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Biomedical subjects

C J Howard

Publications and source records attributed to C J Howard.

At least 91 records · Page 5Linked to original sources
PubMed1996

Bovine gamma/delta TcR+ T lymphocytes are stimulated to proliferate by autologous Theileria annulata-infected cells in the presence of interleukin-2.

An in vitro model system has been developed in which freshly isolated resting WC1+ gamma/delta TcR+ T cells proliferate in response to cells transformed by the protozoan parasite Theileria annulata, providing a strategy in which the basis of activation of naive gamma/delta T cells can be investigated. Irradiated parasite-transformed cells stimulate the proliferation, but not cytolytic activity, of autologous peripheral blood mononuclear cells (PBMC) from non-immune animals. The proliferating cells are mainly WC1+ gamma/delta T cells. The majority of WC1+ gamma/delta T cells in freshly isolated PBMC express CD25 at a low level that increases when stimulated with T. annulata-infected cells. Purified WC1+ gamma/delta T cells fail to proliferate when cultured with irradiated T. annulata-infected cells and produce a small proliferative response to IL-2, but proliferate strongly to irradiated or lightly fixed Theileria-infected cells in combination with IL-2. The Theileria-infected cells express cytokine transcripts encoding IL-1 alpha, IL-1 beta, IL-6 and IL-10, but not IFN gamma, IL-2, IL-4 and IL-7. Purified WC1+ gamma/delta T cells stimulated with T. annulata-infected cells with or without IL-2 fail to produce IL-2 transcripts, but do produce those for TNF alpha. These experiments show that WC1+ gamma/delta T cells recognize a surface determinant on T. annulata-infected cells, that together with a second signal, which can be provided by exogenous IL-2, stimulates their proliferation.

Animals↗
PubMed1996

Identification of a molecule uniquely expressed on a gamma/delta TCR+ subset within bovine intestinal intraepithelial lymphocytes.

An antigen has been identified, recognized by a novel monoclonal antibody CC45, which is expressed by a subpopulation of bovine gamma/delta T-cell receptor-positive (gamma/delta TCR+) T cells restricted in their distribution to the intestinal epithelium. This subset of intestinal intraepithelial lymphocytes (iIEL) which represented 8-29% of gamma/delta TCR+ T cells in the gut epithelium expressed CD45, CD3 and L-selectin; most of these cells were CD2- and CD8-. Electron microscopic studies of CC45+ cells revealed that they were large mononuclear leucocytes containing numerous mitochondria and smooth vesicles; a proportion of these contained membrane-bound dense granules. Immunoprecipitation of 125I-labelled iIEL analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis under reducing and non-reducing conditions revealed polypeptides of 60,000 and 200,000 molecular weights, respectively indicating that the antigen, which appears distinct from molecules described in other species, is expressed on the cell surface as a complex.

Animals↗
PubMed1995

Identification of a novel class of mammalian Fc gamma receptor.

A cDNA encoding an Ig receptor that conferred the ability to bind erythrocytes sensitized with IgG2, but not IgG1, was cloned by screening a cattle alveolar macrophage library, made in the vector pCDM8, expressed in COS-7 cells. A search of the PIR database indicated a greater level of similarity with human Fc alpha R than with any other FcR. The percentage of identical amino acids was 41% and nucleotides 56%. This high similarity is between the extracellular and transmembrane domains; the cytoplasmic tails are unrelated. Similarities with human Fc gamma RI, Fc gamma RII, Fc gamma RIII, Fc epsilon RI, or bovine Fc gamma RII were less than 28%. COS-7 cells transfected with the cloned plasmid bound erythrocytes specifically sensitized with IgG2 but not with IgG1. In tests with heat-aggregated bovine Igs, IgG2 purified from serum bound to transfected COS-7 cells but IgG1 from serum and IgA from tracheobronchial secretions did not. Human serum IgA also failed to bind to transfected COS-7 cells although it did bind to bovine neutrophils and monocytes. Only aggregated bovine IgG2 inhibited the binding of IgG2-sensitized erythrocytes to COS-7 cells transfected with the plasmid. These observations indicate that the FcR bound IgG2 specifically and was not a receptor for IgA. Thus, this receptor, which we have named bovine Fc gamma 2R, represents a novel class of mammalian Fc gamma R, the evolution of which is likely to have been influenced by the truncated hinge of the bovine IgG2 molecule. Analysis of genetic divergence of the genes encoding bovine and human Fc gamma R and Fc alpha R indicated that the novel bovine gene and the human Fc alpha R gene probably evolved from a common ancestor, which is not shared by other Fc gamma R.

