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D Brouty-Boyé

Publications and source records attributed to D Brouty-Boyé.

At least 19 recordsLinked to original sources
PubMed2006

Co expression of SCF and KIT in gastrointestinal stromal tumours (GISTs) suggests an autocrine/paracrine mechanism.

KIT is a tyrosine kinase receptor expressed by several tumours, which has for specific ligand the stem cell factor (SCF). KIT is the main oncogene in gastrointestinal stromal tumours (GISTs), and gain-of-function KIT mutations are present in 70% of these tumours. The aim of the study was to measure and investigate the mechanisms of KIT activation in 80 KIT-positive GIST patients. KIT activation was quantified by detecting phosphotyrosine residues in Western blotting. SCF production was determined by reverse transcriptase-PCR, ELISA and/or immunohistochemistry. Primary cultures established from three GISTs were also analysed. The results show that KIT activation was detected in all cases, even in absence of KIT mutations. The fraction of activated KIT was not correlated with the mutational status of GISTs. Membrane and soluble isoforms of SCF mRNA were present in all GISTs analysed. Additionally, SCF was also detected in up to 93% of GISTs, and seen to be present within GIST cells. Likewise, the two SCF mRNA isoforms were found to be expressed in GIST-derived primary cultures. Thus, KIT activation in GISTs may in part result from the presence of SCF within the tumours.

Autocrine Communication↗
PubMed2005

Importance of stromal determinants in the generation of dendritic and natural killer cells in the human spleen.

Summary The interaction between stroma and blood cells in the human spleen has received little attention, despite their well-defined roles during blood cell development in bone marrow. We have reported previously that human spleen-derived fibroblasts display a differentiated myofibroblast phenotype and constitutively express a biologically active form of membrane interleukin (IL)-15 that can drive co-cultured CD34(+) blood cells to differentiate into activated natural killer (NK) cells. Here, we show that, in addition to NK cells, CD34/fibroblast co-cultures also yield myeloid CD1a(+)CD38(+)CD68(+)CD86(+) HLA-DR(+)CD14(-)CD80(-) dendritic cells (DCs) after 3-4 weeks in culture. We found that DC development depended on endogenously secreted stromal macrophage colony-stimulating factor (M-CSF) and CD40/CD40L interaction rather than on fibroblast- and CD34-derived membrane IL-15. CD1a(+) cells were necessary for co-produced NK cells to acquire lytic functions by a mechanism involving cell-to-cell contact and DC-derived IL-12. This study highlights the importance of spleen myofibroblasts in the in vitro generation of two distinct cell types (DC and NK cells) from the innate immune system and suggests that the human spleen is involved in the generation of NK cells from circulating progenitors.

Antigens, CD1↗
PubMed2004

Inflammatory status and cartilage regenerative potential of synovial fibroblasts from patients with osteoarthritis and chondropathy.

OBJECTIVES: To evaluate the inflammatory status and the cartilage regenerative potential of pathological synovial fibroblasts from patients with osteoarthritis (OA) compared with non-inflamed synovium (NS)-derived cells from patients with chondropathy. METHODS: The inflammatory cell phenotype was investigated based on the constitutive and inducible surface expression and secretion of various effector molecules using flow cytometry or ELISA assays. The capacity of cells to produce cartilage-like extracellular matrix was assessed using acid Alcian blue staining and type II collagen immunostaining after treatment with transforming growth factor beta1 (TGF-beta1). RESULTS: OA and NS fibroblasts consistently expressed CD29, CD44, CD49e, CD54, CD90 and CD106. Expression of high-affinity receptors for IL-4, IL-15, CXCL8 and CXCL12 was also detected but only intracellularly. All types of fibroblasts spontaneously released abundant amounts of CXCL12, CCL2, IL-6 and tissue inhibitor of metalloproteinase 1, while the production of IL-11, TGF-beta1, matrix metalloproteinase 1 (MMP-1) and MMP-9 was detected at moderate levels. Several other secreted factors remained undetectable. No statistically significant differences were noted between the two groups of fibroblasts. Treatment with the proinflammatory cytokine tumour necrosis factor alpha (TNF-alpha) up-regulated the same set of surface and secreted molecules, including CD54, CD106, membrane IL-15, CCL2 and CCL5. Under TGF-beta1 treatment and adipogenic culture conditions, both OA and NS fibroblasts displayed chondrogenic and adipocytic activities that were reduced in OA compared with NS cells. CONCLUSIONS: OA synovial fibroblasts did not display a distinct activated inflammatory phenotype compared with NS cells. However, they did differ in their reduced ability to produce cartilage-like matrix. This difference may be an additional important factor contributing to OA pathogenesis.

