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S M Watt

Publications and source records attributed to S M Watt.

At least 55 records · Page 3Linked to original sources
PubMed1991

CD66 identifies a neutrophil-specific epitope within the hematopoietic system that is expressed by members of the carcinoembryonic antigen family of adhesion molecules.

Preliminary results from the IVth Leucocyte Culture Conference have classified the monoclonal antibody (MoAb), YTH 71.3.2, as CD66. Two other MoAbs, YPC 2/12.1 and CE6/2D3.1, share a common cellular specificity, reacting with cells of the neutrophil series and colonic epithelium. The YTH 71.3.2 and CE6/2D3.1 MoAbs both recognize a similar CD66 defined epitope that is distinct from that identified by YPC 2/12.1. By Western blotting, these antibodies react with different molecular species from cells of different lineages. The antibodies identify 50- to 55-Kd, 80- to 100-Kd, and 130- to 200-Kd components present in a semi-purified carcinoembryonic antigen (CEA) preparation from colonic adenocarcinomas and a 90- to 130-Kd molecule from HL-60 cells. With the colonic cell line, LS174T, YPC2/12.1 stains diffuse bands of 160 to 200 Kd and 90 to 130 Kd with equal intensity, whereas the binding of CE6/2D3.1 and YTH 71.3.2 is biased toward the lower molecular weight set of molecules. Remarkably, all three antibodies recognize CEA-related molecules. Defined analyses using HeLa cells transfected with CEA, NCA(NCA-50/90), and CGM6(NCA-95) cDNAs show that the three MoAbs identify CEA to varying degrees. While YTH 71.3.2 and CE6/2D3.1 also bind to NCA-50/90, YPC 2/12.1 recognizes an epitope expressed by both the NCA-50/90 and NCA-95 molecular species.

Antibodies, Monoclonal↗
PubMed1991

Human thymic epithelial cells are frequently transformed by retroviral vectors encoding simian virus 40.

The thymic microenvironment contains a mixture of phenotypically distinct epithelial cells of varied functions, some of which are unknown. In an attempt to understand their relevance to T cell differentiation in the thymus, human thymic epithelial cell clones from both fetal (SM3-SM5) and postnatal (SM6) thymus were produced by using a defective recombinant retroviral vector encoding the simian virus 40 large T antigen and the neomycin resistance gene. The presence of keratins 8 and 18, desmosomes, and tonofilaments confirmed the epithelial origin of the cell strains. The cells expressed Thy-1 and HLA-Class I at high levels, showed weak-expression antigens defined by TE3B and A2B5, and low to negligible levels of the MR19-defined molecule. When compared with the phenotype of thymic epithelial cells in situ, the cell strains appear to be derived from neuroendocrine components in the outer cortical region of the human thymus. The use of retroviral vectors to transform human thymic epithelium was considerably more efficient than transfection with a plasmid carrying the origin of replication-defective SV40 large T gene. In the latter case, only two cell strains with subcapsular epithelial phenotypes were derived from fetal thymus. With the retroviral vectors, epithelial cell strains could, for the first time, be generated from human postnatal thymus as well as from fetal thymus.

Cell Division↗
PubMed1991

An improved negative immunomagnetic selection strategy for the purification of primitive hemopoietic cells from normal bone marrow.

An improved negative immunomagnetic selection strategy has been devised for the enrichment of primitive hemopoietic cells using the high proliferative potential colony-forming cell (HPP-CFC) assay as an index of stem cell purification. Immunomagnetic selection was carried out using goat anti-rat conjugated M-450 Dynabeads and a cocktail of rat monoclonal antibodies directed against lineage antigens expressed on B-lymphocytes (B220), neutrophils and activated macrophages (7/4), differentiating erythroid cells (YW 25.12.7), and T-lymphocyte subsets (Lyt-2 and L3T4). This negative selection strategy results in the highly reproducible enrichment of HPP-CFC with negligible loss of HPP-CFC at the immunomagnetic selection step. A 30-fold enrichment of HPP-CFC stimulated by interleukin 3 (IL-3) plus colony-stimulating factor (CSF-1), or interleukin 1 alpha (IL-1 alpha) plus IL-3 plus CSF-1, is obtained with simultaneous resolution of HPP-CFC from progenitor cells of low proliferative potential responsive to CSF-1 alone (LPP-CFC). Flow cytometric analysis of these lineage-negative cells reveals that they almost exclusively exhibit the light-scattering characteristics of blast cells and the morphology of a candidate hemopoietic stem cell. Positive fluorescence-activated cell sorting of immunomagnetically pre-enriched normal bone marrow cells using wheat germ agglutinin yields cell preparations with a cloning efficiency of up to 45% and a HPP-CFC content of 20%.

