Biomedical subjects

M R Brandon

Publications and source records attributed to M R Brandon.

At least 109 records · Page 6Linked to original sources

PubMed1987

Immunogold localization of placental lactogen and the SBU-3 antigen by cryoultramicrotomy at implantation in the sheep.

In the sheep, granulated trophectodermal binucleate cells (BNC) appear at implantation 16 days post coitum (dpc) and persist throughout pregnancy. Conventional immunocytochemical techniques at both light and electron microscope levels have indicated the presence of the ovine placental lactogen (oPL) hormone in the granules but no earlier than 22 dpc, when the level was very low. Immunofluorescent studies using glycolmethacrylate sections between 15 and 55 dpc suggest a completely different distribution of oPL restricted to uninucleate cells with none in the BNC. Using the most sensitive method available, immunocytochemistry on ultrathin frozen sections, the results in this paper demonstrate that BNC granules contain oPL at their earliest appearance (16-17 dpc). No significant localization was found in any uninucleate cell. In contrast, another molecule, the SBU-3 antigen, which is demonstrated in BNC granules later in pregnancy, is not present at the earliest stages but appears between 24 and 28 dpc coincident with the development of the foetal cotyledonary villi. The significance of these results for BNC function are discussed briefly.

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PubMed1987

Sheep MHC class II molecules. I. Immunochemical characterization.

The physicochemical features, biosynthesis and glycosylation of sheep class II molecules were investigated using a panel of seven monoclonal antibodies. The class II molecules recognized by different monoclonal antibodies could be differentiated using SDS-PAGE. Two monoclonal antibodies, SBU.II 42-20 and 49-1, reacted with dissociated sheep class II molecules and recognized epitopes on class II alpha and beta polypeptides, respectively. The structure of the sheep class II heterodimer differed from that of mouse and man in that it was unstable in the presence of 1% SDS at 20 degrees and, following reduction, sheep beta polypeptides displayed a marked increase in MW, resulting in the apparent co-migration of reduced alpha and beta polypeptides on SDS-PAGE. This phenomenon was not seen using sheep class II molecules synthesized in the presence of tunicamycin. Pulse-chase analyses of biosynthetically labelled sheep class II molecules suggested the rapid association and glycosylation of sheep class II alpha and beta polypeptides during synthesis. Both alpha and beta polypeptides of sheep class II molecules carried N-linked oligosaccharides of MW 6,000 and 3,000, respectively. However, unlike human class II oligosaccharides, these were exclusively of the complex or sialylated type.

Acetylglucosaminidase↗

PubMed1987

Ontogeny of ovine lymphocytes. II. An immunohistological study on the development of T lymphocytes in the sheep fetal spleen.

The development of T and B lymphocytes in the ovine fetal spleen was studied immunohistologically using a panel of monoclonal antibodies. A specific sequence of appearance of lymphocyte markers on cells was observed. At 43-44 days of gestation, SBU-T1- and SBU-T8-positive lymphocytes were present in low numbers. However, no SBU-T4, 20.96-, 25.69-, 38.38-, or 46.66-positive lymphocytes were seen until 50-55 days of gestation. Surface immunoglobulin (sIg) was first detected on fetal spleen cells at 45-50 days of gestation. SBU-T19 lymphocytes appeared later in gestation, being observed in fetal spleens at 57 days gestational age (g.a.). The distribution of T cells, B cells and MHC antigens in the developing spleen of the ovine fetus is described.

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PubMed1987

Ontogeny of ovine lymphocytes. III. An immunohistological study on the development of T lymphocytes in sheep fetal lymph nodes.

The development of T lymphocytes in ovine fetal lymph nodes was studied immunohistologically using a panel of monoclonal antibodies. T lymphocyte subsets appeared within the ovine fetal lymph node in a specific sequence. SBU-T1- and SBU-T8-positive lymphocytes were seen at Day 47 of gestation. LCA, sIg, MHC I, MHC II, SBU-T6 and 46.66 antigens were also seen within the lymph node at this time. The first SBU-T4-positive cells were seen within the fetal lymph node at Day 50 of gestation, along with the first 20.96-, 25.69- and 38.38-positive cells. SBU-T19 lymphocytes appeared later in gestation, being present in fetal lymph nodes from Day 69 of gestation. The appearance and distribution of T and B cells in the developing lymph nodes of the ovine fetus is described and compared with the ontogeny of lymphocytes in the fetal thymus and spleen.

