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R Borojevic

Publications and source records attributed to R Borojevic.

At least 19 recordsLinked to original sources
PubMed2001

Mast cells can revert dexamethasone-mediated down-regulation of stem cell factor.

Mast cell hyperplasia can be causally related with chronic inflammation and liver fibrosis. Their survival and proliferation is dependent upon locally produced growth factors, the major one being the stem cell factor (SCF). Glucocorticoids can decrease mastocytosis, down-regulating the mast cell production of pro-inflammatory factors or inhibiting the expression of SCF in stroma. We compared dexamethasone effect on SCF expression in co-cultures of mast cells with NIH/3T3 fibroblasts or with primary cultures of activated hepatic stellate cells. Dexamethasone abrogated the NIH/3T3 stroma capacity to sustain mast cell proliferation, but not of hepatic stellate cells, at the post-transcriptional level. Mast cells reverted completely dexamethasone effect on hepatic stellate cells, increasing their SCF synthesis and transport. In both models, dexamethasone inhibited the mast cell spreading on the stroma, which was thus not required for mast cell survival and proliferation. Liver pathologies associated with mast cell hyperplasia are not expected to be sensitive to glucocorticoid treatments.

3T3 Cells↗
PubMed2001

Induction of the lipocyte phenotype in murine hepatic stellate cells: reorganisation of the actin cytoskeleton.

Hepatic stellate cells (HSCs) are intralobular connective tissue cells presenting myofibroblast or lipocyte phenotypes. They participate in the homeostasis of liver extracellular matrix, repair, regeneration and fibrosis under the former phenotype, and control retinol metabolism, storage and release under the latter one. Responding to systemic or local demands, they can convert into the required phenotype with deep modifications of their structures. Using immunofluorescence microscopy and Western blots, we investigated the expression and organisation of actin filaments and of two actin-binding proteins, alpha-actinin and tropomyosin, in the cloned GRX cell line representative of murine HSCs. GRX cells expressing the myofibroblast phenotype showed typical well-organised actin stress-fibres, anchored at the focal adhesions located at the cell periphery. Retinol treatment induced active reorganisation of the cytoskeleton. The major stress fibres were reduced in length, and frequently formed a polygonal meshwork. Subsequently, they fragmented and generated diffuse or granular actin in the perinuclear area, a thin continuous layer around lipid droplets and, in fully converted lipocytes, a peripheral layer of thin actin fibres. alpha-Actinin and tropomyosin were present only in lipocytes, co-distributed with actin in a granular form. Since the cytoskeleton reorganisation preceded lipid accumulation, we conclude that the induction of the lipocyte phenotype represents a full reprogramming of cell gene expression and function. We consider that both the lipocyte and the myofibroblast phenotypes should be considered "activated states" of HSCs, each responding to specific physiological or pathological modifications of liver functions.

Actinin↗
PubMed2001

Intermediate filaments modulation in an in vitro model of the hepatic stellate cell activation or conversion into the lipocyte phenotype.

Hepatic stellate cells are intralobular connective tissue cells expressing the myofibroblast or the lipocyte phenotypes. They participate in homeostasis of the liver extracellular matrix, repair, regeneration, and fibrosis under the former phenotype, and control the retinol metabolism, storage, and release under the latter one. They are heterogeneous in terms of their tissue distribution, function, and expression of cytoskeletal proteins. We have studied the expressions of intermediate filaments in the cloned GRX cell line representative of murine hepatic stellate cells, by immunolabeling, reverse transcription polymerase chain reaction (RT-PCR), immunoprecipitation and Western blots. GRX cells expressed vimentin, desmin, glial fibrillary acidic protein (GFAP), and smooth muscle alpha actin (SM-alphaA). Vimentin, desmin, and SMN-alphaA were expressed in all cultures. GFAP showed a heterogeneous intensity of expression and did not form a filamentous cytoskeletal network, showing a distinct punctuate cytoplasmic distribution. When activated by inflammatory mediators, GRX cells increased expression of desmin and GFAP. Retinol-mediated induction of the lipocyte phenotype elicited a strong decrease of intermediate filament protein expression and the collapse of the filamentous structure of the cytoskeleton. Quiescent hepatic stellate precursors can respond to physiologic or pathologic stimuli, expressing activated myofibroblast or lipocyte phenotypes with distinct patterns of cytoskeleton structure, metabolic function, and interaction with the tissue environment.

Actins↗
PubMed2000

The capacity of connective tissue stromas to sustain myelopoiesis depends both upon the growth factors and the local intercellular environment.

