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Previously, we established a murine model, that involves the engraftment of fully allogeneic T cell depleted donor bone marrow cells in sublethally irradiated and single dose anti-CD3 treated recipient mice. These mice developed permanent stable multilineage mixed chimerism and donor-specific tolerance without graft-versus-host disease. Recently, we have shown that donor-specific tolerance is not induced and/or maintained by clonal anergy, neither by a Th1/Th2 shift, nor by suppressor or other regulatory processes. In the present study, we investigated whether clonal deletion plays a role in tolerance induction in our model. We studied the kinetics of TCRVbeta8(+) T cells in BALB/c (H-2L(d+))-->dm2 (H-2L(d-)) chimeras, in which combination of mouse strains TCRVbeta8 predominates the anti-donor response. We found that TCRVbeta8(+) T cells were specifically deleted. To our surprise, this deletion was also found in mixed chimeras, thymectomized prior to the conditioning regimen. We conclude that clonal deletion plays a role in the establishment and maintenance of donor-specific tolerance, and that the thymus is not required for this process. In addition, confocal laser-scanning microscopy clearly showed the presence of abundant amounts of donor T cells and some donor antigen presenting cells in the small intestine in thymectomized chimeras and not in other organs, suggesting that T cell selection might take place in this organ in the absence of the thymus.

The presence of Chlamydia pneumoniae in the human arterial system has mainly been determined in atherosclerotic plaque, whereas the adventitia has remained relatively unexplored. We assessed the presence of C. pneumoniae in all three vessel wall layers of coronary (n=72) and brachial (n=48) arteries in relation to local atherosclerosis. Immunohistochemical staining of C. pneumoniae was observed in plaque and adventitia. Cells stained for C. pneumoniae were detected in the same areas as cells stained for macrophages in adjacent sections. C. pneumoniae staining in the adventitia was associated with the extent and severity of atherosclerosis. Coronary sections with C. pneumoniae staining in both adventitia and plaque more often contained advanced atherosclerosis than sections with staining only in the adventitia. Staining was observed more often in the coronary artery than in the brachial artery (24/72 vs. 5/48 and 51/72 vs. 8/48 for plaque and adventitia, respectively, P=0.004 and P<0.001). PCR confirmed the presence of C. pneumoniae DNA in the adventitia. In summary, the adventitia of atherosclerotic coronary arteries frequently contains C. pneumoniae that seems to be located within macrophages. These results might indicate a possible route for infected circulating macrophages to home into atherosclerotic lesions in the artery via vasa vasorum.

A procedure in which the "Invitrogen Easy-DNA" kit was followed by a silica-based method for the isolation of DNA was developed for extraction of PCR-inhibitor-free DNA from up to 300 mg of human vessel wall tissue. Optimally designed PCR assays were developed for the detection of at least one infected cell in this amount of tissue. Details of the procedure are given for the detection of DNA of Chlamydia pneumoniae, cytomegalovirus and herpes simplex virus type 1 and type 2 in human vessel walls. The procedure can serve as a reference method or as a gold standard when a high-performance method is needed.

The effect of vitamin A deficiency on the mitogen response of splenic B and T lymphocytes was determined in adult vitamin A-deficient rats. Female weanling Brown Norway/Billingham-Rijswijk (BN/BiRij) and Sprague-Dawley rats were fed a semipurified, essentially vitamin A-free diet, which resulted in clinical symptoms of vitamin A deficiency and severely decreased plasma retinol contents at the age of about 17 and 41 wk for BN/BiRij and Sprague-Dawley rats, respectively. A lower B cell proliferative response after stimulation with lipopolysaccharide in combination with dextran sulfate was observed in vitamin A-deficient rats of both strains, but the T cell proliferative response after concanavalin A stimulation was unchanged. The lower B cell mitogen response was not associated with changes in the cellular composition of the spleen (as analyzed with monoclonal antibodies specific for the various subsets of T and B cells and of macrophages). We suggest that the age at which clinical symptoms of vitamin A deficiency are induced may be an important determinant for the immunological variables affected.

Rats of two strains (BN/BiRij and WAG/Rij) were fed the ethanol-containing Lieber-De Carli liquid diet supplemented with high amounts of vitamin A for 16 months. In contrast to Lieber and co-workers, who showed liver fibrosis developing within 9 months on the same diet in Sprague-Dawley rats, we were unable to demonstrate a histological and biochemical increase in liver collagen in either strain. Steatosis was present to a varying degree in both strains in ethanol-treated rats, but also in control animals. Considerable liver inflammation with focal necrosis accompanied by severe systemic inflammation was observed in 60% of the ethanol-treated WAG rats. This suggests that, at least in rats, the main effects of chronic ethanol consumption on the liver may be secondary to interference with host resistance to infections. The ethanol-high vitamin A Lieber-De Carli liquid diet does not necessarily elicit fibrosis or other characteristic histological abnormalities of human alcoholic liver disease.

