Expression and function of C5a receptor in mouse microvascular endothelial cells

The complement-derived anaphylatoxin, C5a, is a potent phlogistic molecule that mediates its effects by binding to C5a receptor (C5aR; CD88). We now demonstrate specific binding of radiolabeled recombinant mouse C5a to mouse dermal microvascular endothelial cells (MDMEC) with a K(d50) of 3.6 nM and to approximately 15,000-20,000 receptors/cell. Recombinant mC5a competed effectively with binding of [(125)I]rmC5a to MDMEC. Enhanced binding of C5a occurred, as well as increased mRNA for C5aR, after in vitro exposure of MDMEC to LPS, IFN-gamma, or IL-6 in a time- and dose-dependent manner. By confocal microscopy, C5aR could be detected on surfaces of MDMEC using anti-C5aR Ab. In vitro expression of macrophage inflammatory protein-2 (MIP-2) and monocyte chemoattractant protein-1 (MCP-1) by MDMEC was also measured. Exposure of MDMEC to C5a or IL-6 did not result in changes in MIP-2 or MCP-1 production, but initial exposure of MDMEC to IL-6, followed by exposure to C5a, resulted in significantly enhanced production of MIP-2 and MCP-1 (but not TNF-alpha and MIP-1alpha). Although LPS or IFN-gamma alone induced some release of MCP-1 and MIP-2, pre-exposure of these monolayers to LPS or IFN-gamma, followed by addition of C5a, resulted in synergistic production of MIP-2 and MCP-1. Following i.v. infusion of LPS into mice, up-regulation of C5aR occurred in the capillary endothelium of mouse lung, as determined by immunostaining. These results support the hypothesis that C5aR expression on MDMEC and on the microvascular endothelium of lung can be up-regulated, suggesting that C5a in the co-presence of additional agonists may mediate pro-inflammatory effects of endothelial cells.     

Regulatory role of C5a on macrophage migration inhibitory factor release from neutrophils

There is evidence that C5a and macrophage migration inhibitory factor (MIF) both play important roles in experimental sepsis. Humans with sepsis also show elevated levels of both mediators in the blood. Regulation of MIF during sepsis is poorly understood. We now demonstrate that neutrophil depletion greatly reduced serum MIF levels in rats and mice during the onset of sepsis after cecal ligation and puncture. In vitro, C5a induced MIF release from rat and mouse neutrophils. In vivo blockade of C5aR or absence of C5aR led to significantly reduced MIF generation during the onset of sepsis. C5a-induced release in vitro of MIF from neutrophils appeared to be due to up-regulation of MIF in cytoplasmic granules of neutrophils via activation of the protein kinase B signaling pathway together with involvement of PI3K. Our data suggest that C5a plays a role in enhancing MIF release from neutrophils in vitro and during sepsis. These findings represent a previously unrecognized function of C5a and neutrophils in the appearance of MIF in sepsis.     

Evaluation of APOE polymorphisms and the risk for age-related macular degeneration in a Southeastern Brazilian population

