Dunia Ramarli - Academia.edu (original) (raw)
Papers by Dunia Ramarli
<p>LNCaP cells were left untreated or subjected to PSMA cross-linking for the indicated tim... more <p>LNCaP cells were left untreated or subjected to PSMA cross-linking for the indicated times at 37°C. An anti-VCAM-1 mAb was used as the isotype matched control. Panels A and B: p38 and ERK1/2 activation was assessed in crude lysates. Equal amounts (20 µg) of total proteins were boiled in sample buffer and separated by SDS-PAGE. Following immunoblotting with an anti-phospho-p38 or anti-phospho-ERK1/2 mAb, immunoreactive bands were visualized by using horseradish peroxidase-conjugated secondary antibody and the ECL system. Panels C and D: RAC1 and RAS activation was evaluated by a specific assay as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004608#s2" target="_blank">Material and Methods</a> section. Bound active GTP-RAC and GTP-RAS molecules were analyzed by Western blotting using an anti-RAC1 or anti-Ras mAb and visualized as above. Total amount of ERK1/2, p38, RAC1 and RAS in crude lysates are shown as loading control at the bottom of each gel. Results are representative of one of three independent experiments.</p
<p>A) LNCaP proliferation induced by treatment with CCL5 and/or IL-6. <sup>3</sup&... more <p>A) LNCaP proliferation induced by treatment with CCL5 and/or IL-6. <sup>3</sup>H-TdR uptake of cells after 24 h of culture with CCL5 (grey bars), 72 h of culture with IL-6 (hatched bars) or 24 h of culture with combinations of CCL5 and IL-6 (crossed bars) at the indicated concentrations. B) <sup>3</sup>H-TdR uptake of cells after 24 h culture with 5 ng/ml of CCL5 in the presence or in the absence of anti-CCR5 or 7E11c antibodies at the indicated concentrations; C) Western blot analysis of cell extracts (20 µg/lane) of LNCaP cells treated with CCL5 (5 ng/ml) and probed with anti-STAT 5 or with anti-cyclin D1 antibodies. Equal protein loading in all the lanes is confirmed by not specific bands shown in the middle gel. Arrows indicate phosphorylated STAT5 proteins. Western blotting results are representative of two independent experiments. D) <sup>3</sup>H-TdR uptake of cells 72 h after binding or cross-linking of 7E11c or anti-PSMA antibodies. Values represent the mean±SEM of three independent experiments performed in triplicates. Stars on the bars indicate statistical significance as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004608#pone-0004608-g004" target="_blank">Fig. 4</a>.</p
It is well known that the generation of specific cytolytic activity during Mixed Lymphocyte React... more It is well known that the generation of specific cytolytic activity during Mixed Lymphocyte Reaction (MLR) requires cooperation between cytotoxic T cell precursors (CTLp) and T helper cells (1-2). In humans these two cells can be distinguished and separated using the monoclonal antibody 5•9, which reacts with an antigenic determinant present on the helper-inducer containing subset and absent from CTLp (3). On the other hand it has been demonstrated that during MLR, help is also generated for the activation of B cells (4-6). In the present experiments we have used cloned alloreactive T cells to establish whether a single T helper cell can help both B lymphocytes and specific cytotoxic precursors. Materials and Methods Cell Suspensions. Peripheral blood mononuclear cells (PBM) were isolated by Ficoll-Hypaque centrifugation from HLA-A,-B,-C,-DR typed donors. PBM were used as stimulator cells after irradiation (3,000 R from a ~STCs source) or were rosetted with
Prostate tumor specimens express high level of Prostate Specific Membrane Antigen (PMSA) associat... more Prostate tumor specimens express high level of Prostate Specific Membrane Antigen (PMSA) associated in 50%-60% of cases with activation of anti apoptotic signaling pathways, transactivators and cytokines genes. Integrins are aberrantly expressed as their signaling adaptor p130CAS. We have previously demonstrated that clustering PSMA with specific mAbs signals to RAS-RAC-MAPK pathway activation, NF-kB translocation, IL-6 gene expression and proliferation in LNCaP cells. Our recent data show that PSMA activates both AKT/mTOR/BAD and MAPK pathways thereby rescuing LNCaP cells from