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Background Chronic lower limb ischemia develops earlier and more frequently in patients with type 2 diabetes mellitus. Diabetes remains the main cause of lower-extremity non-traumatic amputations. Current medical treatment, based on antiplatelet therapy and statins, has demonstrated deficient improvement of the disease. In recent years, research has shown that it is possible to improve tissue perfusion through therapeutic angiogenesis. Both in animal models and humans, it has been shown that cell therapy can induce therapeutic angiogenesis, making mesenchymal stromal cell-based therapy one of the most promising therapeutic alternatives. The aim of this study is to evaluate the feasibility, safety, and efficacy of cell therapy based on mesenchymal stromal cells derived from adipose tissue intramuscular administration to patients with type 2 diabetes mellitus with critical limb ischemia and without possibility of revascularization. Methods A multicenter, randomized double-blind, placebo-controlled trial has been designed. Ninety eligible patients will be randomly assigned at a ratio 1:1:1 to one of the following: control group (n = 30), low-cell dose treatment group (n = 30), and high-cell dose treatment group (n = 30). Treatment will be administered in a single-dose way and patients will be followed for 12 months. Primary outcome (safety) will be evaluated by measuring the rate of adverse events within the study period. Secondary outcomes (efficacy) will be measured by assessing clinical, analytical, and imaging-test parameters. Tertiary outcome (quality of life) will be evaluated with SF-12 and VascuQol-6 scales. Discussion Chronic lower limb ischemia has limited therapeutic options and constitutes a public health problem in both developed and underdeveloped countries. Given that the current treatment is not established in daily clinical practice, it is essential to provide evidence-based data that allow taking a step forward in its clinical development. Also, the multidisciplinary coordination exercise needed to develop this clinical trial protocol will undoubtfully be useful to conduct academic clinical trials in the field of cell therapy in the near future. Trial registration ClinicalTrials.govNCT04466007. Registered on January 07, 2020. All items from the World Health Organization Trial Registration Data Set are included within the body of the protocol.

Rationale Idiopathic pulmonary fibrosis (IPF) has a dismal prognosis. Mesenchymal stromal cells (MSCs) have shown benefit in other inflammatory diseases. Objectives To evaluate the safety and feasibility of endobronchial administration of bone marrow autologous MSCs (BM-MSC) in patients with mild-to-moderate IPF. Methods A phase I multicentre clinical trial (ClinicalTrials.gov NCT01919827) with a single endobronchial administration of autologous adult BM-MSCs in patients diagnosed with mild-to-moderate IPF. In a first escalating-dose phase, three patients were included sequentially in three dose cohorts (10×106, 50×106 and 100×106 cells). In a second phase, nine patients received the highest tolerated dose. Follow-up with pulmonary function testing, 6-min walk test and St George's Respiratory Questionnaire was done at 1, 2, 3, 6 and 12 months, and with computed tomography at 3, 6 and 12 months. Results 21 bone marrow samples were obtained from 17 patients. Three patients were excluded from treatment due to chromosome aberrations detected in MSCs after culture, and one patient died before treatment. Finally, 13 patients received the BM-MSC infusion. No treatment-related severe adverse events were observed during follow-up. Compared to baseline, the mean forced vital capacity showed an initial decline of 8.1% at 3 months. The number of patients without functional progression was six (46%) at 3 months and three (23%) at 12 months. Conclusions The endobronchial infusion of BM-MSCs did not cause immediate serious adverse events in IPF patients, but a relevant proportion of patients suffered clinical and/or functional progression. Genomic instability of BM-MSCs during culture found in three patients may be troublesome for the use of autologous MSCs in IPF patients., Endobronchial autologous mesenchymal stromal cells (MSCs) did not cause direct serious adverse events in IPF patients. However, significant progression was seen in seven out of 13 patients. Genomic instability of autologous MSCs may limit use in IPF. https://bit.ly/39akv7z

Background: Chronic lower limb ischemia develops earlier and more frequently in patients with type 2 diabetes mellitus. Diabetes remains the main cause of lower-extremity non-traumatic amputations. Current medical treatment, based on antiplatelet therapy and statins, has demonstrated deficient improvement of the disease. In recent years, research has shown that it is possible to improve tissue perfusion through therapeutic angiogenesis. Both in animal models and humans, it has been shown that cell therapy can induce therapeutic angiogenesis, making mesenchymal stromal cell-based therapy one of the most promising therapeutic alternatives. The aim of this study is to evaluate the feasibility, safety and efficacy of cell therapy based on mesenchymal stromal cells derived from adipose tissue intramuscular administration to patients with type 2 diabetes mellitus with critical limb ischemia and without possibility of revascularization. Methods: A multicenter, randomized double-blind, placebo-controlled trial has been designed. 90 eligible patients will be randomly assigned at a ratio 1:1:1 to one of the following: the control group (n=30), low cell dose treatment group (n=30), and high cell dose treatment group (n=30). Treatment will be administered in a single-dose way and patients will be followed for 12 months. Primary outcome (safety) will be evaluated by measuring the rate of adverse events within the study period. Secondary outcomes (efficacy) will be measured by assessing clinical, analytical and imaging-test parameters. Tertiary outcome (quality of life) will be evaluated with SF-12 and VascuQol-6 scales. Discussion: Chronic lower limb ischemia has limited therapeutic options and constitutes a public health problem in both developed and underdeveloped countries. Given that the current treatment is not established in daily clinical practice, it is essential to provide evidence-based data that allow taking a step forward in its clinical development. Also, the multidisciplinary coordination exercise needed to develop this clinical trial protocol will undoubtfully be useful to conduct academic clinical trials in the field of cell therapy in the near future.Trial registration: ClinicalTrials.gov NCT04466007. Registered on January 07, 2020.

