miRNA-7 and miRNA-324-5p regulate alpha9-Integrin expression and exert anti-oncogenic effects in rhabdomyosarcoma.

The prognosis of patients with metastatic rhabdomyosarcoma (RMS), the most common type of soft tissue sarcoma in children, is poor and no strategies have been identified to improve their dismal prognosis. Alpha-9 integrin (ITGA9) plays a particularly crucial role in cancer progression and invasiveness. Despite the consensus on the remarkable pro-oncogenic potential of this protein, the miRNA-mediated regulation of ITGA9 has barely been studied to date. In the present study, miR-7 and miR-324-5p were selected as the best candidates after a screening to find ITGA9 regulators, and their effects on cell proliferation and invasion in RMS are described and characterized for the first time. Interestingly, the overexpression of both miRNA produced a clear impairment of cell proliferation, while miR-7 also induced a remarkable drop in cell invasion. Furthermore, the stable overexpression of both miRNA was found to reduce tumor growth in orthotopic RMS models and miR-7 was able to impair metastatic lung colonization. Consequently, we conclude that miR-7 and miR-324-5p show anti-oncogenic and anti-metastatic potential, thereby opening up the possibility of being used as novel therapeutic tools to avoid RMS progression.

Liver Transplantation for Acute Liver Failure due to Yellow Fever: A Case Report.

Yellow fever is a noncontagious disease caused by an arbovirus in the Flaviviridae family. It is an endemic disease in the tropical forests of Africa and South America, with the mosquito as a vector. Approximately half of those infected will be asymptomatic, while 15% will develop the severe/malignant form of the disease that includes renal and hepatic failure, bleeding, and neurological impairment as the principal symptoms. The lethality of the severe form reaches up to 70%. The objective of this study was to report on the case of a patient who was transferred to the hepatobiliary unit of our service due to acute liver failure due to yellow fever. He was treated with liver transplantation. The patient progressed satisfactorily, being discharged from the intensive care unit in 10 days and discharged from the hospital within 19 days after transplantation. Despite the encouraging result of our team, this has not been applied to other centers that have also performed this modality of treatment; therefore, the question remains as to whether and when to recommend liver transplantation for treatment of severe yellow fever.

Ligand-dependent Hedgehog pathway activation in Rhabdomyosarcoma: the oncogenic role of the ligands.

BACKGROUND: Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children. The Hedgehog (HH) pathway is known to develop an oncogenic role in RMS. However, the molecular mechanism that drives activation of the pathway in RMS is not well understood. METHODS: The expression of HH ligands was studied by qPCR, western blot and immunohistochemistry. Functional and animal model studies were carried out with cells transduced with shRNAs against HH ligands or treated with HH-specific inhibitors (Vismodegib and MEDI-5304). Finally, the molecular characterisation of an off-target effect of Vismodegib was also made. RESULTS: The results showed a prominent expression of HH ligands supporting an autocrine ligand-dependent activation of the pathway. A comparison of pharmacologic Smoothened inhibition (Vismodegib) and HH ligand blocking (MEDI-5304) is also provided. Interestingly, a first description of pernicious off-target effect of Vismodegib is also reported. CONCLUSIONS: The clarification of the HH pathway activation mechanism in RMS opens a door for targeted therapies against HH ligands as a possible alternative in the future development of better treatment protocols. Moreover, the description of a pernicious off-target effect of Vismodegib, via unfolded protein response activation, may mechanistically explain its previously reported inefficiency in several ligand-dependent cancers.

Patient-derived xenografts for childhood solid tumors: a valuable tool to test new drugs and personalize treatments.

The use of preclinical models is essential in translational cancer research and especially important in pediatric cancer given the low incidence of each particular type of cancer. Cell line cultures have led to significant advances in cancer biology. However, cell lines have adapted to growth in artificial culture conditions, thereby undergoing genetic and phenotypic changes which may hinder the translational application. Tumor grafts developed in mice from patient tumor tissues, generally known as patient-derived xenografts (PDXs), are interesting alternative approaches to reproducing the biology of the original tumor. This review is focused on highlighting the interest of PDX models in pediatric cancer research and supporting strategies of personalized medicine. This review provides: (1) a description of the background of PDX in cancer, (2) the particular case of PDX in pediatric cancer, (3) how PDX can improve personalized medicine strategies, (4) new methods to increase engraftment, and, finally, (5) concluding remarks.