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PubMed1987

Ontogeny of ovine lymphocytes. I. An immunohistological study on the development of T lymphocytes in the sheep embryo and fetal thymus.

The time of appearance of lymphocytes expressing T-cell markers and the subsequent development of the fetal thymus were studied in ovine embryos using a panel of monoclonal antibodies. Leucocyte common antigen (LCA) and major histocompatibility complex class I (MHCI) antigens were seen on a small number of cells within the ovine embryo at Day 19 of gestation. SBU-T6 (CD1)-positive cells were found at Day 22 of gestation, while major histocompatibility complex class II (MHC II) antigens were first observed at Day 25 of gestation. Large basophilic cells, weakly staining for SBU-T1 (CD5), were present in the mesenchyme of the neck and in the dorsal mediastinum and mesentery of embryonic sheep of 33 days gestational age (g.a.); however, no SBU-T1-positive cells were detected in the thymus at this time. No SBU-T4 (CD4)- or SBU-T8 (CD8)-positive cells were detected in any organs of embryos of this age. SBU-T4- and SBU-T8-positive cells were first seen in fetal thymi, and elsewhere within the fetus, at 35-38 days g.a. SBU-T19-positive cells were first seen within the fetal thymus at 50-58 days g.a.

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PubMed1987

Sheep MHC class II molecules. II. Identification and characterization of four distinct subsets of sheep MHC class II molecules.

A panel of seven monoclonal antibodies, sequential immunoprecipitation and two-dimensional NEPHGE/SDS-PAGE analyses were used to identify and characterize subsets of sheep MHC class II molecules. Using sequential immunoprecipitation four distinct subsets of class II molecules were identified by the monoclonal antibodies SBU.II 28-1, 37-68, 38-27 and 42-20, while another monoclonal antibody, SBU.II 49-1, recognized all four subsets of class II molecules. These four subpopulations of sheep class II molecules displayed different two-dimensional gel profiles and, using splenocytes from four outbred sheep, the class II molecules recognized by SBU.II 28-1, 37-68 and 42-20 showed structurally detectable allelic polymorphism in their beta polypeptides, but no detectable variation in their alpha polypeptides. In contrast, the class II molecules recognized by SBU.II 38-27 showed allelic variation in both their alpha and beta polypeptides. Two-dimensional (2-D) gel analyses of non-glycosylated class II molecules immunoprecipitated by SBU.II 49-1 suggested that approximately 10-12 different class II molecules were expressed by a single sheep. The results of this study show that sheep express class II molecules that can be divided into four structurally and serologically distinct subsets, and provide additional evidence for the subdivision of the sheep MHC class II genetic region into at least three distinct subregions.

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PubMed1986

Thymocyte subpopulations during early fetal development in sheep.

Phenotypic analysis of thymocytes during fetal development may identify subpopulations which are either absent or difficult to detect in postnatal thymus. A panel of monoclonal antibodies specific for sheep lymphocyte antigens (SBU-T1, -T4, -T8, -T6) was used to identify thymocyte subpopulations in postnatal and fetal sheep. Thymuses were analyzed by two-color immunofluorescence and flow cytometry or by immunohistology. Two-color immunofluorescent staining of postnatal sheep thymus with anti-SBU-T4 and anti-SBU-T8 revealed four relatively distinct subpopulations with particular localizations: a) SBU-T4-T8-, predominantly outer cortex (12%); b) SBU-T4+T8+, inner cortex (74%); c) SBU-T4+T8-, medulla (10%), and d) SBU-T4-T8+, medulla (4%). One- and two-color immunofluorescent analysis of cells from early fetal thymuses demonstrated the appearance of SBU-T8+ cells well before SBU-T4+ cells. Immunohistologic staining of fetal sheep thymus at various stages of gestation (term = 150 days) revealed that lymphoid cells and MHC class II-positive dendritic cells first appeared at 35 days, at which stage the thymic epithelium was weakly positive for class I MHC antigens but negative for class II MHC antigens. The earliest lymphocyte antigens detectable on fetal sheep thymocytes were SBU-LCA and SBU-T1. By 40 days, the antigens SBU-T6, SBU-T4, and SBU-T8 were detectable on a small number of thymocytes; SBU-T8 preceded SBU-T4, and the number of SBU-T8+ thymocytes always exceeded the number of SBU-T4+ thymocytes throughout early gestation. At 50 days, a thymic medulla appeared and thereafter grew rapidly in size. Immunoperoxidase staining of serial sections of the fetal neck revealed cortical-type thymocytes outside the thymus from 40 days onward, before the appearance of a thymic medulla. However, by 60 days, only medullary-type thymocytes were observed either extrathymically or within the interlobular septa of the thymus, indicating that only thymocytes with a medullary phenotype leave the thymus from this stage of gestation.

