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B F Haynes

Publications and source records attributed to B F Haynes.

At least 163 records · Page 9Linked to original sources
PubMed1987

Human thymic epithelial cells produce interleukin 1.

Although the thymus plays a critical role in generation of immunocompetent T lymphocytes, the precise role of the epithelial component of the thymus in the induction of T cell proliferation and maturation remains unknown. Since interleukin 1 (IL 1) is required for mature T cell activation, we have determined whether human thymic epithelial (TE) cells produce IL 1. By using a system for longterm culture of human TE cells, we found that human TE cells produced an IL 1-like factor (TE-IL 1) that augmented the proliferation of C3H/HeJ mouse thymocytes to phytohemagglutinin. IL 1 activity (20 to 200 U/ml) was detected in supernatants of TE cultures from all individuals (2 to 13 yr old) tested. IL 1 activity was also detected in supernatants of TE cultures from a 17-wk fetus but not from a 10-wk fetus. Production of TE-IL 1 was dependent on TE cell density and time in culture with optimal TE-IL 1 activity observed at 10(6) TE cells/ml after 48 to 72 hr of culture. With the use of high performance liquid chromatography, TE-IL 1 chromatographed as a molecule of 18,000 to 20,000 relative molecular mass, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, TE-IL 1 migrated at 15,000 to 17,000 Mr. With the use of isoelectrofocusing gels, charge heterogeneity of TE-IL 1 was demonstrated with two major isoelectric points of 5.7 to 5.8 and 6.9 to 7.0. Polyclonal antibody to human monocyte IL 1 markedly inhibited the TE-IL 1 activity. In indirect immunofluorescence assay of frozen human thymic sections, rabbit anti-IL 1 antibody reacted with epithelial cells in human thymic cortex and medulla. Furthermore, high performance liquid chromatography-purified TE-IL 1 augmented human thymocyte proliferation to suboptimal concentrations of phytohemagglutinin. Thus, thymic epithelial cells are capable of providing an intrathymic source of IL 1-like cytokine (TE-IL 1), which affects thymocyte proliferation. We propose that TE-IL 1 may play an important role in intrathymic proliferation and differentiation of human thymocytes.

Adolescent↗
PubMed1987

Human thymic epithelial cells function as accessory cells for autologous mature thymocyte activation.

Using human thymocytes and autologous thymic epithelial (TE) cells grown in vitro in long-term culture, we have found TE cells can function as accessory cells for mitogen-induced mature thymocyte activation. Tritiated thymidine incorporation, blast formation, and protein synthesis were all induced in accessory cell-depleted thymocytes by autologous TE cells in the presence of suboptimal concentrations of PHA. After 3 days of mitogen stimulation of thymocyte-TE cell cocultures in vitro, thymocyte blasts bound to TE cells and 77 +/- 4% (mean +/- SEM) of TE cells acquired expression of major histocompatibility complex (MHC) class II (DR) antigen. TE accessory cell function for thymocyte activation was dependent on the number of TE cells added to thymocyte cultures, was not dependent on TE cell division, but did require TE cell protein synthesis. In thymocyte separation experiments, the predominant cell type responding to PHA in the presence of TE cells was T6- mature (stage III) thymocytes. Thus, human TE cells are capable of providing signals that lead to mature thymocyte activation.

Antigen-Presenting Cells↗
PubMed1987

Thymocyte binding to human thymic epithelial cells is inhibited by monoclonal antibodies to CD-2 and LFA-3 antigens.

With the use of cultured human thymic epithelial (TE) cells, we have previously shown that thymocytes bind to TE cells in suspension in a rosette-forming assay. To identify cell surface molecules involved in human TE-thymocyte rosette formation, we assayed a large panel of monoclonal antibodies for their ability to inhibit rosette formation. We found anti-CD-2 (LFA-2, T11), and anti-LFA-3 antibodies all inhibited binding of TE cells to thymocytes. By using indirect immunofluorescence assays, we determined that cultured TE cells were 90% LFA-3 positive and CD-2 negative, whereas thymocytes were 10% LFA-3 positive and 98% CD-2 positive. Pretreatment of TE cells with anti-LFA-3 but not anti-LFA-2 inhibited TE-thymocyte binding. In contrast, pretreatment of thymocytes with anti-CD-2 but not anti-LFA-3 antibodies inhibited TE-thymocyte binding. Thus TE cell-thymocyte binding is blocked by antibodies to the CD-2 (T11) antigen on thymocytes and by an antibody to the LFA-3 antigen on TE cells. Because the CD-2 antigen has been implicated in T cell activation, these data suggest that a natural ligand for T cell activation via the CD-2 molecule is present on human thymic epithelial cells.

