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Biomedical subjects

B H Toh

Publications and source records attributed to B H Toh.

At least 127 records · Page 7Linked to original sources
PubMed1984

Vimentin organizing centre in cultured epithelial cells.

Perinuclear buttons were identified in cultured epithelial cell lines, with monoclonal antibodies to vimentin, following treatment with drugs which disrupt intermediate filaments. These buttons appeared after 10 min when cells were exposed to 10 micrograms/ml vinblastine or cyclohexamide. After the drug was removed vimentin-containing filaments radiated from this button through the cytoplasm. In some cells peripheral buttons appeared, up to six in one cell. Within 1 h the filaments had reformed to a normal pattern for intermediate filaments. These buttons did not form in mesenchymal cell lines, mouse; rat and 3T3 fibroblasts. They appeared only in epithelial cell lines. Monoclonal antibodies to keratin and tubulin did not stain these buttons. These appear to be discrete entities separate from the microtubule organizing centre and to be found only in cultured epithelial cell lines. This structure may act as the organization centre for vimentin in cultured epithelial cell lines.

Animals↗
PubMed1984

Thymic sensitivity to sex hormones develops post-natally; an in vivo and an in vitro study.

In vitro thymic organ cultures were used to examine the effects of the sex hormones estradiol and dihydrotestosterone on thymocytes. In contrast with the marked loss of cortical thymocytes seen in vivo with these hormones, no effect was apparent in vitro even at concentrations up to 10(-6) M. The glucocorticoid dexamethasone caused severe depletion in vivo and in vitro. Thymic androgen and estrogen receptors were determined; in the newborn animals up to 2 wk of age, receptor levels were barely detectable. The possibility of indirect modulation of thymic function by steroids in vivo was investigated by culturing thymic lobes in media containing serum from animals treated with these hormones. Only sera from dexamethasone-injected animals caused changes in cell size, number, viability, or phenotype in the culture system. The mechanism for the previously reported effects of sex steroids on the neonatal thymus therefore remains to be elucidated.

Aging↗
PubMed1984

Antinucleolar autoantibodies demonstrated by monolayers of human fibroblasts in sera from patients with systemic lupus erythematosus, progressive systemic sclerosis and chronic active hepatitis.

Antinucleolar antibody (ANoAb) was tested for in sera from 25 patients with systemic lupus erythematosus (SLE), 61 with progressive systemic sclerosis (PSS), 22 with chronic active hepatitis (CAH) and 28 healthy persons, using immunofluorescence reactivity with acetone-fixed monolayers of cultured human fibroblasts, and a procedure to reveal ANoAb when other antinuclear antibodies were concurrently present. ANoAb was found on direct testing in sera from 6 patients with SLE, 15 with PSS and 7 with CAH, but not in any of 28 sera from healthy persons; homogeneously reactive antinuclear antibody was also present in the serum of these 6 cases of SLE, in 6 of the 15 with PSS and in 3 of the 7 with CAH and, in SLE specifically, pre-treatment of fibroblast monolayers with DNase "unmasked" the presence of ANoAb in a further 7 sera which had shown only homogeneous nuclear staining in fibroblasts. ANoAb belonged to the IgG, IgM and IgA class in sera from cases of SLE and PSS, and to only the IgG and IgM class in sera from cases of CAH. ANoAb titres were highest in patients with PSS. ANoAb were sensitive to RNase in 5 cases, to RNase and DNase in 6, and were sensitive to combinations of RNase, DNase, NaC1, and trypsin in the remaining cases. We conclude that (i) fibroblast monolayers are a suitable substrate for the demonstration of ANoAb, (ii) homogeneous staining of cell nuclei may mask ANoAb, so that the incidence of ANoAb becomes higher in SLE than in PSS, (iii) low-titre ANoAb in CAH not visualized in frozen tissue sections may be detected on fibroblast monolayers, and (iv) nucleolar antigens probably include RNA, RNA bound to DNA, and RNA bound to proteins.

Autoantibodies↗
PubMed1983

Cells in neonatal rat hypothalamus primary culture--an immunofluorescence study.