apoptosis. PSMA activity relies on a Filamin A-mediated cooperation with beta1 integrin. PSMA clustering triggers the exposure of HUTS-21 activation epitope on beta1 integrin and the assembling of a complex including PSMA, Filamin A, beta1 integrin, p130CAS and Src1. All these molecules co-localize in the same lipid rafts. PSMA-mediated exposure of HUTS-21 epitope was almost abrogated by Filamin A silencing or Src1 inhibition by PP1. ERK1/2 and AKT phosphorylations were hampered by Filamin A or beta1 silencing or Src1 inhibition. These results first highlights that PSMA/beta1 integrin cooperation regulates apoptosis resistence of LNCaP cells. Moreover they show that the bridging activity of Filamin A can extend the partnership of beta1 integrin to molecules other than growth factors or cytokine receptors
Journal of immunology (Baltimore, Md. : 1950), 1996
In this study, the IFN-gamma induction of MHC class II gene expression in primary cultures of thy... more In this study, the IFN-gamma induction of MHC class II gene expression in primary cultures of thymic epithelial cells (TEC) was analyzed. This cellular system offers the advantage that MHC class II induction is studied in a "physiologic" cell lineage that, as a result of this expression within the thymus, is thought to participate to the selection and maturation of the T cells. It was found that the MHC class II gene expression was associated with the de novo transcription of the gene encoding the CIITA trans-activator, a crucial MHC class II gene regulatory factor. Furthermore, the anatomy of interaction between the MHC class II DRA promoter and corresponding binding factors was analyzed by in vivo DNAse I footprint. It was found that treatment with IFN-gamma induces changes in the occupancy of the DRA gene regulatory sequences by nuclear factors. The resulting occupancy displays strong similarities with the one observed in the MHC class II-constitutive B cells, represent...
preliminary results descriptio
Journal of immunology (Baltimore, Md. : 1950), 1999
The 8B4/20 Ag is a 120-kDa molecule whose expression on human thymocytes varies according to the ... more The 8B4/20 Ag is a 120-kDa molecule whose expression on human thymocytes varies according to the differentiation stage: high density on immature CD3-/low thymocytes, reduced density on CD3medium and double-positive thymocytes, and absent on CD3high and single-positive thymocytes and on circulating T cells. In this paper we present immunological and biochemical evidence demonstrating that 8B4/20 Ag is a variant of CD43. We show that 8B4/20-expressing molecules, which are a subset of the CD43 molecules on thymocytes, are heterogeneous in charge, suggesting varying sialylation levels. The 8B4/20 epitope was mapped to the peripherally exposed N-terminal region of CD43, and the 8B4/20 antigenic determinant was characterized by requirement for the sialic acid exocyclic polyhydroxyl side chain, a feature shared with ligands of CD22. Altogether, 8B4/20-CD43 expression pattern and biochemical characteristics suggest its participation in carbohydrate-based interactions in the thymus. We there...
Biochemical Society symposium, 1986
Recent studies using cloned antigen-specific T lymphocytes and monoclonal antibodies directed at ... more Recent studies using cloned antigen-specific T lymphocytes and monoclonal antibodies directed at their various surface glycoprotein components have led to identification of the human T cell antigen receptor as a surface complex comprised of a clonotypic 90 kDa Ti heterodimer and the invariant 20 and 25 kDa T3 molecules. Approximately 30,000-40,000 Ti and T3 molecules exist on the surface of human T lymphocytes. These glycoproteins are acquired and expressed during late thymic ontogeny, thus providing the structural basis for immunological competence. The alpha and beta subunits of Ti bear no precursor-product relationship to one another and are encoded by separate germline V, D, J and C segments which rearrange during intrathymic differentiation to form an active gene set. Triggering of the T3-Ti receptor complex induces a rapid increase in free cytoplasmic Ca2+ and gives rise to specific antigen-induced proliferation through an autocrine pathway involving endogenous interleukin-2 p...