The use of allogeneic adipose-derived mesenchymal stromal cells (alloADSCs) represents an attractive approach for treating myocardial infarction (MI). Furthermore, adding a natural support improves alloADSCs engraftment and survival in heart tissues, leading to a greater therapeutic effect. We aimed to examine the safety and immunological reaction induced by epicardial implantation of a clinical-grade collagen scaffold (CS) seeded with alloADSCs for its future application in humans. Thus, cellularized scaffolds were myocardially or subcutaneously implanted in immunosuppressed rodent models. The toxicological parameters were not significantly altered, and tumor formation was not found over the short or long term. Furthermore, biodistribution analyses in the infarcted immunocompetent rats displayed cell engraftment in the myocardium but no migration to other organs. The immunogenicity of alloADSC-CS was also evaluated in a preclinical porcine model of chronic MI, no significant humoral or cellular alloreactive responses were found. Moreover, CS cellularized with human ADSCs cocultured with human allogeneic immune cells produced no alloreactive response. Interestingly, alloADSC-CS significantly inhibited lymphocyte responses, confirming its immunomodulatory action. Thus, alloADSC-CS is likely safe and does not elicit any alloreactive immunological response in the host. Moreover, it exerts an immunomodulatory action, which supports its translation to a clinical setting.

Background Mesenchymal stromal cells are a safe and promising option to treat knee osteoarthritis as previously demonstrated in different clinical trials. However, their efficacy, optimal dose and addition of adjuvants must be determined. Here, we evaluated the clinical effects of a dose of 100 × 106 bone marrow mesenchymal stromal cells (BM-MSCs) in combination with Platelet Rich Plasma (PRGF®) as adjuvant in a randomized clinical trial. Methods A phase II, multicenter, randomized clinical trial with active control was conducted. Sixty patients diagnosed with knee OA were randomly assigned to 3 weekly doses of PRGF® or intraarticular administration of 100 × 106 cultured autologous BM-MSCs plus PRGF®. Patients were followed up for 12 months, and pain and function were assessed using VAS and WOMAC and by measuring the knee range of motion range. X-ray and magnetic resonance imaging analyses were performed to analyze joint damage. Results No adverse effects were reported after BM-MSC administration or during follow-up. According to VAS, the mean value (SD) for PRGF® and BM-MSC with PRGF® went from 5 (1.8) to 4.5 (2.2) (p = 0.389) and from 5.3 (1.9) to 3.5 (2.5) (p = 0.01), respectively at 12 months. In WOMAC, the mean (SD) baseline and 12-month overall WOMAC scores in patients treated with PRGF® was 31.9 (16.2) and 22.3 (15.8) respectively (p = 0.002) while that for patients treated with BM-MSC plus PRGF® was 33.4 (18.7) and 23.0 (16.6) (p = 0.053). Although statistical significances between groups have been not detected, only patients being treated with BM-MSC plus PRGF® could be considered as a OA treatment responders following OARSI criteria. X-ray and MRI (WORMS protocol) revealed no changes in knee joint space width or joint damage. Conclusions Treatment with BM-MSC associated with PRGF® was shown to be a viable therapeutic option for osteoarthritis of the knee, with clinical improvement at the end of follow-up. Further phase III clinical trials would be necessary to confirm the efficacy. Trial registration Clinical Trials.gov identifier NCT02365142. Nº EudraCT: 2011-006036-23

Background: Identification of effective treatments in severe cases of COVID-19 requiring mechanical ventilation represents an unmet medical need. Our aim was to determine whether the administration of adipose-tissue derived mesenchymal stromal cells (AT-MSC) is safe and potentially useful in these patients. Methods: Thirteen COVID-19 adult patients under invasive mechanical ventilation who had received previous antiviral and/or anti-inflammatory treatments (including steroids, lopinavir/ritonavir, hydroxychloroquine and/or tocilizumab, among others) were treated with allogeneic AT-MSC. Ten patients received two doses, with the second dose administered a median of 3 days (interquartile range-IQR- 1 day) after the first one. Two patients received a single dose and another patient received 3 doses. Median number of cells per dose was 0.98 × 106 (IQR 0.50 × 106) AT-MSC/kg of recipient's body weight. Potential adverse effects related to cell infusion and clinical outcome were assessed. Additional parameters analyzed included changes in imaging, analytical and inflammatory parameters. Findings: First dose of AT-MSC was administered at a median of 7 days (IQR 12 days) after mechanical ventilation. No adverse events were related to cell therapy. With a median follow-up of 16 days (IQR 9 days) after the first dose, clinical improvement was observed in nine patients (70%). Seven patients were extubated and discharged from ICU while four patients remained intubated (two with an improvement in their ventilatory and radiological parameters and two in stable condition). Two patients died (one due to massive gastrointestinal bleeding unrelated to MSC therapy). Treatment with AT-MSC was followed by a decrease in inflammatory parameters (reduction in C-reactive protein, IL-6, ferritin, LDH and d-dimer) as well as an increase in lymphocytes, particularly in those patients with clinical improvement. Interpretation: Treatment with intravenous administration of AT-MSC in 13 severe COVID-19 pneumonia under mechanical ventilation in a small case series did not induce significant adverse events and was followed by clinical and biological improvement in most subjects. Funding: None. We would like to acknowledge the Instituto de Salud Carlos III (ISCIII) through the project “RD16/0011: Red de Terapia Celular”, from the sub-program RETICS, integrated in the “Plan Estatal de I+D+I 2013-2016” and co-financed by the European Regional Development Fund “A way to make Europe”, groups RD16/0011/0001, -/0002, -/005, -/0013, -/0015, -/0029), the Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, Spain and AvanCell-CM (Red de Investigación de Terapia Celular de la Comunidad de Madrid, Spain), for supporting some personnel and networking activities.

In this work, mesenchymal stem cells derived from adipose tissue (ADSCs) were used for the generation of the human-induced pluripotent stem cell line G15.AO. Cell reprogramming was performed using retroviral vectors containing the Yamanaka factors, and the generated G15.AO hiPSC line showed normal karyotype, silencing of the exogenous reprogramming factors, induction of the typical pluripotency-associated markers, alkaline phosphatase enzymatic activity, and in vivo and in vitro differentiation ability to the three germ layers. This work was supported by the 'Torres Quevedo' Subprogram, Ministry of Economy and Competitiveness (PTQ-11-04777) to JRRM; the Institute of Health Carlos III (PI13/00862) to JRRM, and RETIC (RD12/0019/0031) to FP; and the Fundación Bancaria Caja Navarra (70270) to JRRM.