Antibodies, Monoclonal↗
PubMed1987

Polyspecific reactivity of a murine monoclonal antibody that binds to nuclear matrix-associated, chromatin-bound autoantigens.

To investigate polyspecific autoantibody interactions, we have characterized the binding of a cloned murine monoclonal IgM antibody termed (RTE-23) of strain BALB/c origin. By indirect immunofluorescence this antibody displayed a nuclear speckled and peripheral pattern in interphase cells from human and rodent cell lines. In contrast, in mitotic cells, antibody RTE-23 bound to the periphery of individual chromosomes. Immunoblot analysis of soluble and insoluble nuclear proteins from purified rat fibroblast nuclei showed that antibody RTE-23 bound to molecules of 28, 29, and 33 kDa. Furthermore, antibody RTE-23 demonstrated marked polyspecificity and reacted with cytoskeletal proteins (vimentin, keratin, actin), single-stranded DNA, specific synthetic polynucleotides, and cardiolipin. Antibody RTE-23 also showed a lupus anticoagulant-like activity. Screening of sera of autoimmune disease patients with antinuclear antibodies revealed two patients, both with SLE, whose sera blocked antibody RTE-23 binding to nuclei and recognized nuclear proteins identical to those recognized by antibody RTE-23. These results suggested that antibody RTE-23 displays a pattern of self-antigen binding that is represented as well in SLE patient sera.

Antibodies, Monoclonal↗
PubMed1987

Epithelial-thymocyte interactions in human thymus.

Our data demonstrate that the epithelial component of the human thymic microenvironment is not an inert cell type, but rather is capable of being directly involved in the promotion of both early and late stages of T-cell maturation. Data from our laboratory [54,69], together with the work of Plunkett et al. [61] and Shaw et al. [70] suggest that an endogenous ligand for the CD2 molecule in humans is the LFA-3 molecule. Using an SV40 transformed human thymic epithelial cell line of subcapsular cortical origin, Mizutani et al. have confirmed that thymic epithelial cells bind thymocytes via a CD2/LFA-3 interaction [78]. The data reviewed in this paper suggest that within the thymus one endogenous ligand for the alternative pathway of thymocyte activation via the CD2 molecule is the LFA-3 molecule on TE cells. Following thymocyte binding to TE cells, immature thymocytes are directly activated to proliferate, and their response to both IL1 and IL2 is augmented. Also, following TE-thymocyte binding, TE-IL1 secretion is augmented and TE cell MHC class II antigen expression is induced. Moreover, while undergoing activation, thymocytes appear to be able to modulate their microenvironment milieu of MHC antigens and IL1. Further analysis of the sequelae of TE-thymocyte interactions using phenotypic characterization of thymocytes with anti-T-cell MoAbs, coupled with molecular analysis of thymocyte T-cell receptor genes, should allow for the determination of the precise sequential stages that immature T cells undergo enroute to functional maturity. Understanding these steps in T-cell maturation will be critical to our understanding of the events that transpire in the genesis of autoimmune, lymphoproliferative, and immunodeficiency diseases.

Antibodies, Monoclonal↗
PubMed1987

Human erythrocyte antigens. III. Characterization of a panel of murine monoclonal antibodies that react with human erythrocyte and erythroid precursor membranes.

Human erythrocyte membrane proteins express antigens which serve as markers for erythroid differentiation as well as targets for human blood group alloantibodies. We have produced and characterized a new panel of five monoclonal antibodies to erythrocyte membrane proteins. Three monoclonal antibodies (E3, E4, E5) were specific for erythrocyte glycophorins. One antibody (E3) identified the sialoglycoprotein alpha and beta homologous regions proximal to the plasma membrane, a second antibody (E4) was specific for sialoglycoprotein alpha, while a third (E5) was a sialoglycoprotein-beta-specific antibody. Two antibodies (E6 and TE10) to the 65,000-dalton chymotrypsin cleavage product of band 3 were also produced. These antibodies constitute a new panel of probes for investigation of normal erythroid differentiation, erythroleukemia, and the expression of normal and anomalous blood group antigens.

