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Biomedical subjects

R M Binns

Publications and source records attributed to R M Binns.

At least 37 records · Page 2Linked to original sources
PubMed1995

Inflammatory arthritis: imaging of endothelial cell activation with an indium-111-labeled F(ab')2 fragment of anti-E-selectin monoclonal antibody.

PURPOSE: To use imaging performed with an indium-111-labeled F(ab')2 fragment of monoclonal antibody that recognizes E-selectin (1.2B6) to evaluate inflammation. MATERIALS AND METHODS: After the in vivo immunoreactivity of In-111-F(ab')2 was verified, imaging experiments were conducted on pigs with induced arthritis of the right knee. In-111-labeled 1.2B6-F(ab')2, technetium-99m-hexamethylpropyleneamine oxime-labeled polymorphonuclear leukocytes (PMNs), and iodine-125-MOPC 21-labeled F(ab')2 (the control) were administered by means of intravenous injection. Scintigrams were obtained until 20 hours after injection, and tissue sampling was performed. RESULTS: Scintigrams showed uptake of 1.2B6 in the right knee but only faint filling of the joint space with PMNs. The right knee-to-left knee ratios on images obtained 20 hours after injection of the tracers were 10.8 +/- 3.6 for 1.2B6 and 1.1 +/- 0.4 for PMNs (P < .05). CONCLUSION: Endothelial cell immunotargeting with a radiolabeled anti-E-selectin F(ab')2 monoclonal antibody is feasible for use in imaging of inflammation.

Animals↗
PubMed1995

Lymphocytes migrate from the bronchoalveolar space to regional bronchial lymph nodes.

The fate of lymphocytes in the bronchoalveolar tract was studied in normal young pigs. Peripheral blood lymphocytes were labeled with 51chromium or with fluorescein isothiocyanate (FITC) and instilled into a single segmental bronchus. In the first series of experiments, the radioactivity was determined in several different parts of the lungs, individual bronchial lymph nodes and a series of other lymphoid and nonlymphoid organs. About two-thirds of the radioactivity was still in the lung at 1 d. The draining lymph node also contained high amounts of radioactivity. To exclude that this was caused by labeled cell debris, an immunohistologic technique was used to localize the FITC-labeled cells. There were clearly labeled lymphocytes in the sinusoids of the draining nodes. These lymphocytes were characterized by additional surface staining. B, T, TH, and TS lymphocytes were seen much less often than in the inoculum while the null and gamma delta T cells showed a preference. The lymphocytes reached the bronchial lymph nodes via lymphatics. Lymphocytes in the bronchoalveolar space are not effete cells to be destroyed but return to the immune system. These data provide new interest in the immunologic role of lymphocytes in the bronchoalveolar tract and their kinetics.

Animals↗
PubMed1995

Differences in E-selectin expression and leucocyte infiltration induced by inflammatory agents in a novel subcutaneous sponge matrix model.

We have developed a novel subcutaneous sponge matrix model in major histocompatibility complex (MHC) homozygous SLAb/b inbred pigs to study lymphocyte-endothelial cell interactions during inflammation. Polyether sponges were implanted subcutaneously and left for 12 days before injection of proinflammatory agonists. Implanted sponges became highly vascularized and showed markedly increased uptake of i.v.-injected 51Cr-labelled lymphocytes 5 hr after injection of tumour necrosis factor-alpha (TNF-alpha) (3000 U) or phytohaemagglutinin (PHA) (37 micrograms). Lower levels of traffic were seen in sponges 5 hr after injection with interleukin-1 alpha (IL-1 alpha) (3000 U) and no significant traffic occurred in sponges injected with phorbol 12-myristate 13-acetate (PMA) (15 ng) at 5 hr or PHA at 24 hr (compared to sponges injected with medium alone). Electron microscopy of control sponges revealed low numbers of infiltrating leucocytes and relatively 'flat' endothelium. Many more infiltrating leucocytes were present in PHA-injected sponges. However, no ultrastructural evidence was seen of any significant difference between control and activated endothelium. Immunocytochemistry of frozen sections from sponges showed that E-selectin expression was up-regulated markedly by TNF-alpha and PHA at 5 hr, only moderately by IL-1 alpha at 5 hr, and not at all by PMA at 5 hr. By 24 hr in PHA-injected sponges E-selectin expression had fallen markedly from the level seen at 5 hr. Flow cytometric analysis of cellular infiltrates dispersed from sponges injected with TNF-alpha, PHA, IL-1 alpha or medium alone, revealed differences in lymphocyte subset populations. The infiltrate in sponges injected with TNF-alpha 5 hr before removal was dominated by high numbers of CD2+ lymphocytes, whereas the infiltrate induced by PHA showed relatively higher levels of CD2- CD4-CD8- gamma delta T-cell receptor+ (TCR+) T cells revealed by population-specific monoclonal antibodies (mAb). This model, which permits harvesting of leucocytes and endothelial cells for manipulation in vitro, will be useful for the study of leucocyte-endothelial cell interactions in subacute and chronic inflammation.