Adipocytes↗
PubMed2001

Effects of human fibroblasts from myelometaplasic and non-myelometaplasic hematopoietic tissues on CD34+ stem cells.

Fibroblasts demonstrate different phenotypes and functions according to the tissue of origin and its physiopathologic state. We previously showed that fibroblasts isolated in culture from myelometaplasic (MM) spleen differed phenotypically from fibroblasts from normal bone marrow (BM). We compared the influence of each type of fibroblasts on the behavior of CD34+ stem cells. Expansion of nucleated cells was observed when blood CD34+ cells were co-cultured for 3 weeks with MM spleen-derived fibroblasts in monolayers. Myeloid cell differentiation was also observed as indicated by a decline in CD34+ cells and increases in CD14+, CD15+ and CD41+ cells. This myeloid differentiation was enhanced in the presence of MM spleen compared with normal BM-derived fibroblasts. Similarly, proliferation and differentiation of BM CD34+ cells was better in the presence of BM rather than MM spleen-derived fibroblasts. In addition, fibroblasts from MM spleen also induced a differentiation of CD56+ natural killer (NK) cells whereas BM-derived fibroblasts did not. Overall, the data indicate that cultured fibroblasts from diseased tissue have distinct growth and differentiation regulatory characteristics. They also suggest a role for these cells in hematopoietic disorders.

Antigens, CD34↗
PubMed2000

Chemokines and CD40 expression in human fibroblasts.

Chemokines are cytokines specialized for recruiting leukocytes in inflammatory responses. Recent data indicate that besides macrophages and leukocytes fibroblasts may also be a source of these important immune molecules. We assayed chemokine expression (mRNA/ protein) in cultured fibroblasts isolated from a variety of human tissues and different pathologic states: normal bone marrow vs. myelometaplastic spleen, normal lung vs. metastasis stroma, and normal breast vs. radiation fibrosis and tumor stroma. In all fibroblasts, transcripts for chemokines IL-8, stromal cell-derived factor-1, monocyte chemotactic protein (MCP)-1 and eotaxin were detected. Although the production of IL-8 was abundant in most of the fibroblasts studied, fibroblasts from lung and pathologic breast tissue produced significantly less. Conversely, eotaxin production was low in most fibroblasts except in those isolated from myelometaplastic tissue where it was highly produced. Moreover, chemokines MCP-4, RANTES and macrophage inflammatory protein-1alpha were found to be expressed only in fibroblasts from select tissues. When the expression of CD40, an activating surface molecule for immune cells, was investigated, we found that most of the fibroblasts expressed this antigen. Overall these results indicate that cultured human fibroblasts from various tissues and pathologic settings produce a distinct panel of chemokines and express CD 40, suggesting a possible fundamental role of fibroblasts in immune responses and disease processes.

Breast↗
PubMed1998

Phenotypic diversity in human fibroblasts from myelometaplasic and non-myelometaplasic hematopoietic tissues.

Fibroblasts from a variety of tissues interact with and influence the behavior of the cell types they are associated with by producing specific proteins that mediate these interactions. Thus, it is not surprising that fibroblasts have been shown to differ phenotypically and functionally depending on the tissue they are isolated from and its physiologic state. To study fibroblasts of hematopoietic tissues, cultures were established from human normal bone marrow (BM), and from non-myelometaplasic (NS) and myelometaplasic spleen (MMS) tissues and analyzed for phenotypic characteristics. The results are summarized as follows: (1) cytoskeletal elements: virtually all the MMS fibroblasts were stained positively for alpha-sm-actin while only a small fraction of BM and of NS fibroblasts were positive for this antigen; (2) extracellular matrix elements: MMS fibroblasts stained positively for ED-B fibronectin and tenascin while the other 2 fibroblast cell types did not; (3) cell surface molecules: NS and MMS fibroblasts expressed significantly higher levels of ICAM-1, VLA-4 and CD9 than BM fibroblasts. Moreover, MMS fibroblasts showed a higher expression of ICAM-1 and VLA-4 than NS fibroblasts; and (4) cytokines: IL-II, RANTES and MIP-1alpha were produced in higher amounts by BM than by NS fibroblasts. Conversely, production of GM-CSF, SCF, M-CSF and MCP-1alpha was elevated in NS compared with BM fibroblasts. The production of these cytokines was generally reduced in MMS cells. Overall, our results demonstrate that phenotypic characteristics can be identified to distinguish fibroblasts from normal and pathologic hematopoietic tissues. Such phenotypic characteristics suggest functional differences of each type of fibroblast in their influence on the blood cells with which they are associated.