BRG1/SMARCA4 is essential for neuroblastoma cell viability through modulation of cell death and survival pathways.

Neuroblastoma (NB) is a neoplasm of the sympathetic nervous system, and is the most common solid tumor of infancy. NBs are very heterogeneous, with a clinical course ranging from spontaneous regression to resistance to all current forms of treatment. High-risk patients need intense chemotherapy, and only 30-40% will be cured. Relapsed or metastatic tumors acquire multi-drug resistance, raising the need for alternative treatments. Owing to the diverse mechanisms that are responsible of NB chemoresistance, we aimed to target epigenetic factors that control multiple pathways to bypass therapy resistance. We found that the SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4 (SMARCA4/BRG1) was consistently upregulated in advanced stages of NB, with high BRG1 levels being indicative of poor outcome. Loss-of-function experiments in vitro and in vivo showed that BRG1 is essential for the proliferation of NB cells. Furthermore, whole-genome transcriptome analysis revealed that BRG1 controls the expression of key elements of oncogenic pathways such as PI3K/AKT and BCL2, which offers a promising new combination therapy for high-risk NB.

Impact of model for end-stage liver disease score on long-term survival following liver transplantation for hepatocellular carcinoma.

BACKGROUND AND AIMS: Survival rates after orthotopic liver transplantation (OLT) for hepatocellular carcinoma (HCC) have significantly increased after Milan criteria and Model for End-Stage Liver Disease (MELD) score implementation. However, few studies have reported this survival in countries with organ donor shortages over a period of 10 years and long waiting lists. METHODS: This retrospective analysis of clinical data from 93 consecutive HCC patients who underwent OLT from June 2001 to September 2011 excluded 22 who underwent living donor liver transplantation (LDLT). Seventy-one deceased donor liver transplantations (DDLT) were evaluated before and after the MELD era. Kaplan-Meier analysis was used to plot survival rates. The follow-up was 2 months to 10 years. RESULTS: The overall survival and recurrence rates at 10 years were 67% and 12.2%, respectively. After MELD, patient survival at 5 years decreased from 70% to 64% and the recurrence rate decreased from 15.3% to 12.5%. The most frequent recurrence sites were lung and liver. CONCLUSION: In our center MELD score implementation had a small impact on long-term survival post OLT for HCC.

Notch-mediated induction of N-cadherin and α9-integrin confers higher invasive phenotype on rhabdomyosarcoma cells.

BACKGROUND: Rhabdomyosarcoma (RMS) is the commonest type of soft-tissue sarcoma in children. Patients with metastatic RMS continue to have very poor prognosis. Recently, several works have demonstrated a connection between Notch pathway activation and the regulation of cell motility and invasiveness. However, the molecular mechanisms of this possible relationship remain unclear. METHODS: The Notch pathway was manipulated pharmacologically and genetically. The mRNA changes were analysed by quantitative PCR and protein variations by western blot and immunofluorescence. Finally, the capabilities of RMS cells to adhere, heal a wound and invade were assessed in the presence of neuronal cadherin (N-cadherin)- and α9-integrin-blocking antibodies. RESULTS: Cells treated with γ-secretase inhibitor showed lower adhesion capability and downregulation of N-cadherin and α9-integrin. Genetic manipulation of the Notch pathway led to concomitant variations in N-cadherin and α9-integrin. Treatment with anti-N-cadherin-blocking antibody rendered marked inhibition of cell adhesion and motility, while anti-α9-integrin-blocking antibody exerted a remarkable effect on cell adhesion and invasiveness. CONCLUSION: Neuronal cadherin and α9-integrin are postulated as leading actors in the association between the Notch pathway and promotion of cell adhesion, motility and invasion, pointing to these proteins and the Notch pathway itself as interesting putative targets for new molecular therapies against metastases in RMS.

[Chromosomal translocations in soft tissue sarcomas: from molecular biology to clinical application]. / Translocaciones cromosómicas en los sarcomas de partes blandas: de la biología molecular a la aplicación clínica.

Recent advances in the knowledge of the molecular biology of paediatric sarcomas, especially the characterisation of chromosomal translocations associated specifically with particular types of cancer, have established bases for the introduction of new diagnostic tools. This article reviews the main chromosomal translocations associated with paediatric tumours, and summarises their molecular characteristics regarding their oncogenic capabilities, possible usefulness as a differential diagnostic tools and possible correlation with clinical parameters.

