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B M Dutia

Publications and source records attributed to B M Dutia.

At least 37 records · Page 2Linked to original sources
PubMed1994

Evidence for the expression of two distinct MHC class II DR beta like molecules in the sheep.

This study used monoclonal antibodies to sheep MHC class II molecules as well as an L cell transfectant (T8.1) which expresses DRA and DRB genes to show that two distinct DR beta chains are expressed in the sheep. Two anti-beta chain specific monoclonal antibodies VPM37 and VPM43 react with DR antigen but not DQ antigen by ELISA. These two antibodies do not react with the DR beta chain expressed in the T8.1 cell line. Two-dimensional immunoblotting shows that these antibodies recognize a subgroup of the spots recognized by the DR-specific monoclonal antibody VPM57 which does react with the T8.1 beta chain. Amino-terminal sequence analysis of the alpha chain associated with VPM37 beta chain shows that this alpha chain is homologous to the human DR alpha chain strongly indicating that the beta chain is DR-like. VPM37 and VPM43 are shown to be directed against different epitopes on sheep MHC class II molecules so it is highly unlikely that the data can be explained by the presence of post-translational modifications or the existence of a very common allele. These data provide clear evidence for the expression of two distinct DR beta chains in the sheep.

Amino Acid Sequence↗
PubMed1993

Patterns of major histocompatibility complex class II expression on T cell subsets in different immunological compartments. 1. Expression on resting T cells.

In this study we have investigated the expression of major histocompatibility complex (MHC) class II molecules on T cells from various lymphoid compartments in the sheep. Monoclonal antibodies which react specifically with sheep MHC class II molecules homologous to the human DQ and DR molecules have been characterized. These antibodies have been used, together with the monoclonal antibodies specific for sheep CD4-, CD8- and T19-positive T cells, to quantitate DQ and DR expression on T cell subsets in adult and fetal peripheral blood, afferent lymph, lymph node and efferent lymph. The results show that expression of class II by T cells depends on the age of the animal and the physiological location of the T cell. In fetal blood there is no expression of class II on CD8+ or T19+ cells and very low expression on CD4+ T cells. In adult peripheral blood and efferent lymph a significant proportion of cells express DR but not DQ. A very different situation is found in afferent lymph and the peripheral lymph node: in afferent lymph the majority of T cells in all three subsets express both DQ and DR molecules; in the lymph node over 50% of T cells express DR and 30% are DQ+. These results suggest that within all T cell subsets there is a progression from DQ-DR- to DQ-DR+ and DQ+DR+ which correlates with physiological stages of T cell differentiation in vivo.

Amino Acid Sequence↗
PubMed1993

Patterns of major histocompatibility complex class II expression by T cell subsets in different immunological compartments. 2. Altered expression and cell function following activation in vivo.

This study characterizes antigen-induced phenotypic and functional aspects of major histocompatibility complex (MHC) class II expression on recirculating T cells in efferent lymph. In vivo secondary, but not primary challenge is associated with both kinetic and phenotypic alterations in class II expression by T cells. All three major T cell subsets, CD4+, CD8+ and T19+ (gamma delta T cell receptor), show an approximate four fold increase in the level of MHC class II expression during secondary responses. No changes in B cell expression of class II were seen. Resting efferent lymph T cells are predominantly either class II- or DR+DQ- but this changes to DR+DQ+ after antigenic challenge. The antigen-presenting function of these class II+ T cells was investigated at daily intervals after in vivo antigenic challenge. T cells from non-activated lymph nodes could not induce proliferation of antigen-specific T cells with soluble antigen but were weakly stimulatory in allo-mixed lymphocyte reaction (MLR) at high (> 2:1) stimulator cell ratios. Activated T cells isolated during secondary in vivo responses, and expressing increased quantities of MHC class II, were positive stimulator cells in the MLR. In contrast these cells could not present soluble antigen or trypsin-digested antigen to the T cell lines. In the MLR assays, the relative stimulation by class II+ T cells correlates with the levels of class II expression. We conclude from these experiments that both quantitative and qualitative changes in MHC class II, induced on T cells under physiological conditions, play a role in the regulation of the immune response in vivo but that that role is not simply one of presentation of soluble antigen.

Animals↗
PubMed1993

Comparison of workshop CD45R monoclonal antibodies with OvCD45R monoclonal antibodies in sheep.

