In situ detection of basic fibroblast growth factor by highly specific antibodies.

Basic fibroblast growth factor (bFGF) is thought to be of major importance for fibrosis and angiogenesis. Despite intensive studies dealing with the biochemistry and multiple biologic effects of bFGF, the cellular distribution is virtually unknown. Therefore, using the indirect immunoperoxidase technique, we examined the effect of bFGF on a large pattern of normal, inflammatory, and tumorous human tissues. Staining was performed on cryostat sections with a highly specific affinity-purified antiserum. In normal tissues, especially those of the thymus and placenta, mainly dendritic cells contained the growth factor. High levels of bFGF were also detected in basal cells and gland epithelial cells of skin biopsies. A conspicuous expression was observed in chronic inflammatory tissues corresponding to a generally pronounced proliferation of fibroblasts and endothelial cells in these situations. Tumors revealed a very heterogenous staining pattern. In some lesions, bFGF was predominantly present in infiltrating and endothelial cells. In several, neoplasms tumor cells exhibited an intensive staining. In some, especially vascular tumors, bFGF could not be detected. From the staining results it is concluded that angiogenesis is not simply controlled by the presence of bFGF but is mediated by a balance of several angiogenic inducers and inhibitors.

Ovine haemopoiesis: the development of bone marrow-derived colony-forming cells in vitro in the presence of factors derived from lymphoid cells and helper T-cells.

Techniques for the development of ovine bone marrow-derived haemopoietic progenitor cells and in situ identification of colony morphology are described. Both mitogen stimulated lymphoid cells and antigen stimulated helper T-cells generated potent colony-stimulating activity in conditioned medium. Monocyte/macrophage, neutrophil, eosinophil, basophil/mast cell, neutrophil/monocyte and mixed phenotype colonies developed in stimulated bone marrow cultures in a conditioned medium dose-dependent manner. Neutrophil, monocyte/macrophage and eosinophil colonies were detected in greater numbers than the other types, with mixed colonies representing only around 1% of the total. Eosinophil colonies were particularly abundant when compared to published reports of the numbers obtained with similar cultures of 'normal' mouse or human bone marrow cells. This culture technique will allow a detailed analysis of both ovine colony-stimulating factors and of the distribution of haemopoietic progenitor cells in vivo.

Localization of immunoglobulins in the chicken oviduct.

Studies were made on the distribution of lymphoid tissues and immunoglobulin (Ig: IgA, IgG and IgM)-containing cells (cIg: cIgA, cIgG and cIgM) and the localization of immunoglobulins (Igs) in the oviducal walls of laying hens. Lymphocyte accumulations were occasionally observed, located mainly in the middle infundibulum and in the regions from the isthmus to the vagina. The number of cIgG significantly predominated over that of cIgA or cIgM in the mucosal connective tissue of the magnum and the isthmus. In contrast, in the regions other than the magnum and the isthmus, these three types of cIg were fewer in number. Igs were localized in some superficial epithelial cells (SECs) and glandular cells (GlCs) of the oviduct. Many IgG-containing SECs were found in the infundibulum, the isthmus, and the cranial and major uterus. IgA- or IgM-containing SECs were rare throughout the oviduct. Three types of Ig-containing GlCs were numerously found in the magnum, though lymphocyte accumulations were scarce there. In the isthmus, many IgG-containing GlCs were found, while IgA- or IgM-containing GlCs were rarely observed. Ig-containing GlCs in the magnum were considerably decreased in number after the egg passage. The results suggest that the maternal Igs are transferred to the egg mainly through GlCs in the magnum of the chicken oviduct, and that the oviducal lymphoid tissues have little relationship to the passive immunity.

Reserpine-induced decrease in type I and II corticosteroid receptors in neuronal and lymphoid tissues of adrenalectomized rats.

