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Biomedical subjects

P M Lydyard

Publications and source records attributed to P M Lydyard.

At least 19 recordsLinked to original sources
PubMed1992

Specificity and idiotope expression of IgM produced by CD5+ and CD5- cord blood B-cell clones.

Epstein-Barr virus (EBV)-immortalized monoclonal B-cell lines were established from CD5+ and CD5- cord-blood B cells. IgM from many of both CD5+ and CD5- clones reacted with IgG-Fc, ssDNA, and a variety of other autoantigens. More CD5+ B cells that used light chains of the kappa isotype reacted with IgG-Fc and ssDNA than kappa-bearing CD5- B cells. Because many of the clones reacted with IgG-Fc, they were analyzed for the expression of cross-reactive idiotypes (CRI) associated with rheumatoid factor and cold agglutinin paraproteins using murine antibodies (mAb) recognizing V kappa and VH subgroup-associated determinants. Expression of the V kappa IIIb sub-subgroup-associated idiotope recognized by 17.109 mAb was expressed at significantly higher frequency (32%; p less than 0.05) and IgM antibodies derived from the CD5+ compared with the CD5- clones (5%). Both CD5+ and CD5- clones expressed the RF paraprotein-associated idiotope recognized by G8 mAb to the same extent. Similar results were obtained using binding to SpA as a marker of VH III family usage. Furthermore, no differences in frequency of expression of RF paraprotein-associated idiotopes recognized by B6 and/or D12, and characteristic of some antibodies using VH III family genes, were found between the CD5+ and CD5- populations. Although a higher than expected frequency of VH IV-gene expression was demonstrated (around 30%) in both CD5+ and CD5- cells, there were differences in expression of CRI recognized by mAb Lc1 and R2.1A2 with specificities for two VH IV subfamilies. While some CD5+ and CD5- clones were identified in which their IgM reacted with mAb Lc1, only CD5+ clones were recognized by another mAb R2.1A2. Analysis of the relationships between antigen specificities and V kappa- and VH-family gene usage indicated that auto- or polyreactivity was not associated with V kappa III nor any particular VH family. The higher frequency of the V kappa IIIb sub-subgroup-associated idiotope recognized by 17-109 in the CD5+ clones and the association of CD5+ B cells with the VH IV subfamily recognized by mAb R2.1A2 and 9G4 may suggest that CD5+ B cells in cord blood are expanded as a result of recruitment within the fetal environment.

Animals↗
PubMed1992

The development of monoclonal antibodies to the human mitochondrial 60-kd heat-shock protein, and their use in studying the expression of the protein in rheumatoid arthritis.

OBJECTIVE: To assess the claim that the human 60-kd heat-shock protein (HSP) is highly expressed in the joints of patients with rheumatoid arthritis (RA), but is not readily detected in normal tissues. METHODS: Monoclonal antibodies were raised against the human 60-kd mitochondrial heat-shock protein (P1 protein; hsp60), and their specificity was established. They were then applied to synovial tissue. RESULTS: HSP was expressed similarly in normal, osteoarthritic, and RA synovium. Low levels of hsp60 were detected in synovial fluid by immunoprecipitation. CONCLUSION: Minor differences in the distribution of hsp60 in synovium from RA joints were attributable to increased cellularity and to the disorganization of the tissue architecture.

Animals↗
PubMed1992

Surface expression of heat shock protein 90 by blood mononuclear cells from patients with systemic lupus erythematosus.

