Significance of bursa of Fabricius as target organ in infectious bursal disease of chickens.

The highly virulent strain Cu-1 of infectious bursal disease virus caused 100% mortality in 4-week-old specific pathogen-free chickens. In contrast, chickens infected after bursectomy did not become sick and only showed some discrete and transient necrosis in lymphatic tissues. However, these chickens contained infectious virus and, subsequently, produced specific antibodies. The virus concentrations in the organs studied reached their maximum 2 days postinfection, but were about 1,000 times lower in non-bursectomized animals. It may be assumed that in bursectomized chickens the early events of infection are the same as in non-bursectomized ones. Virus is spread in varius organs, but due to the absence of a sufficient number of susceptible cells, virus multiplication is moderate and can be kept in check by the host defense mechanism. With the occurrence of circulating specific antibodies the virus can be rapidly eliminated. The studies particularly stress that the availability of a large number of highly susceptible cells is a crucial point in acute viral infections.     

The characterization of two monoclonal anti-keratin antibodies and their use in the study of epithelial disorders

The present paper describes the production and characterization of two monoclonal antibodies, K20 and K92. Immunohistological staining showed these two antibodies to be specific for keratinizing epithelium. However, whereas K20 stained all layers of the epidermis K92 reacted with only the suprabasal epidermal layers. Immunoblotting studies with preparations of keratins from both the non-cornified (i.e. the basal, spinous and granular layers) and cornified (stratum corneum) layers of epidermis showed that K20 recognized the 46, 48, 50, 55, 56, 56.5, 59, 61, 62, 64, 65, 66 and 67 kd bands, of which the 50 and 46 kd bands appeared to be masked in tissue sections. In contrast, antibody K92 was more restricted in its activity, recognizing only the 55 and 56 kd bands strongly. These antibodies were used in the study of various epithelial disorders and revealed alterations in the epithelial intermediate filament expression in both benign and malignant disease processes.     

Anti-vimentin monoclonal antibody recognizes a cell with dendritic appearance in the chicken's bursa of Fabricius.

The bursa of Fabricius was studied by immunohistochemical method using anti-vimentin monoclonal antibody (clone 3B4). This monoclonal antibody identified a vimentin positive cell in the medulla of the bursal follicle. During the first 2 weeks of life the vimentin positive cells located along the corticomedullary border and later became prominent in the medulla with the exception of a narrow zone adjacent to the corticomedullary border. After hatching the accumulation of vimentin-type intermediate filaments on one side of the nucleus endowed the vimentin positive cells with a polarized appearance. This "cap-like" vimentin positive area of the cytoplasm determined the position of the major cell process. Within the medulla the Ia positive secretory dendritic cells contained secretory granules in one of the cell processes. The distribution, shape, and polarized appearance of the vimentin positive cells were identical with that of the secretory dendritic cells. Therefore, the anti-vimentin monoclonal antibody proved to be useful for identification of the bursal secretory dendritic cells. During rapid bursal growth the number of secretory dendritic cells increased, possibly, by proliferation of vimentin negative secretory dendritic cell precursors located along the corticomedullary border.     

Fractionation of human bone-marrow mononuclear cells with peanut agglutinin: phenotypic characterization with monoclonal antibodies

Bone marrow mononuclear cells were fractionated by affinity chromatography on immobilized peanut agglutinin (PNA). The resulting PNA+ fraction represented 10% of the total cell number. Twenty percent of the cells within the PNA+ compartment coexpressed the T6, Ia, Mo1, and My4 differentiation antigens and possessed Fc and C3 receptors. The similarity in cell surface antigen phenotype led us to hypothesize that this subset may be a cellular precursor of dendritic cells found in the skin (Langerhans cells) or in the parenchimal organs of the body (D-cells).     

Effect of bursa Fabricius extracts on antibody production in bursectomized or bursal cell autografted chickens.

Restoration and enhancement of immune response against BSA antigen was achieved by 5-day consecutive doses of BF estract from 4-5-week-old chickens, in birds which had been surgically bursectomized or given BF-cell autografts at 17 days of age. A similar 5-day treatment with other tissue extract, i.e. liver, spleen, pancreas or intestine, or with LPS of E. coli, in contrast, failed to provide such restoration or enhancement.     

