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R Borojevic

Publications and source records attributed to R Borojevic.

At least 73 records · Page 4Linked to original sources
PubMed1991

Eosinophil granulocytopoiesis in hepatic periovular granulomas during the chronic phase of experimental murine Schistosomiasis mansoni.

We have observed in hepatic periovular granulomas of C3H mice infected with Schistosoma mansoni, in the chronic phase of the disease (12-19 weeks of infection), groups of early precursors and immature eosinophil granulocytes corresponding, at the ultrastructural level, to promyelocytes and myelocytes. Mitosis was also seen in eosinophil myelocytes. These eosinophil myeloid foci were observed in close contact with macrophages and epithelioid cells, and they were surrounded by an extracellular matrix, rich in collagen fibres. These morphological observations give support to the concept of a peripheral proliferation of eosinophils in chronic schistosomiasis, mediated by a factor secreted by macrophages present in granulomas.

Animals↗
PubMed1991

Eosinophil granulocyte proliferation and differentiation in schistosomal granulomas are controlled by two cytokines.

In parasitic diseases, eosinophilia is controlled at the systemic level by soluble, circulating factors. In addition to their medullar production and migration to tissues involved by parasitosis, eosinophil populations in inflammatory infiltrates may be locally amplified by their in situ proliferation. In granulomas induced in liver tissue by eggs of schistosome worms, eosinophil proliferation and differentiation are observed. We have shown that they were under control of two cytokines, the activity of which can be demonstrated in supernatants of isolated granulomas maintained in culture for 24 hours. One of them has been identified as interleukin-5. The other one is secreted by adherent cells obtained from periovular granulomas, among which macrophages represent more than 99% of cells. It is considered to correspond to the previously described factor, secreted by inflammatory macrophages mobilized on intraperitoneal glass implants in mice with chronic schistosomiasis. In acute schistosomiasis, the activity of the interleukin-5 was predominant, whereas in the chronic phase of the disease, the stimulation of peripheral eosinopoiesis is taken over by the factor secreted by adherent cells. During the progression from the acute to the chronic phase of schistosomiasis, the immune reactivity of the host is down-regulated by T suppressor lymphocyte circuits. In addition, a redistribution of cellular controls of the host reaction to parasites may act as a complementary mechanism for establishment of the viable equilibrium between host and parasite.

Animals↗
PubMed1990

A new approach to the phylogeny of Leishmania: species specificity of glycoconjugate ligands for promastigote internalization into murine macrophages.

Two Leishmania donovani glycoconjugate ligands for the internalization receptor on BALB/c peritoneal macrophages [fucose-mannose ligand (FML) and phosphate mannogalactan ligand (PMGL)] were shown to be species-specific in a comparative phagocytosis-inhibition test. Promastigotes of L. donovani Sudan (LD1S), L. infantum, L. d. donovani, L. major (Jericho and Sudan), L. tropica, L. chagasi, L. mexicana venezuelensis, L. m. mexicana, L. m. amazonensis, L. m. pifanoi, L. m. garnhami, L. braziliensis braziliensis, L. m. amazonensis (Josefa), L. enrietti or L. adleri were incubated with macrophages in the presence of 10 micrograms/ml FML and PMGL purified from L. donovani (LD1S). Parasite internalization was determined and compared with that obtained in control experiments. Specific inhibition of phagocytosis ranged from 83% (L. donovani LD1S) to 7% (L. m. amazonensis). We could distinguish groups of Leishmania consistently with their geographic distribution and the clinical aspects of the disease. Analogous experiments with L. m. amazonensis glycoconjugates showed reciprocal results, with inhibition ranging from 76% (L. m. amazonensis) to 8% (L. donovani LD1S). L. chagasi remained separated from the Old World kala-azar agents. Possible phylogenetic implications of these observations are discussed.

Animals↗
PubMed1990

In vitro induction of the fat-storing phenotype in a liver connective tissue cell line-GRX.