Cells, 2021
One of the mechanisms that characterizes the aging process of different organs is the accumulatio... more One of the mechanisms that characterizes the aging process of different organs is the accumulation of fat. Different authors have demonstrated that adipose tissue replaces the loss of other cell types, deriving from mesenchymal cells. During aging, there is substitution or trans-differentiation of mesenchymal cells with other cells having the same embryological origin. Newly formed adipocytes were also observed in the trabecular matrix of elderly people’s bones, associated with myeloid cells. In this study, we have investigated the relationship between immature myeloid-derived suppressor cells (I-MDSCs) and mesenchymal stem cells (MSCs) in bone marrow (BM) samples harvested from 57 patients subjected to different orthopedic surgeries. Patients aged from 18 to 92 years were considered in order to compare the cellular composition of bone marrow of young and elderly people, considered a biomarker of immunity, inflammation, and bone preservation. The I-MDSC percentage was stable during ...
Primary biliary cirrhosis (PBC) is a chronic cholestatic progressive liver disease frequently ass... more Primary biliary cirrhosis (PBC) is a chronic cholestatic progressive liver disease frequently associated with various immunological abnormalities. We have studied the influence of normal and PBC adherent cells (AC) on peripheral blood lymphocytes (PBL) colony growth both in liquid and soft agar culture. Co-culture experiments with different combinations of AC and PBL of PBC patients and normal donors provided evidence that PBC adherent cells function abnormally. However, this impaired function is not the sole factor involved, since the number of colonies formed by PBC lymphocytes in the presence of normal AC was twice the normal value. The possibility that this increase is correlated with the state of activation of T cells is discussed in the light of the high number of DR-positive T cells found in the PBC patients studied. Finally, none of these immunological abnormalities was related to the stage of the disease.
Summary Thymocytes differentiate in the thymic microenvironment into immunocompetent T cell throu... more Summary Thymocytes differentiate in the thymic microenvironment into immunocompetent T cell through the interaction with a variety of accessory cells, including thymic epithelial cells (TEC) . TEC play an important role in the selection process presenting self antigens in association with major histocompatibilitycomplex(MHC) molecules to the maturingT cells . TheT cell receptor recognizes the selfantigen-MHC complex, but other surface molecules help stabilize this interaction. Thus, the CD2/LFA3 and LFA-1/intercellular adhesion molecule 1 pairs have been shown to participate in the binding between lymphoid cells and TEC. Here we describe an integrin of the very late activation antigen subfamily composed by the known Nt chain and by a novel ci chain. This adhesion molecule is expressed on the surface of medullary TEC and is involved in the adhesion between TEC and thymocytes, but not peripheral blood T lymphocytes.
Please note that technical editing may introduce minor changes to the text and/or graphics, which... more Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. Accepted Manuscript Faraday Discussions
The Journal of experimental medicine, 1991
Thymocytes differentiate in the thymic microenvironment into immunocompetent T cell through the i... more Thymocytes differentiate in the thymic microenvironment into immunocompetent T cell through the interaction with a variety of accessory cells, including thymic epithelial cells (TEC). TEC plays an important role in the selection process presenting self antigens in association with major histocompatibility complex (MHC) molecules to the maturing T cells. The T cell receptor recognizes the self antigen-MHC complex, but other surface molecules help stabilize this interaction. Thus, the CD2/LFA-3 and LFA-1/intercellular adhesion molecule 1 pairs have been shown to participate in the binding between lymphoid cells and TEC. Here we describe an integrin of the very late activation antigen subfamily composed by the known beta 1 chain and by a novel alpha chain. This adhesion molecule is expressed on the surface of medullary TEC and is involved in the adhesion between TEC and thymocytes, but not peripheral blood T lymphocytes.