Adult↗
PubMed1998

Interleukin (IL) 4 and IL-13 act on human lung fibroblasts. Implication in asthma.

Airway hyperresponsiveness leading to subepithelial fibrosis is mediated by inflammatory cells activated by T helper (Th) 2-derived cytokines such as IL-4 and IL-5. By analyzing the phenotype and response of human lung fibroblasts derived from either fetal (ICIG7) or adult (CCL202) tissue as well as from a Th2-type stromal reaction (FPA) to IL-4 and IL-13, we provide evidence that human lung fibroblasts may behave as inflammatory cells upon activation by IL-4 and IL-13. We show that the three types of fibroblasts constitute different populations that display a distinct pattern in cell surface molecule expression and proinflammatory cytokine and chemokine release. All fibroblasts express functional but different IL-4/IL-13 receptors. Thus, while IL-4 receptor (R) alpha and IL-13Ralpha1 chains are present in all the cells, CCL202 and FPA fibroblasts coexpress the IL-13Ralpha2 and the IL-2Rgamma chain, respectively, suggesting the existence of a heterotrimeric receptor (IL-4Ralpha/IL-13Ralpha/IL-2Rgamma) able to bind IL-4 and IL-13. Stimulation with IL-4 or IL-13 triggers in the fibroblasts a differential signal transduction and upregulation in the expression of beta1 integrin and vascular cell adhesion molecule 1 and in the production of IL-6 and monocyte chemoattractant protein 1, two inflammatory cytokines important in the pathogenesis of allergic inflammation. Our results suggest that when activated by IL-4 and IL-13, different subsets of lung fibroblasts may act as effector cells not only in the pathogenesis of asthma but also in lung remodeling processes. They may also differentially contribute to trigger and maintain the recruitment, homing, and activation of inflammatory cells.

Antigens, Surface↗
PubMed1998

IL-15 is produced by a subset of human melanomas, and is involved in the regulation of markers of melanoma progression through juxtacrine loops.

IL-15 is a novel cytokine active through the IL-2R/betagamma. Since several human melanoma cell lines display functional IL-2Rs, we studied the IL-15/melanoma cells interactions. Ten out of 17 melanoma cell lines express the IL-15 transcript and four of them express levels of IL-15 mRNA similar to those detected in control activated monocytes. Nine out of ten cell lines also express two transcripts for the IL-15R alpha originated by the alternative splicing of exon'3'. Two melanoma cell lines, MELP and MELREO, derived from patients with rapidly progressive primary melanomas, co-express the two IL-15 transcripts, originated by alternative splicing of exon 'A'. Intracellular IL-15 protein was only detected in these two cells lines and it is mainly retained in the Endoplasmic Reticulum (ER). However, a small amount of IL-15 is also found in the Golgi apparatus and in the early endosomes, suggesting production and intercellular trafficking of endogenous IL-15 protein. Nevertheless, no biologically active IL-15 could be detected in the supernatant of all melanoma cells. The anti IL-15 blocking mAb M111 causes the up regulation of HLA Class I in dense MELP and MELREO cultures. These data suggest that IL-15 is probably active through juxtacrine loops negatively controlling HLA Class I molecules expression. These data offer, for the first time, a likely explanation to the controversial issue of IL-15 secretion and constitute a natural model for understanding IL-15 routing. Moreover, we identify a subset of melanoma cells producing IL-15, possibly involved in tumor escape mechanisms.

Biomarkers, Tumor↗
PubMed1998

IL-4 and IL-13 specifically increase adhesion molecule and inflammatory cytokine expression in human lung fibroblasts.

Subepithelial fibrosis in the bronchi of asthmatics is the result of an irreversible lung fibroblast activation, triggered by cytokines secreted by IL-4- and IL-5-activated inflammatory cells. Here, we provide evidence that human lung fibroblasts (ICIG7 cells) express a single class of high-affinity IL-4 receptor (IL-4R). This receptor is functional and composed of at least the IL-4Ralpha and IL-13Ralpha1 chains in the absence of the IL-2Rgamma chain. The IL-4Ralpha is efficiently internalized at 37 degrees C within 15 min in the presence of IL-4, whereas this process is slower with IL-13. In ICIG7 cells, IL-4 triggers the tyrosine phosphorylation of at least two proteins (110 and 180 kDa), and up-regulates the transcription of c-fos, c-jun and c-myc proto-oncogenes. In addition, the secretion of several cytokines [IL-6, granulocyte colony stimulating factor and granulocyte macrophage colony stimulating factor (GM-CSF)] as well as the expression of beta1 integrin and VCAM-1 adhesion molecules are augmented by IL-4. IL-13 displays similar biological activities, but less effectively than IL-4. On the other hand, ICIG7 cells could constitute a lung fibroblast population defined by the spontaneous release of several pro-inflammatory cytokines (IL-6, IL-11 and GM-CSF) and cell surface phenotype (CD4 and Thy-1). Through this peculiar cytokine pattern and the IL-4/IL-13-dependent activities, these cells could act as effector cells in the pathogenesis of asthma, triggering and maintaining the recruitment, homing and activation of bone marrow-derived inflammatory cells, and playing a role in the remodeling process of the airways.

