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R M Binns

Publications and source records attributed to R M Binns.

At least 19 recordsLinked to original sources
PubMed1998

Overview of the Second International Workshop to define swine cluster of differentiation (CD) antigens.

The aim of the Second International Swine Cluster of Differentiation (CD) Workshop, supported by the Veterinary Immunology Committee (VIC) of the International Union of Immunological Societies (IUIS), was to standardize the assignment of monoclonal antibodies (mAb) reactive with porcine leukocyte differentiation antigens and to define new antibody clusters. At the summary meeting of the workshop in July, 1995, revisions in the existing nomenclature for Swine CD were approved, so that the rules are now in accord with those for human and ruminant CD. Swine CD numbers will now be given to clusters of mAb to swine orthologues of human CD molecules when homology is proven by (1) suitable tissue distribution and lymphoid cell subset expression, (2) appropriate molecular mass of the antigen recognized by the mAbs, and (3) reactivity of mAbs with the cloned swine gene products, or cross-reactivity of the mAb on the human gene products. In some cases, this reactivity would not be fully proven, mainly due to the lack of cloned gene products; for these CD antigens, the respective clusters will be assigned by the prefix 'w' which will lead to 'wCD' antigens. As a result of the Second International Swine CD Workshop the assignment of 16 mAb to existing CD groups (CD2a, CD4a, CD5a, wCD6, wCD8, CD14, CD18a, wCD21, wCD25) was confirmed, and 2 mAb to existing swine workshop clusters (SWC). More importantly, for the work on the porcine immune system, was the definition of 5 new swine CD antigens, namely CD3 (recognized by 6 new mAb and 3 epitopes), CD16 (1 new mAb), wCD29 (2 mAb), CD45RA (3 mAb) and CD45RC (1 new mAb). Finally, the demarcation of two new SWC molecules in swine, SWC8 (2 mAb) and SWC9 (2 mAb) was confirmed.

Animals↗
PubMed1998

Analysis of monoclonal antibodies that recognize gamma delta T/null cells.

Thirty two monoclonal antibodies (mAbs) from the first round of analysis in the Second International Swine CD Workshop were placed together with additional mAb derived from the first workshop in the null cell panel for further evaluation. Preparations of peripheral blood leukocytes, concanavalin A stimulated peripheral blood mononuclear cells, and spleen cells were used in flow cytometric analyses. Nineteen mAbs identified molecules that were not expressed on null cells, not lineage specific, or recognized activation molecules. Sixteen mAbs including control mAbs were identified that were specific for null cells. One of the latter mAbs, 041 (PGBL22A), that recognizes a determinant on a constant region of porcine gamma delta TcR established the majority of null cells are gamma delta T cells. Use of this mAb in further comparisons demonstrated the gamma delta T cell population is comprised of two major subpopulations, one negative and one positive for CD2. Two color analyses demonstrated that 11 of the mAbs formed a broad cluster that included control mAbs 188 (MAC320) that defined the CD2 negative SWC6 cluster in the first workshop and mAb 122 (CC101) that might recognize an orthologue of bovine WC1 and nine mAbs that recognize determinants on one or more molecules with overlapping patterns of expression on subsets of CD2- gamma delta T cells. Two groups of mAbs formed the previously identified subset clusters SWC4 and SWC5. Two new mAbs formed a third subcluster. Three mAbs did not form clusters. Three mAbs predicted to recognize TcR in the first workshop (020 [PT14A], 021 [PT79A], and 022 [MUC127A]) and mAb PGBL22A were shown to immunoprecipitate a 37, 40 kDa heterodimer.

Animals↗
PubMed1997

Mechanisms for variability in a member of the scavenger-receptor cysteine-rich superfamily.

This study reports a molecular analysis of pig WC1, a new member of the scavenger-receptor cysteine-rich (SRCR) superfamily. The pig WC1 contains up to six extra-cellular SRCR domains, highly homologous to other members of the family. However, the striking feature of the WC1 gene, as for its cattle and sheep homologues, is that it is present as a multigene family showing extensive sequence diversity, for both DNA and predicted protein sequence. The basis of this diversity was examined and was shown to be attributable to several different causes. These included single base-pair changes within SRCR domains, the optional usage of whole domains or exons, including a SRCR domain and the proximal "hinge" region, and alternative isoforms of the putative cytoplasmic tail. These results suggest that WC1 may code for a new, though more primitive type of antigen recognition structure specific for gamma/delta T cells.