Animals↗
PubMed1987

The thymic microenvironment. Characterization of in vitro differentiation of the IT26R21 rat thymic epithelial cell line.

We have previously postulated an in vivo pathway of thymic epithelial (TE) cell maturation in pre- and postnatal thymus, whereby endocrine medullary TE cells terminally differentiate to form Hassall's bodies. Epithelial-cell differentiation has been well documented in vitro using epidermal keratinocytes. Therefore, to characterize TE-cell differentiation in vitro, we observed clones of the rat TE cell line, IT26R21, after 4 and 14 days in culture. We found alterations in cell morphology, the cessation of cell proliferation, and the acquisition of a differentiation antigen defined by monoclonal antibody TE-19 (a marker of terminally differentiated epithelial cells). At light and electron microscopy, we detected progressive TE-cell stratification and squamous-cell formation between 4 and 14 days of culture. Autoradiography on day 14 showed that squamous TE cells in stratified layers did not incorporate tritiated thymidine, while surrounding smaller cells adhering to the substratum continued to synthesize DNA. At indirect immunofluorescence, only 3% of cells reacted with monoclonal antibody TE-19 at day 4, while on day 14, 22% of the TE cells were TE-19 positive (P less than 0.02). Antibody-TE-19 reactivity was limited to stratified, squamous TE cells. Additionally, we isolated a clone of the IT26R21 cell line that did not undergo these changes characteristic of TE cell differentiation. We conclude that IT26R21 TE cells are capable of undergoing programs of both terminal differentiation and cell renewal in vitro.

Animals↗
PubMed1987

Monoclonal antibodies to a human islet cell surface glycoprotein: 4F2 and LC7-2.

Monoclonal antibodies 4F2 and LC7-2 react with a cell surface differentiation antigen expressed by the endocrine cells of the human pancreatic islet, but not by the acinar pancreatic, ductular, vascular, or stromal connective tissue cells. Western immunoblotting procedures demonstrate the reactivity of the monoclonal antibody 4F2 with a 120 kilodalton islet cell protein in detergent-solubilized cell extracts. These two monoclonal antibodies have potential for application in many aspects of islet cell research and diabetes in general.

Antibodies, Monoclonal↗
PubMed1986

C-terminal region of human T cell lymphotropic virus type I (HTLVI) p19 core protein is immunogenic in humans and contains an HTLVI-specific epitope.

To study the human host response to viral structural proteins during HTLV type I infection, five synthetic peptides matching the N-terminal and C-terminal regions of HTLVI p19 core protein were used to identify antigenic sites on p19 that were immunogenic in man. In radioimmunoassay and immunoprecipitation experiments, antibodies in 16 of 18 HTLVI+ patient sera reacted with a synthetic peptide matching the C-terminal 11-amino acid sequence of p19, whereas only two sera contained antibodies that reacted with other N- or C-terminal region p19 synthetic peptides. Polyclonal rabbit antisera to N- and C-terminal peptides reacted with a native viral protein of 19,000 daltons and with gag-encoded precursors of p19. Six monoclonal antibodies against native viral p19 were screened for reactivity to the five synthetic peptides. One of six antibodies (13B12) reacted with the C-terminal synthetic peptide of p19. Antibody 13B12 did not react with HTLVII or HTLVIII proteins or with HTLVIII-infected cells, nor did it cross-react with a wide variety of HTLV-uninfected normal host tissues. Thus, the C-terminus of p19 contains an antigen that is highly immunogenic in most HTLVI-infected patients and is HTLVI specific.

Antibodies, Monoclonal↗
PubMed1986

Characterization of a monoclonal antibody, RTE-21, that binds to keratohyalin granule-associated proteins in epithelial cells of human skin and thymus.

