Granulocyte-macrophage colony stimulatory factor enhances the pro-inflammatory response of interferon-γ-treated macrophages to pseudomonas aeruginosa infection

Pseudomonas aeruginosa is an opportunistic pathogen that can cause severe infections at compromised epithelial surfaces, such those found in burns, wounds, and in lungs damaged by mechanical ventilation or recurrent infections, particularly in cystic fibrosis (CF) patients. CF patients have been proposed to have a Th2 and Th17-biased immune response suggesting that the lack of Th1 and/or over exuberant Th17 responses could contribute to the establishment of chronic P. aeruginosa infection and deterioration of lung function. Accordingly, we have observed that interferon (IFN)-γ production by peripheral blood mononuclear cells from CF patients positively correlated with lung function, particularly in patients chronically infected with P. aeruginosa. In contrast, IL-17A levels tended to correlate negatively with lung function with this trend becoming significant in patients chronically infected with P. aeruginosa. These results are in agreement with IFN-γ and IL-17A playing protective and detrimental roles, respectively, in CF. In order to explore the protective effect of IFN-γ in CF, the effect of IFN-γ alone or in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF), on the ability of human macrophages to control P. aeruginosa growth, resist the cytotoxicity induced by this bacterium or promote inflammation was investigated. Treatment of macrophages with IFN-γ, in the presence and absence of GM-CSF, failed to alter bacterial growth or macrophage survival upon P. aeruginosa infection, but changed the inflammatory potential of macrophages. IFN-γ caused up-regulation of monocyte chemoattractant protein-1 (MCP-1) and TNF-α and down-regulation of IL-10 expression by infected macrophages. GM-CSF in combination with IFN-γ promoted IL-6 production and further reduction of IL-10 synthesis. Comparison of TNF-α vs. IL-10 and IL-6 vs. IL-10 ratios revealed the following hierarchy in regard to the pro-inflammatory potential of human macrophages infected with P. aeruginosa: untreated < treated with GM-CSF < treated with IFN-γ < treated with GM-CSF and IFN-γ

The role of renal hypoperfusion in development of renal microcirculatory dysfunction in endotoxemic rats

To study the role of renal hypoperfusion in development of renal microcirculatory dysfunction in endotoxemic rats. Rats were randomized into four groups: a sham group (n = 6), a lipopolysaccharide (LPS) group (n = 6), a group in which LPS administration was followed by immediate fluid resuscitation which prevented the drop of renal blood flow (EARLY group) (n = 6), and a group in which LPS administration was followed by delayed (i.e., a 2-h delay) fluid resuscitation (LATE group) (n = 6). Renal blood flow was measured using a transit-time ultrasound flow probe. Microvascular perfusion and oxygenation distributions in the renal cortex were assessed using laser speckle imaging and phosphorimetry, respectively. Interleukin (IL)-6, IL-10, and tumor necrosis factor (TNF)-α were measured as markers of systemic inflammation. Furthermore, renal tissue samples were stained for leukocyte infiltration and inducible nitric oxide synthase (iNOS) expression in the kidney. LPS infusion worsened both microvascular perfusion and oxygenation distributions. Fluid resuscitation improved perfusion histograms but not oxygenation histograms. Improvement of microvascular perfusion was more pronounced in the EARLY group compared with the LATE group. Serum cytokine levels decreased in the resuscitated groups, with no difference between the EARLY and LATE groups. However, iNOS expression and leukocyte infiltration in glomeruli were lower in the EARLY group compared with the LATE group. In our model, prevention of endotoxemia-induced systemic hypotension by immediate fluid resuscitation (EARLY group) did not prevent systemic inflammatory activation (IL-6, IL-10, TNF-α) but did reduce renal inflammation (iNOS expression and glomerular leukocyte infiltration). However, it could not prevent reduced renal microvascular oxygenatio

Chondroprotection by urocortin involves blockade of the mechanosensitive ion channel Piezo1