In adults, haemopoiesis is located in the bone marrow, where it is tightly regulated by cytokines and by a physical association of haemopoietic progenitors with the stroma. However, in pathological situations, haemopoiesis can be partly or fully dislodged to peripheral tissues. It is not clear which are the requirements for a given peripheral stroma to sustain haemopoiesis. Using the growth factor-dependent cell line FDC-P1, we have compared the myelopoietic capacities of a murine bone marrow-derived cell line S17, a liver inflammatory granuloma-derived stroma (GR) that sustains haemopoiesis, and normal skin fibroblasts (SF) that sustain neither survival nor proliferation of myeloid cells. All three stromas expressed mRNA for major haemopoietins with the exception of IL-3. Despite the incapacity of SF to sustain FDC-P1 cells, the biologically active GM-CSF could be recovered from all the studied stromas by treatment with high-salt buffers that release non-covalently bound molecules from stroma cells. Glycosaminoglycans purified from stromas had distinct effect on the GM-CSF-mediated proliferation of FDC-P1 cells: those purified from S17 and GR cells were stimulatory, whereas those obtained from SF cells were slightly stimulatory at low concentration, but inhibitory at the higher ones. We conclude that the quality of the stroma pericellular glycoconjugates is determinant for the ability of a given stroma to sustain myelopoiesis, even when biologically active haemopoietins are locally produced.

Animals↗
PubMed2000

Schistosoma mansoni egg-induced hepatic granulomas in mice deficient for the interferon-gamma receptor have altered populations of macrophages, lymphocytes and connective tissue cells.

Systemic production and mobilization of inflammatory cells and formation of hepatic periovular granulomas were studied in Schistosoma mansoni-infected mice with deficient interferon gamma (IFN-gamma) receptor (IFN-gammaR(o/o)). The impaired IFN-gamma signaling did not cause a significant modification of the overall kinetics of inflammatory cells, but mutant mice developed smaller hepatic periovular granulomas with a two-fold reduction in all the cell lineages. In granulomas of normal mice, the fully differentiated macrophages were progressively predominant, whilst in IFN-gammaR(o/o) mice, the granulomas contained a higher percentage of immature and proliferating monocytes. Granulomas of IFN-gammaR(o/o) mice had an enhanced and accelerated fibrotic reaction, corresponding to an increased content of proliferative and activated connective tissue cells. Simultaneously, their granulomas had an increased ratio of T over B cells, with an increase in CD8(+) and a reduction in CD4(+) T cells. The functional IFN-gamma receptor was not required for initial recruitment of monocytes and lymphocytes into granulomas, but it was necessary for the maturation of macrophages, upregulation of major histocompatibility class 2 (MHC-II) expression and consequent stimulation of lymphocyte subpopulations depending upon the MHC-II-mediated antigen presentation.

Animals↗
PubMed1999

Acidic pH modulates the interaction between human granulocyte-macrophage colony-stimulating factor and glycosaminoglycans.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) controls growth and differentiation of hematopoietic cells. Previous reports have indicated that the mitogenic activity of GM-CSF may be modulated by the glycosidic moiety of proteoglycans associated with the membrane of stromal cells. In this work, we have performed in vitro studies of the interaction between GM-CSF and glycosaminoglycans. The addition of heparin promoted a marked blue shift in the fluorescence emission spectrum of GM-CSF as well as a 30-fold increase in the intensity of light scattering, which indicates formation of large molecular weight complexes between the two molecules. Interestingly, heparin-induced changes in the spectral properties of GM-CSF were only observed at acidic pH. The dependence on acidic pH, together with a strict dependence on glycosaminoglycan sulfation and the fact that high ionic strength destabilized the interaction, indicates that the association between GM-CSF and glycosaminoglycans is mediated by electrostatic interactions. These interactions probably involve sulfate groups in the glycosaminoglycans and positively charged histidine residues in GM-CSF. We propose that negatively charged glycolipids present on the plasma membrane of the hematopoietic and/or the stromal cell could promote an acidic microenvironment capable of triggering interaction between GM-CSF and membrane-bound proteoglycans in vivo.

Binding Sites↗
PubMed1999

Extramedullar B lymphopoiesis in liver schistosomal granulomas: presence of the early stages and inhibition of the full B cell differentiation.

