Plasma membrane-associated annexin A6 reduces Ca2+ entry by stabilizing the cortical actin cytoskeleton

The annexins are a family of Ca(2+)- and phospholipid-binding proteins, which interact with membranes upon increase of [Ca(2+)](i) or during cytoplasmic acidification. The transient nature of the membrane binding of annexins complicates the study of their influence on intracellular processes. To address the function of annexins at the plasma membrane (PM), we fused fluorescent protein-tagged annexins A6, A1, and A2 with H- and K-Ras membrane anchors. Stable PM localization of membrane-anchored annexin A6 significantly decreased the store-operated Ca(2+) entry (SOCE), but did not influence the rates of Ca(2+) extrusion. This attenuation was specific for annexin A6 because PM-anchored annexins A1 and A2 did not alter SOCE. Membrane association of annexin A6 was necessary for a measurable decrease of SOCE, because cytoplasmic annexin A6 had no effect on Ca(2+) entry as long as [Ca(2+)](i) was below the threshold of annexin A6-membrane translocation. However, when [Ca(2+)](i) reached the levels necessary for the Ca(2+)-dependent PM association of ectopically expressed wild-type annexin A6, SOCE was also inhibited. Conversely, knockdown of the endogenous annexin A6 in HEK293 cells resulted in an elevated Ca(2+) entry. Constitutive PM localization of annexin A6 caused a rearrangement and accumulation of F-actin at the PM, indicating a stabilized cortical cytoskeleton. Consistent with these findings, disruption of the actin cytoskeleton using latrunculin A abolished the inhibitory effect of PM-anchored annexin A6 on SOCE. In agreement with the inhibitory effect of annexin A6 on SOCE, constitutive PM localization of annexin A6 inhibited cell proliferation. Taken together, our results implicate annexin A6 in the actin-dependent regulation of Ca(2+) entry, with consequences for the rates of cell proliferation.

Dimensional ridge alterations following immediate implant placement in molar extraction sites: a six-month prospective cohort study with surgical re-entry

AIM: To assess dimensional ridge alterations following immediate implant placement in molar extraction sites. MATERIAL AND METHODS: Twelve subjects received 12 immediate transmucosal implants in molar extraction sites. Peri-implant defects were treated according to the principles of Guided Bone Regeneration by means of a deproteinized bone substitute and a bioresorbable collagen membrane. Changes in vertical (IS-BD, CREST-BD) and horizontal distances (EC-I, IC-I) of alveolar bony walls to the bottom of the defects (BD) and to the implant surfaces (I) were compared between implant placement and surgical re-entry at 6 months. RESULTS: The implant survival rate at 6 months was 100%. Statistically significant differences (P<0.01) were observed in the mean changes in vertical distances IS-BD and CREST-BD between baseline and re-entry. At re-entry, all peri-implant marginal defects assessed from the internal socket wall to the implant surface (IC-I) were healed. The residual combined thickness of the buccal wall with the newly formed peri-implant bone at sites with an initial thickness of 1 mm was statistically significantly smaller (P<0.05) compared with that of sites with an initial buccal thickness of 2 mm (2.50 +/- 0.76 vs. 4+/-0 mm). CONCLUSIONS: The marginal defects around immediate implants placed in molar extraction sites were completely filled after 6 months of healing through de novo bone formation. Bone resorption was observed from the external aspects of the buccal and oral socket walls. Dimensional changes of the external socket walls were mostly pronounced at the buccal aspects.

Gender differences in premorbid, entry, treatment, and outcome characteristics in an epidemiological sample of 661 patients with first episode psychosis

Gender differences in psychotic disorder have been observed in terms of illness onset and course; however, past research has been limited by inconsistencies between studies and the lack of epidemiological representative of samples assessed. Thus, the aim of this study was to elucidate gender differences in a treated epidemiological sample of patients with first episode psychosis (FEP).

C-C chemokine receptor 6-regulated entry of TH-17 cells into the CNS through the choroid plexus is required for the initiation of EAE

Interleukin 17-producing T helper cells (T(H)-17 cells) are important in experimental autoimmune encephalomyelitis, but their route of entry into the central nervous system (CNS) and their contribution relative to that of other effector T cells remain to be determined. Here we found that mice lacking CCR6, a chemokine receptor characteristic of T(H)-17 cells, developed T(H)-17 responses but were highly resistant to the induction of experimental autoimmune encephalomyelitis. Disease susceptibility was reconstituted by transfer of wild-type T cells that entered into the CNS before disease onset and triggered massive CCR6-independent recruitment of effector T cells across activated parenchymal vessels. The CCR6 ligand CCL20 was constitutively expressed in epithelial cells of choroid plexus in mice and humans. Our results identify distinct molecular requirements and ports of lymphocyte entry into uninflamed versus inflamed CNS and suggest that the CCR6-CCL20 axis in the choroid plexus controls immune surveillance of the CNS.

How reliable is an undetectable viral load?