Previous studies have indicated that blood mononuclear cells from 15-20% of patients with systemic lupus erythematosus (SLE) carry elevated levels of hsp90, a heat shock protein associated with steroid receptors in cells. We analysed surface expression of hsp90 on mononuclear cells (lymphocytes and monocytes) from patients with SLE by monoclonal antibody AC88 and flow cytometry. Whilst all blood mononuclear cells have intracellular hsp90, a significant proportion of patients with SLE expressed hsp90 on lymphocyte and monocyte surfaces. This was significantly higher on SLE lymphocytes than in laboratory controls and was positively correlated with disease activity. Comparison of total hsp90 with surface hsp90 in the same SLE patients' blood mononuclear samples indicated a correlation with a subgroup of patients. There was no correlation with expression of surface hsp90 by lymphocytes and activation markers. Patients with Sjögren's syndrome, rheumatoid arthritis, dermatomyositis and scleroderma were studied as disease controls and increased levels of shsp90 were detected in only three of the 53 patients studied. It is concluded that surface hsp90 expression is a feature of about 20% of patients with SLE and is correlated with high disease activity. The exposure of this hsp on the surface of some lymphocytes suggests that it is a candidate autoantigen in SLE.

Adult↗
PubMed1992

Repertoire of CD5+ and CD5- cord blood B cells: specificity and expression of VH I and VH III associated idiotopes.

Epstein-Barr (EBV)-immortalized B cell clones were established from CD5+ and CD5- cord blood B cells separated by flow cytometry. We have previously shown that IgM from many of the clones was polyreactive, exhibiting reactivity with a number of autoantigens. In this study, IgM produced by the clones was analysed by MoAb for the expression of cross-reactive idiotypes (CRI) associated with rheumatoid factor paraproteins and from defined VH and V kappa subgroups of immunoglobulin heavy and light chains. IgM produced by clones established from CD5+ and CD5- B cells expressed the VH I associated idiotope G8. Furthermore, IgM produced by both sets of clones exhibited a similar frequency of VH III heavy chain subgroup expression, as determined by reactivity with staphylococcal protein A (SpA) and VH III-associated CRI expression (B6 and/or D12). In contrast, expression of the V kappa III-associated 17.109 CRI was significantly higher in IgM antibodies produced by clones established from CD5+ compared with the CD5- clones (32 versus 5%: P less than 0.05). Analysis of the VH and VL subgroup expression by IgM produced by the CD5+ and CD5- cord blood clones, and their autoantigen reactivity profile did not reveal restriction or selection within CD5+ and CD5- populations. However, our data suggest that differences may exist in the expression of certain germ-line genes between CD5+ and CD5- cord blood B cells and might indicate an expansion of CD5+ B cells within the fetal environment.

Animals↗
PubMed1992

Lymphocytes from patients with rheumatoid arthritis produce agalactosylated IgG in vitro.

The percentage of oligosaccharide chains lacking galactose was measured in IgG obtained from pokeweed mitogen-activated cultures of blood lymphocytes from patients with rheumatoid arthritis and controls. Secreted IgG from rheumatoid arthritis lymphocytes was deficient in galactose compared with IgG from the lymphocytes of controls. This confirms that agalactosylation is a significant feature of the disease and demonstrates that it can occur at the B cell level and is not merely a post-secretory event.

Adult↗
PubMed1992

Anti-Epstein-Barr virus-nuclear antigen-1, -2A and -2B antibodies in rheumatoid arthritis patients and their relatives.

We have examined serum antibodies to Epstein-Barr virus Nuclear Antigen (EBNA)-1, -2A and -2B, in addition to antibodies to viral capsid antigen and early antigen in 100 rheumatoid arthritis patients and 50 of their relatives. Using indirect immunofluorescence on transfected cells and Western-blot technique, we have found increased frequency and titres of antibodies to EBNA-2B in patients and, to a lesser degree, in their family members, whereas other anti-Epstein-Barr virus antibodies appeared to be similar to controls. Cross-inhibition experiments were carried out and show that antibodies to EBNA-2A are distinct from those to -2B, and vice versa.

Adult↗
PubMed1992

Resistance of chronic lymphocytic leukaemia cells to interferon-alpha generated lymphokine activated killer cells.

