HSV Usurps Eukaryotic Initiation Factor 3 Subunit M for Viral Protein Translation: Novel Prevention Target

Prevention of genital herpes is a global health priority. B5, a recently identified ubiquitous human protein, was proposed as a candidate HSV entry receptor. The current studies explored its role in HSV infection. Viral plaque formation was reduced by ∼90% in human cells transfected with small interfering RNA targeting B5 or nectin-1, an established entry receptor. However, the mechanisms were distinct. Silencing of nectin-1 prevented intracellular delivery of viral capsids, nuclear transport of a viral tegument protein, and release of calcium stores required for entry. In contrast, B5 silencing had no effect on these markers of entry, but inhibited viral protein translation. Specifically, viral immediate early genes, ICP0 and ICP4, were transcribed, polyadenylated and transported from the nucleus to the cytoplasm, but the viral transcripts did not associate with ribosomes or polysomes in B5-silenced cells. In contrast, immediate early gene viral transcripts were detected in polysome fractions isolated from control cells. These findings are consistent with sequencing studies demonstrating that B5 is eukaryotic initiation factor 3 subunit m (eIF3m). Although B5 silencing altered the polysome profile of cells, silencing had little effect on cellular RNA or protein expression and was not cytotoxic, suggesting that this subunit is not essential for host cellular protein synthesis. Together these results demonstrate that B5 plays a major role in the initiation of HSV protein translation and could provide a novel target for strategies to prevent primary and recurrent herpetic disease

Biomarkers of Hepatic Fibrosis in Chronic Hepatitis C A Comparison of 10 Biomarkers Using 2 Different Assays for Hyaluronic Acid

Background: Advancing fibrosis is regarded as the most important factor when stratifying patients with chronic hepatitis C for retreatment. Goals: (1) To compare the performance of 10 biomarkers of fibrosis, including patented tests, among patients with chronic hepatitis C and treatment failure; and (2) to assess the impact on biomarker performance of using 2 different assays of hyaluronic acid (HA). Study: For 80 patients, liver histology (Metavir) was compared with biomarker scores using sera obtained within 6 months of liver biopsy (indirect biomarkers: AST:ALT ratio, APRI, Forns index, FIB-4, Fibrometer V3G; direct biomarkers: ELF, Fibrospect II, Hyaluronic acid-HA, Fibrometer V2G, Hepascore). Direct biomarker scores were calculated using 2 validated assays for HA (ELISA and radiometric). Results: Using the ELISA assay for HA to calculate the direct panels, all 10 of the biomarkers exhibited comparable overall discriminatory performance (unweighted Obuchowski measure, ordROC 0.92-0.94, P-value> 0.05) except AST:ALT ratio and APRI (ordROC 0.86-0.88, P-value< 0.05). For the detection of moderate (F2-4) and advanced (F3-4) fibrosis, the AUROC of Fibrometer 2G were significantly higher than AST:ALT ratio and APRI but none of the other biomarkers. Good correlation was observed between the 2 HA assays (intraclass correlation coefficient= 0.873) with the ELISA assay exhibiting superior diagnostic performance (ordROC 0.92 vs. 0.88, P-value= 0.003). Importantly, the performance of many of the direct biomarkers at their diagnostic thresholds was heavily influenced by the choice of HA assay. Conclusions: Although many biomarkers exhibited good diagnostic performance for the detection of advancing fibrosis, our results indicate that diagnostic performance may be significantly affected by the selection of individual component assays

Pediatricians' perspectives on the impact of MRSA in primary care: a qualitative study

<p>Abstract</p> <p>Background</p> <p>The incidence of skin and soft-tissue infections (SSTIs) has rapidly increased among children in primary care settings since the emergence of community-associated methicillin-resistant <it>Staphylococcus aureus </it>(CA-MRSA). Recent treatment recommendations emphasize CA-MRSA as the primary cause, performing incision and drainage (I&D) as the primary therapy, and not prescribing antibiotics for uncomplicated cases. It is unknown how this epidemic has impacted primary care pediatricians in terms of their practice patterns and barriers they face to providing recommended therapies.</p> <p>Methods</p> <p>3 Focus groups among 29 primary care pediatricians in the San Francisco Bay Area were conducted. Transcripts were reviewed and coded into major themes by two investigators using modified grounded theory.</p> <p>Results</p> <p>Substantial changes in clinical practice have occurred since the emergence of CA-MRSA. These include increased office visits for SSTIs, patients with multiple recurrences and transmission within households. Additionally, our participants reported increased visits for mild skin problems due to media reports contributing to fears about CA-MRSA. Participants routinely prescribed antibiotics for SSTIs, however, few performed I&D. Few were aware of recent SSTI treatment recommendations. Barriers to prescribing antibiotics with CA-MRSA activity included concerns about side-effects and lack of local epidemiologic data showing that it is the primary etiology. Barriers to performing I&D included lack of training, resources and skepticism about its necessity. Important clinical challenges included increased time demands for follow-up visits and patient education along with the lack of evidence-based strategies for preventing recurrent inections and household transmission.</p> <p>Conclusion</p> <p>CA-MRSA has influenced the presentation and treatment of SSTIs especially in terms of case numbers and recurrences. Barriers to providing recommended therapies can be addressed through improved dissemination of treatment guidelines and epidemiologic data. Studies are urgently needed toimprove theevidence-base for treatment and prevention strategies.</p

