Synchronized turbo apoptosis induced by cold-shock

In our research on the role of apoptosis in the pathogenesis of the autoimmune disease systemic lupus erythematosus (SLE), we aim to evaluate the effects of early and late apoptotic cells and blebs on antigen presenting cells. This requires the in vitro generation of sufficiently large and homogeneous populations of early and late apoptotic cells. Here, we present a quick method encountered by serendipity that results in highly reproducible synchronized homogeneous apoptotic cell populations. In brief, granulocytic 32Dcl3 cells are incubated on ice for 2 h and subsequently rewarmed at 37°C. After 30–90 min at 37°C more than 80–90% of the cells become early apoptotic (Annexin V positive/propidium iodide negative). After 24 h of rewarming at 37°C 98% of the cells were late apoptotic (secondary necrotic; Annexin V positive/propidium iodide positive). Cells already formed apoptotic blebs at their cell surface after approximately 20 min at 37°C. Inter-nucleosomal chromatin cleavage and caspase activation were other characteristics of this cold-shock-induced process of apoptosis. Consequently, apoptosis could be inhibited by a caspase inhibitor. Finally, SLE-derived anti-chromatin autoantibodies showed a high affinity for apoptotic blebs generated by cold-shock. Overall, cold-shock induced apoptosis is achieved without the addition of toxic compounds or antibodies, and quickly leads to synchronized homogeneous apoptotic cell populations, which can be applied for various research questions addressing apoptosis

Evaluation of risk of falls and orthostatic hypotension in older, long-term topical beta-blocker users

Background: Falls are a serious problem in the elderly, and have recently been described as cardiovascular-mediated side effects of beta-blocker eye drops. Therefore, we investigated the possible association between the long-term use of beta-blockers, prostaglandins and their combinations in eye drops, and falls, dizziness and orthostatic hypotension in older patients. Methods: All participants were long-term users of eye drops containing beta-blockers, prostaglandins or their combinations. They underwent a structured falls interview and blood pressure measurement for testing of orthostatic hypotension. The odds ratio for presence of orthostatic hypotension or a positive falls history according to use of beta-blocker eye drops was calculated with a binary logistic regression analysis. The main outcome measures were a positive falls history and the presence of orthostatic hypotension. Results: In total, 148 of 286 subjects participated. After adjustment for age, gender, and use of fall-risk-increasing drugs other than beta-blocker eye drops, we found no significant difference in fall risk [odds ratio (OR): 0.60; 95% confidence interval (CI): 0.268-1.327] between patients using ophthalmic beta-blockers or a combination of ophthalmic beta-blockers and prostaglandins, and patients using ophthalmic prostaglandins only. Although prevalence of orthostatic hypotension was higher in the beta-blocker group (OR: 1.67; 95% CI: 0.731-3.793) compared to the prostaglandin group, this was a non-significant difference. Conclusions: In our study, we did not find a significant association between long-term use of beta-blockers eye drops and falls, dizziness or orthostatic hypotension in older ophthalmic outpatients, compared to long-term use of prostaglandin eye drops

Complexity of the Inoculum Determines the Rate of Reversion of SIV Gag CD8 T Cell Mutant Virus and Outcome of Infection

Escape mutant (EM) virus that evades CD8+ T cell recognition is frequently observed following infection with HIV-1 or SIV. This EM virus is often less replicatively “fit” compared to wild-type (WT) virus, as demonstrated by reversion to WT upon transmission of HIV to a naïve host and the association of EM virus with lower viral load in vivo in HIV-1 infection. The rate and timing of reversion is, however, highly variable. We quantified reversion to WT of a series of SIV and SHIV viruses containing minor amounts of WT virus in pigtail macaques using a sensitive PCR assay. Infection with mixes of EM and WT virus containing ≥10% WT virus results in immediate and rapid outgrowth of WT virus at SIV Gag CD8 T cell epitopes within 7 days of infection of pigtail macaques with SHIV or SIV. In contrast, infection with biologically passaged SHIVmn229 viruses with much smaller proportions of WT sequence, or a molecular clone of pure EM SIVmac239, demonstrated a delayed or slow pattern of reversion. WT virus was not detectable until ≥8 days after inoculation and took ≥8 weeks to become the dominant quasispecies. A delayed pattern of reversion was associated with significantly lower viral loads. The diversity of the infecting inoculum determines the timing of reversion to WT virus, which in turn predicts the outcome of infection. The delay in reversion of fitness-reducing CD8 T cell escape mutations in some scenarios suggests opportunities to reduce the pathogenicity of HIV during very early infection

Co-expressed immune and metabolic genes in visceral and subcutaneous adipose tissue from severely obese individuals are associated with plasma HDL and glucose levels: a microarray study