Animals↗
PubMed1991

Detection of additional JH/BCL2 translocations in follicular lymphoma.

In vitro enzymatic amplification and direct sequencing were used to detect and characterize t(14;18) recombination junctions in peripheral blood and bone marrow mononuclear cell preparations from patients with follicular lymphoma in remission. Samples from 24/44 patients were found to be positive for translocations involving the major breakpoint region of the BCL2 gene. In samples from seven patients two distinct t(14;18) translocations were shown to be present simultaneously; in one case the second translocation involved the minor cluster region of the BCL2 gene. Biopsy tissue obtained earlier in the course of the disease was available from five of these patients and was shown to contain one of the translocations in each case, but both translocations in only one. Further remission blood and bone marrow samples from the group were also examined. This led to the detection of both translocations in separate samples obtained at different times in a total of four out of the seven cases. In two of the remaining three patients the second translocation could not be amplified from further samples, but in both cases the search led to the identification of a third translocation, again only detectable in a single sample. These findings demonstrate that JH/BCL2 translocations can occur more than once during the course of follicular lymphoma. They suggest that biclonal follicular lymphoma may be more common than has previously been recognized but also raise the possibility that the translocation arises sporadically in the normal lymphoid cells of this group of patients.

Adult↗
PubMed1988

Cell lineage specificity of chromatin configuration around the immunoglobulin heavy chain enhancer.

As tested by DNase I hypersensitivity, the chromatin structure of the IgH enhancer region in human B cell precursor cell lines is in an open or accessible conformation. All T cell lines, with either germline or rearranged IgH genes, were also hypersensitive to DNase I but in contrast to B cell precursors showed no detectable Cmu expression. Normal thymocytes similarly had a hypersensitive IgH enhancer site. In contrast to lymphoid cells, all myeloid cell lines tested, as well as normal granulocytes, were not DNase I hypersensitive and did not express Cmu. A putative lymphomyeloid progenitor cell line KG1, although having a germline configuration of Ig genes, produced Cmu transcripts and was hypersensitive to DNase I in the IgH enhancer region. After induction of myeloid differentiation the Ig enhancer region of KG1 cells is no longer hypersensitive or transcriptionally active. Two HhaI restriction sites on either side of the IgH enhancer were not methylated in all Cmu-expressing lines but methylated in non-expressing cell lines. These results show that an open chromatin structure around the heavy chain enhancer is necessary but insufficient for initiating transcription from unrearranged IgH genes and further suggests this region may be in an open or accessible configuration prior to lineage commitment and closed following adoption of the myeloid lineage.

Blotting, Northern↗
PubMed1988

Structural and partial amino acid sequence analysis of the human hemopoietic progenitor cell antigen CD34.

