Pope Moseley

Activation of heat-shock response by an adenovirus is essential for virus replication.

Successful viral infection requires viruses to redirect host biochemistry to replicate the viral genome, and produce and assemble progeny virions. Cellular heat-shock responses, which are characterized as elevation and relocalization of heat-shock proteins, occur during replication of many viruses. Such responses might be host reactions to the synthesis of foreign protein, or might be irrelevant consequences of the viral need to activate transcription. Alternatively, as heat-shock proteins can facilitate protein folding, activating a heat-shock response might be a specific virus function ensuring proper synthesis of viral proteins and virions. It is not possible to determine whether heat-shock response is essential for virus replication, because the implicated viral genes (such as Ad5 E1A, ref. 10) also control other essential replication steps. Here we report that expression of Gam1, a protein encoded by the avian virus CELO (ref. 11), elevates and relocalizes hsp70 and hsp40. Gam1-negative CELO is replication-defective; however, Gam1 function can be partially replaced by either heat shock or forced hsp40 expression. Thus, an essential function of Gam1 during virus replication is to activate host heat-shock responses with hsp40 as a primary target.

Physiologically relevant increase in temperature causes an increase in intestinal epithelial tight junction permeability

The effects of physiologically relevant increase in temperature (37-41 degrees C) on intestinal epithelial tight junction (TJ) barrier have not been previously studied. Additionally, the role of heat-shock proteins (HSPs) in the regulation of intestinal TJ barrier during heat stress remains unknown. Because heat-induced disturbance of intestinal TJ barrier could lead to endotoxemia and bacterial translocation during physiological thermal stress, the purpose of this study was to investigate the effects of modest, physiologically relevant increases in temperature (37-41 degrees C) on intestinal epithelial TJ barrier and to examine the protective role of HSPs on intestinal TJ barrier. Filter-grown Caco-2 intestinal epithelial cells were used as an in vitro intestinal epithelial model system to assess the effects of heat exposure on intestinal TJ barrier. Exposure of filter-grown Caco-2 monolayers to modest increases in temperatures (37-41 degrees C) resulted in a significant time- and temperature-dependent increases in Caco-2 TJ permeability. Exposure to modest heat (39 or 41 degrees C) resulted in rapid and sustained increases in HSP expression; and inhibition of HSP expression produced a marked increase in heat-induced increase in Caco-2 TJ permeability (P < 0.001). Heat exposure (41 degrees C) resulted in a compensatory increase in Caco-2 occludin protein expression and an increase in junctional localization. Inhibition of HSP expression prevented the compensatory upregulation of occludin protein expression and produced a marked disruption in junctional localization of occludin protein during heat stress. In conclusion, our findings demonstrate for the first time that a modest, physiologically relevant increase in temperature causes an increase in intestinal epithelial TJ permeability. Our data also show that HSPs play an important protective role in preventing the heat-induced disruption of intestinal TJ barrier and suggest that HSP mediated upregulation of occludin expression may be an important mechanism involved in the maintenance of intestinal epithelial TJ barrier function during heat stress.

Heat shock protein 70 regulates cellular redox status by modulating glutathione-related enzyme activities

Abstract Heat shock protein (Hsp) 70 has been reported to protect various cells and tissues from ischemic damage. However, the molecular mechanisms of the protection are incompletely understood. Ischemia induces significant alterations in cellular redox status that plays a critical role in cell survival/death pathways. We investigated the effects of Hsp70 overexpression on cellular redox status in Madin-Darby canine kidney (MDCK) cells under both hypoxic and ischemic conditions with 3 different approaches: reactive oxygen species (ROS) measurement by a fluorescence probe, redox environment evaluation by a hydroxylamine spin probe, and redox status assessment by the glutathione/glutathione disulfide (GSH/GSSG) ratio. Results from each of these approaches showed that the redox status in Hsp70 cells was more reducing than that in control cells under either hypoxic or oxygen and glucose deprivation (OGD) conditions. In order to determine the mechanisms that mediated the alterations in redox state in Hsp70 cells, we measured the activities of glutathione peroxidase (GPx) and glutathione reductase (GR), two GSH-related antioxidant enzymes. We found that OGD exposure increased GPx and GR activities 47% and 55% from their basal levels (no stress) in Hsp70 cells, compared to only 18% and 0% increase in control cells, respectively. These data, for the first time, indicate that Hsp70 modulates the activities of GPx and GR that regulate cellular redox status in response to ischemic stress, which may be important in Hsp70s cytoprotective effects.

Immunologic manifestations of autophagy

The broad immunologic roles of autophagy span innate and adaptive immunity and are often manifested in inflammatory diseases. The immune effects of autophagy partially overlap with its roles in metabolism and cytoplasmic quality control but typically expand further afield to encompass unique immunologic adaptations. One of the best-appreciated manifestations of autophagy is protection against microbial invasion, but this is by no means limited to direct elimination of intracellular pathogens and includes a stratified array of nearly all principal immunologic processes. This Review summarizes the broad immunologic roles of autophagy. Furthermore, it uses the autophagic control of Mycobacterium tuberculosis as a paradigm to illustrate the breadth and complexity of the immune effects of autophagy.