The effect of the biogenic amine depleting drug, reserpine, on the concentration of type II corticosteroid receptors (i.e., glucocorticoid receptors) in neuronal (hippocampus, frontal cortex, hypothalamus), lymphoid (circulating lymphocytes, spleen, thymus) and pituitary tissues as well as hippocampal type I (i.e., mineralocorticoid) receptors was examined in adrenal-intact and adrenalectomized (ADX) rats. Reserpine (2 mg/kg) or vehicle was administered to adrenal-intact rats for 2 consecutive days. Following the second injection rats were ADX and sacrificed 24 h later. Reserpine significantly decreased type I and II hippocampal receptors as well as type II receptors in frontal cortex, hypothalamus, lymphocytes and spleen. Since the reserpine-induced decreases in receptor content could be due to reserpine-induced elevations in circulating corticosterone levels, reserpine (2 mg/kg) or vehicle was administered to 1-day ADX rats which were then sacrificed 2 days later (i.e., 3 days post ADX). A 1-day ADX control group was also included. The 3-day ADX regimen produced significant or nearly significant increases in type II receptors in hippocampus, frontal cortex, hypothalamus, lymphocytes and spleen in vehicle-treated rats. Reserpine attenuated the ADX-induced upregulation of type II receptors in hippocampus, frontal cortex, lymphocytes and spleen, but had no effect on the ADX-induced upregulation of type II receptors in the hypothalamus. The ADX-induced increase in hippocampal type I receptors was not affected by reserpine treatment. In a final experiment, reserpine (2 mg/kg) or vehicle was administered immediately after ADX and rats were sacrificed 24 h later in order to assess the effect of reserpine on basal (i.e., nonupregulated) corticosteroid receptor levels in the absence of circulating corticosterone levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical localization of chromogranin A in normal tissues from laboratory animals.

To analyze the distribution of Chromogranin A in endocrine cells of various species of laboratory animals (dog, gerbil, guinea pig, hamster, monkey, mouse, and fetal, neonatal, and adult rats), normal tissues were stained immunohistochemically with polyclonal anti-bovine Chromogranin A antiserum (SP-1). Selected tissues (pituitary, adrenal, thyroid, parathyroid, pancreas, brain, peripheral nerve, stomach, small and large intestine, bone marrow, spleen, thymus, lymph node, and liver) from these species and from the rabbit were stained with two monoclonal anti-human Chromogranin A antibodies (LK2H10 and PHE5) to compare the immunoreactivities of the monoclonal antibodies and polyclonal antiserum. Staining with the polyclonal antiserum (SP-1) resulted in a broader spectrum of immunoreactivity but had more nonspecific background staining than either monoclonal antibody. Immunoreactivity and staining intensity with SP-1 varied between species, but most endocrine tissues (pituitary cells in the anterior and intermediate lobes, thyroid "C" cells, adrenal medulla, parathyroid, pancreatic islets, and enterochromaffin cells) from most species stained positively. In some species, pancreatic alpha cells stained more intensely, and two populations of adrenal medullary cells with different staining intensities were observed. Sciatic nerve (axonal area) was immunoreactive with monoclonal antibodies and/or the polyclonal antiserum in several species. The spectrum of immunoreactive tissues from fetal and neonatal rats increased with age. There was good cross-reactivity between species with SP-1, but not with either LK2H10 or PHE5. These results indicate that many endocrine cells with secretory granules in laboratory animals express Chromogranin A and that a polyclonal antiserum, such as SP-1, is more sensitive in detecting this protein in various species than monoclonal antibodies such as LK2H10 or PHE5.

Immunoreactivity for IL-1 beta and TNF alpha in human lymphoid and nonlymphoid tissues.

Monoclonal antibodies (MAbs) against two non-cross-reacting antigens of human IL-1 beta (Vhp20 and BRhC3) and human TNF alpha (B154.2 and B154.7) were applied to identify cytokine-containing cells in tissue sections and in cell suspensions. IL-1 beta- or TNF alpha-positive cells were not present in immunostained cytocentrifuge smears prepared from freshly isolated peripheral blood leukocytes, spleen, and lymph node cells. After 18 hours of culture with bacterial endotoxin (LPS), 80% to 90% of blood monocytes, 30% of spleen macrophages, and 2% to 28% of lymph node macrophages were strongly positive for IL-1 beta with either of the MAbs. Furthermore, 25% to 35% of blood monocytes and 6% to 60% of lymph node macrophages were stained for TNF alpha. Cells positive for IL-1 beta or TNF alpha were extremely rare in sections of normal thymus, spleen, and lymph nodes. Immunoreactivity for IL-1 beta or TNF alpha was frequently observed in sections of granulomatous lymphadenitis (N = 11). IL-1 beta or TNF alpha staining was confined to the epithelioid macrophages forming the granuloma, and the intensity of TNF alpha reactivity was generally stronger. The high frequency of cytokine-containing cells in this pathologic condition was confirmed in a cell suspension study showing that 20% of epithelioid macrophages were weakly positive for IL-1 beta and 80% were strongly positive for TNF alpha. The presence of cytokine-containing cells was investigated in cryostat sections of several nonlymphoid organs with normal histologic appearance. IL-1 beta reactivity was not observed in any of the tissues. TNF alpha reactivity was frequently demonstrated in isolated macrophages embedded in the interstitial connective tissue.

