Identification of oncolytic vaccinia restriction factors in canine high-grade mammary tumor cells using single-cell transcriptomics.

Mammary carcinoma, including triple-negative breast carcinomas (TNBC) are tumor-types for which human and canine pathologies are closely related at the molecular level. The efficacy of an oncolytic vaccinia virus (VV) was compared in low-passage primary carcinoma cells from TNBC versus non-TNBC. Non-TNBC cells were 28 fold more sensitive to VV than TNBC cells in which VV replication is impaired. Single-cell RNA-seq performed on two different TNBC cell samples, infected or not with VV, highlighted three distinct populations: naïve cells, bystander cells, defined as cells exposed to the virus but not infected and infected cells. The transcriptomes of these three populations showed striking variations in the modulation of pathways regulated by cytokines and growth factors. We hypothesized that the pool of genes expressed in the bystander populations was enriched in antiviral genes. Bioinformatic analysis suggested that the reduced activity of the virus was associated with a higher mesenchymal status of the cells. In addition, we demonstrated experimentally that high expression of one gene, DDIT4, is detrimental to VV production. Considering that DDIT4 is associated with a poor prognosis in various cancers including TNBC, our data highlight DDIT4 as a candidate resistance marker for oncolytic poxvirus therapy. This information could be used to design new generations of oncolytic poxviruses. Beyond the field of gene therapy, this study demonstrates that single-cell transcriptomics can be used to identify cellular factors influencing viral replication.

Seroprevalance of Crimean-Congo haemorrhagic fever in Bulgarian livestock.

Crimean-Congo haemorrhagic fever (CCHF) is a tick-borne zoonotic disease. Over the past decade, CCHF cases in humans have emerged in Turkey and reemerged in the Balkan countries, Ukraine and Tajikistan. Occupational contact with infected livestock has been recognized as a common cause of the disease. A cross-sectional seroprevalence study in livestock was conducted in farming communities of an endemic area in Bulgaria, southeastern Europe. Overall, 72% of the tested animals were positive for IgG antibodies to CCHF virus. By the time the animals were one-year old almost 50% had serologic evidence of CCHF infection, and by two years already 80% of them had been infected. The data obtained in this study reflect current situation of CCHF virus infection among livestock in Bulgaria. The results showed active CCHF virus circulation that poses risk for humans to be infected during contacts with animals and requires public health awareness.

Myopericarditis and pericarditis in the deployed military member: a retrospective series.

In this study, we investigated the incidence and etiology of pericarditis and myopericarditis of military members deployed to Iraq and Kuwait from 2004 through 2008. The importance of acute pericarditis and myopericarditis in the deployed military service member has resurfaced with the reintroduction of the smallpox vaccination by the U.S. Department of Defense in 2002. There are limited epidemiologic data on acute pericarditis and myopericarditis in the general population. As a primary evacuation node for cardiology patients between 2004 and 2008, the United States Military Hospital Kuwait cardiology clinic was uniquely situated to reliably extrapolate epidemiologic data for U.S. Armed Service Members serving in the Middle East. Between these years, approximately 721,600 service members served in Kuwait and Iraq. A total of 70 cases of pericarditis and 9 cases of myopericarditis were diagnosed. This yields an estimated incidence of 7.4 and 0.95 cases per 100,000 per year for pericarditis and myopericarditis, respectively. A total of eleven patients had received the smallpox vaccine 4 to 30 days before being diagnosed with pericarditis or myopericarditis. Four of the eleven patients (36.3%) had pericarditis, with a mean duration of 28.3 days since vaccination. Seven of these eleven (63.6%) patients had myopericarditis, with a mean duration of 13.7 days since smallpox vaccination. The incidence of pericarditis and myopericarditis was lower than previously reported incidence rates in the population. In all cases of myopericarditis and pericarditis, smallpox vaccination was preferentially related to myopericarditis versus pericarditis.

Virological surveillance of influenza-like illness among children in Ghana, 2008-2010.