The reactivity of the antibovine monoclonal antibodies (mAbs) comprising temporary cluster TC1 was compared with that of two OvCD45R mAbs on sheep cells. Three of the mAbs--CC31, CC99 and CC103--did not cross-react with sheep cells. All the workshop mAbs precipitated two molecules of apparent molecular weight (MW) 200 kDa and 220 kDa while the antisheep CD45R mAb 20-96 precipitated a single band of 220 kDa. Cell surface expression was examined by single colour FACS (fluorescence activated cell sorting) analysis of efferent and afferent lymph cells and peripheral blood lymphocytes and the distribution of the antigens on CD4+, CD8+ and T19+ (WC1) and B cells was determined by two colour fluorescence staining. By cellular distribution and immunohistology the TC1 mAbs could be divided into four distinct groups which differed from a fifth group comprising the two OvCD45R antibodies.

Animals↗
PubMed1993

Analysis of the monoclonal antibodies comprising WC6.

The WC6 antibovine monoclonal antibodies (mAbs) CC98, IL-A114 and IL-A53 were investigated for reactivity in sheep by (fluorescence activated cell sorting) FACS analysis, immunoprecipitation and immunohistology. The mAbs behave identically by all criteria although IL-A114 reacts more weakly than the other mAbs. This probably reflects limited cross-species reactivity. The mAbs stain < 30% of lymphocytes from blood, efferent and afferent lymph and the majority of afferent lymph dendritic cells. They also weakly stain granulocytes. They precipitate molecules of apparent molecular weight 220 kD and 180-190 kD. Sequential immunoprecipitation shows that CC98 antigen is not related to CD45. Immunohistology indicates staining of B cell areas and macrophages in Peyer's patch and lamina propria. The data show that these monoclonal antibodies react with a molecule distinct from OvCD45R.

Animals↗
PubMed1992

Expression and characterization of ovine major histocompatibility complex class II (OLA-DR) genes.

Previous work made use of nucleic acid probes corresponding to different subtypes of the class II regions of the human and murine major histocompatibility complex (MHC) to isolate seven different alpha and 24 different beta genes of the ovine MHC from two cosmid libraries. In an attempt to identify pairs of alpha and beta genes capable of cell surface expression, all permutations of alpha and beta genes were in turn transfected into mouse L-cells. Two pairs of alpha and beta genes co-expressed and stable ovine MHC class II L-cell lines were developed. The expressed alpha genes had previously been defined as DR-alpha homologues (DRA) by differential Southern hybridization to human subtype specific class II probes. The expressed ovine beta genes were also assigned as ovine DR-beta homologues (DRB) on the basis of their sequence having a higher degree of similarity with human DRB than any other subtype. A total of eight out of 23 anti-sheep class II specific monoclonal antibodies were typed OLA-DR specific by FACScan analysis using the L-cell lines.

Amino Acid Sequence↗
PubMed1991

Analysis of the CD1 cluster in sheep.

The anti-CD1 monoclonal antibodies submitted to the 1st International Workshop on Leucocyte Differentiation Antigens of Cattle, Sheep and Goats were tested for their reactivity on sheep skin, thymus and lymph node and for their reactivity with sheep efferent and afferent lymph and peripheral blood. With the exception of 20-27 they all stained that same cell populations. The antibodies precipitated molecules with a heavy chain of 46,000 apparent molecular weight and a light chain of 14,000 apparent molecular weight. VPM5 and CC14 antigens were purified by affinity chromatography. All the antibodies cross-reacted with these molecules. The results show that 20-27 recognises the same molecules as the other antibodies and suggest that 20-27 is a pan CD1 monoclonal antibody and the other monoclonal antibodies are homologues of the human CD1b molecules.

Animals↗
PubMed1991

Identification of a putative cellular receptor for the lentivirus visna virus.

One mechanism by which viral tropism may be controlled is by the expression of a specific virus receptor on the cell surface. This paper reports the identification of a putative cellular receptor for visna virus, the prototype virus of the family Lentiviridae. Using a virus overlay protein blot assay we identified a group of polypeptides of apparent Mr 30K to 33K which interacts with visna virus and is present on permissive but not non-permissive cells. A rat polyclonal anti-ovine major histocompatibility complex (MHC) class II antigen (Ag) serum raised to immunopurified MHC class II Ag, but not preimmune serum, blocked the interaction of visna virus with these polypeptides. In an ELISA, immunopurified MHC class II Ag bound to visna virus but not to bovine parainfluenza 3 virus. Preincubation of visna virus with immunopurified soluble MHC class II Ag resulted in a marked decrease in virus-induced syncytium formation, i.e. preincubation with class II Ag inhibited infection with visna virus, but we have been unable to inhibit infection using class II Ag-specific antisera. These results suggest that ovine MHC class II Ag acts as a component of a cellular receptor for visna virus. This is of particular interest owing to the close similarities between visna virus and human immunodeficiency virus (HIV), and the relationship between MHC class II and CD4, the cellular receptor for HIV. It is also of relevance to recent reports that a growing number of viruses utilize polypeptides of the Ig supergene family as receptors.

