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Biomedical subjects

R W Stoddart

Publications and source records attributed to R W Stoddart.

At least 55 records · Page 3Linked to original sources
PubMed1987

Elimination of the non-specific binding of avidin to tissue sections.

A simple procedure is described for eliminating non-specific staining with avidin-peroxidase conjugates. Murine ovaries were embedded in either paraffin wax or epoxy resin and, after blocking endogenous peroxidase activity, were treated with 10 micrograms/ml biotinylated Pisum sativum agglutinin. Avidin-peroxidase conjugates (5 micrograms/ml), diluted in standard 0.05 M tris-buffered saline, pH 7.6, containing 0.139 M NaCl, produced considerable background coloration and intense mast cell staining in controls without the lectin. This background diminished as the ionic strength of the buffer was raised. At 0.125 M Tris-buffered saline (containing 0.347 M NaCl) the background was completely unstained, with elimination of all binding to mast cells and only minimal loss of specific lectin binding.

Avidin↗
PubMed1987

Proteolysis and lectin histochemistry.

Lectin binding to formalin-fixed, paraffin-embedded tissue can often be enhanced by pre-treatment of the sections with proteolytic enzymes. However, the pattern of staining may be profoundly influenced by the type of enzyme preparation which is used. Sites of binding of thirteen different lectins to murine ovary and thyroid gland were studied after exposure of tissue sections to crude trypsin, purified trypsin, purified alpha-chymotrypsin, pepsin, protease VII, papain, bromelain, thermolysin or elastase. With most lectins, the results obtained were similar regardless of which enzyme was used for proteolytic digestion. However, the pattern of binding of soy bean lectin to the ovary and of concanavalin A and common pea lectin to the thyroid gland was highly dependent upon the enzyme used to pre-treat the sections. In both tissues, the staining pattern seen in untreated frozen sections was similar to that found in formalin-fixed, paraffin-embedded material digested with purified trypsin, but was different from that observed after exposure of processed sections to crude trypsin. The location of binding sites after treatment of paraffin sections with chymotrypsin was the same as that after digestion with crude trypsin. Results obtained after the use of other proteolytic enzymes varied according to the tissue being studied. These findings imply that the effect of treatment with crude trypsin is due to contaminating chymotrypsin, and demonstrate that the use of purified trypsin may have advantages over other proteolytic enzymes in lectin histochemistry. The observations may also apply to other related cytochemical techniques such as immunocytochemistry.

Animals↗
PubMed1986

A post-embedding avidin-biotin peroxidase system to demonstrate the light and electron microscopic localization of lectin binding sites in rat kidney tubules.

A post-embedding method for the light and electron microscopic demonstration of lectin binding sites in rat kidney tubules is described. The use of biotinylated lectins, followed by treatment with avidin peroxidase and the DAB-H2O2 sequence, produced intense staining of acrylic sections at the electron microscope level: brush borders and associated structures, cytoplasmic granules, basal infoldings and basement membrane-plasmalemmal interfaces of proximal tubules bound erythrophytohaemagglutinin, while distal tubules were mainly unstained. At the light microscope level, epoxy resin sections showed a similar staining pattern after etching, as did acrylic resin sections after intensification of the final reaction product. The binding of wheatgerm agglutinin to cytoplasmic granules and brush border structures in the proximal tubules was abolished, at both the light and electron microscope levels, by the competing sugar tri-N-acetylchitotriose. Epoxy resin ultrathin sections required etching before staining was achieved in the electron microscope, and results were far inferior to those obtained with acrylic resin. This method allows rapid and inexpensive screening of large numbers of lectins, if required, at both the light and electron microscope levels, using reagents that are stable for long periods of time.

Animals↗
PubMed1986

The structure of the basement membrane of human lymph node high endothelial venules: an ultrastructural, histochemical and immunocytochemical study.

The structure of the basement membrane of the high endothelium of reactive human lymph nodes was investigated by techniques selective for carbohydrates (periodic acid-Schiff; critical electrolyte concentration staining with Alcian Blue; lectin histochemistry), specific proteins (immunohistochemistry for laminin and fibronectin) and by conventional techniques of light and transmission electron microscopy. Adjacent small lymphocytes were assigned to B and T cell subsets by use of monoclonal antibodies and they were analysed for non-specific esterase, beta-glucuronidase, beta-N-acetylglucaminidase and proteolytic activities. The basement membranes were shown to be distinctive and to contain three layers, of differing laminin, glycosaminoglycan and glycoprotein oligosaccharide content. Certain lymphocytes (probably T) contained enzymes potentially able to degrade some components of these basement membranes.

Basement Membrane↗
PubMed1985

The distribution of cells of the monocytic-lineage in reactive lymph nodes and non-Hodgkin's lymphomata. Characterization using protein histochemistry, lectin binding and monoclonal antibodies.

In this study cells of putative monocytic origin have been investigated in lymph nodes, using a battery of cytochemical investigations on fixed and frozen tissue. In reactive lymph nodes involved in non-Hodgkin's lymphomata six types of presumed monocyte-derived cells have been identified on the basis of morphology and/or cytochemistry. These cells can be divided into two distinct groups. Certain cell types have distinctive distributions in reactive and non-Hodgkin's lymphomata and this taken in conjunction with their morphology and staining properties may give important clues to their function.

