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M J Ratcliffe

Publications and source records attributed to M J Ratcliffe.

At least 55 records · Page 3Linked to original sources
PubMed1989

Generation of immunoglobulin heavy chain diversity subsequent to cell surface immunoglobulin expression in the avian bursa of Fabricius.

The avian bursa of Fabricius represents a site for the generation of antibody diversity. Transfer of neonatal bursal cells into cyclophosphamide-treated neonatal chickens results in reconstitution of recipient bursae with donor-derived bursal stem cells. These stem cells express cell surface IgM and, under conditions of limiting donor bursal stem cell numbers, each reconstituted bursal follicle is colonized by a single precursor cell. The expression of an Ig VH idiotype, CVH-1, was found to be heterogenous within such clonal follicles. The diversity generated within the bursa is subsequently found within the peripheral B cell compartment. Thus, the generation of functional Ig H chain diversity is shown to occur subsequent to Ig H chain rearrangement and expression.

Animals↗
PubMed1989

Bu-2, a novel avian cell surface antigen on B cells and a population of non-lymphoid cells, is expressed homogeneously in germinal centers.

A monoclonal antibody (mAb), Hy30, was generated against bursal cells. Under reducing conditions, it immunoprecipitates a cell surface antigen with an apparent molecular weight of 66 kD that is distinct from immunoglobulin and MHC class II. The antigen recognized by Hy30 is found on greater than 95% of all bursal cells, on a population of thymus resident cells, and on blood, spleen, and marrow mononuclear cells. Immunohistological analysis of tissue sections revealed that the majority of the staining by Hy30 occurs in the B cell areas of the spleen and thymus. Flow cytometric analysis demonstrated that the antigen recognized by Hy30 is found on both immunoglobulin positive (Ig+) and negative (Ig-) cells. Analysis of a chicken rendered B cell deficient by treatment with cyclophosphamide at hatch confirmed this observation. In these birds, the medullary dendritic cells of the bursa and Ig- cells of the "B-cell areas" of the spleen and thymus expressed the Hy30 Ag. Analysis of several avian tumor lines did not suggest an obvious lineage for these Hy30+ Ig- cells. However, analysis of germinal centers suggested that these cells may be tissue macrophages or dendritic cells. This is consistent with previously reported histology of these "B-cell areas". Flow cytometric analysis of the antigen recognized by Hy30 demonstrates that it is distinct from the Bu-1 B-cell surface antigen and we designate it as Bu-2.

Animals↗
PubMed1989

Rationale and statistical methodology for the VISA studies.

After examination of the epanolol ('Visacor') clinical package it became clear that, although efficacy and safety of epanolol were equivalent to efficacy and safety with other antianginal therapies, tolerability was improved. It was decided to initiate 2 studies with 500 patients in each to quantify the improved tolerability and examine patient preference for antianginal treatments. One study was a comparison of epanolol 200mg daily with metoprolol 100mg twice a day and the other compared epanolol 200mg daily with nifedipine retard 20mg twice a day. The rationale, design and statistical methodology are presented, together with a summary of the geographical spread of the study.

Adrenergic beta-Antagonists↗
PubMed1988

Antigen-presenting cell-T cell interaction in the chicken is MHC class II antigen restricted.

The involvement of the MHC in the recognition of Ag by avian T lymphocytes was analyzed. PBL from chickens primed with keyhole limpet hemocyanin in vivo were induced to synthesize DNA in an in vitro response to specific Ag. Responding cells were T cells as judged by immunofluorescence staining. In vivo Ag-primed PBL were stimulated in vitro with specific Ag and further propagated in the presence of IL-2. Subsequent Ag-specific T cell proliferation required the presence of Ag-pulsed peripheral blood adherent cells (APC). T cell responses were restricted by the MHC of the APC; Ag presented by allogeneic APC did not support T cell proliferation. By using MHC-recombinant chicken lines, the gene products controlled by MHC class II loci were shown to restrict the T cell-APC interaction. This conclusion was substantiated by the inhibition of the Ag-specific T cell response by a mAb against chicken MHC class II gene products but not by a mAb against chicken MHC class I gene products.

Animals↗
PubMed1988

The molecular interactions with helper T cells which limit antigen-specific B cell differentiation.

