The neurovascular unit as a selective barrier to polymorphonuclear granulocyte (PMN) infiltration into the brain after ischemic injury

The migration of polymorphonuclear granulocytes (PMN) into the brain parenchyma and release of their abundant proteases are considered the main causes of neuronal cell death and reperfusion injury following ischemia. Yet, therapies targeting PMN egress have been largely ineffective. To address this discrepancy we investigated the temporo-spatial localization of PMNs early after transient ischemia in a murine transient middle cerebral artery occlusion (tMCAO) model and human stroke specimens. Using specific markers that distinguish PMN (Ly6G) from monocytes/macrophages (Ly6C) and that define the cellular and basement membrane boundaries of the neurovascular unit (NVU), histology and confocal microscopy revealed that virtually no PMNs entered the infarcted CNS parenchyma. Regardless of tMCAO duration, PMNs were mainly restricted to luminal surfaces or perivascular spaces of cerebral vessels. Vascular PMN accumulation showed no spatial correlation with increased vessel permeability, enhanced expression of endothelial cell adhesion molecules, platelet aggregation or release of neutrophil extracellular traps. Live cell imaging studies confirmed that oxygen and glucose deprivation followed by reoxygenation fail to induce PMN migration across a brain endothelial monolayer under flow conditions in vitro. The absence of PMN infiltration in infarcted brain tissues was corroborated in 25 human stroke specimens collected at early time points after infarction. Our observations identify the NVU rather than the brain parenchyma as the site of PMN action after CNS ischemia and suggest reappraisal of targets for therapies to reduce reperfusion injury after stroke.

A newly developed in vitro model of the human epithelial airway barrier to study the toxic potential of nanoparticles

The potential health effects of inhaled engineered nanoparticles are almost unknown. To avoid and replace toxicity studies with animals, a triple cell co-culture system composed of epithelial cells, macrophages and dendritic cells was established, which simulates the most important barrier functions of the epithelial airway. Using this model, the toxic potential of titanium dioxide was assessed by measuring the production of reactive oxygen species and the release of tumour necrosis factor alpha. The intracellular localisation of titanium dioxide nanoparticles was analyzed by energy filtering transmission electron microscopy. Titanium dioxide nanoparticles were detected as single particles without membranes and in membrane-bound agglomerates. Cells incubated with titanium dioxide particles showed an elevated production of reactive oxygen species but no increase of the release of tumour necrosis factor alpha. Our in vitro model of the epithelial airway barrier offers a valuable tool to study the interaction of particles with lung cells at a nanostructural level and to investigate the toxic potential of nanoparticles.

In vitro models of the human epithelial airway barrier to study the toxic potential of particulate matter

BACKGROUND: Several epidemiological studies show that inhalation of particulate matter may cause increased pulmonary morbidity and mortality. Of particular interest are the ultrafine particles that are particularly toxic. In addition more and more nanoparticles are released into the environment; however, the potential health effects of these nanoparticles are yet unknown. OBJECTIVES: To avoid particle toxicity studies with animals many cell culture models have been developed during the past years. METHODS: This review focuses on the most commonly used in vitro epithelial airway and alveolar models to study particle-cell interactions and particle toxicity and highlights advantages and disadvantages of the different models. RESULTS/CONCLUSION: There are many lung cell culture models but none of these models seems to be perfect. However, they might be a great tool to perform basic research or toxicity tests. The focus here is on 3D and co-culture models, which seem to be more realistic than monocultures.