BACKGROUND: The global annual attack rate for influenza is estimated to be 10%-20% in children, although limited information exists for Africa. In 2007, Ghana initiated influenza surveillance by routine monitoring of acute respiratory illness to obtain data on circulating strains. We describe influenza surveillance in children <11 years old who had influenza-like illness (ILI) from January 2008 to December 2010. METHODS: Oropharyngeal swabs from pediatric outpatients with ILI attending any of 22 health facilities across the country were submitted. We tested swabs for influenza virus using molecular assays, virus isolation, and hemagglutination assays. RESULTS: Of the 2810 swabs, 636 (23%) were positive for influenza virus. The percentage of positives by gender was similar. The proportion of ILI cases positive for influenza increased with age from 11% (31/275) in infants (aged 0-1 years) to 31% (377/1219) among children aged 5-10 years (P < .001). The majority of cases were influenza A (90%), of which 60% were influenza A(H1N1)pdm09. In all 3 years, influenza activity appeared slightly higher during May through July. CONCLUSIONS: During the 3 years of influenza surveillance in Ghana, children aged <11 years bore a high burden of influenza-associated ILI.

Surveillance of avian influenza viruses in migratory birds in Egypt, 2003-09.

Migratory (particularly aquatic) birds are the major natural reservoirs for type A influenza viruses. However, their role in transmitting highly pathogenic avian influenza (HPAI) viruses is unclear. Egypt is a "funnel" zone of wild bird migration pathways from Central Asia and Europe to Eastern and Central Africa ending in South Africa. We sought to detect and isolate avian influenza viruses in migratory birds in Egypt. During September 2003-February 2009, the US Naval Medical Research Unit Number 3, Cairo, Egypt, in collaboration with the Egyptian Ministry of Environment, obtained cloacal swabs from 7,894 migratory birds captured or shot by hunters in different geographic areas in Egypt. Samples were processed by real-time reverse transcriptase PCR for detection of the influenza A matrix gene. Positive samples were processed for virus isolation in specific-pathogen-free embryonated eggs and isolates were subtyped by PCR and partial sequencing. Ninety-five species of birds were collected. Predominant species were Green-Winged Teal (Anas carolinensis; 32.0%, n=2,528), Northern Shoveler (Anas clypeata; 21.4%, n=1,686), and Northern Pintail (Anas acuta; 11.1%, n=877). Of the 7,894 samples, 745 (9.4%) were positive for the influenza A matrix gene (mainly from the above predominant species). Thirteen of the 745 (1.7%) were H5-positive by PCR (11 were low-pathogenic avian influenza and two were HPAI H5N1). The prevalences of influenza A was among regions were 10-15%, except in Middle Egypt (4%). Thirty-nine influenza isolates were obtained from PCR-positive samples. Seventeen subtypes of avian influenza viruses (including H5N1 and H7N7) were classified from 39 isolates using PCR and partial sequencing. Only one HPAI H5N1 was isolated in February 2006, from a wild resident Great Egret (Ardea alba). No major die-offs or sick migratory birds were detected during the study. We identified avian influenza virus subtypes not previously reported in Egypt. The HPAI H5N1 isolated or detected indicates that migratory birds may play a role in the dispersal of HPAI virus, but a detailed mechanism of this role needs to be elucidated.

A growing global network's role in outbreak response: AFHSC-GEIS 2008-2009.

A cornerstone of effective disease surveillance programs comprises the early identification of infectious threats and the subsequent rapid response to prevent further spread. Effectively identifying, tracking and responding to these threats is often difficult and requires international cooperation due to the rapidity with which diseases cross national borders and spread throughout the global community as a result of travel and migration by humans and animals. From Oct.1, 2008 to Sept. 30, 2009, the United States Department of Defense's (DoD) Armed Forces Health Surveillance Center Global Emerging Infections Surveillance and Response System (AFHSC-GEIS) identified 76 outbreaks in 53 countries. Emerging infectious disease outbreaks were identified by the global network and included a wide spectrum of support activities in collaboration with host country partners, several of which were in direct support of the World Health Organization's (WHO) International Health Regulations (IHR) (2005). The network also supported military forces around the world affected by the novel influenza A/H1N1 pandemic of 2009. With IHR (2005) as the guiding framework for action, the AFHSC-GEIS network of international partners and overseas research laboratories continues to develop into a far-reaching system for identifying, analyzing and responding to emerging disease threats.