Vascular complications after living donor liver transplantation: a Brazilian, single-center experience.

BACKGROUND: In living donor liver transplantation (LDLT), vascular complications are more frequently seen than in deceased donor transplantation. Early arterial, portal vein, or hepatic vein thromboses are complications that can lead to graft loss and patient death. The aim of this study was to assess the incidence, treatment, and outcome of vascular complications after LDLT in a single Brazilian center. METHODS: Between December 2001 and December 2010, we performed 130 LDLT. Sixty-four recipients were children (27 weighing <10 kg). RESULTS: Nine recipients had vascular complications. Hepatic artery thrombosis (HAT) occurred in 4 (3.1%), portal vein thrombosis (PVT) in 3 (2.3%), and hepatic vein thrombosis (HVT) and hepatic arterial stenosis (HAS) in 1 (0.8%) patient each. Complications were identified by Doppler and confirmed by angiography or angiotomography. Patients with HAT were listed for retransplantation. One died before retransplant. Two children were submitted to retransplantation; one is still alive, with neurologic sequelae. One adult with HAT was retransplanted with a deceased donor graft and is doing well 58 months after surgery. Two patients with PVT died as a consequence of graft malfunction. In the other case, portal vein arterialization was performed, but patient died 11 months posttransplant. HVT was detected after cardiac reanimation and was treated with an endovascular stent. This patient died 3 months after LDLT. HAS was diagnosed after liver abscess development and was successfully treated by endovascular angioplasty. No recurrence was observed after 22 months. Follow-up ranged from 9 to 117 months. CONCLUSION: Pediatric patients are more prone to develop vascular complications after LDLT. Long-term survival was statistically lower for recipients with vascular complications (33.3% vs 77.7%; P = .008).

Cellulitis and nodular skin lesions due to Fusarium spp in liver transplant: case report.

Fusariosis is one of the emerging invasive fungal infections over the last decade. However, its recent rise has been in its ability to produce disseminated infection in severely immunosuppressed patients with neutropenia. In solid organ transplantation, fusariosis remains an uncommon picture mainly with nodules, subcutaneous abscesses, ulcers, or necrotic skin lesions resembling erthyma gangrenosum. Herein, we have reported a case of cellulitis, subcutaneous nodules, and abscesses due to Fusarium spp in a liver transplantation patient who was successfully treated with polyenes and surgical resection.

Treatment for recurrent hepatitis C virus infection after liver transplantation.

Cirrhosis due to hepatitis C virus (HCV) infection is the current leading indication for orthotopic liver transplantation (OLT) in the world. This series reports our program's experience with the treatment of HCV infection after the development of histological hepatitis. Between March 2002 and June 2008, patients with recurrent HCV were selected for treatment if the liver biopsy showed at least the F2 degree of Metavir score. HCV viral load was measured at 4, 12 and 24 weeks as well as at the end of treatment and at 6 months thereafter for patients who became HCV RNA negative (sustained virological response [SVR]). In this period, we performed 287 liver transplantations in 279 patients, including 117 (42%) who had HCV cirrhosis as the indication for OLT of whom 25 were eligible for antiviral treatment. Twelve patients completed treatment, 7 remain on treatment, and 6 were discontinued. The principal collateral effect was anemia. Only 1 patient had an episode of acute cellular rejection, which responded to adjustment of immunosuppression. Antiviral treatment in transplanted patients was feasible and did not seem to induce severe immunological effects. Adjuvant therapies to reduce cytopenias are frequently required, principally erythropoietin. The best results were observed with the pegylated interferon alfa (PEG) plus ribavirin (RBV) group: 38.9% of SVR. We recommend antiviral treatment of eligible patients with confirmed HCV recurrence using PEG plus RBV.

[Oxidative stress in a model of experimental diabetic retinopathy: the utility of peroxinytrite scavengers]. / Estrés oxidativo en un modelo de retinopatía diabética experimental II: utilidad de agentes secuestrantes de peroxinitritos.

