Biomedical subjects

H Weedon

Publications and source records attributed to H Weedon.

26 records · Page 2Linked to original sources

PubMed1996

Purification of cord blood lymphocytes.

When cord blood is separated using standard methods based on Ficoll-Hypaque, the mononuclear fraction is contaminated with erythrocytes and also with nucleated cells that do not express the leucocyte marker CD45. The contamination with CD45-negative cells can exceed 50%, and will interfere with phenotypic, mRNA or functional analysis. A large proportion of these cells are erythrocyte precursors. The contaminating cells may be removed by lysis with hypotonic ammonium chloride; when the cells are required for studies which are adversely affected by ammonium chloride (such as antigen processing), high purity can be attained by two rounds of density separation.

Cell Separation↗

PubMed1996

Characterization of human leucocytes bearing the IL-3 receptor.

Human leucocytes from peripheral blood and tonsil were examined for the presence of the IL-3 receptor using monoclonal antibodies directed to epitopes of the alpha and beta chains of the receptor. We found that the beta chain, common to IL-3, IL-5, and GM-CSF, was either present at low levels or not detected on the majority of peripheral blood and tonsil B lymphocytes, while the alpha chain showed a distinct but restricted distribution. In peripheral blood the IL-3R alpha chain was limited to a subpopulation of peripheral B lymphocytes and a population of cells which lack lineage-specific markers. Dimly staining cells were identified as B lymphocytes as they coexpressed CD19, CD20, CD22, CD24, and HLA-DR. A brightly staining population lacks T and B lymphocyte, NK specific, and macrophage lineage markers but expresses CD9, CD45RO, CD26, and, in a proportion of cells, CD36 and CD60. This population remains unclassified. In tonsil tissue IL-3R alpha chain expression was strongest on B lymphocytes present in the T cell rich areas of tonsillar tissue. The IL-3R alpha bearing B tonsil cells included cells in both CD23 and IgD positive and negative populations. The phenotype of the IL-3R alpha positive B cells defines them as a population of B lymphocytes distinct from previously characterized cells in the lymphoid architecture. Lymphoblastoid cell lines with a corresponding phenotype were also identified.

Antigens, CD↗

PubMed1996

Reduced expression of the interleukin-2-receptor gamma chain on cord blood lymphocytes: relationship to functional immaturity of the neonatal immune response.

Mutation of the interleukin-2 (IL-2) receptor gamma chain, which also serves as a component of the receptor complexes for IL-4, 7, 9 and 15, results in severe immune deficiency. We hypothesized that the immunological immaturity of healthy neonates might be associated with low levels of expression of this receptor molecule. Using monoclonal antibody and a highly sensitive immunofluorescence method, we showed that IL-2 receptor gamma chain is expressed at significantly lower levels on cord blood cells compared with adult cells. IL-2-dependent T-cell activation in vitro was reduced in cord blood cells compared with adult cells, but B-cell responses to IL-4 were not obviously impaired. The lower level of expression of the gamma chain and some other cytokine receptor chains may contribute to the immunological immaturity of the newborn, by selectively depressing particular immunological mechanisms.

Adult↗

PubMed1995

Cytokine receptor expression in human lymphoid tissue: analysis by fluorescence microscopy.

A highly-sensitive flourescence method, capable of detecting cytokine receptors present at low concentrations (around 100 molecules per cell) by flow cytometry, was adapted for use on tissue sections. This method was used to examine the expression of several cytokine receptors in lymphoid tissues. IL-2 receptors were distributed broadly, with higher concentrations in T cell areas. IL-1 receptor Type 1 was detected in T cell areas and in the follicular mantle, and was strongly expressed on vascular endothelium. IL-6 receptor was found at very low concentration, both within and outside germinal centres. The gp 130 molecule, which is involved in the functional receptor complex for IL-6 and several other cytokines, was present at higher concentrations, particularly in the germinal centre. Analysis of receptor expression in secondary lymphoid tissue provides evidence bearing on the physiological roles of cytokines, as these tissues contain cells at various stages of physiological activation located in well-defined functional zones.

Antibodies, Monoclonal↗

PubMed1993

Expression of IL-4 receptor on human T and B lymphocytes.