Amino Acid Sequence↗
PubMed1997

Isolation and N-terminal sequence determination of a novel gamma/delta T cell surface antigen.

An antigen complex unique for porcine gamma/delta T cells has previously been identified using the monoclonal antibodies MAC319 and MAC320. Here we use digestion with the proteolytic enzyme bromelain to selectively release the MAC319 antigen as a soluble fragment, for further characterisation. A cytofluorometric inhibition assay was developed to follow the purification of this fragment, as the conformation sensitivity of the MAC319 epitope prevented the use of immunoblotting techniques. The antigen has been purified using a combination of anion-exchange and hydrophobic-interaction columns, followed by separation on a size-exclusion column. Fractions from the size-exclusion column containing the antigen consisted of one major band at Mr 95,000. This species was shown to be specifically absorbed onto MAC319-coupled Sepharose, thereby identifying the MAC319 antigen. N-terminal amino acid sequencing of this band has revealed a previously unidentified sequence. This fragment was also shown to be glycosylated, most likely with a single sugar moiety. Enzymatic removal of the sugars showed that they did not appear to be necessary for binding of the polypeptide to the MAC319 antibody.

Animals↗
PubMed1996

The role of E-selectin in lymphocyte and polymorphonuclear cell recruitment into cutaneous delayed hypersensitivity reactions in sensitized pigs.

We have studied the role of E-selectin in leukocyte accumulation into Ag-specific cutaneous delayed-type hypersensitivity reactions in pigs sensitized to the topical application of 2,4-dinitro-1-fluorobenzene or to the intradermal injection of bacillus Calmette-Guérin. The delayed-type hypersensitivity reactions were shown to be specific for the sensitizing Ag and characterized by the up-regulation of E-selectin, as demonstrated by the uptake of tracer 99mTc-labeled monoclonal anti-E-selectin mAb and entry of 51Cr-labeled PBL and (111)In-labeled polymorphonuclear cells (PMN). Intravenous injection of 5 mg/kg of a F(ab')2 preparation of a monoclonal anti-E-selectin Ab at peak times of leukocyte entry resulted in a significant inhibition of entry of both PMN and lymphocytes. The anti-E-selectin Ab inhibited PMN recruitment by 70 to 90% and lymphocyte recruitment by 50 to 60%. In comparison, an anti-CD18 treatment reduced PMN recruitment by 70 to 90% and lymphocyte recruitment by 60 to 70% in this model. These data confirm an important role for E-selectin in the recruitment of both PMN and lymphocytes to sites of immune-based dermal inflammation.

Age Factors↗
PubMed1996

Improved protocol for colorimetric detection of complement-mediated cytotoxicity based on the measurement of cytoplasmic lactate dehydrogenase activity.

The conventional cytotoxicity detection protocols based on the lactate dehydrogenase (LDH) activity assays rely upon the quantitation of the enzyme activity released into the assay medium upon cell lysis. In the case of complement-mediated cytotoxicity this results in the need to take into account the high and variable background LDH activity from the serum added to the cells. Using primate-derived COS-7 and pig kidney PK15 cell lines we show that, in the case of adherent cells, it is possible to overcome this drawback and to measure cell death caused by complement attack, by quantifying the amount of LDH activity retained by the undamaged cells. The modified assay is therefore quicker to carry out than the conventional procedure and cheaper because fewer control readings must be taken.

Animals↗
PubMed1996

Expression of CD44 isoforms during bleomycin-or radiation-induced pulmonary fibrosis in rats and mini-pigs.

The distribution of CD44s and CD44v molecules in normal and injured lung tissue of rats and mini-pigs was studied by examining the immunohistochemical binding of monoclonal antibodies against CD44 isoforms. We showed that the expression of CD44v and CD44s varies greatly among different pulmonary fibrosis samples and that some tissues express either enhanced expression of CD44s, particularly in the interstitium and on alveolar macrophages, or very low levels of CD44v in the alveolar epithelium. Normal type II pneumocytes expressed the CD44s and CD44v molecules at the basolateral aspect of the cell. Such localisation favours a role for CD44 in epithelial cell-fibroblast interaction during lung development and repair.

Animals↗
PubMed1996

Transfer of T-cell-mediated, antigen-specific delayed type hypersensitivity reactions to naive recipient inbred pigs.

