Open ventral hernia repair with a composite ventral patch : final results of a multicenter prospective study

Background: This study assessed clinical outcomes, including safety and recurrence, from the two-year follow-up of patients who underwent open ventral primary hernia repair with the use of the Parietex (TM) Composite Ventral Patch (PCO-VP). Methods: A prospective single-arm, multicenter study of 126 patients undergoing open ventral hernia repair for umbilical and epigastric hernias with the PCO-VP was performed. Results: One hundred twenty-six subjects (110 with umbilical hernia and 16 with epigastric hernia) with a mean hernia diameter of 1.8cm (0.4-4.0) were treated with PCO-VP. One hundred subjects completed the two-year study. Cumulative hernia recurrence was 3.0% (3/101; 95%CI: 0.0-6.3%) within 24months. Median Numeric Rating Scale pain scores improved from 2 [0-10] at baseline to 0 [0-3] at 1 month (P<0.001) and remained low at 24months 0 [0-6] (P<0.001). 99% (102/103) of the patients were satisfied with their repair at 24months postoperative. Conclusions: The use of PCO-VP to repair primary umbilical and epigastric defects yielded a low recurrence rate, low postoperative and chronic pain, and high satisfaction ratings, confirming that PCO-VP is effective for small ventral hernia repair in the two-year term after implantation. Trial registration: The study was registered publically at clinicaltrials.gov (NCT01848184 registered May 7, 2013)

NF-κB is activated in response to temozolomide in an AKT-dependent manner and confers protection against the growth suppressive effect of the drug.

BACKGROUND: Most DNA-damaging chemotherapeutic agents activate the transcription factor nuclear factor κB (NF-κB). However, NF-κB activation can either protect from or contribute to the growth suppressive effects of the agent. We previously showed that the DNA-methylating drug temozolomide (TMZ) activates AKT, a positive modulator of NF-κB, in a mismatch repair (MMR) system-dependent manner. Here we investigated whether NF-κB is activated by TMZ and whether AKT is involved in this molecular event. We also evaluated the functional consequence of inhibiting NF-κB on tumor cell response to TMZ. METHODS: AKT phosphorylation, NF-κB transcriptional activity, IκB-α degradation, NF-κB2/p52 generation, and RelA and NF-κB2/p52 nuclear translocation were investigated in TMZ-treated MMR-deficient (HCT116, 293TLα-) and/or MMR-proficient (HCT116/3-6, 293TLα+, M10) cells. AKT involvement in TMZ-induced activation of NF-κB was addressed in HCT116/3-6 and M10 cells transiently transfected with AKT1-targeting siRNA or using the isogenic MMR-proficient cell lines pUSE2 and KD12, expressing wild type or kinase-dead mutant AKT1. The effects of inhibiting NF-κB on sensitivity to TMZ were investigated in HCT116/3-6 and M10 cells using the NF-κB inhibitor NEMO-binding domain (NBD) peptide or an anti-RelA siRNA. RESULTS: TMZ enhanced NF-κB transcriptional activity, activated AKT, induced IκB-α degradation and RelA nuclear translocation in HCT116/3-6 and M10 but not in HCT116 cells. In M10 cells, TMZ promoted NF-κB2/p52 generation and nuclear translocation and enhanced the secretion of IL-8 and MCP-1. TMZ induced RelA nuclear translocation also in 293TLα+ but not in 293TLα- cells. AKT1 silencing inhibited TMZ-induced IκB-α degradation and NF-κB2/p52 generation. Up-regulation of NF-κB transcriptional activity and nuclear translocation of RelA and NF-κB2/p52 in response to TMZ were impaired in KD12 cells. RelA silencing in HCT116/3-6 and M10 cells increased TMZ-induced growth suppression. In M10 cells NBD peptide reduced basal NF-κB activity, abrogated TMZ-induced up-regulation of NF-κB activity and increased sensitivity to TMZ. In HCT116/3-6 cells, the combined treatment with NBD peptide and TMZ produced additive growth inhibitory effects. CONCLUSION: NF-κB is activated in response to TMZ in a MMR- and AKT-dependent manner and confers protection against drug-induced cell growth inhibition. Our findings suggest that a clinical benefit could be obtained by combining TMZ with NF-κB inhibitors

Detection of PIGO-Deficient Cells Using Proaerolysin: A Valuable Tool to Investigate Mechanisms of Mutagenesis in the DT40 Cell System