OBJECTIVES: An article by the Swiss AIDS Commission states that patients with stably suppressed viraemia [i.e. several successive HIV-1 RNA plasma concentrations (viral loads, VL) below the limits of detection during 6 months or more of highly active antiretroviral therapy (HAART)] are unlikely to be infectious. Questions then arise: how reliable is the undetectability of the VL, given the history of measures? What factors determine reliability? METHODS: We assessed the probability (henceforth termed reliability) that the n+1 VL would exceed 50 or 1000 HIV-1 RNA copies/mL when the nth one had been <50 copies/mL in 6168 patients of the Swiss HIV Cohort Study who were continuing to take HAART between 2003 and 2007. General estimating equations were used to analyse potential factors of reliability. RESULTS: With a cut-off at 50 copies/mL, reliability was 84.5% (n=1), increasing to 94.5% (n=5). Compliance, the current type of HAART and the first antiretroviral therapy (ART) received (HAART or not) were predictive factors of reliability. With a cut-off at 1000 copies/mL, reliability was 97.5% (n=1), increasing to 99.1% (n=4). Chart review revealed that patients had stopped their treatment, admitted to major problems with compliance or were taking non-HAART ART in 72.2% of these cases. Viral escape caused by resistance was found in 5.6%. No explanation was found in the charts of 22.2% of cases. CONCLUSIONS: After several successive VLs at <50 copies/mL, reliability reaches approximately 94% with a cut-off of 50 copies/mL and approximately 99% with a cut-off at 1000 copies/mL. Compliance is the most important factor predicting reliability.

Conserved leucine residue in the head region of morbillivirus fusion protein regulates the large conformational change during fusion activity

Paramyxovirus cell entry is controlled by the concerted action of two viral envelope glycoproteins, the fusion (F) and the receptor-binding (H) proteins, which together with a cell surface receptor mediate plasma membrane fusion activity. The paramyxovirus F protein belongs to class I viral fusion proteins which typically contain two heptad repeat regions (HR). Particular to paramyxovirus F proteins is a long intervening sequence (IS) located between both HR domains. To investigate the role of the IS domain in regulating fusogenicity, we mutated in the canine distemper virus (CDV) F protein IS domain a highly conserved leucine residue (L372) previously reported to cause a hyperfusogenic phenotype. Beside one F mutant, which elicited significant defects in processing, transport competence, and fusogenicity, all remaining mutants were characterized by enhanced fusion activity despite normal or slightly impaired processing and cell surface targeting. Using anti-CDV-F monoclonal antibodies, modified conformational F states were detected in F mutants compared to the parental protein. Despite these structural differences, coimmunoprecipitation assays did not reveal any drastic modulation in F/H avidity of interaction. However, we found that F mutants had significantly enhanced fusogenicity at low temperature only, suggesting that they folded into conformations requiring less energy to activate fusion. Together, these data provide strong biochemical and functional evidence that the conserved leucine 372 at the base of the HRA coiled-coil of F(wt) controls the stabilization of the prefusogenic state, restraining the conformational switch and thereby preventing extensive cell-cell fusion activity.

The viral RNase E(rns) prevents IFN type-I triggering by pestiviral single- and double-stranded RNAs

Interferon (IFN) type-I is of utmost importance in the innate antiviral defence of eukaryotic cells. The cells express intra- and extracellular receptors that monitor their surroundings for the presence of viral genomes. Bovine viral diarrhoea virus (BVDV), a Pestivirus of the family Flaviviridae, is able to prevent IFN synthesis induced by poly(IC), a synthetic dsRNA. The evasion of innate immunity might be a decisive ability of BVDV to establish persistent infection in its host. We report that ds- as well as ssRNA fragments of viral origin are able to trigger IFN synthesis, and that the viral envelope glycoprotein E(rns), that is also secreted from infected cells, is able to inhibit IFN expression induced by these extracellular viral RNAs. The RNase activity of E(rns) is required for this inhibition, and E(rns) degrades ds- and ssRNA at neutral pH. In addition, cells infected with a cytopathogenic strain of BVDV contain more dsRNA than cells infected with the homologous non-cytopathogenic strain, and the intracellular viral RNA was able to excite the IFN system in a 5'-triphosphate-, i.e. RIG-I-, independent manner. Functionally, E(rns) might represent a decoy receptor that binds and enzymatically degrades viral RNA that otherwise might activate the IFN defence by binding to Toll-like receptors of uninfected cells. Thus, the pestiviral RNase efficiently manipulates the host's self-nonself discrimination to successfully establish and maintain persistence and immunotolerance.