Liver connective tissue cells have been characterized as perisinusoidal myofibroblasts and hepatic lipocytes (Ito cells, fat-storing cells). A concept of a single mesenchymal cell population that may be modulated between these two phenotypes has been postulated. We have previously established a continuous murine cell line, GRX, obtained from fibrotic granulomatous lesions induced by schistosomal infection in mouse liver. This cell line is considered to represent liver myofibroblasts. In the present study we have induced the conversion of these cells into lipocyte (fat storing) phenotype by treatment with insulin and indomethacin. We have quantified the lipid synthesis and the increase of activity of involved enzymes during the induction of the fat-storing phenotype and described modifications of cell organization along this modulation of cell functions.

Animals↗
PubMed1990

Comparative analysis of splenic cell proliferation induced by interleukin 3 and by syngeneic accessory cells (syngeneic mixed leukocyte reaction): evidence that autoreactive T-cell functioning instructs hematopoietic phenomena.

Murine syngeneic mixed leukocyte reaction (SMLR) was studied under totally autologous culture conditions using syngeneic normal mouse serum in the culture. SMLR was detected in splenic, but not in lymph node, nonadherent responding cell populations (NWNAC). In the absence of stimulator, accessory cells (AC), IL3-containing fluids also induced splenic, but not lymph node, NWNAC growth. SMLR-derived supernatants contained IL3, but not IL2, activity, and production of this IL3 activity could be prevented by adding anti-CD4 mAbs to SMLR cultures. Precursor frequencies of both SMLR and IL3 splenic responses were very low and similar, and there was a synergism between IL3 and AC in induction of NWNAC growth. Growth of responding NWNAC was further enhanced by T-cell depletion with anti-Thy1 mAb and complement. Lack of T-cell proliferation in the SMLR was confirmed by BUdR and light protection experiments. Autoradiographs indicated that the same cell type grew in both SMLR and IL3-induced NWNAC cultures. Besides blast cells, cells with the appearance of immature monocytes with 3H-labeled nuclei were found in both kinds of culture. No labeled lymphocytes could be found. Both SMLR and IL3-induced NWNAC cultures contained expanded numbers of M-CSF-responsive monocyte precursors. On the other hand, SMLR- but not IL3-induced cultures contained expanded numbers of IL3-responsive, immature precursors capable of giving rise to large colonies of monocytic-like cells. Although IL2 could not be detected in SMLR supernatants, both cell growth and IL3 production could be blocked with anti-IL2 receptor and anti-IL2 mAbs. Exogenous IL2, on the other hand, enhanced both cell growth and IL3 production in the SMLR. These results indicate that, under totally autologous conditions, CD4+ autoreactive T-cells do not proliferate in the SMLR, but rather instruct the growth of splenic hematopoietic precursors capable of differentiating along the monocytic lineage. Autoreactive T-cell activation in the SMLR seems to involve minimal IL2 production, which is critically necessary for triggering IL3 production in a markedly amplified manner. These results suggest a link between normal regulation of hematopoiesis and MHC-restricted, autoreactive T-cell activation.

Animals↗
PubMed1990

Extramedullar proliferation of eosinophil granulocytes in chronic schistosomiasis mansoni is mediated by a factor secreted by inflammatory macrophages.

In chronic murine schistosomiasis mansoni, extramedullar myelopoiesis of eosinophils has been described, associated with tissue inflammatory infiltrates and periovular granulomas. It may be experimentally induced by intraperitoneal glass implants in mice with chronic schistosomiasis but not in normal mice or in mice with acute schistosomiasis. In vivo studies showed that this induction depended on macrophages adherent to the implants. In an in vitro test with bone marrow cells, we demonstrated that this eosinophil proliferation was mediated by a factor secreted by the mobilized macrophages. In contrast to murine interleukin-5 (IL-5), which induced both proliferation and differentiation of eosinophils, the studied monokine did not induce their maturation. A potent neutralizing monoclonal antibody for murine IL-5 did not abrogate the stimulatory activity of the monokine, indicating that it is a cytokine distinct from IL-5. These data, together with a quantitation of bone marrow, blood, and peripheral eosinophils, indicated that in chronic schistosomiasis, the systemic medullar supply of eosinophils is supplemented in tissues by their local proliferation, mediated by macrophages mobilized in local granulomatous and diffuse inflammatory reactions.

