MicroRNA inhibition using antimiRs in acute human brain tissue sections

Antisense inhibition of microRNAs is an emerging preclinical approach to pharmacoresistant epilepsy. A leading candidate is an "antimiR" targeting microRNA-134 (ant-134), but testing to date has used rodent models. Here, we develop an antimiR testing platform in human brain tissue sections. Brain specimens were obtained from patients undergoing resective surgery to treat pharmacoresistant epilepsy. Neocortical specimens were submerged in modified artificial cerebrospinal fluid (ACSF) and dissected for clinical neuropathological examination, and unused material was transferred for sectioning. Individual sections were incubated in oxygenated ACSF, containing either ant-134 or a nontargeting control antimiR, for 24 h at room temperature. RNA integrity was assessed using BioAnalyzer processing, and individual miRNA levels were measured using quantitative reverse transcriptase polymerase chain reaction. Specimens transported in ACSF could be used for neuropathological diagnosis and had good RNA integrity. Ant-134 mediated a dose-dependent knockdown of miR-134, with approximately 75% reduction of miR-134 at 1 μmol L-1 and 90% reduction at 3 μmol L-1 . These doses did not have off-target effects on expression of a selection of three other miRNAs. This is the first demonstration of ant-134 effects in live human brain tissues. The findings lend further support to the preclinical development of a therapy that targets miR-134 and offer a flexible platform for the preclinical testing of antimiRs, and other antisense oligonucleotide therapeutics, in human brain

Contribution of factor H-Binding protein sequence to the cross-reactivity of meningococcal native outer membrane vesicle vaccines with over-expressed fHbp variant group 1

Factor H-binding protein (fHbp) is an important meningococcal vaccine antigen. Native outer membrane vesicles with over-expressed fHbp (NOMV OE fHbp) have been shown to induce antibodies with broader functional activity than recombinant fHbp (rfHbp). Improved understanding of this broad coverage would facilitate rational vaccine design. We performed a pair-wise analysis of 48 surface-exposed amino acids involved in interacting with factor H, among 383 fHbp variant group 1 sequences. We generated isogenic NOMV-producing meningococcal strains from an African serogroup W isolate, each over-expressing one of four fHbp variant group 1 sequences (ID 1, 5, 9, or 74), including those most common among invasive African meningococcal isolates. Mice were immunised with each NOMV, and sera tested for IgG levels against each of the rfHbp ID and for ability to kill a panel of heterologous meningococcal isolates. At the fH-binding site, ID pairs differed by a maximum of 13 (27%) amino acids. ID 9 shared an amino acid sequence common to 83 ID types. The selected ID types differed by up to 6 amino acids, in the fH-binding site. All NOMV and rfHbp induced high IgG levels against each rfHbp. Serum killing from mice immunised with rfHbp was generally less efficient and more restricted compared to NOMV, which induced antibodies that killed most meningococci tested, with decreased stringency for ID type differences. Breadth of killing was mostly due to anti-fHbp antibodies, with some restriction according to ID type sequence differences. Nevertheless, under our experimental conditions, no relationship between antibody cross-reactivity and variation fH-binding site sequence was identified. NOMV over-expressing different fHbp IDs belonging to variant group 1 induce antibodies with fine specificities against fHbp, and ability to kill broadly meningococci expressing heterologous fHbp IDs. The work reinforces that meningococcal NOMV with OE fHbp is a promising vaccine strategy, and provides a basis for rational selection of antigen sequence types for over-expression on NOMV

Epidermal Growth Factor Receptor (EGFR) gene copy number (GCN) correlates with clinical activity of irinotecan-cetuximab in K-RAS wild-type colorectal cancer: a fluorescence in situ (FISH) and chromogenic in situ hybridization (CISH) analysis