Hypothalami from 1 day neonatal rats were dissociated and cultured for 4-16 days. Using immunofluorescence and antisera against neurofilament (NF) peptides, glial fibrillary acidic protein (GFAP), galactocerebroside and fibronectin we have distinguished neurons, astrocytes, oligodendrocytes and fibroblast-like cells in culture. Astrocytes initially grew as islets of 15-30 cells which dispersed as the culture aged. These cells, together with fibronectin-reactive flat cells, formed a monolayer upon which ovoid and process-bearing cells grew after 4 days in culture. Neurofilament-positive neurons constituted 5-10% of the total cell population. In maturing cultures the number of neurons decreased and fibroblasts increased. Oligodendrocytes represented less than 1% of total cell population. These studies emphasize the necessity of using the complementary techniques of morphology and immunocytochemistry for the characterization of hypothalamic neural cells in vitro.

Animals↗
PubMed1983

Autoantibodies cytotoxic to gastric parietal cells in serum of patients with pernicious anemia.

We measured the complement-dependent cytotoxic activity of serum in 60 patients with pernicious anemia. Canine gastric mucosal cells served as the indicator of cytotoxicity, which was expressed as a percentage of the maximal effect produced by a cytolytic agent (Triton X-100). Serum from patients with pernicious anemia showed a higher average activity (11.8 +/- 10.3 per cent, P less than 0.001) than serum from 29 patients with systemic lupus erythematosus (1.0 +/- 1.8 per cent), 10 with scleroderma (0.1 +/- 0.1 per cent), 10 with rheumatoid arthritis (0.6 +/- 0.6 per cent), 22 with multiple sclerosis (0.4 +/- 1.2 per cent), and 23 with chronic persistent hepatitis (0.03 +/- 0.1 per cent), and serum from 64 healthy persons (0.4 +/- 1.0 per cent). Serum from patients with pernicious anemia was not toxic to canine liver or kidney cells. Absorption with gastric mucosal cells and heat inactivation of complement abolished the cytotoxic reaction. The cytotoxic factor resided in the IgG fraction of immunoglobulin, and the amount of cytotoxicity was proportional to the IgG concentration. Cytotoxic activity correlated with the presence of parietal-cell-surface--reactive autoantibody demonstrated by immunofluorescence. We conclude that cytotoxic autoantibodies to parietal cells may contribute to the loss of such cells from the gastric mucosa of patients with pernicious anemia.

Adult↗
PubMed1983

The behaviour of human ameloblastoma tissue in cell culture.

Human ameloblastoma tissue was investigated using cell culture techniques, transmission electron microscopy and fluorescent microscopy. Cultured cellular morphology was dependent on the type of substratum, with polygonal cells predominant on collagen substrata in contrast to an elongated cellular morphology on glass substrata. The presence of tonofilaments and desmosomes confirmed the epithelial origin of these cells. The distribution of Con A surface receptors and cytoplasmic actin in the same cell was studied using a double fluorochrome technique. Incubation with fluorescein isothiocyanate-labelled Con A at 37 degrees C for increasing time periods resulted in the Con A receptors showing progressive changes in staining patterns from clusters, to caps to perinuclear globules. Sequential changes in cytoplasmic actin, labelled by a specific anti-actin auto-antibody traced with rhodamine-labelled goat anti-human globulin, corresponded to the Con A staining patterns.

Actins↗
PubMed1983

Routine flow cytometric diagnosis of lymphoproliferative disorders.

The results of the analysis of suspected lymphoproliferative disorders using flow cytometry (FACS II) over the past 12 months have been evaluated and assessed with respect to "conventional" microscopical examination. The major advantages in using these methods is the speed, the number of parameters which are capable of measurement, the sensitivity, and the ability to quantitate large numbers of cells for a particular criterion. These qualities have proved extremely helpful in the diagnosis of chronic lymphocytic leukemia, acute lymphoblastic leukemia, lymphoma, and other abnormalities. The combination of flow cytometry and a panel of monoclonal antisera (many of which are available commercially) allows the investigator a system of fine resolution which can confidently identify or characterize an abnormal clone or population of cells. With the increase in advanced flow cytometers, it is suggested that time should be allocated for the routine analysis of possible lymphomas and leukemias to allow quick and accurate evaluations of the disease status.

Antibodies, Monoclonal↗
PubMed1983

Immunofluorescent sites in vero cells infected with the flavivirus Kunjin.

