Regulation of cell death receptor S-nitrosylation and apoptotic signaling by Sorafenib in hepatoblastoma cells.

Nitric oxide (NO) plays a relevant role during cell death regulation in tumor cells. The overexpression of nitric oxide synthase type III (NOS-3) induces oxidative and nitrosative stress, p53 and cell death receptor expression and apoptosis in hepatoblastoma cells. S-nitrosylation of cell death receptor modulates apoptosis. Sorafenib is the unique recommended molecular-targeted drug for the treatment of patients with advanced hepatocellular carcinoma. The present study was addressed to elucidate the potential role of NO during Sorafenib-induced cell death in HepG2 cells. We determined the intra- and extracellular NO concentration, cell death receptor expression and their S-nitrosylation modifications, and apoptotic signaling in Sorafenib-treated HepG2 cells. The effect of NO donors on above parameters has also been determined. Sorafenib induced apoptosis in HepG2 cells. However, low concentration of the drug (10nM) increased cell death receptor expression, as well as caspase-8 and -9 activation, but without activation of downstream apoptotic markers. In contrast, Sorafenib (10µM) reduced upstream apoptotic parameters but increased caspase-3 activation and DNA fragmentation in HepG2 cells. The shift of cell death signaling pathway was associated with a reduction of S-nitrosylation of cell death receptors in Sorafenib-treated cells. The administration of NO donors increased S-nitrosylation of cell death receptors and overall induction of cell death markers in control and Sorafenib-treated cells. In conclusion, Sorafenib induced alteration of cell death receptor S-nitrosylation status which may have a relevant repercussion on cell death signaling in hepatoblastoma cells.

Long-Term Control of Endemic Hospital-Wide Methicillin-Resistant Staphylococcus aureus (MRSA): The Impact of Targeted Active Surveillance for MRSA in Patients and Healthcare Workers

To evaluate the long-term impact of successive interventions on rates of methicillin-resistant Staphylococcus aureus (MRSA) colonization or infection and MRSA bacteremia in an endemic hospital-wide situation. DESIGN:Quasi-experimental, interrupted time-series analysis. The impact of the interventions was analyzed by use of segmented regression. Representative MRSA isolates were typed by use of pulsed-field gel electrophoresis. SETTING:A 950-bed teaching hospital in Seville, Spain. PATIENTS:All patients admitted to the hospital during the period from 1995 through 2008. METHODS:Three successive interventions were studied: (1) contact precautions, with no active surveillance for MRSA; (2) targeted active surveillance for MRSA in patients and healthcare workers in specific wards, prioritized according to clinical epidemiology data; and (3) targeted active surveillance for MRSA in patients admitted from other medical centers. RESULTS:Neither the preintervention rate of MRSA colonization or infection (0.56 cases per 1,000 patient-days [95% confidence interval {CI}, 0.49-0.62 cases per 1,000 patient-days]) nor the slope for the rate of MRSA colonization or infection changed significantly after the first intervention. The rate decreased significantly to 0.28 cases per 1,000 patient-days (95% CI, 0.17-0.40 cases per 1,000 patient-days) after the second intervention and to 0.07 cases per 1,000 patient-days (95% CI, 0.06-0.08 cases per 1,000 patient-days) after the third intervention, and the rate remained at a similar level for 8 years. The MRSA bacteremia rate decreased by 80%, whereas the rate of bacteremia due to methicillin-susceptible S. aureus did not change. Eighty-three percent of the MRSA isolates identified were clonally related. All MRSA isolates obtained from healthcare workers were clonally related to those recovered from patients who were in their care. CONCLUSION:Our data indicate that long-term control of endemic MRSA is feasible in tertiary care centers. The use of targeted active surveillance for MRSA in patients and healthcare workers in specific wards (identified by means of analysis of clinical epidemiology data) and the use of decolonization were key to the success of the program.

Mutation screening of multiple genes in Spanish patients with autosomal recessive retinitis pigmentosa by targeted resequencing.