<p>Abstract</p> <p>Background</p> <p>Excessive accumulation of body fat, in particular in the visceral fat depot, is a major risk factor to develop a variety of diseases such as type 2 diabetes. The mechanisms underlying the increased risk of obese individuals to develop co-morbid diseases are largely unclear.</p> <p>We aimed to identify genes expressed in subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) that are related to blood parameters involved in obesity co-morbidity, such as plasma lipid and glucose levels, and to compare gene expression between the fat depots.</p> <p>Methods</p> <p>Whole-transcriptome SAT and VAT gene expression levels were determined in 75 individuals with a BMI >35 kg/m². Modules of co-expressed genes likely to be functionally related were identified and correlated with BMI, plasma levels of glucose, insulin, HbA_1c, triglycerides, non-esterified fatty acids, ALAT, ASAT, C-reactive protein, and LDL- and HDL cholesterol.</p> <p>Results</p> <p>Of the approximately 70 modules identified in SAT and VAT, three SAT modules were inversely associated with plasma HDL-cholesterol levels, and a fourth module was inversely associated with both plasma glucose and plasma triglyceride levels (p < 5.33 × 10^-5). These modules were markedly enriched in immune and metabolic genes. In VAT, one module was associated with both BMI and insulin, and another with plasma glucose (p < 4.64 × 10^-5). This module was also enriched in inflammatory genes and showed a marked overlap in gene content with the SAT modules related to HDL. Several genes differentially expressed in SAT and VAT were identified.</p> <p>Conclusions</p> <p>In obese subjects, groups of co-expressed genes were identified that correlated with lipid and glucose metabolism parameters; they were enriched with immune genes. A number of genes were identified of which the expression in SAT correlated with plasma HDL cholesterol, while their expression in VAT correlated with plasma glucose. This underlines both the singular importance of these genes for lipid and glucose metabolism and the specific roles of these two fat depots in this respect.</p

The Rotterdam Study: objectives and design update

The Rotterdam Study is a prospective cohort study ongoing since 1990 in the city of Rotterdam in the Netherlands. The study targets cardiovascular, neurological, ophthalmological and endocrine diseases. As of 2008 about 15,000 subjects aged 45 years or over comprise the Rotterdam Study cohort. The findings of the Rotterdam Study have been presented in some 600 research articles and reports (see http://www.epib.nl/rotterdamstudy). This article gives the reasons for the study and its design. It also presents a summary of the major findings and an update of the objectives and methods

Dogs Leaving the ICU Carry a Very Large Multi-Drug Resistant Enterococcal Population with Capacity for Biofilm Formation and Horizontal Gene Transfer

The enterococcal community from feces of seven dogs treated with antibiotics for 2–9 days in the veterinary intensive care unit (ICU) was characterized. Both, culture-based approach and culture-independent 16S rDNA amplicon 454 pyrosequencing, revealed an abnormally large enterococcal community: 1.4±0.8×108 CFU gram−1 of feces and 48.9±11.5% of the total 16,228 sequences, respectively. The diversity of the overall microbial community was very low which likely reflects a high selective antibiotic pressure. The enterococcal diversity based on 210 isolates was also low as represented by Enterococcus faecium (54.6%) and Enterococcus faecalis (45.4%). E. faecium was frequently resistant to enrofloxacin (97.3%), ampicillin (96.5%), tetracycline (84.1%), doxycycline (60.2%), erythromycin (53.1%), gentamicin (48.7%), streptomycin (42.5%), and nitrofurantoin (26.5%). In E. faecalis, resistance was common to tetracycline (59.6%), erythromycin (56.4%), doxycycline (53.2%), and enrofloxacin (31.9%). No resistance was detected to vancomycin, tigecycline, linezolid, and quinupristin/dalfopristin in either species. Many isolates carried virulence traits including gelatinase, aggregation substance, cytolysin, and enterococcal surface protein. All E. faecalis strains were biofilm formers in vitro and this phenotype correlated with the presence of gelE and/or esp. In vitro intra-species conjugation assays demonstrated that E. faecium were capable of transferring tetracycline, doxycycline, streptomycin, gentamicin, and erythromycin resistance traits to human clinical strains. Multi-locus variable number tandem repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE) of E. faecium strains showed very low genotypic diversity. Interestingly, three E. faecium clones were shared among four dogs suggesting their nosocomial origin. Furthermore, multi-locus sequence typing (MLST) of nine representative MLVA types revealed that six sequence types (STs) originating from five dogs were identical or closely related to STs of human clinical isolates and isolates from hospital outbreaks. It is recommended to restrict close physical contact between pets released from the ICU and their owners to avoid potential health risks

The role of neutralizing antibodies in prevention of HIV-1 infection: what can we learn from the mother-to-child transmission context?

