BACKGROUND & AIMS
Hepatitis C virus (HCV) infection leads to progressive liver disease and is associated with a variety of extrahepatic syndromes, including central nervous system (CNS) abnormalities. However, it is unclear whether such cognitive abnormalities are a function of systemic disease, impaired hepatic function, or virus infection of the CNS.
We measured levels of HCV RNA and expression of the viral entry receptor in brain tissue samples from 10 infected individuals (and 3 uninfected individuals, as controls) and human brain microvascular endothelial cells by using quantitative polymerase chain reaction and immunochemical and confocal imaging analyses. HCV pseudoparticles and cell culture–derived HCV were used to study the ability of endothelial cells to support viral entry and replication.
Using quantitative polymerase chain reaction, we detected HCV RNA in brain tissue of infected individuals at significantly lower levels than in liver samples. Brain microvascular endothelia and brain endothelial cells expressed all of the recognized HCV entry receptors. Two independently derived brain endothelial cell lines, hC-MEC/D3 and HBMEC, supported HCV entry and replication. These processes were inhibited by antibodies against the entry factors CD81, scavenger receptor BI, and claudin-1; by interferon; and by reagents that inhibit NS3 protease and NS5B polymerase. HCV infection promotes endothelial permeability and cellular apoptosis.
Human brain endothelial cells express functional receptors that support HCV entry and replication. Virus infection of the CNS might lead to HCV-associated neuropathologies.
Virus Tropism; HCVpp; HCVcc; Neurologic Defect
The widespread use of clopidogrel alone or in combination with aspirin may result in gastrointestinal mucosal injury, clinically represented as recurrent ulceration and bleeding complications. Our recent work suggested that clopidogrel significantly induced human gastric epithelial cell (GES-1) apoptosis and disrupted gastric mucosal barrier, and that a p38 MAPK inhibitor could attenuate such injury. However, their exact mechanisms are largely unknown.
The GES-1 cells were used as a model system, the effects of clopidogrel on the whole gene expression profile were evaluated by human gene expression microarray and gene ontology analysis, changes of the mRNA and protein expression were determined by real-time PCR and Western blot analysis, and cell viability and apoptosis were measured by MTT assay and flow cytometry analysis, respectively.
Gene microarray analysis identified 79 genes that were differentially expressed (P<0.05 and fold-change >3) when cells were treated with or without clopidogrel. Gene ontology analysis revealed that response to stress and cell apoptosis dysfunction were ranked in the top 10 cellular events being affected, and that the major components of endoplasmic reticulum stress-mediated apoptosis pathway – CHOP and TRIB3– were up-regulated in a concentration- and time-dependent manner when cells were treated with clopidogrel. Pathway analysis demonstrated that multiple MAPK kinases were phosphorylated in clopidogrel-treated GES-1 cells, but that only SB-203580 (a p38-specific MAPK inhibitor) attenuated cell apoptosis and CHOP over-expression, both of which were induced by clopidogrel.
Increased endoplasmic reticulum stress response is involved in clopidogrel-induced gastric mucosal injury, acting through p38 MAPK activation.
Chronic hepatitis B continues to be a global problem, with an estimated 240 million cases according to the World Health Organization. Chronic infection with the hepatitis B virus (HBV) is associated with cirrhosis, hepatic decompensation, and hepatocellular carcinoma. There are currently several US Food and Drug Administration-approved medications for treating chronic hepatitis B, with Lamivudine (LAM) being the first oral agent made available. The major problem with LAM is significantly decreased effectiveness over time due to the development of anti-HBV resistance that can lead to virologic and biochemical breakthrough as well as hepatitis B flare, progression of liver disease, and decompensation of pre-existing cirrhosis. Despite its high anti-HBV resistant rate, LAM remains widely used in underdeveloped countries due to its wide availability and low cost compared to other antiviral medications, including those that are more effective. Therefore, it is still clinically important to learn how to prevent and treat LAM resistant strains of HBV. Several regimens with the other available antiviral agents have been studied, including switching to monotherapy with either Adefovir, Entecavir, or Tenofovir, adding Adefovir to LAM, and switching to a combination of Adefovir and Entecavir. This review article will examine molecular mechanisms and diagnosis of LAM anti-HBV resistance, risks for and approaches to reduce LAM anti-HBV resistance, and currently available rescue therapy regimens for LAM resistance.
chronic hepatitis B; nucleot(s)ide analogs; anti-viral resistance; lamivudine; adefovir; entecavir; tenofovir
AIM: To investigate the prevalence and risk factors of polypoid lesions of the gallbladder (PLGs) in petrochemical employees in Ningbo, Zhejiang Province, China.