<p>LNCaP cells were left untreated or subjected to PSMA cross-linking for the indicated tim... more <p>LNCaP cells were left untreated or subjected to PSMA cross-linking for the indicated times at 37°C. An anti-VCAM-1 mAb was used as the isotype matched control. Panels A and B: p38 and ERK1/2 activation was assessed in crude lysates. Equal amounts (20 µg) of total proteins were boiled in sample buffer and separated by SDS-PAGE. Following immunoblotting with an anti-phospho-p38 or anti-phospho-ERK1/2 mAb, immunoreactive bands were visualized by using horseradish peroxidase-conjugated secondary antibody and the ECL system. Panels C and D: RAC1 and RAS activation was evaluated by a specific assay as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004608#s2" target="_blank">Material and Methods</a> section. Bound active GTP-RAC and GTP-RAS molecules were analyzed by Western blotting using an anti-RAC1 or anti-Ras mAb and visualized as above. Total amount of ERK1/2, p38, RAC1 and RAS in crude lysates are shown as loading control at the bottom of each gel. Results are representative of one of three independent experiments.</p
<p>A) LNCaP proliferation induced by treatment with CCL5 and/or IL-6. <sup>3</sup&... more <p>A) LNCaP proliferation induced by treatment with CCL5 and/or IL-6. <sup>3</sup>H-TdR uptake of cells after 24 h of culture with CCL5 (grey bars), 72 h of culture with IL-6 (hatched bars) or 24 h of culture with combinations of CCL5 and IL-6 (crossed bars) at the indicated concentrations. B) <sup>3</sup>H-TdR uptake of cells after 24 h culture with 5 ng/ml of CCL5 in the presence or in the absence of anti-CCR5 or 7E11c antibodies at the indicated concentrations; C) Western blot analysis of cell extracts (20 µg/lane) of LNCaP cells treated with CCL5 (5 ng/ml) and probed with anti-STAT 5 or with anti-cyclin D1 antibodies. Equal protein loading in all the lanes is confirmed by not specific bands shown in the middle gel. Arrows indicate phosphorylated STAT5 proteins. Western blotting results are representative of two independent experiments. D) <sup>3</sup>H-TdR uptake of cells 72 h after binding or cross-linking of 7E11c or anti-PSMA antibodies. Values represent the mean±SEM of three independent experiments performed in triplicates. Stars on the bars indicate statistical significance as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004608#pone-0004608-g004" target="_blank">Fig. 4</a>.</p
It is well known that the generation of specific cytolytic activity during Mixed Lymphocyte React... more It is well known that the generation of specific cytolytic activity during Mixed Lymphocyte Reaction (MLR) requires cooperation between cytotoxic T cell precursors (CTLp) and T helper cells (1-2). In humans these two cells can be distinguished and separated using the monoclonal antibody 5•9, which reacts with an antigenic determinant present on the helper-inducer containing subset and absent from CTLp (3). On the other hand it has been demonstrated that during MLR, help is also generated for the activation of B cells (4-6). In the present experiments we have used cloned alloreactive T cells to establish whether a single T helper cell can help both B lymphocytes and specific cytotoxic precursors. Materials and Methods Cell Suspensions. Peripheral blood mononuclear cells (PBM) were isolated by Ficoll-Hypaque centrifugation from HLA-A,-B,-C,-DR typed donors. PBM were used as stimulator cells after irradiation (3,000 R from a ~STCs source) or were rosetted with
Prostate tumor specimens express high level of Prostate Specific Membrane Antigen (PMSA) associat... more Prostate tumor specimens express high level of Prostate Specific Membrane Antigen (PMSA) associated in 50%-60% of cases with activation of anti apoptotic signaling pathways, transactivators and cytokines genes. Integrins are aberrantly expressed as their signaling adaptor p130CAS. We have previously demonstrated that clustering PSMA with specific mAbs signals to RAS-RAC-MAPK pathway activation, NF-kB translocation, IL-6 gene expression and proliferation in LNCaP cells. Our recent data show that PSMA activates both AKT/mTOR/BAD and MAPK pathways thereby rescuing LNCaP cells from apoptosis. PSMA activity relies on a Filamin