The human thymus microenvironment: heterogeneity detected by monoclonal anti-epithelial cell antibodies.

Monoclonal antibodies were raised against human thymus stromal cells and their specificity for the epithelial component of thymus stroma assessed by double immunofluorescence using anti-keratin antibodies to identify epithelium. Our monoclonal antibodies identify six distinct patterns of epithelial cell antigen expression within the thymus: pan epithelial (antibody IP1); cortex (MR3 and MR6); cortical/medullary junction (IP2); subcapsule and subpopulation of medulla (MR10/MR14); Hassall's corpuscles and adjacent subpopulation of medulla (IP3); Hassall's corpuscles only (MR13/IP4). This heterogeneity of antigen expression suggests that many different epithelial microenvironments exist within the human thymus.     

The bursa of Fabricius: a trapping site for environmental antigens.

The entry of environmental antigens into the lumen of the bursa of Fabricius was prevented by ligating the bursal duct prior to hatching (BDL: bursal duct ligation). The development of 'natural' serum agglutinins for bacteria and heteroerythrocytes was markedly suppressed by BDL. The antigen-specific recovery was observed by simultaneous administration of sterile antigens into the bursal lumen at the time of BDL. These results strongly suggest that the bursa of Fabricius is a major channel through which environmental antigens stimulate the immune system and induce the formation of 'natural' serum agglutinins.     

Production and characterization of monoclonal antibodies to the protective antigen component of Bacillus anthracis toxin.

Thirty-six monoclonal antibodies to the protective antigen protein of Bacillus anthracis exotoxin have been characterized for affinity, antibody subtype, competitive binding to antigenic regions, and ability to neutralize lethal and edema toxin activities. At least 23 antigenic regions were detected on protective antigen by a blocking, enzyme-linked immunosorbent assay. Two clones, 3B6 and 14B7, competed for a single antigenic region and neutralized the activity of both the lethal toxin in vivo (Fisher 344 rat) and the edema toxin in vitro (CHO cells). These two antibodies blocked the binding of 125I-labeled protective antigen to FRL-103 cells. Our results support the proposal that binding of protective antigen to cell receptors is required for expression of toxicity.     

Follicle-associated epithelium and medullary epithelial tissue of the bursa of Fabricius are two different compartments

The bursae of Fabricius from the chicken and turkey were studied by light and electron microscopy and immunohistochemical methods. The study focused on the relationship of follicle-associated epithelium to the medulla. The follicle-associated epithelium was supported by 3 to 5 layers of stratified epithelial cells which were a continuation of the corticomedullary epithelial cells. The follicle-associated epithelium consisted of M cells and scattered secretory dendritic cells. The network of the reticular epithelial cells of the medulla was filled with secretory dendritic cells, B cells, and a few T cells and macrophages. The cellular content of the follicle-associated epithelium and the medulla suggested that they were different cellular compartments. Communication between the follicle associated epithelium and medullary epithelial compartment occurred through the supporting cells of the follicle-associated epithelium. When the supporting layers of the follicle-associated epithelium infolded into the medulla, they formed lamellated epithelial bodies similar to the thymic Hassall bodies. The lamellated bodies enclosed secretory dendritic cells but not lymphocytes. The infolding of supporting cells varied from follicle to follicle. The asynchronization of infolding contributed to heterogeneity of follicle composition. Follicle heterogeneity was demonstrated by differences in reactivity with a battery of monoclonal antibodies. © 1992 Wiley-Liss, Inc.     

Production and characterization of monoclonal antibodies against the lethal factor component of Bacillus anthracis lethal toxin.