Background Mesenchymal stromal cells (MSCs) are a promising option to treat knee osteoarthritis (OA). Their safety and usefulness have been reported in several short-term clinical trials but less information is available on the long-term effects of MSC in patients with osteoarthritis. We have evaluated patients included in our previous randomized clinical trial (CMM-ART, NCT02123368) to determine their long-term clinical effect. Materials A phase I/II multicenter randomized clinical trial with active control was conducted between 2012 and 2014. Thirty patients diagnosed with knee OA were randomly assigned to Control group, intraarticularly administered hyaluronic acid alone, or to two treatment groups, hyaluronic acid together with 10 × 106 or 100 × 106 cultured autologous bone marrow-derived MSCs (BM-MSCs), and followed up for 12 months. After a follow up of 4 years adverse effects and clinical evolution, assessed using VAS and WOMAC scorings are reported. Results No adverse effects were reported after BM-MSCs administration or during the follow-up. BM-MSCs-administered patients improved according to VAS, median value (IQR) for Control, Low-dose and High-dose groups changed from 5 (3, 7), 7 (5, 8) and 6 (4, 8) to 7 (6, 7), 2 (2, 5) and 3 (3, 4), respectively at the end of follow up (Low-dose vs Control group, p = 0.01; High-dose vs Control group, p = 0.004). Patients receiving BM-MSCs also improved clinically according to WOMAC. Control group showed an increase median value of 4 points (− 11;10) while Low-dose and High-dose groups exhibited values of − 18 (− 28;− 9) and − 10 (− 21;− 3) points, respectively (Low-dose vs Control group p = 0.043). No clinical differences between the BM-MSCs receiving groups were found. Conclusions Single intraarticular injection of in vitro expanded autologous BM-MSCs is a safe and feasible procedure that results in long-term clinical and functional improvement of knee OA.

Background Prognosis of patients with glioblastoma multiforme (GBM) remains dismal, with median overall survival (OS) of about 15 months. It is therefore crucial to search alternative strategies that improve these results obtained with conventional treatments. In this context, immunotherapy seems to be a promising therapeutic option. We hypothesized that the addition of tumor lysate-pulsed autologous dendritic cells (DCs) vaccination to maximal safe resection followed by radiotherapy and concomitant and adjuvant temozolomide could improve patients’ survival. Methods We conducted a phase-II clinical trial of autologous DCs vaccination in patients with newly diagnosed patients GBM who were candidates to complete or near complete resection. Candidates were finally included if residual tumor volume was lower than 1 cc on postoperative radiological examination. Autologous DCs were generated from peripheral blood monocytes and pulsed with autologous whole tumor lysate. The vaccination calendar started before radiotherapy and was continued during adjuvant chemotherapy. Progression free survival (PFS) and OS were analyzed with the Kaplan–Meier method. Immune response were assessed in blood samples obtained before each vaccines. Results Thirty-two consecutive patients were screened, one of which was a screening failure due to insufficient resection. Median age was 61 years (range 42–70). Karnofsky performance score (KPS) was 90–100 in 29%, 80 in 35.5% and 60–70 in 35.5% of cases. MGMT (O6-methylguanine-DNA-methyltransferase) promoter was methylated in 45.2% of patients. No severe adverse effects related to immunotherapy were registered. Median PFS was 12.7 months (CI 95% 7–16) and median OS was 23.4 months (95% CI 16–33.1). Increase in post-vaccination tumor specific immune response after vaccines (proliferation or cytokine production) was detected in 11/27 evaluated patients. No correlation between immune response and survival was found. Conclusions Our results suggest that the addition of tumor lysate-pulsed autologous DCs vaccination to tumor resection and combined radio-chemotherapy is feasible and safe. A multicenter randomized clinical trial is warranted to evaluate the potential survival benefit of this therapeutic approach. Trial registration This phase-II trial was registered as EudraCT: 2009-009879-35 and ClinicalTrials.gov Identifier: NCT01006044 retrospectively registered

Purpose The purpose of this study was to develop a culture method for expansion of corneal endothelial cells (CEC) based on the combined activation of PI3K/Akt and Smad2. Methods Morphology, proliferation, and migration of cultured rabbit and nonhuman primate CEC were examined in the presence of the PI3K/Akt activators IGF-1 and heregulin beta in combination with the Smad2 activator activin A. Phenotypic characterization of CEC was performed at the RNA and protein levels. Cell pump function and transepithelial electric resistance were used for in vitro functional assessment of CEC. Finally, ex vivo-expanded rabbit CEC were transplanted into a model of endothelial damage in rabbit corneas. Results Treatment of rabbit and nonhuman primate CEC in vitro with IGF-1, heregulin beta, and activin A induced an upregulation of PI3K/Akt and Smad2 signaling pathways and an increase in proliferation and migration of CEC expressing ZO-1, connexin-43, and Na+/K+-ATPase. Cell pump function evaluation revealed the complete functionality of cultured CEC. Injection of rabbit CEC successfully produced recovery of normal corneal thickness in a rabbit model of endothelial dysfunction. Conclusions We demonstrated that the combined activation of PI3K/Akt and Smad2 results in in vitro expansion of phenotypic and functional CEC. Expanded cells were able to contribute to restoration of corneal endothelium in a rabbit model. These findings may represent a new therapeutic approach for treating corneal endothelial diseases.

Due to the attractive properties of poly(l-lactic acid) (PLLA) for tissue engineering, the aim was to determine the growth and differentiation capacity of mesenchymal stromal cells (MSCs) in PLLA scaffolds and their potential use in the treatment of cartilage diseases. MSCs were cultured in PLLA films and thin porous membranes to study adherence and proliferation. Permeability and porosity were determined for the different scaffolds employed. The optimal conditions for cell seeding were first determined, as well as cell density and distribution inside the PLLA. Scaffolds were then maintained in expansion or chondrogenic differentiation media for 21 days. Apoptosis, proliferation and chondrogenic differentiation was assessed after 21 days in culture by immunohistochemistry. Mechanical characteristics of scaffolds were determined before and after cell seeding. MSCs uniformly adhered to PLLA films as well as to porous membranes. Proliferation was detected only in monolayers of pure PLLA, but was no longer detected after 10 days. Mechanical characterization of PLLA scaffolds showed differences in the apparent compression elastic modulus for the two sizes used. After determining high efficiencies of seeding, the production of extracellular matrix (ECM) was determined and contained aggrecan and collagens type I and X. ECM produced by the cells induced a twofold increase in the apparent elastic modulus of the composite. Biocompatible PLLA scaffolds have been developed that can be efficiently loaded with MSCs. The scaffold supports chondrogenic differentiation and ECM deposition that improves the mechanics of the scaffold. Although this improvement does not met the expectations of a hyaline-like cartilage ECM, in part due to the lack of a mechanical stimulation, their potential use in the treatment of cartilage pathologies encourages to improve the mechanical component., This work has been supported by the Spanish Ministry of Science and Innovation DPI2010-20399-C04-00 project and Instituto de Salud Carlos III RETIC RD06/0014.

