Biomedical subjects

R Borojevic

Publications and source records attributed to R Borojevic.

At least 55 records · Page 3Linked to original sources

PubMed1995

Clonal heterogeneity in murine liver myofibroblasts.

GR primary cells cultures were isolated from hepatic granulomas induced in C3H mice livers by Schistosoma mansoni infection; the GRX continuous cell line was derived from GR cells after long-term culture and a progressive drift towards a rapidly proliferating cell population. These cells were analyzed and compared in terms of their clonal heterogeneity. Clones were classified on the basis of cell substrate, cell-cell adhesion (growth morphology of the clone) and fat droplet accumulation. GR cells were composed of two slow-growing clone types, while GRX cells gave rise to clones with several phenotypes, including the two found in the GR cells. The overall proportion of different clones in the GRX cell population was stable in long-term cultures, as well as after recloning of the highly proliferating, but not the slowly proliferating, clones. We propose that the slow-growing clones are maintained in the overall population by continuous contribution of new slow-growing cells from the rapidly growing ones. The slow-growing clones may represent the basal population of liver connective tissue cells that can be mobilized into injured tissues and that are involved in tissue repair. The highly proliferating clones with a broad capacity of phenotype expression that arise after long-term growth stimulation of the local cell population may represent the hypertrophic connective tissue cells, such as those observed in progressive fibrotic reactions associated with chronic liver tissue inflammation.

Animals↗

PubMed1994

Myelopoietic competence of stroma composed of hepatic granuloma-derived connective tissue cells or skin fibroblasts.

1. Connective tissue cells isolated from hepatic granulomas (GR cells), induced in mouse liver tissue by schistosomal infection, are able to sustain myelopoiesis, while other connective tissue cells such as skin fibroblasts (SF) are not. 2. We compared the ability of SF and GR cells to sustain in vitro proliferation of the FDC-P1 myeloid cell line, dependent upon IL-3 or GM-CSF. 3. Only the GR stroma sustained the proliferation of co-cultured FDC-P1 cells. RT-PCR analysis showed that both cell lines expressed the message for GM-CSF, but not for IL-3. We showed that GM-CSF was produced by, and remained bound to the cell layer through heparan sulfate; this growth factor could be released by high-salt treatment in a biologically active form from both cell types. The same activity could be restored to NaCl-treated GR cells, but not to SF, by incubation with recombinant murine GM-CSF. 4. These results indicate that the ability of connective tissue cells to sustain myelopoiesis depends directly upon the capacity of their heparan sulfate-bearing molecules to bind and present the GM-CSF to the target cells in a biologically active form. Alternatively, a yet unidentified set of cell layer-associated molecules may be required for the positive or negative control of the membrane-bound GM-CSF.

Animals↗

PubMed1994

Increase of B-lymphocyte number and activity during experimental murine schistosomiasis mansoni.

1. In schistosomal infection, the hyperergic acute phase of the disease evolves progressively into the chronic one, with establishment of a relative equilibrium between the parasites and the corresponding host responses. This down-regulation of host reactivity is considered to be under the control of T-lymphocyte circuits. 2. In the present study, we investigated lymphocyte populations in spleens of normal mice and the kinetics of the B-cell number increase in mice in the acute, chronic and late chronic phases of schistosomal infection, and we monitored their proliferation and activity in antibody isotype secretion. 3. We observed polyclonal B-cell activation and modulation of Ig isotype production, compatible with the alternate predominance of TH2 and TH1 lymphocyte subsets, in the acute and the chronic phases of the disease, respectively.

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PubMed1994

In vitro collagen synthesis by liver connective tissue cells isolated from schistosomal granulomas.

Hepatic injury elicits an excessive deposition of extracellular matrix probably due to a loss of control mechanisms in mesenchymal cells in fibrotic lesions, or a local activity of growth factors. To study collagen synthesis in an in vitro model of fibrotic lesions, we isolated liver connective tissue cells (LCTC) from murine schistosomal granulomas in C3H/HeN mice. Collagen was quantified in culture supernatants using a sirius red dye assay. LCTC and skin fibroblasts (SF) secreted similar amounts of collagen per cell and secretion was inversely proportional to the cell density. Cells cultured at low density (10,000 cells/cm2) secreted two- to three-times more collagen per cell when compared to cells grown in high-density cultures (60,000 cells/cm2). Collagen secretion was stimulated by transforming growth factor-beta (TGF-beta) in both cell lines, but the response of LCTC was detected from 1 ng/ml on, while SF responded only to higher concentrations (2.5 and 5 ng/ml). These data do not support the hypothesis that cells from fibrotic livers have lost the normal control mechanisms and suggest that their control is disturbed locally by the presence of peptide growth factors during the development of fibrosis.