Monoclonal antibodies of the CD34 class all recognize a monomeric cell surface antigen of approximately Mr 110,000 which is selectively expressed on human hemopoietic progenitor cells. This structure can be readily surface-labeled with [125I]actoperoxidase and by periodate-[3H]borohydride, but it labels only weakly with [35S]methionine, [35Sl]cysteine, 3H-amino acids, or 3H-mannose, even after prolonged labeling periods. However, the antigen is more efficiently labeled by [3H]glucosamine. Lectin binding studies, sensitivity to certain glycosidases, and gel filtration analysis of glycans released by alkaline hydrolysis indicate that this glycoprotein contains several complex-type N-linked glycans as well as several highly sialylated O-linked glycans. Western blotting experiments show that various CD34 antibodies fail to efficiently detect desialylated and/or de-N-glycosylated forms of the antigen. Experiments involving the use of tunicamycin, together with metabolic labeling studies, strongly suggest that this structure "turns over" very slowly in vivo. The CD34 antigen is not detectably labeled by 32P-phosphate in vivo, nor are immune complexes containing it associated with phosphokinase activity in vitro. Sequential immunoprecipitation and Western blotting studies indicate that this antigen is not a member of the leukosialin/sialophorin family despite the fact that these molecules share several structural similarities. Partial amino acid analysis of highly purified CD34 antigen revealed no significant sequence similarity with any previously described structures.

Amino Acid Sequence↗
PubMed1988

Interleukin-3 and interleukin-1 alpha allow earlier bone marrow progenitors to respond to human colony-stimulating factor 1.

By using human bone marrow cells enriched for early progenitors by selective immunoadsorption and plated at low cell density (10(3) to 10(4) cells/mL/9.6 cm2) in semisolid methylcellulose culture, we have analyzed the cooperative effects of human colony-stimulating factor 1 (CSF-1), granulocyte-macrophage-CSF (GM-CSF), interleukin-1 alpha (IL-1 alpha), and gibbon as well as human recombinant IL-3 on the formation of monocytic colonies. CSF-1 alone stimulated mature monocytic colony formation by human CFU-M. However, in the presence of IL-3 and erythropoietin, CSF-1 stimulated maximal immature monocytic colony formation at low concentrations and inhibited the formation of granulomonocytic, erythrocytic, and mixed colonies. Cultures with CSF-1 and IL-3 contained more immature monocytic colonies than did cultures with CSF-1 alone. IL-1 alpha alone had little effect. However, IL-1 alpha in combination with optimal concentrations of either CSF-1, GM-CSF, or IL-3 increased the number of colonies containing immature or mature monocytic colonies.

Cells, Cultured↗
PubMed1987

Cell-surface markers on haemopoietic precursors. Reagents for the isolation and analysis of progenitor cell subpopulations.

Within the last decade, major advances have been made in the analysis of cell-surface marker expression on haemopoietic progenitor cells as a result of the development of multiparameter cell sorting and monoclonal antibody techniques. Although some controversy exists with regard to the actual identification of the stem cell, markers specific for CFU-s and for particular subsets of progenitor cells have not yet been identified. An analysis of cell-surface markers on haemopoietic progenitor cells is complicated by at least three factors. First, it appears that, in mice, the clonal assays do not adequately identify the haemopoietic stem cell. Complete repopulation of all haemopoietic cell compartments in vivo over an extended period of time appears to be the only reliable method for identifying such a cell. Secondly cell-surface marker distribution on haemopoietic progenitors from normal tissues may be indicative of the cycling status of cells. Thus, expression of markers on progenitors from bone marrow or foetal liver which have been perturbed by drugs or viruses may merely reflect a change in their cycling status following drug or viral insult. Thirdly, substantial loss of cells occurs during the purification of particular cell types. For most cell separation procedures, only a minor proportion of the progenitor cells of interest are recovered and these may not be representative of the progenitor population as a whole. During differentiation to mature cells, antigenic determinants present on early progenitor cells may either be progressively lost or amplified. This differential expression of cell-surface molecules has provided a useful tool for the substantial enrichment of haemopoietic subsets, particularly CFU-E and CFU-s. To date, however, most early haemopoietic progenitor cells detected by in vitro CFC assays (day 8 CFC) cannot be completely segregated from one another. The ability to distinguish between such progenitors during the early stages of lineage commitment would provide a more detailed understanding of the relationship between lymphoid precursors, myeloid precursors and stem cells, and would lead to significant advances in developmental biology. Separation of cells at different stages of differentiation within a given lineage would provide an opportunity for studying regulatory mechanisms involved in gene expression in normal cell populations.

