Biomedical subjects

A W Boyd

Publications and source records attributed to A W Boyd.

At least 55 records · Page 3Linked to original sources

PubMed1993

Interactions between purified human cord blood haemopoietic progenitor cells and accessory cells.

The role of accessory cells in haemopoiesis remains confused. This appears in large part to reflect the use of impure populations of accessory cells and progenitor cells in previous studies. In this study, cell sorter purified populations of both accessory cells and haemopoietic progenitor cells were used to examine interactions between these cell types. We used a double culture protocol in which purified CD34+ cells were cultured with purified NK, T or monocytic cells in the first liquid culture phase after which the cells were transferred to secondary agar cultures to determine the number of colony forming cells (CFC). NK cells co-cultured with CD34+ cells resulted in an increased number of erythroid progenitors with no effect on the number of nonerythroid progenitors. In contrast, there were increased numbers of erythroid and non-erythroid CFC when the CD34+ cells were co-cultured with either purified T cells or monocytes. CD34+ cells cultured with cell-conditioned media derived from NK cells, T cells or monocytes in transwells where the CD34+ cells and the accessory cells were separated by a 0.2 micron membrane, showed no enhancement in CFC. These results suggest that intimate cell-cell contact is required for these events.

Agar↗

PubMed1992

Support of human cord blood progenitor cells on human stromal cell lines transformed by SV40 large T antigen under the influence of an inducible (metallothionein) promoter.

We describe the development of a human bone marrow (BM) culture system which allows study of the interaction of stromal cell lines (SCL) and highly purified hematopoietic progenitor cells. Normal BM stromal cells were electroporated with a plasmid containing the simian virus 40 (SV40) large T antigen (SV40 T Ag) under the control of a synthetic metallothionein promoter (MT4); this construct is designated MT4 SV40 T Ag. SCL in which the rate of proliferation could be controlled by altering the zinc (Zn) concentration were characterized, demonstrating that the SCL were heterogeneous with respect to G-CSF and GM-CSF production. Suppression of SCL proliferation on removal of Zn made it possible to use these lines in coculture with purified CD34+ progenitor cells from umbilical cord blood. The ability to control proliferation of SCL has allowed us to maintain the survival and expansion of colony-forming cells in culture for up to 2 months. These lines have enabled us to test for stromal cell characteristics at a clonal level and provided us with a tool to analyze the events leading to lineage commitment and hematopoietic differentiation, as demonstrated by suppression of hematopoiesis by an antibody directed against the c-kit molecule.

Antigens, CD↗

PubMed1992

Molecular cloning of HEK, the gene encoding a receptor tyrosine kinase expressed by human lymphoid tumor cell lines.

We describe the molecular cloning of a receptor tyrosine kinase from a cell line (LK63) derived from a case of human pre-B-cell leukemia. We have previously shown that a monoclonal antibody (IIIA4) raised against LK63 recognized a glycosylated, cell-surface 135-kDa molecule (HEK), which displayed tyrosine kinase activity in vitro. The HEK protein was purified by using a IIIA4 antibody column and both N-terminal and internal amino acid sequences were obtained. A 51-mer degenerate oligonucleotide based on the internal amino acid sequence was used to screen an LK63-derived lambda gt10 cDNA library under low-stringency hybridization conditions. One clone of 2.5 kilobases (kb) was isolated and characterized and used to rescreen the library under more-stringent hybridization conditions. A 4.5-kb clone containing the entire HEK coding region was isolated and its complete DNA sequence was determined. The 4.5-kb insert was subcloned into the expression vector CDM8 and transfected into COS cells. COS cells transfected with the sense HEK/CDM8 construct stained specifically with the IIIA4 antibody, thereby confirming that the antigen recognized by the IIIA4 antibody and the expressed protein product of the HEK cDNA clone were identical. DNA sequence analysis revealed that HEK is a newly discovered member of the EPH/ELK family of receptor tyrosine kinases. Northern blot analysis of a number of cell lines demonstrated the expression of 5.5- to 6.0-kb HEK transcripts in LK63 and the T-cell lines JM and HSB-2. Southern blot analysis of DNA from LK63 suggested that the HEK gene was neither amplified nor rearranged in the LK63 tumor.

