Bone marrow (BM) acts as a dynamic organ within the bone cavity, responsible for hematopoiesis, s... more Bone marrow (BM) acts as a dynamic organ within the bone cavity, responsible for hematopoiesis, skeletal remodeling, and immune system control. Bone marrow adipose tissue (BMAT) was long simply considered a filler of space, but now it is known that it instead constitutes an essential element of the BM microenvironment that participates in homeostasis, influences bone health and bone remodeling, alters hematopoietic stem cell functions, contributes to the commitment of mesenchymal stem cells, provides effects to immune homeostasis and defense against infections, and participates in energy metabolism and inflammation. BMAT has emerged as a significant contributor to the development and progression of various diseases, shedding light on its complex relationship with health. Notably, BMAT has been implicated in metabolic disorders, hematological malignancies, and skeletal conditions. BMAT has been shown to support the proliferation of tumor cells in acute myeloid leukemia and niche adipocytes have been found to protect cancer cells against chemotherapy, contributing to treatment resistance. Moreover, BMAT’s impact on bone density and remodeling can lead to conditions like osteoporosis, where high levels of BMAT are inversely correlated with bone mineral density, increasing the risk of fractures. BMAT has also been associated with diabetes, obesity, and anorexia nervosa, with varying effects on individuals depending on their weight and health status. Understanding the interaction between adipocytes and different diseases maylead to new therapeutic strategies.
INTRODUCTION Dermal fibrosis and sclerosis are pathologic features shared by Scleroderma-like chr... more INTRODUCTION Dermal fibrosis and sclerosis are pathologic features shared by Scleroderma-like chronic graft-versus-host disease (Scl-cGVHD) and Systemic Scleroderma (SSc). Moreover, in both diseases stimulating anti-PDGF-R antibodies were found, leading to abnormal collagen production by fibroblasts, eventually contributing to organ damage. Targeted therapy with tyrosine kinase inhibitors (TKI) like Imatinib and Nilotinib demonstrated clinical efficacy in Scl-cGVHD; however, the molecular basis underpinning the clinical effects are not fully elucidated. We investigated here a potential terapeutical target of the dermal cGVHD pathophysiology: the cellular and molecular features of pathological skin fibroblasts (GVHD-Fbs) and the efficacy of Nilotinib on fibrosis modulation. MATERIALS AND METHODS Fibroblast cultures (GVHD-Fbs) were obtained from skin biopsies of affected skin from 6 patients with active cGVHD, control fibroblasts are Human Dermal Fibroblasts adult (n-FBS). Fibroblasts were characterized by flow cytometry (FACS CANTO II) for the detection of molecules: CD10, CD14, CD29, CD34, CD44, CD45, CD73, CD90, CD105, CD106, CD117, CD146. In order to evaluate the adipogenic, osteogenic or chondrogenic differentiation cGVHD-Fbs and n-Fbs (n = 3) were cultured in differentiation medium (respectively NH AdipoDiff, NH OsteoDiff, NH ChondroDiff) after four passages. Intracellular lipid droplets indicated adipogenic lineage differentiation. The differentiation potential in the osteogenic lineage was evaluated by calcium accumulation, as assessed by Alizarin Red. The pellet obtained from chondrogenic lineage differentiation was embedded in paraffin, cut in the microtome and the sections placed on a glass slide were stained with Alcian Blue [Junker JP, Cells Tissues Organs, 2010]. For incubation with Nilotinib (Santa Cruz Biotechnology) the 10 mM stock solution was diluted to the final concentration in DMEM supplemented with 0,2% FBS (starvation), added