Clumping factor A of Staphylococcus aureus inhibits phagocytosis by human polymorphonuclear leucocytes

Staphylococcus aureus is a major cause of nosocomial and community-acquired infection. It expresses several factors that promote avoidance of phagocytosis by polymorphonuclear leucocytes. Clumping factor A (ClfA) is a fibrinogen-binding surface protein of S. aureus that is an important virulence factor in several infection models. This study investigated whether ClfA is an antiphagocytic factor, and whether its antiphagocytic properties were based on its ability to bind fibrinogen. In S. aureus, ClfA was shown to be of equal importance to protein A, the antiphagocytic properties of which are well established. ClfA expressed in a surrogate Gram-positive host was also found to be antiphagocytic. A ClfA mutant that was unable to bind fibrinogen had a similar antiphagocytic effect to native ClfA in the absence of fibrinogen. ClfA inhibited phagocytosis in the absence of fibrinogen, and showed enhanced inhibition in the presence of fibrinogen.     

Differential Use of Trails by Forest Mammals and the Implications for Camera-Trap Studies: A Case Study from Belize

Relative abundance indices are often used to compare species abundance between sites. The indices assume that species have similar detection probabilities, or that differences between detection probabilities are known and can be corrected for. Indices often consist of encounter frequencies of footprints, burrows, markings or photo captures along trails or transect lines, but the assumption of equal detection probabilities is rarely validated. This study analyzes detection probabilities of a range of Neotropical mammals on trails in dense secondary forests, using camera-trap and track data. Photo captures of the two large cats, jaguars ( Panthera onca ) and pumas ( Puma concolor ), were correlated solely with trail variables, while photo captures of their potential prey species had no correlation or negative correlation with trail variables. The Neotropical mammals varied greatly in their tendency to follow or cross trails based on footprints surveys. This indicates that camera locations on trails will have varying detection probability for these Neotropical mammals. Even the two similar-sized jaguars and pumas, occupying relatively similar niches, differed subtly in their use of trails. Pumas followed trails more completely while jaguars were more likely to deviate from trails. The ecological significance of these findings is that jaguars seem to be more willing to use the forest matrix away from trails than do pumas. We conclude that trail-based indices, such as photographic captures or tracks along trails, are not appropriate for comparison between Neotropical species, and not even between relatively similar species like jaguars and pumas.     

Connectivity of Caribbean coral populations: complementary insights from empirical and modelled gene flow

Understanding patterns of connectivity among populations of marine organisms is essential for the development of realistic, spatially explicit models of population dynamics. Two approaches, empirical genetic patterns and oceanographic dispersal modelling, have been used to estimate levels of evolutionary connectivity among marine populations but rarely have their potentially complementary insights been combined. Here, a spatially realistic Lagrangian model of larval dispersal and a theoretical genetic model are integrated with the most extensive study of gene flow in a Caribbean marine organism. The 871 genets collected from 26 sites spread over the wider Caribbean subsampled 45.8% of the 1900 potential unique genets in the model. At a coarse scale, significant consensus between modelled estimates of genetic structure and empirical genetic data for populations of the reef-building coral Montastraea annularis is observed. However, modelled and empirical data differ in their estimates of connectivity among northern Mesoamerican reefs indicating that processes other than dispersal may dominate here. Further, the geographic location and porosity of the previously described east-west barrier to gene flow in the Caribbean is refined. A multi-prong approach, integrating genetic data and spatially realistic models of larval dispersal and genetic projection, provides complementary insights into the processes underpinning population connectivity in marine invertebrates on evolutionary timescales.     