Trends and causes of hospitalizations among HIV-infected persons during the late HAART era: what is the impact of CD4 counts and HAART use?

BACKGROUND: Declining rates of hospitalizations occurred shortly after the availability of highly active antiretroviral therapy (HAART). However, trends in the late HAART era are less defined, and data on the impact of CD4 counts and HAART use on hospitalizations are needed. METHODS: We evaluated hospitalization rates from 1999 to 2007 among HIV-infected persons enrolled in a large US military cohort. Poisson regression was used to compare hospitalization rates per year and to examine factors associated with hospitalization. RESULTS: Of the 2429 participants, 822 (34%) were hospitalized at least once with 1770 separate hospital admissions. The rate of hospitalizations (137 per 1000 person-years) was constant over the study period [relative rate (RR) 1.00 per year change, 95% confidence interval: 0.98 to 1.02]. The hospitalization rates due to skin infections (RR: 1.50, P = 0.02), methicillin-resistant staphylococcus aureus (RR: 3.19, P = 0.03), liver disease (RR: 1.71, P = 0.04), and surgery (RR: 1.17, P = 0.04) significantly increased over time, whereas psychological causes (RR: 0.60, P < 0.01) and trauma (RR: 0.54, P < 0.01) decreased. In the multivariate model, higher nadir CD4 (RR: 0.92 per 50 cells, P < 0.01) and higher proximal CD4 counts (RR of 0.71 for 350-499 vs. <350 cells/mm(3) and RR 0.67 for > or = 500 vs. 350 cells/mm(3), both P < 0.01) were associated with lower risk of hospitalization. Risk of hospitalization was constant for proximal CD4 levels above 350 (RR: 0.94 P = 0.51, CD4 > or = 500 vs. 350-499). HAART was associated with a reduced risk of hospitalization among those with a CD4 <350 (RR: 0.72, P = 0.02) but had smaller estimated and nonsignificant effects at higher CD4 levels (RR: 0.81, P = 0.33 and 1.06, P = 0.71 for CD4 350-499 and > or = 500, respectively). CONCLUSIONS: Hospitalizations continue to occur at high rates among HIV-infected persons with increasing rates for skin infections, methicillin-resistant staphylococcus aureus, liver disease, and surgeries. Factors associated with a reduced risk of hospitalization include CD4 counts >350 cells per cubic millimeter and HAART use among patients with a CD4 count <350 cells per cubic millimeter.

Obesity among HIV-infected persons: impact of weight on CD4 cell count.

To assess the effect of obesity on CD4 cell counts, we estimated the association of time-updated BMI categories with CD4 changes among 1001 documented HIV seroconverters. During the pre-highly active antiretroviral therapy (HAART) era, a higher BMI was associated with less reduction in CD4 cell counts over time. However during the HAART era, obese versus normal weight patients had smaller increases in CD4 cell counts (+69 versus +116 cells, P = 0.01). Lower CD4 cell counts may now be another adverse consequence of obesity.

The effect of human immunodeficiency virus on hepatitis B virus serologic status in co-infected adults.

