ß-Sarcoglycan gene transfer decreases fibrosis and restores force in LGMD2E mice.

Limb-girdle muscular dystrophy type 2E (LGMD2E) results from mutations in the ß-sarcoglycan (SGCB) gene causing loss of functional protein and concomitant loss of dystrophin-associated proteins. The disease phenotype is characterized by muscle weakness and wasting, and dystrophic features including muscle fiber necrosis, inflammation and fibrosis. The Sgcb-null mouse recapitulates the clinical phenotype with significant endomysial fibrosis providing a relevant model to test whether gene replacement will be efficacious. We directly addressed this question using a codon optimized human ß-sarcoglycan gene (hSGCB) driven by a muscle-specific tMCK promoter (scAAVrh74.tMCK.hSGCB). Following isolated limb delivery (5 × 10(11) vector genome (vg)), 91.2% of muscle fibers in the lower limb expressed ß-sarcoglycan, restoring assembly of the sarcoglycan complex and protecting the membrane from Evans blue dye leakage. Histological outcomes were significantly improved including decreased central nucleation, normalization of muscle fiber size, decreased macrophages and inflammatory mononuclear cells, and an average of a 43% reduction in collagen deposition in treated muscle compared with untreated muscle at end point. These measures correlated with improvement of tetanic force and resistance to eccentric contraction. In 6-month-old mice, as indicated by collagen staining, scAAVrh74.tMCK.hSGCB treatment reduced fibrosis by 42%. This study demonstrates the potential for gene replacement to reverse debilitating fibrosis, typical of muscular dystrophy, thereby providing compelling evidence for movement to clinical gene replacement for LGMD2E.

Correlation between tumor-associated proteins and response to neoadjuvant treatment in patients with advanced squamous-cell esophageal cancer.

BACKGROUND: Possible predictive markers of response to neoadjuvant radiochemotherapy (NRCT) of esophageal cancer have been identified. PATIENTS AND METHODS: Patient biopsies were obtained from both tumor and normal tissue before the NRCT of locally advanced esophageal squamous cell carcinoma. Protein solutions were separated and immunoblot analysis was performed with heat shock protein (Hsp)16.2, heme-binding protein 2 (SOUL), BCL2-associated X protein (Bax), B-cell-associated leukemia protein 2 (Bcl-2) and heat shock protein 90 (Hsp90) antibodies. Following NRCT, the patients were restaged according to the Response Evaluation Criteria In Solid Tumors (RECIST). Following resections the pathological down-staging was evaluated. RESULTS: Clinical restaging revealed a response rate of 65%. Pathological examination revealed down-staging in 30% and 25% of the cases for the T and N categories respectively. Compared to the normal esophageal mucosa, a decreased expression of Hsp16.2, Hsp90 and SOUL proteins and an increased Bax/Bcl-2 ratio was found in the responding tumors. CONCLUSION: Hsp16,2, Hsp90 and SOUL expression and Bax/ Bcl-2 ratio correlates to the efficacy of NRCT and predict outcome in patients with locally advanced squamous-cell esophageal cancer.

The inhibitory effect of a novel cytotoxic somatostatin analogue AN-162 on experimental glioblastoma.

Glioblastoma multiforme is the most common and most aggressive type of high grade tumor with a poor prognosis upon discovery. Based on earlier promising results earned with AN-162, a doxorubicin molecule linked to somatostatin (SST) analogue RC-160, it was our aim to determine the effect of AN-162 on DBTRG-05 glioblastoma cell line, and to test its efficacy in experimental brain tumors. We detected the expression of mRNA for somatostatin receptor (SSTR) subtypes 2 and 3 in DBTRG-05 cells with RT-PCR. Using ligand competition assay, specific high affinity receptors for somatostatin were found. The MTT assay showed that both AN-162 and doxorubicin (DOX) significantly inhibited cell proliferation and that there was no significant difference between the effects in vitro. Nude mice were xenografted with DBTRG-05 glioblastoma tumors. AN-162 showed a significant inhibition of tumor growth compared with the control group and the groups treated with equimolar doses of doxorubicin, somatostatin analogue RC-160, or the unconjugated mixture of doxorubicin plus RC-160. The tumor doubling time in the group of animals treated with AN-162 was extended and was significantly different from doubling times in the control group and in the other treatment groups. Our study clearly demonstrates a potent inhibitory effect of AN-162 in experimental glioblastoma, thus suggesting the possibility of its utilization in patients suffering from malignant brain cancer.

TIP47 protects mitochondrial membrane integrity and inhibits oxidative-stress-induced cell death.

We found that overexpression of tail interacting protein of 47 kDa (TIP47), but not its truncated form (t-TIP47) protected NIH3T3 cells from hydrogen-peroxide-induced cell death, prevented the hydrogen-peroxide-induced mitochondrial depolarization determined by 5,50,6,60-tetrachloro-1,10,3,30-tetraethyl-benzimidazolylcarbocyanine iodide (JC1), while suppression of TIP47 in HeLa cells facilitated oxidative-stress-induced cell death. TIP47 was located to the cytoplasm of untreated cells, but some was associated to mitochondria in oxidative stress. Recombinant TIP47, but not t-TIP47 increased the mitochondrial membrane potential (Deltapsi), and partially prevented Ca2+ induced depolarization. It is assumed that TIP47 can bind to mitochondria in oxidative stress, and inhibit mitochondria mediated cell death by protecting mitochondrial membrane integrity.

The effect of carotid stenting on rheological parameters, free radical production and platelet aggregation.

INTRODUCTION: Carotid artery stenting has become a possible treatment of significant carotid stenosis. The risk of stent occlusion and restenosis might be increased by abnormal rheological conditions amplified platelet aggregation and free radical production during the operation. AIMS: The aim of our study was to assess the changes in hemorheological parameters, platelet aggregation, and catalase activity after endovascular treatment of carotid stenosis. METHODS: 18 patients (11 men, ages 68 +/- 9 years and 7 women, ages 62 +/- 8 years) suffering from significant carotid stenosis and treated with carotid endovascular intervention were examined. Alteration in hemorheological parameters as well as epinephrine-, ADP-, and collagen-induced platelet aggregation were evaluated. Antioxidant reserve was characterized by the determination of catalase activity. The measurements were carried out directly before and after the procedure and 1, 2, 5 days and 1 month following the intervention. Preceding the operation the patients were administered a maximum dose (300 mg) of clopidogrel. RESULTS: Hematocrit, plasma fibrinogen concentration (PFC) and whole blood-, and plasma viscosity values (WBV and PV) significantly decreased immediately after stenting (p<0.001). By the fifth day following the intervention the PFC, WBV, PV, red blood cell (RBC) aggregation and ADP-induced platelet aggregation significantly increased (p<0.0001) compared to values measured postprocedurally. At 1 month follow-up these parameters, except whole blood viscosity, decreased significantly compared to measurements made on the 5th day. On the other hand, catalase activity showed significant elevation by the end of the first month. CONCLUSION: Hemorheological parameters and platelet aggregation showed specific changes following carotid stenting. Abnormal changes of the rheological conditions and increasing platelet activation are the most pronounced in the first week following stenting, which may lead to early stent occlusion. Oxidative stress production returned to baseline levels only by the end of the first month.