A Randomized Trial to Assess Anti-HIV Activity in Female Genital Tract Secretions and Soluble Mucosal Immunity Following Application of 1% Tenofovir Gel

Preclinical and early phase clinical microbicide studies have not consistently predicted the outcome of efficacy trials. To address this gap, candidate biomarkers of microbicide pharmacodynamics and safety were evaluated in a double-blind, placebo-controlled trial of tenofovir gel, the first microbicide to demonstrate significant protection against HIV acquisition.30 women were randomized to apply a single daily dose of tenofovir or placebo gel for 14 consecutive days. Anti-HIV activity was measured in cervicovaginal lavage (CVL) on Days 0, 3, 7, 14 and 21 by luciferase assay as a surrogate marker of pharmacodynamics. Endogenous activity against E. coli and HSV-2 and concentrations of immune mediators were quantified in CVL as candidate biomarkers of safety. Tenofovir levels were measured in CVL and blood.A significant increase in anti-HIV activity was detected in CVL from women who applied tenofovir gel compared to their endogenous anti-HIV activity in genital tract secretions on Day 0 and compared to activity in CVL from women in the placebo group. The activity correlated significantly with CVL concentration of tenofovir (r = 0.6, p<0.001) and fit a sigmoid E(max) pharmacodynamic model. Anti-HIV activity in CVL from women who applied tenofovir persisted when virus was introduced in semen, whereas endogenous anti-HIV activity decreased. Tenofovir did not trigger an inflammatory response or induce sustained loss in endogenous antimicrobial activity or immune mediators.Tenofovir gel had no deleterious impact on soluble mucosal immunity. The increased anti-HIV activity in CVL, which persisted in the presence of semen and correlated with tenofovir concentration, is consistent with the efficacy observed in a recent clinical trial. These results promote quantified CVL anti-HIV activity as a surrogate of tissue pharmacodynamics and as a potential biomarker of adherence to product. This simple, feasible and inexpensive bioassay may promote the development of models more predictive of microbicide efficacy.ClinicalTrials.gov NCT00594373

An Important Role for Syndecan-1 in Herpes Simplex Virus Type-1 Induced Cell-to-Cell Fusion and Virus Spread

Herpes simplex virus type-1 (HSV-1) is a common human pathogen that relies heavily on cell-to-cell spread for establishing a lifelong latent infection. Molecular aspects of HSV-1 entry into host cells have been well studied; however, the molecular details of the spread of the virus from cell-to-cell remain poorly understood. In the past, the role of heparan sulfate proteoglycans (HSPG) during HSV-1 infection has focused solely on the role of HS chains as an attachment receptor for the virus, while the core protein has been assumed to perform a passive role of only carrying the HS chains. Likewise, very little is known about the involvement of any specific HSPGs in HSV-1 lifecycle. Here we demonstrate that a HSPG, syndecan-1, plays an important role in HSV-1 induced membrane fusion and cell-to-cell spread. Interestingly, the functions of syndecan-1 in fusion and spread are independent of the presence of HS on the core protein. Using a mutant CHO-K1 cell line that lacks all glycosaminoglycans (GAGs) on its surface (CHO-745) we demonstrate that the core protein of syndecan-1 possesses the ability to modulate membrane fusion and viral spread. Altogether, we identify a new role for syndecan-1 in HSV-1 pathogenesis and demonstrate HS-independent functions of its core protein in viral spread

Increased prevalence of methicillin-resistant Staphylococcus aureus nasal colonization in household contacts of children with community acquired disease

<p>Abstract</p> <p>Background</p> <p>To measure Methicillin-resistant <it>Staphylococcus aureus </it>(MRSA) nasal colonization prevalence in household contacts of children with current community associated (CA)-MRSA infections (study group) in comparison with a group of household contacts of children without suspected <it>Staphylococcus aureus </it>infection (a control group).</p> <p>Methods</p> <p>This is a cross sectional study. Cultures of the anterior nares were taken. Relatedness of isolated strains was tested using pulse field gel electrophoresis (PFGE).</p> <p>Results</p> <p>The prevalence of MRSA colonization in the study group was significantly higher than in the control group (18/77 (23%) vs 3/77 (3.9%); p ≤ 0.001). The prevalence of SA colonization was 28/77 (36%) in the study group and 16/77 (21%) in the control group (p = 0.032). The prevalence of SA nasal colonization among patients was 6/24 (25%); one with methicillin-susceptible <it>S. aureus </it>(MSSA) and 5 with MRSA. In the study (patient) group, 14/24 (58%) families had at least one household member who was colonized with MRSA compared to 2/29 (6.9%) in the control group (p = 0.001). Of 69 total isolates tested by PFGE, 40 (58%) were related to USA300. Panton-Valetine leukocidin (PVL) genes were detected in 30/52 (58%) tested isolates. Among the families with ≥1 contact colonized with MRSA, similar PFGE profiles were found between the index patient and a contact in 10/14 families.</p> <p>Conclusions</p> <p>Prevalence of asymptomatic nasal carriage of MRSA is higher among household contacts of patients with CA-MRSA disease than control group. Decolonizing such carriers may help prevent recurrent CA-MRSA infections.</p