BACKGROUND: Factors associated with serologic hepatitis B virus (HBV) outcomes in HIV-infected individuals remain incompletely understood, yet such knowledge may lead to improvements in the prevention and treatment of chronic HBV infection. METHODS AND FINDINGS: HBV-HIV co-infected cohort participants were retrospectively analyzed. HBV serologic outcomes were classified as chronic, resolved, and isolated-HBcAb. Chronic HBV (CHBV) was defined as the presence of HBsAg on two or more occasions at least six months apart. Risk factors for HBV serologic outcome were assessed using logistic regression. Of 2037 participants with HBV infection, 281 (14%) had CHBV. Overall the proportions of HBV infections classified as CHBV were 11%, 16%, and 19% for CD4 cell count strata of > or =500, 200-499, and <200, respectively (p<0.0001). Risk of CHBV was increased for those with HBV infection occurring after HIV diagnosis (OR 2.62; 95% CI 1.78-3.85). This included the subset with CD4 count > or =500 cells/microL where 21% of those with HBV after HIV diagnosis had CHBV compared with 9% for all other cases of HBV infection in this stratum (p = 0.0004). Prior receipt of HAART was associated with improved HBV serologic outcome overall (p = 0.012), and specifically among those with HBV after HIV (p = 0.002). In those with HBV after HIV, HAART was associated with reduced risk of CHBV overall (OR 0.18; 95% CI 0.04-0.79); including reduced risk in the subsets with CD4 > or =350 cells/microL (p<0.001) and CD4 > or =500 cells/microL (p = 0.01) where no cases of CHBV were seen in those with a recent history of HAART use. CONCLUSIONS: Clinical indicators of immunologic status in HIV-infected individuals, such as CD4 cell count, are associated with HBV serologic outcome. These data suggest that immunologic preservation through the increased use of HAART to improve functional anti-HBV immunity, whether by improved access to care or earlier initiation of therapy, would likely improve HBV infection outcomes in HIV-infected individuals.

Epidemiology of Hepatitis B virus infection in a US cohort of HIV-infected individuals during the past 20 years.

BACKGROUND: The epidemiologic trends of hepatitis B virus (HBV) infection in human immunodeficiency virus (HIV)-infected patients over the past 20 years are largely unknown. METHODS: Prevalence and risk factors for HBV infection overall, at the time of HIV infection, and after HIV infection were examined in an ongoing observational HIV cohort study. Risk factors for HBV infection at the time of diagnosis of HIV infection were evaluated using logistic regression, and risk of incident HBV infection after diagnosis of HIV infection was evaluated using Cox proportional hazards models. RESULTS: Of the 2769 evaluable participants, 1078 (39%) had HBV infection, of whom 117 (11%) had chronic HBV infection. The yearly cross-sectional prevalence of HBV infection decreased from a peak of 49% in 1995 to 36% in 2008 (P < .001). The prevalence of HBV infection at the time of diagnosis of HIV infection decreased during 1989-2008 from 34% to 9% (P < .001). The incidence of HBV infection after diagnosis of HIV infection decreased from 4.0 cases per 100 person-years during the pre-highly active antiretroviral therapy (HAART) era to 1.1 cases per 100 person-years during the HAART era (P < .001); however, this incidence remained unchanged during 2000-2008 (P = .49), with >20% of HBV infections occurring after HIV infection being chronic. Decreased risk of HBV infection after diagnosis of HIV infection was associated with higher CD4 cell count and the use of HBV-active HAART. Receipt of 1 dose of HBV vaccine was not associated with reduced risk of HBV infection after diagnosis of HIV infection. CONCLUSIONS: Although the burden of HBV infection overall is slowly decreasing among HIV-infected individuals, the persistent rate of HBV infection after diagnosis of HIV infection raises concern that more-effective prevention strategies may be needed to significantly reduce the prevalence of HBV infection in this patient population.

Anal cancers among HIV-infected persons: HAART is not slowing rising incidence.