Bovine viral diarrhea virus in free-ranging wild ruminants in Switzerland: low prevalence of infection despite regular interactions with domestic livestock

ABSTRACT: BACKGROUND: In the frame of an eradication program for bovine viral diarrhea (BVD) in Swiss livestock, the question was raised whether free-ranging wildlife could threaten the success of this sanitary measure. Therefore, we conducted serological and virological investigations on BVD virus (BVDV) infections in the four indigenous wild ruminant species (roe deer, red deer, Alpine chamois and Alpine ibex) from 2009 to 2011, and gathered information on interactions between wild and domestic ruminants in an alpine environment by questionnaire survey. RESULTS: Thirty-two sera out of 1'877 (1.7%, 95% confidence interval [CI] 1.2-2.4) were seropositive for BVDV, and a BVDV1 sub genotype h virus was found in a seropositive chamois (0.05%, 95% CI 0.001-0.3). The seropositive animals originated from sub-alpine or alpine regions and significantly more seropositive red deer, chamois and ibex than roe deer were found. There were no statistically significant differences between sampling units, age classes, genders, and sampling years. The obtained prevalences were significantly lower than those documented in livestock, and most positive wild ruminants were found in proximity of domestic outbreaks. Additionally, BVDV seroprevalence in ibex was significantly lower than previously reported from Switzerland. The survey on interspecific interactions revealed that interactions expected to allow BVDV transmission, from physical contacts to non-simultaneous use of the same areas, regularly occur on pastures among all investigated ruminant species. Interactions involving cervids were more often observed with cattle than with small ruminants, chamois were observed with all three domestic species, and ibex interacted mostly with small ruminants. Interactions related to the use of anthropogenic food sources were frequently observed, especially between red deer and cattle in wintertime. CONCLUSIONS: To our knowledge, this is the first report of BVDV RNA isolated from an Alpine chamois. Nevertheless, our results suggest that BVDV infections are only sporadic in Swiss wild ruminants, despite regular occurrence of interactions with potentially infected livestock. Overall, serological, virological and ethological data indicate that wildlife is currently an incidental spill-over host and not a reservoir for BVDV in Switzerland.

Antibody Response to Inactivated Viral Vaccines Administered to Calves at Weaning

Respiratory disease in beef calves has been associated with the stress of weaning. Management practices commonly delay vaccination of calves to this time, and weaning stress could potentially suppress the immune response. To reduce this stress we have been experimenting with a procedure termed “pasture weaning” in which the dams are removed and the calves remain on pasture. Observation suggests that calves weaned with this approach adapt to the weaned state much better than those held in drylot. Consequently, one would expect less stress-mediated effects including those on the immune system. Calves were weaned and assigned to groups that were pasture or drylot weaned, and calves within the groups were vaccinated with one of two inactivated virus vaccines by either the intramuscular or subcutaneous route. Weaning placement did not affect antibody responses to the viruses included in the vaccines. The route of administration did not influence responses with subcutaneous injection inducing responses equivalent to the intramuscular site. Utilization of this route for vaccination could be advantageous because it precludes the tissue damage and hidden abscessation that sometimes results from intramuscular injections. A distinct difference was noted in the immunogenicity of the vaccines with the Vira Shield product yielding significantly better responses to all viral entities.

Antibody Responses of Young Calves to Inactivated Viral Vaccines

Three commercial inactivated virus vaccines were evaluated for immunogenicity in young calves with residual maternal antibodies. Groups of 30 calves were administered each of the vaccines at the start of the experimentation and were administered a second dose 32 days later. Serum was obtained from these calves and 30 calves in a nonvaccinated control group prior to vaccination and at 32, 61, 99 and 125 days thereafter. Antibody responses to viruses in two of the vaccines were extremely limited. The third vaccine overcame suppression by maternal antibodies and elicited responses clearly differentiated from antibody levels in the control group of calves.

Cost-effectiveness of point-of-care viral load monitoring of antiretroviral therapy in resource-limited settings: mathematical modelling study

BACKGROUND Monitoring of HIV viral load in patients on combination antiretroviral therapy (ART) is not generally available in resource-limited settings. We examined the cost-effectiveness of qualitative point-of-care viral load tests (POC-VL) in sub-Saharan Africa. DESIGN Mathematical model based on longitudinal data from the Gugulethu and Khayelitsha township ART programmes in Cape Town, South Africa. METHODS Cohorts of patients on ART monitored by POC-VL, CD4 cell count or clinically were simulated. Scenario A considered the more accurate detection of treatment failure with POC-VL only, and scenario B also considered the effect on HIV transmission. Scenario C further assumed that the risk of virologic failure is halved with POC-VL due to improved adherence. We estimated the change in costs per quality-adjusted life-year gained (incremental cost-effectiveness ratios, ICERs) of POC-VL compared with CD4 and clinical monitoring. RESULTS POC-VL tests with detection limits less than 1000 copies/ml increased costs due to unnecessary switches to second-line ART, without improving survival. Assuming POC-VL unit costs between US$5 and US$20 and detection limits between 1000 and 10,000 copies/ml, the ICER of POC-VL was US$4010-US$9230 compared with clinical and US$5960-US$25540 compared with CD4 cell count monitoring. In Scenario B, the corresponding ICERs were US$2450-US$5830 and US$2230-US$10380. In Scenario C, the ICER ranged between US$960 and US$2500 compared with clinical monitoring and between cost-saving and US$2460 compared with CD4 monitoring. CONCLUSION The cost-effectiveness of POC-VL for monitoring ART is improved by a higher detection limit, by taking the reduction in new HIV infections into account and assuming that failure of first-line ART is reduced due to targeted adherence counselling.