Animals↗
PubMed1995

Porcine E-selectin: cloning and functional characterization.

E-selectin, a member of the selectin family, is believed to play an important role in mediating the initial adhesive events between leucocytes and the endothelium during inflammation. A monoclonal antibody against human E-selectin was found to cross-react with the porcine equivalent, a glycoprotein of 92,000 MW. Isolation of the cDNA for porcine E-selectin showed that it was 71% homologous with human E-selectin but had two less short consensus repeats. The porcine adhesion molecule could also support the adhesion of both porcine and human neutrophils. Expression of E-selectin on interleukin-1 alpha (IL-1 alpha)- or tumour necrosis factor-alpha (TNF-alpha)-activated porcine aortic endothelial cells in culture was prolonged, persisting for up to 48 hr. Binding studies using a chimeric molecule consisting of the lectin domain of porcine E-selectin and the epidermal growth factor (EGF) domain of human E-selectin fused to the human IgG constant region, further characterized porcine E-selectin as recognizing mainly granulocytic leucocytes and a subpopulation of lymphocytes.

Animals↗
PubMed1995

Genetically determined CD45 variant of value in leucocyte tracing in vivo in the pig.

This paper describes a monoclonal antibody (mAb), anti-pig CD45 (MAC323), that is directed against a polymorphic determinant. A monomorphic anti-pig CD45 mAb (K252.1E4) bound strongly to leucocytes from both MAC323+ and MAC323- pigs, demonstrating the absence of the epitope rather than the CD45 molecule. The MAC323 determinant was present on all leucocytes in positive pigs, exhibiting different expression levels on subsets (monocytes > lymphocytes > polymorphs), but was absent on red blood cells. Pigs lacking this determinant were healthy and grew normally. Careful selection of male and female SLAb/b pigs produced families that were either positive or negative for this epitope. Interbreeding within these families identified the genetic segregation of this variation, which is consistent with the CD45(323) epitope being inherited as a simple dominant autosomal gene. The lack of this determinant in certain lines of inbred pigs has been used to study the homing, lifespan and tissue distribution of donor unlabelled MAC323+ leucocytes and their subsets (using single- and two-colour immunocytological techniques) in MAC323- recipients after either intravenous injection or exchange transfusion. These results, identifying trafficking areas and subsets in constitutive lymphoid and inflammatory tissues, obtained using this genetic marker, extend those obtained previously using fluorescein isothiocyanate (FITC)-labelled donor cells.

Animals↗
PubMed1995

Major long-term changes in gamma delta T-cell receptor-positive and CD2+ T-cell subsets after neonatal thymectomy in the pig: a longitudinal study lasting nearly 2 years.

Blood leucocyte subsets in neonatally (20-day-old) thymectomized (Tx) and sham-thymectomized (STx) pigs were analysed 13 times over nearly 2 years. Tx piglets showed a persistent selective leucopenia, due mainly to a approximately 95% reduction in gamma delta null T cells which fell, with a circulating half-life of approximately 2 weeks, to approximately 0.3 x 10(6)/ml. This residual population was extrathymic in origin since it increased numerically at least approximately eightfold as the Tx pigs grew. Changes in other subsets were complex and affected by antigenic experience associated with weaning and with a change of accommodation at approximately 4 months postoperation (p.o.). Most major populations were increased long-term after thymectomy, especially after 3 months p.o. [i.e. surface (s)IgM+ B cells and CD2+, CD8+, major histocompatibility complex (MHC) class II+, CD4+ and double-positive CD4+ CD8+ T-cell subsets]. However, during the first 3 months, thymectomy caused a significant delay in development of CD8high and CD4+ T cells and, after 4 months p.o., a continuing lack of CD4only (single-positive) T cells. Fortuitous environmental antigenic stimulation caused a major transient lymphocytosis, with counts increasing 1.2-fold in STx and 3.5-fold in Tx pigs. This was largely due to an increase in CD2+ CD8+ MHC class II+ T cells, particularly in Tx pigs. The small residual thymus-independent gamma delta null subset also increased, while gamma delta T cells in STx pigs actually decreased. Evolving changes in expression of CD45, CD45R, CD44, CD18 and very late antigen type-4 (VLA-4) also occurred following thymectomy. Thus, the most persistent long-term effect of thymectomy, other than the lack of gamma delta null T cells, was the markedly increased numbers of double-positive (CD4+ CD8low) T cells, most of which expressed MHC class II and higher levels of adhesion molecules.