There is a need for comparative studies to determine which cell types are better candidates to remedy ischemia. Here, we compared human AC133+ cells and multipotent adult progenitor cells (hMAPC) in a mouse model reminiscent of critical limb ischemia. hMAPC or hAC133+ cell transplantation induced a significant improvement in tissue perfusion (measured by microPET) 15 days posttransplantation compared to controls. This improvement persisted for 30 days in hMAPC-treated but not in hAC133+-injected animals. While transplantation of hAC133+ cells promoted capillary growth, hMAPC transplantation also induced collateral expansion, decreased muscle necrosis/fibrosis, and improved muscle regeneration. Incorporation of differentiated hAC133+ or hMAPC progeny into new vessels was limited; however, a paracrine angio/arteriogenic effect was demonstrated in animals treated with hMAPC. Accordingly, hMAPC-conditioned, but not hAC133+-conditioned, media stimulated vascular cell proliferation and prevented myoblast, endothelial, and smooth muscle cell apoptosis in vitro. Our study suggests that although hAC133+ cell and hMAPC transplantation both contribute to vascular regeneration in ischemic limbs, hMAPC exert a more robust effect through trophic mechanisms, which translated into collateral and muscle fiber regeneration. This, in turn, conferred tissue protection and regeneration with longer term functional improvement.

BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent stem cells. Based on their properties, several clinical trials have been designed to explore their potential therapeutic effect. Fetal calf serum (FCS, commonly used for in vitro expansion) is an undesirable source of xenogeneic antigens and bears the risk of transmitting contaminations. As an alternative for FCS, platelet lysate (PL) and both autologous and allogeneic human serum have been proposed. The aim of this study is to compare the culture of bone marrow (BM)-derived MSCs in the presence of different serum supplements to determine the effect on cell growth, differentiation potential, and immunologic function. STUDY DESIGN AND METHODS: MSCs from BM of healthy volunteer donors were grown in the presence of 10% FCS supplemented with 1 ng/mL basic fibroblast growth factor (bFGF), 10% human serum supplemented with 1 ng/mL bFGF, 5% PL, and PL 5% supplemented with 1 ng/mL bFGF (PL plus bFGF). RESULTS: MSCs that expanded in either medium showed a comparable morphology, phenotype, and proliferative and differentiation capacity. While the presence of MSCs in vitro significantly decreased CD3/CD28-mediated T-cell activation, this effect was significantly higher in MSCs cultured with human serum. Production of interferon-γ was inhibited by cocultured media with MSCs while MSCs also induced a significant inhibition of cell cycle in T cells. DISCUSSION: In conclusion, PL or autologous serum could offer an alternative to the use of FCS in MSC expansion for clinical use maintaining the same growing potential, phenotype, immunomodulatory properties, and differentiation potential.

The aim of this study was to assess the physiological response of Anguilla anguilla to propanil and the degree of recovery after being moved to clean water. Preliminary acute toxicity test was carried out in the laboratory and the median lethal concentration (LC50) at 96 h was calculated as 31.33 mg/L (29.60–33.59 mg/L). NOEC and LOEC values (at 96 h) were also calculated as 20 and 25 mg/L, respectively. The fish were exposed to 0.63 and 3.16 mg/L of propanil for 72 h and allowed to recover for 144 h. Total proteins (TPs), γ -glutamil transpeptidase ( γ -GT), alanin aminotransferase (AlAT), alkaline phosphatase (AP), lactate dehydrogenase (LDH) and water content (WC) were assayed in muscle and liver tissues, liver somatic index (LSI) was also determined. Liver TPs and γ -GT activity decreased after propanil exposure while AlAT and LDH increased. Muscular AP, AlAT and proteins decreased in intoxicated eels while LDH and γ -GT activities increased. WC increased in both tissues after herbicide exposure as well as LSI. These results revealed that propanil affects the intermediary metabolism of A. anguilla and that the assayed enzymes can be used as good biomarkers of herbicide contamination. However a longer recovery period should be necessary to re-establish eel physiology. The parameters measured in the present study can be used as herbicide toxicity indicators and are recommended for environmental monitoring assessments.

In order to determine new signal transduction pathways implicated in chronic myeloid leukaemia (CML), we performed a gene expression profile comparison between CD34+ cells from CML patients and healthy donors. Functional studies were performed using the Mo7e and Mo7e-p210 cell lines. Expression of CCND1 (Cyclin D1), as well as the chaperone HSPA8, which is important for regulation of CCND1, were significantly upregulated in CD34+ CML cells. Upregulation of HSPA8 was dependent, at least in part, on STAT5 (signal transducer and activator of transcrition 5)-dependent transcriptional activation, as demonstrated by chromatin immunoprecipitation. The presence of HSPA8 in the nuclear protein fraction as well as its binding to CCND1 suggests that it may contribute to stabilization of the CCND1/CDK4 complex, which, in turn, may participate in proliferation of CML cells. Treatment of CML cells with the specific HSPA8 inhibitor 15-deoxyspergualin induced inhibition of CML cell viability but did not induce apoptosis. In conclusion, our studies suggest that STAT5-mediated activation of HSPA8 induces nuclear translocation and activation of the CCND1/CDK4 complex leading to increased proliferation of CML cells, deciphering a new pathway implicated in CML and supporting a potential role of chaperone inhibitors in the treatment of CML.

The aim of our study was to determine the potential mechanism(s) implicated in Imatinib resistance in patients with Ph + ALL. Resistance of Ph + ALL cells to Imatinib-induced apoptosis was associated with lack of inhibition of Akt phosphorylation. Addition of the PI3K inhibitor LY294002 to Imatinib significantly increased apoptosis of Ph + ALL cells. Interestingly, expression of PTEN was reduced in Ph + ALL cells which was due to PTEN promoter hypermethylation. Treatment of Ph + ALL cells with 5-Aza-2′-deoxycytidine was associated with an increased expression of PTEN and an increase in cell apoptosis. These results suggest that Imatinib resistance in patients with ALL may be dependent at least in part to PTEN down-regulation due to the abnormal promoter hypermethylation and support the potential role of de-methylating agents for the treatment of patients with Ph + ALL.

