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This study characterizes the prevalence of reverse-transcriptase polymerase chain reaction results positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among patients presenting with influenzalike illness who underwent nasopharyngeal swab testing for influenza and respiratory syncytial virus over 4 days in March 2020.

Hepatic stellate cells (HSCs) are well-characterized non-parenchymal cells of the liver with established roles in fibrosis, repair and immunity (1). During liver injury, quiescent HSCs undergo activation, secreting a repertoire of molecules involved in cell proliferation, chemotaxis, inflammation and fibrosis, among others. Although their role in fibrogenesis is well established, the contributions of HSCs to acute hepatocellular damage and tissue homeostasis are not well understood. Models to manipulate HSC function or number offer an appealing strategy to clarify this issue. However, only two models have been established to deplete HSCs in vivo thus far, by using gliotoxin (2) or gliotoxin-coupled antibodies against synaptophysin (3, 4). Gliotoxin reportedly induces selective apoptosis of HSCs by subverting NF-κB-mediated survival (2), and can reduce fibrosis and enhance resolution in experimental models, especially when targeted using HSC-specific antibodies (3–5). However, gliotoxin also has broad actions in vivo and in culture, targeting not only HSCs but also immune and endothelial cells and hepatocytes (5, 6). An alternative strategy is to ectopically express the herpes simplex virus-thymidine kinase (HSV-Tk) gene in target cells, which renders them susceptible to killing by the antiviral agent ganciclovir (GCV), but only when the cells are proliferating. This possibility was first reported as an anti-cancer approach (7) and further refined (8) in murine sarcoma and lymphoma cells, provoking both apoptotic and non-apoptotic cell death (9, 10). The approach has also been reported in liver injury models and in cultured HSCs (11), but has not been used to deplete HSCs in vivo (12). We have exploited this strategy by using mice expressing the HSV-Tk gene driven by the GFAP promoter, which is a marker of HSCs in rodent liver (1). The approach has uncovered a novel and unexpected role for HSCs in amplifying acute liver injury.

There is scarce information about the link between specific single-nucleotide polymorphisms (SNPs) and risk of liver disease among Latinos, despite the disproportionate burden of disease among this population. Our aim was to investigate nine SNPs in or near the following genes: PNPLA3, LYPLAL1, PPP1R3B, GCKR, NCAN, IRS1, PPARG, and ADIPOR2 and examine their association with persistently elevated alanine aminotransferase (ALT) or aspartate aminotransferase (AST) levels in Mexican adults. Data and samples were collected from 741 participants in the Mexican Health Worker Cohort Study, in Cuernavaca, Mexico. We identified 207 cases who had persistently elevated levels of ALT or AST (≥40 U/L) and 534 controls with at least two consecutive normal ALT or AST results in a 6 month period, during 2004-2006 and 2011-2013. TaqMan assays were used to genotype the SNPs. The risk allele of PNPLA3 rs738409 was found to be associated with persistently elevated levels of ALT or AST, adjusting for age, sex, BMI, type 2 diabetes, and ancestry: (OR 2.28, 95% CI 1.13, 4.58). A significant association was found between the LYPLAL1, PPP1R3B, and GCKR risk alleles and elevated ALT or AST levels among overweight/obese adults. These results suggest that among Mexicans, the PNPLA3 (rs738409), LYPLAL1 (rs12137855), PPP1R3B (rs4240624), and GCKR (rs780094) polymorphisms may be associated with a greater risk of chronic liver disease among overweight adults. This study is the first to examine these nine SNPs in a sample of adults in Mexico.

Inflammatory bowel disease (IBD) patients display elevated levels of intraluminal nitric oxide (NO). NO can react with other molecules to form toxic compounds, which has led to the idea that NO may be an important mediator of IBD. However, the cellular source of NO and how its production is regulated in the intestine are unclear. In this study we aimed to determine if intestinal myofibroblasts produce NO in response to the IBD-associated cytokines IL-1β, TNFα, and IFNγ. Intestinal myofibroblasts were isolated from mice and found to express inducible nitric oxide synthase (iNOS) mRNA, but not endothelial NOS or neuronal NOS. Individual treatment of myofibroblasts with IL-1β, TNFα, or IFNγ had no effect on NO production, but stimulation with combinations of these cytokines synergistically increased iNOS mRNA and protein expression. Treatment with TNFα or IFNγ increased cell surface expression of IFNγRI or TNFRII, respectively, suggesting that these cytokines act in concert to prime NO production by myofibroblasts. Impairment of NF-κB activity with a small molecule inhibitor was sufficient to prevent increased expression of IFNγRI or TNFRII, and inhibition of Akt, JAK/STAT, or NF-κB blocked nearly all NO production induced by combinatorial cytokine treatment. These data indicate that intestinal myofibroblasts require stimulation by multiple cytokines to produce NO and that these cytokines act through a novel pathway involving reciprocal cytokine receptor regulation and signaling by Akt, JAK/STAT, and NF-κB.

The contraction of hepatic stellate cells has been proposed to mediate fibrosis by regulating sinusoidal blood flow and extracellular matrix remodeling. Abundant data from diverse, yet complementary, experimental methods support a robust model for the regulation of contractile force generation by stellate cells. In this model, soluble factors associated with liver injury, including endothelin 1 and nitric oxide, are transduced primarily through Rho signaling pathways that promote the myosin II-powered generation of contractile force by stellate cells. The enhanced knowledge of the role and differential regulation of stellate cell contraction may facilitate the discovery of new and targeted strategies for the prevention and treatment of hepatic fibrosis.