Animals↗
PubMed1994

Noninvasive imaging of E-selectin expression by activated endothelium in urate crystal-induced arthritis.

OBJECTIVE: To assess the expression of the cytokine-inducible endothelial leukocyte adhesion molecule E-selectin during the evolution of urate crystal-induced arthritis, using a recently described radiolabeled monoclonal antibody (MAb) imaging technique. METHODS: Monosodium urate (MSU) crystals and saline alone were injected respectively into the right (inflamed) and left (control) knees of 3 young pigs. Four hours later, 111In-labeled 1.2B6 F(ab')2 (anti-E-selectin MAb) and 125I-labeled MOPC 21 F(ab')2 (control MAb) were injected intravenously. Uptake of 1.2B6 in inflamed and control joints was assessed by scintigraphy 7 and 24 hours after intraarticular injection of MSU crystals. Immunohistochemistry studies and radioactivity counting of tissues were performed postmortem to confirm the observations from scintigraphy. RESULTS: MAb 1.2B6 F(ab')2 scintigraphic images of the knees revealed a significantly increased uptake in the right (inflamed) knee at 7 and 24 hours postinjection, particularly over the joint space. These in vivo images were consistent with E-selectin expression in the inflamed tissue detected by immunohistochemistry and with radioactivity counts postmortem. The synovial localization ratio (inflamed:control synovium counts) was 25.4 +/- 9.7 (mean +/- SD) for the anti-E-selectin MAb compared with 2.5 +/- 0.9 for the control MAb (P < 0.05, by paired Student's t-test). CONCLUSION: E-selectin is expressed by synovial endothelium during the evolution of urate crystal-induced arthritis and can be detected noninvasively using a radiolabeled MAb. This E-selectin imaging technique has considerable potential for the noninvasive assessment of endothelial activation in arthritis and other inflammatory rheumatic diseases.

Animals↗
PubMed1994

Adhesion molecule expression and infiltrating maternal leucocyte phenotypes during blastocyst implantation in the pig.

During the implantation period (days 14 to 30 post coitum) in Large White pigs substantial numbers of endometrial sub-epithelial leucocytes were observed from about day 18 p.c. These were predominantly MHC Class II+, CD45+ and reactive for an accessory cell determinant carried by polymorphs and macrophages. Maternal lymphocytes of the CD2+, CD4-, CD8-, and a gamma delta TcR+ subtype identified by the mAb 86D, were also present. Few CD4+, CD8+, or double-positive lymphocytes were seen. CD2-CD4-CD8- gamma delta TcR+ "Null" cells were seen in stroma adjacent to endometrial glands, as were CD2+ lymphocytes. There was no apparent up-regulation of CD18, VLA-4 or CD25. E-selectin could not be detected on endothelium within uterine tissues or on trophectoderm, nor was a ligand for L-selectin detectable. The luminal aspect of the uterine epithelium was strongly reactive with anti-CD15 mAb, which was specifically blocked by lacto-N-fucopentaose III, indicating the presence of the LewisX determinant.

Animals↗
PubMed1994

Infiltrating gamma delta T-cells and selectin endothelial ligands in the cutaneous phytohaemagglutinin-induced inflammatory reaction.

The 24 h phytohaemagglutinin-induced skin inflammatory site (intradermal and subcutaneous) was studied in inbred MHC-homozygous (SLAb/b) pigs and it was found, by immunohistology, that the predominant lymphocytes in the infiltrate are CD2-CD4-CD8-sIg-T-cells, the Null/gamma delta T-cell family, identified using the monoclonal antibodies (mAbs) MAC320 and MAC319 (which recognises a subset of MAC320+ cells). A large percentage of the infiltrating cells expressed the gamma delta T-cell receptor phenotype identified by binding of the mAb 86D. Fewer CD2+, CD8+ and CD4+ cells were present and surface immunoglobulin positive (sIg+) cells were virtually absent in the infiltrate. Areas of lymphocytic infiltration were associated with endothelial activation as determined by expression of the E-selectin and a ligand for the L-selectin.

Animals↗
PubMed1994

The Null/gamma delta TCR+ T cell family in the pig.