Helper T (Th) cell-dependent activation requirements for 2,4,6-trinitrophenyl (TNP)-specific resting B cells obtained from mice transgenic for Sp-6 mu, kappa genes were analyzed. Carrier-specific T cell help required linked recognition of TNP carrier and was functionally restricted by the B cell major histocompatibility complex. However, histoincompatible T cell-B cell conjugates formed by bridging surface immunoglobulin and Th cell receptor for antigen (TcR) through TNP-conjugated anti-TcR antibodies resulted in the efficient differentiation of TNP-specific B cells. Thus, Th cell-dependent cognate recognition of B cells is not obligatory. Specific conjugate formation could be obviated by using unconjugated fragments of anti-TcR antibodies. If dimeric, these fragments supported the Th cell-dependent differentiation of co-cultured histoincompatible resting B cells. Unconjugated monomeric fragments were ineffective, demonstrating the necessity for TcR cross-linking. Resting B cells from Sp-6+ mice rendered TNP-conjugated monomeric fragments of anti-TcR antibodies effectively multivalent, thereby satisfying conditions for the activation of co-cultured Th cells. The results demonstrate that Th cells do not transduce activation signals through TcR recognition of B cell membrane-associated ligand which limit the induction of B cell differentiation. Cross-linking of TcR on Th cells is required, sufficient and can be induced through interaction with the antigen-specific B cell surface.

Animals↗
PubMed1987

A re-evaluation of the function of the bursa of Fabricius.

We will briefly outline mammalian B cell ontogeny to provide a comparison with the avian model. The earliest defined stage of mammalian B cell development is the pre-B cell (itself derived from a multipotent stem cell) which expresses heavy chains within the cytoplasm and is a large, rapidly dividing cell. This cell drops out of division, reduces in size and, over about 24 hours, rearranges Ig light chain V region gene. As a consequence of light chain rearrangement and expression, intact IgM is expressed on the cell surface and the cell leaves the bone marrow as a small virgin B lymphocyte with the capacity to respond to antigen (Opstelten and Osmond 1983). This pathway occurs throughout the life of the animal and so there is constant production of B cells from the bone marrow which are recently derived from sIg- precursors, which have, in turn, recently rearranged their Ig V region genes. Thus there is a constant influx of new V region gene combinations into the peripheral mammalian B cell pool. Furthermore, the mammalian B cell repertoire is very large with estimated germ line diversity approaching 5 X 10(7) as a consequence of various germ-line V region recombinations (Honjo 1983). The pre-bursal stem cell is present in the periphery of the embryo from before day 8 to about day 16 - 17 of embryonation. Most colonisation of bursal follicles probably occurs prior to day 13. Individual bursal follicles are populated by a low number (2-3) of precursor cells. These rapidly become committed for the expression of particular V region genes, probably as a consequence of productive V region recombination. This recombination is restricted to the embryo and may not require the bursal microenvironment for its induction. The available germ-line repertoire of heavy and light chain V region in the chicken is very limited and little diversity can be generated from this initial recombination event (see Weill et al 1986). By day 12 of embryonation, sIg+ cells are present in the bursa and from this time sIg+ cells rapidly divide within the medulla of the bursal follicle. By about day 18 of embryonation, there are no pre-bursal cells in the periphery and sIg+ cells begin to seed from the bursa. It is from about this time that the cortex appears within the bursal follicle and it is tempting to suggest that cells migrate from the medulla to the cortex where further cell division occurs prior to export into the periphery.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1986

Avian B cell precursors: surface immunoglobulin expression is an early, possibly bursa-independent event.