METHODS: All active and retired employees aged 20-90 years (n = 11098) of a refinery and chemical plant in eastern China were requested to participate in a health survey. The participants were subjected to interview, physical examination, laboratory assessments and ultrasonography. All the participants were invited to have a physical examination after a face-to-face interview. Fasting blood samples were obtained from the antecubital vein, and the samples were used for the analysis of biochemical values. Abdominal ultrasonography was conducted.
RESULTS: A total of 10461 (7331 men and 3130 women) current and former petrochemical employees attended for screening. The overall prevalence of post-cholecystectomy, gallstones and PLGs was 0.9%, 5.2% and 7.4%, respectively. Compared with the increased prevalence of either gallstones or post-cholecystectomy in older persons, PLGs were more common in the middle-aged, peaking in those aged 40-59 years. Excluding the patients with gallstones, gallstones mixed with PLGs, or those who had undergone cholecystectomy, in the remaining 9828 participants, the prevalence of PLGs in men (8.9%) was significantly higher than that in women (5.5%, P < 0.001). The analyzed risk factors with increased OR for the development of PLGs were male gender (OR = 1.799, P < 0.001), age ≥ 30 years (OR = 2.699, P < 0.001) and hepatitis B surface antigen (HBsAg) positivity (OR = 1.374, P = 0.006).
CONCLUSION: PLGs are not rare among Chinese petrochemical employees. Male gender, HBsAg positivity, and middle age are risk factors for developing PLGs.
Prevalence; Risk factors; Polypoid lesions; Gallbladder; Chinese
Pasireotide (SOM230), a novel multireceptor ligand somatostatin analog (SSA), binds with high affinity to four of the five somatostatin receptor subtypes (sst1–3, 5). This study evaluated the safety, tolerability, pharmacokinetics, and pharmacodynamics profiles of pasireotide long-acting release (LAR) formulation in patients with advanced gastroenteropancreatic neuroendocrine tumor (GEP NET) refractory to other SSAs.
In this randomized, multicenter, open-label, phase II study, patients with biopsy-proven primary or metastatic GEP NET refractory to available SSAs were randomly assigned 1:1:1 to receive pasireotide LAR by deep intragluteal injection at a dose of 20, 40, or 60 mg once every 28 days for 3 months.
Forty-two patients received pasireotide LAR. Adverse events were reported by 34 (81 %) patients, with the most frequently reported including diarrhea, fatigue, abdominal pain, and nausea. Mean fasting glucose levels were increased compared with baseline at all points throughout the study. After the third injection of pasireotide LAR, the median trough plasma concentrations on day 84 were 4.82, 12.0, and 19.7 ng/mL in the 20-, 40-, and 60-mg treatment groups, respectively. Drug accumulation was limited for each dose based on the increase in trough concentrations after the first to third injections (accumulation ratios were approximately 1 from all dose levels).
This study demonstrated that a new, once-monthly, intramuscular LAR formulation of pasireotide was well tolerated in patients with advanced GEP NET. Steady state levels of plasma pasireotide were achieved after three injections.
Pasireotide (SOM230); Gastroenteropancreatic neuroendocrine tumor (GEP NET); Safety; Pharmacokinetics; Pharmacodynamics
The transmembrane domain proteins of the claudin superfamily are the major structural components of cellular tight junctions. One family member, claudin-1, also associates with tetraspanin CD81 as part of a receptor complex that is essential for hepatitis C virus (HCV) infection of the liver. To understand the molecular basis of claudin-1/CD81 association we previously produced and purified milligram quantities of functional, full-length CD81, which binds a soluble form of HCV E2 glycoprotein (sE2). Here we report the production, purification and characterization of claudin-1. Both yeast membrane-bound and detergent-extracted, purified claudin-1 were antigenic and recognized by specific antibodies. Analytical ultracentrifugation demonstrated that extraction with n-octyl-β-d-glucopyranoside yielded monodispersed, dimeric pools of claudin-1 while extraction with profoldin-8 or n-decylphosphocholine yielded a dynamic mixture of claudin-1 oligomers. Neither form bound sE2 in line with literature expectations, while further functional analysis was hampered by the finding that incorporation of claudin-1 into proteoliposomes rendered them intractable to study. Dynamic light scattering demonstrated that claudin-1 oligomers associate with CD81 in vitro in a defined molar ratio of 1∶2 and that complex formation was enhanced by the presence of cholesteryl hemisuccinate. Attempts to assay the complex biologically were limited by our finding that claudin-1 affects the properties of proteoliposomes. We conclude that recombinant, correctly-folded, full-length claudin-1 can be produced in yeast membranes, that it can be extracted in different oligomeric forms that do not bind sE2 and that a dynamic preparation can form a specific complex with CD81 in vitro in the absence of any other cellular components. These findings pave the way for the structural characterization of claudin-1 alone and in complex with CD81.
