Defining the molecular role of gp91phox in the immune manifestation of acute allergic asthma using a preclinical murine model

<p>Abstract</p> <p>Objective</p> <p>The phenomena manifested during inflammation require interplay between circulating effector cells, local resident cells, soluble mediators and genetic host factors to establish, develop and maintain itself. Of the molecues involed in the initiation and perpetuation of acute allergic inflammation in asthma, the involvement of effector cells in redox reactions for producing O₂^-(superoxide anion) through the mediation of NADPH oxidase is a critical step. Prior data suggest that reactive oxygen species (ROS) produced by NADPH oxidase homologues in non-phagocytic cells play an important role in the regulation of signal transduction, while macrophages use a membrane-associated NADPH oxidase to generate an array of oxidizing intermediates which inactivate MMPs on or near them.</p> <p>Materials and Methods and Treatment</p> <p>To clarify the role of gp91phox subunit of NADPH oxidase in the development and progression of an acute allergic asthma phenotype, we induced allergen dependent inflammation in a gp91<it>^phox</it>-/- single knockout and a gp91phox-/-MMP-12-/- double knockout mouse models.</p> <p>Results</p> <p>In the knockout mice, both inflammation and airway hyperreactivity were more extensive than in wildtype mice post-OVA. Although OVA-specific IgE in plasma were comparable in wildtype and knockout mice, enhanced inflammatory cell recruitment from circulation and cytokine release in lung and BALf, accompanied by higher airway resistance as well as Penh in response to methacholine, indicate a regulatory role for NADPH oxidase in development of allergic asthma. While T cell mediated functions like Th2 cytokine secretion, and proliferation to OVA were upregulated synchronous with the overall robustness of the asthma phenotype, macrophage upregulation in functions such as proliferation, and mixed lymphocyte reaction indicate a regulatory role for gp91phox and an overall non-involvement or synergistic involvement of MMP12 in the response pathway (comparing data from gp91phox-/- and gp91phox-/-MMP-12-/- mice).</p

Epigenomic analysis of aberrantly methylated genes in colorectal cancer identifies genes commonly affected by epigenetic alterations.

Methylation profiling based on bead-chip arrays is an effective method for screening aberrantly methylated genes in CRC. In addition, we identified novel methylated genes that are candidate diagnostic or prognostic markers for CRC

Shunting Inhibition Controls the Gain Modulation Mediated by Asynchronous Neurotransmitter Release in Early Development

The sensitivity of a neuron to its input can be modulated in several ways. Changes in the slope of the neuronal input-output curve depend on factors such as shunting inhibition, background noise, frequency-dependent synaptic excitation, and balanced excitation and inhibition. However, in early development GABAergic interneurons are excitatory and other mechanisms such as asynchronous transmitter release might contribute to regulating neuronal sensitivity. We modeled both phasic and asynchronous synaptic transmission in early development to study the impact of activity-dependent noise and short-term plasticity on the synaptic gain. Asynchronous release decreased or increased the gain depending on the membrane conductance. In the high shunt regime, excitatory input due to asynchronous release was divisive, whereas in the low shunt regime it had a nearly multiplicative effect on the firing rate. In addition, sensitivity to correlated inputs was influenced by shunting and asynchronous release in opposite ways. Thus, asynchronous release can regulate the information flow at synapses and its impact can be flexibly modulated by the membrane conductance

Bacteriophage Lysin Mediates the Binding of Streptococcus mitis to Human Platelets through Interaction with Fibrinogen

The binding of bacteria to human platelets is a likely central mechanism in the pathogenesis of infective endocarditis. We have previously found that platelet binding by Streptococcus mitis SF100 is mediated by surface components encoded by a lysogenic bacteriophage, SM1. We now demonstrate that SM1-encoded lysin contributes to platelet binding via its direct interaction with fibrinogen. Far Western blotting of platelets revealed that fibrinogen was the major membrane-associated protein bound by lysin. Analysis of lysin binding with purified fibrinogen in vitro confirmed that these proteins could bind directly, and that this interaction was both saturable and inhibitable. Lysin bound both the Aα and Bβ chains of fibrinogen, but not the γ subunit. Binding of lysin to the Bβ chain was further localized to a region within the fibrinogen D fragment. Disruption of the SF100 lysin gene resulted in an 83±3.1% reduction (mean ± SD) in binding to immobilized fibrinogen by this mutant strain (PS1006). Preincubation of this isogenic mutant with purified lysin restored fibrinogen binding to wild type levels. When tested in a co-infection model of endocarditis, loss of lysin expression resulted in a significant reduction in virulence, as measured by achievable bacterial densities (CFU/g) within vegetations, kidneys, and spleens. These results indicate that bacteriophage-encoded lysin is a multifunctional protein, representing a new class of fibrinogen-binding proteins. Lysin appears to be cell wall-associated through its interaction with choline. Once on the bacterial surface, lysin can bind fibrinogen directly, which appears to be an important interaction for the pathogenesis of endocarditis

Genomic analysis and relatedness of P2-like phages of the Burkholderia cepacia complex