Retinitis Pigmentosa (RP) is a heterogeneous group of inherited retinal dystrophies characterised ultimately by the loss of photoreceptor cells. RP is the leading cause of visual loss in individuals younger than 60 years, with a prevalence of about 1 in 4000. The molecular genetic diagnosis of autosomal recessive RP (arRP) is challenging due to the large genetic and clinical heterogeneity. Traditional methods for sequencing arRP genes are often laborious and not easily available and a screening technique that enables the rapid detection of the genetic cause would be very helpful in the clinical practice. The goal of this study was to develop and apply microarray-based resequencing technology capable of detecting both known and novel mutations on a single high-throughput platform. Hence, the coding regions and exon/intron boundaries of 16 arRP genes were resequenced using microarrays in 102 Spanish patients with clinical diagnosis of arRP. All the detected variations were confirmed by direct sequencing and potential pathogenicity was assessed by functional predictions and frequency in controls. For validation purposes 4 positive controls for variants consisting of previously identified changes were hybridized on the array. As a result of the screening, we detected 44 variants, of which 15 are very likely pathogenic detected in 14 arRP families (14%). Finally, the design of this array can easily be transformed in an equivalent diagnostic system based on targeted enrichment followed by next generation sequencing.

Clinical pharmacogenomic testing of KRAS, BRAF and EGFR mutations by high resolution melting analysis and ultra-deep pyrosequencing

BACKGROUND: Epidermal growth factor receptor (EGFR) and its downstream factors KRAS and BRAF are mutated in several types of cancer, affecting the clinical response to EGFR inhibitors. Mutations in the EGFR kinase domain predict sensitivity to the tyrosine kinase inhibitors gefitinib and erlotinib in lung adenocarcinoma, while activating point mutations in KRAS and BRAF confer resistance to the anti-EGFR monoclonal antibody cetuximab in colorectal cancer. The development of new generation methods for systematic mutation screening of these genes will allow more appropriate therapeutic choices. METHODS: We describe a high resolution melting (HRM) assay for mutation detection in EGFR exons 19-21, KRAS codon 12/13 and BRAF V600 using formalin-fixed paraffin-embedded samples. Somatic variation of KRAS exon 2 was also analysed by massively parallel pyrosequencing of amplicons with the GS Junior 454 platform. RESULTS: We tested 120 routine diagnostic specimens from patients with colorectal or lung cancer. Mutations in KRAS, BRAF and EGFR were observed in 41.9%, 13.0% and 11.1% of the overall samples, respectively, being mutually exclusive. For KRAS, six types of substitutions were detected (17 G12D, 9 G13D, 7 G12C, 2 G12A, 2 G12V, 2 G12S), while V600E accounted for all the BRAF activating mutations. Regarding EGFR, two cases showed exon 19 deletions (delE746-A750 and delE746-T751insA) and another two substitutions in exon 21 (one showed L858R with the resistance mutation T590M in exon 20, and the other had P848L mutation). Consistent with earlier reports, our results show that KRAS and BRAF mutation frequencies in colorectal cancer were 44.3% and 13.0%, respectively, while EGFR mutations were detected in 11.1% of the lung cancer specimens. Ultra-deep amplicon pyrosequencing successfully validated the HRM results and allowed detection and quantitation of KRAS somatic mutations. CONCLUSIONS: HRM is a rapid and sensitive method for moderate-throughput cost-effective screening of oncogene mutations in clinical samples. Rather than Sanger sequence validation, next-generation sequencing technology results in more accurate quantitative results in somatic variation and can be achieved at a higher throughput scale.

The Chemotherapeutic Drug 5-Fluorouracil Promotes PKR-Mediated Apoptosis in a p53-Independent Manner in Colon and Breast Cancer Cells

The chemotherapeutic drug 5-FU is widely used in the treatment of a range of cancers, but resistance to the drug remains a major clinical problem. Since defects in the mediators of apoptosis may account for chemo-resistance, the identification of new targets involved in 5-FU-induced apoptosis is of main clinical interest. We have identified the ds-RNA-dependent protein kinase (PKR)as a key molecular target of 5-FU involved in apoptosis induction in human colon and breast cancer cell lines. PKR distribution and activation, apoptosis induction and cytotoxic effects were analyzed during 5-FU and 5-FU/IFNalpha treatment in several colon and breast cancer cell lines with different p53 status. PKR protein was activated by 5-FU treatment in a p53-independent manner,inducing phosphorylation of the protein synthesis translation initiation factor eIF-2alpha and cell death by apoptosis. Furthermore, PKR interference promoted a decreased response to 5-FU treatment and those cells were not affected by the synergistic antitumor activity of 5-FU/IFNalpha combination. These results, taken together, provide evidence that PKR is a key molecular target of 5-FU with potential relevance in the clinical use of this drug.

