Identification of a C3bi-specific membrane complement receptor that is expressed on lymphocytes, monocytes, neutrophils, and erythrocytes

Cells expressing a membrane C receptor (CR(3)) specific for C3b-inactivator- cleaved C3b (C3bi) were identified by rosette assay with C3bi-coated sheep erythrocytes (EC3bi) or C3bi-coated fluorescent microspheres (C3bi-ms). C3bi- ms, probably because of their smaller size, bound to a higher proportion of cells than did EC3bi. C3bi-ms bound to greater than 90 percent of mature neutrophils, 85 percent of monocytes, 92 percent of erythrocytes, and 12 percent of peripheral blood lymphocytes. Binding of C3bi-ms to neutrophils, monocytes, and erythrocytes was inhibited by fluid-phase C3bi, Fab anti-C3c, or Fab anti-C3d but was not inhibited by F(ab’)(2) anti-CR(1) (C3b receptor) or F(ab’)(2) anti-CR(2) (C3d receptor) nor by fluid-phase C3b, C3c, or C3d. This indicated that monocytes, neutrophils, and erythrocytes expressed C3bi receptors (CR(3)) that were separate and distinct from CR(1) and CR(2) and specific for a site in the C3 molecule that was only exposed subsequently to cleavage of C3b by C3b inactivator and that was either destroyed, covered, or liberated by cleavage of C3bi into C3c and C3d fragments. Lymphocytes differed from these other cell types in that they expressed CR2 in addition to CRa. Lymphocyte C3bi-ms rosettes were inhibited from 50 to 84 percent by F(ab’)(2)-anti-CR(2) or fluid-phase C3d, whereas C3d-ms rosettes were inhibited completely by F(ab’)(2) anti-CR(2), fluid-phase C3bi, or fluid- phase C3d. Thus, with lymphocytes, C3bi was bound to CR(3), and in addition was bound to CR(2) by way of the intact d region of the C3bi molecule. In studies of the acquisition of C receptors occurring during myeloid cell maturation, the ability to rosette with C3bi-coated particles was detected readily with immature low-density cells, whereas this ability was nearly undetectable with high density mature polymorphonuclear cells. This absence of C3bi binding to polymorphs was not due to a loss of the CR(3) but instead was due to the maturation-linked acquisition of the abiity to secrete elastase that cleaved reagent particle-bound C3bi into CR(3)-unreactive C3d. Neither neutrophils nor monocytes bound C3d-coated particles at any stage of maturation. Assay of CR(3) with mature neutrophils required inhibition of neutrophil elastase with either soybean trypsin inhibitor or anti-elastase antibodies, and the amounts of these elastase inhibitors required to allow EC3bi rosette formation increased with neutrophil maturation. Because lymphocytes bound C3bi to CR(2) as well as to CR(3), specific assay of lymphocyte CR(3) required saturation of membrane CR(2) with Fab’ anti-CR(2) before assay for rosettes with C3bi-ms. Only 3.5 percent of anti-CR(2)- treated peripheral blood lymphocytes bound C3bi-ms. Therefore, among normal blood lymphocytes the majority of the 12 percent C3bi-ms-binding cells expressed only CR(2) (8.5 percent), and the small proportion of C3bi-ms- binding cells that expressed CR(3) (3.5 percent) represented a distinct subset from the CR2(+) cells. Double-label assay indicated that 3.0 percent out of 3.5 percent of these CR(3)-bearing lymphocytes were B cells because they expressed membrane immunoglobulins. Of the remaining CR(3)(+) cells, 0.2 percent expressed either Leu-1 or 3A1 T cell antigens, and 0.6 percent expressed the OKM-1 monocyte-null lymphocyte determinant

Characterization of the lymphocyte membrane receptor for factor H (β1H- globulin) with an antibody to anti-factor H idiotype

Antibody to the binding site (idiotype) of anti-factor H was shown to have specificity for both B lymphocyte membrane H receptors and C3b. Goat F(ab’)(2) anti-human H was purified by absorption and elution from H agarose and used for rabbit immunization to produce anti-anti-H (aaH). After absorption with nonimmune goat IgG, (125)I-labeled aaH bound to B lymphocytes and to sheep erythrocytes coated with C3b (EC3b) but did not bind to T lymphocytes or to EC3d. All B cell- and C3b-specific activities of the aaH were removed and subsequently recovered by absorption and elution of the antibody from either C3-agarose or goat-anti-H-agarose. This indicated that the aaH probably recognized a single common antigenic structure that was shared by anti-H, C3b, and the membranes of B cells. Affinity-purified aaH resembled H in that it bound to B cells, blocked the uptake of H onto B cell H receptors, and triggered B cells to release endogenous factor I (C3b inactivator). In addition, aaH functioned with factor I as either a cofactor for cleavage of fluid-phase C3b or a potentiator for cleavage of bound C3b. This same spectrum of C3 binding functions could not be demonstrated with either sheep anti-C3b or rabbit-anti-C3c. Analysis by sodium dodecyl sulfate- polyacrylamide get electrophoresis of the [(3)H]leucine intrinsically labeled B cell proteins reactive with the purified aaH revealed proteins of 100,000 M(r) and 50,000 M(r) without reduction, and after complete reduction of disulfide bonds, a single protein band of 50,000 M(r). This same protein molecular weight profile was also demonstrated with labeled B cell proteins that were absorbed and eluted from H-agarose. Thus, aaH is apparently specific for both B cell H receptors and C3b. However, because parallel analysis of C3b confirmed its known 115,000- and 75,000-M(r) polypeptide chain structure, the H receptor is probably not C3b and shares only the structure of the H binding site with C3b

Tissue resolved, gene structure refined equine transcriptome.