The role of skin and thymic epithelium in the promotion of T-cell activation and maturation is currently an area of great interest. In thymus, epithelium is located in the cortex, medulla, and in medullary epithelial swirls called Hassall's bodies. During studies of antigens shared by skin and thymic epithelial cells, we produced a murine monoclonal antibody (RTE-21) raised against the rat thymic epithelial cell line, IT26R21, that identified an antigen present in terminally differentiated epithelium of normal human skin and thymus. In indirect immunofluorescence studies, antibody RTE-21 identified cytoplasmic granules located in the stratum granulosum of normal human skin, Hassall's bodies of human thymus, and in a subset of cells of the IT26R21 rat thymic epithelial cell line. Moreover, the granular reactivity pattern of antibody RTE-21 in the stratum granulosum could be extracted by 1 M potassium phosphate (1 M KPO4). A 1 M KPO4 extract of epidermis containing keratohyalin granule proteins was dialyzed against distilled water, solubilized in reduced sample buffer and subjected to polyacrylamide gel electrophoresis and Western immunoblot analysis. In this assay, antibody RTE-21 recognized proteins of 70, 36, 34, and 30 kDa. Using antibody RTE-21 and indirect immunofluorescence, we demonstrated that keratohyalin granules in human thymus were localized exclusively to Hassall's bodies. These data support the notion that human thymic Hassall's bodies result from terminal differentiation of medullary thymic epithelium. Thus, antibody RTE-21 should be an important probe for the study of skin and thymic epithelial cell maturation in vitro and in vivo.

Antibodies, Monoclonal↗
PubMed1986

Human medullary thymocyte p80 antigen and In(Lu)-related p80 antigen reside on the same protein.

Study of human T lymphocyte differentiation antigens with monoclonal antibodies has led to the identification of two antigens shared by erythrocytes and leukocytes. The protein (p80) defined by A1G3 antibody has previously been shown to be acquired during human intrathymic T-cell maturation. The antigen defined by A3D8 antibody has been demonstrated also to reside on an 80 kd protein; expression of the A3D8 antigen on erythrocytes and a subset of leukocytes is regulated by the rare In(Lu) gene. In this study, we demonstrate that the antigens defined by the A1G3 and A3D8 antibodies reside on the same protein and represent closely related but nonidentical epitopes on the p80 molecule. Expression of the A1G3 antigen on erythrocytes and a subset of leukocytes is also down-regulated by the In(Lu) gene. The possible role of the In(Lu) gene in thymocyte differentiation is discussed.

Antibodies, Monoclonal↗
PubMed1986

Diagnostic and therapeutic approach to the patient with vasculitis.

This article presents an approach to the diagnosis and therapy of patients with vasculitis. Effective treatment of patients with vasculitis requires a systemic approach to diagnosis and classification of disease, evaluation of the extent of organ system involvement, and an exclusion of underlying treatable diseases.

Adrenal Cortex Hormones↗
PubMed1986

Human thymocytes bind to autologous and allogeneic thymic epithelial cells in vitro.

The thymus plays a critical role in the generation of immunocompetent T lymphocytes. In the thymus, lymphocytes are in close contact with epithelial cells, and this contact is necessary for T-cell maturation. Using cultured human thymic epithelial (TE) cells, we have found that human thymocytes bind to human TE cells in vitro. Thymocytes bound to both allogeneic and autologous TE cells and to the epidermoid carcinoma cell line A431 but did not bind to epidermal keratinocytes or to thymic fibroblasts. Thymocyte binding to TE cells was trypsin- and cytochalasin B-sensitive. Indirect immunofluorescence assays showed that both mature (T6-, T3+) and immature (T6+, T3-) thymocytes bound TE cells. In our system, TE-thymocyte binding was not inhibited by antibodies to class I or class II major histocompatibility antigens. In vitro binding of thymocytes to TE cells may represent a correlate of in vivo TE-thymocyte interactions and provides a model system for the study of human intrathymic T-lymphocyte maturation and activation.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1986

Immunoglobulin and T-cell receptor gene rearrangement and expression in human lymphoid leukemia cells at different stages of maturation.