Amino Acid Sequence↗

PubMed1992

Isolation and characterization of a novel receptor-type protein tyrosine kinase (hek) from a human pre-B cell line.

In this report we describe the identification and characterization of a novel tumor-associated receptor-type tyrosine kinase (hek). We produced a monoclonal antibody (III.A4) that detected a novel glycoprotein on the immunizing pre-B cell acute lymphoblastic leukemia cell line (LK63). This antigen was shown to be expressed sporadically on hemopoietic tumor cell lines and on ex vivo tumors. However, using antibody staining, the molecule was undetectable on normal tissues. Further biochemical characterization showed this molecule (hek) to be a phosphoroprotein. This observation taken together with the tumor-associated nature of hek expression suggested that hek might be a receptor-type protein tyrosine kinase. This was demonstrated by affinity purification of hek. In in vitro kinase experiments the purified hek protein was autophosphorylated on tyrosine and also mediated tyrosine phosphorylation of casein. Purified hek was subjected to N-terminal amino acid sequence analysis which showed that hek had a unique N terminus. Amino acid sequence determination of peptides from a V8 protease digest of hek yielded one 21-amino acid stretch of sequence which showed close homology with the eph subfamily of protein tyrosine kinases. These studies show hek to be a novel human tumor-associated protein tyrosine kinase, which by analogy with previously characterized protein tyrosine kinase proto-oncogenes, may have a role in tumorigenesis.

Amino Acid Sequence↗

PubMed1992

Marrow proliferation and the appearance of giant neutrophils in response to recombinant human granulocyte colony stimulating factor (rhG-CSF).

During a study of recombinant human granulocyte colony stimulating factor (rhG-CSF) administration, 15 patients received twice daily i.v. infusions and nine patients received daily s.c. infusions of rhG-CSF for 5 d prior to cytotoxic therapy, and then a second course subsequent to melphalan administration. There was a striking dose-related neutrophilia and the appearance in the blood of early myeloid cells that express the intercellular adhesion molecule CD54. In addition, giant neutrophils or macropolycytes were observed in the peripheral blood of all patients. These cells were evident on the display of the Technicon H*1 as a population of large peroxidase positive cells, and using Feulgen staining these cells were shown to be tetraploid. Bone marrow kinetics studies performed on Day 4 or 5 indicated an increase in the proportion of bone marrow cells in S phase, G2 and mitosis, reflecting a proliferative response of the marrow. Large myeloid precursors and occasional binucleate promyelocytes were seen in the bone marrows done on Days 14 and 18 but not on Day 5. These findings indicate that administered G-CSF has both quantitative and qualitative effects on myeloid cells in vivo.

Bone Marrow↗

PubMed1992

Adhesion molecules are expressed in the human retina and choroid.

Immunoperoxidase staining of samples of normal human retina and choroid was performed to demonstrate selected adhesion molecules in vivo. Intercellular adhesion molecule-1 (ICAM-1), lymphocyte function-associated antigen-3 (LFA-3) and endothelial leucocyte adhesion molecule-1 (ELAM-1) were expressed on retinal vascular endothelium and ICAM-1 and ELAM-1 in the region of the external limiting membrane. The very late antigen-2 (VLA-2) was found around the retinal ganglion cells and in the inner and outer fibre layers. The endothelium on the choriocapillaris showed moderate staining of ICAM-1, and slight staining of LFA-3 and ELAM-1. The presence of these molecules in this distribution strongly suggests they have an important role in the pathogenesis of immunologically mediated ocular conditions.

Antibodies, Monoclonal↗

PubMed1992

The effect of cytokines on the expression of MHC antigens and ICAM-1 by normal and transformed synoviocytes.