The sites of replication and of accumulation of viral macromolecules were examined using fluorescent antibodies to viral products and to cell organelles. Synthesis of envelope protein and its accumulation in a narrow rim around the nucleus were detected at 4 hours post infection; concurrently, a progressive change was observed in the rough endoplasmic reticulum from a fine to a coarse network emanating from around the nucleus. This change in the network was visible by light microscopy. The immunofluorescent sites of viral RNA synthesis, located by use of anti-double stranded RNA, extended from the perinuclear region in another fine network which included many small foci or vesicles; these sites were also visible by light microscopy late in infection. None of these changes were associated with any visible redistribution of actin, intermediate filaments or microtubules, and no nuclear involvement was detected. However, when microtubules were disrupted by vinblastine treatment of cells, the distribution of the immunofluorescent sites of viral RNA synthesis was modified and the virus yield was reduced by at least 10-fold. These results confirmed our biochemical studies showing separation of viral sites of RNA synthesis and translation, and the accumulation of envelope protein in nuclear-associated membranes. The relevance of these observations is discussed in relation to the reports of specific membrane structures induced in flavivirus-infected cells.

Animals↗
PubMed1983

Flow microfluorometry detects IgM autoantibody to oligodendrocytes in multiple sclerosis.

Freshly isolated canine oligodendrocytes were reacted by indirect membrane immunofluorescence with 44 sera from patients with multiple sclerosis (MS). Using analysis by flow microfluorometry (FMF), we found significant IgM antibody binding to the surfaces of oligodendrocytes in the MS sera. The fluorescence intensity of cell surface reactions for MS sera (F.I. greater than 40 = 37.2 +/- 21.34%) was significantly different (P less than 0.001) to that for 53 sera from normal subjects (10.0 +/- 8.97%), 15 sera from patients with Murray Valley encephalitis (6.18 +/- 5.3%), 22 with brain tumours (6.18 +/- 5.3%), 25 with SLE (13.42 +/- 11.65%), 7 with GBS (9.77 +/- 3.9%) and 7 miscellaneous neurological disorders (6.87 +/- 3.04%). Cell surface binding was restricted to oligodendrocytes and was absorbed out by oligodendrocytes but not by liver cells or kidney cells. Oligodendrocytes were identified by conventional histology, phase-contrast optics, electron microscopy (EM) and by cell surface reactions with anti-galactocerebroside, a specific immunocytological marker for oligodendrocytes. A cell sort of the single 0 degree FMF scatter peak followed by EM examination confirmed that the reactive cells consisted exclusively of oligodendrocytes. Viability of oligodendrocytes before and after the staining reactions, was greater than 80% as assessed by trypan blue and fluorescein diacetate exclusion. The possibility that immune reactions mediated by the surface-reactive antibody with readily accessible cell surface autoantigens in vivo may contribute to the loss of oligodendrocytes and demyelination in MS is raised.

Adolescent↗
PubMed1983

Growth and gliotic response of astrocytes in vitro.

We investigated the development of astrocytes in mechanically dissociated primary cultures of foetal and neonatal rat brain grown in different culture media using immunofluorescence and antiserum to glial fibrillary acidic protein (GFAP). GFAP-positive cells developed at a time corresponding to the 16th day of embryonic development and initially grew more slowly in Dulbecco's modified Eagle's Medium (DMEM) than in Medium 199 (M199) or Modified Eagle's Medium (MEM), but as the cultures matured a greater proportion of GFAP-positive cells was obtained in DMEM, resulting in relatively pure populations of GFAP-positive cells after 1-2 months in vitro. GFAP-positive cells comprised process-bearing and fibroblastoid cell types and cells with intermediate morphology. Cultures in DMEM were also characterized by the appearance after 8-18 days in vitro of thick long bands of glial processes surrounding cyst-like spaces. The morphological change may represent a gliotic reaction to necrosis in vitro.

Animals↗
PubMed1983

Neonatal rat hypothalamus cell culture: neuron subpopulations secrete immunoreactive beta-endorphin but not immunoreactive ACTH.