Nosocomial Outbreak of Infection With Pan-Drug-Resistant Acinetobacter baumannii in a Tertiary Care University Hospital

OBJECTIVE To describe what is, to our knowledge, the first nosocomial outbreak of infection with pan-drug-resistant (including colistin-resistant) Acinetobacter baumannii, to determine the risk factors associated with these types of infections, and to determine their clinical impact. DESIGN Nested case-control cohort study and a clinical-microbiological study. SETTING A 1,521-bed tertiary care university hospital in Seville, Spain. PATIENTS Case patients were inpatients who had a pan-drug-resistant A. baumannii isolate recovered from a clinical or surveillance sample obtained at least 48 hours after admission to an intensive care unit (ICU) during the time of the epidemic outbreak. Control patients were patients who were admitted to any of the "boxes" (ie, rooms that partition off a distinct area for a patient's bed and the equipment needed to care for the patient) of an ICU for at least 48 hours during the time of the epidemic outbreak. RESULTS All the clinical isolates had similar antibiotic susceptibility patterns (ie, they were resistant to all the antibiotics tested, including colistin), and, on the basis of repetitive extragenic palindromic-polymerase chain reaction, it was determined that all of them were of the same clone. The previous use of quinolones and glycopeptides and an ICU stay were associated with the acquisition of infection or colonization with pan-drug-resistant A. baumannii. To control this outbreak, we implemented the following multicomponent intervention program: the performance of environmental decontamination of the ICUs involved, an environmental survey, a revision of cleaning protocols, active surveillance for colonization with pan-drug-resistant A. baumannii, educational programs for the staff, and the display of posters that illustrate contact isolation measures and antimicrobial use recommendations. CONCLUSIONS We were not able to identify the common source for these cases of infection, but the adopted measures have proven to be effective at controlling the outbreak.

Prevention of the renarrowing of coronary arteries using drug-eluting stents in the perioperative period: an update.

The management of patients scheduled for surgery with a coronary stent, and receiving 1 or more antiplatelet drugs, has many controversies. The premature discontinuation of antiplatelet drugs substantially increases the risk of stent thrombosis (ST), myocardial infarction, and cardiac death, and surgery under an altered platelet function could also lead to an increased risk of bleeding in the perioperative period. Because of the conflict in the recommendations, this article reviews the current antiplatelet protocols after positioning a coronary stent, the evidence of increased risk of ST associated with the withdrawal of antiplatelet drugs and increased bleeding risk associated with its maintenance, the different perioperative antiplatelet protocols when patients are scheduled for surgery or need an urgent operation, and the therapeutic options if excessive bleeding occurs.

Assessment of drug-induced hepatotoxicity in clinical practice: a challenge for gastroenterologists.

Currently, pharmaceutical preparations are serious contributors to liver disease; hepatotoxicity ranking as the most frequent cause for acute liver failure and post-commercialization regulatory decisions. The diagnosis of hepatotoxicity remains a difficult task because of the lack of reliable markers for use in general clinical practice. To incriminate any given drug in an episode of liver dysfunction is a step-by-step process that requires a high degree of suspicion, compatible chronology, awareness of the drug's hepatotoxic potential, the exclusion of alternative causes of liver damage and the ability to detect the presence of subtle data that favors a toxic etiology. This process is time-consuming and the final result is frequently inaccurate. Diagnostic algorithms may add consistency to the diagnostic process by translating the suspicion into a quantitative score. Such scales are useful since they provide a framework that emphasizes the features that merit attention in cases of suspected hepatic adverse reaction as well. Current efforts in collecting bona fide cases of drug-induced hepatotoxicity will make refinements of existing scales feasible. It is now relatively easy to accommodate relevant data within the scoring system and to delete low-impact items. Efforts should also be directed toward the development of an abridged instrument for use in evaluating suspected drug-induced hepatotoxicity at the very beginning of the diagnosis and treatment process when clinical decisions need to be made. The instrument chosen would enable a confident diagnosis to be made on admission of the patient and treatment to be fine-tuned as further information is collected.

Early estimates of the effectiveness of the 2011/12 influenza vaccine in the population targeted for vaccination in Spain, 25 December 2011 to 19 February 2012.