We report the expression on synovial cells of cell surface molecules known to be involved in T cell activation by antigen presenting cells. Normal human synovial fibroblasts and a human synovial cell line transformed with the SV40 large T antigen were used for in vitro stimulation studies with recombinant cytokines. We demonstrate an increase in MHC-A, B, C expression in normal synovial cells in response to recombinant interferon gamma (r gamma IFN), tumour necrosis factor alpha and beta (rTNF alpha and beta) and interleukin-1 (rIL-1 alpha). Intercellular adhesion molecular-1 (ICAM-1) expression was increased in parallel with MHC Class I. The combination of r gamma IFN and rTNF alpha was additive in its effect on ICAM-1 expression. Northern blot analysis suggests that ICAM-1 expression in synovial cells is controlled at the level of transcription. In contrast, MHC Class II (HLA-DR) was only significantly induced by r gamma IFN. Other stimuli including interleukin-4 (IL-4), interleukin 6 (IL-6), granulocyte macrophage colony stimulating factor (GM-CSF) and prostaglandin E2 (PGE2) did not affect the expression of ICAM-1 or MHC Class I and II. Leucocyte function antigen 3 (LFA-3) expression was not affected by any of the stimuli tested. Immunoperoxidase staining of rheumatoid synovial tissue confirmed enhanced in vivo expression of ICAM-1 in rheumatoid arthritis. These changes are discussed in the context of T cell activation in inflammatory arthritis.

Arthritis, Rheumatoid↗

PubMed1992

The effect of recombinant stem cell factor (SCF) on purified CD34-positive human umbilical cord blood progenitor cells.

We describe the effect of soluble c-kit ligand (stem cell factor, SCF) on highly purified CD34-positive hemopoietic progenitors from human umbilical cord blood. Progenitor cells were purified from cord blood mononuclear cells by immune rosetting with lineage specific antibodies and subsequent sorting of the rosette-negative population for CD34(BI3C5)-positive cells. This procedure enriched greater than 100-fold for colony forming cells (CFC). Using optimal concentrations of colony-stimulating factors (CSF) without added SCF approximately 2.5% of cells formed colonies. SCF also had CSF activity on this population, up to 0.5% of cells forming small colonies in response to SCF alone. In contrast, the addition of SCF to optimal concentrations of the other growth factors produced a greater than 10-fold increase in colony number. However, the most notable effect was an approximately 100-fold increase in the number of cells in each colony. Equally striking was the very high proportion (50-80%) of mixed colonies (CFU-MIX). These findings suggest the progenitor cell pool in cord blood is skewed towards very early cells. However, when day 14 colonies formed in response to SCF and other factors were assessed for their re-cloning potential they did not contain significant numbers of CFC, implying that SCF did not support the self-renewal of these CD34 positive cord blood progenitor cells. These findings support a role for SCF as an enhancing factor for hemopoietic progenitor cells but it does not promote self-renewal in these populations.

Antigens, CD↗

PubMed1991

Inhibition of intercellular adhesion molecule 1-dependent biological activities by a synthetic peptide analog.

We have used a combination of hydropathy analysis of the intercellular adhesion molecule 1 (ICAM-1) sequence and dot-matrix comparison of the sequence with the homologous, but functionally distinct, protein myelin-associated glycoprotein to identify a putative functional binding region. One polar, and presumably surface-exposed, region of ICAM-1 showed no significant identity with myelin-associated glycoprotein. A synthetic peptide analog based on the sequence of this region (JF9) mimicked the inhibitory effects of the anti-ICAM-1 monoclonal antibody WEHI-CAM-1. These included inhibition of ICAM-1-dependent homotypic aggregation of Raji Burkitt lymphoma and phorbol-ester treated U937 cells at concentrations as low as 80 micrograms/ml (24 microM). In addition, at a concentration of 100 micrograms/ml, the peptide analog effectively inhibited cytotoxic cell activity, an ICAM-1-dependent effector function of the immune response. This simple method of sequence analysis may have general applicability to the identification of functional domains in homologous, but functionally distinct, proteins such as the translated products of gene families.

Amino Acid Sequence↗

PubMed1991

Human interleukin-4: an immunomodulator with potential therapeutic applications.