The aim of this nonrandomized, open label, phase 1 clinical trial was to evaluate the safety and the feasibility of the treatment with autologous bone marrow–derived endothelial progenitor cells (EPC) in decompensated liver cirrhosis. In addition, the changes in liver function and hepatic venous pressure gradient (HVPG) and their relation with the characteristics of the cellular product were analyzed. Twelve patients with Child-Pugh ≥8 liver cirrhosis underwent bone marrow harvest for ex vivo differentiation of EPC. The final product was administered through the hepatic artery in a single administration. Patients underwent clinical and radiologic follow-up for 12 months. The phenotype and the ability to produce cytokines and growth factors of the final cellular suspension were analyzed. Eleven patients were treated (feasibility 91%). No treatment-related severe adverse events were observed as consequence of any study procedure or treatment. Model for end-stage liver disease score improved significantly ( P 0.042) in the first 90 days after cells administration and 5 of the 9 patients alive at 90 days showed a decreased of HVPG. There was a direct correlation between the expression of acetylated-low density lipoprotein and von Willebrand factor in the cellular product and the improvement in liver function and HVPG. The treatment with EPCs in patients with decompensated liver cirrhosis is safe and feasible and might have therapeutic potential. Patients receiving a higher amount of functionally active EPC showed an improvement of liver function and portal hypertension suggesting that the potential usefulness of these cells for the treatment of liver cirrhosis deserves further evaluation.

Most human cancers derive from a single cell targeted by genetic and epigenetic alterations that initiate malignant transformation. Progressively, these early cancer cells give rise to different generations of daughter cells that accumulate additional mutations, acting in concert to drive the full neoplastic phenotype. As we have currently deciphered many of the gene pathways disrupted in cancer, our knowledge about the nature of the normal cells susceptible to transformation upon mutation has remained more elusive. Adult stem cells are those that show long-term replicative potential, together with the capacities of self-renewal and multi-lineage differentiation. These stem cell properties are tightly regulated in normal development, yet their alteration may be a critical issue for tumorigenesis. This concept has arisen from the striking degree of similarity noted between somatic stem cells and cancer cells, including the fundamental abilities to self-renew and differentiate. Given these shared attributes, it has been proposed that cancers are caused by transforming mutations occurring in tissue-specific stem cells. This hypothesis has been functionally supported by the observation that among all cancer cells within a particular tumor, only a minute cell fraction has the exclusive potential to regenerate the entire tumor cell population; these cells with stem-like properties have been termed cancer stem cells. Cancer stem cells can originate from mutation in normal somatic stem cells that deregulate their physiological programs. Alternatively, mutations may target more committed progenitor cells or even mature cells, which become reprogrammed to acquire stem-like functions. In any case, mutated genes should promote expansion of stem/progenitor cells, thus increasing their predisposition to cancer development by expanding self-renewal and pluripotency over their normal tendency towards relative quiescency and proper differentiation.

In this study, we report the case of a Philadelphia (Ph) positive chronic myelogenous leukemia (CML) patient with the presence of p190 and p210 BCR-ABL1 mRNA fusion transcripts derived from e1a2 and b3a2 BCR-ABL1 genomic rearrangements, respectively. The presence of e1a2 BCR-ABL1 genomic rearrangement was seen in 2 different clones, one with the rearrangement and another one with the rearrangement and deletion of the BCR gene of the non-rearranged chromosome 22. After treatment with imatinib, the p210 transcript could not be detected, whereas p190 was still present 6 months after initiation of imatinib therapy and progression to blast phase. The absence of p210 transcript post treatment indicates that the clone with b3a2 responded to imatinib and that the observed resistance was associated to cells harboring the e1a2 genomic rearrangement. Despite resistance of this patient to imatinib, no evidence of mutations in the kinase domain of ABL1 was found. Loss of normal BCR in one cell clone may contribute to the resistance to imatinib due to the lack of BCR mediated inhibition of BCR-ABL1.

European eels (Anguilla anguilla) were exposed to sublethal thiobencarb concentrations in a continuous flow-through system for 4 days. Brain, muscle, and gill acetylcholinesterase (AChE) activities were evaluated after 2, 12, 24, 48, 72, and 96 h herbicide exposure. Thiobencarb induced significant inhibitory effects on the total and specific AChE activity of A. anguilla, ranging from >30–40% inhibition in eel brain and gills to >50% inhibition in muscle tissue 2 h after the initial exposure. In a second experiment, eels were exposed to thiobencarb for 96 h and then allowed a period of recovery in pesticide-free water. Following 1 week of recovery, the AChE activity of affected eels approached control level. Fish transferred to clean water recovered specific AChE activities in the studied tissues slowly in the next 8 days (without toxicant), reaching about 60–80% of the normal levels in brain and muscle tissue around the 4th day after the beginning of the recovery period (in clean water). Specific AChE activity in the gills from eels transferred to clean water recovered its normal value while total AChE activity remained depressed (29%) in this tissue 8 days later. Many behavioral changes such as lowering of motility with loss of equilibrium, uncoordinated movements, and increase in the levels of respiratory frequency were observed during exposure to the herbicide.

ヨーロッパウナギ Anguilla anguilla の代謝系へのチオベンカルブの影響と, 中毒からの回復とを評価した. 暴露期間ならびにそれに続く非汚染水中での8日間のウナギの血漿, 筋肉, 肝臓についてグリコーゲン, 脂質およびタンパクを測定した. 暴露により血糖値の上昇が認められたが, 肝臓と筋肉におけるグリコーゲンレベルは著しく低下した. 血中の脂質は増加したが, 肝臓での脂質は顕著に減少した. タンパク質の易動化は, とくに筋肉組織で認められた. ほとんどの代謝障害は, 非汚染水へ移した8日後まで継続した. 以上のチオベンカルブの代謝系への影響はある種のストレス症状であり, 魚類の農薬ストレスを代償しようとする生理的適応の具体例と考えられた.