We present early estimates of influenza vaccine effectiveness (VE) in the population targeted for vaccination, during 25 December 2011 to 19 February 2012. The adjusted VE was 55% (95% CI: 3 to 79) against any type of influenza virus and 54% (95% CI: 1 to 79) against influenza A(H3N2) virus. This suggests a moderate protective effect of the vaccine in the targeted population in a late influenza epidemic with limited match between vaccine and circulating strains.

Trends in qualifying biomarkers in drug safety. Consensus of the 2011 meeting of the spanish society of clinical pharmacology.

In this paper we discuss the consensus view on the use of qualifying biomarkers in drug safety, raised within the frame of the XXIV meeting of the Spanish Society of Clinical Pharmacology held in Málaga (Spain) in October, 2011. The widespread use of biomarkers as surrogate endpoints is a goal that scientists have long been pursuing. Thirty years ago, when molecular pharmacogenomics evolved, we anticipated that these genetic biomarkers would soon obviate the routine use of drug therapies in a way that patients should adapt to the therapy rather than the opposite. This expected revolution in routine clinical practice never took place as quickly nor with the intensity as initially expected. The concerted action of operating multicenter networks holds great promise for future studies to identify biomarkers related to drug toxicity and to provide better insight into the underlying pathogenesis. Today some pharmacogenomic advances are already widely accepted, but pharmacogenomics still needs further development to elaborate more precise algorithms and many barriers to implementing individualized medicine exist. We briefly discuss our view about these barriers and we provide suggestions and areas of focus to advance in the field.

Genetic variations in drug-induced liver injury (DILI): resolving the puzzle.

Despite stringent requirements for drug development imposed by regulatory agencies, drug-induced liver injury (DILI) is an increasing health problem and a significant cause for failure to approve drugs, market withdrawal of commercialized medications, and adoption of regulatory measures. The pathogenesis is yet undefined, though the rare occurrence of idiosyncratic DILI (1/100,000–1/10,000) and the fact that hepatotoxicity often recurs after re-exposure to the culprit drug under different environmental conditions strongly points toward a major role for genetic variations in the underlying mechanism and susceptibility. Pharmacogenetic studies in DILI have to a large extent focused on genes involved in drug metabolism, as polymorphisms in these genes may generate increased plasma drug concentrations as well as lower clearance rates when treated with standard medication doses. A range of studies have identified a number of genetic variants in drug metabolism Phase I, II, and III genes, including cytochrome P450 (CYP) 2E1, N-acetyltransferase 2, UDP-glucuronosyltransferase 2B7, glutathione S-transferase M1/T1, ABCB11, and ABCC2, that enhance DILI susceptibility (Andrade et al., 2009; Agundez et al., 2011). Several metabolic gene variants, such as CYP2E1c1 and NAT2 slow, have been associated with DILI induced by specific drugs based on individual drug metabolism information. Others, such as GSTM1 and T1 null alleles have been associated with enhanced risk of DILI development induced by a large range of drugs. Hence, these variants appear to have a more general role in DILI susceptibility due to their role in reducing the cell's antioxidative capacity (Lucena et al., 2008). Mitochondrial superoxide dismutase (SOD2) and glutathione peroxidase 1 (GPX1) are two additional enzymes involved in combating oxidative stress, with specific genetic variants shown to enhance the risk of developing DILI

Comparison of drug survival rates for tumor necrosis factor antagonists in rheumatoid arthritis.

BACKGROUND Persistence of anti-tumor necrosis factor (TNF) therapy in rheumatoid arthritis (RA) is an overall marker of treatment success. OBJECTIVE To assess the survival of anti-TNF treatment and to define the potential predictors of drug discontinuation in RA, in order to verify the adequacy of current practices. DESIGN An observational, descriptive, longitudinal, retrospective study. SETTING The Hospital Clínico Universitario de Valladolid, Valladolid, Spain. PATIENTS RA patients treated with anti-TNF therapy between January 2011 and January 2012. MEASUREMENTS Demographic information and therapy assessments were gathered from medical and pharmaceutical records. Data is expressed as means (standard deviations) for quantitative variables and frequency distribution for qualitative variables. Kaplan-Meier survival analysis was used to assess persistence, and Cox multivariate regression models were used to assess potential predictors of treatment discontinuation. RESULTS In total, 126 treatment series with infliximab (n = 53), etanercept (n = 51) or adalimumab (n = 22) were administered to 91 patients. Infliximab has mostly been used as a first-line treatment, but it was the drug with the shortest time until a change of treatment. Significant predictors of drug survival were: age; the anti-TNF agent; and the previous response to an anti-TNF drug. LIMITATION The small sample size. CONCLUSION The overall efficacy of anti-TNF drugs diminishes with time, with infliximab having the shortest time until a change of treatment. The management of biologic therapy in patients with RA should be reconsidered in order to achieve disease control with a reduction in costs.