Cryoablation is one of the local therapies for hepatocellular carcinoma (HCC), but its safety and effect has not been studied in patients with Child class A or B and Barcelona Clinic Liver Cancer (BCLC) stage C HCC. Metastasis-associated in colon cancer-1 (MACC1) overexpression has been associated with poor prognosis of HCC, but its predictive value to post-cryoablation outcomes remains unknown in patients with BCLC stage C HCC.
This study assessed the safety and outcomes of cryoablation measured by time to progression (TTP) and overall survival (OS), and predictive value of MACC1 mRNA and protein overexpression in tumorous tissue to post-cryoablation outcomes in 120 advanced HCC patients with child-pugh class A or B by quantitative polymerase chain reaction and immunohistochemical staining. The potenial correlation of MACC1 and c-Met expression to tumor cell proliferation and apoptosis was also analyzed.
The cryoablation in patients with advanced unresectable HCC resulted in a median TTP and OS of 5.5 (4.2- 6.7) months and 10.5 (9.0-12.0) months, respectively and no significant complications, comparable to the historical report for RFA therapy. The MACC1 mRNA and nuclear protein expression was significantly increased in tumorous tissues in these patients than that in normal liver tissue controls. Higher expression of MACC1 mRNA and nuclear protein in tumorous tissues in these patients was associated with shorter post cryoablation median TTP and OS than that with lower MACC1 expression.
Cryoablation is a safe and effective therapeutic option for patients with advanced HCC and Child-pugh class A or B cirrhosis; and a higher intratumoral expression of MACC1 or nuclear translocation predicts poor outcomes of cryotherapy in these patients.
Cryoablation; Metastasis-associated in colon cancer-1 (MACC1); Advanced hepatocellular carcinoma; Efficacy; Safety; Outcomes
Genetic variability is a hallmark of RNA virus populations. However, transmission to a new host often results in a marked decrease in population diversity. This genetic bottlenecking is observed during hepatitis C virus (HCV) transmission and can arise via a selective sweep or through the founder effect. To model HCV transmission, we utilized chimeric SCID/Alb-uPA mice with transplanted human hepatocytes and infected them with a human serum HCV inoculum. E1E2 glycoprotein gene sequences in the donor inoculum and recipient mice were determined following single-genome amplification (SGA). In independent experiments, using mice with liver cells grafted from different sources, an E1E2 variant undetectable in the source inoculum was selected for during transmission. Bayesian coalescent analyses indicated that this variant arose in the inoculum pretransmission. Transmitted variants that established initial infection harbored key substitutions in E1E2 outside HVR1. Notably, all posttransmission E1E2s had lost a potential N-linked glycosylation site (PNGS) in E2. In lentiviral pseudoparticle assays, the major posttransmission E1E2 variant conferred an increased capacity for entry compared to the major variant present in the inoculum. Together, these data demonstrate that increased envelope glycoprotein fitness can drive selective outgrowth of minor variants posttransmission and that loss of a PNGS is integral to this improved phenotype. Mathematical modeling of the dynamics of competing HCV variants indicated that relatively modest differences in glycoprotein fitness can result in marked shifts in virus population composition. Overall, these data provide important insights into the dynamics and selection of HCV populations during transmission.
Hepatitis C virus (HCV) leads to progressive liver disease and hepatocellular carcinoma. Current treatments are only partially effective, and new therapies targeting viral and host pathways are required. Virus entry into a host cell provides a conserved target for therapeutic intervention. Tetraspanin CD81, scavenger receptor class B member I, and the tight-junction proteins claudin-1 and occludin have been identified as essential entry receptors. Limited information is available on the role of receptor trafficking in HCV entry. We demonstrate here that anti-CD81 antibodies inhibit HCV infection at late times after virus internalization, suggesting a role for intracellular CD81 in HCV infection. Several tetraspanins have been reported to internalize via motifs in their C-terminal cytoplasmic domains; however, CD81 lacks such motifs, leading several laboratories to suggest a limited role for CD81 endocytosis in HCV entry. We demonstrate CD81 internalization via a clathrin- and dynamin-dependent process, independent of its cytoplasmic domain, suggesting a role for associated partner proteins in regulating CD81 trafficking. Live cell imaging demonstrates CD81 and claudin-1 coendocytosis and fusion with Rab5 expressing endosomes, supporting a role for this receptor complex in HCV internalization. Receptor-specific antibodies and HCV particles increase CD81 and claudin-1 endocytosis, supporting a model wherein HCV stimulates receptor trafficking to promote particle internalization.