Chemotaxis (i.e., directed migration) of hepatic stellate cells to areas of inflammation is a requisite event in the liver's response to injury. Previous studies of signaling pathways that regulate stellate cell migration suggest a key role for focal adhesions, but the exact function of these protein complexes in motility remains unclear. Focal adhesions attach a cell to its substrate and therefore must be regulated in a highly coordinated manner during migration. To test the hypothesis that focal adhesion turnover is an essential early event for chemotaxis in stellate cells, we employed a live-cell imaging technique in which chemotaxis was induced by locally stimulating the tips of rat stellate cell protrusions with platelet-derived growth factor-BB (PDGF). Focal adhesions were visualized with an antibody directed against vinculin, a structural component of the focal adhesion complex. PDGF triggered rapid disassembly of adhesions within 6.25 min, subsequent reassembly by 12.5 min, and continued adhesion assembly in concert with the spreading protrusion until the completion of chemotaxis. Blockade of adhesion disassembly by growing cells on fibronectin or treatment with nocodazole prevented a chemotactic response to PDGF. Augmentation of adhesion disassembly with ML-7 enhanced the chemotactic response to PDGF. These data suggest that focal adhesion disassembly is an essential early event in stellate cell chemotaxis in response to PDGF.

Growth cones are highly polarized and dynamic structures confined to the tips of axons. The polarity of growth cones is in part maintained by suppression of protrusive activity from the distal axon shaft, a process termed axon consolidation. The mechanistic basis of axon consolidation that contributes to the maintenance of growth cone polarity is not clear. We report that inhibition of RhoA-kinase (ROCK) or myosin II resulted in unstable consolidation of the distal axon as evidenced by increased filopodial and lamellipodial extension. Furthermore, when ROCK or myosin II was inhibited lamellipodia formed at the growth cone migrated onto the axon shaft. Analysis of EYFP-actin dynamics in the distal axon revealed that ROCK negatively regulates actin polymerization and initiation of protrusive structures from spontaneously formed axonal F-actin patches, the latter being an effect attributable to ROCK-mediated regulation of myosin II. Inhibition of ROCK or myosin II blocked growth cone turning toward NGF by preventing suppression of protrusive activity away from the source of NGF, resulting in aborted turning responses. These data elucidate the mechanism of growth cone polarity, provide evidence that consolidation of the distal axon is a component of guidance, and identify ROCK as a negative regulator of F-actin polymerization underlying protrusive activity in the distal axon.

The contractile force generated by hepatic stellate cells in response to endothelin-1 contributes to sinusoidal blood flow regulation and hepatic fibrosis. This study's aim was to directly test the widely held view that changes in cytosolic Ca2+ concentration ([Ca2+]i) mediate stellate cell force generation. Contractile force generation by primary cultures of rat hepatic stellate cells grown in three-dimensional collagen gels was directly and quantitatively measured using a force transducer. Stellate cell [Ca2+]i, myosin activation, and migration were quantified using standard techniques. [Ca2+]i was modulated using ionomycin, BAPTA, KCl, and removal of extracellular Ca2+. Removal of extracellular Ca2+ did not alter endothelin-1-stimulated force development or [Ca2+]i. Ionomycin, a Ca2+ ionophore, triggered an increase in [Ca2+]i that was three times greater than that stimulated by endothelin-1, but only induced 16% of the force and 38% of the myosin regulatory light chain (MLC) phosphorylation induced by endothelin-1. Physiological increases in [Ca2+]i induced by hyperkalemia had no effect on contractile force. Loading BAPTA, a Ca2+ chelator, in stellate cells completely blocked endothelin-1-induced increases in [Ca2+]i but had no effect on endothelin-1-stimulated force generation or MLC phosphorylation. In contrast, Y-27632, a selective rho-associated kinase inhibitor, inhibited endothelin-1-stimulated force generation by at least 70% and MLC phosphorylation by at least 80%. Taken together, these observations indicate that changes in [Ca2+]i are neither necessary nor sufficient for contractile force generation by rat stellate cells. Our results challenge the current model of contractile regulation in hepatic stellate cells and have important implications for our understanding of hepatic pathophysiology.

Intestinal response to injury requires coordinated regulation of the tension exerted by subepithelial myofibroblasts (SEM). However, the signals governing relaxation of intestinal SEM have not been investigated. Our aim was to test the hypothesis that signal transduction pathways initiated by C-type natriuretic peptide (CNP) induce intestinal SEM relaxation. We directly quantified the effects of CNP on isometric tension exerted by cultured human colonic SEM. We also measured the effects of CNP on cGMP content, myosin regulatory light chain (MLC) phosphorylation, and cytosolic Ca2+concentration. CNP induced relaxation of SEM within 10 s. By 10 min, relaxation reached a plateau that was sustained for 2 h. CNP-induced relaxation was saturable, with a maximal decrease in tension (51.7 ± 3.8 dyn) observed at 250 nM. SEM relaxation in response to CNP constituted ∼23% of total basal tension. CNP increased intracellular cGMP content and reduced MLC phosphorylation. Effects of CNP on cGMP and MLC exhibited the same dose dependence as CNP-induced relaxation. MLC phosphorylation decreased within 2 min of CNP exposure and was sustained for at least 45 min. CNP also stimulated a large transient increase in cytosolic Ca2+concentration that occurred within 30 s and was nearly complete by 1 min. We also observed that calyculin-A, a potent inhibitor of MLC phosphatase, completely abolished the reduction in MLC phosphorylation induced by CNP. These results suggest that CNP induces intestinal SEM relaxation through cGMP-associated reductions in MLC phosphorylation. Moreover, these findings raise the possibility that CNP plays a role in intestinal wound healing.