Inflammatory granulomatous reactions in liver elicited by schistosomal infection have been shown to function as active extramedullar myelopoietic sites, producing potentially all the myeloid lineages. We have now addressed the question of the extramedullar B lymphopoiesis in these sites. We have shown the presence of early B cell precursors (pro-B cells) in the granulomas by immunophenotyping. Their total number in the liver was equivalent to the pro-B cells in the bone marrow of one femur. In agreement with their phenotype, the RT-PCR analysis showed that these cells expressed RAG-1 and lambda5 genes. However, the conversion of the pro-B to pre-B cells was not observed and no clonogenic B cell precursors could be detected in semi-solid cultures stimulated by IL-7. The granulomatous stroma was shown to produce IL-7 and express c-kit, and was able to sustain the full B lymphopoiesis in vitro. Conversely, the granuloma supernatant was shown to inhibit actively the development of B lymphocytes. We conclude that the granuloma environment elicits homing and proliferation of totipotent hematopoietic precursors, and that it is permissive for early commitment to the B cell lineage, but the full extramedullar production of B cell is abrogated by soluble factors produced inside the granulomas.

Animals↗
PubMed1999

Replication-defective adenovirus infection reduces Helicobacter felis colonization in the mouse in a gamma interferon- and interleukin-12-dependent manner.

Helicobacter infection leads to chronic inflammation of the stomach. Although the infection persists in spite of an immune response, animal studies have shown that adjuvant-based oral vaccines can protect against infection and even eliminate established infection. These vaccines are thought to induce a Th2 immune response, counterbalancing the Th1 response seen with natural infections. As a prelude to using adenovirus vectors carrying cytokine genes to modulate the immune response to established Helicobacter felis infection, we first examined the effect of the replication-defective adenovirus (RDA) vector itself. C57BL/6 mice chronically infected with H. felis (8 to 10 weeks) received intramuscular injections of RDA. The effect of RDA on the severity of H. felis colonization and the degree of gastric inflammation was assessed 2 weeks later. RDA caused a significant decrease in H. felis colonization without significantly altering the associated inflammation. RDA did not alter the H. felis-specific immunoglobulin G1 (IgG1), IgG2a, and IgA responses in the serum but was associated with an increase in gamma interferon (IFN-gamma)-producing CD8(+) spleen cells. To determine if IFN-gamma or Th1 cytokines were involved in the response to RDA, we examined RDA treatment of H. felis infection in mice lacking either IFN-gamma or interleukin-12 (IL-12). RDA failed to alter H. felis colonization in either of these two mouse strains. Thus, viral infection of mice chronically infected with H. felis led to a significant decrease in H. felis colonization in an IFN-gamma- and IL-12-dependent manner. These results demonstrate that Th1 responses associated with systemic viral infection can influence an established H. felis infection.

Adenoviridae Infections↗
PubMed1998

Primmorphs generated from dissociated cells of the sponge Suberites domuncula: a model system for studies of cell proliferation and cell death.

Sponges (Porifera) represent the lowest metazoan phylum; they have been shown to be provided with the characteristic metazoan structural and functional molecules. One autapomorphic character of sponges is the presence of high levels of telomerase activity in all cells (or almost all cells, including somatic cells). In spite of this fact previous attempts to cultivate sponge cells remained unsuccessful. It was found that dissociated sponge cells do not replicate DNA and lose their telomerase activity. In addition, no nutrients or metabolites have been detected that would stimulate sponge cells to divide. In the present study we report the culture conditions required for the formation of multicellular aggregates from dissociated single cells of Suberites domuncula, termed primmorphs. These primmorphs are formed in seawater without addition of further supplements, and have an organised tissue-like structure; they have been cultured for more than 5 months. Cross-sections revealed a distinct external layer covered by a continuous pinacoderm, and a central zone composed primarily of spherulous cells. After their association into primmorphs, the cells turn from the telomerase-negative state into the telomerase-positive state; a telomerase level of 4.7 total product generated (TPG) units/5 x 10(3) cell equivalents has been determined. Moreover, a major fraction of the cells in the primmorphs undergoes DNA synthesis and hence has the capacity to grow. Applying the BrdU-labelling and detection assay it is demonstrated that up to 33.8% of the cells in the primmorphs are labelled with BrdU after an incubation period of 12 h. It is proposed that the primmorph system described here is a powerful novel model system to study basic mechanisms of cell proliferation and cell interaction, as well as of morphogenesis, ageing and apoptosis.

Animals↗
PubMed1998

Inhibition by the anti-mitotic drug doxorubicin of platelet-activating-factor-induced late eosinophil accumulation in rats.

