Leonardo Feldman

Cardiomyogenic differentiation of human bone marrow mesenchymal cells: Role of cardiac extract from neonatal rat cardiomyocytes.

Bone marrow mesenchymal stromal cells (BM-MSCs) with regenerative potential have been identified in heart. Whether these cells become new cardiac lineage cells by phenomena of transdifferentiation or fusion is also being investigated. Although, these mechanisms give cardiomyocytes, it has to be considered that MSCs transplantation could carry out ossification and calcification processes. An alternative might be the use of myocytes; however, the problem is the arrythmia. For those reasons, is that we investigated how to obtain cardiomyocyte-like cells from human MSCs (hMSCs). The aim of the present work was to evaluate a nuclear reprogramming of the hMSCs by a neonatal rat cardiomyocytes extract (EX) using Streptolysin O (SLO) treatment. hMSCs treated with 57.5ng/ml SLO presented ball-like, stick-like and myotube-like morphology. In the absence of cardiomyogenic stimuli, hMSCs expressed markers of cardiac phenotype-like sarcomeric alpha-actinin, connexin-43 and GATA-4. However, when hMSCs were treated with SLO+EX or 10 microM of 5-azacytidine (5-AZA), the expression of these markers were significantly increased and furthermore, expressed SERCA-2, cardiac Troponin I, beta-MyHC, desmin, MLC-2a and MLC-2v thus showing the phenotype of mature cardiomyocytes. PCR analysis showed that cardiomyocyte-related genes, such as beta1-adrenergic receptor (beta1-AR), MLC-2a and cardiac Troponin T, were expressed after SLO+EX treatment like with 5-AZA. We concluded that the extract of neonatal rat cardiomyocytes could promote a nuclear modification of hMSCs to cardiomyogenic-like cells differentiation. Since the 5-AZA treatment appears to be genotoxic and taking into account the obtained results, the nuclear reprogramming by cell extract may be an approach leading to the identification of soluble factors that drives the reprogramming.

Allogeneic hematopoietic stem cell transplantation in pediatric myelodysplastic syndromes: a multicenter experience from Argentina.

Allogeneic hematopoietic stem cell transplantation (AHSCT) represents the only curative treatment for the majority of pediatric patients with Myelodysplastic Syndrome (MDS). We aimed to evaluate overall survival (OS), disease‐free survival (DFS), non‐relapse mortality (NRM) and relapse incidence in children who underwent AHSCT for MDS in six institutions from Argentina.

Freezing the graft is not necessary for autotransplants for plasma cell myeloma and lymphomas

We studied rates of granulocyte and platelets recovery in 359 consecutive subjects receiving blood cell infusions in the context of autotransplants for plasma cell myeloma (N = 216) and lymphomas (N = 143). Blood cells were mobilised with filgrastim given for 4–5 days and collected after a median of 2 (range, 1–2) apheresis. Apheresis products were stored at 4° C for a median of 3 days (range, 2–6 days). Most subjects received carmustine, etoposide, cytarabine and melphalan (BEAM), cyclophosphamide, carmustine and etoposide (CBV) or high-dose melphalan. Filgrastim was given post transplant to 319 subjects. Median numbers of mononuclear cells collected was 31 × 10E + 6/kg (interquartile range (IQR) 37 × 10E + 6 cells/kg). Median numbers of CD34-positive cells collected was 3.6 × 10E + 6/kg (IQR 3.8 × 10E + 6/Kg). Median viability after collection was 90% (IQR 7%) after storage, 88% (IQR 12%). A total of 255 of 256 evaluable subjects recovered bone marrow function and there was no late bone marrow failure. Median interval to neutrophils >0.5 × E + 9/L was 13 days (range, 9–39 days) and to platelets >20 × 10E + 9/L, 16 days (range, 7–83 days). These rates and ranges seem comparable to those reported after autotransplants of frozen blood cells. There was no correlation between numbers of storage days at 4 °C and viability afte storage (r = −0.018, p = 0.14)) nor rates of recovery of neutrophils (r = −0.054, p = 0.52) or platelets (r = 0.116, p = 0.14). Blood cells collected for autotransplant can be stored at 4 °C for 6 d. This method is simple, inexpensive and widely applicable.