Etiologic diagnoses of lower respiratory tract infections (LRTI) have been relying primarily on bacterial cultures that often fail to return useful results in time. Although DNA-based assays are more sensitive than bacterial cultures in detecting pathogens, the molecular results are often inconsistent and challenged by doubts on false positives, such as those due to system- and environment-derived contaminations. Here we report a nationwide cohort study on 2986 suspected LRTI patients across P. R. China. We compared the performance of a DNA-based assay qLAMP (quantitative Loop-mediated isothermal AMPlification) with that of standard bacterial cultures in detecting a panel of eight common respiratory bacterial pathogens from sputum samples. Our qLAMP assay detects the panel of pathogens in 1047(69.28%) patients from 1533 qualified patients at the end. We found that the bacterial titer quantified based on qLAMP is a predictor of probability that the bacterium in the sample can be detected in culture assay. The relatedness of the two assays fits a logistic regression curve. We used a piecewise linear function to define breakpoints where latent pathogen abruptly change its competitive relationship with others in the panel. These breakpoints, where pathogens start to propagate abnormally, are used as cutoffs to eliminate the influence of contaminations from normal flora. With help of the cutoffs derived from statistical analysis, we are able to identify causative pathogens in 750 (48.92%) patients from qualified patients. In conclusion, qLAMP is a reliable method in quantifying bacterial titer. Despite the fact that there are always latent bacteria contaminated in sputum samples, we can identify causative pathogens based on cutoffs derived from statistical analysis of competitive relationship.
Pasireotide (SOM230) is a multireceptor-targeted somatostatin analog with high binding affinity for four of the five somatostatin receptor subtypes (sst1,2,3 and sst5), and potential clinical activity in several neuroendocrine and oncologic conditions, including acromegaly, Cushing’s disease, and neuroendocrine tumors (NET). This manuscript reports the first-in-man dose-escalation study of pasireotide, evaluating its safety, tolerability, and pharmacokinetics (PK) in healthy male volunteers. A single dose of pasireotide 1–1200 μg was administered subcutaneously in four to eight subjects per dose level, with two additional subjects per cohort administered placebo. PK and safety evaluations were carried out over 7 days post-dose. Growth hormone (GH) suppression was evaluated using a GH-releasing hormone stimulation test on Day –1 and Day 1 at 3–5 hours post-injection. Seventy-two subjects completed the study. Pasireotide was well tolerated with no serious adverse events observed at any dose. Transient elevations in blood glucose levels were observed 2–6 hours after administration of pasireotide at doses between 200 μg and 1200 μg, but this resolved without intervention by 23 hours post-dosing. The maximum tolerable dose was not established within the tested range. Pasireotide demonstrated a favorable PK profile with fast absorption (tmax: 0.25–0.5 hours), low clearance (CL/F: 8–13 L/hour), long effective elimination half-life (mean t½,β: 7–11 hours), and a proportional dose-exposure relationship. GH suppression of 79%–96% was observed at single pasireotide doses between 200 μg and 1200 μg. In conclusion, pasireotide demonstrated favorable safety, tolerability, and PK profiles, as well as promising activity in suppressing the release of GH. The efficacy and safety of pasireotide is currently being evaluated in patients with acromegaly, Cushing’s disease, NET, and various non-neuroendocrine disorders.
pasireotide; safety; tolerability; pharmacokinetics; healthy volunteers
Background & Aims
Hepatitis C virus (HCV) establishes chronic infections in 3% of the world's population. Infection leads to progressive liver disease; hepatocytes are the major site of viral replication in vivo. However, chronic infection is associated with a variety of extrahepatic syndromes, including central nervous system (CNS) abnormalities. We therefore screened a series of neural and brain-derived cell lines for their ability to support HCV entry and replication.
We used a panel of neural-derived cell lines, HCV pseudoparticles (HCVpp), and an infectious, HCV JFH-1 cell-culture system (HCVcc) to assess viral tropism.