Platelet-activating factor (PAF) has been shown, in the rat model of pleural inflammation, to induce the generation of an intermediate proteic factor able to cause eosinophil proliferation in vitro. This study was undertaken to investigate the effect of the anti-mitotic compound doxorubicin on PAF-induced eosinophilia in rats, in order to evaluate the contribution of local cell proliferation to this phenomenon. The late eosinophil infiltration caused by another chemoattractant leukotriene B4 was used for comparison. We observed that local treatment with doxorubicin (20 and 40 microg/cavity), given 6 h after PAF (1 microg/cavity), suppressed the eosinophil accumulation within 24 h, whilst only the higher dose was effective when the drug was given 12 h post-PAF. An effect on chemotaxis was ruled out, since local doxorubicin (40 microg/cavity) failed to modify the eosinophil migration noted 24 h after leukotriene B4 (0.5 microg/cavity) and the neutrophil/eosinophil infiltration noted at 6 h after PAF injection. Transfer of the pleural fluids collected 6 h after PAF from donors to recipient rats caused significant eosinophil accumulation in the recipient rats, an effect which was inhibited by the co-administration of doxorubicin (40 microg/cavity). No inhibitory effect was noted when the drug was given 6 h after the pleural fluids were transferred. We also found no change in the number of blood or bone marrow eosinophils after PAF stimulation. We conclude that doxorubicin selectively impaired the late eosinophil accumulation triggered by PAF in the pleural cavity of rats, clearly indicating that local cell proliferation seems to contribute to the development of this inflammatory response.

Animals↗
PubMed1998

Sponges (Porifera) model systems to study the shift from immortal to senescent somatic cells: the telomerase activity in somatic cells.

Sponges (Porifera) represent the lowest metazoan phylum, characterized by a pronounced plasticity in the determination of cell lineages. In a first approach to elucidate the molecular mechanisms controlling the switch from the cell lineage with a putative indefinite growth capacity to senescent, somatic cells, the activity of the telomerase as an indicator for immortality has been determined. The studies were performed with the marine demosponges Suberites domuncula and Geodia cydonium. It was found that the activity for the telomerase in the tissue of both sponges is high; a quantitative analysis revealed that the extract from S. domuncula contained 10.3 TPG units per 5000 cell equivalents and the one from G. cydonium 8.3 TPG units; hence the activity reached approximately 30-20% of the activity seen in telomerase-positive reference cells. In contrast, dissociated spherulous cells from G. cydonium, after an incubation period of 24 h, contained no detectable telomerase activity. From earlier studies it is known that isolated sponge cells do not proliferate. Based on these findings it is assumed that the separation of the senescent sponge cell lineage from the immortal germ/somatic cell lineage is triggered by the loss of contact with cell adhesion factors. First evidence is included which suggests that the final progress of the senescent, telomerase-negative cells to cell death is caused by apoptosis.

Animals↗
PubMed1998

Murine schistosomiasis mansoni: experimental analysis of bone marrow and peripheral myelopoiesis.

In schistosomiasis a systemic hyperplasia of the monomacrophagic cell lineage is associated with its mild modifications in myelograms and hemograms. We monitored the in vitro proliferation of myeloid precursors obtained from bone marrow, blood, spleen, and liver. The macrophage colony-forming unit (M-CFU) numbers were stable in bone marrow but increased progressively in spleen and in liver, reaching in each organ the values equivalent to one femur. The bone marrow had an increased production and enhanced capacity to release M-CFU. Their quantitative increase in blood and in peripheral tissues of schistosome-infected mice was associated with their qualitative modifications: augmented proliferative capacity, enhanced adhesion, and accelerated differentiation. The accelerated release of monomacrophage progenitors and their enhanced proliferation in peripheral tissues potentially account for the relatively low involvement of the bone marrow and for an efficient in situ production of phagocytes, which participate in host reactions to parasites.

Animals↗
PubMed1998

Experimental murine schistosomiasis mansoni: modulation of the B-1 lymphocyte distribution and phenotype expression.