BackgroundTranscriptome interpretation relies on a good-quality reference transcriptome for accurate quantification of gene expression as well as functional analysis of genetic variants. The current annotation of the horse genome lacks the specificity and sensitivity necessary to assess gene expression especially at the isoform level, and suffers from insufficient annotation of untranslated regions (UTR) usage. We built an annotation pipeline for horse and used it to integrate 1.9 billion reads from multiple RNA-seq data sets into a new refined transcriptome.ResultsThis equine transcriptome integrates eight different tissues from 59 individuals and improves gene structure and isoform resolution, while providing considerable tissue-specific information. We utilized four levels of transcript filtration in our pipeline, aimed at producing several transcriptome versions that are suitable for different downstream analyses. Our most refined transcriptome includes 36,876 genes and 76,125 isoforms, with 6474 candidate transcriptional loci novel to the equine transcriptome.ConclusionsWe have employed a variety of descriptive statistics and figures that demonstrate the quality and content of the transcriptome. The equine transcriptomes that are provided by this pipeline show the best tissue-specific resolution of any equine transcriptome to date and are flexible for several downstream analyses. We encourage the integration of further equine transcriptomes with our annotation pipeline to continue and improve the equine transcriptome

A Collaborative Approach to Developing a Model for Oil Spill Policy Decision Support: Building a better model while learning together

The Washington State Department of Ecology is developing a quantitative model to evaluate risk of oil spills in Washington waters. The model provides a long-term resource for evaluating oil spill policy and oil spill risks in Washington waters. To do so, it must produce understandable and accessible information for effective decision-making support and characterize risk in a way that addresses the concerns of tribes and stakeholders. Guided by the analytic-deliberative process recommended by the National Research Council, our team approached model development with a focus on collaboration and empirical rigor. Between the summer of 2020 and fall of 2021, we held 22 public events on model development topics, while simultaneously researching and developing a model framework. This approach brought community perspectives and values to the table, and informed our decisions on key structural aspects of the model. The analytic-deliberative process provided a framework for integrating feedback from tribes and stakeholders. Our open-ended approach to outreach helped us establish the structure and underpinnings of the model. However, it also limited our ability to provide timely opportunities for feedback on some details of the model. This presentation will cover our approach to consulting with Tribes, our outreach with stakeholders, survey results from participants, and lessons learned from our process. We will present statistics on participation levels over the course of outreach, most discussed topics, and how feedback informed model structure. We will discuss how the lessons from this process are informing outreach planning for the first two analysis projects we will conduct using the quantitative oil spill risk model

A Descriptive Review of the Methodologies Used in Household Surveys on Medicine Utilization

Background: Studies carried out in the community enable researchers to understand access to medicines, affordability, and barriers to use from the consumer's point of view, and may stimulate the development of adequate medicines policies. The aim of the present article was to describe methodological and analytical aspects of quantitative studies on medicine utilization carried out at the household level. Methods: Systematic review of original papers with data collected in studies in which the household was a sampling unit, published between 1995 and 2008. The electronic review was carried out in Medline/Pubmed, Scielo and Lilacs. The reference lists of the papers identified were examined, as well as other publications by their authors. Studies on the utilization of specific pharmacological groups, or those including only respondents with a given disease were excluded. Results: Out of 4852 papers initially identified in the literature search, 61 fulfilled our inclusion criteria. Most studies were carried out in Europe and North America and used a cross-sectional approach. More than 80% used face-to-face interviews for data collection, and the most frequently used recall period for assessing medicine utilization was 14–15 days. In 59% of the studies, interviewers were trained to request the packaging of the medicines reported by the subjects; medical prescriptions were requested less frequently (15% of the studies). Conclusion: These data will be useful for updating researchers on what methods their peers are currently using. Such information may help overcome challenges in the planning and analyses of future studies. Moreover, this publication may contribute to the improvement of the quality of medicine use data obtained in household surveys

Modulating attentional load affects numerosity estimation: evidence against a pre-attentive subitizing mechanism

Traditionally, the visual enumeration of a small number of items (1 to about 4), referred to as subitizing, has been thought of as a parallel and pre-attentive process and functionally different from the serial attentive enumeration of larger numerosities. We tested this hypothesis by employing a dual task paradigm that systematically manipulated the attentional resources available to an enumeration task. Enumeration accuracy for small numerosities was severely decreased as more attentional resources were taken away from the numerical task, challenging the traditionally held notion of subitizing as a pre-attentive, capacity-independent process. Judgement of larger numerosities was also affected by dual task conditions and attentional load. These results challenge the proposal that small numerosities are enumerated by a mechanism separate from large numerosities and support the idea of a single, attention-demanding enumeration mechanism

On the upstream mobility scheme for two-phase flow in porous media

When neglecting capillarity, two-phase incompressible flow in porous media is modelled as a scalar nonlinear hyperbolic conservation law. A change in the rock type results in a change of the flux function. Discretizing in one-dimensional with a finite volume method, we investigate two numerical fluxes, an extension of the Godunov flux and the upstream mobility flux, the latter being widely used in hydrogeology and petroleum engineering. Then, in the case of a changing rock type, one can give examples when the upstream mobility flux does not give the right answer.Comment: A preprint to be published in Computational Geoscience