Amino Acid Sequence↗
PubMed1995

Role of T-lymphocyte subsets in recovery from respiratory syncytial virus infection in calves.

The role of T-cell subsets in respiratory syncytial virus (RSV) infection was investigated by using monoclonal antibodies (MAbs) to selectively deplete gnotobiotic calves of CD4+, CD8+, or WC1+ gamma delta T-cell receptor+ lymphocytes. Injection of these MAbs produced specific reductions of the target cell populations in the circulation and tissues. Ten days after RSV infection, immunoglobulin M (IgM), IgG1, and IgA antibodies were detected in sera and lung washings from control calves. Depletion of CD8+ T cells had no effect on either the serum or local antibody responses to RSV, whereas depletion of CD4+ T cells suppressed the antibody responses in two of three calves. The IgM and IgA responses were significantly increased in the lung washings of calves from which WC1+ T cells were depleted. Depletion of CD4+ or WC1+ T cells caused no significant delay in virus clearance, although an increase in the extent of pneumonic consolidation was observed in anti-CD4-treated calves. Nasopharyngeal excretion of RSV was prolonged in calves depleted of CD8+ T cells, and virus was isolated in high titers from lung washings of these animals 10 days after infection, whereas virus had been cleared from lung washings of all other animals. The delayed virus clearance was associated with an increase in the severity of pneumonic consolidation in three of four of the calves from which CD8+ T cells were depleted. This study shows that CD8+ T cells play a dominant role in the recovery of calves from RSV infection.

Animals↗
PubMed1995

CD45RO expression on bovine T cells: relation to biological function.

The 180,000 MW isoform of CD45 (CD45RO) has been identified in cattle with a novel monoclonal antibody (mAb) (IL-A116). This has allowed a more precise analysis of T-cell function in relation to CD45 isoform expression. Within the CD4+ and CD8+ T-cell populations, CD45RO+ and CD45RO- subsets were evident. Most CD4+ and CD8+ T cells that expressed the CD45RO isoform did not express the 220,000 and 205,000 MW isoforms recognized by mAb CC76. In contrast, the WC1+, CD2-, CD4-, CD8-, gamma delta T-cell receptor (TCR)+ T cells in bovine peripheral blood mononuclear cells (PBMC) were all CD45RO+. Monocytes and granulocytes were CD45RO+ but B cells were CD45RO-. Sorting experiments with CD4+ T cells from an immunized calf demonstrated that proliferative responses to ovalbumin (OVA) were entirely within the CD45RO+ subset. Following stimulation with concanavalin A (Con A) the CD45RO- subset of CD4+ T cells produced transcripts for interleukin-2 (IL-2) but not IL-4 or interferon-gamma (IFN-gamma), while the CD45RO+ subset produced mRNA for IL-2, IL-4 and IFN-gamma. Biologically active IL-2 was present in supernatants from both CD45RO+ and CD45RO-, CD4+ T cells, and IFN-gamma protein was identified by ELISA in supernatants from the CD45RO+ subset, confirming the production of cytokines implied by polymerase chain reaction (PCR). In contrast, sorting experiments with CD8+ T cells from animals immune to the protozoan parasite Theileria parva revealed substantial numbers of cytotoxic T-lymphocyte precursors in both the CD45RO+ and CD45RO- subsets. Thus it appears that although all antigenically primed CD4+ T cells remain CD45RO+, and expression of this molecule consequently identifies memory cells within PBMC, antigenically primed CD8+ T cells down-regulate CD45RO expression after activation.