Animals↗
PubMed1990

Monoclonal antibodies to the sheep analogues of human CD45 (leucocyte common antigen), MHC class I and CD5. Differential expression after lymphocyte activation in vivo.

This paper describes three anti-sheep monoclonal antibodies. The tissue distribution and apparent molecular weight of the antigens detected by these antibodies is consistent with them reacting with sheep leucocyte common antigen (CD45 (VPM18], MHC class I (VPM19) and CD5 (VPM29). An ELISA method is described that permits the cross-reactivity of different antibodies to be assessed, this confirms the identity of the antigens detected by VPM18, VPM19 and VPM29. This method is also of value as either a positive or a negative screen in the construction of further monoclonals. A study of the expression of these three antigens on efferent lymph small lymphocytes and antigen-activated lymphoblasts shows that the density of CD45 on lymphoblasts (activated either in vivo or in vitro) is approximately half that of small lymphocytes whereas the density of MHC class I is the same in both populations. Furthermore, about 75% of small lymphocytes express CD5 but less than 10% of lymphoblasts are positive. Cell membrane CD5 expression is lost on lymphocyte activation. It does not seem to be linked to cell membranes via phosphatidylinositol and the loss is not due to the breaking of that link.

Animals↗
PubMed1990

Cytopathic and non-cytopathic biotypes of border disease virus induce polypeptides of different molecular weight with common antigenic determinants.

Ten monoclonal antibodies have been raised against lysates of cells infected with cytopathic border disease virus (BDV). These antibodies all recognize non-cytopathic BDV and react with a number of different strains of bovine viral diarrhoea virus (BVDV). Studies with radiolabelled cell lysates show that all the antibodies precipitate two polypeptides of apparent Mr 80,000 and 130,000 from cells infected with cytopathic virus and a single polypeptide of apparent Mr 120,000 from cells infected with non-cytopathic virus. Two of the monoclonal antibodies react on immunoblots and show the same pattern of reactivity indicating that these three polypeptides are antigenically related.

Animals↗
PubMed1990

Characterization of monoclonal antibodies specific for alpha- and beta-chains of sheep MHC class II.

In order to identify and characterize the sheep major histocompatibility complex (MHC) class II molecules, a panel of 19 monoclonal antibodies (mAb) has been raised following immunization of mice with a variety of class II antigen preparations. Antibodies were selected by ELISA using immunopurified sheep class II as antigen and further screened for the ability to react on immunoblots. Nine mAb reacted with the beta-chain and four reacted with the alpha-chain. The chain specificity of the remainder could not be determined as they did not blot. The anti-alpha and anti-beta mAb all reacted with deglycosylated class II. With the exception of one anti-alpha and one anti-beta antibody, the antibodies reacted with both efferent lymph cells and peripheral blood mononuclear cells (PBMC). Three of the anti-sheep class II mAb reacted with human PBMC.

Animals↗
PubMed1989

Characterization of sheep afferent lymph dendritic cells and their role in antigen carriage.

We have ablated peripheral lymph nodes in sheep and subsequently cannulated the pseudo-afferent lymphatic vessel that arises as a consequence of afferent lymphatic vessels reanastomosing with the former efferent duct. This technique allows the collection of lymph with a cellular composition that resembles true afferent fluid, and in particular, containing 1-10% dendritic cells. A 16-h collection of this lymph may contain between 10(6) and 10(7) dendritic cells. This dendritic cell population may be enriched to greater than 75% by a single-density gradient centrifugation step. We have generated a mAb that recognizes sheep CD1. This monoclonal not only reacts with afferent dendritic cells, but with dendritic cells in the skin and paracortical T cell areas of lymph nodes. The expression of CD1 suggests afferent dendritic cells are related to skin Langerhans' cells and other dendritic cells that act as accessory cells for T cell responses. Consistent with this is the high level of expression by dendritic cells of molecules involved in antigen recognition by T cells, including MHC class I and class II. Afferent dendritic cells express high levels of the cellular adhesion molecule LFA-3, and at the same time express a ligand for this molecule, namely CD2. The accessory functions of afferent dendritic cells resemble those displayed by mature Langerhans' cells and by lymph node interdigitating cells. These include clustering with resting T cells and stimulating their proliferation in a primary response to antigen. Afferent dendritic cells are capable of acquiring soluble protein antigen in vivo or in vitro and presenting the material directly to autologous T cells in an antigen-specific manner. We conclude that afferent dendritic cells represent a lymph-borne Langerhans' cell involved in antigen carriage to the lymph node.

