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OBJECTIVE: To investigate morphological changes in the thymus, the subpopulation composition of lymphocytes and its non-lymphoid cells in dextran-induced experimental acute ulcerative colitis and in different periods of chronic ulcerative colitis. MATERIAL AND METHODS: Acute and chronic ulcerative colitis was simulated in C57BL/6 mice, by replacing drinking water with a 1% aqueous dextran sulfate sodium solution. Thymic changes were morphometrically assessed; the number and absolute area of thymic corpuscles and epithelial cells were calculated; and the subpopulation composition of lymphocytes and thymic stromal cells was determined using flow cytofluorimetry; the Kruskal-Wallis test and the Mann-Whitney test were used to compare the groups. RESULTS: In acute catarrhal and ulcerative colitis, there was acute accidental thymic involution with devastation of the cortical substance and with a decline in its volume fraction, with an increase in the levels of cells dying through the mechanism of apoptosis, and with a decrease in the absolute number of lymphocytes, T-helper cells, cytotoxic T-cells, regulatory T-lymphocytes, B-lymphocytes, and dendritic cells, with a rise in the index of the area of thymic corpuscles and in the content of late-phase corpuscles among them, and with the appearance of thymic corpuscles as cyst-like cavities. In chronic ulcerative colitis, the cortex was expanded and the area of thymic corpuscles and the count of medullary epithelial cells increased. The cyst-like thymic corpuscles formed clusters, the count of dendritic cells increased in early-stage chronic ulcerative colitis, but the levels of macrophages decreased in both periods of its development. CONCLUSION: There is acute accidental involution and thymic hyperplasia with an increase in medullary epithelial cells and thymic corpuscles consisting of cytokeratin 19+ in the epithelial cells in experimental acute and chronic ulcerative colitis, respectively. The more pronounced epithelial cell response found in end-stage experimental chronic ulcerative colitis reflects the enhanced differentiation of regulatory T-lymphocytes and the larger number of which is observed in peripheral blood and in the focus of inflammation in patients with ulcerative colitis, according to the literature.

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SETTING: Sofia State Hospital for Tuberculosis Treatment, Bulgaria. OBJECTIVE: To investigate the morphology of two 'heteroresistant' clinical isolates and one non-heteroresistant isolate, all isolated from newly diagnosed tuberculosis (TB) patients, as well as the reference strain H37Rv. Heteroresistant isolates contained clonally-related sensitive and drug-resistant organisms which could subsequently be separated using drug-containing primary cultures and had been isolated from patients originally diagnosed with susceptible TB by the 1% proportion method. Mycobacterial cultures were evaluated by transmission electron microscopy after 25 days of cultivation in Dubos broth. RESULTS: In contrast to H37Rv and the non-heteroresistant isolate, the bacterial populations in both heteroresistant isolates demonstrated distinct pleomorphic variability and coexistence of both classical and cell-wall deficient forms. Electron micrographs of mutants resistant to streptomycin and isoniazid showed predominance of atypical granular L-forms, which formed L-type colonies on Dubos agar. CONCLUSION: The L-form transformation processes, observed both in clinical heteroresistant isolates containing mixed populations of Mycobacterium tuberculosis organisms with different resistance gene genotypes and in the isolated resistant (mutant) clones, indicate a possible link between resistance and cell-wall deficient L-phase states and suggest one of the possible mechanisms by which resistant mutants are able to survive in vivo.

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To study the in vivo interaction between lentinan-stimulated alveolar macrophages and Mycobacterium tuberculosis were used rats intranasally infected with 2 x 10(8) CFU. Before infection animals were treated intranasally with lentinan at a dose of 1 mg/kg (administrated three times at 2 days periods). Samples of broncho-alveolar lavage fluid were obtained from rats at different intervals -3, 24 and 72 h after infection. The process of phagocytosis in vivo was observed by light and electron microscopy, as well as acid phosphatase cytochemistry methods. Bactericidal activity of alveolar macrophages following the same intranasal installation of lentinan was assessed by in vitro "killing" ability test against M. tuberculosis. Nitrite production by lentinan activated alveolar macrophages was measured 24 h after ex vivo culture of these cells. It was demonstrated that lentinan induces high level of alveolar macrophage activation manifested through enhanced bactericidal effect against M. tuberculosis, which correlates with the induction of reactive nitrogen intermediates, increased activity of lysosomal enzymes (acid phosphatase), and with effective phagolysosomal fusion followed by destruction of Mycobacteria.