Transforming growth factor-|[beta]|1 functional polymorphisms in myeloablative sibling hematopoietic stem cell transplantation

Hematopoietic stem cell transplantation (HSCT) with sibling donors (s.d.) is a life-saving intervention for patients with hematological malignancies. Numerous genetic factors have a role in transplant outcome. Several functional polymorphisms have been identified in TGF-β1 gene, such as single-nucleotide polymorphism (SNP) at +29C>T within exon 1. Two hundred and forty five patient/donor pairs who underwent a s.d. HSCT in our centers were genotyped for this SNP. In the myeloablative cohort, +29CC donors were associated with an increase in severe chronic GvHD (32% vs 16%, hazard ratio (HR) 9.0, P=0.02). Regarding survival outcomes, +29CC patients developed higher non relapse mortality (NRM) (1–5 years CC 28–32% vs TC/TT 7–10%; HR 5.1, P=0.01). Recipients of +29TT donors experienced a higher relapse rate (1–5 years TT 37–51% vs TC 19–25% vs CC 13%–19%; HR 2.4, P=0.01) with a decreased overall survival (OS) (1–5 years TT 69–50% vs TC/CC 77–69%; HR 1.9, P=0.05). Similar to previous myeloablative unrelated donors HSCT results, we confirmed that +29CC patients had higher NRM. In addition we found that +29TT donors might be associated with a higher relapse rate and lower OS. These results should be confirmed in larger series. Identification of these SNPs will allow personalizing transplant conditioning and immunosuppressant regimens, as well as assisting in the choice of the most appropriate donor.

Evaluation of Bone Mineral Density in Patients with Type 1 Gaucher Disease in Argentina.

The purpose of this study was to evaluate the frequency of osteoporosis (OP) in patients with Gaucher disease (GD) in Argentina. GD patients from 28 centers were consecutively included from April 2012 to 2014. Bone mineral density (BMD) was determined by dual X-ray absorptiometry in the lumbar spine and the femoral neck or the total proximal femur for patients ≥20 yr of age, and by whole-body scan in the lumbar spine in patients <20 yr of age. In children, mineral density was calculated using the chronological age and Z height. OP diagnosis was determined following adult and pediatric official position of the International Society for Clinical Densitometry. A total of 116 patients were included, of which 62 (53.5%) were women. The median age was 25.8 yr. All patients received enzyme replacement therapy, with a median time of 9.4 yr. Normal BMD was found in 89 patients (76.7%), whereas low bone mass (LBM) or osteopenia was found in 15 patients (13%) and OP in 12 patients (10.3%). The analysis of the pediatric population revealed that 4 patients (9.3%) had LBM and 3 (7%) had OP (Z-score ≤ -2 + fractures height-adjusted by Z), whereas in the adult population (n = 73), 11 patients (15%) had LBM or osteopenia and 9 (12.3%) had OP. Bone marrow infiltration and the presence of fractures were significantly correlated with the presence of OP (p = 0.04 and <0.001, respectively). This is the first study in Argentina and in the region describing the frequency of OP or LBM in GD patients treated with imiglucerase using the official position of the International Society for Clinical Densitometry.