Cell Adhesion Molecules↗
PubMed1997

Discrimination of fibroblast subtypes by multivariate analysis of gene expression.

Fibroblasts and myofibroblasts from normal, fibrotic or tumoral breast tissues present multiple quantitative differences in gene expression even when grown in isolation. We were therefore prompted to investigate whether one could recognize various subtypes by their constitutive-gene expression profile. Quantitative autoradiographic data for 34 constitutively expressed transcripts were submitted to multivariate analysis of variance, followed by discriminant analysis and single linkage cluster analysis. Models assuming up to 8 putative fibroblast subtypes (among fibroblasts or myofibroblasts from breast skin, normal mammary stroma, tumor-adjacent "normal" stroma, post-radiation fibrosis lesions and benign or malignant tumors) and an epithelial-cell group used as an internal control resulted in 100% correct classification. Myofibroblasts from various origins clustered close to, although distinctly apart from, their corresponding alpha-smooth-muscle-actin-negative counterparts. Malignant tumor fibroblasts were phenotypically more distant from normal cells compared with other pathological types. Our results support the hypothesis of co-adaptive transformation of stromal and epithelial tissues during breast tumoral development and suggest that different types of fibroblasts give rise to different types of myofibroblasts. Discriminant analysis of quantitative molecular variation may be considered for the development of a powerful artificial-intelligence method for cell typing and should be particularly useful when no reliable discrete molecular markers are available.

Breast↗
PubMed1995

Quantitative variation of proto-oncogene and cytokine gene expression in isolated breast fibroblasts.

Transcripts coding for transcription factors (RB, P53, FOS, MYC, MYB, ERBA, REL), growth factors (FGF1, FGF2, INT2, TGFA, TGFB, PDGF, IGF1, IGF2), interleukins, (IL1, IL2, IL3, IL4, IL6, TNF), growth-factor receptors or cytosolic protein kinases (RAF, PIM, FES, MET, SRC, ROS, TRK, KIT, CSFR, IGFR, PDGFR, EGFR, NEU) were quantified in cultured human mammary fibroblasts from normal tissues, benign tumours, carcinomas and post-radiation fibrosis lesions by slot-blot autoradiography and image analysis. The effects of a differentiating agent (cholera toxin) and of a tumour promoter (12-O-tetradecanoyl-phorbol-13-acetate) were also examined. The drugs modulated the levels of the anti-oncogene transcripts (RB, P53) and of ERBA, REL, RAF, MET, ROS, TRK, CSFR, EGFR, NEU, FGF1, INT2, IGF1, IL1, IL2, IL4 and IL6. Apart from this variation, there were multiple differences in gene expression among normal and pathological cells (concerning all but P53, TGFB and interleukin transcripts) and between sub-types defined by the presence of alpha-sm-actin (myofibroblasts) or EDB-fibronectin (RAF, ROS, FES, KIT, IGFR, NEU, INT2, TGFB, PDGF, IGFs, ILs). It appears, therefore, that mammary stroma progress irreversibly along with the epithelium during tumoral development, and that breast cancer is not only a multi-gene but also a multi-tissue phenotype.

Breast↗
PubMed1995

Relationship of multidrug resistance to rhodamine-123 selectivity between carcinoma and normal epithelial cells: taxol and vinblastine modulate drug efflux.