The very large population of highly thymus-dependent, non-adherent 'Null' blood lymphocytes in young pigs, which lack most surface markers characteristic of classical T-cell subsets, have recently been shown to bear several novel population- and subset-specific antigens and gamma delta T-cell receptors. These T-lymphocytes differ radically from conventional CD2-positive T-cells in their origins, biological lifespan, tissue distribution, migration behaviour, cell surface phenotype, immunological responsiveness and mechanisms of trafficking through constitutive lymphoid tissues and inflammatory lesions. Elucidation of the roles of these enigmatic cells and their characteristic surface macromolecules in the initiation and regulation of immune functions promises to reveal much of fascination.

Animals↗
PubMed1994

Lymphoid tissue structure and lymphocyte trafficking in the pig.

The organised lymphoid tissues of the pig, though conventionally mammalian in most respects, show several distinctive properties in their structure and physiology. Specialised antigen-presenting lymphoid organs function at three compartmental levels: the body surfaces, their draining lymph nodes and the spleen in the bloodstream. Other organs act as lymphocytic depots and sites of phagocytosis of debris. Pig lymphocytes recirculate continuously through these organs and through different forms of inflammatory change, experimentally induced by mitogens and cytokines, using a spectrum of distinctively different physiological mechanisms. These tissues in the young pig differ both in the resting and activated cell subsets and the molecules involved, many of which remain to be completely defined. Intriguing insights are evident in the subtlety of regulation of the specificity, level and foetal ontogeny of trafficking mechanisms in these different tissues, though they are as yet poorly explained. This subtle molecular physiology is only now emerging because appropriate monoclonal antibody reagents are being developed and rigorous attention is being paid to the use of gentle physiological methods in experiments in vivo.

Animals↗
PubMed1994

The immune system of the respiratory tract in pigs.

Although the lung is not a lymphoid organ it contains large numbers of lymphocytes. These can be found in different compartments: (1) the pulmonary intravascular pool, which is organ-specific and shows a unique migration pattern; (2) the interstitial lymphocyte pool, which is equivalent in size to the whole blood pool; (3) the bronchus-associated lymphoid tissue (BALT) which develops as a result of microbial stimulation; (4) the intraepithelial and lamina propria lymphocytes of the bronchi, with their typical subset composition; (5) the lymphocytes in the bronchoalveolar space, which can be sampled by bronchoalveolar lavage. The size and kinetics of the lymphocyte pools have been studied in the pig in more detail than in most other species. Despite this organotypic compartmentalisation of the pulmonary lymphoid cells in the pig, the lung is part of the integrated mucosal immune system, as shown by protective oral immunisation against the lung-pathogenic bacteria Actinobacillus pleuropneumoniae. The lung immune system in the pig is not only of veterinary interest, but also a relevant model for the human respiratory tract.

Actinobacillus Infections↗
PubMed1994

Summary of first round cluster analysis: complete antibody panel.

The reactivities of 141 monoclonal antibodies (mAbs) with 60 target cell types or lines were analysed by flow cytometry and the data subjected to statistical clustering. mAbs were assigned to 23 clusters by statistical similarity and information on specificity supplied by the contributor. Clustered mAbs were examined in more detail in the second round of workshop analyses.

Animals↗
PubMed1994

Report on the behaviour of monoclonal antibodies in the First International Pig CD Workshop identifying the Null cell families.

Clustering analyses were carried out on data from five independent laboratories testing 22 monoclonal antibodies (mAbs) reacting with CD2-sIg-lymphocytes on 14 pig blood and/or tissue lymphoid target cells using cytofluorometry. This was coupled with extensive further studies on blood lymphocytes from normal and thymectomised SLAb/b inbred pigs. These mAbs formed two groups: those mainly identifying the large blood-borne thymus-dependent Null T cells (N) and those reacting with tissue and a small number of blood-borne thymus-independent lymphocytes (N'). Based on their tissue cell reaction patterns, the 10 N' mAbs formed three main groups: N'1A and B; N'2A and B; and N'3. The 12N mAbs fell into four groups N4-N7; N6 was divided into subgroups A-D. One N' (032) and two N mAbs (010 and 063) were unclustered. Based on these data, swine workshop cluster numbers were designated to groups N5 (021, 022 and 059) as SWC4, N6 (061 and 117) as SWC5 and N7 (020 and 141) as SWC6, the latter exceptionally as a single antibody, MAC320, since it is the 'type' mAb identifying effectively all blood Null T lymphocytes. Future research and workshops will have to define with a wider range of techniques the relationships, molecular properties and functional roles of the several new, perhaps novel, antigens identified by this family of fascinating, as yet still poorly defined, mAbs.

Animals↗
PubMed1994

Analyses of monoclonal antibodies reactive with porcine CD44 and CD45.