Abstract B66: Bone marrow microenvironment of advanced breast cancer patients without bone metastasis favors the cancer cell colonization

Bone metastasis is the major cause of death for advanced breast cancer patients (BCP). It is a multistep process that includes tumor cell mobilization, intravasation, survival in the circulation, extravasation and proliferation in bone marrow (BM) or bone. Accumulating evidence suggests that BM-mesenchymal stromal cells (MStC) play a critical role in BC-cell (BCC) colonization of the BM/bone. Despite increasing knowledge, the beginning of bone metastatic process in advanced BCP without BCC in the BM/bone has been poorly studied. So, this work was performed to evaluate the levels of OPG, RANKL, TRAIL, SDF-1, PDGF-AB, stanniocalcin-1 and MIF in peripheral blood (PB) and BM-plasma and BM-conditioned media (CM) of colony forming unit-fibroblastic cultures (CFU-F, day 14) from advanced BCP (IDC, without BM/bone metastasis) and healthy volunteers (HV). Also, we investigated the expression of membrane-RANKL (mRANKL) and SDF-1 in BM-MStC and their specific receptors in primary BC-tissue from these patients and MCF-7 and MDA-MB231 cells. At the end, we evaluated the effect of BM-plasma and CFU-F-CM over the migration and proliferation of both BCC lines. Methodology: Soluble factors were studied by ELISA. SDF-1, m-RANKL, CXCR-4 and RANK expression was analyzed by immunochemistry. Migration was performed over 14hs in transwells seeded with BCC exposed to BM-plasma and CFU-F-CM. Proliferation by MTS: after arrest, BCC were incubated for 48hs with 10% of BM-plasma or 100% or 50% of these CM with or without 1.25% FBS. Results: Significant difference in the OPG, RANKL, SDF-1 and MIF values in PB-plasma was found between both groups (BCP vs HV, X±SE, pg/ml): 2,005±195.90 vs 1,100±124.10 (p=0.001), 130.20±23.63 vs ≤31.25, 117±25 vs 254±28 (p<0.05) and 4,564±591 vs 2,265±402 (p<0.05), respectively. PDGF-AB level in BCP-BM-plasma was significant higher than HV-value (X±SE, pg/ml): 4,468±746 vs 2,528±421. 100% of BM-MStC expressed SDF-1 and mRANKL in both groups, but we observed higher mRANKL expression/MStC from BCP-BM compared to HV-values (++++vs++). The primary tissue-BCC expressed CXCR-4 and RANK (++) but we did not observe expression of them in the epithelial cells of non-malignan tissue. Moreover, 50% of BCC of both lines expressed CXCR-4 and 100% RANK. In addition, the BM-plasma and the CFU-F-CM from BCP induced a higher migration increase of MCF-7 and MDA-MB231cells compared with HV-values (p<0.05 in both lines and p<0.0001 and p=0.0356, respectively). We have not observed proliferation effect by CFU-F-CM over any of the lines, but we did observe a significant higher proliferation of MDA-MB231 cells when we used the BM-plasma from BCP compared with HV (p=0.0434). Conclusions: Data suggests that the high PB-RANKL and MIF levels in these BCP could play a role in the intravasation of BCC into the blood vasculature, binding to their R (RANK and CXCR-4, respectively) expressed in them. MIF and OPG not only are pro-angiogenic factors, but also, they have an anti-apoptotic effect over BCC favoring the circulating BCC survival. In addition, PDGF-AB could be responsible of higher proliferation of MDA-MB231 cells when we used the BM-plasma from BCP compared with HV, inducing a favorable BM microenvironment to seed and proliferation of circulating BCC. Moreover, the BM-MStC from BCP could enhance the migration of circulating BCC to BM/bone and the likely association between RANK present in BCC and the mRANKL in BM-MStC could promote the tumor cell proliferation favoring the bone metastatic process. Our findings suggest that the BM microenvironment of advanced BCP without BM/bone metastasis may induce a premetastatic niche to BCC colonization. Citation Format: Leandro M. Martinez, Valeria B. Fernandez Vallone, Vivian Labovsky, Hosoon Choi, Leonardo Feldman, Raul H. Bordenave, Emilio Batagelj, Federico Dimase, Ana Rodriguez Villafane, Norma A. Chasseing. Bone marrow microenvironment of advanced breast cancer patients without bone metastasis favors the cancer cell colonization. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B66.