ALBERT

Description: 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 (〉4) GVHD-Fbs, collagen mRNA levels were similar to n-FBS (p=0.6 for COL1α1; p=0.4 for COL1α2). As expected, TGFβ boosted collagen expression in n-FBS, but it did not increase COL1α1 and COL1α2 mRNA levels in GVHD-Fbs. Therapeutic doses of Nilotinib (1μM) were able to reduce expression of COL1α1 and COL1α2 mRNA by 86,5% and 49%, respectively (p

Description: Introduction Chronic Graft Versus Host Disease (cGVHD) is a major complication of allogeneic stem-cell transplantation and is characterized by frequent multi-organ involvement that resembles the autoimmune diseases. Donor-derived CD4+ and CD8+ T lymphocytes have classically been considered to be the main effector cells mediating GVHD pathogenesis. Indeed, removal of T cells from transplant inocula almost completely prevents GVHD developing, at the price of increased incidences of graft rejection and disease recurrence. However recent studies suggest that B cells might also play an important role in the biology of cGVHD. The role of Treg lymphocytes in the pathogenesis of cGVHD is still controversial and the tyrosine kinase inhibitor′s (TKI) role in the modulation of this pathway is not yet fully characterized. In vitro data confirm that TKIs regulates both innate and adaptive immune response by interacting with many cell population such as T-cells, B-cells, dendritic cells, mast cells and macrophages. According to these observations, we investigated the TKI′s immunomodulatory effects (Nilotinib, Dasatinib, Imatinib, Ponatinib) on lymphocyte populations. Materials and Methods Peripheral blood mononuclear cells were isolated by density gradient centrifugation using Ficoll-Biocoll. Cells were cultured in RPMI 1640 at a concentration 1x106 cell/well. Nilotinib, Imatinib, Dasatinib and Ponatinib were added to cell cultures at serial concentration (Imatinib:1μM,10μM,50μM; Nilotinib:0.5μM,2μM,10μM; Dasatinib:50nM,100nM,200nM; Ponatinib:1nM,10nM,50nM,100nM) on the first day. Six-color flow cytometry analysis (Facs Canto II) was performed on the cells harvested after 96 h cultures using conjugated antibodies (CD3,CD4,CD16,CD56,CD3,CD25,CD19,CD45RA,FoxP3,CD127,7-Aminoactinomycin-D), for cell cycle analysis cells were stained with propidium iodide. For cytokine analysis, supernatants were collected and analyzed for cytokines according to the instruction of Bio-Plex Pro Human Cytokine 17-plex Assay with Bio-Plex (Bio-Rad). Results A significant decrease of cytotoxic T cells viability was observed when cells were cultured in presence of Imatinib (50μM,p

Description: Shwachman-Diamond syndrome (SDS) is one of the more common inherited bone marrow failure syndromes (IBMFS). Almost 90% of patients with SDS present mutations in the Shwachman-Bodian-Diamond syndrome gene (SBDS) which encodes for the homonymous small protein involved in ribogenesis. SDS is a multiple-organ disease mostly characterized by exocrine pancreas insufficiency, bone malformations, and more importantly bone marrow failure. Most patients with SDS present severe neutropenia, whereas thrombocytopenia and anemia are less frequent. Furthermore, 15-20% of patients develop myelodysplastic syndrome with high risk of acute myeloid leukemia (AML). STAT3 pathway is upregulated both in primary SDS leukocytes and immortalized B cells. Being STAT3 a key regulator of interleukin-6 (IL-6), we postulated that STAT3 hyper-activation could lead to a dysregulation of the IL-6 signaling cascade. Increased levels of IL-6 have been found in pediatric patients with AML and it has been associated with poorer outcomes in these patients, highlighting IL-6 as a cytokine potentially involved in the development of AML. Thus, our hypothesis is that STAT3-IL6 axis may contribute to leukemogenesis in SDS. Almost 55% of patients with SDS carry a specific nonsense mutations, namely the c.183-184TA〉CT, which cause a premature termination codon (PTC). Ataluren (PTC124, PTC Therapeutics Inc, NJ) is a small PTC suppressor molecule already approved by the European Medicines Agency as a therapeutic option for Duchenne muscular dystrophy. Interestingly, we recently reported that ataluren can restore SBDS expression in bone marrow progenitors and in peripheral blood mononuclear cells isolated from patients with SDS. Moreover, we have shown that ataluren can reduce mTOR hyper-phosphorylation and excessive apoptotic rate observed in SDS leukocytes. More importantly, we reported that ataluren can improve myeloid differentiation in a small cohort of patients (Bezzerri et al, Am J Hematol 2018). In this further analysis considering an enlarged cohort of 20 SDS patients carrying nonsense mutations we found the following: Ataluren can significantly improve both myeloid colony-forming unit-granulocyte/macrophage (CFU-GM) and colony-forming unit granulocyte, erythrocyte, monocyte, megakaryocyte (CFU-GEMM) generation from bone marrow mononuclear stem cells obtained from an enlarged cohort of 20 patients with SDS carrying nonsense mutations. Ataluren indeed almost doubled the number of CFU-GM and CFU-GEMM after 7 and 14 days of treatment.Colony-forming unit erythroid (CFU-E) generation was not affected by the treatment.Ataluren induces neutrophil maturation in SDS bone marrow mononuclear stem cells (mean increase of 61% CD16+ CD11b+ cells over untreated controls) after 24-48 hours of treatment.Consistently with STAT3 hyper-activation observed in SDS cells, here we show that patients with SDS present a significantly increased level of IL-6 in plasma (4.3-fold higher expression than the healthy control group). Also lymphoblastoid cell lines (LCL) and primary bone marrow mesenchymal stromal cells (MSC) obtained from patients with SDS show increased IL-6 release in culture supernatants compared to healthy controls (2.5-fold and 6.8-fold higher levels, respectively).Of note, ataluren can reduce IL-6 expression in SDS cells restoring normal levels both in LCL and MSC. In conclusion, these new data support the enrollment of patients for the first clinical trial for this drug in SDS. Furthermore, this study could pave the way for the use of ataluren for other nonsense mutation-mediated IBMFS where STAT3-IL6 axis and similar pro-leukemic pathways are involved. Disclosures Bezzerri: Marco Cipolli, Valentino Bezzerri, Baroukh Maurice Assael: Patents & Royalties: WO2018/050706 A1 "Method of treatment of Shwachman-Diamond syndrome". Cipolli:Marco Cipolli, Valentino Bezzerri, Baroukh Maurice Assael: Patents & Royalties: WO2018/050706 A1 "Method of treatment of Shwachman-Diamond syndrome".