Two independently derived neuroepithelioma cell lines (SK-N-MC and SK-PN-DW) permitted HCVpp entry. In contrast, several neuroblastoma, glioma, and astrocytoma cell lines were refractory to HCVpp infection. HCVcc infected the neuroepithelioma cell lines and established a productive infection. Permissive neuroepithelioma cells expressed CD81, scavenger receptor BI (SR-BI), and the tight junction proteins Claudin-1 (CLDN1) and occludin, whereas non-permissive neural cell lines lacked CLDN1 and in some cases SR-BI. HCVpp infection of the neuroepithelioma cells was neutralized by antibodies to CD81, SR-BI, CLDN1 and HCV E2. Furthermore, anti-CD81, interferon and the anti-NS3 protease inhibitor VX-950 significantly reduced HCVcc infection of neuroepithelioma and hepatoma cells.
Neuroepithelioma-derived cell lines express functional receptors that support HCV entry at comparable levels to that of hepatoma cells. HCV infection in vitro is not restricted to hepatic-derived cells, so HCV might infect cells of the CNS in vivo.
OCLN; neurotropism; brain; therapy; replicon; Huh-7; VX-950
2009 pandemic H1N1 (pH1N1) influenza posed an increased risk of severe illness among pregnant women. Data on risk factors associated with death of pregnant women and neonates with pH1N1 infections are limited outside of developed countries.
Retrospective observational study in 394 severe or critical pregnant women admitted to a hospital with pH1N1 influenza from Sep. 1, 2009 to Dec. 31, 2009. rRT-PCR testing was used to confirm infection. In-hospital mortality was the primary endpoint of this study. Univariable logistic analysis and multivariate logistic regression analysis were used to investigate the potential factors on admission that might be associated with the maternal and neonatal mortality.
394 pregnant women were included, 286 were infected with pH1N1 in the third trimester. 351 had pneumonia, and 77 died. A PaO2/FiO2 ≤ 200 (odds ratio (OR), 27.16; 95% confidence interval (CI), 2.64-279.70) and higher BMI (i.e. ≥ 30) on admission (OR, 1.26; 95% CI, 1.09 to 1.47) were independent risk factors for maternal death. Of 211 deliveries, 146 neonates survived. Premature delivery (OR, 4.17; 95% CI, 1.19-14.56) was associated neonatal mortality. Among 186 patients who received mechanical ventilation, 83 patients were treated with non-invasive ventilation (NIV) and 38 were successful with NIV. The death rate was lower among patients who initially received NIV than those who were initially intubated (24/83, 28.9% vs 43/87, 49.4%; p = 0.006). Septic shock was an independent risk factor for failure of NIV.
Severe hypoxemia and higher BMI on admission were associated with adverse outcomes for pregnant women. Preterm delivery was a risk factor for neonatal death among pregnant women with pH1N1 influenza infection. NIV may be useful in selected pregnant women without septic shock.
Pregnant women; Neonate; Pandemic H1N1 influenza; Mortality; Non-invasive ventilation
There is limited data on the clinical outcome of patients with pandemic H1N1 (pH1N1) pneumonia who received oseltamivir treatment, especially when the treatment was administered more than 48 hours after symptom onset.
During the pandemic in 2009, a cohort of pH1N1 influenza pneumonia was built in China, and their clinical information was collected systematically, and analyzed with Cox models.
920 adults and 541 children with pneumonia who didn't receive corticosteroids were analyzed. In-hospital mortality was higher in adults who did not receive antiviral therapy (18.2%) than those with who received oseltamivir ≤ 2days (2.9%), between 2–5 days (4.6%) and >5 days after illness onset (4.9%), p<0.01. A similar trend was observed in pediatric patients. Cox regression showed that at 60 days after symptoms onset, 11 patients (10.8%) who did not receive antivirals died versus 4 (1.8%), 18 (3.3%), and 23 (3.7%) patients whose oseltamivir treatment was started ≤ 2days, between 2–5days, and >5 days, respectively. For males patients, aged ≥ 14 years and baseline PaO2/FiO2<200, oseltamivir administration reduced the mortality risk by 92.1%, 88% and 83.5%, respectively. Higher doses of oseltamivir (>3.8 mg/kg/d) did not improve clinical outcome (mortality, higher dose 2.5% vs standard dose 2.8%, p>0.05).
Antiviral therapy might reduce mortality of patients with pH1N1 pneumonia, even when initiated more than 48 hours after onset of illness. Greater protective effects might be in males, patients aged 14–60 years, and patients with PaO2/FiO2<200.