Animals↗
PubMed1990

Identification of cells responsible for synthesis of sulphated glycosaminoglycans in schistosome-induced hepatic granulomas.

Sulphated glycosaminoglycans were isolated from schistosome-induced hepatic granuloma and from the pericellular, intracellular and extracellular compartments of two murine cell lines derived from granulomas: the primary cell line GR, and the permanent cell line GRX, established spontaneously from GR. The glycosaminoglycans composition in the whole granuloma was similar to that observed in the intracellular and extracellular compartments of GR cells. This result suggests that GR cells may be the major cell population involved in the synthesis and accumulation of glycosaminoglycans in the granulomas, and play an important role in the process of hepatic fibrosis. The conversion of the primary cell line GR into the established GRX cells did not modify the ratios that prevail among different glycosaminoglycans of the cell surface. However, it decreased the synthesis and secretion of glycosaminoglycans, reduced the proportion of iduronic acid units in the chondroitin sulphate, and increased the proportion of heparan sulphate in intracellular and extracellular pools. These characteristics of the GRX cells are similar to those observed in long-term cultures of smooth-muscle cells. In agreement with the general phenomenon of progressive de-differentiation during in-vitro culture of primary cell lines, these data indicate that the connective tissue cells of liver may belong to the myofibroblastic cell lineage.

Animals↗
PubMed1989

Retinoid-mediated induction of the fat-storing phenotype in a liver connective tissue cell line (GRX).

The GRX cell line is derived from murine liver connective tissue cells. It has myofibroblastic characteristics and can be induced to display a phenotype analogous to fat-storing (Ito) cells. Retinol-mediated induction of the fat-storing phenotype was studied in vitro. Based on the incorporation of radiolabelled acetate into cell lipids, cholesterol synthesis increased and phospholipid synthesis was modified shortly after the beginning of the induction, indicating an activation of pre-existing metabolic pathways. Triacylglycerol synthesis was increased only after a delay of 4 d, indicating the de novo induction of enzymes necessary for triacylglycerol metabolism. Retinol incorporation and conversion into retinyl esters were also considerably increased by previous incubation with retinoids. Retinoid-induced changes in GRX cells provide a model for studying in vitro the interconversion of liver connective tissue cells between the myofibroblastic and fat-storing phenotypes. This interconversion is considered to be one of the major control points of normal homeostasis and of pathological modifications of liver connective tissue.

Acetates↗
PubMed1989

Schistosomiasis and in vitro transdifferentiation of murine peritoneal macrophages into fibroblastic cells.

We developed a method for avoiding contamination by fibroblasts when cultures of peritoneal cells are initiated. Macrophages were identified by immunogold detection [light microscope, transmission (TEM) and scanning (SEM) electron microscopes] of membrane antigens (Mac-1+, Thy-1,2-), non-specific esterase activity and ultrastructural features (TEM). As compared with controls, the yield of peritoneal macrophages was 2- and 12-fold higher, respectively, in acutely and chronically infected mice. In all, 30 "chronic", 18 "acute" and 18 control cultures were followed up. At a given cell-density seeding, the decline of control, "acute" and "chronic" cultures starts at about day 10, 15, and 27, respectively. In "chronic" cultures only, fibroblast-like cells appear from day 6 onwards; their number increases with time. Cells showing characters intermediary between macrophages and fibroblasts were observed. We suggest that fibroblast-like cells result from the in vitro transdifferentiation of a limited number of in vivo committed macrophages.

Animals↗
PubMed1989

Quantification of attached cells in tissue culture plates and on microcarriers.