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PubMed1986

Three distinct subpopulations of sheep T lymphocytes.

Monoclonal antibodies reactive with distinct T lymphocyte subpopulations have been described in man, mouse and rat and structural analyses of these antigens have demonstrated a high degree of evolutionary conservation. This report describes the reactivity of three monoclonal antibodies (mAb), 19-19, alpha SBU-T4 and alpha SBU-T8, which define T cell subpopulations in the sheep. The mAb alpha SBU-T4 and alpha SBU-T8 define the sheep CD4 and CD8 molecules, respectively. These two antigens show similar tissue distributions, molecular weights and fluorescence-activated cell sorter profiles to human, mouse and rat CD4 and CD8 molecules. The mAb 19-19 is reactive with a subpopulation of T lymphocytes which displays a tissue distribution unlike that reported for a T cell subset in any other species. 19-19 stains 7% of efferent lymph lymphocytes, 15% of peripheral blood lymphocytes but only 1-3% of lymph node lymphocytes. Two-color immunofluorescence demonstrates that the 19-19+ T cell subset is SBU-T4- and SBU-T8-, and thus defines a third T cell subpopulation in sheep. Immunohistology on frozen lymph node tissue sections localizes 19-19 mAb-reactive cells to the subcapsular region of the lymph node and lymph node trabeculae. Only 1% of thymocytes are 19-19+ and these cells are located mainly in the medulla and often arranged as foci around blood vessels. The 19-19 mAb immunoprecipitates from sheep lymphocytes an antigen with an apparent molecular mass of 215 kDa under both reducing and nonreducing conditions. It is concluded that alpha SBU-T4 and alpha SBU-T8 recognize the sheep homologues of the human T4 and T8 antigens, respectively, whereas 19-19 recognizes an antigen (termed SBU-T19) which has not been reported in any other species.

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PubMed1986

Immunogold co-localization of ovine placental lactogen and the antigen recognized by the SBU-3 monoclonal antibody in sheep placental granules.

Ovine placental lactogen and the SBU-3 antigen (derived from a trophoblast membrane preparation), two proteins of widely different structure, function and destination, were shown by ultrastructural immunogold techniques to localize in identical structures in the sheep placentome throughout most of pregnancy. Both were restricted to the ultrastructurally similar membrane-bounded granules in the chorionic fetal binucleate cell and the syncytium at the fetomaternal interface. The Golgi body from which the granules form was also doubly labelled but only in the binucleate cell, never the syncytium. This provides further evidence that the binucleate cells migrate and fuse to form the syncytium. The two proteins were homogeneously distributed in the granules and would be released together by exocytosis. Only the lactogen reaches the fetal and maternal circulations so the SBU-3 may have some more local function. In early pregnancy the SBU-3 antigen is found by itself in the granules, indicating that the association with the lactogenic hormone is not obligatory. Neither antigen was found consistently in the otherwise ultrastructurally similar interplacentomal binucleate cell granules, corroborating the presence of two functional populations of binucleate cells.

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PubMed1986

Light and electron microscope immunocytochemical studies on the role of binucleate cells in villus growth in goat placentomes.

The monoclonal antibody SBU-3 exclusively labels granules and Golgi cisternae in fetal trophectodermal binucleate cells but only similar granules in the adjacent syncytium in goat placentomes. This localisation supports previous morphological evidence that binucleate cells migrate and form the syncytium by fusion. SBU-3 reactivity is not demonstrable in binucleate cells or syncytium before the start of placentomal fetal chorionic villus formation (30-34 days post coitum (d.p.c.)) but then rises rapidly to a peak during the period of maximal villus growth into the caruncle (36-95 d.p.c.). During this period the highest reactivity and numbers of granules are concentrated in the most extensive syncytium and largest binucleate cells at the advancing tips of the villi. After the villi have reached maximum length (90-100 d.p.c.) the SBU-3 reactivity decreases and the binucleate cell distribution becomes more diffuse, although even at term (145-150 d.p.c.) SBU-3 positive granules are present in binucleate cells and the syncytium. In the interplacentomal regions, where no chorionic villi form, no binucleate cells or syncytium show any SBU-3 activity at any stage of pregnancy despite having equivalent ultrastructure (including granules) to their placentomal neighbours. These results support the concept of two populations of binucleate cells of equivalent ultrastructure but different granule content and function in the goat placenta. The distribution of the SBU-3 positive population suggests a role in placentomal villus formation.