<p>Abstract</p> <p>Background</p> <p>The <it>Burkholderia cepacia </it>complex (BCC) is comprised of at least seventeen Gram-negative species that cause infections in cystic fibrosis patients. Because BCC bacteria are broadly antibiotic resistant, phage therapy is currently being investigated as a possible alternative treatment for these infections. The purpose of our study was to sequence and characterize three novel BCC-specific phages: KS5 (vB_BceM-KS5 or vB_BmuZ-ATCC 17616), KS14 (vB_BceM-KS14) and KL3 (vB_BamM-KL3 or vB_BceZ-CEP511).</p> <p>Results</p> <p>KS5, KS14 and KL3 are myoviruses with the A1 morphotype. The genomes of these phages are between 32317 and 40555 base pairs in length and are predicted to encode between 44 and 52 proteins. These phages have over 50% of their proteins in common with enterobacteria phage P2 and so can be classified as members of the <it>Peduovirinae </it>subfamily and the "P2-like viruses" genus. The BCC phage proteins similar to those encoded by P2 are predominantly structural components involved in virion morphogenesis. As prophages, KS5 and KL3 integrate into an AMP nucleosidase gene and a threonine tRNA gene, respectively. Unlike other P2-like viruses, the KS14 prophage is maintained as a plasmid. The P2 <it>E+E' </it>translational frameshift site is conserved among these three phages and so they are predicted to use frameshifting for expression of two of their tail proteins. The <it>lysBC </it>genes of KS14 and KL3 are similar to those of P2, but in KS5 the organization of these genes suggests that they may have been acquired via horizontal transfer from a phage similar to λ. KS5 contains two sequence elements that are unique among these three phages: an IS<it>Bmu</it>2-like insertion sequence and a reverse transcriptase gene. KL3 encodes an EcoRII-C endonuclease/methylase pair and Vsr endonuclease that are predicted to function during the lytic cycle to cleave non-self DNA, protect the phage genome and repair methylation-induced mutations.</p> <p>Conclusions</p> <p>KS5, KS14 and KL3 are the first BCC-specific phages to be identified as P2-like. As KS14 has previously been shown to be active against <it>Burkholderia cenocepacia in vivo</it>, genomic characterization of these phages is a crucial first step in the development of these and similar phages for clinical use against the BCC.</p

Funduscopy in Adult Zebrafish and Its Application to Isolate Mutant Strains with Ocular Defects

Funduscopy is one of the most commonly used diagnostic tools in the ophthalmic practice, allowing for a ready assessment of pathological changes in the retinal vasculature and the outer retina. This non-invasive technique has so far been rarely used in animal model for ophthalmic diseases, albeit its potential as a screening assay in genetic screens. The zebrafish (Danio rerio) is well suited for such genetic screens for ocular alterations. Therefore we developed funduscopy in adult zebrafish and employed it as a screening tool to find alterations in the anterior segment and the fundus of the eye of genetically modified adult animals. A stereomicroscope with coaxial reflected light illumination was used to obtain fundus color images of the zebrafish. In order to find lens and retinal alterations, a pilot screen of 299 families of the F3 generation of ENU-treated adult zebrafish was carried out. Images of the fundus of the eye and the anterior segment can be rapidly obtained and be used to identify alterations in genetically modified animals. A number of putative mutants with cataracts, defects in the cornea, eye pigmentation, ocular vessels and retina were identified. This easily implemented method can also be used to obtain fundus images from rodent retinas. In summary, we present funduscopy as a valuable tool to analyse ocular abnormalities in adult zebrafish and other small animal models. A proof of principle screen identified a number of putative mutants, making funduscopy based screens in zebrafish feasible

ICAR: endoscopic skull‐base surgery

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Outcomes and treatment strategies for autoimmunity and hyperinflammation in patients with RAG deficiency

BACKGROUND: While autoimmunity and hyperinflammation secondary to recombinase activating gene (RAG) deficiency have been associated with delayed diagnosis and even death, our current understanding is limited primarily to small case series. OBJECTIVE: Understand the frequency, severity, and treatment responsiveness of autoimmunity and hyperinflammation in RAG deficiency. METHODS: In reviewing the literature and our own database, we identified 85 patients with RAG deficiency, reported between 2001 and 2016, and compiled the largest case series to date of 63 patients with prominent autoimmune and/or hyperinflammatory pathology. RESULTS: Diagnosis of RAG deficiency was delayed a median of 5 years from the first clinical signs of immune dysregulation. The majority of patients (55.6%) presented with more than one autoimmune or hyperinflammatory complication, with the most common etiologies being cytopenias (84.1%), granulomas (23.8%), and inflammatory skin disorders (19.0%). Infections, including live viral vaccinations, closely preceded the onset of autoimmunity in 28.6% of cases. Autoimmune cytopenias had early onset (median 1.9, 2.1, and 2.6 years for autoimmune hemolytic anemia (AIHA), immune thrombocytopenia (ITP) and autoimmune neutropenia (AN), respectively) and were refractory to intravenous immunoglobulin, steroids, and rituximab in the majority of cases (64.7%, 73.7%, and 71.4% for AIHA, ITP, and AN, respectively). Evans syndrome specifically was associated with lack of response to first-line therapy. Treatment-refractory autoimmunity/hyperinflammation prompted hematopoietic stem cell transplantation in 20 patients. CONCLUSIONS: Autoimmunity/hyperinflammation can be a presenting sign of RAG deficiency and should prompt further evaluation. Multi-lineage cytopenias are often refractory to immunosuppressive treatment and may require hematopoietic cell transplantation for definitive management