attempted to identify and define subgroups of patients participating in cardiac rehabilitation (CR) whose CR goal profiles were similar, what factors influence the definition of targets for these goals, and how the definition of goals influenced the short-term and medium-term effects of CR. METHODS: Data were obtained from 2,182 patients participating in 17 CR centers in Germany from 2001 to 2003. The data collection instruments included a patient questionnaire on general health status and a case report form completed by a physician. RESULTS: A latent class analysis led to a 3-cluster solution including a patient cluster of "risk factor patients" for whom the CR goals were primarily reduction of blood pressure and cholesterol level; a patient cluster of "multimorbidity patients" with numerous interdisciplinary CR goals; and a patient cluster for which the goals were relatively undefined. These clusters were derived only from the analysis of the CR goals defined by physicians rather than from the analysis of the baseline health status. Goal setting in the psychologic area was greater than 1.5 times more likely to occur for men than for women. The setting of goals had no independent influence on the effects of CR. CONCLUSION: Findings suggest that the cognitive illness perception of the treating physician affects goal setting in CR.

Clinical and mechanistic aspects of glucocorticoid-induced chemotherapy resistance in the majority of solid tumors.

BACKGROUND: Glucocorticoids have been used widely in conjunction with cancer therapy due to their ability to induce apoptosis in hematological cells and to prevent nausea and emesis. However, recent data including ours, suggest induction of therapy-resistance by glucocorticoids in solid tumors, although it is unclear whether this happens only in few carcinomas or is a more common cell type specific phenomenon. MATERIAL AND METHODS: We performed an overall statistical analysis of our new and recent data with 157 tumor probes evaluated in vitro, ex vivo and in vivo. The effect of glucocorticoids on apoptosis, viability and cell cycle progression under diverse clinically important questions was examined. RESULTS: New in vivo results demonstrate glucocorticoid-induced chemotherapy resistance in xenografted prostate cancer. In an overall statistical analysis we found glucocorticoid-induced resistance in 89% of 157 analysed tumor samples. Resistance is common for several cytotoxic treatments and for several glucocorticoid-derivatives and due to an inhibition of apoptosis, promotion of viability and cell cycle progression. Resistance occurred at clinically achievable peak plasma levels of patients under anti-emetic glucocorticoid therapy and below, lasted for a long time, after one single dose, but was reversible upon removal of glucocorticoids. Two nonsteroidal alternative anti-emetic agents did not counteract anticancer treatment and may be sufficient to replace glucocorticoids in cotreatment of carcinoma patients. CONCLUSION: These data demonstrate the need for prospective clinical studies as well as for detailed mechanistic studies of GC-induced cell-type specific pro- and anti-apoptotic signalling.

Comparative cytotoxicity of alkyl gallates on mouse tumor cell lines and isolated rat hepatocytes.

Alkyl esters of gallic acid inhibited the respiration rate of mouse sarcoma 786A and mouse mammary adenocarcinoma TA3 cell lines and its multiresistant variant TA3-MTX-R more effectively than gallic acid, both in the absence and in the presence of the uncoupler CCCP. The order of inhibition of the respiration rate by gallates in intact cells was n-octyl- approximately iso-amyl- approximately n-amyl- approximately iso-butyl->n-butyl->iso-propyl->n-propyl-gallate>>gallic acid. Sarcoma 786A was significantly more susceptible to all seven esters than the TA3 cell line. Respiration rates of the TA3-MTX-R cell line showed almost the same sensitivity to these esters as the TA3 cell line. However, hepatocytes were significantly less sensitive than all tumor cells tested. These alkyl gallates blocked mitochondrial electron flow, mainly at the NADH-CoQ segment, preventing ATP synthesis, which would lead to cellular death. These esters also inhibited, in the same order of potencies as respiration, the growth of 786A, TA3 and TA3-MTX-R cells in culture. In mice carrying TA3 or TA3-MTX-R tumor cells, an important decrease of the tumor growth rate and an increase of survival were observed when mice were treated with iso-butyl gallate alone or in combination with doxorubicin. These results indicate that alkyl gallates are selectively cytotoxic to tumor cells, which may be due to the mitochondrial dysfunctions of these cells.

In vitro generated human memory-like T cells are CD95 type II cells and resistant towards CD95-mediated apoptosis.

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.