Twenty-six monoclonal antibodies (mAbs), assigned to the CD44/CD45 section of the First International Swine CD Workshop, were compared for their reactivity against a selected group of target cells by one- and two-color flow cytometric analysis. Based on staining and reactivity patterns the 26 mAbs were assigned to six groups, group F1 mAbs were designated CD44 mAbs; and groups F2 and F3 as CD45 mAbs. With the information available, a CD designation could not be given to the mAbs in groups F4, F5 or F6 consisting of four, three and four mAbs each, respectively. The reactivity of all six mAbs in group F1 (MAC35, 25-32, PORC24A, H22A, BAG40A, and BAT31A) was blocked by soluble porcine CD44. One mAb in this group (MAC325) reacted with a cell surface protein with a molecular weight of 80 kDa and was designated as CD44; the other five mAbs were designated as wCD44 because no molecular weight was known. Blocking experiments utilizing a cross reactive anti-human CD44 (mAb Z062) allowed the definition of the wCD44a epitope recognized by mAbs PORC24A and H22A. The group F2 mAbs (74-9-3; MAC323; K252.1E4; and 2A5) were designated as CD45 based on their broad reactivity pattern with lymphoid and myeloid cells and their ability to immunoprecipitate three polypeptides with an apparent molecular weight of 226, 210 and 190 kDa. The F3 mAbs (MAC327; MAC326; 3a56 and -a2) were designated as CD45R based on their restricted reactivity against lymphoid and myeloid target cells, and their ability to immunoprecipitate either two polypeptides with an apparent molecular weight of 226 and 210 kDa (mAbs MAC327 and MAC326) or a single polypeptide with an apparent molecular weight of 210 kDa (mAbs-a2 and 3a56). Sequential immunoprecipitation analyses confirmed the relatedness of the F2 and F3 group mAbs. The work conducted for this first workshop led to the definition of six mAbs specific for CD44, four mAbs specific for CD45, and four mAbs specific for CD45R which should prove to be very valuable reagents for the study of the porcine immune system.

Animals↗
PubMed1994

Biochemical analysis of molecules reactive with monoclonal antibodies specific for porcine CD45.

The apparent molecular weight of molecules reactive with eight mAbs, which were identified during the First International Swine CD Workshop as putatively specific for porcine CD45, was determined by immunoprecipitation analysis of 125I surface-labeled peripheral blood mononuclear cells. Four putative CD45 specific mAbs (K252.1E4, MAC323, 74-9-3 and 2A5) precipitated three polypeptides of 226, 210 and 190 kDa. Two putative CD45R specific mAbs (MAC326 and MAC327) precipitated two polypeptides with an apparent molecular weight of 226 and 210 kDa, while another two (3a56 and -a2) precipitated a single polypeptide of 210 kDa. Sequential immunoprecipitations analyses indicated that the comigrating molecules precipitated by the CD45 and CD45R were the same. The results from this study confirmed the assignment of mAbs K252.1E4, MAC323, 74-9-3 and 2A5 as CD45, and mAbs MAC326, MAC327, 3a56 and -a2, as CD45R. These CD45 and CD45R specific mAbs should prove to be valuable reagents for the study of the porcine immune system.

Animals↗
PubMed1994

Characterization of E-selectin expression in vivo with use of a radiolabeled monoclonal antibody.

We have studied endothelial luminal surface expression of E-selectin in vivo in the pig. Intravenous interleukin-1 (IL-1, 5-micrograms/kg bolus +/- 50-ng.kg-1 x min-1 infusion for 2 h) induced E-selectin expression in many organs, as shown by immunostaining and selective clearance of intravenous 111In- or 99mTc-labeled anti-E-selectin monoclonal antibody (MAb 1.2B6) compared with radiolabeled immunoglobulin G1 control. Specific clearance of MAb 1.2B6 commenced 30-45 min after intravenous IL-1. Skin sites injected with IL-1, tumor necrosis factor, phytohemagglutinin, or phorbol myristate acetate at various times (45 min-24 h) before exsanguination showed specific accumulation of MAb 1.2B6 when 99mTc-MAb 1.2B6 and 111In-control immunoglobulin G1 were injected intravenously 10 min before exsanguination. This was maximal in 2-h IL-1 and tumor necrosis factor lesions and after 9 h in phytohemagglutinin and phorbol myristate acetate lesions. This novel approach has allowed us to quantify changes in vascular luminal expression of E-selectin in models of inflammation involving systemic and localized endothelial cell activation and has considerable potential for analyzing these changes in relation to leukocyte traffic and other manifestations of inflammatory responses in vivo.

Animals↗