Although hepatic myofibroblast (HMF) migration contributes to the development of fibrosis, the mechanisms coordinating this movement are uncertain. We determined the effects of lysophosphatidic acid (LPA) and platelet-derived growth factor-BB (PDGF) on actin polymerization, FAK tyrosine phosphorylation, and migration of cultured human HMFs. LPA (0.4-100 microM) stimulated migration, FAK tyrosine phosphorylation, and stress fiber assembly with a sigmoidal dose response. PDGF (1-250 ng/ml) stimulated migration, FAK tyrosine phosphorylation, and actin polymerization with a bell-shape dose-response characterized by a maximum at 10-25 ng/ml. Concentrations of cytochalasin D, which abolished FAK tyrosine phosphorylation, also blocked LPA- and PDGF-induced migration. A dose of 1-10 ng/ml PDGF acted synergistically with LPA (10 microM) to stimulate FAK tyrosine phosphorylation and migration, whereas higher concentrations of PDGF (100-250 ng/ml) inhibited FAK tyrosine phosphorylation and migration in response to LPA (10 microM). These data indicate that PDGF and LPA coordinately govern the migration of HMFs by differentially regulating FAK and suggest a novel model in which PDGF, acting as an amplifier/attenuator of LPA-induced signaling, facilitates HMF accumulation within injured areas of the liver.

A decision-analysis model was developed to compare the strategies to maintain hepatitis B virus (HBV) immunity in hemodialysis patients who responded to the primary HBV vaccine. Our hypothesis is that the routine, annual administration of the vaccine booster to all hemodialysis patients (non-screening strategy) is more cost-effective than the current strategy of vaccination based on anti-HBs titers (screening strategy). Under baseline assumptions, the screening strategy was less costly and was associated with fewer HBV infections than the non-screening strategy. The results of our model did not support our hypothesis, and indicate that regularly screening patients for HBV immunity before revaccination is less costly and more effective than the empiric vaccination of hemodialysis patients.

Although the migration of hepatic myofibroblasts (HMFs) contributes to the development of fibrosis, the signals regulating migration of these cells are poorly understood. In this study, we tested the hypothesis that HMF migration is stimulated by platelet-derived growth factor-BB (PDGF-BB) through p38 mitogen-activated protein (MAP) kinase and extracellular signal-regulated kinase (ERK) signaling pathways. This hypothesis was addressed by directly visualizing the migration of cultured human HMFs into a wound. PDGF-BB stimulated membrane ruffling, migration, and proliferation. PDGF-BB also induced activation of p38 MAP kinase, its downstream effector, heat shock protein (HSP) 27, ERK 1 and ERK 2, and p125 focal adhesion kinase (FAK). Selective antagonism of p38 MAP kinase blocked PDGF-BB-stimulated HSP 27 phosphorylation, membrane ruffling, and migration, but did not alter PDGF-BB-induced proliferation. Selective antagonism of ERK kinase inhibited PDGF-BB-induced ERK phosphorylation and proliferation, but did not affect PDGF-BB-stimulated migration. Concentrations of PDGF-BB that stimulated migration and proliferation did not influence myosin-dependent contractility. Neither selective inhibition of p38 MAP kinase nor ERKs altered PDGF-BB-induced activation of FAK. In conclusion, these results provide novel evidence indicating that (1) HMF migration is stimulated by PDGF-BB through the regulation of membrane ruffling by a p38 MAP kinase signaling pathway, (2) whereas p38 MAP kinase mediates PDGF-BB-stimulated migration, but not proliferation, ERKs mediate PDGF-induced proliferation, but not migration, and (3) increases in myosin-dependent contractility are not required for PDGF-BB-stimulated migration.

The role of G protein-coupled receptors and their ligands in intestinal epithelial cell signaling and proliferation is poorly understood. Here, we demonstrate that arginine vasopressin (AVP) induces multiple intracellular signal transduction pathways in rat intestinal epithelial IEC-18 cells via a V1Areceptor. Addition of AVP to these cells induces a rapid and transient increase in cytosolic Ca2+concentration and promotes protein kinase D (PKD) activation through a protein kinase C (PKC)-dependent pathway, as revealed by in vitro kinase assays and immunoblotting with an antibody that recognizes autophosphorylated PKD at Ser916. AVP also stimulates the tyrosine phosphorylation of the nonreceptor tyrosine kinase proline-rich tyrosine kinase 2 (Pyk2) and promotes Src family kinase phosphorylation at Tyr418, indicative of Src activation. AVP induces extracellular signal-related kinase (ERK)-1 (p44mapk) and ERK-2 (p42mapk) activation, a response prevented by treatment with mitogen-activated protein kinase kinase (MEK) inhibitors (PD-98059 and U-0126), specific PKC inhibitors (GF-I and Ro-31-8220), depletion of Ca2+(EGTA and thapsigargin), selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (tyrphostin AG-1478, compound 56), or the selective Src family kinase inhibitor PP-2. Furthermore, AVP acts as a potent growth factor for IEC-18 cells, inducing DNA synthesis and cell proliferation through ERK-, Ca2+-, PKC-, EGFR tyrosine kinase-, and Src-dependent pathways.