Recent studies have shown that, when used in early stage disease, interferon-alpha (IFN-alpha) can produce a fall in the number of malignant cells in the peripheral blood of patients with B-CLL. In this study, we investigated the effect of IFN-alpha on natural killer (NK) cell and lymphokine-activated cell (LAK) activity in patients with B-CLL. In vitro, IFN-alpha (500 U/ml for 18 hours) induced LAK activity in patients with B-CLL (27.7 +/- 9.9%, n = 20), and IL-2 (500 U/ml for 5 days) produced similar activity (35.9 +/- 8.8%, n = 7). Despite the induction of LAK activity by IFN-alpha and IL2 in patients with B-CLL, the malignant cells remained resistant to both allogeneic and autologous LAK effectors. NK activity in patients with B-CLL is also low (23.1 +/- 7.2%, n = 20), and B-CLL cells were resistant to NK cell activity. In cold target competition assays, CLL cells did not compete with labelled K562 or Daudi targets in the NK and LAK assays, suggesting that the malignant cells are not recognised by the effector cells, and this may be related to low level of expression of the adhesion receptors, LFA-1 and ICAM-1. Finally, CLL cells were also resistant to antibody dependent cell mediated cytotoxicity, but were susceptible to antibody dependent complement mediated lysis. These results suggest that it is unlikely that the effects of IFN-alpha in B-CLL are due to the enhancement of NK or LAK activity.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1992

Cytokine induction of leucocyte adhesion molecule-1 (LAM- 1) expression on chronic lymphocytic leukaemia cells.

Leucocyte adhesion molecule 1 (LAM-1) participates in the binding of human leucocytes to high endothelial venules in peripheral lymph nodes. Other adhesion receptors which are involved include CD44 and the integrin family, CD11/CD18. In this study, B-cell chronic lymphocytic leukemia (B-CLL) cells were examined for the expression of these adhesion molecules, and for the way in which cytokines are able to modulate the levels of these receptors. B-CLL cells express significant but variable levels of LAM-1 and high levels of CD44. In contrast, these cells exhibit very low or absent amounts of surface CD11a, CD11b, or CD11c. Most CLL cells expressed no detectable levels of intercellular adhesion molecule-1 but some cases show levels of up to 30%. Following 24 h incubation with interferon alpha (500 U/ml), surface LAM-1 expression on peripheral blood E-negative cells from CLL patients rose to 330 +/- 127% of levels on control cells incubated with medium alone (n = 13, p less than 0.0005). Interleukin 4 (1 ng/ml) and interferon gamma (100 U/ml) also increased surface LAM-1 levels on these cells to 218 +/- 119% (n = 8, p less than 0.001) and 245 +/- 116% (n = 5, p less than 0.001) of control levels respectively. Induction of LAM-1 expression occurred over 48 h (greater than 50% of the increase was seen in the first 24 h) in a dose-dependent manner and required protein synthesis. The induction of LAM-1 expression on the malignant cells may, by altering the homing behaviour of these cells, relate to the reduction in peripheral leukaemic cells seen following treatment with interferon alpha in CLL.

Cell Adhesion Molecules↗
PubMed1991

Selective expression of a VHIV subfamily of immunoglobulin genes in human CD5+ B lymphocytes from cord blood.

Human B lymphocytes expressing the CD5 surface antigen (CD5+ B cells) constitute a subset capable of producing polyspecific antibodies recognizing a variety of self antigens. The repertoire of antibodies produced by CD5+ and CD5- B cells is different. However, it is not yet established whether this distribution is reflected in different immunoglobulin variable region gene (IgV) use. Rearrangement of heavy chain IgV (IgVH) genes represents one of the first identifiable stages in the maturation of B cells, and occurs in a developmentally ordered fashion. The repertoire of IgVH gene expression is highly restricted during fetal life but diversifies progressively after birth. A high frequency of VH gene use from the relatively small VHIV gene family has previously been demonstrated in human fetal liver B cells. In the present study, 102 B cell lines established by Epstein-Barr Virus-transformation of separated CD5+ and CD5- cord blood B cells, were examined for the frequency of IgV expression using monoclonal antibodies to cross-reactive idiotypes (CRI). The results demonstrate a relatively high frequency of VHIV gene use (30%) in B cells from cord blood. Furthermore, two mutually exclusive CRI associated with distinct subgroups of the VHIV family are segregated in their association with either subset of B cells. One CRI is exclusively expressed in lines established from CD5+ B cells while the other is associated with lines established from CD5- B cells.