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In vivo cell interactions between Staphylococcus aureus and rat alveolar macrophages were investigated after intranasal inoculation during a 30-days period of examination. Some dynamic characteristics of microorganisms in the macrophages were examined by electron microscopy and acid phosphatase cytochemistry. It was found that at earlier infection intervals (days 3 and 7) the ingested cocci were sequestered in phagosomes and phagolysosomes and later many of the microbial cells were digested. An interesting finding was the intracellular appearance of cell wall-defective forms (L-forms) of S. aureus at later intervals (days 14 and 30 after challenge). Infection kinetics were evaluated by isolation and enumeration of colony-forming units of S. aureus from bronchoalveolar fluid and by assessment of blood and bronchoalveolar total and differential leukocyte counts. The results indicate that induction and survival of S. aureus L-forms may occur spontaneously in vivo. This phenomenon could explain some of the mechanisms, provoking the latent and relapsing lung infections.

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Groups of rats were injected intraperitoneally with cell wall-deficient (L) forms of Streptococcus pyogenes, with their parental (S) forms, as well as with a combined inoculum of both forms (S+L). Peritoneal exudate samples were harvested on days 1, 3, 7, 15 and 30 after challenge and were investigated by microbiological, electron microscopic, cytometric and biochemical methods. Parental S forms were isolated from peritoneal exudate samples up to day 15 post infection, while L form cultures were isolated until the end of the examined interval. Electron microscopic examination revealed continuous adhesion of L forms on the macrophage surface as well as intracellular persistence inside them. It was demonstrated that the intraperitoneal inflammatory response to L form infection was higher than to the other infections and the monocyte-macrophage populations were predominant. The established atypical behaviour and long survival of S. pyogenes L forms in the rat's peritoneum could explain some of the mechanisms of the pathogens' persistence as well as the reasons for chronic streptococcal infections.

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The interaction between stable protoplast L forms of Staphylococcus aureus and the alveolar wall of infected rats was observed in the course of experimental pulmonary infection (days 3, 7, 14, and 30 p.i.). The L forms were successfully cultivated from bronchoalveolar lavage samples taken throughout the tested interval. The ultrastructural results demonstrated the ability of the L forms to invade the alveolar wall where they established, grew and reproduced mainly in the interstitium. The infection caused lung lesions: granulomas, focal fibroses and destruction of type I alveolar epithelial cells.

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After co-cultivation of Mobiluncus curtisii, an obligate non-sporeforming anaerobe, with free living amebae from the Acanthamoeba spp. under aerobic conditions, internalization, multiplication and persistence of bacterial cells were established for at least 4-6 weeks. Under the same conditions and media without viable amebae, the cells of M. curtisii did not replicate and died in 4-7 days. The infection of amebae occurred with 10 to 100 bacteria per ml of co-cultivation media. In 7-14 days the amount of bacterial cells increased to 1x10(5)-1x10(6) CFU/mL. Electron microscopic examinations revealed bacteria within vacuoles in the amebae and intracellular replication. These results suggest a previously undescribed mechanism for spread, replication and persistence of obligately anaerobe bacteria in the environment and new possible sources, reservoirs and transfer mechanisms of infections caused by obligate anaerobe bacteria.

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Experimental infections were induced with different bacterial forms of Listeria monocytogenes: parental (S-forms), protoplastic (L-forms) and combined inoculum of both forms by i.p. injection of rats. The parental bacterial forms (S-forms) were isolated up to 7 days after challenge from the peritoneal cavity and the liver, while the L-forms were isolated up to 60 days from the peritoneal cavity. Continuous adhesion of L-forms on the peritoneal macrophage surface was found by scanning-electron microscopy. Erythrocyte and leucocyte count as well as some clinical chemistry parameters were measured during infections. They showed different dynamics in the three experimental groups. Histomorphological changes in the liver (microabscesses and mononuclear cellular granulomas) of infected animals were observed. They were less intensive and appeared later in rats infected with L-forms. The experiments demonstrated that infections caused by parental bacterial forms and by combined inoculum took an acute course, while the infection caused by L-forms could be distinguished as a prolonged and persistent one.