OBJECTIVE: To evaluate the incidence rates of anal cancer over the HIV epidemic and assess the impact of HAART use on anal cancer events. METHODS: We evaluated the incidence of and factors associated with anal cancer using longitudinal data from the prospective U.S. Military Natural History Study (1985-2008). Poisson regression and Cox proportional hazard models were utilized. RESULTS: Among 4506 HIV-infected men with 37 806 person-years of follow-up, anal cancer rates (per 100 000 person-years) increased five-fold, from 11 in the pre-HAART to 55 in the HAART era (P = 0.02). Rates continued to increase, reaching 128 in 2006-2008. Persons with HIV infection for more than 15 years had a 12-fold higher rate than those with less than 5 years (348 vs. 28, P < 0.01). At cancer diagnosis (n = 19), median age was 42 years, median CD4 cell count was 432 cells/microl, 74% had a CD4 nadir cell count less than 200 cells/microl, 42% had a prior AIDS event, and 74% had received HAART. From separate models, prior AIDS event (hazard ratio 3.88, P = 0.01) and lower CD4 nadir (hazard ratio 0.85 per 50 cell, P = 0.03) were associated with anal cancer, with a trend for a history of gonorrhea (hazard ratio 2.43, P = 0.07). Duration of HAART use was not associated with a reduced risk of anal cancer (hazard ratio 0.94, P = 0.42). CONCLUSION: Incidence rates of anal cancer have progressively increased during the HIV epidemic. Persons with a longer duration of HIV infection have a substantially higher rate of anal cancer. As HIV-infected persons are experiencing longer life expectancies and HAART does not appear protective of anal cancer, studies on preventive strategies are needed.

Silencing of hSlo potassium channels in human osteosarcoma cells promotes tumorigenesis.

Potassium channels, the most diverse superfamily of ion channels, have recently emerged as regulators of carcinogenesis, thus introducing possible new therapeutic strategies in the fight against cancer. In particular, the large conductance Ca(2+)-activated K(+) channels, often referred to as BK channels, are at the crossroads of several tumor-associated processes such as cell proliferation, survival, secretion and migration. Despite the high BK channel expression in osteosarcoma (OS), their function has not yet been investigated in this malignant bone pathology. Here, using stable RNA interference to reduce the expression of hSlo, the human pore-forming alpha-subunit of the BK channel, in human Cal72 OS cells, we show that BK channels play a functional role in carcinogenesis. Our results reveal for the first time that BK channels exhibit antitumoral properties in OS in vivo and affect the tumor microenvironment through the modulation of both chemokine expression and leukocyte infiltration.

Car-Parrinello treatment for an approximate density-functional theory method.

The authors formulate a Car-Parrinello treatment for the density-functional-based tight-binding method with and without self-consistent charge corrections. This method avoids the numerical solution of the secular equations, the principal drawback for large systems if the linear combination of atomic orbital ansatz is used. The formalism is applicable to finite systems and for supercells using periodic boundary conditions within the Gamma-point approximation. They show that the methodology allows the application of modern computational techniques such as sparse matrix storage and massive parallelization in a straightforward way. All present bottlenecks concerning computer time and consumption of memory and memory bandwidth can be removed. They illustrate the performance of the method by direct comparison with Born-Oppenheimer molecular dynamics calculations. Water molecules, benzene, the C(60) fullerene, and liquid water have been selected as benchmark systems.

TRAF1 regulates recruitment of lymphocytes and, to a lesser extent, neutrophils, myeloid dendritic cells and monocytes to the lung airways following lipopolysaccharide inhalation.

Inhaled lipopolysaccharide (LPS) induces an inflammatory response that may contribute to the pathogenesis of asthma and other airway diseases. Here we investigate the role of tumour necrosis factor (TNF) receptor-associated factor 1 (TRAF1) in leucocyte recruitment using a model of LPS-induced lung inflammation in mice. TRAF1(-/-) mice are completely deficient in the recruitment of lymphocytes to the lower respiratory tract after inhalation of LPS. Although TRAF1(-/-) mice display normal early accumulation of neutrophils, dendritic cells and monocytes in the alveolar airspace, they have a significantly reduced recruitment of these cells by 24 hr after inhalation of LPS when compared to wild-type (WT) mice. Despite normal expression of the pro-inflammatory cytokines TNF, interleukin-1 (IL-1) and IL-6 after LPS treatment, TRAF1(-/-) mice displayed decreased expression of intercellular adhesion molecule 1, vascular cell adhesion molecule 1, CCL17 and CCL20 in the lungs, when compared to LPS-treated WT mice. These results suggest that TRAF1 facilitates LPS-induced leucocyte recruitment into the lung airways by augmenting the expression of chemokines and adhesion molecules. Mice lacking TNF receptor 1 (TNFR1) but not TNFR2 show a phenotype similar to the TRAF1(-/-) mice, suggesting that TRAF1 may act downstream of TNFR1. Significantly, we use bone marrow chimeras to demonstrate that expression of TRAF1 by cells resident in the lungs, but not by circulating leucocytes, is necessary for efficient LPS-induced recruitment of leucocytes to the lung airways.