PURPOSE: The retina is the neurosensorial tissue of the eye and is extremely rich in polyunsaturated lipid membranes. This feature makes it especially sensitive to oxygen and/or nitrogen activated species and lipid peroxidation. Several authors have postulated the importance of superoxide (O2-) and peroxynitrite production in the development of diabetic complications. In the present study, we have used two different antioxidants, ebselen and lutein, that present as a common feature their peroxynitrite scavenging capacity, to ameliorate the oxidative stress that exists in the retina in diabetic patients. METHODS: Hyperglycemia was accomplished by the intraperitoneal injection of Alloxan in a mouse model of diabetic retinopathy. Malondialdehyde (MDA) and glutathione (GSH) concentrations in eye homogenates (without the lens) were determined. We also recorded serial electroretinograms (ERG) and measured latency and implicit times. RESULTS: The MDA concentration increased and the GSH concentration decreased in the eyes of the diabetic animals. Treatment with ebselen and lutein restored the MDA and GSH concentrations to control values. Latency and implicit times were not affected by the diabetes. CONCLUSION: New studies are required to better understand the protective mechanism of ebselen and lutein in this model of experimental diabetic retinopathy.

[Intratesticular calcifications: clinical significance]. / Calcificaciones intratesticulares: significación clínica.

OBJECTIVE: [corrected] To evaluate the clinical-pathological significance of the intratesticular calcification. MATERIAL AND METHODS: We analyzed by scrotal U.S., transducer 6.5 Mhz, the patients that consulted about testicular painful, infertility or intrascrotal deformity. Biopsy was effectuated when detected size modification of testicles, and central or focal calcifications. Also was done the classics tumoral markers. We founds 16 patients with microlithiasis over 24 testicles with these pathology. Previous antecedents: infertility 6 cases, testicular devolvulations surgery 5c, bilateral orchidopexy 3c and unilateral epididymitis 2c. The calcifications was classified in: peripheric or central follow-up 26.4 months. RESULTS: Associated pathology: a) Bilateral cryptorchidism: 4 testicles. b) Testicular tumors: 5 testicles. These tumors were biopsed: 3 seminomas, 1 embryonary carcinoma and 1 ca in situ. Both with negative biologic markers. The association with testicular tumor had an incidence of 20.83%. The central localization was detected in the 5 tumors, while the peripheric were 4 testis (cryptorchidism) and 15 with aspect of "snow storm". Histopathologically was observed eosinophilic bodies, with calcified nucleus, but not a exclusive features. CONCLUSIONS: Is a benign condition, casual, whose clinical significance is a still enigma. The association with testicular tumor is 20.83% and associated pathology is 33%. We proposed a testicular biopsy in cases of microlithiasis focal, central or with previous pathology.

Beta-amyloid-induced activation of caspase-3 in primary cultures of rat neurons.

It is known that beta-amyloid peptide (Abeta) contributes to the neurodegeneration in Alzheimer's disease (AD) and operates through activation of an apoptotic pathway. Apoptotic signal is driven by a family of cysteine proteases called caspases. The beta-amyloid precursor protein (APP) is directly and efficiently cleaved by caspases during apoptosis, resulting in elevated beta-amyloid peptide formation. Cerebellar neurons from rat pups were treated with the aged Abeta(25-35) at 1 and 5 microM and fluorescence assays of caspase activity performed over 4 days. We observed an increase in caspase activity after 48 h treatment in both 1 and 5 microM treated cells, then (72-96 h) caspase activity decreased to control values. The data presented support the hypothesis that Abeta(25-35)-induced apoptosis is mediated by the activation of Caspase-3 and that this is a transient effect.

Reduction of brain antioxidant defense upon treatment with butylated hydroxyanisole (BHA) and Sudan III in Syrian golden hamster.

Treatment with the antioxidant butylated hydroxyanisole (BHA) or the azo dye Sudan III during two weeks led to changes in the brain enzymatic antioxidant defense of Syrian golden hamsters. BHA was able to induce liver superoxide dismutase (SOD) 2-fold but had no effect on the brain SOD activity, whereas SOD activity was reduced to 50% in brain and remained unchanged in liver with Sudan III. These two substances are known inducers of DT-diaphorase and in fact this enzymatic activity was induced 4- and 6-fold in liver with BHA and Sudan III, respectively. However, BHA promoted a significant 40% reduction, whereas no change was observed with Sudan III in brain DT-diaphorase activity. Glutathione(GSH)-related enzymatic activities were also assayed in brain and liver. No induction was observed with BHA or Sudan III for any of the activities tested in hamster brain: GSH S-transferase (GST), GSH peroxidase (GSH-Px) and glutathione disulfide (GSSG) reductase (GR). Only 1.3- and 1.4-fold increases of GST and GR activities were observed in liver and no change in any of these enzymatic activities in brain with BHA; a partial limitation of permeability to BHA of the blood-brain barrier may explain this results. Furthermore, Sudan III promoted reductions in all these GSH-related enzymatic activities in brain and liver. The possible explanations for these results are discussed.