Double immunocytochemical labeling of cell and tissue samples with monoclonal anti-bromodeoxyuridine.

We describe a new monoclonal antibody (designated Bu20a) against bromodeoxyuridine (BrdU). This antibody was selected by screening against human tissues using the APAAP technique, and shows no crossreactivity with normal nuclei. It stains BrdU incorporated into the nuclei of a wide range of cell types, including human tonsil lymphoid cells, normal mouse tissues, and human tumors growing in nude mice. A double-labeling technique is described using this antibody in which cell smears or tissue sections are first labeled by an immunoperoxidase procedure for a cellular antigen (e.g., mouse or human histocompatibility class II antigen, T-lymphocyte antigen, keratin) and BrdU is then detected by indirect immunofluorescence. This procedure, which was applied to a variety of human and animal cells and tissues, is of wide potential value in analyzing the phenotype of S-phase cells and in co-localizing antigen expression and BrdU incorporation in tissue sections.

Distribution of injected anti-Thy-1 monoclonal antibodies in mouse lymphatic organs: evidence for penetration of the cortical blood-thymus barrier, and for intravascular antibody-binding onto lymphocytes.

The localization of monoclonal anti-Thy-1 binding in mouse thymus, spleen, and lymph nodes was studied at early intervals after intravenous (i.v.), intraperitoneal (i.p.) and subcutaneous (s.c.) injection of a single dose of the monoclonal antibody (MAb). Five minutes after i.v. injection, anti-Thy-1 was bound to cortical thymocytes surrounding capillaries in the thymic cortex, to thymic cells beneath the thymic capsule and to medullary thymocytes around venules of the thymus medulla. When anti-Thy-1 was injected i.p. or s.c. the MAb was first deposited in capillary walls in the thymus cortex and did not appear on thymocytes outside of capillaries until 60 min after injection. These findings suggest that thymic cortical capillaries are permeable for anti-Thy-1 MAb contrary to the generally accepted principle of a blood thymus barrier to antigens in thymic cortex. Some cortex capillaries also became permeable for peroxidase when injected 15 min after anti-Thy-1 MAb. Anti-Thy-1 MAb penetration into spleen white pulp and lymph node paracortex occurs along the circulatory pathway of the vascular system in the spleen and of lymphatics in lymph nodes. But those lymphocytes with a strong anti-Thy-1 MAb loading always appeared along the pathways of lymphocyte circulation indicating that the most intense contact between anti-Thy-1 MAb and T-lymphocytes occurs not in the lymphatic organs but during the intravascular period of recirculation of lymphocytes.

In vivo immunohistochemical identification of tumor necrosis factor/cachectin in human lymphoid tissue.

To find an alternative approach to the in vivo detection of tumor necrosis factor/cachectin (TNF alpha), an immunohistochemical method to identify TNF alpha in histologic sections was developed. This method employs the streptavidin-biotin immunoperoxidase technique, and TNF alpha-specific monoclonal and polyclonal antibodies, on cryostat sections of fresh frozen human lymphoid tissue. Staining was evident in most specimens displaying follicular hyperplasia, but was absent from histologically normal tissue. Both tingible body macrophages and follicular dendritic reticulum cells appeared from phenotype analysis in serial sections and by double staining experiments to constitute the main source of TNF alpha. This technique complements other systemically oriented assays that may fail to detect significant in vivo TNF alpha production and activity at a cellular level.

Improved detection of aneuploidy in malignant melanoma using multiparameter flow cytometry for S100 protein and DNA content.