<p>Abstract</p> <p>Background</p> <p>K-RAS wild type colorectal tumors show an improved response rate to anti-EGFR monoclonal antibodies. Nevertheless 70% to 40% of these patients still does not seem to benefit from this therapeutic approach. FISH EGFR GCN has been previously demonstrated to correlate with clinical outcome of colorectal cancer treated with anti-EGFR monoclonal antibodies. CISH also seemed able to provide accurate EGFR GCN information with the advantage of a simpler and reproducible technique involving immunohistochemistry and light microscopy. Based on these findings we investigated the correlation between both FISH and CISH EGFR GCN and clinical outcome in K-RAS wild-type colorectal cancer treated with irinotecan-cetuximab.</p> <p>Methods</p> <p>Patients with advanced K-RAS wild-type, colorectal cancer receiving irinotecan-cetuximab after failure of irinotecan-based chemotherapy were eligible.</p> <p>A cut-off value for EGFR GCN of 2.6 and 2.12 for FISH and CISH respectively was derived from ROC curve analysis.</p> <p>Results</p> <p>Forty-four patients were available for analysis. We observed a partial remission in 9 (60%) and 2 (9%) cases with a FISH EGFR GCN ≥ 2.6 and < 2.6 respectively (p = 0.002) and in 10 (36%) and 1 (6%) cases with a CISH EGFR GCN ≥ 2.12 and < 2.12 respectively (p = 0.03). Median TTP was 7.7 and 6.4 months in patients showing increased FISH and CISH EGFR GCN whereas it was 2.9 and 3.1 months in those with low FISH and CISH EGFR GCN (p = 0.04 and 0.02 respectively).</p> <p>Conclusion</p> <p>FISH and CISH EGFR GCN may both represent effective tools for a further patients selection in K-RAS wild-type colorectal cancer treated with cetuximab.</p

A systematic analysis of host factors reveals a Med23-interferon-λ regulatory axis against herpes simplex virus type 1 replication

Herpes simplex virus type 1 (HSV-1) is a neurotropic virus causing vesicular oral or genital skin lesions, meningitis and other diseases particularly harmful in immunocompromised individuals. To comprehensively investigate the complex interaction between HSV-1 and its host we combined two genome-scale screens for host factors (HFs) involved in virus replication. A yeast two-hybrid screen for protein interactions and a RNA interference (RNAi) screen with a druggable genome small interfering RNA (siRNA) library confirmed existing and identified novel HFs which functionally influence HSV-1 infection. Bioinformatic analyses found the 358 HFs were enriched for several pathways and multi-protein complexes. Of particular interest was the identification of Med23 as a strongly anti-viral component of the largely pro-viral Mediator complex, which links specific transcription factors to RNA polymerase II. The anti-viral effect of Med23 on HSV-1 replication was confirmed in gain-of-function gene overexpression experiments, and this inhibitory effect was specific to HSV-1, as a range of other viruses including Vaccinia virus and Semliki Forest virus were unaffected by Med23 depletion. We found Med23 significantly upregulated expression of the type III interferon family (IFN-λ) at the mRNA and protein level by directly interacting with the transcription factor IRF7. The synergistic effect of Med23 and IRF7 on IFN-λ induction suggests this is the major transcription factor for IFN-λ expression. Genotypic analysis of patients suffering recurrent orofacial HSV-1 outbreaks, previously shown to be deficient in IFN-λ secretion, found a significant correlation with a single nucleotide polymorphism in the IFN-λ3 (IL28b) promoter strongly linked to Hepatitis C disease and treatment outcome. This paper describes a link between Med23 and IFN-λ, provides evidence for the crucial role of IFN-λ in HSV-1 immune control, and highlights the power of integrative genome-scale approaches to identify HFs critical for disease progression and outcome

Down-Regulation of Serum/Glucocorticoid Regulated Kinase 1 in Colorectal Tumours Is Largely Independent of Promoter Hypermethylation

Background: We have previously shown that serum/glucocorticoid regulated kinase 1 (SGK1) is down-regulated in colorectal cancers (CRC) with respect to normal tissue. As hyper-methylation of promoter regions is a well-known mechanism of gene silencing in cancer, we tested whether the SGK1 promoter region was methylated in colonic tumour samples. Methodology/Principal Findings: We investigated the methylation profile of the two CpG islands present in the promoter region of SGK1 in a panel of 5 colorectal cancer cell lines by sequencing clones of bisulphite-treated DNA samples. We further confirmed our findings in a panel of 10 normal and 10 tumour colonic tissue samples of human origin. We observed CpG methylation only in the smaller and more distal CpG island in the promoter region of SGK1 in both normal and tumour samples of colonic origin. We further identified a single nucleotide polymorphism (SNP, rs1743963) which affects methylation of the corresponding CpG. Conclusions/Significance: Our results show that even though partial methylation of the promoter region of SGK1 is present