Animals↗
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

Systemic vaccination with inactivated bovine virus diarrhoea virus protects against respiratory challenge.

Inactivated bovine virus diarrhoea virus, strain 11249nc, inoculated subcutaneously three times with Quil-A into calves protected against intranasal challenge with the same strain. Virus was isolated from nasopharyngeal swabs taken 4 to 8 days post challenge and blood taken 4 to 6 days post challenge from control calves but not from vaccinated calves. A second strain of virus, Ky1203nc, was selected on the basis of previously established data on its antigenicity and the amount of viral antigen produced by five cell cultures compared using an ELISA. Cultures of one cell line, MDBK, yielded a greater amount of viral antigen than the others. Strain Ky1203nc grown in MDBK cells was inactivated with beta-propiolactone, mixed with adjuvant and used as a vaccine inoculated into calves subcutaneously three times. All of 5 calves were protected against intranasal challenge with a heterologous strain. In contrast virus was isolated from nasopharyngeal swabs taken from 5 control calves and from the blood of 4 controls. All 5 control calves, but none of the vaccinates, had a leukopenia after challenge. We conclude that the selected strain and system of vaccine preparation provide an effective means of protecting calves against respiratory infection and that live vaccines are not required to protect against challenge via the respiratory tract.

Administration, Intranasal↗
PubMed1994

The Arabidopsis ribonuclease gene RNS1 is tightly controlled in response to phosphate limitation.

Two stimuli that have been associated with nutrient remobilization in plants are phosphate (P(i)) starvation and senescence. Little is known about how the nutrient remobilization machinery is induced at the molecular level, but in the case of P(i) starvation, ribonucleases are considered to play important roles in the remobilization process. Here, the control of two closely related ribonuclease genes of Arabidopsis, RNS1 and RNS3 is investigated. The RNS1 gene is sharply induced during starvation for P(i), an effect specific among the major macronutrients, whereas RNS3 transcript levels remain relatively constant. RNS1 and RNS3 produced in yeast co-migrate with Arabidopsis ribonuclease activities that exhibit the same induction properties as the transcripts in both wild-type plants and the pho1 mutant, which is defective in xylem loading of P(i). In contrast to what occurs during P(i) starvation, both RNS1 and RNS3 are modestly induced during senescence, indicating that the two stimuli could trigger different signal transduction pathways. The characterization of RNS1, in particular, provides an important first step towards elucidating the mechanisms by which plants sense and respond to P(i) limitation, a prominent condition in many soil types.

Amino Acid Sequence↗
PubMed1994

Detection of bovine viral diarrhoea virus p80 protein in subpopulations of bovine leukocytes.

Flow cytometry and two-colour immunofluorescence were used to detect cytoplasmic bovine viral diarrhoea virus (BVDV) antigen in leukocytes from viraemic cattle. Monoclonal antibody to the p80 protein of BVDV, a non-structural viral antigen, was used to identify the subpopulations of leukocytes in which viral protein synthesis had occurred. Viral antigen was detected in 23% of peripheral blood mononuclear cells. Monocytes were found to have the highest frequency of infection (35%). A higher proportion of CD2+ T cells (23%) were infected, compared with B cells (11%) or WC1+ gamma delta T cells (11%). No significant differences in percentages of different leukocyte subpopulations in blood were detected in persistently viraemic animals compared with controls.

Animals↗
PubMed1994

Identification of three distinct allelic forms of bovine CD4.