Tannic acid as a prefixative for EM purposes was introduced by Futaesaku et al. (1972). The fixative creates conditions for enhancing electron density of different protein materials. By using a mixture of tannic acid and glutaraldehyde as prefixative, followed by a routine procedure of postfixation (OsO4) and poststaining (uranylacetate and lead citrate), membrane bound antibodies not conjugated with electron dense markers are made visible under the electron microscope.

Rabbit T lymphocytes were characterized using fluorochrome-labelled antisera against thymus cell determinants and fluorochrome-labelled lectins. Three anti-T cell antisera were used, detecting different antigenic determinants on the majority of T cells. Only small subpopulations were found stained by one of the three antisera. After dexamethasone (DX) treatment, the proportion of T cells was significantly increased in bone marrow and appendix, presumably by different mechanisms. Cells binding peanut agglutinin, mainly belonging to the T cell population, were present in small numbers in thymus, spleen, lymph node, and appendix from normal as well as from DX-treated animals. In bone marrow, however, a large PNA+ population showing neither T nor B cell surface properties was observed. After treatment with neuraminidase (NA), PNA binding sites were exposed on B as well as on an increased number of T cells. It is suggested, therefore, that T and B cells retained their PNA receptors during maturation. Masking of these receptors will take place before differentiation of T cells is initiated within the thymus. After NA treatment, also binding sites for Helix pomatia agglutinin were exposed on T cells and to different extents, revealing subsets of negative, weakly and strongly positive HPA cells. HPA weakly positive cells form the major population present in the thymus, while HPA strongly positive cells constitute the major population of the T cells in spleen and lymph node. The exposure of receptors for PNA of HPA appears not to be related to the steroid sensitivity of the cells.

The increased lipid fluidity of rabbit lymphocyte membranes resulting from treatment of the cells with soybean lecithin liposomes is shown to affect the capping kinetics of thymocytes, but not of peripheral T lymphocytes. Capping is induced by first treating the cells in the cold with a rat anti-rabbit T cell serum (ATS) followed by incubation at 37 degrees C with fluorescent (FITC) goat anti-rat serum. Untreated thymocytes belonging to subclasses with high proportions of mature cells are shown to cap slower than immature thymocytes. Interaction with soybean lecithin liposomes results in an enhancement of this capping kinetics, the strongest effect being observed in the case of the more mature thymocytes. This effect is not found with (mature) lymphocytes isolated from the mesenteric lymph nodes. The capping of these cells is strongly affected by cytoskeleton-inhibiting drugs, in contradiction to the capping of immature thymocytes. From these experiments we conclude that both the membrane fluidity and the action of the cytoskeleton are of importance in ATS capping of rabbit T lymphocytes dependent on the differentiation stage of the cells.

Five cases of monophasic and 7 cases of biphasic spindle-cell carcinomas were analyzed immunohistochemically for the presence of vimentin and keratin type intermediate filaments in the pleomorphic spindle cells. Vimentin reactivity proved to be a consistent feature but keratin reactivity was more variable, this latter filament being lost in two cases initially presenting as pure squamous cell carcinomas showing dedifferentiation towards a pure monophasic spindle-cell tumour when recurring. The converse was also noted: acquisition of keratin in a monophasic spindle-cell tumour that recurred as squamous cell carcinoma. These results were considered to support the concept that spindle-cell tumours of the upper aerodigestive tract are a peculiar type of carcinoma and not a product of a pluripotent stem cell exhibiting bidirectional differentiation. Diagnostic implications are as follows: keratin positivity in a spindle-cell tumour substantiates its carcinomatous nature but its absence does not rule out a diagnosis of spindle-cell carcinoma.