Preferential retention and cytotoxicity of Rhodamine-123 (Rho-123) was originally reported in a number of carcinoma cell types isolated from a variety of tissues as compared to normal epithelial cells from a limited number of other tissues. In the present study, we have examined Rho-123 selectivity in normal and tumor cell lines isolated from the same tissue source, i.e., human breast. We found that: (a) in matched pairs of normal and carcinoma breast cells, Rho-123 displays no preferential retention in either cell type; (b) there is no preferential toxicity in carcinoma as compared to normal breast cells; in fact, one of the carcinoma cell lines (MDA-MB231) shows moderate resistance to this dye; (c) all of the human breast cell lines do not express P-glycoprotein-mediated multidrug resistance; (d) the normal monkey kidney epithelial cell line CV-1, which was originally used as a model to demonstrate the relative resistance of normal epithelial cells to this drug, is found to express high levels of the mdr-1 gene, is resistant to other multidrug-resistant drugs (taxol and vinblastine), and its resistance to Rho-123 as well as decreased Rho-123 retention can be reversed by verapamil; and (e) taxol and vinblastine are found to block increased Rho-123 efflux in CV-1 cells. Thus, overall the data suggest that preferential retention and cytotoxicity of Rho-123 in carcinoma versus normal epithelial cells is related to the differential expression of the mdr-1 gene.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Evaluation of quantitative variation in gene expression.

We investigate the behaviour of the gene-expression rate as a statistical variable using autoradiographic data for 39 transcripts from a heterogeneous set of 80 breast-tissue cultures. Despite standardization, the data distributions of all transcripts showed intervals of normality and intervals of systematic departure from normality which most frequently resulted in a significant skewness and/or kurtosis. Non-normal shapes are attributed to modulation of gene expression. This statistical particularity creates difficulties in the evaluation of differences among specimens. Using classical parametric and non-parametric procedures for normal and non-normal variation, respectively, we demonstrate that large differences in optical density are neither necessary nor sufficient for associating expression rates with biological factors. The transcripts coding for the metalloprotease stromelysin-3 (ST3) and for the receptor to insulin-like growth factors (IGFR) are used as examples and their variation is presented in detail. ST3 expression appeared to be specifically associated with mammary stroma fibroblasts derived from post-radiation fibrosis lesions. IGFR was expressed at higher rates in mammary gland and skin fibroblasts than in mammary epithelial cells and was subject to frequent and strong modulation.

Animals↗
PubMed1994

Fibroblast-mediated differentiation in human breast carcinoma cells (MCF-7) grown as nodules in vitro.

The growth of cells in 3-dimensional form as nodules in vitro facilitates studies of in vivo cellular interactions. Taking advantage of this technique, human breast carcinoma cells (MCF-7) were co-cultured with stromal fibroblasts isolated from either normal or tumorous breast tissue to study the influence of such fibroblasts on tumor-cell growth and differentiation. Ten days after co-culture of carcinoma cells with fibroblasts from normal tissue at a 1:10 ratio, the size of nodules began to increase and stabilize by day 30 while the fibroblast number decreased and finally disappeared. Concurrently, the carcinoma cells underwent a progressive redifferentiation process which histologically resulted in the appearance of highly developed papillar and tubular structures after 2 months in culture. The production of mucins was further evidence that these cells had undergone differentiation. By contrast, when MCF-7 cells were grown alone or with fibroblasts isolated from a breast carcinoma, the nodules continued to exhibit their characteristic histodedifferentiation properties and did not grow. The re-establishment of a normal epithelial state of differentiation in MCF-7 carcinoma nodules indicates that the phenotypic characteristics of tumor cells are reversible and are influenced or controlled by the stromal environment by which these tumor cells are surrounded or in contact with. Overall, our results open the possibility of exploiting the effects that connective tissue cells have on tumor-cell differentiation for use in prevention and treatment of cancer.

Breast Neoplasms↗
PubMed1994

Myofibroblast and concurrent ED-B fibronectin phenotype in human stromal cells cultured from non-malignant and malignant breast tissue.

Primary cultures of stromal cells from non-malignant and malignant breast tissues contained myofibroblasts based on immunoreactivity to alpha-smooth muscle (alpha-sm) actin. The proportions of these cells were variable among cultures from non-malignant origin while consistently high in cultures from carcinomas. High expression of ED-B fibronectin and of type V collagen was observed in myofibroblast-containing cultures. While cells from non-malignant tissues grew relatively steadily, the proliferation of carcinoma-derived cells declined during serial subculturing. In both types of cultures, alpha-sm actin and ED-B fibronectin expression decreased with increasing passage numbers. Epidermal growth factor (EGF), fibroblast growth factor b (FGFb), transforming growth factor alpha (TGF alpha) and platelet-derived growth factor (PDGF) showed consistent mitogenic effects. Addition of FGFb prolonged culture growth and allowed alpha-sm actin and ED-B fibronectin expression to persist. These results demonstrate similar phenotypic modulations in stromal cells from non-malignant and malignant breast tissues that may reflect a common stromal response to various tissue injuries, including neoplasia.

Actins↗