The CD4-specific monoclonal antibody (mAb) CC26, when tested on a total of 143 cattle, failed to react with T cells from 16% of animals and gave reduced intensity staining in a further 35% of animals. The results of family studies with groups of half- and full-siblings indicated that CC26 recognizes an allele of CD4 which is co-dominantly expressed in heterozygous animals. This was confirmed by sequential immunoprecipitation and by selecting transfectants expressing the CC26+ and CC26- allelic forms of CD4 following transfection of genomic DNA from a heterozygous animal. Biochemical studies also revealed an allelic difference in the relative molecular weight (M(r)) of the CD4 molecule, one allele giving 49,000/52,000 MW bands and the other 52,000/57,000 MW bands in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Treatment of CD4+ cell lines with tunicamycin resulted in the appearance of a 47,000 MW band for both allelic forms indicating that the difference in M(r) is due to glycosylation. All of the CC26+ alleles examined were of the low molecular weight form (M(r)low) whereas both M(r)low and M(r)high alleles were represented in CC26- animals. Thus, on the basis of M(r) and reactivity with mAb CC26, three allelic forms of bovine CD4 can be identified, namely CC26+ M(r)low, CC26- M(r)low and CC26- M(r)high; it is proposed that these alleles are designated CD4.1, CD4.2 and CD4.3, respectively. The allelic difference detected by CC26 was present in both Bos taurus and B. indicus cattle indicating that it had arisen prior to divergence of these subspecies. The M(r)high allele (CD4.3) was detected only in B. indicus animals.

Alleles↗
PubMed1994

Expression on porcine gamma delta lymphocytes of a phylogenetically conserved surface antigen previously restricted in expression to ruminant gamma delta T lymphocytes.

A 180,000 MW molecule has been identified on porcine leucocytes that is the homologue of the 215,000/300,000 MW WC1 (T19) leucocyte antigen previously considered to be restricted to ruminants. In ruminants the WC1 molecule is expressed by a T-cell subpopulation that is CD2-CD4-CD8-CD5+ and that is gamma delta T-cell receptor positive (TcR+). In pigs, the 180,000 MW molecule, identified by a new monoclonal antibody CC101, is expressed by a gamma delta TcR+ T-cell subpopulation that is also CD2-CD4-CD8-. The p180+ cells are a major T-cell subpopulation comprising approximately 40% of the peripheral blood mononuclear cells from 6-9-month-old pigs. Expression of p180 identifies the majority of the CD2-CD4-CD8- T cells in porcine blood. The p180+ T cells have a distribution in lymphoid tissues that is distinct from that of T cells that express the CD2, CD4 or CD8 molecules. They are evident particularly in the thymic medulla, the epithelium, lamina propria and interfollicular areas of the small intestine, and the superficial dermis of the skin, but largely absent from conventional T-dependent areas of secondary lymphoid tissue.

Animals↗
PubMed1993

Initial statistical clustering: definition of temporary clusters of monoclonal antibodies for the duration of the workshop.

Statistical clustering was performed on flow cytometry data generated from the reactivities of 189 mAbs with 56 cell preparations or cell lines (targets). Six targets were excluded from the statistical clustering because of non-reproducibility of data from the internal controls. Thirty-six temporary clusters (TC) were formed which were subjected to further examination in the second phase of the workshop.

Animals↗
PubMed1993

Final clustering.

Final statistical cluster analysis was performed on 51 target cells. These included three new complete datasets that were submitted to the workshop in time for the final cluster analysis. Two human cell lines that were used for the initial temporary clustering were omitted from the final targets. Most temporary clusters remained unchanged but several, previously loosely clustered, mAbs no longer formed clusters.

Animals↗
PubMed1993

A new bovine leukocyte antigen cluster comprising two monoclonal antibodies, CC43 and CC118, possibly related to CD1.

Two mAbs, CC43 (012) and CC118 (040), clustered together when the flow cytometric data for staining of target cells was analysed statistically. The antigen recognized was present on immature thymocytes but not mature T cells; it was expressed by B cells and monocytes but not by granulocytes. Two molecules were evident by SDS-PAGE run under reducing conditions. One had an M(r) of 44 kDa, the other an M(r) of 12 kDa. The smaller molecule was probably beta 2-microglobulin; the larger one was distinct from bovine CD1b and MHC Class I. The mAb may recognize the product of a previously unrecognized bovine CD1 gene and should be considered as a novel bovine mAb cluster. The molecule appeared to be polymorphic and distinct alleles may be expressed on Bos taurus and Bos indicus.

Animals↗