A-mediated cooperation with beta1 integrin. PSMA clustering triggers the exposure of HUTS-21 activation epitope on beta1 integrin and the assembling of a complex including PSMA, Filamin A, beta1 integrin, p130CAS and Src1. All these molecules co-localize in the same lipid rafts. PSMA-mediated exposure of HUTS-21 epitope was almost abrogated by Filamin A silencing or Src1 inhibition by PP1. ERK1/2 and AKT phosphorylations were hampered by Filamin A or beta1 silencing or Src1 inhibition. These results first highlights that PSMA/beta1 integrin cooperation regulates apoptosis resistence of LNCaP cells. Moreover they show that the bridging activity of Filamin A can extend the partnership of beta1 integrin to molecules other than growth factors or cytokine receptors
Journal of immunology (Baltimore, Md. : 1950), 1996
In this study, the IFN-gamma induction of MHC class II gene expression in primary cultures of thy... more In this study, the IFN-gamma induction of MHC class II gene expression in primary cultures of thymic epithelial cells (TEC) was analyzed. This cellular system offers the advantage that MHC class II induction is studied in a "physiologic" cell lineage that, as a result of this expression within the thymus, is thought to participate to the selection and maturation of the T cells. It was found that the MHC class II gene expression was associated with the de novo transcription of the gene encoding the CIITA trans-activator, a crucial MHC class II gene regulatory factor. Furthermore, the anatomy of interaction between the MHC class II DRA promoter and corresponding binding factors was analyzed by in vivo DNAse I footprint. It was found that treatment with IFN-gamma induces changes in the occupancy of the DRA gene regulatory sequences by nuclear factors. The resulting occupancy displays strong similarities with the one observed in the MHC class II-constitutive B cells, represent...
preliminary results descriptio
Journal of immunology (Baltimore, Md. : 1950), 1999
The 8B4/20 Ag is a 120-kDa molecule whose expression on human thymocytes varies according to the ... more The 8B4/20 Ag is a 120-kDa molecule whose expression on human thymocytes varies according to the differentiation stage: high density on immature CD3-/low thymocytes, reduced density on CD3medium and double-positive thymocytes, and absent on CD3high and single-positive thymocytes and on circulating T cells. In this paper we present immunological and biochemical evidence demonstrating that 8B4/20 Ag is a variant of CD43. We show that 8B4/20-expressing molecules, which are a subset of the CD43 molecules on thymocytes, are heterogeneous in charge, suggesting varying sialylation levels. The 8B4/20 epitope was mapped to the peripherally exposed N-terminal region of CD43, and the 8B4/20 antigenic determinant was characterized by requirement for the sialic acid exocyclic polyhydroxyl side chain, a feature shared with ligands of CD22. Altogether, 8B4/20-CD43 expression pattern and biochemical characteristics suggest its participation in carbohydrate-based interactions in the thymus. We there...
Biochemical Society symposium, 1986
Recent studies using cloned antigen-specific T lymphocytes and monoclonal antibodies directed at ... more Recent studies using cloned antigen-specific T lymphocytes and monoclonal antibodies directed at their various surface glycoprotein components have led to identification of the human T cell antigen receptor as a surface complex comprised of a clonotypic 90 kDa Ti heterodimer and the invariant 20 and 25 kDa T3 molecules. Approximately 30,000-40,000 Ti and T3 molecules exist on the surface of human T lymphocytes. These glycoproteins are acquired and expressed during late thymic ontogeny, thus providing the structural basis for immunological competence. The alpha and beta subunits of Ti bear no precursor-product relationship to one another and are encoded by separate germline V, D, J and C segments which rearrange during intrathymic differentiation to form an active gene set. Triggering of the T3-Ti receptor complex induces a rapid increase in free cytoplasmic Ca2+ and gives rise to specific antigen-induced proliferation through an autocrine pathway involving endogenous interleukin-2 p...