A method for quantification of anchorage-dependent cells in culture on plane surfaces or on microcarriers is proposed. It is based on Coomassie brilliant blue R-250 adsorption, followed by elution of the dye and measurement by spectrophotometry at 595 nm. A linear correlation (r = 0.988 to 0.996) was observed between absorbance and cell number along a large range of cell densities. This technique may be used for monitoring cell growth, from seeding of initial inoculi to scaling up of cultures in bioreactors.

Cell Count↗
PubMed1989

Patterns of sulfated glycosaminoglycan synthesis and accumulation in hepatic granulomas induced by schistosomal infection.

We have characterized sulfated glycosaminoglycans of periovular granulomas induced in mouse liver by experimental infection with Schistosoma mansoni and determined parameters of their synthesis and accumulation by metabolic incorporation of 35S. The major component of glycosaminoglycans isolated from granulomas was dermatan sulfate and the minor component was heparan sulfate. A similar proportion was observed among newly synthesized 35S-labeled glycosaminoglycans, with a slight increase in the relative amount of heparan sulfate. Neither qualitative nor quantitative differences were observed between glycosaminoglycans isolated from granulomas of the acute and the chronic phase of the disease. In contrast, collagen content of granulomas increased eightfold during evolution of the disease from the acute to the chronic phase. It may be concluded that different mechanisms control glycosaminoglycan and collagen synthesis in schistosomal granulomas, as well as the ratio between these components in the extracellular matrix. This is consistent with the loose organization of the extracellular matrix in acute inflammatory reactions and its dense organization in the chronic reactions.

Animals↗
PubMed1989

Inhibition of Leishmania donovani promastigote internalization into murine macrophages by chemically defined parasite glycoconjugate ligands.

Leishmania donovani, the agent of human visceral leishmaniasis, is an intracellular parasite that must be recognized and internalized by host macrophages to complete its biological cycle. In a search for possible ligands for macrophage surface receptors, glycoconjugates were obtained from Leishmania promastigotes by aqueous, phenol-aqueous, and alkaline extraction. A fucose-mannose glycoproteic ligand, a lipopeptidephosphoglycan, and a phosphate mannogalactan ligand were purified from promastigotes and analyzed for their chemical contents, with special attention to their glycidic moieties. Sugars that were identified as components of these glycoconjugates were tested for their capacity to inhibit promastigote internalization by BALB/c peritoneal macrophages in vitro. Neutral hexoses showed little inhibitory activity; fucose, charged monosaccharides, and a mannose polymer showed the highest activity. Two of the glycoconjugates (fucose-mannose glycoproteic ligand and phosphate mannogalactan ligand) purified from promastigotes were potent inhibitors of internalization, 75% inhibition being obtained at concentrations of 6 to 10 micrograms/ml. The simultaneous presence of both ligands in low concentrations yielded an increase in inhibitory activity above that found for each ligand alone, indicating that promastigotes may use at least two receptor sites for penetration into macrophages. These ligands are specific inhibitors of L. donovani promastigote phagocytosis, since 10 micrograms of each ligand per ml interfered neither with internalization of yeast cells nor with phagocytosis of Leishmania adleri promastigotes.

Animals↗
PubMed1989

Experimental murine schistosomiasis mansoni: hyperplasia of the mono-macrophage cell lineage and stimulation of myeloid proliferation by peripheral macrophages.

1. Normal and schistosome-infected mice were similar in terms of the total number of bone marrow myeloid cell precursors and their proliferative capacity in vitro when stimulated with supernatants of L-929 cells containing M-CSF. 2. Delayed differentiation of bone marrow neutrophil granulocytes and blood monocytosis of infected animals were consistent with a modification in the differentiation of bone marrow myeloid precursors, favoring the production of a mono-macrophage cell lineage. 3. Macrophages isolated from periovular granulomas secreted a considerable stimulatory activity for the proliferation of the mono-macrophagic cell lineage, whereas peritoneal macrophages from the same animals had only a very low stimulatory activity. 4. We conclude that systemic hyperplasia of mono-macrophagic cells in schistosomiasis may be related to their increased release from the bone marrow and to their peripheral amplification in inflammatory tissue infiltrate as a consequence of the local production of stimulatory activity for their proliferation.