This study aimed to evaluate the role of APOE polymorphisms (rs429358 and rs7412) in the risk of age-related macular degeneration in a sample of the Southeastern Brazilian population. Seven hundred and five unrelated individuals were analyzed, 334 with age-related macular degeneration (case group), and 371 without the disease (control group). In the case group, patients were further stratified according to disease phenotypes, divided into dry and wet age-related macular degeneration, and non-advanced and advanced age-related macular degeneration. APOE polymorphisms (rs429358 and rs7412) were evaluated through polymerase chain reaction and direct sequencing. In the comparison of cases vs. controls, none of the associations reached statistical significance, considering the Bonferroni-adjusted P-value, although there was a suggestive protection for the E3/E4 genotype (OR = 0.626; P-value = 0.037) and E4 carriers (OR = 0.6515; P-value = 0.047). Statistically significant protection for both the E3/E4 genotype and E4 carriers was observed in the comparisons: advanced age-related macular degeneration vs. controls (OR = 0.3665, P-value = 0.491 × 10⁻³ and OR = 0.4031, P-value = 0.814 × 10⁻³, respectively), advanced age-related macular degeneration vs. non-advanced age-related macular degeneration (OR = 0.2529, P-value = 0.659 × 10⁻⁴ and OR = 0.2692, P-value = 0.631 × 10⁻⁴, respectively). In the comparison of wet age-related macular degeneration vs. control, protection was statistically significant only for E3/E4 (OR = 0.4052, P-value = 0.001). None of the comparisons demonstrated any significant association for E2 genotypes or E2 carriers in age-related macular degeneration risk in this study. Findings suggest a protective role of the E4 haplotype in the APOE gene in the risk for advanced and wet forms of age-related macular degeneration, in a sample of the Brazilian population. To our knowledge, this is the first Brazilian study to show the association between APOE polymorphisms and age-related macular degeneration.     

Changes in the novel orphan, C5a receptor (C5L2), during experimental sepsis and sepsis in humans

Sepsis is associated with extensive complement activation, compromising innate immune defenses, especially in neutrophils (PMN). Recently, a second C5a receptor (C5L2) was detected on PMN without evidence of intracellular signaling. The current study was designed to determine changes in C5L2 in blood PMN during sepsis. In vitro exposure of PMN to C5a, but not to fMLP, led to reduced content of C5L2. Following cecal ligation and puncture-induced sepsis in rats, PMN demonstrated a time-dependent decrease in C5L2. In vivo blockade of C5a during experimental sepsis resulted in preservation of C5L2. Similarly, PMN from patients with progressive sepsis showed significantly reduced C5L2 expression (n = 26), which was virtually abolished in patients who developed multiorgan failure (n = 10). In contrast, sepsis survivors exhibited retention of C5L2 (n = 12/13). The data suggest that C5L2 on PMN diminishes during sepsis due to systemic generation of C5a, which is associated with a poor prognosis.     

Are there valid proxy measures of clinical behaviour? a systematic review

Background

Accurate measures of health professionals' clinical practice are critically important to guide health policy decisions, as well as for professional self-evaluation and for research-based investigation of clinical practice and process of care. It is often not feasible or ethical to measure behaviour through direct observation, and rigorous behavioural measures are difficult and costly to use. The aim of this review was to identify the current evidence relating to the relationships between proxy measures and direct measures of clinical behaviour. In particular, the accuracy of medical record review, clinician self-reported and patient-reported behaviour was assessed relative to directly observed behaviour.

Methods

We searched: PsycINFO; MEDLINE; EMBASE; CINAHL; Cochrane Central Register of Controlled Trials; science/social science citation index; Current contents (social & behavioural med/clinical med); ISI conference proceedings; and Index to Theses. Inclusion criteria: empirical, quantitative studies; and examining clinical behaviours. An independent, direct measure of behaviour (by standardised patient, other trained observer or by video/audio recording) was considered the 'gold standard' for comparison. Proxy measures of behaviour included: retrospective self-report; patient-report; or chart-review. All titles, abstracts, and full text articles retrieved by electronic searching were screened for inclusion and abstracted independently by two reviewers. Disagreements were resolved by discussion with a third reviewer where necessary.

Results

Fifteen reports originating from 11 studies met the inclusion criteria. The method of direct measurement was by standardised patient in six reports, trained observer in three reports, and audio/video recording in six reports. Multiple proxy measures of behaviour were compared in five of 15 reports. Only four of 15 reports used appropriate statistical methods to compare measures. Some direct measures failed to meet our validity criteria. The accuracy of patient report and chart review as proxy measures varied considerably across a wide range of clinical actions. The evidence for clinician self-report was inconclusive.