Protozoa in the phylum Apicomplexa are a large group of obligate intracellular parasites. Toxoplasma gondii and other apicomplexan parasites, such as Plasmodium falciparum, cause diseases by reiterating their lytic cycle, comprising host cell invasion, parasite replication, and parasite egress. The successful completion of the lytic cycle requires that the parasite senses changes in its environment and switches between the non-motile (for intracellular replication) and motile (for invasion and egress) states appropriately. Although the signaling pathway that regulates the motile state switch is critical to the pathogenesis of the diseases caused by these parasites, it is not well understood. Here we report a previously unknown mechanism of regulating the motility activation in Toxoplasma, mediated by a protein lysine methyltransferase, AKMT (for Apical complex lysine (K) methyltransferase). AKMT depletion greatly inhibits activation of motility, compromises parasite invasion and egress, and thus severely impairs the lytic cycle. Interestingly, AKMT redistributes from the apical complex to the parasite body rapidly in the presence of egress-stimulating signals that increase [Ca2+] in the parasite cytoplasm, suggesting that AKMT regulation of parasite motility might be accomplished by the precise temporal control of its localization in response to environmental changes.
Toxoplasma gondii is one of the most successful human parasites, infecting ∼20% of the total world population. It is the most common cause of congenital neurological defects in humans, and an agent for devastating opportunistic infections in immunocompromised patients. To cause diseases, Toxoplasma gondii and other related parasites, such as Plasmodium falciparum, must reiterate their lytic cycle, comprising host cell infection, intracellular replication and parasite egress. At each step of the lytic cycle, the parasite tightly regulates its motility, being completely immotile while intracellular, and becoming highly motile as it leaves the host cell. Changes in local ionic conditions are known to trigger this rapid transition from immotile to motile. In this study, we report a previously unknown mechanism of regulating the motility activation in Toxoplasma, mediated by a novel protein lysine methyltransferase, AKMT (for Apical complex lysine (K) methyltransferase). The depletion of this protein greatly inhibits the parasite's ability to invade into and egress from the host cell due to impaired motility activation. Interestingly, the localization of AKMT in the parasite is sensitive to egress-stimulating signals, suggesting that AKMT regulation of parasite motility might be accomplished by the precise temporal control of its localization in response to environmental changes.
Hepatitis C virus (HCV) can initiate infection by cell-free particle and cell-cell contact-dependent transmission. In this study we use a novel infectious coculture system to examine these alternative modes of infection. Cell-to-cell transmission is relatively resistant to anti-HCV glycoprotein monoclonal antibodies and polyclonal immunoglobulin isolated from infected individuals, providing an effective strategy for escaping host humoral immune responses. Chimeric viruses expressing the structural proteins representing the seven major HCV genotypes demonstrate neutralizing antibody-resistant cell-to-cell transmission. HCV entry is a multistep process involving numerous receptors. In this study we demonstrate that, in contrast to earlier reports, CD81 and the tight-junction components claudin-1 and occludin are all essential for both cell-free and cell-to-cell viral transmission. However, scavenger receptor BI (SR-BI) has a more prominent role in cell-to-cell transmission of the virus, with SR-BI-specific antibodies and small-molecule inhibitors showing preferential inhibition of this infection route. These observations highlight the importance of targeting host cell receptors, in particular SR-BI, to control viral infection and spread in the liver.
The incidence of HCC is rising worldwide. Studies on ethnicity-based clinical presentation of HCC remain limited. The aim is to compare the clinical presentation and stage of HCC between Asian-Americans and non-Asian-Americans. This retrospective study assessed ethnicity-based differences in HCC presentation, including demographics, laboratory results, diagnosis of underlying liver disease, and stage of HCC. Of 276 patients, 162 were Asian-Americans and 114 were non-Asian-Americans. Compared to non-Asian-Americans, Asian-Americans had a significantly higher incidence of history of hepatitis B virus (HBV) infection (55.0% vs. 4.9%, P < 0.001), family history of HBV infection (12.5% vs. 0.0%, P < 0.001) and HCC (15.2% vs. 2.9%, P = 0.002), but lower incidence of history of hepatitis C virus (HCV) infection (37.5% vs. 61.6%, P < 0.001). At diagnosis of HCC, Asian-American patients had a significantly lower frequency of hepatic encephalopathy (8.9% vs. 29.3%, P = 0.001), and ascites (26.7% vs. 57.3%, P < 0.001). Asian-Americans had lower Child-Pugh scores (class A: 62.0% vs. 31.4%, P < 0.001), and MELD scores (9.2 ± 4.4 vs. 12.0 ± 6.4, P = 0.02), and presented with a lower stage of HCC by Okuda staging (I: 43.8% vs. 22.8%, P = 0.001). Asian-American patients with HCC presented with a higher incidence of history and family history of HBV infection, lower incidence of hepatic decompensation, lower Child and MELD scores, and an early stage HCC disease.