We evaluated the feasibility, safety, and efficacy of the administration of 4 sequential doses (intravenously administered on days 1, 4, 11, and 18) of cryopreserved bone marrow–derived mesenchymal stromal cells (MSC) expanded with platelet lysate and obtained from third-party donors as a second-line treatment for steroid-refractory acute graft-versus-host (aGVHD) disease in a series of 25 patients. All patients received at least 2 doses of MSC, whereas 21 received 3 doses and 18 received the initially planned 4 doses. Because of the achievement of partial response, 4 patients received additional doses of MSC. Median single cell dose administered was 1.1 × 106 MSC/kg of recipient body weight. There were no adverse events related to the MSC infusion in the 99 procedures performed, with the exception of a cardiac ischemic event that occurred twice in a patient with prior history of cardiac ischemia. Response to MSC at 60 days after the first dose was evaluable in 24 patients. Seventeen patients (71%) responded (11 complete and 6 partial responses), with a median time to response of 28 days after the first MSC dose, whereas 7 patients did not respond. In summary, we can conclude that sequential cryopreserved third-party MSC therapy administered on days 1, 4, 11, and 18 is a safe procedure for patients with steroid-refractory aGVHD. This strategy may provide a high rate of overall responses of aGVHD with a low toxicity profile.

OBJECTIVE: Uncontrolled studies of mesenchymal stem cells (MSCs) in multiple sclerosis suggested some beneficial effect. In this randomized, double-blind, placebo-controlled, crossover phase II study we investigated their safety and efficacy in relapsing-remitting multiple sclerosis patients. Efficacy was evaluated in terms of cumulative number of gadolinium-enhancing lesions (GEL) on magnetic resonance imaging (MRI) at 6 months and at the end of the study. METHODS: Patients unresponsive to conventional therapy, defined by at least 1 relapse and/or GEL on MRI scan in past 12 months, disease duration 2 to 10 years and Expanded Disability Status Scale (EDSS) 3.0-6.5 were randomized to receive IV 1-2×10(6) bone-marrow-derived-MSCs/Kg or placebo. After 6 months, the treatment was reversed and patients were followed-up for another 6 months. Secondary endpoints were clinical outcomes (relapses and disability by EDSS and MS Functional Composite), and several brain MRI and optical coherence tomography measures. Immunological tests were explored to assess the immunomodulatory effects. RESULTS: At baseline 9 patients were randomized to receive MSCs (n = 5) or placebo (n = 4). One patient on placebo withdrew after having 3 relapses in the first 5 months. We did not identify any serious adverse events. At 6 months, patients treated with MSCs had a trend to lower mean cumulative number of GEL (3.1, 95% CI = 1.1-8.8 vs 12.3, 95% CI = 4.4-34.5, p = 0.064), and at the end of study to reduced mean GEL (-2.8±5.9 vs 3±5.4, p = 0.075). No significant treatment differences were detected in the secondary endpoints. We observed a non-significant decrease of the frequency of Th1 (CD4+ IFN-γ+) cells in blood of MSCs treated patients. CONCLUSION: Bone-marrow-MSCs are safe and may reduce inflammatory MRI parameters supporting their immunomodulatory properties. ClinicalTrials.gov NCT01228266.

Patients with mantle cell lymphoma (MCL) may present with either nodal or leukemic disease. The molecular determinants underlying this different biologic behavior are not known. This study compared the pattern of genetic abnormalities in patients with nodal and leukemic phases of MCL using comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) for specific gene loci. Although both leukemic and nodal MCL showed similar genomic patterns of losses (involving 6q, 11q22-q23, 13q14, and 17p13) and gains (affecting 3q and 8q), genomic loss of chromosome 8p occurred more frequently in patients with leukemic disease (79% versus 11%,P

We have developed a new nonradioactive assay to identify human low-density lipoprotein receptor defects. It is based on the incubation of cultured cells with colloidal gold-LDL conjugates and quantitation of the gold associated with the cells by electrothermal atomic absorption spectrometry. After an oxidative treatment with nitric and hydrochloric acids, the biological matrix interferes neither with the gold recovery nor with the gold measurements, which are linear, at least from 0.15 to 3 ng of gold. When cells expressing a functional LDL receptor are incubated with increasing amounts of colloidal-gold LDL conjugates, the obtained saturation curve parallels that described when [125I]LDL is used as ligand. Moreover, this new assay allows us to clearly distinguish among fibroblasts from normal subjects or from heterozygous or homozygous patients of familial hypercholesterolemia, a very common autosomal disease. The assay is easy to perform, is sensitive, and avoids the use of radioactive compounds. Therefore, it could be successfully employed in the clinical diagnosis of this disease. Furthermore, since the methodology developed here can be applied to quantify the association of other gold-conjugated ligands to cells, it could have a widespread use in a variety of clinical and basic research studies.

Bcr-Abl–expressing leukemic cells are highly resistant to apoptosis induced by chemotherapeutic drugs. Although a number of signaling molecules have been shown to be activated by the Bcr-Abl kinase, the antiapoptotic pathway triggered by this oncogene has not been elucidated. Here, we show that the interleukin 3-independent expression of the antiapoptotic protein, Bcl-xL, is induced by Bcr-Abl through activation of signal transducer and activator of transcription (Stat)5. Inhibition of the Bcr-Abl kinase activity in Bcr-Abl–expressing cell lines and CD34+ cells from chronic myelogenous leukemia (CML) patients induces apoptosis by suppressing the capacity of Stat5 to interact with the bcl-x promoter. Interestingly, after inhibition of the Bcr-Abl kinase, the expression of Bcl-xL is downregulated more rapidly in chronic phase than in blast crisis CML cells, suggesting an involvement of this protein in disease progression. Overall, we describe a novel antiapoptotic pathway triggered by Bcr-Abl that may contribute to the resistance of CML cells to undergo apoptosis.

Acute and chronic toxicity tests with the rice herbicides molinate (S‐ethyl hexahydro‐1H‐azepine‐1‐carbothioate) and thiobencarb (S‐4‐chlorobenzyl diethyl thiocarbamate) were conducted on the rotifer Brachionus calyciflorus. The 24‐hr static LC50's were 11.37 and 47.82 mg.L‐1 for molinate and thiobencarb, respectively. The effects of sublethal levels of both herbicides on the rotifer B. calyciflorus were evaluated using some demographic parameters: intrinsic rate of natural increase (r), generation time (T) and net reproductive rate (Ro). All the parameters studied decreased with increasing toxicant concentrations. The maximum acceptable toxicant concentration (MATC) was calculated for both herbicides using the intrisic rate of natural increase (r) as parameter of evaluation and the chronic data was used to calculate aplication factors (AF) of 0.062 and 0.066 mg.L‐1 for molinate and thiobencarb, respectively. Finally, the EC50 values were derived for the selected life history parameters (Ro, T, r...