Temporal disease trajectories condensed from population-wide registry data covering 6.2 million patients

A key prerequisite for precision medicine is the estimation of disease progression from the current patient state. Disease correlations and temporal disease progression (trajectories) have mainly been analysed with focus on a small number of diseases or using large-scale approaches without time consideration, exceeding a few years. So far, no large-scale studies have focused on defining a comprehensive set of disease trajectories. Here we present a discovery-driven analysis of temporal disease progression patterns using data from an electronic health registry covering the whole population of Denmark. We use the entire spectrum of diseases and convert 14.9 years of registry data on 6.2 million patients into 1,171 significant trajectories. We group these into patterns centred on a small number of key diagnoses such as chronic obstructive pulmonary disease (COPD) and gout, which are central to disease progression and hence important to diagnose early to mitigate the risk of adverse outcomes. We suggest such trajectory analyses may be useful for predicting and preventing future diseases of individual patients.

Soluble CD14 Levels, Interleukin-6, and Mortality Among Prevalent Hemodialysis Patients

BACKGROUND CD14 is a pattern-recognition receptor that has a central immunomodulatory role in proinflammatory signaling in response to a variety of ligands, including endotoxin. CD14 protein is present in 2 forms: soluble (sCD14) and membrane bound. Here, we studied the implications of increased sCD14 levels in hemodialysis patients. We hypothesized that sCD14 level increase may link to cytokine activation and protein-energy wasting, predisposing to increased mortality risk. STUDY DESIGN Prospective observational study of prevalent hemodialysis patients. SETTING & PARTICIPANTS 211 prevalent hemodialysis patients, median age of 65 years, with 29 months of vintage dialysis time followed up for mortality for a median of 31 months. PREDICTORS Tertiles of baseline circulating sCD14 levels corresponding to less than 2.84, 2.85 to 3.62, and greater than 3.63 microg/mL. OUTCOME The major outcome of interest was all-cause mortality. MEASUREMENTS sCD14 and endotoxin, together with other markers of inflammation and protein-energy wasting. RESULTS Median sCD14 level was 3.2 microg/mL (25th to 75th percentile, 2.7 to 3.9). sCD14 level correlated positively with C-reactive protein, interleukin 6, endotoxin, and pentraxin 3 levels and negatively with serum albumin level, muscle mass, and handgrip strength. Patients with increased sCD14 levels had lower body mass index and increased prevalence of muscle atrophy. Patients within the highest sCD14 tertile had a crude morality hazard ratio of 1.94 (95% confidence interval, 1.13 to 3.32) that persisted after adjustment for multiple confounders (hazard ratio, 3.11; 95% confidence interval, 1.49 to 6.46). In patients with persistent inflammation, the presence of a concurrent sCD14 level increase gradually increased mortality risk, but this effect was less than multiplicative and failed to show a statistical interaction. LIMITATIONS Those inherent to an observational study. CONCLUSIONS sCD14 level is associated with inflammation and protein-energy wasting in hemodialysis patients. It is a strong and independent predictor of mortality that warrants further assessment in the clinical setting regarding its usefulness as a complementary prognosticator to other general inflammatory markers.

Interleukin-6 Markedly Decreases Skeletal Muscle Protein Turnover and Increases Nonmuscle Amino Acid Utilization in Healthy Individuals

CONTEXT IL-6 is a key modulator of immune function and suggested to be involved in skeletal muscle wasting as seen in sepsis. OBJECTIVE Our objective was to determine the role of IL-6 in human in vivo systemic and skeletal muscle amino acid metabolism and protein turnover. SUBJECTS AND METHODS There were 12 healthy men infused for 3 h with saline (saline, n = 6) or recombinant human IL (rhIL)-6 (n = 6). Systemic and muscle protein turnover was determined with a combination of tracer dilution methodology, primed constant infusion of L-[ring-(2)H(5)]phenylalanine, and femoral arterial-venous blood differences and m. vastus lateralis biopsies after 2-h basal, 3-h infusion, and 3 h after infusion. RESULTS The IL-6 concentration after 30-min infusion was approximately 4 (saline) and 140 pg/ml (rhIL-6). Three-hour rhIL-6 infusion caused an approximate 50% decrease in muscle protein turnover, albeit synthesis was more suppressed than breakdown, causing a small increase in net muscle protein breakdown. Furthermore, rhIL-6 decreased arterial amino acid concentration with 20-40%, despite the increase net release from muscle. CONCLUSIONS We demonstrated that IL-6 profoundly alters amino acid turnover. A substantial decrease in plasma amino acids was observed with a concomitant 50% decrease in muscle protein turnover, however, modest increase in net muscle degradation. We hypothesize that the profound reduction in muscle protein turnover and modest increase in net degradation are primarily caused by the reduced plasma amino acid availability and not directly mediated by IL-6.