Asian-Americans; Ethnicity; Hepatocellular carcinoma (HCC); Staging HCC; Cirrhosis; Hepatitis B; Hepatitis C
The asymmetric unit of the title complex, [Ag4W2S8(C25H22P2)3]·2C3H7NO, tris[μ2-bis(diphenylphosphino)methane]-3:6κ2
S-disulfido-1κS,2κS-tetrasilver(I)ditungsten(VI) N,N-dimethylformamide disolvate, contains two [WS4]2− anions, four silver cations, three bidentate–bridging bis(diphenylphosphino)methane (dppm) ligands and two N,N-dimethylformamide (DMF) solvent molecules. The coordination geometry of each Ag atom is distorted tetrahedral. Two Ag ions are coordinated by μ2-S and μ5-S atoms, and by two P atoms from two dppm ligands, while the other two Ag atoms are coordinated by μ2-S, μ3-S and μ5-S atoms, and by one P atom from a dppm ligand.
In the title complex, [Ag(ClO4)(CH3OH)(C18H15P)2], the angles around the central Ag+ ion indicate that it is in a distorted tetrahedral coordination. The coordination sphere of silver is formed by two P atoms of two triphenylphosphine ligands, one O atom of a perchlorate anion and one O atom of a methanol molecule. The crystal structure is stablized by a bifurcated intermolecular O—H⋯O hydrogen bond, involving the O—H donor from methanol and two acceptor O atoms from the perchlorate anion, so forming a zigzag chain propagating in .
In the title complex, [Ag(C18H15P)3(ClO4)], the silver coordination environment is dominated by the distorted P3AgO tetrahedron in which Ag—O = 2.608 (12) Å and the Ag—P bond lengths are 2.5663 (17), 2.5076(16) and 2.5450 (17) Å. The perchlorate O-atoms are disordered over two positions in a 0.584 (14):0.416 (14) ratio.
Hepatitis C virus (HCV) entry occurs via a pH- and clathrin-dependent endocytic pathway and requires a number of cellular factors, including CD81, the tight-junction proteins claudin 1 (CLDN1) and occludin, and scavenger receptor class B member I (SR-BI). HCV tropism is restricted to the liver, where hepatocytes are tightly packed. Here, we demonstrate that SR-BI and CLDN1 expression is modulated in confluent human hepatoma cells, with both receptors being enriched at cell-cell junctions. Cellular contact increased HCV pseudoparticle (HCVpp) and HCV particle (HCVcc) infection and accelerated the internalization of cell-bound HCVcc, suggesting that the cell contact modulation of receptor levels may facilitate the assembly of receptor complexes required for virus internalization. CLDN1 overexpression in subconfluent cells was unable to recapitulate this effect, whereas increased SR-BI expression enhanced HCVpp entry and HCVcc internalization, demonstrating a rate-limiting role for SR-BI in HCV internalization.
Toxoplasma gondii is among the most successful parasites, with nearly half of the human population chronically infected. T. gondii has five sHsps [small Hsps (heat-shock proteins)] located in different subcellular compartments. Among them, Hsp20 showed to be localized at the periphery of the parasite body. sHsps are widespread, constituting the most poorly conserved family of molecular chaperones. The presence of sHsps in membrane structures is unusual.
The localization of Hsp20 was further analysed using high-resolution fluorescent light microscopy as well as electron microscopy, which revealed that Hsp20 is associated with the outer surface of the IMC (inner membrane complex), in a set of discontinuous stripes following the same spiralling trajectories as the subpellicular microtubules. The detergent extraction profile of Hsp20 was similar to that of GAP45 [45 kDa GAP (gliding-associated protein)], a glideosome protein associated with the IMC, but was different from that of IMC1 protein. Although we were unable to detect interacting protein partners of Hsp20 either in normal or stressed tachyzoites, an interaction of Hsp20 with phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate phospholipids could be observed.
Hsp20 was shown to be associated with a specialized membranous structure of the parasite, the IMC. This discontinuous striped-arrangement is unique in T. gondii, indicating that the topology of the outer leaflet of the IMC is not homogeneous.