The EVIDENT-trial: protocol and rationale of a multicenter randomized controlled trial testing the effectiveness of an online-based psychological intervention

BACKGROUND: Depressive disorders are among the leading causes of worldwide disability with mild to moderate forms of depression being particularly common. Low-intensity treatments such as online psychological treatments may be an effective way to treat mild to moderate depressive symptoms and prevent the emergence or relapse of major depression. METHODS/DESIGN: This study is a currently recruiting multicentre parallel-groups pragmatic randomized-controlled single-blind trial. A total of 1000 participants with mild to moderate symptoms of depression from various settings including in- and outpatient services will be randomized to an online psychological treatment or care as usual (CAU). We hypothesize that the intervention will be superior to CAU in reducing depressive symptoms assessed with the Personal Health Questionnaire (PHQ-9, primary outcome measure) following the intervention (12 wks) and at follow-up (24 and 48 wks). Further outcome parameters include quality of life, use of health care resources and attitude towards online psychological treatments. DISCUSSION: The study will yield meaningful answers to the question of whether online psychological treatment can contribute to the effective and efficient prevention and treatment of mild to moderate depression on a population level with a low barrier to entry. TRIAL REGISTRATION: Trial Registration Number: NCT01636752.

Experimental adaptation of wild-type canine distemper virus (CDV) to the human entry receptor CD150.

Canine distemper virus (CDV), a close relative of measles virus (MV), is widespread and well known for its broad host range. When the goal of measles eradication may be achieved, and when measles vaccination will be stopped, CDV might eventually cross the species barrier to humans and emerge as a new human pathogen. In order to get an impression how fast such alterations may occur, we characterized required adaptive mutations to the human entry receptors CD150 (SLAM) and nectin-4 as first step to infect human target cells. Recombinant wild-type CDV-A75/17(red) adapted quickly to growth in human H358 epithelial cells expressing human nectin-4. Sequencing of the viral attachment proteins (hemagglutinin, H, and fusion protein, F) genes revealed that no adaptive alteration was required to utilize human nectin-4. In contrast, the virus replicated only to low titres (10(2) pfu/ml) in Vero cells expressing human CD150 (Vero-hSLAM). After three passages using these cells virus was adapted to human CD150 and replicated to high titres (10(5) pfu/ml). Sequence analyses revealed that only one amino acid exchange in the H-protein at position 540 Asp→Gly (D540G) was required for functional adaptation to human CD150. Structural modelling suggests that the adaptive mutation D540G in H reflects the sequence alteration from canine to human CD150 at position 70 and 71 from Pro to Leu (P70L) and Gly to Glu (G71E), and compensates for the gain of a negative charge in the human CD150 molecule. Using this model system our data indicate that only a minimal alteration, in this case one adaptive mutation, is required for adaptation of CDV to the human entry receptors, and help to understand the molecular basis why this adaptive mutation occurs.

New exposure system to evaluate the toxicity of (scooter) exhaust emissions in lung cells in vitro

A constantly growing number of scooters produce an increasing amount of potentially harmful emissions. Due to their engine technology, two-stroke scooters emit huge amounts of adverse substances, which can induce adverse pulmonary and cardiovascular health effects. The aim of this study was to develop a system to expose a characterized triple cell coculture model of the human epithelial airway barrier, to freshly produced and characterized total scooter exhaust emissions. In exposure chambers, cell cultures were exposed for 1 and 2 h to 1:100 diluted exhaust emissions and in the reference chamber to filtered ambient air, both controlled at 5% CO(2), 85% relative humidity, and 37 degrees C. The postexposure time was 0-24 h. Cytotoxicity, used to validate the exposure system, was significantly increased in exposed cell cultures after 8 h postexposure time. (Pro-) inflammatory chemo- and cytokine concentrations in the medium of exposed cells were significantly higher at the 12 h postexposure time point. It was shown that the described exposure system (with 2 h exposure duration, 8 and 24 h postexposure time, dilution of 1:100, flow of 2 L/min as optimal exposure conditions) can be used to evaluate the toxic potential of total exhaust emissions.

Xenograft rejection: IgG1, complement and NK cells team up to activate and destroy the endothelium

Acute vascular rejection represents a formidable barrier to clinical xenotransplantation and it is known that this type of rejection can also be initiated by xenoreactive antibodies that have limited complement-activating ability. Using a sophisticated mouse model, a recent study has provided in vivo evidence for the existence of an IgG(1)-mediated vascular rejection, which uniquely depends on both the activation of complement and interactions with FcgammaRIII on natural killer (NK) cells.