Animals↗
PubMed1987

Cloning of human thymic subcapsular cortex epithelial cells with T-lymphocyte binding sites and hemopoietic growth factor activity.

The thymic microenvironment involves complex cell interactions among different types of epithelial cells, macrophages, tissue histiocytes, and immature and maturing T cells. We describe the isolation of a subset of thymic epithelial cells by selective primary culture followed by cotransfection with a simian virus 40 replication-origin-defective mutant and pSV2neo plasmid. The cloned cells have the composite immunophenotype that is unique to thymic subcapsular epithelial cells, suggesting that they may provide a model system in vitro for analyzing the earliest steps in T-cell differentiation. This possibility is supported by the finding that these epithelial cells express LFA-3-associated binding sites for T cells, secrete a macrophage hemopoietic growth factor, and synergize with macrophages in the production of interleukin 1.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1987

Expression of HPCA-1 and HLA-DR antigens on growth factor- and stroma-dependent colony forming cells.

The expression of HLA-DR and HPCA-1 antigens (recognized by the L243 and BI.3C5 antibodies respectively) on adult human bone marrow cells was examined by fluorescence activated cell sorting and colony assays. Nearly all the (day 14) lineage restricted and multipotential colony forming cells analysed in methylcellulose cultures in the presence of added growth factors express HLA-DR and HPCA-1 determinants. Two colour cell sorting reveals that the lineage restricted HLA-DR positive progenitors express variable levels of BI.3C5 positivity whereas most of the multipotential progenitors, the multi-CFC or CFU-GEMM, are highly BI.3C5 positive. The isolated HLA-DR and BI.3C5 positive populations also contain haemopoietic precursors which adhere to and form colonies on pre-formed stromal layers. Thus, haemopoietic progenitors assayed in both types of culture system can be analysed and enriched by simultaneous two-colour sorting using anti-HLA-DR and BI.3C5 monoclonal antibodies. Similarities in the antigenic phenotype of such cells, however, precludes the use of these reagents for segregating growth factor-dependent from stroma-dependent progenitors.

Adult↗
PubMed1987

Distribution and epitope analysis of the cell membrane glycoprotein (HPCA-1) associated with human hemopoietic progenitor cells.

Monoclonal antibodies My10, BI.3C5, 12.8, and ICH3 identify a monomeric cell surface glycoprotein (HPCA-1) of 100-120 kD, which is selectively expressed on human hemopoietic progenitor cells. Other tissues are nonreactive with the exception of capillary endothelia and basement membrane in some sites. In addition, the antigen can be detected on cell lines that exhibit characteristics associated with early T cell precursors. HPCA-1 is therefore associated with myeloid, B, and T lineage precursors. Sequential immunoprecipitation and Western blotting studies demonstrate that BI.3C5, ICH3, My10, and an antibody directed against endothelial cells, 188.27, all react with the same glycoprotein species, although the epitopes involved may be distinct. The epitope recognized by BI.3C5 is sialic acid dependent, whereas that recognized by ICH3 is not. The My10 epitope has partial sensitivity to neuraminidase. Competitive/additive binding experiments suggest that these epitopes, although probably distinct, may be closely associated.

Antibodies, Monoclonal↗
PubMed1986

Co-ordinate expression of BI.3C5 and HLA-DR antigens on haemopoietic progenitors from chronic myeloid leukaemia.

Haemopoietic cells isolated from the peripheral blood of patients with chronic myeloid leukaemia (CML), have been extensively purified and enriched using either Percoll density gradients or Percoll density gradients combined with elutriation. The quantitative expression of the BI.3C5 associated antigen and the co-expression of BI.3C5 and HLA-DR antigens on these two populations has been studied using either single or simultaneous two colour FACS sorting, following by in-vitro culture for single and multilineage haemopoietic progenitors thus obtained. The data show that the CFU-GEMM are always found in the most strongly BI.3C5 positive fraction, irrespective of the separation procedure and that the bulk of the CFU-GEMM co-express BI.3C5 and HLA-DR. The cell types initiating these CFU-GEMM are morphologically immature blasts. The more mature cells of the myelomonocytic and erythroid lineages forming single lineage colony types show variable BI.3C5 expression, although most are HLA-DR positive. Such enriched populations of malignant progenitors could provide a useful source of material to study both gene expression and the molecular mechanisms underlying malignant transformation.