Conversion of plasma VLDL and IDL precursors into various LDL subpopulations using density gradient ultracentrifugation

The contribution of very low density lipoproteins (VLDL) and intermediate density lipoproteins (IDL) to various low density lipoprotein (LDL) subfractions was examined in three normal subjects and two with familial combined hyperlipidemia. Autologous VLDL + IDL (d less than 1.019 g/ml) or VLDL only (d less than 1.006 g/ml; one subject only) were isolated by sequential ultracentrifugation, iodinated, and injected into each subject. The appearance, distribution, and subsequent disappearance of radioactivity into LDL density subpopulations was characterized using density gradient ultracentrifugation. These techniques help determine the contribution of precursors to various LDL subpopulations defined uniquely for each subject. The results from these studies have suggested: 1) it took up to several days of intravascular processing of precursor-derived LDL before it resembled the distribution of the 'steady-state' plasma LDL protein; 2) plasma VLDL and IDL precursors contributed rapidly to a broad density range of LDL; 3) the radiolabeled plasma precursors did not always contribute to all LDL density subfractions within an individual in proportion to their relative LDL protein mass as determined by density gradient ultracentrifugation; 4) with time, the distribution of the precursor-derived LDL became more buoyant or more dense than distribution of the LDL protein mass; and 5) the kinetic characteristics of precursor-derived particles within LDL changed within a relatively narrow density range and were not always related to the LDL density heterogeneity of each subject. These studies emphasize the complexities of apoB metabolism and the need to design studies to carefully examine the production of various LDL subpopulations, the kinetic fate and interconversions among the subpopulations, and ultimately, their relationship to the development of atherosclerosis.     

The putative brain photoperiodic photoreceptors in the vetch aphid,Megoura viciae

In an attempt to identify the brain photoreceptors that mediate the photoperiodic response of the vetch aphid, Megoura viciae, we utilised immunocytochemical techniques and employed 20 antibodies directed against invertebrate and vertebrate opsins and phototransduction proteins. A sub-set of these antibodies (to Drosophila rhodopsin 1: RH1-1; vertebrate cone opsins: COS-1; CERN-874; CERN-933; vertebrate rod opsin: CERN-901; vertebrate arrestin: AB-Arr; vertebrate transducin+arrestin+rhodopsin kinase+cGMP phosphodiesterase: CERN-911; and vertebrate cellular retinoid binding protein: CRALBP) consistently labelled an anterior ventral neuropile region of the protocerebrum. These anatomical findings, coupled with previous localised illumination and micro-lesion studies, provide strong evidence that this region of the aphid brain houses the photoperiodic photoreceptors. The present study also confirms that the medial (Group I) neurosecretory cells are not the photoperiodic photoreceptors.     

Metabolism of 125I-atrial natriuretic factor by vascular smooth muscle cells. Evidence for a peptidase that specifically removes the COOH-terminal tripeptide

The addition of 200 pM monoiodinated human atrial natriuretic factor-(99-126) (125I-hANF) to cultured bovine aortic smooth muscle cells at 37 degrees C resulted in a rapid clearance from the medium (t1/2 approximately 7.5 min). Within 5 min, [125I]iodotyrosine126 (125I-Y), Arg125-[125I]iodotyrosine126 (125I-RY) and Phe124-Arg-[125]iodotyrosine126 (125I-FRY) appeared in the medium. The identities of these degradation products were confirmed by 1) retention time on high performance liquid chromatography (HPLC) relative to standards, 2) products generated by digestion with aminopeptidase M, and 3) the absence of the Met110. Preincubation of the cells with ammonium chloride or chloroquine resulted in a significant increase in the intracellular accumulation of radiolabel, indicative of endocytosis and rapid delivery of 125I-hANF to an acidic intracellular compartment (endosome and/or lysosome). Neither ammonium chloride, chloroquine, nor excess unlabeled hANF blocked the rapid appearance in the medium of 125I-RY or 125I-FRY. Bestatin inhibited the generation of 125I-RY, with a concomitant increase in 125I-FRY, suggesting that the 125I-RY is produced by aminopeptidase action on 125I-FRY. The endopeptidase 24.11 (enkephalinase) inhibitor, SCH 39370, did not inhibit the formation of 125I-FRY. These results provide evidence of a peptidase capable of specifically removing the COOH-terminal tripeptide from 125I-hANF. The COOH-terminal tripeptide, Phe124-Arg-Tyr126, was also isolated from cell digests of hANF by HPLC and its identity confirmed by amino acid analysis. Since it is generally believed that the COOH-terminal tripeptide is critical to many of atrial natriuretic factor-(99-126)'s bioactivities, this enzyme may be involved in the inactivation of atrial natriuretic factor-(99-126) in target tissues.     