The avian bursa of Fabricius contains about 1 X 10(4) discrete follicles, each of which is colonized by a small number of lymphoid progenitor cells during embryonic life. We have previously shown (J.R.L. Pink et al., Eur. J. Immunol. 1985. 15:617) that all, or almost all B cell progenitors in the bursae of 4-day-old chicks express cell surface IgM. In this report, we have analyzed the distribution of cell surface (s)IgM-1 allotypes within individual follicles of (M-1a/M-1b) allotype heterozygous birds. Although the majority of follicles contained a mixture of sIgM-1a+ and sIgM-1b+ cells, a significant proportion of isolated follicles contained exclusively sIgM-1a+ or sIgM-1b+ cells. Statistical analysis of the frequency of such "M-1a" and "M-1b" follicles demonstrated that the sIg+ B cells in the bursae of 4-8-week-old birds are derived from 2-4 allotypically committed precursor cells per follicle. Since we have previously shown that each bursal follicle is colonized by 2-5 pre-bursal stem cells, these cells must be committed to the eventual expression of one or other allotypic haplotype before they have undergone extensive proliferation within the bursa. In addition, we show that almost all B progenitor cells from the bursae of chicks which had been allotype suppressed as embryos were committed to synthesis of the nonsuppressed allotype, showing that this commitment was essentially complete at the time of suppression (i.e. before 19 days of incubation). Finally the bone marrow of 16-day embryos was used to reconstitute the bursal lymphocytes of cyclophosphamide-treated host embryos. Reconstitution was inhibited by anti-Ig antiserum indicating that most 16-day embryonic BM-derived bursal cell precursors also express sIgM. These results raise the possibility that expression of sIgM may be controlled by a "biological clock" rather than by any inductive capacity of the bursal microenvironment. Furthermore, these results provide further evidence that in normal birds a self-renewing sIg+ B cell population in the hatched chicken is the sole source of B cells in the adult.

Animals↗
PubMed1986

Chicken T-cell growth factor: use in the generation of a long-term cultured T-cell line and biochemical characterization.

Supernatants from concanavalin A (Con A)-stimulated chicken spleen cells were used to generate a long-term cultured cell line from antigen-primed chicken peripheral blood leukocytes. This line has been kept in continuous proliferation in vitro for more than 25 weeks. Morphologically these cells were lymphoblastoid and expressed class I and class II antigens of the major histocompatibility complex as well as T-cell (but not B-cell or macrophage) antigens. In addition they contained no peroxidase or non-specific esterase activity, neither were they phagocytic. Proliferation of the line was totally dependent on exogenous T-cell growth factor (TCGF) activity provided by the Con-A-stimulated spleen cell supernatant, comparable with the proliferation of Con-A-induced T-cell blasts. TCGF activity from the supernatant was absorbed both by the long-term cultured T cells and by Con A blasts, demonstrating the presence of receptors for the same TCGF species on the two populations. We have used the long-term cultured cell line to characterize chicken TCGF further. The molecular weight of the biologically active fractions found by gel filtration on Sephadex G-100 was approximately 13,000 and isoelectric focusing showed chicken TCGF to have a pI of pH 5.9. We propose that the TCGF described here is the chicken analogue to the mammalian interleukin 2.

Animals↗
PubMed1985

Immunoglobulin-bearing stem cells for clones of B (bursa-derived) lymphocytes.

Treatment of neonatal chickens with cyclophosphamide depletes bursal lymphocytes while maintaining the bursal epithelium intact. The bursae of normal young chickens contain "bursal stem cells" which can reconstitute the lymphoid compartment in the bursa of the cyclophosphamide-treated recipient. Using bursal stem cells from IgM allotype-heterozygous donors we show that most bursal follicles in the reconstituted host are colonized by single stem cells which are committed to the expression of one or other IgM allotype. In addition we show that the reconstituting bursal stem cells express allelically excluded surface IgM at the time of transfer. Our results suggest that B lymphocyte numbers in hatched chickens are maintained by self-renewal of committed precursors rather than by de novo production from multipotential stem cells.

Animals↗
PubMed1984

Acquired immunity to heavy infection with Mycobacterium bovis bacillus Calmette-Guérin and its relationship to the development of nonspecific unresponsiveness in vitro.

Mice heavily infected with Mycobacterium bovis bacillus Calmette-Guérin (BCG) rapidly generated an acquired cellular immune response to this infection, as characterized primarily by the emergence of a splenic T-cell population capable of passively transferring substantial levels of adoptive protection against a challenge infection with M. tuberculosis. The emergence of this protective T-cell population was temporally associated with considerable levels of DNA synthesis in vivo in both the spleen and liver, and with the development of an acquired capacity within the animal to express very high levels of nonspecific resistance to secondary intracellular bacterial infection. Concomitant with the emergence of this acquired response, splenic T cells from infected animals became severely unresponsive to blastogenic in vitro stimulation with the mitogen phytohemagglutinin, and possessed the capacity to suppress the responsiveness of normal T cells in cocultures. Both the unresponsiveness of T cells from infected mice and their immunosuppressive activity in vitro could be essentially ablated by supplementation of the tissue culture medium with a supernatant containing very high titers of the T-cell growth factor interleukin 2 (IL-2). Furthermore, T cells harvested from these animals at the peak of in vitro unresponsiveness exhibited a substantial capacity to absorb or consume IL-2 from IL-2-containing supernatants. It is hypothesized, on the basis of these findings, that mice heavily infected with BCG acquire an IL-2-dependent T-cell population within the spleen in response to this infection, and that the observed in vitro blastogenic unresponsiveness of spleen cells which contain this population may be an artefactual effect arising from the reduction or consumption of available IL-2 within the sustaining culture medium. The relevance of these findings is discussed with particular regard to clinical situations, such as lepromatous leprosy, in which restorative strategies involving the in vivo use of IL-2 are presently being postulated.