DNA aneuploidy has been demonstrated to be an independent parameter of prognostic significance in malignant melanomas. In order to improve the detection of DNA aneuploidy in malignant melanomas, and to minimize diploid non-tumor cells in the sample, we developed a two-color staining strategy for S100 protein and DNA content in paraffin embedded samples. The ability to detect aneuploidy, defined as DNA ploidy index less than or equal to 0.90 or greater than or equal to 1.10, in 37 stage I malignant melanoma samples by flow cytometry analysis was significantly improved from 10.8% of cases using traditional one-color analysis for DNA content only (propidium iodide) to 32.4% of cases using two-color analysis for simultaneous measurement of both DNA (propidium iodide) and S100 protein (fluorescein conjugated antibody) (Chi-square with Yates' correction; p less than 0.05). The largest increase in sensitivity was found in level I and II melanomas less than or equal to 0.76 mm in thickness. In addition, we report the new observation that multiple S100 protein-positive subpopulations were found significantly more frequently in malignant melanomas (23/37 cases) than in compound melanocytic nevi (5/22 cases) (Chi-square with Yates' correction; p less than 0.01). These findings suggest that there is a previously unsuspected degree of tumor heterogeneity even in thin, presumably early, malignant melanomas.

Antisera to the secretory component recognize the murine Fc receptor for IgA.

Studies were undertaken to determine a possible structural relationship between the secretory component (SC) and the receptor for IgA (Fc alpha R). An IgA-mediated rosetting technique was used to assess the presence of Fc alpha R+ cells in various lymphoid tissues from normal BALB/c mice and mice bearing an IgA plasmacytoma (MOPC 315). Tissues from the MOPC 315-bearing BALB/c mice were found to have a significantly higher percentage of Fc alpha R+ cells; thus, nonadherent spleen cells from MOPC 315-bearing mice were used as a source of Fc alpha R+ cells in these studies. The cells were preincubated with anti-SC and then assayed for the ability of IgA to bind to the Fc alpha R. Antisera to SC from various species inhibited the formation of IgA-mediated rosettes, although preincubation of the Fc alpha R+ cells with antisera directed against other cell surface molecules (e.g., Thy1.2, Lyt1, Lyt2, Fc gamma R, MHC class I and II) or preimmune sera had no significant effect on IgA-mediated rosette formation. Preabsorption of the anti-SC with secretory IgA or with free SC removed the inhibitory effect; preabsorption with myeloma IgA had no effect. These data suggest that SC and Fc alpha R are related serologically and may be structurally related, possible in the IgA-binding region.

Regulation of expression of a human intercellular adhesion molecule (ICAM-1) during lymphohematopoietic differentiation.

The intercellular adhesion molecule (ICAM-1) is a cell-surface molecule which binds to leukocyte function antigen-1 (LFA-1) and regulates both leukocyte adhesion to endothelial cells and immune functions requiring cell-cell contact. Membrane expression of ICAM-1 is highly regulated on all hematopoietic lineages. Cell membrane antigen is significantly expressed on a small subset of bone marrow (BM) progenitors but is weak or absent on all cell lineages once they enter the circulation. However, strong expression on tissue macrophages and germinal center B cells suggested that activated cells may show upregulated expression. When B cells, T cells, macrophages, or granulocytes were activated in vitro by suitable mitogens, ICAM-1 expression was induced in all cases. Parallel studies of hematopoietic tumors demonstrated a heterogeneity of expression which correlated with expression on their normal cellular counterparts. In particular, a striking correlation between expression on B-cell tumors and corresponding stages of B-cell differentiation was noted. The widely varying expression of ICAM-1 contrasts with LFA-1 which, while variable, is nevertheless significantly positive at all stages of differentiation. This suggests that the major regulation of homotypic adhesion mediated by the LFA-1/ICAM-1 linkage occurs through control of ICAM-1 expression. In keeping with this notion, ICAM-1 expression was also correlated with the "adhesiveness" of B-lymphoid tumors. Large solitary lymphoma masses showed intense expression of ICAM-1. Conversely, chronic lymphocytic leukemia (CLL) cells and lymphoma cells from tumors exhibiting diffuse, widespread lymph node disease showed weak expression. These observations are discussed in relation to the role of ICAM-1 in regulation of lymphoid recirculation and the biology of lymphoid tumors.

VH repertoire in progeny of long term lymphoid-cultured cells used to reconstitute immunodeficient mice.