Osteoarthritis (OA) is characterised by progressive destruction of articular cartilage and chondrocyte cell death. Here, we show the expression of the endogenous peptide urocortin1 (Ucn1) and two receptor subtypes, CRF-R1 and CRF-R2, in primary human articular chondrocytes (AC) and demonstrate its role as an autocrine/paracrine pro-survival factor. This effect could only be removed using the CRF-R1 selective antagonist CP-154526, suggesting Ucn1 acts through CRF-R1 when promoting chondrocyte survival. This cell death was characterised by an increase in p53 expression, and cleavage of caspase 9 and 3. Antagonism of CRF-R1 with CP-154526 caused an accumulation of intracellular calcium (Ca2+) over time and cell death. These effects could be prevented with the non-selective cation channel blocker Gadolinium (Gd3+). Therefore, opening of a non-selective cation channel causes cell death and Ucn1 maintains this channel in a closed conformation. This channel was identified to be the mechanosensitive channel Piezo1. We go on to determine that this channel inhibition by Ucn1 is mediated initially by an increase in cyclic adenosine monophosphate (cAMP) and a subsequent inactivation of phospholipase A2 (PLA2), whose metabolites are known to modulate ion channels. Knowledge of these novel pathways may present opportunities for interventions that could abrogate the progression of OA

Factors Contributing to the Biofilm-Deficient Phenotype of Staphylococcus aureus sarA Mutants

Mutation of sarA in Staphylococcus aureus results in a reduced capacity to form a biofilm, but the mechanistic basis for this remains unknown. Previous transcriptional profiling experiments identified a number of genes that are differentially expressed both in a biofilm and in a sarA mutant. This included genes involved in acid tolerance and the production of nucleolytic and proteolytic exoenzymes. Based on this we generated mutations in alsSD, nuc and sspA in the S. aureus clinical isolate UAMS-1 and its isogenic sarA mutant and assessed the impact on biofilm formation. Because expression of alsSD was increased in a biofilm but decreased in a sarA mutant, we also generated a plasmid construct that allowed expression of alsSD in a sarA mutant. Mutation of alsSD limited biofilm formation, but not to the degree observed with the corresponding sarA mutant, and restoration of alsSD expression did not restore the ability to form a biofilm. In contrast, concomitant mutation of sarA and nuc significantly enhanced biofilm formation by comparison to the sarA mutant. Although mutation of sspA had no significant impact on the ability of a sarA mutant to form a biofilm, a combination of protease inhibitors (E-64, 1-10-phenanthroline, and dichloroisocoumarin) that was shown to inhibit the production of multiple extracellular proteases without inhibiting growth was also shown to enhance the ability of a sarA mutant to form a biofilm. This effect was evident only when all three inhibitors were used concurrently. This suggests that the reduced capacity of a sarA mutant to form a biofilm involves extracellular proteases of all three classes (serine, cysteine and metalloproteases). Inclusion of protease inhibitors also enhanced biofilm formation in a sarA/nuc mutant, with the combined effect of mutating nuc and adding protease inhibitors resulting in a level of biofilm formation with the sarA mutant that approached that of the UAMS-1 parent strain. These results demonstrate that the inability of a sarA mutant to repress production of extracellular nuclease and multiple proteases have independent but cumulative effects that make a significant contribution to the biofilm-deficient phenotype of an S. aureus sarA mutant

Drosophila Nociceptors Mediate Larval Aversion to Dry Surface Environments Utilizing Both the Painless TRP Channel and the DEG/ENaC Subunit, PPK1

A subset of sensory neurons embedded within the Drosophila larval body wall have been characterized as high-threshold polymodal nociceptors capable of responding to noxious heat and noxious mechanical stimulation. They are also sensitized by UV-induced tissue damage leading to both thermal hyperalgesia and allodynia very similar to that observed in vertebrate nociceptors. We show that the class IV multiple-dendritic(mdIV) nociceptors are also required for a normal larval aversion to locomotion on to a dry surface environment. Drosophila melanogaster larvae are acutely susceptible to desiccation displaying a strong aversion to locomotion on dry surfaces severely limiting the distance of movement away from a moist food source. Transgenic inactivation of mdIV nociceptor neurons resulted in larvae moving inappropriately into regions of low humidity at the top of the vial reflected as an increased overall pupation height and larval desiccation. This larval lethal desiccation phenotype was not observed in wild-type controls and was completely suppressed by growth in conditions of high humidity. Transgenic hyperactivation of mdIV nociceptors caused a reciprocal hypersensitivity to dry surfaces resulting in drastically decreased pupation height but did not induce the writhing nocifensive response previously associated with mdIV nociceptor activation by noxious heat or harsh mechanical stimuli. Larvae carrying mutations in either the Drosophila TRP channel, Painless, or the degenerin/epithelial sodium channel subunit Pickpocket1(PPK1), both expressed in mdIV nociceptors, showed the same inappropriate increased pupation height and lethal desiccation observed with mdIV nociceptor inactivation. Larval aversion to dry surfaces appears to utilize the same or overlapping sensory transduction pathways activated by noxious heat and harsh mechanical stimulation but with strikingly different sensitivities and disparate physiological responses