Using inbred major histocompatibility complex-homozygous SLAb/b pigs, delayed-type hypersensitivity (DTH) reactions against either intradermal tuberculin (PPD) or topical 2,4-dinitro-1-fluorobenzene (DNFB) were transferred specifically by the intravenous injection of approximately 6 x 10(8) blood lymphocytes kg-1 bodyweight from donors sensitised, respectively, either with BCG or with DNFB into three-week-old piglets from an inbred litter. This antigen-specific, passively acquired sensitivity was revealed by three measures of DTH reactivity: first, macroscopic inflammation, which developed at the rate and intensity expected for actively acquired sensitivity to DNFB or PPD in older pigs; secondly, similarly enhanced local specific uptake of intravenously injected 51Cr-labelled normal lymphocytes (more than 35-fold for each); and, thirdly, histological evidence of markedly increased local infiltration of CD45+ lymphocytes and polymorphs, endothelial activation and the expression of adhesion molecules.

Animals↗
PubMed1996

Constitutive and inflammatory lymphocyte trafficking.

Here we provide a brief overview of lymphocyte trafficking with particular emphasis on the current state of knowledge in the pig. We discuss how the emphasis of research has changed since early studies in the 1960s and outline the current hypothesis of a multistep cascade for lymphocyte migration through specialized endothelia. During the last several years our research has focused mainly on lymphocyte migration in vivo. The inbred Babraham herd of MHC homozygous Large White pigs has allowed study of entry of either labelled (FITC or 51Cr) or unlabelled CD45 allotype-different donor lymphocytes and their subsets into various lymphoid, non-lymphoid and inflammatory tissues. The findings are considered under three different categories. Firstly, constitutive lymphocyte entry via 'high endothelial venules' (HEV-mediated), secondly, non-HEV-mediated lymphocyte homing and thirdly, lymphocyte entry into several models of inflammation with particular reference to the role of E-selectin. These findings demonstrate and underline the complexity and heterogeneity of lymphocyte homing, both at the whole population and subset level and yet, whilst a major step forward, the current hypotheses are perhaps too simple to explain much of this heterogeneity.

Animals↗
PubMed1996

Characterization of E-selectin expression, leucocyte traffic and clinical sequelae in urate crystal-induced inflammation: an insight into gout.

The self-limiting response to urate crystals allows the exploration of events involved in both the onset and resolution of gout. Using i.v. injected radiolabelled anti-E-selectin monoclonal antibody 1.2b6 together with differentially radiolabelled neutrophils, mononuclear cells and albumin, we have characterized the expression of E-selectin in relation to leucocyte traffic, microvascular permeability and clinical sequelae following intracutaneous injection of monosodium urate crystals. We found that the inflammatory response in this model involved several distinct phases. First, E-selectin expression increased over 2-6 h in the context of increases in neutrophil and mononuclear cell accumulation, and albumin leakage. Secondly, leucocyte accumulation rapidly declined despite persisting E-selectin expression. Thirdly, E-selectin expression peaked at approximately 8 h and then fell despite an increase in clinically detectable erythema and induration. Lastly, these clinical manifestations of inflammation resolved despite the continued presence of urate crystals in the tissues. The further dissection of mechanisms regulating these phases will lead to a better understanding of events in both the pathogenesis and resolution of gout. Of broader significance, this inflammatory model may yield information about the protective events that underly resolution of inflammation, and provide insights into factors which determine chronicity.

Albumins↗
PubMed1996

In vivo E-selectin upregulation correlates early with infiltration of PMN, later with PBL entry: MAbs block both.

The endothelial molecule E-selectin binds most leukocyte subsets in vitro. Yet its role in regulating the very different kinetics of inflammatory infiltration of different leukocyte subsets in vivo is unclear. The kinetics of E-selectin upregulation and polymorphonuclear leukocyte (PMN) and blood lymphocyte (PBL) localization in inflammation induced by interleukin-1 alpha (IL-1 alpha), tumor necrosis factor-alpha (TNF-alpha), phytohemagglutinin (PHA), and phorbol myristate acetate (PMA) were investigated in a well-established inbred pig trafficking model. They differed markedly both for these three labeled indicators of inflammation and in each of the four inflammatory processes. In each, E-selectin upregulation correlated with early PMN entry and later with PBL infiltration but was more protracted than both. The importance of E-selectin was confirmed by marked inhibition of PMN and PBL entry (up to > 60%) by F(ab')2 anti-E-selectin. Involvement of other molecules was illustrated by similar or greater inhibition with anti-CD18 F(ab')2. We conclude that, like CD18, E-selectin is necessary for most PMN and PBL infiltration but alone is insufficient, consistent with the involvement of several alternative multistep molecular mechanisms in this entry.