Primary cultures of dissociated hypothalamic cells were prepared from 1-day-old rat neonates. Studies with cell-specific antisera revealed the presence of neurons, glial cells, oligodendrocytes and fibroblast-like cells. By immunohistochemistry, two morphologically distinct cells in culture were positive for immunoreactive beta-endorphin (IR-beta-EP). The medium derived from these cultures contained radioimmunoassayable IR-beta-EP, but not IR-ACTH. These data suggest that, in rat neonatal hypothalamic cultures, two subpopulations of cells exist which store and secrete IR-beta-EP, but not IR-ACTH.

Adrenocorticotropic Hormone↗
PubMed1983

Expression of glial fibrillary acidic protein, actin, fibronectin and factor VIII antigen in human astrocytomas.

Frozen sections and cell cultures of 50 human astrocytomas, fetal and adult human brain were examined for immunofluorescence reactivity with antisera to glial fibrillary acidic protein (GFAP), actin, fibronectin and factor VIII antigen. In frozen tissue sections GFAP expression was restricted to normal and neoplastic astrocytes while fibronectin and factor VIII antigen were localized to blood vessels. In primary cell culture, 80-100% neoplastic astrocytes expressed GFAP but not fibronectin or factor VIII antigen while actin was present as diffuse cytoplasmic staining of the cell body and cell processes. By the 5th-6th passage in vitro, GFAP immunoreactivity was lost while fibronectin and actin cables were prominently expressed. Factor VIII antigen remained negative throughout serial subculture. In double fluorochrome experiments, GFAP positive cells did not express fibronectin or actin cables, while GFAP negative cells expressed fibronectin and had prominent actin cables. Our results suggest a change in population of astrocytoma cells with increasing passage in vitro, reflecting either an overgrowth of tumour glioblasts or dedifferentiation of tumour astrocytes.

Actins↗
PubMed1983

Fibroblast monolayers as substrates for the demonstration and analysis of anti-nuclear autoantibodies.

Monolayers of acetone-fixed cultured fibroblasts were compared with frozen tissue sections as substrates for the immunofluorescent demonstration of anti-nuclear autoantibodies (ANA) in 8 sera. Fibroblast monolayers gave more readily recognizable nuclear staining patterns and more intense staining reactions. Pretreatment of acetone-fixed monolayers with DNase, RNase, trypsin, 2M NaCl or 0.2 HCl before exposure to test sera, permitted analysis of the nature of the ANA-reactive nuclear antigens and unmasked "hidden" anti-nucleolar and possibly antinuclear matrix autoantibodies in 4 sera with homogeneous ANA reactivity. The observations suggest that autoantibodies to nucleoli may be commoner than hitherto reported and that fibroblast monolayers may prove to be a useful ancillary substrate for the diagnosis and analysis of ANA.

Animals↗
PubMed1983

ELISA assay for IgG autoantibody to G-actin: comparison of chronic active hepatitis and acute viral hepatitis.

An ELISA was developed to test for IgG autoantibody to rabbit skeletal muscle G-actin in liver disease. To express results of the ELISA, we used a single reference serum with known high anti-actin activity, and assessed binding of test sera as a percentage of that displayed by the reference antiserum. Antibody to G-actin was measured in 40 sera from patients with chronic active hepatitis (CAH), 39 from acute viral hepatitis A (Hep A), 46 from acute viral hepatitis B (Hep B), 23 from non-A, non-B hepatitis (Hep non-A, non-B), 31 from systemic lupus erythematosus (SLE), 21 from scleroderma and 93 from normal persons. The results were compared with those obtained using indirect immunofluorescence tests with frozen sections of rodent stomach or fibroblast monolayers as substrates. Anti-G-actin activity of serum was significantly higher (P less than 0.001) in CAH (mean 63% +/- 23, range 18-110) than in controls (mean 15% +/- 12, range 0-51), systemic lupus erythematosus (mean 25% +/- 16, range 8-76), scleroderma (mean 23% +/- 13, range 3-59), Hep A (mean 24% +/- 11, range 6-25), Hep B (17% +/- 8, range 2-36), or Hep non-A, non-B (mean 23% +/- 11, range 7-53). There was no significant difference (P greater than 0.05) in the occurrence of anti-G-actin activity of serum in Hep A, Hep B or non-A, non-B compared with controls. At an ELISA reading of greater than or equal to 40% (mean + 2 s.d. of controls) the assay detected autoantibodies to G-actin in 85% of CAH, compared to 80% on fibroblast monolayers and 70% on rodent stomach. The ELISA described in the present study is a simple, sensitive and quantitative assay for autoantibody to G-actin. It should prove useful in assessing subspecificities of actin antibodies in liver diseases, and in differential diagnosis, particularly CAH from acute viral hepatitis.