Antigens, CD↗
PubMed1991

Human B cells cannot be triggered to kill target cells through their Fc gamma RII or Fc epsilon RII receptors.

There has been some controversy as to whether or not B cells can kill target cells through their Fc receptors. To address this, we have examined the ability of human B cells from a variety of sources to lyse hybridoma cells with specificity for either the B cell Fc gamma RII or Fc epsilon RII using a reverse killing assay, as well as their ability to lyse opsonized chicken erythrocytes using a classic ADCC assay. Tonsil B cells, chronic lymphocytic leukemia B cells, and Epstein-Barr virus-induced B cells, even after preactivation with a cocktail of cytokines, all failed to lyse any of these targets. We conclude that Fc gamma RII and Fc epsilon RII on human B cells are not cytotoxic trigger molecules.

Animals↗
PubMed1991

Auto- and polyreactivity of IgM from CD5+ and CD5- cord blood B cells.

The presence of the CD5 (67 kDa) molecule on the surface of B cells has been considered a marker for cells producing auto- and polyreactive antibodies. Cord blood B lymphocytes (rich in CD5+ B cells) have been sorted into CD5 positive and negative populations by flow cytometry using monoclonal antibodies to CD20 and CD5. Clones of these populations were obtained by immortalization with Epstein-Barr virus. Clones derived from both CD5+ and CD5- B cells produced IgM which was auto- and polyreactive with a higher frequency of these specificities in the CD5+ population. These data indicate that expression of surface CD5 on cord blood B cells is not a definitive marker of an auto/polyreactive population.

Antibodies, Monoclonal↗
PubMed1991

Sequence analysis and idiotypic relationships of BEG-2, a human fetal antibody reactive with DNA.

Monoclonal antibody (mAb) BEG-2 is a dsDNA binding IgM lambda derived from a 12-week human fetus. Two binding site idiotypes (BEG-2 Id alpha and BEG-2 Id beta) have been defined with the use of polyclonal rabbit anti-idiotypic anti-serum. BEG-2 Id alpha is located on the lambda light chain and has been described previously. The BEG-2 Id beta is present on the mu heavy chain. By means of a direct binding ELISA, BEG-2 Id beta has been identified on EBV-derived mAbs from human fetal liver or spleen (5%), human cord blood (2.7%) and adult peripheral blood (1%). In addition, the Id is present on 8.5% of adult spleen-derived hybridoma antibodies and 6% of RA synovium-derived hybridoma antibodies. In all populations the presence of the Id is strongly associated with binding to DNA and other polyanions. Competition assays indicated that the Id was located at or near the antigen-binding site on these molecules. To explore the structural basis of this binding, a major part of the BEG-2 heavy chain was sequenced and found to be encoded by a member of the VH4 family joined to a variant of JH5 by a very short Diversity or N region. Of the BEG-2 Id beta positive mAbs for which the VH family has been determined, five are encoded by VH4 and two are encoded by VH6, but none is encoded by other families. Thus, the BEG-2 Id beta identifies a set of polyreactive antibodies that are common in fetal life, persist into adulthood and are encoded by VH6 and, a subset of VH4 genes.

Amino Acid Sequence↗
PubMed1991

Do gamma delta T cells play an important role in autoimmune disease?