Animals↗
PubMed1984

Heterogeneity in the response of T cells to antigens presented by B lymphoma cells.

Proliferation of antigen-specific T-cell populations was induced in cultures stimulated with antigen and a suitable source of antigen-presenting cells. Soluble (keyhole limpet hemocyanin) and particulate (horse red blood cells) antigens were presented by irradiated spleen cells and by a variety of B-lymphoma-cell lines, providing support for antigen-specific H-2-restricted T-cell responses. A marked heterogeneity was demonstrated, however, in the capacity of T-cell lines to proliferate in response to antigen presented by the B-lymphoma cells. T-cell populations were prepared from the lymph nodes of antigen-primed mice and restimulated in vitro in the presence of antigen and irradiated spleen cells. During the first six in vitro restimulations, these T-cell populations maintained the capacity to respond to antigen presented either by irradiated spleen cells or by B-lymphoma cells. Continued growth of these T-cell populations, again in the presence of antigen and irradiated spleen cells, resulted in the generation of T-cell lines which had lost the ability to respond to antigen presented by B-lymphoma cells. These lines however, fully retained the capacity to proliferate in the presence of antigen and irradiated spleen cells. T-cell clones derived from one of these lines were also unable to respond to antigen presented by B-lymphoma cells but again proliferated in the presence of antigen and irradiated spleen cells. Supernatants containing high levels of IL-1, IL-2, or IL-3 activity failed to reconstituted the antigen-specific response of T-cell lines which had lost the capacity to respond to antigen presented by B-lymphoma cells. Furthermore, titrated numbers of irradiated spleen cells, while having the capacity to support T-cell proliferation themselves, failed to synergize with B-lymphoma cells in the support of antigen-specific T-cell proliferation. Thus we have defined populations of antigen-specific, H-2-restricted T cells which do not recognize antigen presented by B-lymphoma cells and can therefore discriminate between different antigen-presenting cell types.

Animals↗
PubMed1984

Proliferation of chicken peripheral blood leukocytes in response to pokeweed mitogen is macrophage dependent.

Chicken peripheral blood leukocytes (PBL) proliferate in vitro in response to a wide range of pokeweed mitogen (PWM) concentrations. The PWM response was not influenced by the major histocompatibility complex (MHC) haplotype nor by the intrinsic responsiveness of the PBL to concanavalin A (Con A). The results indicate that PWM under our conditions stimulates B-lineage cells, although a T-cell subset is also clearly induced to division. The PBL from B-cell-depleted animals gave substantially lower responses than those from normal controls. Pokeweed mitogen stimulation of PBL was adherent cell dependent. Thus the low PWM response of adherent cell-depleted PBL was reconstituted by the addition of irradiated unseparated PBL. Furthermore, purified irradiated adherent cells containing greater than 90% macrophages were also able to reconstitute PWM responses. Finally, we have shown that PWM was able to induce large numbers of B cells to produce cytoplasmic immunoglobulin. However, only a minor proportion of such cells were induced to immunoglobulin secretion.

Animals↗
PubMed1984

T-dependent activation of resting B cells mediated by concanavalin A.

In cultures containing long-term cultured lines of antigen-specific helper T (Th) cells, normal unprimed B cells and concanavalin A (Con A), induction of B cells to immunoglobulin secretion and DNA synthesis was observed. The plaque-forming cell (PFC) response was large (frequently greater than 75 000 PFC/10(6) input B cells) demonstrating the polyspecific nature of the response. Con A-mediated maturation and induction to DNA synthesis of responding B cells was completely Th cell dependent and inhibited with methyl-alpha-D-mannoside. Both resting and blasted B cells, separated by Percoll density centrifugation, were induced to DNA synthesis and immunoglobulin secretion. Responses were completely unrestricted by the B cell major histocompatibility complex, even at the level of the resting B cell. The polyclonal nature of the response taken together with the Con A-mediated bypassing of T cell specificity and restricting haplotype indicates that this response is analogous to lectin-mediated cytotoxicity.