Human interleukin-4 (IL-4) is a 20kDa cytokine produced by activated T cells and has an extensive range of stimulatory and inhibitory effects on the wide range of cells which express its receptor. It specifically promotes the immunoglobulin class switch to IgE and IgG4 and potently co-stimulates with CD40 monoclonal antibodies the long term proliferation of human B cells. It has variable effects on T cells, but predominantly has inhibitory actions on monocytes suggesting a potential therapeutic role as an anti-inflammatory agent. There is evidence for indirect anti-cancer activity of IL-4 both in animal models and in in vitro studies on human tumour infiltrating lymphocytes. In addition, IL-4 directly inhibits the in vitro proliferation of the majority of B cell neoplasms. Phase I studies of IL-4 in patients with cancer have commenced and promising observations have been made in patients with haematological malignancies receiving low, well-tolerated doses.

Adjuvants, Immunologic↗

PubMed1991

Arg-Gly-Asp-containing peptides expose novel collagen receptors on fibroblasts: implications for wound healing.

Integrins are a family of cell-surface receptors intimately involved in the interactions of cells with their extracellular matrix. These receptors comprise an alpha and beta subunit in noncovalent association and many have been shown to recognize and bind an arginine-glycine-aspartate (RGD) sequence contained within their specific extracellular matrix ligand. Fibroblasts express integrin receptors belonging to two major subfamilies. Some of the members within the subfamily defined by beta 1 (VLA) are receptors for collagen but, perhaps surprisingly, the other major subfamily of integrins on fibroblasts--that defined by the alpha chain of the vitronectin receptor, alpha v--all appear to bind primarily vitronectin and/or fibronectin. In the present study we show that RGD-containing peptides expose cryptic binding sites on the alpha v-associated integrins enabling them to function as collagen receptors. The addition of RGD-containing peptides to fibroblasts cultured on type I collagen induced dramatic cell elongation and, when the cells were contained within collagen matrices, the peptides induced marked contraction of the gels. These processes were inhibited by Fab fragments of a monoclonal antibody against an alpha v integrin. Also, alpha v-associated integrins from cell lysates bound to collagen I affinity columns in the presence, but not in the absence, of RGD-containing peptides. These data suggest a novel regulatory control for integrin function. In addition, because the cryptic collagen receptors were shown to be implicated in the contraction of collagen gels, the generation of such binding forces suggests that this may be the major biological role for these integrins in processes such as wound healing.

Amino Acid Sequence↗

PubMed1991

Isolation and characterization of the promoter region of the human intercellular adhesion molecule-1 gene.

We have isolated and restriction enzyme-mapped a human genomic DNA clone encompassing the first two exons and the 5' flanking sequence of the human intercellular adhesion molecule-1 (ICAM-1) gene. The transcription initiation site was identified using primer extension analysis, and 1.7 kb of DNA upstream of the transcription initiation site was sequenced. The 5' region and first exon of the ICAM-1 gene was found to be a CpG island as it was (i) (G + C)-rich with a high frequency of the dinucleotide CpG and (ii) hypomethylated irrespective of the level of ICAM-1 expression in the tissues examined. These features of the ICAM-1 promoter are similar to the promoters of many 'housekeeping' genes. However, consensus sequences for several potential regulatory elements were found in the 5'-flanking sequence, an observation in keeping with the pattern of strongly regulated ICAM-1 expression. Examination of the chromatin structure upstream of the ICAM-1 gene revealed the presence of a constitutive DNase I-hypersensitive site 1.5 kb upstream of the transcription initiation site. Direct evidence that the upstream region constitutes a promoter element was demonstrated in transient transfection assays. A series of chloramphenicol acetyltransferase gene (CAT) constructs containing 5' fragments ranging in size from 1054 to 310 bp had equivalent levels of promoter activity when transfected into HeLa cells. Using a CAT construct containing a 447 bp ICAM-1 promoter fragment, we demonstrate an increase in transcription in response to interferon gamma (IFN-gamma), suggesting that this proximal region of the promoter is responsible, at least in part, for IFN-gamma induction of ICAM-1 expression.

Amino Acid Sequence↗

PubMed1991

The expression of adhesion molecules in the human retina and choroid.