Background: Although the contractile, migratory, and proliferative responses of subepithelial myofibroblasts to injury have been postulated to be important events in intestinal wound healing, contractile force generation and migration by these cells has not been investigated previously, and the signals that regulate proliferation by these cells are poorly understood. Aims: The primary aim of this study was to test the hypothesis that the inflammatory mediator endothelin-1 modulates contraction, migration, and proliferation of intestinal myofibroblasts. We also sought to examine the signal transduction pathways which might underlie these putative effects. Methods: Contraction, migration, proliferation, cytosolic [Ca 2+ ], and myosin phosphorylation were measured in human colonic subepithelial myofibroblasts in the absence and presence of endothelin receptor agonists and antagonists. Results: Endothelin-1, but not interleukin 1α, interleukin 6, interleukin 8, interleukin 10, or tumour necrosis factor α, induced a rapid and robust generation of contractile force, which was associated with an increase in cytosolic [Ca 2+ ] and myosin phosphorylation. Inhibition of rho associated kinase reduced endothelin-1 stimulated myosin phosphorylation and contractile force development. Endothelin-1 stimulated migration with a dose-response relationship similar to that observed for contraction. Endothelin A and B receptors mediated contraction while migration was mediated predominantly through endothelin B receptors. Platelet derived growth factor and serum, but not endothelin-1, induced proliferation. Conclusions: Endothelin-1 stimulates colonic subepithelial myofibroblast contraction and migration via endothelin receptor mediated myosin phosphorylation. These results support an important role for subepithelial myofibroblasts in the injury response of the gut and consequently intestinal wound repair.

Although migration of stellate cells during hepatic injury is essential for wound-healing and fibrosis of the liver, the extracellular and intracellular signals that regulate stellate cell migration are incompletely understood. In this study we tested the hypothesis that wound-induced migration of stellate cells is modulated by endothelin-1 (ET-1) through rho-kinase-mediated alterations in the acto-myosin cytoskeleton. To address this hypothesis, a method was established for direct visualization of wound-induced migration of culture-activated stellate cells with subcellular resolution. Migration in response to wounding was characterized by (1) plasma membrane ruffling and protrusion into the wound, (2) lamellipodia formation at the leading edge, (3) focal adhesion and stress fiber assembly, and (4) myosin reorganization. Exogenous ET-1 accelerated wound-induced migration of stellate cells, but did not alter wound-induced proliferation. Experiments using ET-1 antagonists in the absence of exogenous ET-1 showed that wound-induced migration was also stimulated by endogenous ET-1. Selective inhibition of rho-associated kinase decelerated migration in response to wounding. Moreover, inhibition of rho-associated kinase was distinguished by abrogation of focal adhesion formation, stress fiber assembly, and myosin reorganization. This study shows that rho-kinase-dependent alterations in the acto-myosin cytoskeleton contribute to wound-induced stellate cell migration, which is accelerated by both exogenous and endogenous ET-1. Consequently, these results provide important new evidence suggesting that, migration of stellate cells is modulated by paracrine and autocrine ET-1 stimulation via the action of rho-kinase on the acto-myosin cytoskeleton.

The present study examined the roles of peroxisome proliferator-activated receptors (PPAR) in activation of hepatic stellate cells (HSC), a pivotal event in liver fibrogenesis. RNase protection assay detected mRNA for PPARgamma1 but not that for the adipocyte-specific gamma2 isoform in HSC isolated from sham-operated rats, whereas the transcripts for neither isoforms were detectable in HSC from cholestatic liver fibrosis induced by bile duct ligation (BDL). Semi-quantitative reverse transcriptase-polymerase chain reaction confirmed a 70% reduction in PPARgamma mRNA level in HSC from BDL. Nuclear extracts from BDL cells showed an expected diminution of binding to PPAR-responsive element, whereas NF-kappaB and AP-1 binding were increased. Treatment of cultured-activated HSC with ligands for PPARgamma (10 microm 15-deoxy-Delta(12,14)-PGJ(2) (15dPGJ(2)); 0.1 approximately 10 microm BRL49653) inhibited DNA and collagen synthesis without affecting the cell viability. Suppression of HSC collagen by 15dPGJ(2) was abrogated 70% by the concomitant treatment with a PPARgamma antagonist (GW9662). HSC DNA and collagen synthesis were inhibited by WY14643 at the concentrations known to activate both PPARalpha and gamma (>100 microm) but not at those that only activate PPARalpha (

The experiments presented here were designed to examine the contribution of the extracellular signal-regulated mitogen-activated protein kinases (ERKs) to the tyrosine phosphorylation of the focal adhesion proteins p125Fak, p130Cas , and paxillin induced by G protein-coupled receptors (GPCRs) and tyrosine kinase receptors in Swiss 3T3 cells. Stimulation of these cells with bombesin, lysophosphatidic acid (LPA), endothelin, and platelet-derived growth factor (PDGF) led to a marked increase in the tyrosine phosphorylation of these focal adhesion proteins and in ERK activation. Exposure of the cells to two structurally unrelated mitogen-activated protein kinase or ERK kinase (MEK) inhibitors, PD98059 and U0126, completely abrogated ERK activation but did not prevent tyrosine phosphorylation of p125Fak, p130Cas, and paxillin. Furthermore, different dose-response relationships were obtained for tyrosine phosphorylation of focal adhesion proteins and for ERK activation in response to PDGF. Putative upstream events in the activation of focal adhesion proteins including actin cytoskeletal reorganization and myosin light chain (MLC) phosphorylation were also not prevented by inhibition of ERK activation. Thus, our results demonstrate that the activation of the ERK pathway is not necessary for the increase of the tyrosine phosphorylation of p125Fak, p130Cas, and paxillin induced by either GPCRs or tyrosine kinase receptors in Swiss 3T3 cells. J. Cell. Physiol. 183:208–220, 2000. © 2000 Wiley-Liss, Inc.