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PubMed1994

Experimental murine Leishmania donovani infection: immunoprotection by the fucose-mannose ligand (FML).

The fucose-mannose ligand (FML) of Leishmania donovani is a complex glycoprotein fraction present in pro- and amastigotes, that interferes with parasite-macrophage interactions in vitro. In the present study, we have tested the potential immunoprotective effect of FML on L. donovani infection in inbred female BALB/c mice. The protection schemes included three weekly intraperitoneal administrations of FML, supplemented or not with saponin. Mice were challenged by intravenous injections of 2 x 10(7) amastigotes of Leishmania donovani (LD-1S/MHOM/SD/00-strain 1S) obtained from CB hamsters' infected spleens. After 15 days of infection, we monitored the splenocyte proliferative response to FML in vitro by ELISA for specific antibody response, and by parasite quantification as "Leishman-Donovan Units" in liver. A significant (P < 0.001) protective effect of FML with saponin, but not of FML or saponin alone, was shown by the reduction of parasite burden in liver and by the enhancement of splenocyte proliferation. The antibody response, very low at 15 days of infection in both untreated and control animals, showed a pronounced increase (P < 0.001) in animals sensitized with FML/saponin. Taken together, our results represent a 79.1 and 89.1% increase in specific proliferative and antibody responses, respectively, and an 84.4% protection in reduction of parasite liver burden. The protective potential was specifically due to FML (P < 0.001). Under the present conditions, no toxic or nonspecific effect could be attributed to saponin. A detailed study of the molecular events related to vaccination against murine visceral leishmaniasis with total and fractionated FML is currently underway.

Animals↗

PubMed1993

Leishmania donovani surface glycoconjugate GP36 is the major immunogen component of the fucose-mannose ligand (FML).

Leishmania donovani promastigote glycoconjugate ligands, studied in our laboratory, that interact with the internalization receptors on BALB/c macrophages: the 'fucose mannose ligand' (FML), the 'phosphate mannogalactan ligand' (PMGL), and the 'lipopeptidephosphoglycan' (LPPD), interfered also with interaction between amastigotes and host cells in vitro. Among the three compounds studied, the FML was shown to be the most potent inhibitor of both promastigote and amastigote internalization, and to be present on parasite surface during the vertebrate-host cycle. The FML, but not the other two glycoconjugates, is a potent immunogen in rabbits (ELISA, agglutination and immuno-blots). Rabbit hyperimmune sera recognized essentially the 36 kDa band of FML. Mouse monoclonal antibodies against FML recognized either the 36 kDa or the 55 kDa band. No cross-reactivity between these two FML components was detected. No antigenic similarity could be detected between the 36 and 55 kDa bands of FML and the 'GP63' (promastigote surface proteinase) major surface leishmanial antigen. The 36 kDa-glycoprotein was identified as the major FML antigenic fraction and designated 'GP36'. The integrity of the glycidic moiety was necessary for its antigenicity. This L. donovani surface glycoprotein is apparently one of the major molecules involved in interactions between the parasite and the vertebrate host.

Animals↗

PubMed1993

Eosinophil granulocyte proliferation induced by an intermediate factor generated in the pleural cavity of rats injected with platelet-activating factor-acether.

In previous research, we have observed that intrathoracic administration of platelet-activating factor-acether (PAF) promoted a delayed eosinophilia in the pleural cavity of rats that lasted for at least 96 h. We investigated the ability of pleural washings from rats previously injected with PAF (1 micrograms/cavity) to stimulate in vitro murine hematopoietic eosinophil proliferation. We observed that pleural fluid sustained eosinophil proliferation but not differentiation, under conditions in which PAF itself had no effect. The phenomenon lasted for 3 days and was maximal on the 1st day of culture. Treatment with neutralizing antibodies against interleukin (IL)-5, granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL-3, alone or in combination, did not modify the eosinophil proliferation induced by PAF pleural fluid, suggesting that these cytokines may not be involved in the studied phenomenon. We conclude that the rat pleural fluid obtained 6 h after PAF administration induces eosinophil proliferation in vitro by a mechanism probably independent of IL-5, GM-CSF or IL-3.