chaperone; gliding-associated protein; inner membrane complex; small heat-shock protein (sHsp); Toxoplasma gondii; transfection
Viruses initiate infection by attaching to molecules or receptors at the cell surface. Hepatitis C virus (HCV) enters cells via a multistep process involving tetraspanin CD81, scavenger receptor class B member I, and the tight junction proteins Claudin-1 and Occludin. CD81 and scavenger receptor class B member I interact with HCV-encoded glycoproteins, suggesting an initial role in mediating virus attachment. In contrast, there are minimal data supporting Claudin-1 association with HCV particles, raising questions as to its role in the virus internalization process. In the present study we demonstrate a relationship between receptor active Claudins and their association and organization with CD81 at the plasma membrane by fluorescence resonance energy transfer and stoichiometric imaging methodologies. Mutation of residues 32 and 48 in the Claudin-1 first extracellular loop ablates CD81 association and HCV receptor activity. Furthermore, mutation of the same residues in the receptor-inactive Claudin-7 molecule enabled CD81 complex formation and virus entry, demonstrating an essential role for Claudin-CD81 complexes in HCV infection. Importantly, Claudin-1 associated with CD81 at the basolateral membrane of polarized HepG2 cells, whereas tight junction-associated pools of Claudin-1 demonstrated a minimal association with CD81. In summary, we demonstrate an essential role for Claudin-CD81 complexes in HCV infection and their localization at the basolateral surface of polarized hepatoma cells, consistent with virus entry into the liver via the sinusoidal blood and association with basal expressed forms of the receptors.
Fluorescence Resonance Energy Transfer (FRET); Receptor Structure-Function; Receptors; Tight Junction; Virus Entry
Toxoplasma gondii is a leading cause of congenital birth defects, as well as a cause for ocular and neurological diseases in humans. Its cytoskeleton is essential for parasite replication and invasion and contains many unique structures that are potential drug targets. Therefore, the biogenesis of the cytoskeletal structure of T. gondii is not only important for its pathogenesis, but also of interest to cell biology in general. Previously, we and others identified a new T. gondii cytoskeletal protein, TgMORN1, which is recruited to the basal complex at the very beginning of daughter formation. However, its function remained largely unknown. In this study, we generated a knock-out mutant of TgMORN1 (ΔTgMORN1) using a Cre-LoxP based approach. We found that the structure of the basal complex was grossly affected in ΔTgMORN1 parasites, which also displayed defects in cytokinesis. Moreover, ΔTgMORN1 parasites showed significant growth impairment in vitro, and this translated into greatly attenuated virulence in mice. Therefore, our results demonstrate that TgMORN1 is required for maintaining the structural integrity of the parasite posterior end, and provide direct evidence that cytoskeleton integrity is essential for parasite virulence and pathogenesis.
The disease toxoplasmosis is the result of uncontrolled growth and proliferation of the intracellular parasite Toxoplasma gondii, which is pathogenic for most warm-blooded animals. If growth of the parasite is blocked, then it does not cause disease, even though it may persist in the host as a chronic infection. Proper assembly of the cytoskeleton of T. gondii is known to be essential for its growth, and consequently required for virulence. In this study, we investigated the function of a novel cytoskeletal protein, TgMORN1, in T. gondii. TgMORN1 is a major component of the basal complex, a novel cytoskeletal assembly located at the posterior end of the parasite. We found that TgMORN1 is required for maintaining the structural integrity of the parasite posterior end and is important for ensuring successful separation of daughters at late stage of parasite replication. In addition, infection with parasites deficient in TgMORN1 not only failed to kill mice but also provided protective immunity against a lethal challenge infection, indicating the importance of TgMORN1 in T. gondii growth both in vitro and in vivo.
Developing a quantifiable in vitro model of steatosis is critical in understanding the pathogenesis of nonalcoholic fatty liver disease (NAFLD) and searchingfor effective therapies. Using an ORO-based colorimetric measurement, we developed a convenient assay to qualify the degree of OA-induced steatosis in HepG2 cells. We demonstrated that in the absence of exogenous inflammatory mediators, OA-induced steatosis was associated with increased production and secretion of tumor necrosis factor alpha and decreased expression of peroxisome proliferators-activated receptor α in HepG2 cells. OA-induced steatosis was also associated with increased lipid peroxidation, apoptosis, but decreased proliferation in these cells. The increased lipid peroxidation was related to decreased SOD-1, a free radical scavenger enzyme; while increased apoptosis was related to increased active caspase-9. The decreased proliferation mediated by OA-induced steatosis was associated with increased production of p27 with unchanged alanine transaminase (ALT) level in the culture medium, indicating OA-induced steatosis alters cell cycle progression without direct toxicity to these cells. In conclusion, the present study developed a colorimetric assay that accurately quantifies OA-induced steatosis in HepG2 cells. In the absence of exogenous inflammatory mediators, OA-induced steatosis results in a series of pathophysilogical changes in HepG2 cells, indicating direct pathogenic roles of hepatocytes in NAFLD.
Steatosis; tumor necrosis factor α; peroxisome proliferators-activated receptor α; apoptosis; lipid peroxidation; cell proliferation