Contraction is an essential component of many fundamental processes in non-muscle cells. Fibroblast contraction mediates orientation of collagen fibres in connective tissue (Harris et al. 1981) and contraction of granulation tissue (Majno et al. 1971). More complex cellular behaviours such as chemotaxis and cytokinesis are mediated by localized contractions within cells (Mabuchi & Okuno, 1977; Jay et al. 1995; Post et al. 1995). Among the myosin superfamily of motor proteins, only myosin II forms bipolar filaments, which can contract cytoplasm in a manner analogous to muscle. Non-muscle myosin II, like smooth muscle myosin II, is activated through phosphorylation of LC20, primarily at Ser19 and to a lesser extent at Thr18 (Sellers & Adelstein, 1987). In smooth muscle, LC20 is phosphorylated predominantly by Ca2+-calmodulin-activated myosin light chain kinase (MLCK) (Somlyo & Somlyo, 1994). However, it has remained unclear if this smooth muscle paradigm of [Ca2+]i-regulated myosin II based contractility also applies to non-muscle cells. Several lines of evidence support the widely held view that non-muscle cell contraction is regulated similarly to the better characterized process of smooth muscle contraction (i.e. through Ca2+-calmodulin stimulated LC20 phosphorylation). First, MLCK activity endogenous to both fibroblasts and endothelial cells is necessary and sufficient to mediate Ca2+-dependent LC20 phosphorylation and contraction in detergent-permeabilized cell models (Cande & Ezzell, 1986; Wysolmerski & Lagunoff, 1991). Second, MLCK inhibitors decrease LC20 phosphorylation and contraction in non-muscle cells (Lamb et al. 1988; Giuliano et al. 1992; Chrzanowska-Wodnicka & Burridge, 1996). Finally, in some cell types, Ca2+ ionophores stimulate structural alterations resembling cell contraction (Winter et al. 1991; Garcia et al. 1997). However, these studies should be interpreted cautiously for several reasons: (1) permeabilization of cells results in the loss of endogenous soluble molecules (Wysolmerski & Lagunoff, 1991) that may have important roles in the regulation of contraction, (2) protein kinase inhibitors thought to have specificity for MLCK may, in fact, exert their effects through inhibition of other kinases, and (3) cell shape reflects a balance between cytoplasmic contraction and resisting forces from cell adhesion and cytoplasmic stiffness (Chicurel et al. 1998). Therefore the morphological alterations caused by Ca2+ ionophores do not necessarily reflect contraction or [Ca2+]i-stimulated activation of myosin. To investigate whether the smooth muscle paradigm for the regulation of contraction also applies to non-muscle cells, we used a model system in which chicken embryo fibroblasts (CEF) contract in response to fetal bovine serum (FBS) stimulation. FBS, like most agonists for non-muscle or smooth muscle contraction, stimulates (1) a rise in [Ca2+]i (McNeil et al. 1985), (2) phosphorylation of LC20 (Giuliano et al. 1992; Kolodney & Elson, 1993), and (3) generation of contractile force (Kolodney & Elson, 1993). We quantified fibroblast isometric force generation by attaching a population of cells, cultured within a collagen matrix, to an isometric force transducer (Kolodney & Elson, 1995), thereby allowing direct measurement of cellular force generation. We examined the putative role of [Ca2+]i as the primary regulator of contractility in CEF by either buffering [Ca2+]i or selectively permeabilizing cells to extracellular Ca2+ and measuring the effects of these interventions on LC20 phosphorylation and force generation.

Hepatic stellate cell activation, thought to play a key role in fibrosis of the liver, is characterized by changes in cellular morphology. The intracellular signals regulating morphological alterations associated with stellate cell activation are uncertain. The ras-like guanosine triphosphate-binding protein, rho, has recently emerged as an important regulator of the actin cytoskeleton, and consequently cell morphology. The aim of this study was to test the hypothesis that rho signaling pathways direct activation-associated morphological changes in stellate cells by regulating the actin cytoskeleton. The morphology and actin cytoskeleton of primary rat hepatic stellate cells were studied with phase contrast, differential interference contrast, and epifluorescence microscopy. Immunohistochemistry and immunoblot analysis were used to examine rho expression and activity, respectively. Quiescent and activated stellate cells were investigated in the absence and presence of C3 transferase, a bacterial toxin that specifically inhibits rho. Stellate cell activation was characterized by the development of prominent intracellular fibers, and the loss of dendrite-like processes and perinuclear retinoid droplets. Moreover, activation was accompanied by the formation of prominent actin stress fibers and focal adhesions. Both rho expression and activity were demonstrated in stellate cells. C3 transferase blocked and reversed, both activation-associated morphological alterations and activation-associated changes in the actin cytoskeleton, in quiescent and activated stellate cells, respectively. These results indicate that rho directs activation-associated changes in rat hepatic stellate cell morphology via regulation of the actin cytoskeleton.

Contraction of intestinal myofibroblasts (IMF) contributes to the development of strictures and fistulas seen in inflammatory bowel disease, but the mechanisms that regulate tension within these cells are poorly understood. In this study we investigated the role of nitric oxide (NO) signaling in C-type natriuretic peptide (CNP)-induced relaxation of IMF. We found that treatment with ODQ, a soluble guanylyl cyclase (sGC) inhibitor, or NG-nitro-l-arginine (l-NNA) or NG-monomethyl-l-arginine (l-NMMA), inhibitors of NO production, all impaired the relaxation of human and mouse IMF in response to CNP. ODQ, l-NNA, and l-NMMA also prevented CNP-induced elevations in cGMP concentrations, and l-NNA or l-NMMA blocked CNP-induced decreases in myosin light phosphorylation. IMF isolated from transgenic mice deficient in inducible nitric oxide synthase (iNOS) had reduced relaxation responses to CNP compared with IMF from control mice and were insensitive to the effects of ODQ, l-NNA, and l-NMMA on CNP treatment. Together these data indicate that stimulation of sGC though NO produced by iNOS activation is required for maximal CNP-induced relaxation in IMF.