Animals↗

PubMed1993

Cell membrane-associated proteoglycans mediate extramedullar myeloid proliferation in granulomatous inflammatory reactions to schistosome eggs.

In chronic murine schistosomiasis, extramedullar myelopoiesis was observed, with proliferation of myeloid cells in liver parenchyma and in periovular granulomas. We have studied the question of whether cells obtained from granulomatous connective tissue may act as myelopoietic stroma, supporting long-term myeloid proliferation. Primary cell lines (GR) were obtained in vitro from periovular granulomas, induced in mouse livers by Schistosoma mansoni infection. These cells were characterized as myofibroblasts, and represent liver connective tissue cells involved in fibro-granulomatous reactions. They were able to sustain survival and proliferation of the multipotent myeloid cell lines FDC-P1 and DA-1 (dependent on interleukin-3 and/or granulocyte-macrophage colony stimulating factor, GM-CSF) without the addition of exogenous growth factors. This stimulation was dependent upon myeloid cell attachment to the GR cell layer; GR cell-conditioned medium had no activity. Primary murine skin fibroblasts could not sustain myelopoiesis. The endogenous growth-factor was identified as GM-CSF by neutralization assays with monoclonal antibodies. The stimulation of myelopoiesis occurred also when GR cells had been fixed with glutardialdehyde. The observed stimulatory activity was dependent upon heparan sulphate proteoglycans (HSPGs) associated with GR cell membranes. It could be dislodged from the cell layer with heparin or a high salt buffer. Our results indicate a molecular interaction between endogenous growth-factor and HSPGs; this interaction may be responsible for the stabilization and presentation of growth factors in myelopoietic stromas, mediating extramedullar proliferation of myeloid cells in periovular granulomas.

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PubMed1993

Experimental schistosomiasis mansoni: characterization of connective tissue cells in hepatic periovular granulomas.

Connective tissue cells present in periovular granulomas, induced in mice livers by schistosomal infection, were studied in situ and in an in vitro culture after cell migration from explanted granulomas. They were compared to cells of the adjacent hepatic tissue. Connective tissue cells in granulomas and granuloma-derived primary cell lines were characterized as myofibroblasts. The presence of lipid droplets in cells of early granulomas, and comparison with adjacent perisinusoidal cells, indicated their origin through activation and mobilization of lipocytes from the adjacent hepatic tissue. This origin was confirmed in long-standing cultures of granuloma-derived cells, in the stationary phase of growth, in which myofibroblasts could return spontaneously into the fat-storing phenotype.

Animals↗

PubMed1992

Phospholipid modifications during conversion of hepatic myofibroblasts into lipocytes (Ito-cells).

Connective tissue cells of liver parenchyma (perisinusoidal myofibroblasts) can be induced to express the lipocyte (Ito cell) phenotype. We have studied phospholipid synthesis and phosphate incorporation during this in vitro conversion, induced by insulin and/or indomethacin, in the established murine cell line GRX. Phospholipid synthesis, measured by [14C]acetate incorporation, was increased after a full induction of the lipocyte phenotype. The 32Pi incorporation into phospholipids was increased from the beginning of induction. Phosphatidic acid and phosphatidylinositol synthesis were increased early in the induction, whilst the increase of major constitutive phospholipids was significant only after the full lipocyte phenotype induction. The presence of unsaturated fatty acids in phospholipids was increased in lipocytes. Linoleic acid was present only in diacylglycerols and in phosphatidylinositol. Since we have shown previously that linoleic acid was not present in triacylglycerols, this result indicates the importance of future studies on activation of phosphatidylinositol cycles in induction of lipocyte phenotype in liver connective tissue cells.

Acetates↗

PubMed1992

Proteoglycans synthesized by the hepatic granulomas isolated from schistosome-infected mice and by the granuloma-derived connective tissue cell lines.