This study examines the expression of some selected adhesion molecules likely to be important in the migration of leucocytes across the blood-retinal and blood-aqueous barriers in samples of normal human retina and choroid by immunoperoxidase staining. ICAM-1, LFA-3 and ELAM-1 were expressed on retinal endothelium and ICAM-1, ELAM-1 and CD44 in the region of the external limiting membrane. VLA-2 was found around the retinal ganglion cells and in the inner and outer fibre layers and is likely to be important in maintaining the structural integrity of the retina. The endothelium on the choriocapillaris showed moderate staining of ICAM-1, and slight staining of LFA-3 and ELAM-1. These molecules may have an important functional role in the pathogenesis of many immunological ocular conditions, including uveoretinitis and corneal allograft rejection.

Antibodies, Monoclonal↗

PubMed1991

Analysis of adhesion molecules in the immunopathogenesis of giant cell arteritis.

To explore the role of adhesion molecules in mediating mononuclear cell localisation, development of the granulomatous reaction, and cell mediated damage to the arterial wall in giant cell arteritis, 17 temporal artery biopsy specimens were examined. Eleven showed the histological features of giant cell arteritis and six showed no evidence of arteritis. All were examined for the expression of LFA-3, ICAM-1 and its receptor LFA-1, and HLA-DR. Temporal arteries with early features of arteritis, as well as histologically unaffected skip areas, showed a regional induction of ICAM-1 expression, but not HLA-DR, on smooth muscle cells of the media. ICAM-1 expression was detected in areas where a clinically important mononuclear cell infiltrate had not yet developed. In more florid cases of giant cell arteritis there was an additional widespread induction of ICAM-1 expression on intimal myofibroblasts. Strong expression of ICAM-1, HLA-DR, and LFA-3 was found on macrophages, epithelioid cells, and giant cells comprising the granulomatous lesion. The pattern of expression of these adhesion molecules suggests that they have a role in leucocyte traffic into the vascular lesion as well as in mediating the intercellular interactions which constitute the granulomatous response.

Aged↗

PubMed1990

Knob-independent cytoadherence of Plasmodium falciparum to the leukocyte differentiation antigen CD36.

The survival of Plasmodium falciparum-infected erythrocytes is enhanced by the sequestration of mature trophozoites and schizonts from the peripheral circulation. Cytoadherence of infected erythrocytes in vivo is associated with the presence of knobs on the erythrocyte surface, but we and others have shown recently that cytoadherence to C32 melanoma cells may occur in vitro in the absence of knobs. We show here that a knobless clone of P. falciparum adheres to the leukocyte differentiation antigen, CD36, suggesting that binding to CD36 is independent of the presence of knobs on the surface of the infected erythrocyte. This clone showed little cytoadherence to immobilized thrombospondin or to endothelial cells expressing the intercellular adhesion molecule 1. Furthermore, an Mr approximately 300-kD trypsin-sensitive protein doublet was immunoprecipitated from knobless trophozoite-infected erythrocytes. Finding a P. falciparum erythrocyte membrane protein 1 (PfEMP1)-like molecule on these infected erythrocytes is consistent with a role for PfEMP1 in cytoadherence to CD36 and C32 melanoma cells.

Animals↗

PubMed1990

Blood leukocytes bind platelet glycoprotein (IIb-IIIa)' but do not express the vitronectin receptor.

Within the integrin family of Arg-Gly-Asp(RGD)-binding adhesion receptors, the subfamily defined by the beta chain known as beta-3 or glycoprotein (GP)IIIa is known to contain two individual receptors. These are the GPIIb-IIIa complex of platelets, where the alpha chain of the heterodimer is GPIIb, and the vitronectin receptor (VnR) containing the alpha V subunit. The presence of either GPIIb-IIIa and/or the VnR on blood leukocytes has been controversial. We have investigated this problem by performing immunoprecipitation and immunoblotting studies with rabbit and monoclonal antibodies (mAb) to each of the subunits of GPIIb-IIIa and the VnR. On cultured cells of different origin, it was established that almost all expressed the VnR but none had GPIIb-IIIa, and the only polypeptide associated with beta 3 was alpha V. Platelets expressed predominantly GPIIb-IIIa, and weakly, the VnR. Monocytes and neutrophils freshly isolated from blood did not express the VnR but bore on their surface a modified form of GPIIb-IIIa. This molecule appeared identical to GPIIb-IIIa but an epitope on GPIIb was masked on the intact cell and was only revealed after immunoblotting. We have termed this modified form of GPIIb-IIIa, GP(IIb-IIIa)'. With differentiation in culture, monocytes rapidly lost surface GP(IIb-IIIa)' and concurrently began to express the VnR. Evidence is presented that GP(IIb-IIIa)' is derived from particles released by activated platelets and is bound firmly to the leukocyte membrane. Its primary function does not seem to be to mediate attachment to matrix proteins; thus, although U937 cells bearing platelet-derived GP(IIb-IIIa)' bound fibrinogen in an RGD-dependent manner, isolated blood monocytes did not. It is suggested that this transfer of membrane proteins from platelets to monocytes and neutrophils may regulate the expression of the leukocyte VnR and also serve as a means of facilitating leukocyte procoagulant activity.