The study of individuals who are able to achieve durable control over human immunodeficiency virus (HIV) replication in the absence of antiretroviral therapy has become increasingly important in light of recent vaccine trials which have failed to induce protective immunity. Understanding the correlates of protection in these HIV “controllers” would aid in both the rational design of potential vaccine candidates and the testing of their efficacy. Cell-mediated immune responses, in particular HIV-specific CD8+ T-cell responses, have been shown to be critical in decreasing the initial viremia after acute infection and determining the chronic infection set point (32, 33, 43). These responses have proven to be “too little and too late” to prevent the establishment of chronic infection; however, studies of HIV-specific CD8+ T-cell responses in controllers suggest that strong, polyfunctional responses may be important in long-term virologic control (1, 6, 11, 15, 16, 44). In addition, the association of controller status with class I HLA alleles, predominantly HLA-B27 and HLA-B57, has been well documented in multiple cohorts (2, 9, 13, 39). The maintenance of robust HIV-specific CD4+ T-cell responses may also favor long-term control of HIV replication in untreated persons. One of the primary roles of CD4+ T cells is to provide “help” to CD8+ T cells. Many studies have shown that proper functioning of CD4+ T cells is necessary for high-quality antigen-specific CD8+ T-cell responses (3, 28, 29, 38, 58). HIV-specific CD4+ T cells in the peripheral blood of long-term nonprogressors (LTNP) have been shown to be polyfunctional, producing both gamma interferon (IFN-γ) and interleukin-2 (IL-2), whereas those from progressors tend to be monofunctional, secreting only IFN-γ (7, 14, 21, 22, 44). Additionally, the ability of CD4+ T cells to proliferate appears to be preserved in controllers (46, 56). The maturation status of CD4+ T cells that function in the context of HIV infection may also be important, as individuals who are able to preserve central memory T cells and sustain an activated effector memory CD4+ T-cell population are better able to suppress viral replication (45). Much less is known about the association of class II HLA alleles and controller status; however, a few studies have cited a relationship between HLA-DRB1*13 and/or HLA-DQB1*06 and HIV control (10, 31, 36, 55). Previously, we have shown that CD8+ T cells from the rectal mucosa of controllers with protective class I alleles (HLA-B13, -B27, -B57, -B58, and -B81) are highly polyfunctional compared to CD8+ T cells from either controllers or noncontrollers (NC) lacking these alleles (16). Additionally, we found that mucosal CD8+ T-cell responses from individuals who had protective class I alleles in combination with the class II alleles HLA-DRB1*13 and/or HLA-DQB1*06 were of greater magnitude than mucosal CD8+ T-cell responses from those who had protective class I alleles alone (16). Therefore, we wanted to specifically examine HIV-specific mucosal CD4+ T-cell responses among controllers with and without these potentially protective class II HLA alleles. Our hypothesis was that controllers, particularly those who possessed HLA-DRB1*13 and/or HLA-DQB1*06, would have more robust and polyfunctional HIV-specific CD4+ T-cell responses than noncontrollers or subjects on highly active antiretroviral therapy (HAART) and that these responses would correlate with strong CD8+ T-cell responses in the same individuals. We found that, indeed, controllers generally had stronger HIV-specific CD4+ T-cell responses than other groups in rectal mucosa and that, among controllers, those with the haplotype HLA-DRB1*13/HLA-DQB1*06 had particularly high percentages of polyfunctional HIV-specific CD4+ T cells in rectal mucosa. Furthermore, the proportion of polyfunctional mucosal CD4+ T cells directly correlated with the total magnitude of the mucosal CD8+ T-cell response. These data collectively suggest that the preservation or expansion of both HIV-specific CD4+ and CD8+ T cells may be an important mechanism whereby certain controllers maintain durable control of HIV replication.

Previous studies have suggested that polyfunctional mucosal CD8 + T-cell responses may be a correlate of protection in HIV controllers. Mucosal T-cell breadth and/or specificity may also contribute to defining protective responses. In this study, rectal CD8 + T-cell responses to HIV Gag, Env, and Nef were mapped at the peptide level in four subject groups: elite controllers ( n = 16; viral load [VL], n = 14; VL, 75 to 2,000 copies/ml), noncontrollers ( n = 14; VL, >10,000 copies/ml), and antiretroviral-drug-treated subjects ( n = 8; VL, + T-cell responses were generally shared by blood and mucosa, although there were exceptions. In HIV controllers, responses to HLA-B27- and HLA-B57-restricted epitopes were common to both tissues, and their magnitude (in spot-forming cells [SFC] per million) was significantly greater than those of responses restricted by other alleles. Furthermore, peptides recognized by T cells in both blood and rectal mucosa, termed “concordant,” elicited higher median numbers of SFC than discordant responses. In magnitude as well as breadth, HIV Gag-specific responses, particularly those targeting p24 and p7, dominated in controllers. Responses in noncontrollers were more evenly distributed among epitopes in Gag, Env, and Nef. Viremic controllers showed significantly broader mucosal Gag-specific responses than other groups. Taken together, these findings demonstrate that (i) Gag-specific responses dominate in mucosal tissues of HIV controllers; (ii) there is extensive overlap between CD8 + T cells in blood and mucosal tissues, with responses to immunodominant epitopes generally shared by both sites; and (iii) mucosal T-cell response breadth alone cannot account for immune control.