The effect of two sublethal fenitrothion concentrations (0.02 and 0.04 mg/L) on the energy metabolism of the european eel Anguilla anguilla and its recovery from intoxication was investigated. Analysis of various parameters such as glycogen, proteins and total lipids was made on liver and muscle eel tissues after 2, 8, 12, 24, 32, 48, 56, 72 and 96 hr of fenitrothion (0,0-dimethyl 0-3-methyl-4-nitrophenyl phosphorothioate) exposure. Subsequently, the fish were allowed recovery periods of 8, 12, 24, 48, 72, 96, 144 and 192 hr in clean water, and the same parameters were again evaluated. The results obtained during the exposure to the pesticide as well as during the recovery phase were used to calculate the caloric content in both tissues of A. anguilla. A reduction in energy reserves in the selected tissues was observed after exposure to both fenitrothion concentrations and the caloric content in those animals was lower than in the controls. Most of the metabolic disorders did not persist after allowing recovery in clean water during a week.

The feeding behavior of the cladocera Daphnia magna was studied when subjected to a short‐term exposure to the acaricide tetradifon (4‐chlorophenyl 2,4,5‐trichlorophenyl sulfone). Test species were exposed to sublethal levels of tetradifon and the effect of the toxicant on filtration and ingestion rates was determined. The experiments were performed using the unicellular algae Nannochloris oculata in a density of 5×105 cells/mL as food for the organisms. Rates of filtration and ingestion of D. magna declinedwith increasing toxicant concentrations. The effective tetradifon concentrations in D. magna at which feeding rate were reduced to 50% of that in controls (EC50) were 0.02 and 0.24 mg/L for filtration and ingestion rates respectively.

Eels of species Anguilla anguilla were exposed to sublethal concentrations of fenitrothion in a flow-through test system. Bioaccumulation and elimination of the toxicant in the branchial tissue of the eels were studied. Steady-state was reached after 48 hr of exposure to 0.02 ppm of toxicant, and earlier (24 hr) exposed to 0.04 ppm. Steady-state was maintained until the end of the exposure in both experiments. The highest bioconcentration factor (BCF) value was calculated in animals exposed to 0.04ppm of toxicant, showing the relationship between the bioconcencentration into gills and the water disponibility. When the animals were transferred to clean water (depuration phase) they showed a rapid tendency to eliminate the pesticide. Pharmaco-kinetics parameters for fenitrothion in eel gills (K 1 , K 1 and T 1 /2) were calculated. They showed relatively low K 2 (0.0014 and 0.029 hr -1 ) and higher half-lives. Paralelly, total protein content in the gills was monitored during exposure and depuration phases. A depletion of total proteins of a 70% was observed in gill tissue at the end of the pesticide exposure. Although a tendency to recover protein levels was observed during the depuration phases, the depletion was maintained until the end of the experiments. The results showed a stress situation as a consequence of fenitrothion exposure which indicates the use of proteins as response to the increased energy requirements in this tissue.

European eel (Anguilla anguilla) were exposed to sublethal fenitrothion concentrations in a continuous flow-through system for 4 days. Brain acetylcholinesterase (AChE) activity was evaluated after 2, 8, 12, 24, 32, 48, 56, 72, and 96 h pesticide exposure. Results indicated that AChE activity in eel brains decreased as the concentration of fenitrothion increased. The pesticide induced significant inhibitory effects on the AChE activity ofA. anguilla,ranging from >40% inhibition at a sublethal concentration of 0.02 ppm to >60% inhibition at a sublethal concentration of 0.04 ppm. Eel were exposed to both fenitrothion concentrations for 96 h and then allowed a period of recovery in pesticide-free water. Samples were removed at 8, 12, 24, 48, 72, 96, 144, and 192 h and eel brain AChE activity was evaluated. Following 1 week of recovery, the AChE activity of those animals previously exposed to 0.02 and 0.04 ppm fenitrothion was still different from that of the controls. So, the AChE activity of eel brains at the end of the recovery phase remained significantly depressed.

European eels (Anguilla anguilla) were exposed to sublethal fenitrothion concentrations (0.02 and 0.04 mg/liter) in a continuous flow-through system for 4 days. Gill Mg2+- and Na+,K+-ATPase activities were evaluated after 2, 8, 12, 24, 32, 48, 56, 72, and 96 h of pesticide exposure. Results indicated that ATPase activity in gill tissue decreased as concentration of fenitrothion increased. Pesticide induced significant inhibitory effects on the Na+, K+-ATPase activity of A. anguilla, ranging from >56% inhibition at a sublethal concentration of 0.02 ppm to >73% inhibition at a sublethal concentration of 0.04 ppm. Eels were exposed to both fenitrothion concentrations for 96 h and then allowed a period of recovery in pesticide-free water. Samples were removed at 8, 12, 24, 48, 72, 96, 144, and 192 h and eel gill ATPase activity was evaluated. Following 1 week of recovery, the Na+,K+-ATPase activity for those animals previously exposed to 0.04 ppm fenitrothion was still different from that of the controls.

European eel (Anguilla anguilla) were exposed to sublethal fenitrothion concentrations in a continuous flow-through system for 4 days. Muscle acetylcholinesterase (AChE) activity was evaluated after 2, 8, 12, 24, 32, 48, 56, 72 and 96 hours pesticide exposure. Results showed that AChE activity in eel muscle tissue decreased as concentration of fenitrothion increased. Pesticide induced significant inhibitory effects on the AChE activity of A. anguilla ranging from35% inhibition at sublethal concentration of 0.02 ppm to44% inhibition at sublethal concentration of 0.04 ppm. Eel were exposed to both fenitrothion concentrations for 96 hours and then allowed a period of recovery in pesticide-free water. Samples were taken out at 8, 12, 24, 48, 72, 96, 144 and 192 hours and eel muscle AChE activity was evaluated. Following 1 week of recovery, the AChE activity for those animals previously exposed to 0.02 and 0.04 ppm fenitrothion was still different from the controls. So, the AChE activity of eel muscle at the end of the recovery phases remained significantly depressed.