The expression of the interleukin-4 receptor on human blood and tonsil lymphocytes has been studied using a monoclonal antibody and high-sensitivity immunofluorescence flow cytometry. While no receptor expression could be detected on circulating or tonsil T cells, a subset of B cells was shown to express the receptor. The IL-4R-positive B cells in tonsil had a phenotype suggesting that they included both germinal centre B cells and B cells outside the germinal centre. The subset of B cells in the blood that expressed the receptor included CD23-positive B cells. Activation of tonsil B cells using anti-IgM, IL-4, IL-2, or combinations of these reagents led to increases in IL-4R expression, but these changes were small compared to changes in the expression of IL-2R p55 (CD25), a known marker of activation. Similarly, activation of T cells led to low-level expression of IL-4R, with IL-4 itself up-regulating IL-4R, especially in CD4 cells. The majority of chronic lymphocytic leukaemia samples were positive for IL-4R expression, whilst most other leukemic samples were negative.

ADP-ribosyl Cyclase↗

PubMed1993

Expression of membrane receptor for tumour necrosis factor on human blood lymphocytes.

Using a monoclonal antibody against the human p75 tumour necrosis factor receptor (TNFR-I) combined with a high-sensitivity immunofluorescence flow cytometric procedure, a proportion of peripheral blood lymphocytes can be shown to express TNFR-I constitutively. Approximately 50% of peripheral blood lymphocytes consisting mostly of CD4 cells and including most CD45R0-positive cells, express TNFR-I. Receptor expression is increased by a variety of activation signals. Only a minority (up to 30%) of tonsil B cells express measurable levels of TNFR-I. The tonsil B cells which express TNFR-I include both cells with a germinal centre cell phenotype and cells with the phenotype of the follicular mantle zone. Activation of B cells with anti-immunoglobulin, alone or in combination with interleukin-4 or interleukin-2, increases receptor expression, particularly in cells with the phenotype of mantle zone cells. The functional significance of constitutive expression of TNFR by blood and tissue lymphocytes is discussed.

Antibodies, Monoclonal↗

PubMed1991

Preparation and characterization of a chimeric CD19 monoclonal antibody.

FMC63 is an IgG2a mouse monoclonal antibody belonging to the CD19 cluster. CD19 antibodies react with a 95kDa protein expressed by cells of the B lymphocyte lineage, from pre-B cells to mature B lymphocytes. CD19 antibodies have been suggested as candidates for immunological attack on leukaemic and lymphoma cells of the B lineage because the antigen is restricted to the B lineage. With the potential use of FMC63 in immunotherapy in mind, we have produced a mouse-human chimera in which the genes coding for the VDJ region of the heavy chain and the VJ region of the light chain derive from the FMC63 mouse hybridoma, while the C region genes code for human IgG1. The genes have been transfected back into a mouse myeloma line, which secretes low levels of immunoglobulin. (Ig). This Ig was purified and biotinylated in order to determine the specificity of the antibody. The chimeric antibody has a reaction profile concordant with the original FMC63 antibody, but has the properties of a human IgG1, including the ability to fix human complement. However, the antibody is not cytotoxic in vitro in the presence of complement or cells capable of mediating antibody-dependent cellular cytotoxicity. Possible reasons for this and ways of using the antibody are discussed.

Animals↗

PubMed1991

Expression of IL-2 receptor p55 and p75 chains by human B lymphocytes: effects of activation and differentiation.

Whilst B cells in human blood can be shown to express interleukin-2 receptor (IL-2R) p55 and p75 chains, using a high-sensitivity immunofluorescence procedure, fresh tonsil B cells did not show detectable levels of expression. Culture of tonsil B cells led to low levels of expression of the p55 chain of the IL-2R, an effect which was dependent on protein synthesis. The level of expression of IL-2R chains could be modulated by culturing in the presence of a number of factors which activate B cells. p55 levels were more readily modulated than p75 levels. IL-4 and combinations of IL-4 with anti-IgM, IL-2 or tumour necrosis factor-beta (TNF-beta) modulated p55 levels, but IL-5 did not. Changes in IL-2R expression were small when compared with other B-cell activation markers such as CD23. When unfractionated tonsil cells were activated with a polyclonal stimulus, the major change was the expression of p55 by T-cell blasts--p75 expression remained low in T and B cells, and p55 expression by B cells remained low.

B-Lymphocytes↗