The role of macrophages in mitogen-induced rabbit T cell proliferation has been investigated. The blastogenic response to the 3 mitogens, PHA, ConA and oxidative treatment by neuraminidase and galactose oxidase (NaGo) was tested. T cell proliferation was reduced by removal of low density or plastic adherent cells, including macrophages, and could be enhanced by the addition of peritoneal resident macrophages, indicating a macrophage requirement for rabbit T cell proliferation. However, PHA-induced proliferation could not be raised to the level expected. It was found that catalase and especially 2-ME could considerably enhance macrophage dependent proliferation, even at low macrophage concentrations. It is concluded therefore, that macrophages not only support but also suppress lymphocyte proliferation, namely by non-specific damage to lymphocytes through release of radicals and hydrogen peroxide. In addition, peritoneal, but not lymph node macrophages were found to suppress lymphocyte proliferation by prostaglandin production, although to a lesser extent. Experiments, done in the presence of blockers of macrophage-mediated suppression, showed that macrophages were able to magnify the PHA-induced T cell proliferation to the expected values. The experiments thus show that unactivated macrophages support and suppress lymphocyte proliferation at the same time.

The authors have investigated a possible relationship between tumor cells of malignant fibrous histiocytomas (MFHs) and histiocytes. This relationship was studied by means of immunophenotyping using monoclonal antibodies specific for the monocyte cell lineage (FMC-17, Mac-1, OKM-1, Leu-M1, and lysozyme) and mono- and polyclonal antibodies specific for fibroblasts (respectively, FIB-86 and FSG). The immunophenotypes of the MFH tumor cells were compared with those of tumor cells of "true" histiocytic tumors. Monocyte lineage-specific determinants could be demonstrated in varying amounts on cells of the "true" histiocytic tumors but not on cells of MFH or other soft-tissue tumors. The reverse was true for determinants on fibroblasts. The absence of these determinants on malignant histiocytes, and their presence on MFH (and also on benign fibrous histiocytomas, fibrosarcomas, schwannomas, osteosarcomas, hemangiosarcomas, leio- and rhabdomyosarcomas) supported the conclusion that MFH tumor cells originate from mesenchymal cells which do not belong to the mononuclear phagocytic system. Subdivision of the MFH tumors revealed that the storiform-pleomorphic subtypes could express HLA-Dr/Ia antigens, like histiocytic tumors. The inflammatory cell subtype, however, lacked these antigens.

K-type pyruvate kinase was purified from human kidney by immunoadsorbant chromatography. Monoclonal antibodies secreting hybridomas were made using conventional techniques. Two clones were established which produced antibodies against K-type not cross-reacting with the other pyruvate kinase isoenzymes, named the M, L and R-types. The specificity of the monoclonal antibodies was proven by enzyme-linked immunosorbent assay, immunoprecipitation and immunoblotting experiments. The M- and K-isoenzymes are produced from the same gene probably by alternative splicing, and all differences between both enzymes originate from one exon coding for 45 amino acids (Noguchi et al. J. Biol. Chem. 261, 13807-13812 (1986]. The monoclonal antibodies are specific for K-type under denaturing conditions. Thus, it is likely that these antibodies recognize (a) continuous epitope(s), of which at least some amino acids are coded in the K-specific exon. The monoclonal antibodies could be successfully used in immunohistochemical studies. Neurons and astrocytes in brain, Kupffer cells in liver, connective tissue cells and vascular smooth muscle cells showed immunoreactivity. However, striated muscle cells in skeletal muscle and heart and hepatocytes were not immunoreactive. Other types of glial cells, e.g., oligodendrocytes and microglia, so far studied, showed no reaction either.

Twenty-two human sarcomas were grafted subcutaneously into nude mice. Twelve tumors grew successfully. Nine of these 12 tumors had an aneuploid DNA content, whereas only 1 of 10 nonsuccessful tumors was aneuploid. The 12 sarcomas included two leiomyosarcomas, two malignant schwannomas, one synovial sarcoma, and seven malignant fibrous histiocytomas (MFHs). With light and electron microscopic and immunolabeling studies the original and xenografted tumors (the latter for at least two generations) were histopathologically compared. The xenografted leiomyosarcomas showed ultrastructurally a more pronounced leiomyodifferentiation, and one of the malignant schwannomas a more pronounced schwannian differentiation. The second malignant schwannoma and the synovial sarcoma, however, remained unchanged. Five storiform pleomorphic MFHs expressed features that were not observed in the original tumors. Tumor cells of three of these xenografted sarcomas showed leiomyogenic differentiation (filamentous densities, pinocytotic vescicles, and desmin immunoreactivity), whereas cells of the two others demonstrated schwannian differentiation (long cytoplasmic processes, basal lamina). A xenografted myxoid MFH and a pleomorphic MFH gave rise to pleomorphic sarcomas composed of undifferentiated cells. It appeared that under transplantation conditions tumor cells of storiform pleomorphic MFH can differentiate into various directions.