Amino Acid Sequence↗

PubMed1990

The response of human B cells to interleukin 4 is determined by their stage of activation and differentiation.

The effect of purified recombinant human interleukin 4 (IL-4) on proliferation and IgM secretion of normal and malignant human B cells was studied. IL-4 was found to co-stimulate the proliferation of splenic B cells in the presence of anti-Ig coupled to polyacrylamide beads (anti-Ig beads) for a period of 4 days. In contrast, IL-4 had little co-stimulatory effect on the proliferative response of splenic B cells to the more potent mitogen Staphylococcus aureus Cowan strain 1 (SAC). Moreover, IL-4 inhibited interleukin 2 (IL-2)-induced proliferation of cells co-stimulated with SAC. Mitogen-induced pre-activation of B cells in the presence of IL-4 resulted in a reduction in subsequent IL-2-induced IgM secretion without significantly affecting proliferation. Human B-cell tumours were also cultured over a 2-3 day period in the presence of anti-Ig beads plus IL-2, or IL-4 or both IL-2 and IL-4. IL-4 inhibited IL-2-induced proliferation in all cases of B-cell chronic lymphocytic leukaemia (B-CLL) and the majority of cases of low-grade lymphoma (LGL) and hairy cell leukaemia (HCL). These findings suggest that IL-4 has stimulatory actions on resting B cells, most evident in the presence of submaximal co-mitogenic signals, and inhibitory actions on activated B cells, especially antagonism of the effects of IL-2.

Antibodies, Anti-Idiotypic↗

PubMed1990

Cloned stromal cell lines derived from human Whitlock/Witte-type long-term bone marrow cultures.

We report a human bone marrow culture technique that initially parallels the murine Whitlock/Witte culture system. As in the murine system, B cells predominate over other cell types, and all differentiation stages from pre-B to plasma cell are observed. Although these human long-term cultures pass through stages resembling phases I to III of murine Whitlock/Witte cultures, no outgrowth of nonadherent cells was seen after cultures had reached the "crisis" phase unless Epstein-Barr virus (EBV)-transformants appeared. The stromal cells persisted well beyond crisis, but they could not be maintained and passaged as cell lines, limiting their use in molecular analysis. Transfection of these stromal cells with plasmid DNA containing the simian virus 40 (SV40) early region yielded 124 cloned cell lines. Analysis of these lines showed that all expressed SV40 large T antigen, but they retained most phenotypic markers found on non-transformed stromal cells. When adherent and T-cell-depleted bone marrow cells were cultured on either nontransformed stromal layers or transformed cell lines they proliferated actively and soon yielded predominantly lymphoid nonadherent populations. Moreover, prolonged survival of acute lymphoblastic leukemia cells of pre-B phenotype was regularly achieved on both normal and transformed adherent cell layers. Although the liquid culture system favored lymphocytes, transformed stroma supported colony formation by both human and murine hemopoietic progenitors when marrow was added in agar medium. This was not explained by colony-stimulating factor (CSF) production, because striking heterogeneity in the levels of granulocyte CSF (G-CSF) and granulocyte-macrophage CSF (GM-CSF) secretion by the lines was noted. Some lines that did not produce detectable CSF demonstrated good support of fresh bone marrow growth and acute lymphoblastic leukemia (ALL) cell survival. The heterogeneity of these cell lines and their capacity to support hemopoiesis suggest that they will be useful in studying the molecular basis of in vitro lymphohemopoiesis in man.

Age Factors↗