VH gene utilization in the progeny of long term lymphoid-cultured cells used for reconstitution of severe combined immunodeficient mice under varying conditions was determined. Hybridomas made from the spleens of these animals were evaluated for clonality and donor origin and a panel of 146 independent hybridomas were subsequently examined for VH expression. Hybridomas derived from the spleens of SCID mice reconstituted with fresh cells, used as a control, utilized VH families in proportion to their numerical representation in the genome. However, hybridomas from the spleens of mice reconstituted with long term cultured cells utilized a predominance of the two VH gene families most proximal to JH, characteristic of cells early in B lymphocyte development. Coinjection of thymocytes with cultured fetal liver cells, to provide good levels of T lymphocytes, did not alter this pattern of VH utilization. Irradiation (3 Gy) of the mice before cultured cell injection, which leads to more complete reconstitution of the B cell compartment, was effective in removing this bias in the VH repertoire. Hybridomas derived from these mice expressed their VH genes more in proportion to family size, characteristic of cells later in B lymphocyte development. In this manner, long term lymphoid-cultured cells can be used to study the transitions that occur in VH repertoire expression which appear to be mediated by either B lymphocyte developmental microenvironment or population size.

Extracellular matrix of lymphoid tissues in the chick.

We describe the immunohistochemical distribution of components of the extracellular matrix of the chick lymphoid system. In the thymus, basement membranes of epithelial cells bordering the lobules were intensely stained by laminin antibodies; fibronectin antibodies labeled the capsule and the septal matrix, and similar reactivity was seen with tropoelastin and gp 115 antibodies. No positivity was detected with any of the antibodies within the cortical parenchymal cells. Laminin was not detected in the medullary parenchyma, whereas fibronectin was present as coarse fibers. Tropoelastin and gp 115 appeared as a finer and more diffuse meshwork. In the bursa, laminin antibodies outlined the epithelial cells separating the cortex from the medulla. Fibronectin, tropoelastin, and gp 115 antibody stained the interfollicular septa and the cortical matrix, although to a different extent. Laminin was also detected in association with the interfollicular epithelium (IFE) basement membrane, whereas no staining was found underneath the follicle-associated epithelium (FAE). FAE cells not only lack a proper basement membrane but are also not separated from medullary lymphocytes by any of the other extracellular matrix components were investigated. Consequently, medullary lymphocytes are not sequestered, and can come easily into contact with antigens present in the intestinal lumen. All four antibodies stained the spleen capsule and spleen blood vessels, tropoelastin and gp 115 antibodies giving the strongest reactivity. A fine trabecular staining pattern was detected with gp 115 antibodies in the white pulp.

[Study of the DNA structure of peripheral blood leukocytes, proliferating lymphoid and tumor cells according to the rate of alkaline denaturation of DNA lysates]. / Izuchenie struktury DNK leikotsitov perifericheskoi krovi, proliferiruiushchikh limfoidnykh i opukholevykh kletok po skorosti shchelochnoi denaturatsii DNK lizatov.

Direct fluorometry was used to identify quantitative differences in the DNA structure of human peripheral blood leukocytes and proliferating lymphoid cells. The differences should be taken into account in the study of varied damaging actions.

B-cell differentiation in the chicken: expression of immunoglobulin genes in the bursal and peripheral lymphocytes.

We have studied the expression of immunoglobulin genes in the chicken B-cell precursors, and of a B-cell surface marker (Bu-1) on the bursal and peripheral B cells during normal ontogeny. Since there is no way of distinguishing the precursor cells from the more mature bursal lymphocytes on the basis of surface markers, we chose to study the total bursal lymphocyte population at ages when the numbers of the various precursor cells (bursal, early post-bursal, and post-bursal stem cells) in the bursa are estimated to be at their highest. Thereafter, comparisons with the more mature lymphocytes in the peripheral organs were made. As a result, levels of the lambda and mu transcripts and expression of Bu-1 antigen in the chicken B-cell precursors were found to be unchanged during the post-hatching period. In the light of these experiments, the later events of B-cell differentiation, i.e. the development from the bursal to post-bursal B lymphocytes, occurs without the lambda, mu, and Bu-1 gene loci involved. On the other hand, the higher level of lambda and mu expression in the splenic B lymphocytes indicates that the post-bursal stem cells mature into highly active plasma cells after seeding to the peripheral organs.