Characterization of coagulase-negative staphylococcal isolates from blood with reduced susceptibility to glycopeptides and therapeutic options

<p>Abstract</p> <p>Background</p> <p>Coagulase-negative staphylococci (CoNS) are a major cause of nosocomial blood stream infection, especially in critically ill and haematology patients. CoNS are usually multidrug-resistant and glycopeptide antibiotics have been to date considered the drugs of choice for treatment. The aim of this study was to characterize CoNS with reduced susceptibility to glycopeptides causing blood stream infection (BSI) in critically ill and haematology patients at the University Hospital Tor Vergata, Rome, Italy, in 2007.</p> <p>Methods</p> <p>Hospital microbiology records for transplant haematology and ICU were reviewed to identify CoNS with elevated MICs for glycopeptides, and isolates were matched to clinical records to determine whether the isolates caused a BSI. The isolates were tested for susceptibility to new drugs daptomicin and tigecycline and the genetic relationship was assessed using f-AFLP.</p> <p>Results</p> <p>Of a total of 17,418 blood cultures, 1,609 were positive for CoNS and of these, 87 (5.4%) displayed reduced susceptibility to glycopeptides. Clinical review revealed that in 13 cases (7 in haematology and 6 in ICU), CoNS with reduced susceptibility to glycopeptides were responsible for a BSI. <it>Staphylococcus epidermidis </it>was the causative organism in 11 instances and <it>Staphylococcus haemolyticus </it>in 2. The incidence of oxacillin resistance was high (77%), although all isolates remained susceptible to linezolid, daptomycin and tigecycline. Fingerprinting of CoNS identified one clonal relationship between two isolates.</p> <p>Conclusion</p> <p>Multi-resistant CoNS with reduced susceptibility to glycopeptides, although still relatively infrequent in our hospital, are emerging pathogens of clinical concern. Surveillance by antibiotyping with attention to multi-resistant profile, and warning to clinicians, is necessary.</p

BRCA2 polymorphic stop codon K3326X and the risk of breast, prostate, and ovarian cancers

Background: The K3326X variant in BRCA2 (BRCA2*c.9976A&gt;T; p.Lys3326*; rs11571833) has been found to be associated with small increased risks of breast cancer. However, it is not clear to what extent linkage disequilibrium with fully pathogenic mutations might account for this association. There is scant information about the effect of K3326X in other hormone-related cancers. Methods: Using weighted logistic regression, we analyzed data from the large iCOGS study including 76 637 cancer case patients and 83 796 control patients to estimate odds ratios (ORw) and 95% confidence intervals (CIs) for K3326X variant carriers in relation to breast, ovarian, and prostate cancer risks, with weights defined as probability of not having a pathogenic BRCA2 variant. Using Cox proportional hazards modeling, we also examined the associations of K3326X with breast and ovarian cancer risks among 7183 BRCA1 variant carriers. All statistical tests were two-sided. Results: The K3326X variant was associated with breast (ORw = 1.28, 95% CI = 1.17 to 1.40, P = 5.9x10- 6) and invasive ovarian cancer (ORw = 1.26, 95% CI = 1.10 to 1.43, P = 3.8x10-3). These associations were stronger for serous ovarian cancer and for estrogen receptor–negative breast cancer (ORw = 1.46, 95% CI = 1.2 to 1.70, P = 3.4x10-5 and ORw = 1.50, 95% CI = 1.28 to 1.76, P = 4.1x10-5, respectively). For BRCA1 mutation carriers, there was a statistically significant inverse association of the K3326X variant with risk of ovarian cancer (HR = 0.43, 95% CI = 0.22 to 0.84, P = .013) but no association with breast cancer. No association with prostate cancer was observed. Conclusions: Our study provides evidence that the K3326X variant is associated with risk of developing breast and ovarian cancers independent of other pathogenic variants in BRCA2. Further studies are needed to determine the biological mechanism of action responsible for these associations