to cell cultures at a concentration of 1 μM or 2 μM for 48h, which covered the mean plasma levels in cGVHD patients after standard doses. In subsets of experiments, after starvation, fibroblasts were stimulated with recombinant TGFβ at 10 ng/ml (GIBCO, Invitrogen). After incubation, total RNA was isolated and reverse transcribed. Gene expression was quantified by real-time PCR using the Sybr Green Mix for qPCR. Specific primer pairs for COL1α1 and COL1α2 were designed with the Primer 3 software. The transcript levels were normalized for the expression of GAPDH constitutive gene. Differences were calculated with the threshold cycle (Ct) and the comparative Ct method for relative quantification. RESULTS GVHD-Fbs are morphologically and phenotypically similar to normal fibroblasts (n-FBS). GVHD-FBS did not show a different immunophenotype from n-Fbs, both in early and late culture passages. Also, no differences were noted between GVHD-Fbs and n-FBS in terms of multilineage differentiation capacity towards the adipogenic, osteogenic and chondrogenic lineage. Gene expression of COL1α1 and COL1α2 in GVHD-Fbs was respectively 4 and 1,6 times higher compared to n-FBS (p = 0.02). However, the increased collagen expression was exclusive of early-passage GVHD-Fbs; in late-passage…
1. Hoy SM. Obinutuzumab: a review of its use in patients with chronic lymphocytic leukaemia. Drug... more 1. Hoy SM. Obinutuzumab: a review of its use in patients with chronic lymphocytic leukaemia. Drugs. 2015;75(3):285–96. 2. Goede V, Fischer K, Busch R, Engelke A, Eichhorst B, Wendtner CM, et al. Obinutuzumab plus chlorambucil in patients with CLL and coexisting conditions. N Engl J Med. 2014;370(12):1101–10. 3. Fischer K, Al-Sawaf O, Bahlo J, Fink AM, Tandon M, Dixon M, et al. Venetoclax and obinutuzumab in patients with CLL and coexisting conditions. N Engl J Med. 2019;380(23):2225–36. 4. Moreno C, Greil R, Demirkan F, Tedeschi A, Anz B, Larratt L, et al. Ibrutinib plus obinutuzumab versus chlorambucil plus obinutuzumab in firstline treatment of chronic lymphocytic leukaemia (iLLUMINATE): a multicentre, randomised, open-label, phase 3 trial. Lancet Oncol. 2019;20(1): 43–56. 5. Reynaud Q, Durieu I, Dutertre M, Ledochowski S, Durupt S, Michallet AS, et al. Efficacy and safety of rituximab in auto-immune hemolytic anemia: a meta-analysis of 21 studies. Autoimmun Rev. 2015;14(4):304–13. 6. Dierickx D, Kentos A, Delannoy A. The role of rituximab in adults with warm antibody autoimmune hemolytic anemia. Blood. 2015;125(21):3223–9. 7. Narat S, Gandla J, Hoffbrand AV, Hughes RG, Mehta AB. Rituximab in the treatment of refractory autoimmune cytopenias in adults. Haematologica. 2005;90(9):1273–4. 8. D’Arena G, Laurenti L, Capalbo S, D’Arco AM, De Filippi R, Marcacci G, et al. Rituximab therapy for chronic lymphocytic leukemia-association autoimmune hemolytic anemia. Am J Hematol. 2006;81(8):598–602. 9. Chugh S, Darvish-Kazem S, Lim W, Crowther MA, Ghanima W, Wang G, et al. Rituximab plus standard of care for treatment of primary immune thrombocytopenia: a systematic review and meta-analysis. Lancet Haematol. 2015;2(2):e75–e81. 10. D’Arena G, Capalbo S, Laurenti L, Del Poeta G, Nunziata G, Deaglio S, et al. Chronic lymphocytic leukemia-associated immune thrombocytopenia treated with rituximab: a retrospective study of 21 patients. Eur J Haematol. 2010;85(6):502–7. 11. Arnold DM, Dentali F, Crowther MA, Meyer RM, Cook RJ, Sigouin C, et al. Systematic review: efficacy and safety of rituximab for adults with idiopathic thrombocytopenic purpura. Ann Intern Med. 2007;146(1):25. 12. Lucchini E, Zaja F, Bussel J. Rituximab in the treatment of immune thrombocytopenia: What is the role of this agent in 2019? Haematologica. 2019;104(6):1124–35.