Animals↗
PubMed1989

In vivo modulation of CD1 and MHC class II expression by sheep afferent lymph dendritic cells. Comparison of primary and secondary immune responses.

The experiments described in this article characterize the phenotypic and functional changes in afferent lymph cell populations that occur as a result of in vivo immune stimulation. During the primary immune response (in antigen-naive sheep) there are very transient increases in level of CD1 expression by subpopulations of dendritic cells (DC) but no alterations in cell kinetics or MHC class II expression. In contrast, secondary antigenic challenge (in primed sheep) into the drainage area of an afferent lymphatic causes profound changes in the cell output, characterized by a greater than threefold drop in total cell output on days 1-3 followed by an approximate fivefold rise on day 5. There is also a substantial increase in both the proportion of MHC class II-positive T lymphocytes (from 28 to 54%) and in the quantitative expression of class II by both DC and lymphocytes. Class II expression by DC increases five- to sixfold by day 5, while the level of expression of class II on lymphocytes approximately doubles. The increase in CD1 expression during the secondary response is more prolonged than during the primary response, being detectable between days 2 and 6 after challenge. The rise in class II affects the whole DC population, in contrast to CD1 where the increase affects only a subpopulation of cells. In terms of functional properties, afferent lymph DC isolated during a primary response show no alteration of their activity, whereas DC taken 4-5 d after secondary challenge are up to fivefold more active in their ability to present soluble antigen to primed autologous T cells and to antigen-specific cell lines as well as to stimulate in the MLR. The relative expression of class II correlates temporally with an increased capacity of DC to present antigen. Monoclonal anti-class II antibodies totally inhibit the in vitro assays but anti-CD1 antibodies have no effect. The previous paper has demonstrated that afferent DC can associate with antigen in vivo and can present that antigen to antigen-specific T cells. This article extends our knowledge of DC biology and demonstrates that DC, activated during secondary in vivo immune responses, have an enhanced ability to present an antigen, unrelated to that used for challenge, to specific T cell lines. This enhancement correlates directly with quantitative variation of expressed class II and not CD1 and suggests that this variation in class II expression plays a physiological role in in vivo immune regulation.

Animals↗
PubMed1987

Improved method for the measurement of ribonucleotide reductase activity.

An improved method for the measurement of herpes simplex virus type 1 encoded ribonucleotide reductase has been developed. The enzyme which catalyses the conversion of ribonucleoside diphosphates to deoxyribonucleoside diphosphates was determined by first converting the ribonucleotide substrate and deoxyribonucleotide product to the corresponding nucleosides by treatment with snake venom phosphodiesterase. Then nucleosides were separated by HPLC and measured by flow through scintillation counting and by monitoring their absorbance at 254 nm. Under the conditions used in the experiment cytidine and deoxcytidine, the derivitised substrate and product respectively, eluted from the column at approximately 4 min 33 s and 6 min 24 s. Peak heights and areas were automatically calculated by computer to ascertain the amount of product formed and thus quantitate the assay. Automation of the assay from sample injection to analysis provides a significant saving in time and an improvement in the efficiency of measurement of ribonucleotide reductase activity over other published methods.

Chromatography, High Pressure Liquid↗
PubMed1986

Monoclonal antibodies to sheep lymphocytes. I. Identification of MHC class II molecules on lymphoid tissue and changes in the level of class II expression on lymph-borne cells following antigen stimulation in vivo.

A rat monoclonal antibody is described that recognizes sheep MHC Class II antigen and appears to recognize a determinant that is non-polymorphic. This antibody precipitates polypeptides of apparent molecular weight 33,000 and 28,000/29,000 (under non-reducing conditions). The binding of this antibody was inhibited by an anti-human DP, DQ, DR beta-chain monoclonal (DA6.231) and its reactivity on murine cells suggests specificity for the I-E gene product. Using this antibody we have studied the distribution of Class II molecules in sheep and also the variations in Class II expression by cells in both peripheral and efferent lymph, produced as a result of in vivo antigen inoculation.

Animals↗