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Scanning electron microscopy (SEM) investigations on the interactions between peritoneal macrophages from Lewis lung carcinoma (LLC)-bearing mice and LLC tumour cells during 21 days after tumour implantation were carried out. The action of lipopolysaccharide (LPS)-containing cytoplasmic membranes (CM), from the stable protoplast type L-form of Escherichia coli, on the activity of in vitro phagocytosis was studied; CM induced a continuous increase in macrophage numbers. Activation of macrophage surfaces in healthy and tumour-bearing mice was established. Lamelipods, pseudopods and migration fringes 14 days after CM application were seen. Crater-like cavities deeply in the macrophage cells as well as adherent or prominent engulfed tumour cells within macrophages were observed during in vitro interaction with LLC cells. Macrophages from tumour-bearing mice without CM treatment showed less activation evaluated by SEM during earlier stages of tumour growth. The SEM investigation proved the temporary stimulating effect of E. coli L-form CM on the cell surface activation of peritoneal macrophages in healthy and LLC-bearing mice.

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The polysaccharide components (mannan and glucan) in the cell wall of Candida boidinii M 363 grown on methanol and glucose as control were investigated using electron microscopy, cytochemical and biochemical methods. An ultrastructural rearrangement of the polymers in the cell wall of yeasts cultivated on methanol in comparison to those cultivated on glucose was established. The morphological changes correlate to the quantitative changes in the polysaccharide constituents of the cell wall. The forming and the role of thiosemihydrocarbazide (TSHC)--negative zones in the Candida boidinii cell wall cultivated on methanol media are discussed.

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The presence of protein A in the stable L-form of S. aureus BM 3041 was proved. Its amino acid composition and electrophoretic characteristics were compared with the parent strain. The location of protein A on the cytoplasmic membranes of the L-form cells, as well as on the extracellular membranes were established by immunoelectron microscopy. In the cells of the parent bacterium its location on the cell wall was observed.

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Experiments in order to induce food allergy were carried out in guinea pigs. The sensitization with egg albumin, pasteurized cow milk and bovine serum albumin provoked anaphylactic shock. The passive cutaneous anaphylaxis, serum antibodies, liver cytochrome P-450 concentration and the anaphylactic shock were determined. Some correlation between the mortality, anaphylactic antibodies and cytochrome P-450 monooxygenase system was established. The morphology of the jejunal mucosa, the activities of the 5 disaccharidases, the number of immunoglobulin secreting cells (Ig SC) and the mastocytes were investigated in 35 patients with food allergy. Normal mucosa was found in 28 cases as well as a significant decrease of the lactase, sucrase and trehalase activities. An increase of IgM and IgG secreting cells and of mastocytes, different electron microscopic changes in the enterocytes (an increased number of lysosomes, appearance of vesicles in cytoplasma, shortening, enlargement and uneven distribution of microvilli) as well as symptoms of functional activity in the plasmocytes and some others were also revealed. The experimental model obtained is similar to that one in humans according to the enteral way of sensitization the high selectivity of the allergic reaction which is of reagin type as the immunoglobulin changes are involved.

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Electron microscopical investigations on in vitro and in vivo interactions of normal Staphylococcus aureus cells with rat peritoneal macrophages showed that these bacteria were rapidly endocytosed and digested even in the absence of specific antibodies. In contrast to the parental strains oxacilin-induced and stable variante lacking a cell wall (L-forms) were ingested without subsequent formation of phagolysomes and digestive vacuoles. The intracytoplasmic L-form bodies retained their characteristic ultrastructure, i.e. no visible alterations occurred. Some morphological aspects of the L-forms and their persistence in macrophages 7 days after intraperitoneal administration of L-form to rats, suggest the possibility of their intracellular survival.

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