Actins↗
PubMed1983

Parietal cell surface reactive autoantibody in pernicious anaemia demonstrated by indirect membrane immunofluorescence.

We examined, in a 'double blind' study, 60 sera from patients with pernicious anaemia for immunofluorescence reactivity with the surface membranes of viable parietal cells isolated from dog stomachs. Fifty-three sera (88%) gave an IgG autoantibody reaction with the surface membranes of parietal cells. Surface staining was also seen with parietal cells from monkey, pig, rat and mouse. The parietal cell surface reactive autoantibody was not found in any of 14 sera from patients with chronic active hepatitis, 10 from patients with systemic lupus erythematosus and 50 from healthy persons. The surface reactivity autoantibody was present in 13 of 14 sera without parietal cell microsomal antibody, 28 of 31 sera without intrinsic factor antibody and in four of four sera without microsomal and intrinsic factor antibodies. Absorption with parietal cell enriched gastric mucosal cells neutralized the activity of the surface reactive but not the microsomal antibody and cross absorption with gastric microsomes neutralized the activity of the microsomal but not the surface reactive antibody. Surface staining of parietal cells was not abolished by absorption with dog or rat hepatocytes, dog or rat kidney cells, human fibroblasts or human AB red blood cells. The results suggest that the parietal cell surface reactive antibody is probably different from the microsomal antibody. Immune reactions of the cell surface reactive antibody with parietal cell surface antigens may play a role in the pathogenesis of the gastric lesion in pernicious anaemia.

Adult↗
PubMed1983

A monoclonal IgM smooth muscle antibody reactive with fibroblast stress fibres produced by immunization with Treponema pallidum.

A monoclonal IgM smooth muscle antibody secreted by a hybrid (MMI-1) of mouse plasmacytoma NS-1 with spleen cells from mouse immunized with Treponema pallidum was detected by indirect immunofluorescence tests on frozen tissue sections and on acetone fixed monolayers of rat and human fibroblasts. The antibody did not react with acetone fixed smears of T. pallidum but reacted with smooth muscle fibres and with striations of skeletal and cardiac muscle. In non-muscle cells, the antibody stained liver in a 'polygonal' pattern, thymus with accentuated staining of the thymic medulla, renal glomeruli and the brush border and peritubular fibrils of renal tubules. In fibroblast monolayers, the antibody stained stress fibres in an interrupted pattern. Immunoblotting with muscle proteins and the antibody showed labelling of a 100K molecule. The cellular distribution of the mouse monoclonal antibody is similar to that obtained with anti-actin antibody suggesting that the corresponding antigen may be an actin binding protein.

Actins↗
PubMed1983

Dexamethasone induces different cellular protein synthetic responses in PNA+ and PNA- mouse thymocyte subpopulations.

Thymocytes from adrenalectomized BALB/c male mice were separated by peanut agglutination (PNA) into cortical, corticosensitive, PNA+ cells and larger, medullary, corticoresistant, PNA- cells; the extent of cross-contamination of PNA+ and PNA- cells, and vice versa, was checked by flow microfluorometry. Glucocorticoid receptor profiles were established with 3H-dexamethasone as probe; no differences in receptor affinity or cellular concentration, or in cytoplasmic and nuclear compartmentalization were seen between PNA+ and PNA- cells. On two-dimensional gel electrophoresis, PNA+ and PNA- thymocytes from oil-injected (control) adrenalectomized mice showed patterns of incorporation of 35S-methionine into protein that differed in at least 12 spots, as revealed by autoradiography. PNA+ and PNA- cells from mice treated with submaximal (6 micrograms/day) or near-maximal thymolytic doses of dexamethasone (20 micrograms/day) were also examined by two-dimensional gel electrophoresis. Both PNA+ and PNA- cells showed substantial, overlapping dexamethasone-induced changes in protein synthetic profiles.

Animals↗