International audienceIn most viral infections, protection through existing vaccines is linked to the presence of vaccine-induced neutralizing antibodies (NAbs). However, more than 30 years after the identification of AIDS, the design of an immunogen able to induce antibodies that would neutralize the highly diverse HIV-1 variants remains one of the most puzzling challenges of the human microbiology. The role of antibodies in protection against HIV-1 can be studied in a natural situation that is the mother-to-child transmission (MTCT) context. Indeed, at least at the end of pregnancy, maternal antibodies of the IgG class are passively transferred to the fetus protecting the neonate from new infections during the first weeks or months of life. During the last few years, strong data, presented in this review, have suggested that some NAbs might confer protection toward neonatal HIV-1 infection. In cases of transmission, it has been shown that the viral population that is transmitted from the mother to the infant is usually homogeneous, genetically restricted and resistant to the maternal HIV-1-specific antibodies. Although the breath of neutralization was not associated with protection, it has not been excluded that NAbs toward specific HIV-1 strains might be associated with a lower rate of MTCT. A better identification of the antibody specificities that could mediate protection toward MTCT of HIV-1 would provide important insights into the antibody responses that would be useful for vaccine development. The most convincing data suggesting that NAbs migh confer protection against HIV-1 infection have been obtained by experiments of passive immunization of newborn macaques with the first generation of human monoclonal broadly neutralizing antibodies (HuMoNAbs). However, these studies, which included only a few selected subtype B challenge viruses, provide data limited to protection against a very restricted number of isolates and therefore have limitations in addressing the hypervariability of HIV-1. The recent identification of highly potent second-generation cross-clade HuMoNAbs provides a new opportunity to evaluate the efficacy of passive immunization to prevent MTCT of HIV-1

Electron spin resonance in membrane research: protein–lipid interactions from challenging beginnings to state of the art

Conventional electron paramagnetic resonance (EPR) spectra of lipids that are spin-labelled close to the terminal methyl end of the acyl chains are able to resolve the lipids directly contacting the protein from those in the fluid bilayer regions of the membrane. This allows determination of both the stoichiometry of lipid–protein interaction (i.e., number of lipid sites at the protein perimeter) and the selectivity of the protein for different lipid species (i.e., association constants relative to the background lipid). Spin-label EPR data are summarised for 20 or more different transmembrane peptides and proteins, and 7 distinct species of lipids. Lineshape simulations of the two-component conventional spin-label EPR spectra allow estimation of the rate at which protein-associated lipids exchange with those in the bulk fluid regions of the membrane. For lipids that do not display a selectivity for the protein, the intrinsic off-rates for exchange are in the region of 10 MHz: less than 10× slower than the rates of diffusive exchange in fluid lipid membranes. Lipids with an affinity for the protein, relative to the background lipid, have off-rates for leaving the protein that are correspondingly slower. Non-linear EPR, which depends on saturation of the spectrum at high radiation intensities, is optimally sensitive to dynamics on the timescale of spin-lattice relaxation, i.e., the microsecond regime. Both progressive saturation and saturation transfer EPR experiments provide definitive evidence that lipids at the protein interface are exchanging on this timescale. The sensitivity of non-linear EPR to low frequencies of spin exchange also allows the location of spin-labelled membrane protein residues relative to those of spin-labelled lipids, in double-labelling experiments

Recurrent Signature Patterns in HIV-1 B Clade Envelope Glycoproteins Associated with either Early or Chronic Infections

Here we have identified HIV-1 B clade Envelope (Env) amino acid signatures from early in infection that may be favored at transmission, as well as patterns of recurrent mutation in chronic infection that may reflect common pathways of immune evasion. To accomplish this, we compared thousands of sequences derived by single genome amplification from several hundred individuals that were sampled either early in infection or were chronically infected. Samples were divided at the outset into hypothesis-forming and validation sets, and we used phylogenetically corrected statistical strategies to identify signatures, systematically scanning all of Env. Signatures included single amino acids, glycosylation motifs, and multi-site patterns based on functional or structural groupings of amino acids. We identified signatures near the CCR5 co-receptor-binding region, near the CD4 binding site, and in the signal peptide and cytoplasmic domain, which may influence Env expression and processing. Two signatures patterns associated with transmission were particularly interesting. The first was the most statistically robust signature, located in position 12 in the signal peptide. The second was the loss of an N-linked glycosylation site at positions 413–415; the presence of this site has been recently found to be associated with escape from potent and broad neutralizing antibodies, consistent with enabling a common pathway for immune escape during chronic infection. Its recurrent loss in early infection suggests it may impact fitness at the time of transmission or during early viral expansion. The signature patterns we identified implicate Env expression levels in selection at viral transmission or in early expansion, and suggest that immune evasion patterns that recur in many individuals during chronic infection when antibodies are present can be selected against when the infection is being established prior to the adaptive immune response

Gender differences in the use of cardiovascular interventions in HIV-positive persons; the D:A:D Study

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