A T cell subpopulation using an alternative receptor heterodimer (gamma delta T cells) contributes 0.5-10% to the T cell population in the normal peripheral blood. One subset of gamma delta T cells (V gamma 9+/V delta 2+/ C gamma 1) preferentially recognizes mycobacterial antigens, which are the triggering antigens in the rat adjuvant arthritis (AA), an animal model with some similarities with rheumatoid arthritis (RA). Here we summarize data on the prevalence of gamma delta T cells in peripheral blood, synovial fluid and synovial membranes of RA patients which show that gamma delta T cells, in particular the subset reactive with mycobacterial antigens in normal individuals, are rare in all studied compartments. Furthermore, there was no enrichment of gamma delta T cells in the peripheral blood of patients with ankylosing spondylitis or systemic lupus erythematosus, autoimmune diseases with involvement of the joints. In the AA model depletion of the "conventional" alpha beta T cells completely prevented the induction of the disease and very effectively improved ongoing arthritis even though substantial numbers of gamma delta T cells were present in these animals. Taken together, our data do not support the notion of a significant contribution of gamma delta T cells to chronic joint inflammation in the autoimmune diseases studied or in adjuvant arthritis.

Animals↗
PubMed1991

Regulation of interleukin-1 and tumour necrosis factor gene expression in myelomonocytic cell lines by 1,25-dihydroxyvitamin D3.

1,25-dihydroxyvitamin D3[1,25-(OH)2D3] is capable of regulating cells in the immune system and affects cytokine production by both T lymphocytes and by monocytes. We examined the effects of 1,25-(OH)2D3 on the regulation of interleukin-1 beta (IL-1 beta) and tumour necrosis factor-alpha (TNF-alpha) genes in HL-60 and U937 cells. 1,25-(OH)2D3 alone only induced low level expression of the genes for these cytokines. Phorbol 12-myristate 13-acetate (PMA) strongly induced the transcription of these genes, whilst the addition of 1,25-(OH)2D3 to PMA-stimulated cells caused a further dose-dependent synergistic increase in the mRNA for both cytokines in U937 cells. In PMA-stimulated HL-60 cells, 1,25-(OH)2D3 increased the mRNA for IL-1 beta but not that for TNA-alpha, These differences may be related to the different stage of myeloid differentiation in HL-60 and U937 cells.

Blotting, Northern↗
PubMed1990

The role of gamma delta T cells in the normal and disordered immune system.

A small population of T cells does not express the conventional T cell receptor characterized by the alpha and beta polypeptide chains (TCR alpha beta) but instead, two polypeptides termed gamma and delta (TCR gamma delta). This alternative receptor is able to recognize antigen. It appears early in T cell ontogeny, but its role in the thymus prior to the availability of TCR alpha beta remains unclear. In selected sites such as skin or gut TCR gamma delta predominates in mice which might suggest a role of gamma delta T cells in the first line of defense against infection. gamma delta T cells secrete lymphokines and display cytotoxic activity. However, their activation requirements may differ from what is known for alpha beta T cells since MHC-nonrestricted and also CD4 and CD8 negative gamma delta T cells have been described. Preferential activation by mycobacterial antigens possibly indicates a special repertoire of the gamma delta T cells. In various diseases slightly increased numbers of gamma delta T cells were found, but these preliminary studies have not yet provided evidence for a major pathogenetic role of gamma delta T cells.

Animals↗
PubMed1990

Polymorphism and expression of the galactosyltransferase-associated protein kinase gene in normal individuals and galactosylation-defective rheumatoid arthritis patients.

We used restriction endonuclease digestion of leukocyte DNA to assess the structural integrity of an N-acetylglucosamine beta 1----4 galactosyltransferase (GalTase)-associated (GTA) protein kinase gene in rheumatoid arthritis (RA) patients. This analysis provides evidence that the gross structure of the GTA protein kinase gene locus remains intact in patients with defective galactosylation and that this gene locus is polymorphic both in normal individuals and in patients with RA, although no polymorphisms unique to RA patients were observed. Initial data on the expression of this gene indicate that comparable levels of GTA protein kinase messenger RNA are present in the lymphocytes of normal individuals and RA patients, irrespective of whether lymphocytes were obtained from patients with decreased or normal levels of galactosylation.

Acetylglucosamine↗