Regulatory Coordination between Two Major Intracellular Homeostatic Systems: HEAT SHOCK RESPONSE AND AUTOPHAGY*

Background: Both autophagy and the heat stress response represent protein management alternatives for the stressed cell. Their inter-relationship is not known. Results: Heat shock factor-1 knockdown increases and HSP70 overexpression inhibits autophagy in cell culture model. Preactivation of heat shock inhibits exercise-induced autophagy in humans. Conclusion: Heat shock response plays a negative role in autophagy regulation. Significance: Heat shock response regulates autophagy. The eukaryotic cell depends on multitiered homeostatic systems ensuring maintenance of proteostasis, organellar integrity, function and turnover, and overall cellular viability. At the two opposite ends of the homeostatic system spectrum are heat shock response and autophagy. Here, we tested whether there are interactions between these homeostatic systems, one universally operational in all prokaryotic and eukaryotic cells, and the other one (autophagy) is limited to eukaryotes. We found that heat shock response regulates autophagy. The interaction between the two systems was demonstrated by testing the role of HSF-1, the central regulator of heat shock gene expression. Knockdown of HSF-1 increased the LC3 lipidation associated with formation of autophagosomal organelles, whereas depletion of HSF-1 potentiated both starvation- and rapamycin-induced autophagy. HSP70 expression but not expression of its ATPase mutant inhibited starvation or rapamycin-induced autophagy. We also show that exercise induces autophagy in humans. As predicted by our in vitro studies, glutamine supplementation as a conditioning stimulus prior to exercise significantly increased HSP70 protein expression and prevented the expected exercise induction of autophagy. Our data demonstrate for the first time that heat shock response, from the top of its regulatory cascade (HSF-1) down to the execution stages delivered by HSP70, controls autophagy thus connecting and coordinating the two extreme ends of the homeostatic systems in the eukaryotic cell.

Thermotolerance and heat acclimation may share a common mechanism in humans

Thermotolerance and heat acclimation are key adaptation processes that have been hitherto viewed as separate phenomena. Here, we provide evidence that these processes may share a common basis, as both may potentially be governed by the heat shock response. We evaluated the effects of a heat shock response-inhibitor (quercetin; 2,000 mg/day) on established markers of thermotolerance [gastrointestinal barrier permeability, plasma TNF-α, IL-6, and IL-10 concentrations, and leukocyte heat shock protein 70 (HSP70) content]. Heat acclimation reduced body temperatures, heart rate, and physiological strain during exercise/heat stress) in male subjects (n = 8) completing a 7-day heat acclimation protocol. These same subjects completed an identical protocol under placebo supplementation (placebo). Gastrointestinal barrier permeability and TNF-α were increased on the 1st day of exercise/heat stress in quercetin; no differences in these variables were reported in placebo. Exercise HSP70 responses were increased, and plasma cytokines (IL-6, IL-10) were decreased on the 7th day of heat acclimation in placebo; with concomitant reductions in exercise body temperatures, heart rate, and physiological strain. In contrast, gastrointestinal barrier permeability remained elevated, HSP70 was not increased, and IL-6, IL-10, and exercise body temperatures were not reduced on the 7th day of heat acclimation in quercetin. While exercise heart rate and physiological strain were reduced in quercetin, this occurred later in exercise than with placebo. Consistent with the concept that thermotolerance and heat acclimation are related through the heat shock response, repeated exercise/heat stress increases cytoprotective HSP70 and reduces circulating cytokines, contributing to reductions in cellular and systemic markers of heat strain. Exercising under a heat shock response-inhibitor prevents both cellular and systemic heat adaptations.

Interleukin-6 modulates hepatic and muscle protein synthesis during hemodialysis

Increased demand for amino acids to sustain acute-phase protein synthesis could be the stimulus for the increased muscle protein catabolism during hemodialysis (HD). This could be attenuated by intradialytic amino-acid infusion. To test this, we measured the fractional synthesis rates of albumin, fibrinogen, and muscle protein in eight patients with end-stage renal disease at baseline before dialysis and during HD without or with amino-acid infusion. The percentage change in the fractional synthesis rates of albumin, fibrinogen, and muscle protein from baseline was significantly higher during HD with amino-acid infusion than without amino-acid infusion. Leg muscle proteolysis was significantly increased during unsupplemented HD compared with baseline, but this was not decreased by amino-acid infusion. Arteriovenous balance studies across the leg showed a net efflux of interleukin-6 (IL-6) from the muscle into the vein during HD. The fractional synthesis rate of albumin, fibrinogen, and muscle protein correlated with each other and with the IL-6 efflux from the leg. Leg muscle protein catabolism was positively related to IL-6 release from the leg and not associated with amino-acid availability. Our results show that intradialytic cytokine activation and not amino-acid depletion is the major protein catabolic signal during HD.