Effects of HIV type-1 immune selection on susceptability to integrase inhibitor resistance

BACKGROUND: All site-specific interactions between HIV type-1 (HIV-1) subtype, human leukocyte antigen (HLA)-associated immune selection and integrase inhibitor resistance are not completely understood. We examined naturally occurring polymorphisms in HIV-1 integrase sequences from 342 antiretroviral-naive individuals from the Western Australian HIV Cohort Study and the Swiss HIV Cohort Study. METHODS: Standard bulk sequencing and sequence-based typing were used to generate integrase sequences and high-resolution HLA genotypes, respectively. Viral residues were examined with respect to drug resistance mutations and CD8(+) T-cell escape mutations. RESULTS: In both predominantly subtype B cohorts, 12 of 38 sites that mediate integrase inhibitor resistance mutations were absolutely conserved, and these included the primary resistance mutations. There were 18 codons with non-primary drug resistance-associated substitutions at rates of up to 58.8% and eight sites with alternative polymorphisms. Five viral residues were potentially subject to dual-drug and HLA-associated immune selection in which both selective pressures either drove the same amino acid substitution (codons 72, 157 and 163) or HLA alleles were associated with an alternative polymorphism that would alter the genetic barrier to resistance (codons 125 and 193). The common polymorphism T125A, which was characteristic of non-subtype B and was also associated with carriage of HLA-B*57/*5801, increased the mutational barrier to the resistance mutation T125K. CONCLUSIONS: Primary integrase inhibitor resistance mutations were not detected in the absence of drug exposure in keeping with sites of high constraint. Viral polymorphisms caused by immune selection and/or associated with non-subtype B might alter the genetic barrier to some non-primary resistance-associated mutations.

The cobblers stick to their lasts: pollinators prefer native over alien plant species in a multi-species experiment

The majority of plant species rely, at least partly, on animals for pollination. Our knowledge on whether pollinator visitation differs between native and alien plant species, and between invasive and non-invasive alien species is still limited. Additionally, because numerous invasive plant species are escapees from horticulture, the transition from human-assisted occurrence in urbanized habitats to unassisted persistence and spread in (semi-)natural habitats requires study. To address whether pollinator visitation differs between native, invasive alien and non-invasive alien species, we did pollinator observations for a total of 17 plant species representing five plant families. To test whether pollinator visitation to the three groups of species during the initial stage of invasion depends on habitat type, we did the study in three urbanized habitats and three semi-natural grasslands, using single potted plants. Native plants had more but smaller flower units than alien plants, and invasive alien plants had more but smaller flowers than non-invasive alien plants. After accounting for these differences in floral display, pollinator visitation was higher for native than for alien plant species, but did not differ between invasive and non-invasive alien plant species. Pollinator visitation was on average higher in semi-natural than in urbanized habitats, irrespective of origin or status of the plant species. This might suggest that once an alien species has managed to escape from urbanized into more natural habitats, pollinator limitation will not be a major barrier to establishment and invasion.

Mechanism for active membrane fusion triggering by morbillivirus attachment protein.

The paramyxovirus entry machinery consists of two glycoproteins that tightly cooperate to achieve membrane fusion for cell entry: the tetrameric attachment protein (HN, H, or G, depending on the paramyxovirus genus) and the trimeric fusion protein (F). Here, we explore whether receptor-induced conformational changes within morbillivirus H proteins promote membrane fusion by a mechanism requiring the active destabilization of prefusion F or by the dissociation of prefusion F from intracellularly preformed glycoprotein complexes. To properly probe F conformations, we identified anti-F monoclonal antibodies (MAbs) that recognize conformation-dependent epitopes. Through heat treatment as a surrogate for H-mediated F triggering, we demonstrate with these MAbs that the morbillivirus F trimer contains a sufficiently high inherent activation energy barrier to maintain the metastable prefusion state even in the absence of H. This notion was further validated by exploring the conformational states of destabilized F mutants and stabilized soluble F variants combined with the use of a membrane fusion inhibitor (3g). Taken together, our findings reveal that the morbillivirus H protein must lower the activation energy barrier of metastable prefusion F for fusion triggering.