The lethal toxin of Bacillus anthracis consists of two components, protective antigen and lethal factor. Protective antigen is cleaved after binding to cell receptors, yielding a receptor-bound fragment that binds lethal factor. Sixty-one monoclonal antibodies to the lethal factor protein have been characterized for specificity, antibody subtype, and ability to neutralize lethal toxin. Three monoclonal antibodies (10G3, 2E7, and 3F6) neutralized lethal toxin in Fisher 344 rats. However, in a macrophage cytolysis assay, monoclonal antibodies 10G3, 2E7, 10G4, 10D4, 13D10, and 1D8, but not 3F6, were found to neutralize lethal toxin. Binding studies showed that five of the monoclonal antibodies that neutralized lethal toxin in the macrophage assay (10G3, 2E7, 10G4, 10D4, and 13D10) did so by inhibiting the binding of lethal factor to the protective antigen fragment bound to cells. Monoclonal antibody 1D8, which was also able to neutralize lethal toxin activity after lethal factor was prebound to cell-bound protective antigen, only partially inhibited binding of lethal factor to protective antigen. Monoclonal antibody 3F6 did not inhibit the binding of lethal factor to protective antigen. A competitive-binding enzyme-linked immunosorbent assay showed that at least four different antigenic regions on lethal factor were recognized by these seven neutralizing hybridomas. The anomalous behavior of 3F6 suggests that it may induce a conformational change in lethal factor. Differences in neutralizing activity of monoclonal antibodies were related to their relative affinity and epitope specificity and the type of assay.     

Development, characterization and use of monoclonal antibodies against sTRAIL: measurement of sTRAIL by ELISA.

Two monoclonal antibodies against tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL), designated VI10E and III6F, have been generated. These antibodies were useful in flow cytometry analysis, immunohistochemistry, immunoprecipitation and in the development of an immunoassay for the detection of soluble TRAIL (sTRAIL)in biological samples. The immunoassay was based on two monoclonal antibodies against TRAIL. VI10E was used as the capture antibody and bound TRAIL was detected with anti-TRAIL from R&D Systems which was digoxigenin (DIG)-labeled. This enzyme-linked immunosorbent assay (ELISA) was specific for TRAIL since a panel of other cytokines did not affect the signal. The immunoassay was suitable for the detection of sTRAIL in human serum and plasma samples, cell culture supernatants and cell lysates. In a preliminary screening, it was found that serum samples from human immunodeficiency virus (HIV)-infected patients contained sTRAIL, and all these positive samples were found in the AIDS group. Using the immunoassay, it was found that phytohaemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) to produce significant amounts of sTRAIL, the levels of which increased with exposure time. Thus, the immunoassay for TRAIL presented here represents a useful tool for measuring sTRAIL in various biological samples. It will also permit studies of release mechanisms as well as possible functions of the soluble form of this molecule.     

Epitope mapping of horseradish peroxidase with use of monoclonal antibodies.

Nine mouse monoclonal antibodies (MAbs) to horseradish peroxidase (HRP) were used to develop an epitope map of the enzyme. The results of a competitive binding assay indicated three distinct patterns of reactivity. Two groups of MAbs (I and III) recognized epitopes located in separate antigenic regions on the molecule; another (II) bound to sites that overlapped with epitopes in either region I or III. Further definition of these regions was obtained by analyzing the MAbs for their binding to isolated heme, other peroxidases and heme-containing proteins, and to denatured and apo-HRP. None of the group I MAbs bound heme, suggesting that this region was removed from the active site of the enzyme. All of the group II and III MAbs bound heme as well as the other peroxidases and heme-containing proteins, indicating that they recognized heme-associated epitopes at or near the active site. Only one MAb (2A2) in groups II and III bound to apo-HRP but not to denatured HRP; it was also the only MAb in the entire panel that inhibited the catalytic activity of HRP. This suggests that the epitope recognized by 2A2 involves both the heme moiety and a conformationally dependent protein determinant near the active site.     

Influence of the bursa of Fabricius on the pathogenesis of Marek''s disease.

A series of experiments was conducted to study the influence of embryonal bursectomy (EBX) on the early and late pathogenesis of Marek's disease (MD). The early lytic infection in the lymphoid organs normally associated with oncogenic MD virus infection in intact chickens was not seen in EBX chickens. Therefore, the damage to the immune system was minimal. EBX chickens also had lower viremia levels, higher lymphocyte responses to mitogens, and a lower or delayed MD mortality when compared with intact chickens. Furthermore, it was shown that although vaccination with SB-1 by itself did not protect against a highly virulent MD transplantable tumor, the combination of EBX and vaccination gave significant protection. All these effects could be explained by an enhanced immune response in EBX birds. In contrast, the pathogenesis of nononcogenic MD virus was not influenced by EBX. The possible mechanism(s) involved in these observed effects of EBX on MD pathogenesis are discussed.     