The use of probes to genes (IG and TCRB) encoding immunoglobulins (IG) and the beta chain of the T-cell antigen receptor (TCRB), respectively, have become a sensitive means to assess clonality and lineage in lymphoid malignancies. It has become apparent that some individual cases show rearrangements of both IG and TCRB genes. In an attempt to more accurately define cell lineage we have analyzed cells from patients with B- or T-cell leukemia (n = 26) at various stages of maturation with probes to two additional TCR genes, TCRG and TCRA (encoding the TCR gamma and alpha chains, respectively), as well as the IG heavy chain joining region (IGHJ) and TCRB genes. On Southern blot analysis, the mature T-cell leukemia cells studied had rearranged TCRG and TCRB while IGHJ remained as in the germ line. The mature B-cell leukemia cells studied had rearranged IGHJ with germ-line TCRG and TCRB. These data suggest that, in the majority of more mature leukemias, cells have rearranged IG or TCR genes but not both. In contrast, cells from five of nine precursor B-cell leukemia patients and cell lines from one of four precursor T-cell leukemia patients had rearranged both IGHJ and TCR genes. TCRG and TCRB mRNAs were expressed in the cells of precursor T- but not B-cell leukemia patients studied. The spectrum of leukemia cells studied within the T-cell series permitted an assessment of the order of TCR gene rearrangements. Two of 13 patients had cells with germ-line TCRG and TCRB, 2 patients had cells with rearranged TCRG alone, and the remainder had cells with rearranged TCRG and TCRB. TCRG and TCRB mRNAs were expressed in precursor T-cell leukemia cells, whereas TCRB and TCRA were expressed in mature T-cell leukemia cells. These results parallel observations from mouse studies on gene expression and support the view of a hierarchy of TCR gene rearrangements in T-lymphocyte ontogeny. TCRG genes are rearranged first, subsequently TCRB genes are rearranged, followed by TCRA gene activation.

Cell Differentiation↗
PubMed1986

The human thymic microenvironment: thymic epithelium contains specific keratins associated with early and late stages of epidermal keratinocyte maturation.

Normal T-cell development is dependent on interactions with the thymic microenvironment; thymic epithelial cells are thought to play a key role in the induction of thymocyte maturation, both through direct contact and, indirectly, via thymic hormone secretion. It has been postulated that thymic epithelial cells progress through an antigenically defined pathway of differentiation similar to that of epidermal keratinocytes. As keratins vary according to epithelial cell type and the stage of epithelial cell maturation, we used a panel of monoclonal antibodies against keratins to study specific types of keratin intermediate filaments within human thymic epithelium. The demonstration in human thymus of keratins previously shown to be associated with distinct stages of epidermal keratinocytic maturation would support the hypothesis that thymic epithelial cells undergo sequential stages of differentiation. Two-dimensional immunoblot analysis of cytoskeletal extracts from human thymus revealed that thymic epithelium contains the following keratins: 1-2, 5, 6, 7, 8, 10, 13, 14, 15, 16, and 17 (molecular masses, 65-67, 58, 56, 54, 52, 56.5, 51, 50, 50', 48, and 46 kilodaltons, respectively). Thus, in thymic epithelium, we found keratins previously observed in epidermal basal cells (5, 14, 15), as well as keratins specific for terminally differentiated keratinocytes in supra-basal epidermis (1-2, 10). Indirect immunofluorescence (IF) performed on fetal and postnatal human thymus demonstrated that keratin epitopes recognized by antibodies AE-3, 35 beta H11, and RTE-23 are present on epithelial cells of the subcapsular cortex, the cortex, the medulla, and Hassall's bodies. In contrast, antibodies AE-1 and RTE-22 reacted primarily with neuroendocrine thymic epithelium (subcapsular cortex, medulla, Hassall's bodies). The epithelial reactivity of antibody AE-2 was limited to epithelial cells in Hassall's bodies and did not appear until 16 weeks of fetal gestation i.e., when Hassall's bodies first formed. Two-dimensional gel analysis of thymic keratins demonstrated that antibody AE-2 identified only the keratins with molecular masses of 56.6 and 65-67 kilodaltons (10 and 1-2 respectively) in thymus. These data, together with the selective reactivity of AE-2 with Hassall's bodies in fluorescence assays, demonstrate the localization in Hassall's bodies of the high-molecular-weight keratins associated with the late stages of epidermal cell maturation. In summary, we demonstrated that human thymic epithelium contains specific keratins found in multiple epithelial types as well as keratins associated with both early and late stages of epidermal cell differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies, Monoclonal↗