[Embryonic stem cells and epidermal commitment]. / Cellules souches embryonnaires et lignage épidermique.

Embryonic stem (ES) cells can be cultured indefinitely, differentiated into many cell types in vitro, thus providing a potentially unlimited supply of cells for cell-based therapy. We recently reported the efficient derivation of ectodermal and epidermal cells from murine ES cells. These differentiated ES cells are able to form, in culture, a multilayered epidermis coupled with an underlying dermal compartment, similar to native skin. This model demons- trates that ES cells have the potential to recapitulate the reciprocal instructive ectodermal-mesodermal commitments, characteristic of embryonic skin formation, clarifies the role of the morphogen BMP-4 in the binary neuroectodermal choice and provides a powerful tool for the study of molecular mechanisms controlling skin development and multipotent epidermal stem cell properties. Its potential for cutaneous cell therapy and dermatocosmetological applications is discussed.

Planar tetracoordinate carbons in cyclic hydrocarbons.

[structure: see text] A series of cyclic hydrocarbons containing a planar tetracoordinate carbon atom is proposed. To rationalize the electronic factors contributing to the stability of these molecules, an analysis of the molecular orbitals and the induced magnetic field is presented.

T cell-specific expression of the human TNF-alpha gene involves a functional and highly conserved chromatin signature in intron 3.

Using a phylogenetic approach, we identified highly conserved sequences within intron 3 of the human TNF-alpha gene. These sequences form cell type-specific DNase I hypersensitivity sites and display cell type-specific DNA-protein contacts in in vivo genomic footprints. Consistent with these results, intron 3 confers specific activity upon a TNF-alpha reporter gene in Jurkat T cells, but not THP-1 monocytic cells. Thus, using a combinatorial approach of phylogenetic analysis, DNase I hypersensitivity analysis, in vivo footprinting, and transfection analysis, we demonstrate that intronic regulatory elements are involved in the cell type-specific regulation of TNF-alpha gene expression.

Regulation of tumor necrosis factor alpha gene expression by mycobacteria involves the assembly of a unique enhanceosome dependent on the coactivator proteins CBP/p300.

Tumor necrosis factor alpha (TNF-alpha) plays an important role in host containment of infection by Mycobacterium tuberculosis, one of the leading causes of death by an infectious agent globally. Using the pathogenic M. tuberculosis strain H37Rv, we present evidence that upon stimulation of monocytic cells by M. tuberculosis a unique TNF-alpha enhanceosome is formed, and it is distinct from the TNF-alpha enhanceosome that forms in T cells stimulated by antigen engagement or virus infection. A distinct set of activators including ATF-2, c-jun, Ets, Sp1, Egr-1 and the coactivator proteins CBP/p300 are recruited to the TNF-alpha promoter after stimulation with M. tuberculosis. Furthermore, the formation of this enhanceosome is dependent on inducer-specific helical phasing relationships between transcription factor binding sites. We also show that the transcriptional activity of CBP/p300 is potentiated by mycobacterial stimulation of monocytes. The identification of TNF-alpha regulatory elements and coactivators involved in M. tuberculosis-stimulated gene expression thus provides potential selective molecular targets in the modulation of TNF-alpha gene expression in the setting of mycobacterial infection.