There exists a unique group of persons who are able to durably control HIV in the absence of therapy. The mechanisms of control in these persons remain poorly defined. In this study, we examined CD8+ T-cell responses in blood and rectal mucosa from 17 “elite controllers” (viral load < 75 copies/mL), 11 “viremic controllers” (75-2000 copies/mL), 14 noncontrollers (> 10 000 copies/mL), and 10 antiretroviral-treated persons (< 75 copies/mL). Production of interferon-γ, interleukin-2, tumor necrosis factor-α, macrophage inflammatory protein-1β, and CD107a by CD8+ T cells in response to HIV-1 Gag stimulation was measured using flow cytometry. Our hypothesis was that “polyfunctional” T cells producing multiple antiviral factors would be most abundant in mucosal tissues of HIV controllers. Mucosal CD8+ T-cell responses were significantly stronger and more complex in controllers than in antiretroviral-suppressed persons (P = .0004). The frequency of 4-function responses in rectal mucosa was higher in controllers than in noncontrollers and patients on therapy (P < .0001). Mucosal responses in controllers were frequently stronger and more complex than blood responses. These findings demonstrate that many controllers mount strong, complex HIV-specific T-cell responses in rectal mucosa. These responses may play an important role in mucosal immune surveillance, as suggested by their relative enrichment among persons who control HIV in the absence of therapy.

Hepatic stellate cells play an essential role in the liver's injury response. Although stellate cells are defined by the presence of cytoplasmic protrusions, the function of these characteristic structures has been obscure. We hypothesized that stellate cell protrusions act by coupling injury-associated stimuli to chemotaxis. To test this hypothesis, we developed an assay for directly visualizing the response of living stellate cells in early primary culture to local stimulation of the tips of protrusions with platelet-derived growth factor-BB (PDGF). Stellate cells exhibited elongate protrusions containing actin, myosin, and tubulin. PDGF, but not cytochrome C, localized at a protrusion tip induced a coordinated series of morphological events—cell spreading at the tip, movement of the cell body toward the PDGF, and retraction of trailing protrusions— that resulted in chemotaxis. Soluble PDGF and AG 1296, a receptor tyrosine kinase inhibitor, both reduced stellate cell chemotaxis. PDGF-induced chemotaxis was associated with an early and transient increase in myosin phosphorylation within the spreading lamella. We observed that blebbistatin, a myosin II inhibitor, completely and reversibly blocked protrusion-mediated lamella formation and chemotaxis. Moreover, blockade of MRLC phosphorylation with the myosin light chain kinase inhibitor, ML-7, or the rho kinase inhibitor, Y-27632, blocked lamella formation, myosin phosphorylation within the protrusion, and chemotaxis. Conclusion: These results support a model in which protrusions permit stellate cells to promptly detect PDGF distant from their cell bodies and transduce this signal into mechanical forces that propel the cell toward the site of injury. (HEPATOLOGY 2007.)

Although hepatic myofibroblast migration plays a key role in the liver's injury response, the signal transduction pathways mediating the migration of this cell type are uncertain. Recently, we reported that lysophosphatidic acid (LPA) stimulates the migration of hepatic myofibroblasts. The goal of this study was to test the hypothesis that rho and p38 MAP kinase signaling pathways mediate LPA-stimulated hepatic myofibroblast migration. We measured migration, myosin regulatory light chain and p38 MAP kinase phosphorylation, and contractile force generation by human hepatic myofibroblasts. LPA stimulated migration in a dose-dependent and saturable manner that was partially blocked by Y-27632, a rho-associated kinase inhibitor, as well as by SB-202190, a p38 MAP kinase inhibitor. LPA also induced myosin regulatory light chain phosphorylation and contractile force generation in a Y-27632 dependent, and SB-202190 independent fashion. Moreover, LPA stimulated a dose-dependent and saturable phosphorylation of p38 MAP kinase, which was not altered by Y-27632 or C3 transferase, a rho inactivator. These novel results suggest that LPA stimulates hepatic myofibroblast migration via distinct pathways that signal through rho and p38 MAP kinase.

Growth cone motility and guidance depend on the dynamic reorganization of filamentous actin (F-actin). In the growth cone, F-actin undergoes turnover, which is the exchange of actin subunits from existing filaments. However, the function of F-actin turnover is not clear. We used jasplakinolide (jasp), a cell-permeable macrocyclic peptide that inhibits F-actin turnover, to study the role of F-actin turnover in axon extension. Treatment with jasp caused axon retraction, demonstrating that axon extension requires F-actin turnover. The retraction of axons in response to the inhibition of F-actin turnover was dependent on myosin activity and regulated by RhoA and myosin light chain kinase. Significantly, the endogenous myosin-based contractility was sufficient to cause axon retraction, because jasp did not alter myosin activity. Based on these observations, we asked whether guidance cues that cause axon retraction (ephrin-A2) inhibit F-actin turnover. Axon retraction in response to ephrin-A2 correlated with decreased F-actin turnover and required RhoA activity. These observations demonstrate that axon extension depends on an interaction between endogenous myosin-driven contractility and F-actin turnover, and that guidance cues that cause axon retraction inhibit F-actin turnover.

Treatment of intact Swiss 3T3 cells with calyculin-A, an inhibitor of myosin light chain (MLC) phosphatase, induces tyrosine phosphorylation of p125(Fak) in a sharply concentration- and time-dependent manner. Maximal stimulation was 4.2 +/- 2.1-fold (n = 14). The stimulatory effect of calyculin-A was observed at low nanomolar concentrations (10 nM); at higher concentrations (10 nM) tyrosine phosphorylation of p125(Fak) was strikingly decreased. Calyculin-A induced tyrosine phosphorylation of p125(Fak) through a protein kinase C- and Ca(2+)-independent pathway. Exposure to either cytochalasin-D or latrunculin-A, which disrupt actin organization by different mechanisms, abolished tyrosine phosphorylation of p125(Fak) in response to calyculin-A. Treatment with high concentrations of platelet-derived growth factor (20 ng/ml) which also disrupt actin stress fibers, completely inhibited tyrosine phosphorylation of p125(Fak) in response to calyculin-A. This agent also induced tyrosine phosphorylation of the focal adhesion-associated proteins p130(Cas) and paxillin. These tyrosine phosphorylation events were associated with a striking increase in the assembly of focal adhesions. The Rho kinase (ROK) inhibitor HA1077 that blocked focal adhesion formation by bombesin, had no effect on the focal adhesion assembly induced by calyculin-A. Thus, calyculin-A induces transient focal adhesion assembly and tyrosine phosphorylation of p125(Fak), p130(Cas), and paxillin, acting downstream of ROK.