Reduced Gray to White Matter Tissue Intensity Contrast in Schizophrenia

BACKGROUND: While numerous structural magnetic resonance imaging (MRI) studies revealed changes of brain volume or density, cortical thickness and fibre integrity in schizophrenia, the effect of tissue alterations on the contrast properties of neural structures has so far remained mostly unexplored. METHODS: Whole brain high-resolution MRI at 3 Tesla was used to investigate tissue contrast and cortical thickness in patients with schizophrenia and healthy controls. RESULTS: Patients showed significantly decreased gray to white matter contrast in large portions throughout the cortical mantle with preponderance in inferior, middle, superior and medial temporal areas as well as in lateral and medial frontal regions. The extent of these intensity contrast changes exceeded the extent of cortical thinning. Further, contrast changes remained significant after controlling for cortical thickness measurements. CONCLUSIONS: Our findings clearly emphasize the presence of schizophrenia related brain tissue changes that alter the imaging properties of brain structures. Intensity contrast measurements might not only serve as a highly sensitive metric but also as a potential indicator of a distinct pathological process that might be independent from volume or thickness alterations

A Novel Core Genome-Encoded Superantigen Contributes to Lethality of Community-Associated MRSA Necrotizing Pneumonia

Bacterial superantigens (SAg) stimulate T-cell hyper-activation resulting in immune modulation and severe systemic illnesses such as Staphylococcus aureus toxic shock syndrome. However, all known S. aureus SAgs are encoded by mobile genetic elements and are made by only a proportion of strains. Here, we report the discovery of a novel SAg staphylococcal enterotoxin-like toxin X (SElX) encoded in the core genome of 95% of phylogenetically diverse S. aureus strains from human and animal infections, including the epidemic community-associated methicillin-resistant S. aureus (CA-MRSA) USA300 clone. SElX has a unique predicted structure characterized by a truncated SAg B-domain, but exhibits the characteristic biological activities of a SAg including Vβ-specific T-cell mitogenicity, pyrogenicity and endotoxin enhancement. In addition, SElX is expressed by clinical isolates in vitro, and during human, bovine, and ovine infections, consistent with a broad role in S. aureus infections of multiple host species. Phylogenetic analysis suggests that the selx gene was acquired horizontally by a progenitor of the S. aureus species, followed by allelic diversification by point mutation and assortative recombination resulting in at least 17 different alleles among the major pathogenic clones. Of note, SElX variants made by human- or ruminant-specific S. aureus clones demonstrated overlapping but distinct Vβ activation profiles for human and bovine lymphocytes, indicating functional diversification of SElX in different host species. Importantly, SElX made by CA-MRSA USA300 contributed to lethality in a rabbit model of necrotizing pneumonia revealing a novel virulence determinant of CA-MRSA disease pathogenesis. Taken together, we report the discovery and characterization of a unique core genome-encoded superantigen, providing new insights into the evolution of pathogenic S. aureus and the molecular basis for severe infections caused by the CA-MRSA USA300 epidemic clone

Importance of the Global Regulators Agr and SaeRS in the Pathogenesis of CA-MRSA USA300 Infection

CA-MRSA infection, driven by the emergence of the USA300 genetic background, has become epidemic in the United States. USA300 isolates are hypervirulent, compared with other CA- and HA-MRSA strains, in experimental models of necrotizing pneumonia and skin infection. Interestingly, USA300 isolates also have increased expression of core genomic global regulatory and virulence factor genes, including agr and saeRS. To test the hypothesis that agr and saeRS promote the observed hypervirulent phenotype of USA300, isogenic deletion mutants of each were constructed in USA300. The effects of gene deletion on expression and protein abundance of selected downstream virulence genes were assessed by semiquantitative real-time reverse-transcriptase PCR (qRT-PCR) and western blot, respectively. The effects of gene deletion were also assessed in mouse models of necrotizing pneumonia and skin infection. Deletion of saeRS, and, to a lesser extent, agr, resulted in attenuated expression of the genes encoding α-hemolysin (hla) and the Panton-Valentine leukocidin (lukSF-PV). Despite the differences in hla transcription, the toxin was undetectable in culture supernatants of either of the deletion mutants. Deletion of agr, but not saeRS, markedly increased the expression of the gene encoding protein A (spa), which correlated with increased protein abundance. Each deletion mutant demonstrated significant attenuation of virulence, compared with wild-type USA300, in mouse models of necrotizing pneumonia and skin infection. We conclude that agr and saeRS each independently contribute to the remarkable virulence of USA300, likely by means of their effects on expression of secreted toxins