Cytotoxic T cell responses in HLA-A2.1 transgenic mice. Recognition of HLA alloantigens and utilization of HLA-A2.1 as a restriction element.

Previous studies have indicated that the frequency of murine CTL precursors (CTLp) for human class I molecules is one to two orders of magnitude lower than that for murine class I alloantigens, and that this is due to species-specific structural differences between these molecules. Transgenic mice expressing the human class I MHC Ag HLA-A2.1 were used to examine changes in the frequency of class I HLA-specific precursors after T cell differentiation in an HLA-A2.1 positive environment. The HLA-A2.1 gene product was expressed at levels comparable to those of the endogenous H-2Db molecule in thymus, bone marrow, and spleen. By limiting dilution analysis, it was observed that the frequencies of CTLp in transgenic mice responding to the human alloantigens HLA-B7 or HLA-A2.2 were comparable to or lower than those in normal C57BL/6 mice, regardless of whether the Ag was presented on human or murine cells. Thus, expression of a human class I molecule in these animals did not result in an expansion of the number of CTLp specific for other human class I Ag. In addition, the frequency of HLA-A2.1-restricted, influenza specific CTLp was substantially lower than the frequency of H-2b restricted CTLp, indicating a poor utilization of HLA-A2.1 as a restricting element. Finally, the frequencies of CTLp for HLA-A2.1 expressed on syngeneic murine tumor cells were decreased significantly. Thus, expression of HLA-A2.1 in these animals appeared to induced tolerance to this Ag. Interestingly, however, these mice were not tolerant to the HLA-A2.1 molecule expressed on human cells. This indicates that the HLA-A2.1 associated epitopes expressed on murine and human cells differ and suggests that, under these circumstances, HLA-A2.1 acts as a restricting element for human nominal Ag. These results are discussed in the context of current models of T cell repertoire development.

Cellular localization of simian immunodeficiency virus in lymphoid tissues. I. Immunohistochemistry and electron microscopy.

Simian immunodeficiency virus (SIV) is a lentivirus with genetic relatedness to the human immunodeficiency viruses (HIV-1 and HIV-2). It induces a fatal syndrome in rhesus monkeys that closely parallels the clinical course of AIDS in humans. The authors used double-labeling immunohistochemical procedures on rhesus lymph node and spleen taken during different time periods after SIV infection to localize the p27 gag protein to specific cellular immunophenotypes. In animals with follicular hyperplasia, viral protein was found associated predominantly with follicular dendritic cells. Many of these cells showed ultrastructural alterations consisting of swollen dendritic processes containing electron-dense material. Lentiviral particles were found associated with this cell type only rarely. In lymphoid tissues with other histopathologic changes, macrophages and multinucleate giant cells were the predominant cell types containing detectable quantities of viral protein; smaller numbers of p27+ lymphocytes were present. Ultrastructurally, viral particles were found within the extracellular space adjacent to tissue macrophages and within membrane-bound vacuoles of giant cells and tissue macrophages. These results show that certain histologic patterns seen during the course of infection correlate with the localization of viral antigen to specific cellular immunophenotypes and that during the disease course, viral protein is preferentially localized in sections of lymph node and spleen to cells of the macrophage and dendritic cell lineages.

Comparison of flow cytometric data obtained using fresh and paraffin-embedded lymphoid tissue.

Flow cytometric (FCM) DNA analysis was carried out on 24 lymph nodes: 13 from benign reactive hyperplasias and 11 from non-Hodgkin's lymphomas. FCM was performed on two types of samples: (1) fresh cell suspensions and (2) suspensions prepared from formalin-fixed, paraffin-embedded sections. FCM of fresh samples detected aneuploidy in 23 of the 24 cases while FCM of paraffin-embedded samples detected aneuploidy in only 6 of the 24 cases. Those six cases were lymphomas considered histologically as having a poor prognosis. Only one case, a lymphoma, was euploid with both methods. The coefficients of variance determined in each case for both methods were found to be within "normal ranges," but were greater in the paraffin-embedded specimens. The results suggest that FCM DNA analysis of formalin-fixed, paraffin-embedded sections does not have as great a resolution capacity as does analysis of fresh cell suspensions, since the former failed to detect cell populations that had a small degree of aneuploidy (close to the 2n population).