Methyl-CpG binding protein 2 (MeCP2) is a ubiquitous transcriptional regulator. The study of this... more Methyl-CpG binding protein 2 (MeCP2) is a ubiquitous transcriptional regulator. The study of this protein has been mainly focused on the central nervous system because alterations of its expression are associated with neurological disorders such as Rett syndrome. However, young patients with Rett syndrome also suffer from osteoporosis, suggesting a role of MeCP2 in the differentiation of human bone marrow mesenchymal stromal cells (hBMSCs), the precursors of osteoblasts and adipocytes. Here, we report an in vitro downregulation of MeCP2 in hBMSCs undergoing adipogenic differentiation (AD) and in adipocytes of human and rat bone marrow tissue samples. This modulation does not depend on MeCP2 DNA methylation nor on mRNA levels but on differentially expressed miRNAs during AD. MiRNA profiling revealed that miR-422a and miR-483-5p are upregulated in hBMSC-derived adipocytes compared to their precursors. MiR-483-5p, but not miR-422a, is also up-regulated in hBMSC-derived osteoblasts, sug...
Background The exposure of breast cancer to extremely low frequency magnetic fields (ELF-MFs) res... more Background The exposure of breast cancer to extremely low frequency magnetic fields (ELF-MFs) results in various biological responses. Some studies have suggested a possible cancer-enhancing effect, while others showed a possible therapeutic role. This study investigated the effects of in vitro exposure to 50 Hz ELF-MF for up to 24 h on the viability and cellular response of MDA-MB-231 and MCF-7 breast cancer cell lines and MCF-10A breast cell line. Methods and results The breast cell lines were exposed to 50 Hz ELF-MF at flux densities of 0.1 mT and 1.0 mT and were examined 96 h after the beginning of ELF-MF exposure. The duration of 50 Hz ELF-MF exposure influenced the cell viability and proliferation of both the tumor and nontumorigenic breast cell lines. In particular, short-term exposure (4–8 h, 0.1 mT and 1.0 mT) led to an increase in viability in breast cancer cells, while long and high exposure (24 h, 1.0 mT) led to a decrease in viability and proliferation in all cell lines...
Shwachman-Diamond syndrome (SDS) is one of the most commonly inherited bone marrow failure syndro... more Shwachman-Diamond syndrome (SDS) is one of the most commonly inherited bone marrow failure syndromes (IBMFS). In SDS, bone marrow is hypocellular, with marked neutropenia. Moreover, SDS patients have a high risk of developing myelodysplastic syndrome (MDS), which in turn increases the risk of acute myeloid leukemia (AML) from an early age. Most SDS patients are heterozygous for the c.183-184TA>CT (K62X) SBDS nonsense mutation. Fortunately, a plethora of translational read-through inducing drugs (TRIDs) have been developed and tested for several rare inherited diseases due to nonsense mutations so far. The authors previously demonstrated that ataluren (PTC124) can restore full-length SBDS protein expression in bone marrow stem cells isolated from SDS patients carrying the nonsense mutation K62X. In this study, the authors evaluated the effect of a panel of ataluren analogues in restoring SBDS protein resynthesis and function both in hematological and non-hematological SDS cells. B...
Ciliary neurotrophic factor (CNTF) is a pleiotropic cytokine that signals through a receptor comp... more Ciliary neurotrophic factor (CNTF) is a pleiotropic cytokine that signals through a receptor complex containing a specific subunit, CNTF receptor α (CNTFRα). The two molecules are constitutively expressed in key structures for human placental growth and differentiation. The possible role of CNTF in enhancing cell proliferation and/or invasion during placental development and remodelling was investigated using HTR-8/SVneo and BeWo cells, taken respectively as cytotrophoblast and syncytiotrophoblast models. In both cell lines, treatment with human recombinant (hr) CNTF activated JAK2/STAT3 signalling and inhibited the ERK pathway. Interestingly, in HTR-8/SVneo cells, 50 ng hrCNTF induced significant downregulation of matrix metalloprotease (MMP)-1 and significant upregulation of MMP-9. Moreover, pharmacological inhibition of JAK2/STAT3 signalling by AG490 and curcumin resulted in MMP-9 downregulation; it activated the ERK signalling pathway and upregulated MMP-1 expression. Collective...