Conclusion

Valid measures of clinical behaviour are of fundamental importance to accurately identify gaps in care delivery, improve quality of care, and ultimately to improve patient care. However, the evidence base for three commonly used proxy measures of clinicians' behaviour is very limited. Further research is needed to better establish the methods of development, application, and analysis for a range of both direct and proxy measures of behaviour.     

Characterization of a p75(NTR) apoptotic signaling pathway using a novel cellular model.

The p75 neurotrophin receptor (p75(NTR)) belongs to the tumor necrosis factor receptor/nerve growth factor receptor superfamily. In some cells derived from neuronal tissues it causes cell death through a poorly characterized pathway. We developed a neuronal system using conditionally immortalized striatal neurons, in which the expression of p75(NTR) is inducibly controlled by the ecdysone receptor. In these cells p75(NTR) induces apoptosis through its death domain in a nerve growth factor-independent manner. Caspases 9, 6, and 3 are activated by receptor expression indicating the activation of the common effector pathway of apoptosis. Cell death is blocked by a dominant negative form of caspase 9 and Bcl-X(L) consistent with a pathway that involves mitochondria. Significantly, the viral flice inhibitory protein E8 protects from p75(NTR)-induced cell death indicating that death effector domains are involved. A p75(NTR) construct with a deleted death domain dominantly interferes with p75(NTR) signaling, implying that receptor multimerization is required. However, in contrast to the other receptors of the family, p75(NTR)-mediated apoptosis does not involve the adaptor proteins Fas-associated death domain protein or tumor necrosis factor-associated death domain protein, and the apical caspase 8 is not activated. We conclude that p75(NTR) signals apoptosis by similar mechanisms as other death receptors but uses different adaptors and apical caspases.     

Friend or foe? Evolutionary history of glycoside hydrolase family 32 genes encoding for sucrolytic activity in fungi and its implications for plant-fungal symbioses

Background  

Many fungi are obligate biotrophs of plants, growing in live plant tissues, gaining direct access to recently photosynthesized carbon. Photosynthate within plants is transported from source to sink tissues as sucrose, which is hydrolyzed by plant glycosyl hydrolase family 32 enzymes (GH32) into its constituent monosaccharides to meet plant cellular demands. A number of plant pathogenic fungi also use GH32 enzymes to access plant-derived sucrose, but less is known about the sucrose utilization ability of mutualistic and commensal plant biotrophic fungi, such as mycorrhizal and endophytic fungi. The aim of this study was to explore the distribution and abundance of GH32 genes in fungi to understand how sucrose utilization is structured within and among major ecological guilds and evolutionary lineages. Using bioinformatic and PCR-based analyses, we tested for GH32 gene presence in all available fungal genomes and an additional 149 species representing a broad phylogenetic and ecological range of biotrophic fungi.     

Generation of C5a in the absence of C3: a new complement activation pathway

Complement-mediated tissue injury in humans occurs upon deposition of immune complexes, such as in autoimmune diseases and acute respiratory distress syndrome. Acute lung inflammatory injury in wild-type and C3-/- mice after deposition of IgG immune complexes was of equivalent intensity and was C5a dependent, but injury was greatly attenuated in Hc-/- mice (Hc encodes C5). Injury in lungs of C3-/- mice and C5a levels in bronchoalveolar lavage (BAL) fluids from these mice were greatly reduced in the presence of antithrombin III (ATIII) or hirudin but were not reduced in similarly treated C3+/+ mice. Plasma from C3-/- mice contained threefold higher levels of thrombin activity compared to plasma from C3+/+ mice. There were higher levels of F2 mRNA (encoding prothrombin) as well as prothrombin and thrombin protein in liver of C3-/- mice compared to C3+/+ mice. A potent solid-phase C5 convertase was generated using plasma from either C3+/+ or C3-/- mice. Human C5 incubated with thrombin generated C5a that was biologically active. These data suggest that, in the genetic absence of C3, thrombin substitutes for the C3-dependent C5 convertase. This linkage between the complement and coagulation pathways may represent a new pathway of complement activation.