Efficacy of the antioxidant ebselen in experimental uveitis.

Inflammation results in the production of free radicals. In a model of experimental uveitis upon subcutaneous injection of endotoxin to Lewis rats, i.e., endotoxin-induced experimental uveitis (EIU), we have evaluated the status of the antioxidant capacity of ocular tissues. EIU results in a decrease of glutathione (GSH) content and glutathione peroxidase (GPx) activity in whole eye homogenates 24-h after endotoxin administration. Furthermore, an increase in malondialdehyde (MDA) content was observed in these same samples, thus confirming the involvement of oxidative stress in the pathophysiology of the process. In view of the ability of the antioxidant ebselen as GPx enzyme mimic, we tested the effect of the oral treatment with two doses of 100 mg/kg body weight of ebselen (first dose administered at the same time of endotoxin, and the second after 12 h). Ebselen administration normalized the GSH and MDA contents and protected the GPx activity of the EIU rat eyes. The GPx activity in the eye homogenate of the treated rats could be completely acounted for by the ebselen-dependent GPx-like activity, i.e., GPx activity measured in the acidic supernatant of the homogenate after neutralization. Unmodified ebselen was detected in whole eye homogenates, thus it shows for the first time the penetration of ebselen through the blood-aqueous and blood-retina barrier. The results herein may allow the proposal of ebselen as a suitable antiinflammatory agent in ocular tissues.

Evaluation of antioxidants, protein, and lipid oxidation products in blood from sporadic amyotrophic lateral sclerosis patients.

Several parameters indicators of oxidative stress were evaluated in blood from individuals with the sporadic form of amyotrophic lateral sclerosis (SALS) and compared to healthy controls. Plasma levels of 2-thiobarbituric-reactive substances (TBARS), products of lipid peroxidation, were significantly higher (p < 0.03) in the SALS patients compared to controls. The concentration of plasma antioxidants (alpha-tocopherol, beta-carotene, ubiquinol-10 and glutathione) and the activity of red blood cell CuZn superoxide dismutase were not significantly different between the groups. The ratio TBARS/alpha-tocopherol was 47% higher in the SALS individuals than in controls. Protein thiols and protein-associated carbonyls in red blood cell membranes and supernates were similar for both groups. A positive correlation (r2 = 0.91) was found between the concentration of protein-associated carbonyls in red blood cells and the onset of clinical symptoms. These findings are in agreement with several reports showing higher levels of oxidative damage to cell components in ALS.

Phenytoin-induced glutathione depletion in rat peripheral nerve.

Administration of high doses (150-250 mg/kg body weight) of phenytoin (DPH) promote a 40% decrease in glutathione (GSH) content of rat sciatic nerve. This DPH-induced GSH depletion is accompanied with an electrophysiological impairment of peripheral neuromuscular function. H7 (20 mg/kg body weight IP, 30 min prior to DPH), a protein kinase C inhibitor, was able to prevent the DPH-induced GSH depletion only at the lower DPH dose used. This same inhibitor completely prevented the electrophysiological impairment at the lower DPH dose, and only partially at the higher DPH dose used. These results confirm the hypothesis of a DPH-dependent activation of PKC (that might be triggered by, or be the consequence of, the reduction of the intracellular antioxidant GSH), as one of the pathophysiological mechanisms involved in DPH-induced neurotoxicity.

Decreased glutathione peroxidase activity in sciatic nerve of alloxan-induced diabetic mice and its correlation with blood glucose levels.

The effect of alloxan-induced diabetes on glutathione peroxidase (GSH-Px) activity in sciatic nerve of mice has been studied. We have found, 7 days after alloxan treatment, a significant decrease in this enzymatic activity in the cytosol of sciatic nerve of diabetic mice, and moreover, that these changes remained unaltered up to 21 days after alloxan injection. No modification in the glutathione content of sciatic nerve of diabetic mice was observed throughout the experiment when compared with controls. The decrease in GSH-Px activity in this tissue shows a good correlation with the increase of blood glucose levels throughout the experiment. It is hypothesized whether a combination of mechanisms could be involved in this decrease of GSH-Px activity and if oxygen radicals might be the common mediators of these processes.