Selected ABCB1, ABCB4 and ABCC2 Polymorphisms Do Not Enhance the Risk of Drug-Induced Hepatotoxicity in a Spanish Cohort.

BACKGROUND AND AIMS Flawed ABC transporter functions may contribute to increased risk of drug-induced liver injury (DILI). We aimed to analyse the influence of genetic variations in ABC transporters on the risk of DILI development and clinical presentations in a large Spanish DILI cohort. METHODS A total of ten polymorphisms in ABCB1 (1236T>C, 2677G>T,A, 3435T>C), ABCB4 (1954A>G) and ABCC2 (-1774G>del, -1549A>G, -24C>T, 1249G>A, 3972C>T and 4544G>A) were genotyped using Taqman 5' allelic discrimination assays or sequencing in 141 Spanish DILI patients and 161 controls. The influence of specific genotypes, alleles and haplotypes on the risk of DILI development and clinical presentations was analysed. RESULTS None of the individual polymorphisms or haplotypes was found to be associated with DILI development. Carriers homozygous for the ABCC2 -1774del allele were however only found in DILI patients. Hence, this genotype could potentially be associated with increased risk, though its low frequency in our Spanish cohort prevented a final conclusion. Furthermore, carriers homozygous for the ABCC2 -1774G/-1549A/-24T/1249G/3972T/4544G haplotype were found to have a higher propensity for total bilirubin elevations when developing DILI. CONCLUSIONS Our findings do not support a role for the analysed polymorphisms in the ABCB1, ABCB4 and ABCC2 transporter genes in DILI development in Spanish patients. The ABCC2 -1774deldel genotype was however restricted to DILI cases and could potentially contribute to enhanced DILI susceptibility.

The use of liver biopsy evaluation in discrimination of idiopathic autoimmune hepatitis versus drug-induced liver injury.

Distinguishing drug-induced liver injury (DILI) from idiopathic autoimmune hepatitis (AIH) can be challenging. We performed a standardized histologic evaluation to explore potential hallmarks to differentiate AIH versus DILI. Biopsies from patients with clinically well-characterized DILI [n = 35, including 19 hepatocellular injury (HC) and 16 cholestatic/mixed injury (CS)] and AIH (n = 28) were evaluated for Ishak scores, prominent inflammatory cell types in portal and intra-acinar areas, the presence or absence of emperipolesis, rosette formation, and cholestasis in a blinded fashion by four experienced hepatopathologists. Histologic diagnosis was concordant with clinical diagnosis in 65% of cases; but agreement on final diagnosis among the four pathologists was complete in only 46% of cases. Interface hepatitis, focal necrosis, and portal inflammation were present in all evaluated cases, but were more severe in AIH (P < 0.05) than DILI (HC). Portal and intra-acinar plasma cells, rosette formation, and emperiopolesis were features that favored AIH (P < 0.02). A model combining portal inflammation, portal plasma cells, intra-acinar lymphocytes and eosinophils, rosette formation, and canalicular cholestasis yielded an area under the receiver operating characteristic curve (AUROC) of 0.90 in predicting DILI (HC) versus AIH. All Ishak inflammation scores were more severe in AIH than DILI (CS) (P ≤ 0.05). The four AIH-favoring features listed above were consistently more prevalent in AIH, whereas portal neutrophils and intracellular (hepatocellular) cholestasis were more prevalent in DILI (CS) (P < 0.02). The combination of portal inflammation, fibrosis, portal neutrophils and plasma cells, and intracellular (hepatocellular) cholestasis yielded an AUC of 0.91 in predicting DILI (CS) versus AIH. Conclusion: Although an overlap of histologic findings exists for AIH and DILI, sufficient differences exist so that pathologists can use the pattern of injury to suggest the correct diagnosis.