The present communication deals with the effect of diazinon (0.042 mg/L) on the energy metabolism of the european eel Anguilla anguilla. Analysis of various parameters such as glycogen, lactate and glucose was made on different eel tissues after 6, 24, 48, 72 and 96 hr of diazinon exposure. Liver and muscle glycogen content decreased significantly (p < 0.05) from 6 to 96 hr pesticide exposure. Mean blood glucose values were elevated significantly after exposure to this sublethal diazinon concentration. Liver, blood and muscle lactate levels in fish increased during all exposure time being the highest at 96 hr exposure. Diazinon intoxicated fish showed signs of pesticide poisoning as muscular twitching, gyrating swimming movements and diminished sensory activity but survived the stipulated exposure period. The results are discussed in relation to the stress effect produced by the pesticide and the related responses of the fish.

AIM: To assess cultured limbal epithelial stem cell transplantation in patients with limbal stem cell deficiency by analyzing and quantifying corneal neovascularization.METHODS: This retrospective, interventional case series included eight eyes with total limbal stem cell deficiency. Ex vivo limbal epithelial stem cells were cultured on human amniotic membrane using an animal-free culture method. The clinical parameters of limbal stem cell deficiency, impression cytology, and quantification of corneal neovascularization were evaluated before and after cultured limbal stem cell transplantation. The area of corneal neovascularization, vessel caliber (VC), and invasive area (IA) were analyzed before and after stem cell transplantation by image analysis software. Best-corrected visual acuity (BCVA), epithelial transparency, and impression cytology were also measured.RESULTS: One year after surgery, successful cases showed a reduction (improvement) of all three parameters of corneal neovascularization [neovascular area (NA), VC, IA], while failed cases did not. NA decreased a mean of 32.31% (P=0.035), invasion area 29.37% (P=0.018) and VC 14.29% (P=0.072). BCVA improved in all eyes (mean follow-up, 76±21mo). Epithelial transparency improved significantly from 2.00±0.93 to 0.88±1.25 (P=0.014). Impression cytology showed that three cases failed after limbal epithelial stem cell therapy before 1y of follow-up.CONCLUSION: This method of analyzing and monitoring surface vessels is useful for evaluating the epithelial status during follow-up, as successful cases showed a bigger reduction in corneal neovascularization parameters than failed cases. Using this method, successful cases could be differentiated from failed cases.

The binding and endocytic uptake of low-density lipoprotein (LDL) particles by cells, transiently or permanently transfected with the human LDL receptor cDNA, was investigated, under different situations, using colloidal gold–LDL conjugates. The amount of gold associated with the various cells, which bind and internalize LDL to different extents, was estimated by inductively coupled plasma–MS. In all cases, the existing differences in LDL binding and uptake were clearly detectable with this procedure. We conclude, therefore, that inductively coupled plasma–MS provides an appropriate assay system for the rapid quantitation of these processes. This procedure also recognizes differences in LDL receptor expression in human lymphocytes and, therefore, it could be of value for the differential diagnosis of LDL receptor defects in familial hypercholesterolemia in various cell types. In addition, this easily performed methodology can also be applied to a variety of other problems requiring quantitation of colloidal gold associated with cells.

Eels were exposed to a sublethal diazinon concentration of 0.042 mg/L for exposure times of 6, 24, 48, 72 and 96 hours. Biochemical analyses of blood composition, such as plasma glucose, total plasma cholesterol and triglycerides, plasma lactate, plasma urea and uric acid, showed significant differences between treated and control animals. Plasma glucose and lactate increased after 6 hours exposure to the insecticide. Plasma cholesterol and triglycerides content decreased during 96 hours treatment. Urea levels increased at 72 hours while uric acid content decreased significantly at 24, 72 and 96 hours exposure to the pesticide. The observed effects of diazinon on eel metabolism suggested that the treated fish was faced with a serious metabolic crisis, and the fish looked for alternative methods of metabolism to overcome the toxic stress.

The present communication deals with the effect of fenitrothion (0.04 ppm) on the carbohydrate metabolism of the european eel Anguilla anguilla and its recovery from intoxication. Analysis of various parameters such as glycogen, lactate and glucose was made on different eel tissues after 2, 8, 12, 24, 32, 48, 56, 72 and 96 hr of fenitrothion exposure. Subsequently, the fish were allowed recovery periods of 8, 12, 24, 48, 72, 96, 144 and 192 hr in clean water, and the same parameters were evaluated. Muscle glycogen content decreased significantly during the exposure time, while blood glucose levels increased markedly. Gill, muscle and blood lactate values increased during pesticide exposure. Most of the metabolic disorders did not persist after allowing recovery in clean water during less than a week. The observed effects of fenitrothion on fish metabolism are discussed in relation to a stress syndrome and probable reasons for alterations have been exposed.

Previous works on endosulfan eel toxicology in this laboratory demonstrated that 0.041 mg/liter of endosulfan was the 50% lethal concentration of 96 hr exposure. Eels of species Anguilla anguilla were exposed to two sublethal endosulfan concentrations: 8.2 μg/liter (1/5 LC50) and 4.1 μg/liter (1/10 LC50), and the experiment was done at different exposure times: 12, 24, 48, 72, and 96 hr. Muscle glycogen content decreased significantly (P < 0.05) at 24, 48, 72, and 96 hr exposure to 8.2 μg/liter pesticide; levels did not decline at any time when animals were exposed to 4.1 μg/liter. Muscle lactate levels in fish did not change significantly while lactate levels in eel blood increased at 12, 24, 48, 72, and 96 hr exposure to 8.2 μg/liter. Mean blood glucose values were elevated after exposure to both endosulfan concentrations. The results are discussed in relation to the stress effect produced by the pesticide and the related responses of the fish.

Previous works on endosulfan toxicology of Anguilla anguilla in our laboratory showed that 41μg/l endosulfan was the 50% lethal concentration at 96 hr exposure. The eels were exposed to two sublethal endosulfan concentrations: 8.2 μg/l ( 1 5 lc 50) and 4.1 μg/l ( 1 10 lc 50), during different exposure times: 12, 24,48,72 and 96 hr. Glycogen and lactate concentrations were determined in liver tissue after endosulfan exposure. At the same time, we evaluated glucose and lactate levels in eel blood. Liver glycogen content decreased during the exposure time, while lactate levels increased in both liver and blood tissues. Glucose blood concentration increased after pesticide intoxication. The results are discussed in relation to the stress effect produced by the pesticide and the related responses of the fish.