Cells, 2021
One of the mechanisms that characterizes the aging process of different organs is the accumulatio... more One of the mechanisms that characterizes the aging process of different organs is the accumulation of fat. Different authors have demonstrated that adipose tissue replaces the loss of other cell types, deriving from mesenchymal cells. During aging, there is substitution or trans-differentiation of mesenchymal cells with other cells having the same embryological origin. Newly formed adipocytes were also observed in the trabecular matrix of elderly people’s bones, associated with myeloid cells. In this study, we have investigated the relationship between immature myeloid-derived suppressor cells (I-MDSCs) and mesenchymal stem cells (MSCs) in bone marrow (BM) samples harvested from 57 patients subjected to different orthopedic surgeries. Patients aged from 18 to 92 years were considered in order to compare the cellular composition of bone marrow of young and elderly people, considered a biomarker of immunity, inflammation, and bone preservation. The I-MDSC percentage was stable during ...
Primary biliary cirrhosis (PBC) is a chronic cholestatic progressive liver disease frequently ass... more Primary biliary cirrhosis (PBC) is a chronic cholestatic progressive liver disease frequently associated with various immunological abnormalities. We have studied the influence of normal and PBC adherent cells (AC) on peripheral blood lymphocytes (PBL) colony growth both in liquid and soft agar culture. Co-culture experiments with different combinations of AC and PBL of PBC patients and normal donors provided evidence that PBC adherent cells function abnormally. However, this impaired function is not the sole factor involved, since the number of colonies formed by PBC lymphocytes in the presence of normal AC was twice the normal value. The possibility that this increase is correlated with the state of activation of T cells is discussed in the light of the high number of DR-positive T cells found in the PBC patients studied. Finally, none of these immunological abnormalities was related to the stage of the disease.
Summary Thymocytes differentiate in the thymic microenvironment into immunocompetent T cell throu... more Summary Thymocytes differentiate in the thymic microenvironment into immunocompetent T cell through the interaction with a variety of accessory cells, including thymic epithelial cells (TEC) . TEC play an important role in the selection process presenting self antigens in association with major histocompatibilitycomplex(MHC) molecules to the maturingT cells . TheT cell receptor recognizes the selfantigen-MHC complex, but other surface molecules help stabilize this interaction. Thus, the CD2/LFA3 and LFA-1/intercellular adhesion molecule 1 pairs have been shown to participate in the binding between lymphoid cells and TEC. Here we describe an integrin of the very late activation antigen subfamily composed by the known Nt chain and by a novel ci chain. This adhesion molecule is expressed on the surface of medullary TEC and is involved in the adhesion between TEC and thymocytes, but not peripheral blood T lymphocytes.
Please note that technical editing may introduce minor changes to the text and/or graphics, which... more Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. Accepted Manuscript Faraday Discussions
The Journal of experimental medicine, 1991
Thymocytes differentiate in the thymic microenvironment into immunocompetent T cell through the i... more Thymocytes differentiate in the thymic microenvironment into immunocompetent T cell through the interaction with a variety of accessory cells, including thymic epithelial cells (TEC). TEC plays an important role in the selection process presenting self antigens in association with major histocompatibility complex (MHC) molecules to the maturing T cells. The T cell receptor recognizes the self antigen-MHC complex, but other surface molecules help stabilize this interaction. Thus, the CD2/LFA-3 and LFA-1/intercellular adhesion molecule 1 pairs have been shown to participate in the binding between lymphoid cells and TEC. Here we describe an integrin of the very late activation antigen subfamily composed by the known beta 1 chain and by a novel alpha chain. This adhesion molecule is expressed on the surface of medullary TEC and is involved in the adhesion between TEC and thymocytes, but not peripheral blood T lymphocytes.