Hip fracture risk in relation to vitamin D supplementation and serum 25-hydroxyvitamin D levels: a systematic review and meta-analysis of randomised controlled trials and observational studies

<p>Abstract</p> <p>Background</p> <p>Vitamin D supplementation for fracture prevention is widespread despite conflicting interpretation of relevant randomised controlled trial (RCT) evidence. This study summarises quantitatively the current evidence from RCTs and observational studies regarding vitamin D, parathyroid hormone (PTH) and hip fracture risk.</p> <p>Methods</p> <p>We undertook separate meta-analyses of RCTs examining vitamin D supplementation and hip fracture, and observational studies of serum vitamin D status (25-hydroxyvitamin D (25(OH)D) level), PTH and hip fracture. Results from RCTs were combined using the reported hazard ratios/relative risks (RR). Results from case-control studies were combined using the ratio of 25(OH)D and PTH measurements of hip fracture cases compared with controls. Original published studies of vitamin D, PTH and hip fracture were identified through PubMed and Web of Science databases, searches of reference lists and forward citations of key papers.</p> <p>Results</p> <p>The seven eligible RCTs identified showed no significant difference in hip fracture risk in those randomised to cholecalciferol or ergocalciferol supplementation versus placebo/control (RR = 1.13[95%CI 0.98-1.29]; 801 cases), with no significant difference between trials of <800 IU/day and ≥800 IU/day. The 17 identified case-control studies found 33% lower serum 25(OH)D levels in cases compared to controls, based on 1903 cases. This difference was significantly greater in studies with population-based compared to hospital-based controls (χ²₁(heterogeneity) = 51.02, p < 0.001) and significant heterogeneity was present overall (χ²₁₆(heterogeneity) = 137.9, p < 0.001). Serum PTH levels in hip fracture cases did not differ significantly from controls, based on ten case-control studies with 905 cases (χ²₉(heterogeneity) = 149.68, p < 0.001).</p> <p>Conclusions</p> <p>Neither higher nor lower dose vitamin D supplementation prevented hip fracture. Randomised and observational data on vitamin D and hip fracture appear to differ. The reason for this is unclear; one possible explanation is uncontrolled confounding in observational studies. Post-fracture PTH levels are unrelated to hip fracture risk.</p

The theory of expanded, extended, and enhanced opportunities for youth physical activity promotion

Background Physical activity interventions targeting children and adolescents (≤18 years) often focus on complex intra- and inter-personal behavioral constructs, social-ecological frameworks, or some combination of both. Recently published meta-analytical reviews and large-scale randomized controlled trials have demonstrated that these intervention approaches have largely produced minimal or no improvements in young people\u27s physical activity levels. Discussion In this paper, we propose that the main reason for previous studies\u27 limited effects is that fundamental mechanisms that lead to change in youth physical activity have often been overlooked or misunderstood. Evidence from observational and experimental studies is presented to support the development of a new theory positing that the primary mechanisms of change in many youth physical activity interventions are approaches that fall into one of the following three categories: (a) the expansion of opportunities for youth to be active by the inclusion of a new occasion to be active, (b) the extension of an existing physical activity opportunity by increasing the amount of time allocated for that opportunity, and/or (c) the enhancement of existing physical activity opportunities through strategies designed to increase physical activity above routine practice. Their application and considerations for intervention design and interpretation are presented. Summary The utility of these mechanisms, referred to as the Theory of Expanded, Extended, and Enhanced Opportunities (TEO), is demonstrated in their parsimony, logical appeal, support with empirical evidence, and the direct and immediate application to numerous settings and contexts. The TEO offers a new way to understand youth physical activity behaviors and provides a common taxonomy by which interventionists can identify appropriate targets for interventions across different settings and contexts. We believe the formalization of the TEO concepts will propel them to the forefront in the design of future intervention studies and through their use, lead to a greater impact on youth activity behaviors than what has been demonstrated in previous studies