Nonsense-mediated mRNA decay (NMD) restricts replication of mammalian RNA viruses

A genome-wide siRNA screen against host factors that affect the infection of Semliki Forest virus (SFV), a positive-strand (+)RNA virus, revealed that components of the nonsense-mediated mRNA decay (NMD) pathway restrict early, post-entry steps of the infection cycle. In HeLa cells and primary human fibroblasts, knockdown of UPF1, SMG5 and SMG7 leads to increased levels of viral proteins and RNA and to higher titers of released virus. The inhibitory effect of NMD was stronger when the efficiency of virus replication was impaired by mutations or deletions in the replicase proteins. Accordingly, impairing NMD resulted in a more than 20-fold increased production of these attenuated viruses. Our data suggest that intrinsic features of genomic and sub-genomic viral mRNAs, most likely the extended 3'-UTR length, make them susceptible to NMD. The fact that SFV replication is entirely cytoplasmic strongly suggests that degradation of the viral RNA occurs through the exon junction complex (EJC)-independent mode of NMD. Collectively, our findings uncover a new biological function for NMD as an intrinsic barrier to the translation of early viral proteins and the amplification of (+)RNA viruses in animal cells. Thus, in addition to its role in mRNA surveillance and post-transcriptional gene regulation, NMD also contributes to protect cells from RNA viruses.

Using multiple landscape genetic approaches to test the validity of genetic clusters in a species characterized by an isolation-by-distance pattern

Bayesian clustering methods are typically used to identify barriers to gene flow, but they are prone to deduce artificial subdivisions in a study population characterized by an isolation-by-distance pattern (IbD). Here we analysed the landscape genetic structure of a population of wild boars (Sus scrofa) from south-western Germany. Two clustering methods inferred the presence of the same genetic discontinuity. However, the population in question was characterized by a strong IbD pattern. While landscape-resistance modelling failed to identify landscape features that influenced wild boar movement, partial Mantel tests and multiple regression of distance matrices (MRDMs) suggested that the empirically inferred clusters were separated by a genuine barrier. When simulating random lines bisecting the study area, 60% of the unique barriers represented, according to partial Mantel tests and MRDMs, significant obstacles to gene flow. By contrast, the random-lines simulation showed that the boundaries of the inferred empirical clusters corresponded to the most important genetic discontinuity in the study area. Given the degree of habitat fragmentation separating the two empirical partitions, it is likely that the clustering programs correctly identified a barrier to gene flow. The differing results between the work published here and other studies suggest that it will be very difficult to draw general conclusions about habitat permeability in wild boar from individual studies.

DNase 2 is the main DNA-degrading enzyme of the stratum corneum

The cornified layer, the stratum corneum, of the epidermis is an efficient barrier to the passage of genetic material, i.e. nucleic acids. It contains enzymes that degrade RNA and DNA which originate from either the living part of the epidermis or from infectious agents of the environment. However, the molecular identities of these nucleases are only incompletely known at present. Here we performed biochemical and genetic experiments to determine the main DNase activity of the stratum corneum. DNA degradation assays and zymographic analyses identified the acid endonucleases L-DNase II, which is derived from serpinB1, and DNase 2 as candidate DNases of the cornified layer of the epidermis. siRNA-mediated knockdown of serpinB1 in human in vitro skin models and the investigation of mice deficient in serpinB1a demonstrated that serpinB1-derived L-DNase II is dispensable for epidermal DNase activity. By contrast, knockdown of DNase 2, also known as DNase 2a, reduced DNase activity in human in vitro skin models. Moreover, the genetic ablation of DNase 2a in the mouse was associated with the lack of acid DNase activity in the stratum corneum in vivo. The degradation of endogenous DNA in the course of cornification of keratinocytes was not impaired by the absence of DNase 2. Taken together, these data identify DNase 2 as the predominant DNase on the mammalian skin surface and indicate that its activity is primarily targeted to exogenous DNA.