Antibodies, Monoclonal↗
PubMed1985

Systemic infection with Trichosporon capitatum in two patients with acute leukaemia.

Trichosporon capitatum was isolated from the blood of two patients with acute leukaemia who were undergoing induction chemotherapy. Both patients died of their infections, and the fungus was cultured from their tissues after death. Systemic infection was proved by demonstrating the same pattern of fluorescein-labelled lectin staining of fungal elements in the tissues as was shown by the fungal isolates.

Adult↗
PubMed1985

Disseminated zygomycosis associated with erythroleukaemia: confirmation by lectin stains.

Zygomycosis is not often diagnosed in the United Kingdom, and so the possible importance of the findings in a patient with disseminated zygomycosis who had been treated with chemotherapy for erythroleukaemia was not appreciated until histological examination of specimens obtained at necropsy provided a presumptive diagnosis. No attempt had therefore been made to identify the organism by culture, and lectin binding methods were used to try to compensate for this. The characteristics of the hyphae on staining with lectins were similar to those previously shown in Rhizopus oryzae and were unlike those of a wide range of other hyphal fungi. Although definite speciation of the fungus was not achieved, these findings confirm that this was a case of zygomycosis and would seem to represent the first such reported confirmation in the absence of culture.

Aged↗
PubMed1984

The migration of lymphocytes across specialized vascular endothelium: VIII. Physical and chemical conditions influencing the surface morphology of lymphocytes and their ability to enter lymph nodes.

The introductory review amplifies the finding that simply holding lymphocytes in vitro reversibly compromises their ability to enter lymph nodes from the blood, although entry into the spleen is unaffected. The differential migration of T and B lymphocytes from the blood, lymphocyte traffic in athymic rats, and the secretion of a sulphated glycoconjugate by high endothelial cells in lymph nodes are also discussed. Original data are presented concerning the effects of varying the conditions under which lymphocytes are held in vitro (time, temperature, medium, centrifugation) on their ability to enter lymph nodes and also on their surface morphology. In general, conditions that reduced the number of microvilli and induced surface blebbing also tended to affect the delicate function of crossing specialized vascular endothelium; but there was no simple relationship between morphology and migratory behavior. The localization of lymphocytes to the bone marrow was augmented by holding them in vitro, and this effect was greater after holding at room temperature (RT) than at 0 degree C, in contrast to impaired entry into lymph nodes. Small amounts of heparin (10 units) injected along with lymphocytes significantly reduced early localization in lymph nodes. These findings have practical implications for the design of lymphocyte traffic experiments and are relevant to the mechanism of lymphocyte attachment to vascular endothelium, since the well-known effect of trypsinizing lymphocytes can be reproduced by maintenance in vitro.

Animals↗
PubMed1982

Studies of lectin binding to normal and neoplastic lymphoid tissues. I. Normal nodes and Hodgkin's disease.

Lectins are proteins which have the ability to interact specifically with carbohydrate residues of glycoproteins and other glycoconjugates. The staining patterns of 10 fluorescein conjugated lectins (F-Con A, F-LCA, F-RCA, F-WGA, F-PHA, F-PWM, F-LTA, F-SBA, F-PNA, F-DB) and a protease inhibitor (F-LA) have been studied in histological sections of 11 normal or reactive lymph nodes and 6 nodes and one skin biopsy involved by Hodgkin's disease. On the basis of the patterns of lectin binding, and current knowledge of their saccharide specificities, we found that within germinal centres there is an orderly carbohydrate rich extracellular matrix which contains a higher concentration of GlcNAc and terminal Gal residues than the surface membranes of component cells. This suggests active secretion rather than simple membrane shedding, and it is possible that this pericellular domain plays a part in the regulation of the proliferative response, or controls migration of lymphocytes in and out of the germinal centre. Lectin binding in Reed-Sternberg cells suggests that the huge nucleoli contain glycoconjugates of diverse structure, which may be linked with their failure to undergo cytokinesis.

Fluoresceins↗
PubMed1982

Studies of lectin binding to normal and neoplastic lymph nodes. II. Non-Hodgkin's lymphoma.

Fluorescein conjugated lectins have been used as histochemical stains in lymph node sections from 22 patients with non-Hodgkin's Lymphoma. Variations in the distribution and structure of glycoprotein sequences between the different types of lymphoma, and also normal nodes, have been detected. The lectin-binding patterns of neoplastic lymphocytes of small cell lymphomas, both follicular and diffuse, suggested a predominance of sialylated glycopeptides, as in normal small lymphocytes of the mantle zone of germinal centres. In contrast, the staining patterns of large cell follicular and diffuse lymphomas showed a greater diversity of carbohydrate structure, with enhanced cytoplasmic staining and increased numbers of incomplete oligosaccharide sequences. Heterogeneity of staining, together with reduced sialic acid expression at all cellular sites was a common feature of lymphoblastic lymphomas and seemed to be linked with a poor prognosis. The extracellular matrix of small cell follicular lymphomas showed altered saccharide content, but retained some degree of organization. The large cell follicular lymphomas were characterized by a prominent disorderly matrix, with staining characteristics which suggested shedding of surface membrane from component cells. The loss or disordered production of the normal extracellular matrix may reflect a breakdown of control mechanisms within neoplastic follicles.