Animals↗
PubMed1984

Allotype suppression in the chicken. V. Abnormal isotype ratios of chronically suppressed IgM and IgG allotypes.

Chronic allotype suppression was generated in M-1 (C mu), G-1 (C gamma) heterozygous B14 line chickens either by embryonal injection or by maternal transfer of anti-M-1b allotype antibodies. Using a sensitive radioimmunoassay to detect the suppressed IgM-1b, a striking disparity was observed between the degree of suppression of the IgM-1b allotype and that of the genetically linked IgG-1i allotype. The amount of suppressed IgM-1b ranged between 0.2 and 3% of total serum IgM in chronically suppressed birds. The levels of the genetically linked IgG-1i, however, comprised 5 to 25% of total serum IgG in such birds. The allotypes measured in suppressed chickens were qualitatively identical to those in normal, nonsuppressed birds by serological criteria. These results are discussed within the context of isotype regulation in the chicken.

Alleles↗
PubMed1983

Two classes of bystander B cell response: activation requirements reflect those of B cells in general.

Bystander anti-sheep red blood cell (SRBC) antibody responses were induced in cultures containing unprimed B cells, SRBC, particulate or soluble eliciting antigen, and helper T (Th) cells primed to the eliciting antigen. The use of long-term cultured Th cells, depleted of alloreactive cells, allowed direct assessment of the requirements for major histocompatibility complex- (MHC) restricted interactions in the cellular events leading to bystander B cell activation. H-2 restricted Th cell activation limited all bystander B cell responses; however, the requirement for H-2 restricted Th-B cell interactions varied with the state of activation of the responding B cell population before assay. Thus, we defined two classes of bystander B cell response. Activation of small (i.e., resting) SRBC-specific bystander B cells, purified by density gradient centrifugation, was completely restricted by B cell MHC. The requirement for high concentrations of eliciting antigen suggests that nonspecific binding of soluble or particulate antigen to the responding B cell surface plays a mandatory role leading to the activation of resting bystander B cells. The concomitant requirement for a restricted Th-B cell interaction suggests that this role is one of focusing antigen-specific, H-2 restricted T cell help to the resting B cell. In contrast, bystander responses, unrestricted by B cell MHC, were generated exclusively from large (i.e., blasted) B cells limited only by Th cell activation. This class of SRBC-specific bystander response correlated with an SRBC-dependent increase in the number of cells secreting antibodies that recognized degraded mouse RBC. Thus, we propose that the H-2 unrestricted anti-SRBC response is derived from the amplification of B cells activated in vivo to degraded homologous RBC. Both classes of anti-SRBC response were enhanced by the presence of the bystander antigen. This phenomenom is discussed in terms of a mitotic advantage provided by the interaction of antigen with surface immunoglobulin on specific bystander B cells.

Animals↗
PubMed1981

Allotype suppression in the chicken. II. Suppression in homozygous chickens with antiallotype antibody and allotype-disparate B cells.

Injection of heterozygous (M-1a/M-1b, G1g/G-1i) B 14-line chickens with antisera directed against either IgM-1a or IgM-1b induced suppression of the relevant IgM-1 and genetically linked IgG-1 allotypes, whereas a mixture of anti-M-1a and anti-M-1b antibodies failed to produce allotype suppression. Injection of anti-M-1 antiserum into M-1 homozygous chickens induced only a transient delay of a few days in the appearance and rise of serum IgM-1 levels. However, suppression of host allotypes was induced by injecting M-1, G-1 homozygous neonatal or embryonal recipients with anti-M-1 antisera together with B locus- histocompatible allotype-disparate spleen, bone marrow or bursal cells. The active cell type were donor B cells, which established chimerism in the injected host, whereas peripheral blood T lymphocytes from agammaglobulinemic donors were ineffective. Allotype suppression was attributed to a homeostatic control mechanism which is exerted by normal B cells (but not T cells) over B cell recruitment in anti-M-1 antibody-treated, immature hosts.

Animals↗