While isogenic DT40 cell lines deficient in DNA repair pathways are a great tool to understand the DNA damage response to genotoxic agents by a comparison of cell toxicity in mutants and parental DT40 cells, no convenient mutation assay for mutagens currently exists for this reverse-genetic system. Here we establish a proaerolysin (PA) selection-based mutation assay in DT40 cells to identify glycosylphosphatidylinositol (GPI)-anchor deficient cells. Using PA, we detected an increase in the number of PA-resistant DT40 cells exposed to MMS for 24 hours followed by a 5-day period of phenotype expression. GPI anchor synthesis is catalyzed by a series of phosphatidylinositol glycan complementation groups (PIGs). The PIG-O gene is on the sex chromosome (Chromosome Z) in chicken cells and is critical for GPI anchor synthesis at the intermediate step. Among all the mutations detected in the sequence levels observed in DT40 cells exposed to MMS at 100 µM, we identified that ∼55% of the mutations are located at A:T sites with a high frequency of A to T transversion mutations. In contrast, we observed no transition mutations out of 18 mutations. This novel assay for DT40 cells provides a valuable tool to investigate the mode of action of mutations caused by reactive agents using a series of isogenic mutant DT40 cells

Transglutaminase and coeliac disease: Endomysial reactivity and small bowel expression

This study was aimed at verifying whether tissue transglultaminase (tTG) is the sole autoantigen eliciting anti-endomysial antibodies in coeliac disease (CoD) and investigating tIG expression in normal and coeliac mucosa. Twelve anti-endomysial-positive coeliac sera and 12 anti-endomysial-negative control sera (10 microl, diluted 1:5-1:400 in PBS pH 7.3) were preincubated with 10, 20 or 50 microg guinea pig liver tTG at 4 degrees C overnight. Monkey oesophagus tissue slides were then tested with tTG-preincubated and non-preincubated sera to search for IgA anti-endomysial reactivity by indirect immunofluorescence. Moreover, six sections of monkey oesophagus were incubated with an anti-tTG mouse MoAb, six sections with an anti-cytokeratin mouse MoAb and six sections with only 3% bovine serum albumin. FInally, endoscopic duodenal biopsy sections obtained from 12 patients affected by untreated coD, six patients affected by treaded CoD and 10 biopsied controls were immunohistochemically stained with a peroxidase-conjugated anti-tTG MoAb. Our results show that (i) preincubation with tTG abolished endomysial immunofluorescence in most, but not all, coeliac sera; (ii) the incubation of anti-tTG MoAb with sections of monkey oesophagus resulted in an immunofluorescence staining pattern similar but not identifical to that of anti-endomysial-positive coeliac sera; (iii) although tTG expression was present at muscularis mucosae and pericryptal fibroblast in both normal and coeliac musosa, it was slithtly more marked and evident in the latter. Although our absorption experiment was performed with guinea pig liver tTG, we confirm that tTG is the predominant antigen of endomysial antibodies, but we speculate that, at least in some patients, it is not the only one

NF-kB is activated in response to temozolomide and confers protection against drug-induced cell growth inhibition

We previously demonstrated that AKT is activated by the methylating agent temozolomide (TMZ) in mismatch repair (MMR)-proficient but not in MMR-deficient cells, and protects against drug-induced cytotoxicity. In this study we investigated whether activation of NF-kB occurs downstream of AKT in TMZ-treated cells. Moreover, we explored the role of NF-kB activation in cell response to the drug. The MMR-proficient melanoma cell line M10, the MMR-proficient colon carcinoma cell line HCT116/3-6 and its MMR-deficient counterpart HCT116 were incubated with 50 microM TMZ + 10 microM O6-benzylguanine (BG), to inhibit the repair of methyl adducts at O6-guanine. After 48 h of culture, phosphorylation of AKT on Ser473 and IkB-alfa degradation were evaluated by Western blotting, while NF-kB activation was determined using a dual-luciferase reporter assay. NF-kB activation was also assessed in MCF-7/KD12 and MCF-7/pUSE cell lines, generated by stable transfection of MMR-proficient MCF-7 cells with a vector encoding a dominant negative mutant form of AKT1, or with the empty vector, respectively. The levels of two transcriptional targets of NF-kB, i.e. IL-8 and MCP-1, were determined in control and TMZ-treated M10 cells. To assess the effect of NF-kB inhibition on cell sensitivity to TMZ, M10 and HCT116/3-6 cells were transfected with siRNAs directed against the p65 subunit of NF-kB and after 24 h incubated with graded concentrations of TMZ + BG. Cell proliferation was evaluated after 5 days of culture by the MTT assay. M10 cells were also exposed to 50 microM NEMO peptide (an inhibitor of NF-kB function) for 24 h and then tested for sensitivity to TMZ + BG as described above. TMZ treatment induced phosphorylation of AKT on Ser473, IkB-alfa degradation and NF-kB activation in M10 and HCT116/3-6 cells but not in HCT116 cells. NF-kB activation was impaired in MCF-7/KD12 cells. Increased secretion of IL-8 and MCP-1 was observed in TMZ-treated M10 cells as compared with control cells. Transfection of M10 and HCT116/3-6 cells with siRNAs against NF-kB significantly increased the growth suppressive effects of TMZ. Inhibition of NF-kB by NEMO peptide in M10 cells also potentiated the antiproliferative activity of TMZ. Our results demonstrate that NF-kB is activated in response to TMZ in a MMR- and AKT-dependent manner, and that pharmacological inhibition of NF-kB might represent an efficient strategy to increase melanoma cell sensitivity to TMZ