Bone marrow (BM) acts as a dynamic organ within the bone cavity, responsible for hematopoiesis, s... more Bone marrow (BM) acts as a dynamic organ within the bone cavity, responsible for hematopoiesis, skeletal remodeling, and immune system control. Bone marrow adipose tissue (BMAT) was long simply considered a filler of space, but now it is known that it instead constitutes an essential element of the BM microenvironment that participates in homeostasis, influences bone health and bone remodeling, alters hematopoietic stem cell functions, contributes to the commitment of mesenchymal stem cells, provides effects to immune homeostasis and defense against infections, and participates in energy metabolism and inflammation. BMAT has emerged as a significant contributor to the development and progression of various diseases, shedding light on its complex relationship with health. Notably, BMAT has been implicated in metabolic disorders, hematological malignancies, and skeletal conditions. BMAT has been shown to support the proliferation of tumor cells in acute myeloid leukemia and niche adipocytes have been found to protect cancer cells against chemotherapy, contributing to treatment resistance. Moreover, BMAT’s impact on bone density and remodeling can lead to conditions like osteoporosis, where high levels of BMAT are inversely correlated with bone mineral density, increasing the risk of fractures. BMAT has also been associated with diabetes, obesity, and anorexia nervosa, with varying effects on individuals depending on their weight and health status. Understanding the interaction between adipocytes and different diseases maylead to new therapeutic strategies.
INTRODUCTION Dermal fibrosis and sclerosis are pathologic features shared by Scleroderma-like chr... more INTRODUCTION Dermal fibrosis and sclerosis are pathologic features shared by Scleroderma-like chronic graft-versus-host disease (Scl-cGVHD) and Systemic Scleroderma (SSc). Moreover, in both diseases stimulating anti-PDGF-R antibodies were found, leading to abnormal collagen production by fibroblasts, eventually contributing to organ damage. Targeted therapy with tyrosine kinase inhibitors (TKI) like Imatinib and Nilotinib demonstrated clinical efficacy in Scl-cGVHD; however, the molecular basis underpinning the clinical effects are not fully elucidated. We investigated here a potential terapeutical target of the dermal cGVHD pathophysiology: the cellular and molecular features of pathological skin fibroblasts (GVHD-Fbs) and the efficacy of Nilotinib on fibrosis modulation. MATERIALS AND METHODS Fibroblast cultures (GVHD-Fbs) were obtained from skin biopsies of affected skin from 6 patients with active cGVHD, control fibroblasts are Human Dermal Fibroblasts adult (n-FBS). Fibroblasts were characterized by flow cytometry (FACS CANTO II) for the detection of molecules: CD10, CD14, CD29, CD34, CD44, CD45, CD73, CD90, CD105, CD106, CD117, CD146. In order to evaluate the adipogenic, osteogenic or chondrogenic differentiation cGVHD-Fbs and n-Fbs (n = 3) were cultured in differentiation medium (respectively NH AdipoDiff, NH OsteoDiff, NH ChondroDiff) after four passages. Intracellular lipid droplets indicated adipogenic lineage differentiation. The differentiation potential in the osteogenic lineage was evaluated by calcium accumulation, as assessed by Alizarin Red. The pellet obtained from chondrogenic lineage differentiation was embedded in paraffin, cut in the microtome and the sections placed on a glass slide were stained with Alcian Blue [Junker JP, Cells Tissues Organs, 2010]. For incubation with Nilotinib (Santa Cruz Biotechnology) the 10 mM stock solution was diluted to the final concentration in DMEM supplemented with 0,2% FBS (starvation), added