Antibodies, Monoclonal↗
PubMed1986

Lineage promiscuity in hemopoietic differentiation and leukemia.

An increasing number of reports document instances in which individual leukemic cells coexpress markers normally believed to be restricted to a single lineage. This has been interpreted by McCulloch and colleagues as aberrant programming or lineage infidelity and contrasts with earlier suggestions that lineage fidelity of gene expression was usually maintained in leukemia. We argue that several examples of infidelity are suspect on technical grounds, whereas others are bona fide and require explanation, eg, partial rearrangements and expression of Ig heavy-chain and/or T cell receptor genes in inappropriate cells and terminal deoxynucleotidyl transferase in leukemic myeloblasts. Individual examples of truly aberrant gene expression may well occur in leukemia but with insufficient regularity to be of general significance. We suggest that verifiable and consistent examples of apparent lineage infidelity do not reflect genetic misprogramming but rather the existence of a transient phase of limited promiscuity of gene expression occurring in normal biopotential or multipotential progenitors and able to be preserved as a relic in leukemic blast cell populations that are in maturation arrest. This alternative explanation has interesting implications for mechanisms of hematopoietic differentiation and leads to some testable predictions.

Animals↗
PubMed1986

Divergent molecular phenotypes of KG1 and KG1a myeloid cell lines.

The cell line KG1 derived from a patient with erythroleukemia in myeloblastic relapse has the composite phenotype and functional repertoire of myeloblasts. In marked contrast, its subline KG1a has lost myeloid features, acquired new karyotypic markers, and has three characteristics associated with immature T cells: low-level expression of the T cell receptor beta mRNA (but not alpha) transcribed from a germline gene; high-level expression of T3 delta mRNA and intracellular, but not cell surface, T3 protein; and expression of the CD7/gp40 T cell-associated membrane antigen. Both KG1 and KG1a transcribe unrearranged IgH genes. These data suggest that either the KG1 cell line was derived from a common myeloid-lymphoid progenitor or that the KG1a subline phenotype is aberrant.

Antibodies, Monoclonal↗
PubMed1984

Homeostasis of mucosal cholesterol in the small intestine of the rat.

This study was undertaken to investigate the capacity of the intestinal mucosa to maintain a constant cholesterol content under conditions where mucosal uptake or cholesterol transport into the lymph were manipulated. Two series of bile-diverted unanaesthetised rats were infused intraduodenally with saline, triolein emulsified with Pluronic F68, or taurocholate with or without added tomatine. Pluronic F68 is a nontoxic detergent which promotes mucosal uptake of polar lipids but not cholesterol. Tomatine is a cholesterol-binding saponin. One series of rats was used for measuring mucosal cholesterol content, DNA and protein after the test infusions. A second series of rats had the thoracic lymph duct cannulated but otherwise remained the same as the first series. The second series was used for measuring the effect of the different infusions on mass cholesterol output into lymph. Mucosal cholesterol content of rats that were not fed decreased with bile-diversion and was restored with taurocholate infusion. This suggested a contribution of luminal cholesterol to the mucosal cholesterol pool. However, evidence for a contribution from the lumen was provided by only one of two groups of rats given infusions which did not promote mucosal uptake of cholesterol. First, addition of tomatine to the taurocholate infusate prevented both the increase in lymph output of cholesterol and the increased mucosal cholesterol content shown in rats given taurocholate alone. Second, in another group of rats in which mucosal uptake of cholesterol was prevented, i.e. in rats given Pluronic F68-triolein emulsions, the increased fat absorption was accompanied by a marked increase in cholesterol output into lymph without a concomitant decrease in mucosal cholesterol content.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