It is recommended that patients on hemodialysis (HD) therapy undergo regular screening for hepatitis C virus (HCV) infection by using alanine aminotransferase (ALT) values. However, the utility of using ALT values in this setting is unknown. The aim of this prospective study at the University of California Los Angeles Hepatitis Screening Program is to determine the sensitivity, specificity, and predictive values of an elevated ALT level for the diagnosis of HCV infection in HD patients. We screened 2,440 HD patients from 39 dialysis centers for viral infection by using hepatitis antibody serological testing and ALT values. We found the sensitivity and specificity of a newly elevated ALT level for acute HCV infection to be 83% and 90%, respectively. According to Bayes’ theorem, the positive predictive value was 4% and the positive likelihood ratio was 8.74. For chronic HCV infection, the sensitivity of a newly elevated aminotransferase level was 21%, and specificity was 91%. The positive predictive value was 16% (according to Bayes’ theorem), and the positive likelihood ratio was 2.47. The negative predictive value of a newly elevated aminotransferase value was 99% for acute HCV infection and 94% for chronic HCV infection. Our results indicate that although a newly elevated aminotransferase level is sensitive and specific for acute HCV infection, its positive predictive value is inadequate. A newly elevated aminotransferase level was neither sensitive nor positively predictive of chronic infection. Therefore, an elevated ALT level is an ineffective method for screening for HCV infection in HD patients.

Serial serum samples and pretreatment and post-treatment liver tissue from patients with chronic hepatitis C virus (HCV) infection were tested for HCV RNA by reverse-transcription polymerase chain reaction and branched DNA signal amplification assays. At the end of treatment with interferon-α (IFNα), 4 of 5 patients showing no biochemical response (in alanine aminotransferase activity), 4 of 5 with transient responses, and 1 of 5 showing complete and sustained responses had HCV RNA detectable in serum. The corresponding numbers for liver tissue were 5, 5, and 0 (of 4). However, all 5 complete responders had virological relapses wdthin 6 months. Biochemical response may not reflect virological profile during IFNα treatment of HCV.

Mammalian plasma membranes, including the myocardial sarcolemma, are abundantly glycosylated. Sialic acid is a ubiquitous anionic sugar found at the periphery of sarcolemmal glycoconjugates. The physiological role of this sugar is not clear, but neuraminidase, which specifically hydrolyzes sialic acid from the sarcolemma, has been found to increase calcium exchange, cause electrophysiological abnormalities, and enhance the transient (T) calcium current in cardiac myocytes. The purpose of this study was to better characterize the effect of neuraminidase on cellular calcium (Ca) and contractile function. Neuraminidase removed up to 57% of total sialic acid from the cells. 45Ca exchange was measured and neuraminidase was found to increase cell calcium proportional to the amount of sialic acid removed (18.6 +/- 0.8 mmol/kg dry w, maximally). Over 80% of the increment in calcium remained rapidly exchangeable (t1/2 less than 15 s) under non-perfusion limited conditions and was inhibited by cations (La greater than Cd greater than Mn greater than Mg) and nifedipine. Using a video-monitoring system, neuraminidase was observed to transiently increase cell shortening during contraction (30 +/- 9%), with progression to arrhythmias followed by cessation of contraction. These results indicate that neuraminidase, probably by removing sarcolemmal sialic acid residues, greatly augments cellular calcium in cultured cardiac myocytes. Most of the increment in Ca induced by neuraminidase was very rapidly exchangeable and most likely mediated by a Ca specific mechanism. Additionally, neuraminidase treatment altered contractile function in a manner consistent with elevated cellular Ca. Despite the many-fold increase in cellular Ca induced by sialic acid removal, cells recovered and demonstrated rhythmic contractions upon return to control incubation conditions.

Sialic acid, an anionic sugar moiety found peripherally on membrane glycoconjugates, is specifically hydrolyzed from the cell surface by neuraminidase. Because neuraminidase has previously been demonstrated to augment myocardial cell calcium content, the effects of neuraminidase on Ca channel function were studied on voltage-clamped guinea pig ventricular myocytes. In 25-50% of cells, neuraminidase treatment (0.12 U/ml for 20 min) enhanced current through the transient (T) Ca channel by 304 +/- 35% without significantly altering the magnitude of the long-lasting (L) Ca channel current. Exposure to neuraminidase did not affect the voltage dependence of activation or inactivation, nor did it affect the selective inhibition of the T-channel current by amiloride or the L-channel current by nifedipine. After neuraminidase treatment, the T-channel current inactivated more rapidly (time constant decreasing from 8.9 +/- 0.9 to 7.7 +/- 0.6 ms), whereas there was no change in the rate of inactivation of the L-channel current. Neuraminidase treatment removed approximately 20% of the total cellular sialic acid. These results indicate that neuraminidase treatment selectively modulates the function of the T Ca channel in ventricular myocytes, possibly through removal of sarcolemmal sialic acid, suggesting that glycosylation of membrane macromolecules may influence membrane function.