We studied the B-1 lymphocyte involvement in host reactions to parasites in the murine model of schistosomiasis. No modifications were observed in the prepostural phase of the disease. From the acute phase on, we observed sequentially an increase of Mac1- B-1 cells in the spleen, followed by their appearance in Peyer's patches and in mesenteric ganglia, suggesting that a fraction of splenic B-1 cells might follow this pathway of migration, acquiring progressively the Mac1 expression. These results are consistent with a primary activation of the splenic B cell compartment, with the subsequent mobilization of B-1 cells into the tissue involved by parasites. Conversely, we found no evidence of an increase of B-1 cells in the peritoneum, nor a mobilization of B-1 cells expressing the peritoneal phenotype (CD5lo, IgMhi) into the tissues involved by infection, despite the general inflammatory reactivity of peritoneal cells. In schistosomiasis, the peritoneal cavity B-1 cells on one side, and those involved in inflammatory reactions to parasites in the spleen, Peyer's patches, and mesenteric ganglia on the other, represent two distinct B-1 lymphocyte pools.

Animals↗
PubMed1998

Retinol uptake and metabolism, and cellular retinol binding protein expression in an in vitro model of hepatic stellate cells.

Liver is a major site of retinoid metabolism and storage, and more than 80% of the liver retinoids are stored in hepatic stellate cells. These cells represent less than 1% of the total liver protein, reaching a very high relative intracellular retinoid concentration. The plasma level of retinol is maintained close to 2 microM, and hepatic stellate cells have to be able both to uptake or to release retinol depending upon the extracellular retinol status. In view of their paucity in the liver tissue, stellate cells have been studied in primary cultures, in which they loose rapidly the stored lipids and retinol, and convert spontaneously into the activated myofibroblast phenotype, turning a long-term study of their retinol metabolism impossible. We have analyzed the retinol metabolism in the established GRX cell line, representative of stellate cells. We showed that this cell line behaves very similarly, with respect the retinol uptake and release, to primary cultures of hepatic stellate cells. Moreover, we showed that the cellular retinol binding protein (CRBP-I) expression in these cells, relevant for both uptake and esterification of retinol, responds to the extracellular retinol status, and is correlated to the retinol binding capacity of the cytosol. Its expression is not associated with the overall induction of the lipocyte phenotype by other agents. We conclude that the GRX cell line represents an in vitro model of hepatic stellate cells, and responds very efficiently to wide variations of the extracellular retinol status by autonomous controls of its uptake, storage or release.

Adipocytes↗
PubMed1998

Protein malnutrition: some aspects of the in vitro adhesion of peritoneal mouse macrophages.

Protein calorie malnutrition and disease are frequently associated. Protein malnutrition modifies both the specific and nonspecific resistance of the organism to infectious agents. The exact mechanisms underlying these findings are not clear. Cellular adhesion is a crucial step in the process of phagocytosis as well as cellular migration. The effect of a low-protein diet on adhesion of macrophages was studied using an experimental murine model. We used malnourished mice that had lost 30% of their initial body weight. We then injected them with a suspension of sodium caseinate and harvested the peritoneal macrophages after 5 days. The cells were then allowed to adhere to cover slips in the presence or absence of 10% fetal calf serum (FCS) in the medium for time periods of 30, 60, 90 and 120 min. Macrophage adhesion to glass slips whose surface had been covered with type I collagen was performed only for 90 min. The expression of fibronectin was studied using an immunohistochemical technique only in the 90-min assay. The results indicate that (1) protein malnutrition impairs the activation potential of macrophages, decreasing their adhesion and expression of fibronectin; (2) when FCS is present in the medium, there is a decrease in the number of adhered cells.

Animals↗
PubMed1998

Origin of the metazoan bodyplan: characterization and functional testing of the promoter of the homeobox gene EmH-3 from the freshwater sponge Ephydatia muelleri in mouse 3T3 cells.

Porifera [sponges] represent the lowest metazoan phylum, probably already existing prior to the 'Cambrian explosion'. Based on amino acid sequences deduced from cDNAs that code for structural proteins, the monophyly of Metazoa was established. Now we analyzed for the first time a promoter of a sponge gene for its activity in a heterologous cell system from higher Metazoa. The promoter of the homeobox gene EmH-3 was cloned and sequenced from a genomic library of the freshwater sponge Ephydatia muelleri. For the determination of functional promoter activity, transient transfection experiments in mouse NIH 3T3 cells were performed; the promoter was fused with the luciferase reporter gene. The data revealed that a 401 nt long promoter fragment, comprising several binding elements for metazoan transcription factors, showed the highest activity, while the 175 bp long promoter segment, comprising solely the TATA- and Cap boxes, showed only 25% of that activity. This result demonstrates that the sponge promoter is activated by factors present in mammalian cells and supports the view that Porifera, together with the other metazoan phyla, are of monophyletic origin.

3T3 Cells↗