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PubMed1986

Four antigens expressed on most ovine cell types.

Monoclonal antibodies have been produced that bind to four different antigens expressed on the surface of ovine lymphocytes as well as to a variety of other ovine cell types. These antigens have been characterized with respect to their tissue distribution and immunochemistry. The timing of appearance of these antigens within the ovine embryo is reported.

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PubMed1986

Comparative studies on the distribution of binucleate cells in the placentae of the deer and cow using the monoclonal antibody, SBU-3.

A monoclonal antibody SBU-3 raised against an antigen expressed by the binucleate cells in the trophoblast of the sheep placentome crossreacts with an antigen expressed by binucleate cells in the placenta of the deer and the cow. This monoclonal antibody has been used to study the distribution of this subpopulation of binucleate cells in the deer and cow during fetal development. In the deer at less than 30 days of gestation, SBU-3-positive binucleate cells were observed in the trophoblast of the chorionic villi and in the trophoblast at the interplacentomal areas. With the advancement of pregnancy, the placentome increased in size and SBU-3-positive binucleate cells were observed to localise mainly at the tips of the chorionic villi and were arranged in clusters. There was evidence of SBU-3-positive binucleate cell migration into the epithelium of the caruncular crypt, but there was no fusion of migrated cells to form a syncytial layer. Such a pattern of SBU-3-positive cell distribution was seen up to 95 days of gestation, the oldest placenta available for study. In the cow at 28-34 days of gestation, SBU-3-positive binucleate cells were seen in the trophoblast located in close apposition to the caruncular epithelium. From 35-240 days, there was an increase in the number of SBU-3-positive binucleate cells in the placentomal trophoblast. As in the deer, SBU-3-positive binucleate cells migrated into the epithelium of the caruncular crypt, and these cells did not fuse to form a syncytial layer. By 241-283 days, SBU-3-positive binucleate cells present in the placentomal trophoblast were stained less intensely. Many of the binucleate cells which had migrated into the epithelium of the caruncular crypts had pyknotic nuclei and scanty cytoplasm containing weakly stained SBU-3-positive product. It is concluded that the monoclonal antibody SBU-3 recognises an antigen common to ruminants which is expressed only by binucleate cells in the placenta. In the deer and the cow, granule transfer seems to be the primary function of binucleate cell migration. The histological classification of the mature placenta of the deer and the cow is epitheliochorial.

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PubMed1985

Characterization of a monoclonal antibody (SBU-1) made to the thymic rudiment of sheep.

A monoclonal antibody (SBU-1) was raised to sheep thymic rudiment by fusion of NSI myeloma cells with spleen cells from BALB/c mice immunized with thymic rudiment isolated from fetal sheep between 25-30 days of gestation. By employing the indirect immunoperoxidase technique the antigen recognized by SBU-1 was found to be present in the epithelial reticular cells of the fetal sheep thymus. The intensity of staining decreased as gestation progressed. In the adult thymus the antigen was mainly restricted to Hassall's corpuscles and occasional epithelial cells in the medulla. In addition, the antigen was also shown to be present in epithelial cells of the small intestine, the bronchiole, the keratinized epithelium of the rumen, and the epithelial cells of the kidney tubules. By use of immunofluorescence the antigen was shown to be present in most of the cells of wool follicles and the cortex of developing wool fibers. Western blotting of SBU-1 against the low-sulfur alpha-keratin proteins of wool confirmed that the antigen recognized by SBU-1 belongs to a family of keratins. It was concluded that SBU-1 was raised against alpha-keratin expressed by the epithelial cells of the thymic rudiment and that the expression of this antigen on the reticular network of the thymus declined with advancement of pregnancy.

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PubMed1985

Sheep lymphocyte antigens (OLA). I. Major histocompatibility complex class I molecules.