to cell cultures at a concentration of 1 μM or 2 μM for 48h, which covered the mean plasma levels in cGVHD patients after standard doses. In subsets of experiments, after starvation, fibroblasts were stimulated with recombinant TGFβ at 10 ng/ml (GIBCO, Invitrogen). After incubation, total RNA was isolated and reverse transcribed. Gene expression was quantified by real-time PCR using the Sybr Green Mix for qPCR. Specific primer pairs for COL1α1 and COL1α2 were designed with the Primer 3 software. The transcript levels were normalized for the expression of GAPDH constitutive gene. Differences were calculated with the threshold cycle (Ct) and the comparative Ct method for relative quantification. RESULTS GVHD-Fbs are morphologically and phenotypically similar to normal fibroblasts (n-FBS). GVHD-FBS did not show a different immunophenotype from n-Fbs, both in early and late culture passages. Also, no differences were noted between GVHD-Fbs and n-FBS in terms of multilineage differentiation capacity towards the adipogenic, osteogenic and chondrogenic lineage. Gene expression of COL1α1 and COL1α2 in GVHD-Fbs was respectively 4 and 1,6 times higher compared to n-FBS (p = 0.02). However, the increased collagen expression was exclusive of early-passage GVHD-Fbs; in late-passage…
1. Hoy SM. Obinutuzumab: a review of its use in patients with chronic lymphocytic leukaemia. Drug... more 1. Hoy SM. Obinutuzumab: a review of its use in patients with chronic lymphocytic leukaemia. Drugs. 2015;75(3):285–96. 2. Goede V, Fischer K, Busch R, Engelke A, Eichhorst B, Wendtner CM, et al. Obinutuzumab plus chlorambucil in patients with CLL and coexisting conditions. N Engl J Med. 2014;370(12):1101–10. 3. Fischer K, Al-Sawaf O, Bahlo J, Fink AM, Tandon M, Dixon M, et al. Venetoclax and obinutuzumab in patients with CLL and coexisting conditions. N Engl J Med. 2019;380(23):2225–36. 4. Moreno C, Greil R, Demirkan F, Tedeschi A, Anz B, Larratt L, et al. Ibrutinib plus obinutuzumab versus chlorambucil plus obinutuzumab in firstline treatment of chronic lymphocytic leukaemia (iLLUMINATE): a multicentre, randomised, open-label, phase 3 trial. Lancet Oncol. 2019;20(1): 43–56. 5. Reynaud Q, Durieu I, Dutertre M, Ledochowski S, Durupt S, Michallet AS, et al. Efficacy and safety of rituximab in auto-immune hemolytic anemia: a meta-analysis of 21 studies. Autoimmun Rev. 2015;14(4):304–13. 6. Dierickx D, Kentos A, Delannoy A. The role of rituximab in adults with warm antibody autoimmune hemolytic anemia. Blood. 2015;125(21):3223–9. 7. Narat S, Gandla J, Hoffbrand AV, Hughes RG, Mehta AB. Rituximab in the treatment of refractory autoimmune cytopenias in adults. Haematologica. 2005;90(9):1273–4. 8. D’Arena G, Laurenti L, Capalbo S, D’Arco AM, De Filippi R, Marcacci G, et al. Rituximab therapy for chronic lymphocytic leukemia-association autoimmune hemolytic anemia. Am J Hematol. 2006;81(8):598–602. 9. Chugh S, Darvish-Kazem S, Lim W, Crowther MA, Ghanima W, Wang G, et al. Rituximab plus standard of care for treatment of primary immune thrombocytopenia: a systematic review and meta-analysis. Lancet Haematol. 2015;2(2):e75–e81. 10. D’Arena G, Capalbo S, Laurenti L, Del Poeta G, Nunziata G, Deaglio S, et al. Chronic lymphocytic leukemia-associated immune thrombocytopenia treated with rituximab: a retrospective study of 21 patients. Eur J Haematol. 2010;85(6):502–7. 11. Arnold DM, Dentali F, Crowther MA, Meyer RM, Cook RJ, Sigouin C, et al. Systematic review: efficacy and safety of rituximab for adults with idiopathic thrombocytopenic purpura. Ann Intern Med. 2007;146(1):25. 12. Lucchini E, Zaja F, Bussel J. Rituximab in the treatment of immune thrombocytopenia: What is the role of this agent in 2019? Haematologica. 2019;104(6):1124–35.