Proteoglycans synthesized in vitro by periovular granulomas isolated from livers of schistosome-infected mice were compared with those produced by granuloma-derived cell lines: the primary cell line GR and the permanent cell line GRX. Proteoglycans were metabolically labelled with 35S-sulfate and extracted with 4 M guanidine-HCl containing 2.0% Triton X-100, in the presence of proteinase inhibitors. The radiolabelled proteoglycans were purified and characterized by anion-exchange, gel-filtration and affinity-column chromatography. Heparan sulfate proteoglycans (HS-PGs) and chondroitin sulfate/dermatan sulfate-containing proteoglycans (CS/DS-PGs) were detected in both the culture medium and the cell-associated fractions obtained from GR cells. More than 90% of the cell-associated HS-PG from these cells contained a hydrophobic portion, as evidenced by their ability to bind to octyl-Sepharose. In contrast, among the secreted proteoglycans, it was the CS/DS-PG and not the HS-PG that bound to this resin. The major fractions of cell-associated and secreted proteoglycans from GRX cells were HS-PGs. Similar to HS-PGs from GR cells, 50% of the cell-associated HS-PG bound to octyl-Sepharose, while only 20% of secreted proteoglycans (HS-PGs) bound to this resin. The proteoglycans purified from the whole granuloma were composed mainly of DS-PG, of a size and hydrophobicity similar to the CS/DS-PG from GR cells. Possible correlations among the structure, secretion, distribution and function of proteoglycans in granulomatous reactions are discussed.

Animals↗

PubMed1992

Membrane-associated and secreted proteoglycans from a continuous cell line derived from fibrotic schistosomal granulomas.

Proteoglycans were isolated from a continuous murine cell line (GRX) established from fibrotic granulomas induced in mouse liver by schistosomal infection, representative of liver connective tissue cells. The proteoglycans were labelled with 35SO4, extracted by guanidine-HCl + Triton X-100 in the presence of proteinase inhibitors, and purified by anion-exchange, gel-filtration and affinity-column chromatography. The major fractions of cell-associated and secreted proteoglycans are heparan sulfate proteoglycans. Gel-filtration chromatography on Sephacryl S-400 revealed Kav values of 0.20 and 0.30 for the cell-associated and secreted heparan sulfate proteoglycans, respectively. About 50% of the cell-associated heparan sulfate proteoglycans contained hydrophobic regions, as evidenced by their ability to bind to octyl-Sepharose, while only about 20% of secreted proteoglycans bound to this resin. In addition, no proteoglycan was competitively displaced from the cell surface by heparin. Taken together with other reports on proteoglycan synthesis by a variety of cell types in culture, these observations suggest that cell-surface heparan sulfate proteoglycans possibly contain a hydrophobic domain that functions as a membrane anchor in their attachment to cells. Addition of beta-D-xyloside to the cultures greatly enhanced the release of 35S-dermatan sulfate to the medium. Interestingly, dermatan sulfate is the major glycosaminoglycan found in the schistosoma-induced granuloma, from which the GRX cell line is derived. These studies provide the first biochemical description of the proteoglycans produced by a liver connective tissue cell line derived from schistosomal granulomas.

Animals↗

PubMed1992

Collagen synthesis in an established liver connective tissue cell line (GRX) during induction of the fat-storing phenotype.

We have studied collagen synthesis and secretion in an established liver connective tissue cell line (GRX) that can be induced in vitro to express either the myofibroblastic or the fat-storing (lipocyte) phenotype. In lipocytes, collagen synthesis was reduced. Their intracellular collagen degradation corresponded to 15% of newly synthesized collagen. In myofibroblasts, collagen synthesis was high but its secretion was considerably altered by intracellular collagen degradation, which attained up to 60% of newly synthesized collagen. In this in vitro model, we have provided direct evidence that hepatic lipocytes, involved mainly in lipid and retinol metabolism, have a low basal level of collagen synthesis. Myofibroblastic phenotype correlates with increased collagen synthesis and may be directly related to increased collagen deposition in hepatic fibrosis. Modulation of the phenotype of liver connective tissue cells are possibly one of the major points of control in normal and pathological deposition of collagen in liver parenchyma.