Three monoclonal antibodies, SBU.I 41-17, 41-19 and 41-28, have been produced which recognize sheep Class I major histocompatibility complex antigens (OLA). All three antibodies are able to precipitate a heavy chain of 44,000 MW and a smaller beta 2-microglobulin of 12,000 MW from 125I-surface labelled lymphocytes. The antibodies have been used to localize OLA Class I antigens in lymphoid and non-lymphoid tissues using indirect immunoperoxidase histological staining and cytofluorograph analyses. Evidence suggests that the three antibodies are directed against monomorphic determinants but that they recognize different epitopes.

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PubMed1985

Analysis of lymphopoietic stem cells with a monoclonal antibody to the rat transferrin receptor.

A mouse monoclonal IgG2a antibody, designated MRC OX-26, is shown to be specific for the rat transferrin receptor, but does not block transferrin binding. The antibody labelled a myeloma, three leukaemia cell lines and normal dividing cells of various types, but also bound to a number of nondividing normal tissues. No labelling of lymphopoietic stem cells could be detected, even though approximately 25% of bone marrow and over 95% of fetal liver cells were clearly labelled.

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PubMed1985

The sheep analogue of leucocyte common antigen (LCA).

The tissue distribution and immunochemical properties of antigens recognized by two monoclonal antibodies 1.11.32 and 1.28.124 define the sheep analogue of the leucocyte common antigen found in rat, man and mouse. Histological and immunofluorescent studies show that this antigen is found on all lymphocytes, as well as other leucocytes but is absent from non-leucocytic cells. Immunochemical data show that a series of proteins of high molecular weight (190,000-225,000) are recognized, and histological studies show the presence of this antigen on a subpopulation of fetal liver cells as early as Day 27-30 of gestation, and on all fetal thymocytes from Day 40 of gestation.

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PubMed1985

Characterization of two sheep lymphocyte differentiation antigens, SBU-T1 and SBU-T6.

The monoclonal antibodies 25.91 and 20.27 define two lymphocyte cell surface antigens of sheep. 25.91 is reactive with 60-80% of lymphocytes and 98% of thymocytes, and only stains surface immunoglobulin-negative peripheral lymphocytes. 25.91 immuno-precipitates a 67,000 MW protein from lymphocyte lysates under both reducing and non-reducing conditions, whereas immunoprecipitation of thymocyte lysates reveals a 67,000, 62,000 MW complex. The tissue distribution and molecular weight analysis reported here for the antigen recognized by 25.91 indicate that this antigen is the sheep homologue of the human T1 and mouse Ly 1 antigens. The monoclonal antibody 20.27 is reactive with 80% of thymocytes and the majority of cell surface immunoglobulin-positive peripheral blood lymphocytes (B cells), but is unreactive with peripheral blood T cells. 20-27 also stains Langerhans cells in skin tissue sections and large dendritic-like cells in the paracortex sections and large dendritic-like cells in the paracortex of lymph node tissue sections. Immunoperoxidase staining of thymus tissue sections with 20.27 shows intense staining of cortical thymocytes and an absence of staining within the medulla. Molecular weight analysis of the 20.27 antigen reveals two major bands of 46,000 and 12,000 MW under both reducing and non-reducing conditions. The 20.27 antigen has properties resembling MHC class I-like antigens such as T6 in the human and TL in the mouse.

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PubMed1985

Surface antigens, SBU-T4 and SBU-T8, of sheep T lymphocyte subsets defined by monoclonal antibodies.

Monoclonal antibodies have been used to characterize molecules found on the surfaces of T cells of sheep. SBU-T4 and STU-T8 are present on 80-85% of thymocytes, absent from B lymphocytes, and present on two-thirds and one-third of lymph node T lymphocytes, respectively. Double-labelling of T lymphocytes shows that the populations recognized are mutually exclusive. Immunoprecipitation of the SBU-T4 antigen from thymocytes and T lymphocytes reveals a molecule which migrates on SDS-PAGE as a single band of 56,000 MW under both reducing and non-reducing conditions. Under reducing conditions, the SBU-T8 molecule migrates as two bands of 33,000 and 36,000 MW when immunoprecipitated from thymocytes, but only the higher MW band is present following immunoprecipitation from peripheral lymphocytes. Under non-reducing conditions, SBU-T8 migrates as dimers and other multiples. Based on immunofluorescent, immunochemical and immunohistological data, these molecules are considered to be the structural analogues of the marker molecules used to delineate T-helper and T-cytotoxic/suppressor lymphocyte subsets of other species.

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