Methyl-CpG binding protein 2 (MeCP2) is a ubiquitous transcriptional regulator. The study of this... more Methyl-CpG binding protein 2 (MeCP2) is a ubiquitous transcriptional regulator. The study of this protein has been mainly focused on the central nervous system because alterations of its expression are associated with neurological disorders such as Rett syndrome. However, young patients with Rett syndrome also suffer from osteoporosis, suggesting a role of MeCP2 in the differentiation of human bone marrow mesenchymal stromal cells (hBMSCs), the precursors of osteoblasts and adipocytes. Here, we report an in vitro downregulation of MeCP2 in hBMSCs undergoing adipogenic differentiation (AD) and in adipocytes of human and rat bone marrow tissue samples. This modulation does not depend on MeCP2 DNA methylation nor on mRNA levels but on differentially expressed miRNAs during AD. MiRNA profiling revealed that miR-422a and miR-483-5p are upregulated in hBMSC-derived adipocytes compared to their precursors. MiR-483-5p, but not miR-422a, is also up-regulated in hBMSC-derived osteoblasts, sug...
Background The exposure of breast cancer to extremely low frequency magnetic fields (ELF-MFs) res... more Background The exposure of breast cancer to extremely low frequency magnetic fields (ELF-MFs) results in various biological responses. Some studies have suggested a possible cancer-enhancing effect, while others showed a possible therapeutic role. This study investigated the effects of in vitro exposure to 50 Hz ELF-MF for up to 24 h on the viability and cellular response of MDA-MB-231 and MCF-7 breast cancer cell lines and MCF-10A breast cell line. Methods and results The breast cell lines were exposed to 50 Hz ELF-MF at flux densities of 0.1 mT and 1.0 mT and were examined 96 h after the beginning of ELF-MF exposure. The duration of 50 Hz ELF-MF exposure influenced the cell viability and proliferation of both the tumor and nontumorigenic breast cell lines. In particular, short-term exposure (4–8 h, 0.1 mT and 1.0 mT) led to an increase in viability in breast cancer cells, while long and high exposure (24 h, 1.0 mT) led to a decrease in viability and proliferation in all cell lines...
Shwachman-Diamond syndrome (SDS) is one of the most commonly inherited bone marrow failure syndro... more Shwachman-Diamond syndrome (SDS) is one of the most commonly inherited bone marrow failure syndromes (IBMFS). In SDS, bone marrow is hypocellular, with marked neutropenia. Moreover, SDS patients have a high risk of developing myelodysplastic syndrome (MDS), which in turn increases the risk of acute myeloid leukemia (AML) from an early age. Most SDS patients are heterozygous for the c.183-184TA>CT (K62X) SBDS nonsense mutation. Fortunately, a plethora of translational read-through inducing drugs (TRIDs) have been developed and tested for several rare inherited diseases due to nonsense mutations so far. The authors previously demonstrated that ataluren (PTC124) can restore full-length SBDS protein expression in bone marrow stem cells isolated from SDS patients carrying the nonsense mutation K62X. In this study, the authors evaluated the effect of a panel of ataluren analogues in restoring SBDS protein resynthesis and function both in hematological and non-hematological SDS cells. B...
Ciliary neurotrophic factor (CNTF) is a pleiotropic cytokine that signals through a receptor comp... more Ciliary neurotrophic factor (CNTF) is a pleiotropic cytokine that signals through a receptor complex containing a specific subunit, CNTF receptor α (CNTFRα). The two molecules are constitutively expressed in key structures for human placental growth and differentiation. The possible role of CNTF in enhancing cell proliferation and/or invasion during placental development and remodelling was investigated using HTR-8/SVneo and BeWo cells, taken respectively as cytotrophoblast and syncytiotrophoblast models. In both cell lines, treatment with human recombinant (hr) CNTF activated JAK2/STAT3 signalling and inhibited the ERK pathway. Interestingly, in HTR-8/SVneo cells, 50 ng hrCNTF induced significant downregulation of matrix metalloprotease (MMP)-1 and significant upregulation of MMP-9. Moreover, pharmacological inhibition of JAK2/STAT3 signalling by AG490 and curcumin resulted in MMP-9 downregulation; it activated the ERK signalling pathway and upregulated MMP-1 expression. Collective...
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