Nurse cells of the bursa of Fabricius: Do they exist?

Cell-cell interactions in B lymphocyte development have so far been incompletely characterized, mostly due to lack of a special organ for B cell maturation in the mammalian species. Certain well-known lymphostromal interactions in the thymus have raised the question whether similar interactions with nurse cells would also operate in the development of B cells. We have tested this hypothesis in the chicken bursa of Fabricius, an organ specific for the B cell maturation. To identify possible nurse cells, with viable lymphocytes enclosed, the cells in the bursa of Fabricius were dispersed with collagenase and trypsin. Light and electron microscopic examination of bursa cell suspensions showed four types of aggregates, identified by low magnification light microscopy as potential nurse cell-like complexes. Electron microscopy revealed that all aggregates consisted of epithelial cells, and complexes of epithelial cells with lymphocytes enclosed were not observed. These findings indicate that interactions similar to those seen in the avian and mammalian thymus between epithelial nurse cells and T lymphocytes are not a part of the avian B cell differentiation process.     

Plasmodium falciparum: characterization of defined antigens by monoclonal antibodies.

Monoclonal antibodies directed against Plasmodium falciparum detect stage-specific, species-specific and common antigenic determinants of Plasmodia. These antibodies provide new tools for purification and characterization of Plasmodium falciparum antigens in relation to future procedures for immunoprophylaxis.     

Production,characterization and use of monoclonal antibodies to grapevine virus A

Summary Four stable hybridoma cell lines secreting monoclonal antibodies to grapevine closterovirus A (GVA) were obtained by fusing spleen cells of immunized BALB/c mice with mouse myeloma cell line Sp 2/0-Ag 14. In ELISA all MAbs reacted with virus in leaf extracts fromNicotiana benthamiana, glass-house-, field-, or in vitro-grown grapevines, or with cortical shavings from mature grape canes. In IEM tests, only one of the MAbs (PA 3.F 5) decorated virus particles on the entire surface. This MAb was likely induced by a surface antigenic determinant, whereas the other three MAbs (PA 3.D 11, PA 3.C 6, and PA 3.B 9) were originated by cryptotopes. Coupling polyclonal antibodies for coating protein A-sensitized plates, and monoclonal antibody conjugates for antigen detection, gave highly efficient and reproducible results for identification of GVA in field-grown grapevines.     

Cell surface glycoproteins of rabbit lymphocytes: characterization with monoclonal antibodies

The structural characteristics of antigens recognized by a panel of monoclonal antibodies prepared against a rabbit T-lymphocyte cell line have been investigated. Those antigens which could be isolated using immunoadsorbents prepared from the monoclonal antibodies had mol. wts of 42,000, 90,000 and 120,000. The 42,000 mol. wt molecule is similar or identical to a rabbit class I major histocompatibility complex antigen and its characterization has been reported elsewhere. Three different 90,000 mol. wt proteins can be distinguished by their reactivity with lectins and by sequential immunoprecipitation. The 120,000 mol. wt protein is a very abundant surface glycoprotein that appears to be a specific marker for T-cells in the rabbit. It is the immunodominant antigen in a lentil lectin bound glycoprotein pool. Over half of the antibodies were directed against this antigen. All antigens detected by the panel of monoclonal antibodies have been detected on normal lymphoid cells.     

Histiocytosis-X: in situ characterization of cutaneous infiltrates with monoclonal antibodies

Cutaneous lesions in three cases of histiocytosis-X were studied by light microscopy, electron microscopy, and immunoperoxidase technics. In each case, Birbeck granule-containing histiocytosis-X cells infiltrated the epidermis and were apposed to lymphocytes. The histiocytosis-X cells and normal Langerhans cells stained with anti-T6 and anti-I1 (Ia-like) antibodies but were negative with anti-T3, anti-T8, anti-M1, and anti-lysozyme antibodies. In addition, the histiocytosis-X cells also stained with anti-T4 antibodies, which react with T-cells associated with helper/inducer phenotype. This study supports the hypothesis that histiocytosis-X cells are abnormal Langerhans cells. The presence of T4/T6-positive cells in cutaneous disease may be a marker for abnormal Langerhans cells.