Animals↗
PubMed1988

Experimental murine schistosomiasis mansoni: inhibition of neutrophil granulocyte inflammatory reaction.

1. The mobilization of neutrophils into the peritoneal cavity following stimulation with sterile mineral oil was studied in normal and schistosome-infected mice. This response was correlated with changes in blood neutrophil counts and with the production and release of medullar granulocytes. 2. The peritoneal neutrophil inflammatory reaction was considerably reduced in schistosome-infected mice. The medullar pool of mature neutrophils was totally depleted, in spite of an increased number of immature neutrophil precursors. 3. We conclude that the decreased inflammatory response of neutrophils during schistosomiasis is due to the delay of their maturation in the bone marrow. A similar delay has been observed in schistosomal patients. A causal relationship between delay of neutrophil maturation and sensitivity of schistosomal patients to chronic microbial infections is suggested by these data.

Animals↗
PubMed1987

In vitro formation of fibrous septa by liver connective tissue cells.

Active fibrous septa are a common feature in liver fibrosis and cirrhosis. Their etiology and formation were studied using cultures of tissue fragments or cells included in collagen gels. Liver fragments obtained from patients with cirrhosis or severe schistosomal fibrosis were able to reorganize the gel and to form discrete, interconnecting fibrous septa composed of parallel arrays of collagen, subsequently colonized by migrating connective tissue cells. The same was obtained in cultures of fibrogranulomatous lesions isolated from schistosome-infected mice livers. However, fragments of normal human and murine liver tissue did not show the capacity to form fibrous septa. Septa formation was also obtained in cultures of cell spheroids formed by liver connective tissue cells isolated from human fibrotic or cirrhotic liver tissues, but not with spheroids of normal skin fibroblasts or smooth muscle cells. This experimental model may represent the fibrous septa formation in vivo, depending on the activity of liver connective tissue cells. The ability of tissue fragments or cell spheroids to form septa in collagen gels might reflect the degree of fibrosis present in the liver tissue in vivo.

Animals↗
PubMed1986

Sulfated glycosaminoglycans synthesized by human smooth muscle cells isolated from different organs.

The sulfated glycosaminoglycans synthesized by human smooth muscle cells isolated from different organs were identified on the basis of electrophoretic mobility, enzymatic degradation with specific mucopolysaccharidases and by the type of degradation products formed. The results obtained indicated that chondroitin sulfate and heparan sulfate were the main glycosaminoglycans found, that most of the labeled glycosaminoglycans were found in the pericellular pool, and that no marked differences were observed in the sulfated glycosaminoglycan composition of the smooth muscle cells obtained from different organs. 'Liver connective tissue cells', isolated from pathological livers (which had been shown to possess biochemical and physiological features typical of smooth muscle cells) showed a pattern of glycosaminoglycan synthesis similar to that of the smooth muscle cells.

Cell Line↗
PubMed1986

Schistosoma mansoni: control of extramedullar eosinophil myelopoiesis in chronically infected mice by inflammatory macrophages.

Extramedullar proliferation of eosinophil granulocytes can be induced in mice chronically infected with Schistosoma mansoni, by intraperitoneal implants of glass coverslips. Immature eosinophils are located in discrete foci on glass implants; they are not correlated with the eosinophil population of the peritoneal cavity, where only mature eosinophils can be observed. The same induction of eosinophil proliferation can be obtained in normal mice, by the transfer of macrophages elicited by glass implants in mice with chronic schistosomiasis. This induction could not be done with cells mobilized in normal mice, either after transplant into normal mice or into schistosome infected ones. Stimulated macrophages of mice with chronic schistosomiasis have a capacity to induce peripherical proliferation of eosinophil granulocytes. This capacity is independent of the quality of the intraperitoneal environment. It can be expressed after transferring macrophages elicited in schistosome infected mice into normal mice.

Animals↗