Suppression of a pokeweed mitogen-stimulated plaque-forming cell response by a human B lymphocyte-derived aggregated IgG-stimulated suppressor factor: suppressive B cell factor (SBF)

The mechanisms whereby formed immune complexes (IC) or immunoglobulin aggregates can suppress further antibody production were explored by culturing normal human peripheral blood mononuclear leukocytes (PBL) with heat-aggregated IgG (HAIgG) and collecting the culture supernatants at 24 hr. These supernatants were found to suppress a pokeweed mitogen (PWM)-induced rheumatoid factor plaque-forming cell (RF-PFC) response in normal individuals. PWM-induced anti-trinitrophenylated sheep red blood cell (TNP-SRBC) PFC were also inhibited by suppressor supernatants from HAIgG-stimulated PBL, suggesting that the polyclonal PFC response was inhibited by a suppressor factor. The suppressor factor inhibited PWM stimulated RF-PFC throughout the culture period, but suppression was maximal at the peak of the RF-PFC response. Suppressor factor was only effective at the initiation of cultures, suggesting that it inhibited early events in the PWM-stimulated RF-PFC response. Molecular weight determination of the suppressor factor by differential membrane fractionation suggested a m.w. range of 30,000 to 50,000, and chromatography on Sephadex G-100 showed a peak activity at an approximate m.w. of 32,000. Studies suggested the factor was not an interferon. Depletion of T lymphocytes by E rosetting and macrophages/monocytes by G-10 adherence did not affect the generation of suppressor factor. Depletion of T lymphocytes (OKT4, OKT8) and NK cells (Leu-11b) by antibody-dependent, complement-mediated cytotoxicity also did not affect the generation of suppressor factor. Depletion of B lymphocytes with OKB7 resulted in the generation of significantly less suppressor factor. Suppression produced by unstimulated purified B lymphocytes was approximately one-half that seen when B lymphocytes were stimulated with HAIgG. Differential membrane fractionation studies suggested that only HAIgG-stimulated B cell cultures contained peak activity in the 30,000 to 50,000 m.w. fraction. Supernatants from unstimulated purified T cells also generated suppression, which was approximately one-half of that seen with HAIgG-stimulated B cells, but no increase in suppressor activity was seen in T cell cultures after incubation with HAIgG. These studies demonstrate that HAIgG is capable of stimulating B lymphocytes to produce a lymphokine, suppressive B cell factor (SBF), which is capable of suppressing a polyclonal PFC response. SBF may be important in feedback control of human immunoglobulin production.     

Development and Validation of the Keele Musculoskeletal Patient Reported Outcome Measure (MSK-PROM)

Objective

To develop and validate a patient report outcome measure (PROM) for clinical practice that can monitor health status of patients with a range of musculoskeletal (MSK) disorders.

Methods

Constructs for inclusion in the MSK-PROM were identified from a consensus process involving patients with musculoskeletal conditions, clinicians, purchasers of healthcare services, and primary care researchers. Psychometric properties of the brief tool, including face and construct validity, repeatability and responsiveness were assessed in a sample of patients with musculoskeletal pain consulting physiotherapy services in the United Kingdom (n=425).

Results

The consensus process identified 10 prioritised domains for monitoring musculoskeletal health status: pain intensity, quality of life, physical capacity, interference with social/leisure activities, emotional well-being, severity of most difficult thing, activities and roles, understanding independence, and overall impact. As the EuroQol (EQ-5D-5L) is a widely adopted PROMs tool and covers the first four domains listed, to reduce patient burden to a minimum the MSK-PROM was designed to capture the remaining six prioritised domains which are not measured by the EQ-5D-5L. The tool demonstrated excellent reliability, construct validity, responsiveness and acceptability to patients and clinicians for use in clinical practice.

Conclusion

We have validated a brief patient reported outcome measure (MSK-PROM) for use in clinical practice to measure musculoskeletal health status and monitor outcomes over time using domains that are meaningful to patients and sensitive to change. Further work will establish whether the MSK-PROM is useful in other musculoskeletal healthcare settings.