Animals↗

PubMed1992

Schistosoma mansoni: peritoneal plasmacytogenesis and polypoid transformation of mesenteric milky spots in infected mice.

We have studied inflammatory reactions in the mesenteric tissue of mice infected with Schistosoma mansoni. Perivascular tissue contained diffuse infiltrates of macrophages, eosinophilic granulocytes and lymphocytes. Angiogenesis in the perivascular adipose tissue was associated with superficial plasmacytogenic foci. Polypoid structures were occasionally formed adjacent to inflammatory foci in the adipose tissue, organized around loops of capillaries, with terminal formation of a glomerular capillary network embedded in connective tissue, covered by plasmacytes. We conclude that these structures are specialized milky spots dedicated to active plasmacytogenesis and antibody secretion into the peritoneal cavity of schistosome-infected mice.

Adipose Tissue↗

PubMed1992

Experimental murine schistosomiasis mansoni: establishment of the chronic phase of the disease.

After the acute hyperergic phase of schistosomal infection, the chronic phase of the disease corresponds to the establishment of a relative equilibrium between the host and the parasite. This involves: (1) A shift from the predominance of the TH2 response observed in the acute phase, to the predominance of the TH1 response in the chronic phase of the disease, with modification of lymphokine and immunoglobulin secretions patterns. (2) Redistribution of hosts responses to parasite, with predominance of systemic controls in the acute phase, and a shift towards local tissue responses in the chronic phase. This redistribution relieves the hyperergic response involving the whole body of the host, and delimits cellular and molecular reactions to parasites to only those tissues that are directly involved by the adult parasites and their eggs. Mobilization of eosinophil granulocytes in schistosomal periovular granulomas is one of examples of this redistribution.

Animals↗

PubMed1992

Milky spots reaction to schistosomal mansoni infection.

Milky spots (MS), considered by the authors as a Coelomatic Lympho-myelopoietic Organ (CLMO), present a strong reactivity during experimental schistosomal mansoni infection, characterized by an increase of lymphocytes, macrophages, plasmocytes, mast cells, neutrophils and expression of eosinophil metaplasia. Intraperitoneal injection of purified Schistosoma mansoni (Sm) eggs provoked a rise in the number and size of MS, which developed the sessile marginal and pedunculated types. The authors conclude that egg antigens are, at least partially, responsible for MS reactivity during Sm infection.

Animals↗

PubMed1992

Effects of retinol on proliferation, cell adherence and extracellular matrix synthesis in a liver myofibroblast or lipocyte cell line (GRX).

We have studied the effect of retinol on an established murine cell line (GRX), representative of liver connective tissue cells. This cell line has myofibroblast characteristics; under retinol treatment it is induced into the lipocyte (Ito-cell) phenotype. Retinol decreased the proliferation rate in the entire cell population. It increased cell adherence to the substrate, which was correlated with the increased secretion of fibronectin. Collagen secretion was specifically decreased, whilst the total protein secretion remained stable. Heparan sulphate was decreased in the pericellular compartment, but other glycosaminoglycans were not affected by retinol treatment. Modulations of pericellular components induced by retinol may alter the relations among liver mesenchymal cells, and may be related to vitamin-A-induced modifications of the homoeostasis of hepatic connective tissue and hepatic fibrosis.

Animals↗

PubMed1991

Neutral lipid synthesis and accumulation during in vitro induction of the lipocyte phenotype in hepatic connective tissue cells.

Connective tissue cells of liver parenchyma are known as hepatic myofibroblasts and lipocytes (fat-storing cells, Ito-cells). They are considered to belong to a single cell lineage, that may switch between these two phenotypes. We have studied cellular and molecular parameters and controls of this switch in the murine GRX cell line, established from liver fibro-granulomatous lesions induced by schistosomal infection. Accumulation of neutral lipids (triacylglycerols, monoalkyl-diacylglycerol, cholesterol) was monitored. It was dependent upon induction with indomethacin. Insulin alone did not induce lipid accumulation in GRX cells, but in cells induced by indomethacin it increased the quantity of stored lipids. We propose that hepatic lipocytes are not cells directly involved in energy storage, but that they represent a particular cell population specialized in storage and in controls of the homoeostasis of lipid-soluble substances at the systemic level.

Animals↗