Fluoresceins↗
PubMed1982

Lectin staining of carbohydrates of haemic cells. III. The cells of Hodgkin's disease and other lymphomas.

When stained for reactive sialyl groups with fluorescein-labelled Aprotinin (FLA), lymphocytes of three diffuse lymphomas were uniformly faintly fluorescent. The nodules of a nodular lymphocytic lymphoma showed dimly fluorescing lymphocytes surrounded by brightly fluorescing, apparently normal cells. The spleens of eight patients with Hodgkin's disease showed involvement in six cases. With FLA, the two uninvolved spleens contained only brightly fluorescing lymphocytes, whereas the foci of Hodgkin's lesions in the six spleens and in eight involved lymph nodes from a further eight patients contained varying proportions of brightly and dimly fluorescing lymphoid cells. Mononuclear Hodgkin's cells and bi- or multinucleated Reed-Sternberg cells fluoresced faintly. Fluorescein-labelled Ricinus communis agglutinin (FL-RCA) for galactose, and Concanavalin A (FL-Con A) for mannose or glucose, showed eosinophils, reticulin and collagen fibres especially in nodular sclerosing Hodgkin's disease, whereas all lymphocytes, Hodgkin and Reed-Sternberg cells stained faintly with either lectin. The reduction of reactive sialyl groups in malignant lymphocytes of lymphomas and Hodgkin's lesions is similar to that in lymphocytic leukaemias. It is suggested that in Hodgkin's disease these lymphocytes together with the Hodgkin and Reed-Sternberg cells represent the malignant component, whereas the brightly fluorescent normal lymphocytes, together with histiocytes, eosinophils (and neutrophils) represent a reactive component in the lesions. Similarly, the reactive lymphocytes in sarcoid lesions and sinus histiocytosis were brightly fluorescing.

Aprotinin↗
PubMed1980

Lectin staining of carbohydrates of haemic cells; the cells of normal blood and bone marrow and of the myeloid leukaemias.

Plant proteins and aprotinin (a protein of beef lung), labelled with fluorescein isothiocyanate, were used as histochemical tools for the demonstration of carbohydrates. Sialic acid (or glucuronate) was stained with aprotinin (FLA); galactose was stained with Ricinus communis agglutinin (FL-RCA) and mannose (or glucose) with Concanavalin A (FL-Con-A). Normal human bone marrow and blood were examined as were the cells of patients with acute and chronic myelogenous leukaemia. The plasmalemma, cytoplasm and nuclear membrane of the cells of the normal granulocytic series were stained well with FLA, but the corresponding leukaemic cells fluoresced less intensely. Chromatin was weakly stained in both normal and leukaemic cells. FLA-RCA and FL-Con A stained the plasmalemma, cytoplasm and nuclear membrane weakly, but did not demonstrate chromatin. There was no detectable difference between normal and leukaemic cells. Eosinophil and basophil granules--in contrast to those of the neutrophils--stained well with all three compounds, in both the normal and leukaemic cells. In megakaryocytes and platelets the plasmalemma and cytoplasm were well stained with FLA. The cytoplasm of megakaryocytes and the plasmalemma of platelets stained particularly well with FL-RCA. The cytoplasm of both platelets and megakaryocytes showed up strongly with FL-Con A. In the erythroblastic series all three compounds stained the plasmalemma. The remaining cellular components were weakly stained, except the chromatin; that of the late erythroblasts showed up particularly well with FLA. Lymphocytes, monocytes and reticulin cells of the bone marrow were also stained with all three reagents. Reticulin fibres were stained strongly with FLA and FL-Con-A.

Aprotinin↗
PubMed1980

Lectin staining of carbohydrates of haemic cells. II. The cells of normal lymphoid origin, of lymphatic leukaemias and related diseases.

Normal and leukaemic lymphoid cells, both human and murine, were stained for specific carbohydrates with three fluorescein-labelled lectins: Aprotinin for sialyl (or uronyl) groups: Ricinus agglutinin for galactosyl groups; and Concanavalin A for mannosyl (or glucosyl) groups. The method gives permanent preparations of sections from methanol fixed, paraffin embedded tissues, from blood and bone marrow films or touch preparations of lymph nodes that were methanol fixed. Whereas normal lymphocytes and lymphoblasts reacted strongly for sialyl groups, lymphoblasts of acute lymphoblastic leukaemia and lymphocytes of chronic lymphocytic leukaemia gave a much weaker reaction. The same was the case of the lymphocytes of the Sézary variant and the lymphocytes of macroglobulinaemia. The fine processes of the cells of hairy cell leukaemia stained well for sialyl groups. No obvious differences were detected between normal monocytes and the cells of monocytic leukaemia, nor between normal plasma cells and those of myeloma.

Animals↗