Animals↗
PubMed1995

Clearance pathways of soluble immune complexes in the pig. Insights into the adaptive nature of antigen clearance in humans.

Efficient delivery of immune complexes (ICs) to the mononuclear phagocytic system, and subsequent IC processing, may prevent their potentially harmful effects in other tissues and may also be important in the development of humoral immune responses. In mice, rabbits, and primates, the liver and spleen are the main sites of IC clearance. It has been demonstrated previously that the pulmonary capillaries in the pig are lined with macrophages and that certain particulates, including bacteria, localize to this organ. In this study, we used gamma scintigraphy to explore the sites and kinetics of clearance of soluble IC comprising 123I-labeled hepatitis B surface Ag (HBsAg):porcine anti-HBsAg in the Large White pig. At t = 10 min after i.v. injection, 43 +/- 5% (mean +/- SE) IC localized in the lungs, and 36 +/- 6% counts in the liver. At t = 85 min, values were: lungs, 15 +/- 4% and liver, 29 +/- 2%. Findings were similar following intraarterial injection. Complement depletion resulted in more rapid initial IC clearance (t1/2 = 5 min), reduced lung uptake (23 +/- 3% at 10 min), and impaired IC catabolism. In normal animals, 5 to 7% injected IC bound to PBMCs, but no E binding was seen. A fall in PBMC numbers (46 to 59% of baseline), was observed following IC injection. These findings contrast with our previous observations using analogous IC in humans, in which we did not observe any change in peripheral blood leukocyte counts consequent upon complex processing, suggesting that in humans, Es may function as a buffering system for complement-bearing IC in the circulation, preventing their interaction with leukocytes bearing complement and FcR, and the potential activation of these cells.

Animals↗
PubMed1995

The behaviour of monoclonal antibodies in the First International Pig CD Workshop reacting with gamma delta/Null T lymphocytes in the blood of SLAb/b line pigs.

Further studies were carried out on the monoclonal antibodies (mAbs) from the First International Swine CD Workshop which react with gamma delta Null T-lymphocytes, defined by the binding of mAb w020/141 (MAC320) as Swine Workshop Cluster number SWC6. Studies were also carried out on several other mAbs from the same workshop which identify other CD antigens, but whose binding is not restricted exclusively to gamma delta Null T-lymphocytes. The first group consists of 11 mAbs (w021, w022, w059-w065, w105 and w117) and the second group of 18 mAbs (w008, w026, w056, w067-w071, w080, w091-w094, w110, w111, w118, w119 and w121). All mAbs were characterised by binding to peripheral blood lymphocytes (PBL) from normal, sham-thymectomized (STx) and thymectomized (Tx) pigs of the Babraham SLAb/b line and by their overlap, using two-colour immunofluorescence with biotinylated mAb MAC320 (w020/141), which identifies all gamma delta Null cell T-lymphocytes and the Null cell subpopulation identified by MAC319 (w021). The Null cell-specific mAbs were also used in inhibition studies of MAC319 and MAC320 binding and by staining PBL with pairs of mAbs together with either MAC319 or MAC320. Based on these data we suggest a putative relationship of the Null cell subsets defined by these mAbs with each other.

Animals↗
PubMed1995

Induced tolerance and chimaerism in human fetuses using coelocentesis: a medical opportunity to avert genetic disease?

Coelocentesis offers a new opportunity for gaining access to the coelomic cavity of human embryos from 28 days post-fertilization (42 days menstrual age). With this technique, cells can be extracted from the cavity for the genetic typing of embryos in early pregnancy. Coelocentesis may also offer a unique opportunity of inducing tolerance to foreign grafts and chimaerism in these human embryos by replacing donor cells into the coelomic cavity. This cavity appears to be closely associated with the fetal haemopoietic system. The optimal age to inject stem cells designed to produce chimaerism may be at 5-6 weeks embryonic age, and these grafted cells may induce tolerance later in gestation. Two successive coelocenteses would be needed, the first to extract fetal cells to type the fetus, and a second within a few days to inject the donor cells into the coelomic cavity. Alternatively, non-invasive methods of diagnosis such as lower uterine pole extramembranous sampling of fetal trophoblast, or the extraction of fetal cells from maternal blood, could be combined with coelocentesis. If tolerance and chimaerism can be established, repeated tissue grafts could be carried out during fetal life and after birth, so that disorders caused by single or multiple gene defects in the haemopoietic system and other organs may be corrected.

Chimera↗