What new cell biology findings could bring to therapeutics: is it time for a phenome-project in Toxoplasma gondii?

"If you know the enemy and know yourself, you need not fear the result of a hundred battles. If you know yourself but not the enemy, for every victory gained you will also suffer a defeat" (SunTzu the Art of War, 544-496 BC). Although written for the managing of conflicts and winning clear victories, this basic guideline can be directly transferred to our battle against apicomplexan parasites and how to focus future basic research in order to transfer the gained knowledge to a therapeutic intervention stratey. Over the last two decades the establishment of several key-technologies, by different groups working on Toxoplasma gondii, made this important human pathogen accessible to modern approaches in molecular cell biology. In fact more and more researchers get attracted to this easy accessible model organism to study specific biological questions, unique to apicomplexans. This fascinating, unique biology might provide us with new therapeutic options in our battle against apicomplexan parasites by finding its Achilles' heel. In this article we argue that in the absence of a powerful high throughput technology for the characterisation of essential gene of interests a coordinated effort should be undertaken to convert our knowledge of the genome into one of the phenome.

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.

Safety and Efficacy of the Newer Biological Therapeutics in the Treatment of Rheumatoid Arthritis

Biological therapies have been a major advance in RA treatment. However, remission or response is not achieved in all patients. Therefore, new drugs seem necessary. Most recent trials have focused in the development of three different groups of molecules: those against commercialized targets but minimizing side effects or improving administration, others molecules against new targets, and a third group including small molecules. Some of them have been shown to be clinically efficacious and safe in RA patients, including: two new anti-TNF therapies (golimumab and certolizumab pegol), three anti-CD (ocrelizumab, ofatumumab and a SMIP), subcutaneous abatacept, anti-IL17 therapy, tasocitinib and fostamatinib disodium. Therefore, a wide spectrum of new RA therapeutics are promising, but more studies are necessary to confirm these results.

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.

Precursor ion scans for the targeted detection of stable-isotope-labeled peptides.

Stable isotope labels are routinely introduced into proteomes for quantification purposes. Full labeling of cells in varying biological states, followed by sample mixing, fractionation and intensive data acquisition, is used to obtain accurate large-scale quantification of total protein levels. However, biological processes often affect only a small group of proteins for a short time, resulting in changes that are difficult to detect against the total proteome background. An alternative approach could be the targeted analysis of the proteins synthesized in response to a given biological stimulus. Such proteins can be pulse-labeled with a stable isotope by metabolic incorporation of 'heavy' amino acids. In this study we investigated the specific detection and identification of labeled proteins using acquisition methods based on Precursor Ion Scans (PIS) on a triple-quadrupole ion trap mass spectrometer. PIS-based methods were set to detect unique immonium ions originating from labeled peptides. Different labels and methods were tested in standard mixtures to optimize performance. We showed that, in comparison with an untargeted analysis on the same instrument, the approach allowed a several-fold increase in the specificity of detection of labeled proteins over unlabeled ones. The technique was applied to the identification of proteins secreted by human cells into growth media containing bovine serum proteins, allowing the preferential detection of labeled cellular proteins over unlabeled bovine ones. However, compared with untargeted acquisitions on two different instruments, the PIS-based strategy showed some limitations in sensitivity. We discuss possible perspectives of the technique.

The potential for vaccination in leprosy elimination: new tools for targeted interventions

Despite the huge effort and massive advances toward the elimination of leprosy over the last two decades, the disease has proven stubborn; new case detection rates have stabilised over the last few years and leprosy remains endemic in a number of localised regions. The American Leprosy Missions and Infectious Disease Research Institute have undertaken a large research effort aimed at developing new tools and a vaccine to continue the push for leprosy elimination. In this paper, we outline our strategy for the integration of rapid diagnostic tests and lab-based assays to facilitate the detection of early or asymptomatic leprosy cases, as well as the efficient and focused implementation of chemoprophylaxis and immunisation to intervene in leprosy development and transmission.

The Wnt inhibitory factor 1 (WIF1) is targeted in glioblastoma and has a tumor suppressing function potentially by induction of senescence.

Gene expression-based prediction of genomic copy number aberrations in the chromosomal region 12q13 to 12q15 that is flanked by MDM2 and CDK4 identified Wnt inhibitory factor 1 (WIF1) as a candidate tumor suppressor gene in glioblastoma. WIF1 encodes a secreted Wnt antagonist and was strongly downregulated in most glioblastomas as compared with normal brain, implying deregulation of Wnt signaling, which is associated with cancer. WIF1 silencing was mediated by deletion (7/69, 10%) or epigenetic silencing by promoter hypermethylation (29/110, 26%). Co-amplification of MDM2 and CDK4 that is present in 10% of glioblastomas was associated in most cases with deletion of the whole genomic region enclosed, including the WIF1 locus. This interesting pathogenetic constellation targets the RB and p53 tumor suppressor pathways in tandem, while simultaneously activating oncogenic Wnt signaling. Ectopic expression of WIF1 in glioblastoma cell lines revealed a dose-dependent decrease of Wnt pathway activity. Furthermore, WIF1 expression inhibited cell proliferation in vitro, reduced anchorage-independent growth in soft agar, and completely abolished tumorigenicity in vivo. Interestingly, WIF1 overexpression in glioblastoma cells induced a senescence-like phenotype that was dose dependent. These results provide evidence that WIF1 has tumor suppressing properties. Downregulation of WIF1 in 75% of glioblastomas indicates frequent involvement of aberrant Wnt signaling and, hence, may render glioblastomas sensitive to inhibitors of Wnt signaling, potentially by diverting the tumor cells into a senescence-like state.

Dipeptidyl peptidase IV and its inhibitors: therapeutics for type 2 diabetes and what else?

The proline-specific dipeptidyl aminopeptidase IV (DPP IV, DPP-4, CD26), widely expressed in mammalians, releases X-Pro/Ala dipeptides from the N-terminus of peptides. DPP IV is responsible of the degradation of the incretin peptide hormones regulating blood glucose levels. Several families of DPP IV inhibitors have been synthesized and evaluated. Their positive effects on the degradation of the incretins and the control of blood glucose levels have been demonstrated in biological models and in clinical trials. Presently, several DPP IV inhibitors, the "gliptins", are approved for type 2 diabetes or are under clinical evaluation. However, the gliptins may also be of therapeutic interest for other diseases beyond the inhibition of incretin degradation. In this Perspective, the biological functions and potential substrates of DPP IV enzymes are reviewed and the characteristics of the DPP IV inhibitors are discussed in view of type 2 diabetes and further therapeutic interest.

Effectiveness of olive oil for the prevention of pressure ulcers caused in immobilized patients within the scope of primary health care: study protocol for a randomized controlled trial.

BACKGROUND Pressure ulcers are considered an important issue, mainly affecting immobilized older patients. These pressure ulcers increase the care burden for the professional health service staff as well as pharmaceutical expenditure. There are a number of studies on the effectiveness of different products used for the prevention of pressure ulcers; however, most of these studies were carried out at a hospital level, basically using hyperoxygenated fatty acids (HOFA). There are no studies focused specifically on the use of olive-oil-based products and therefore this research is intended to find the most cost-effective treatment and achieve an alternative treatment. METHODS/DESIGN The main objective is to assess the effectiveness of olive oil, comparing it with HOFA, to treat immobilized patients at home who are at risk of pressure ulcers. As a secondary objective, the cost-effectiveness balance of this new application with regard to the HOFA will be assessed. The study is designed as a noninferiority, triple-blinded, parallel, multi-center, randomized clinical trial. The scope of the study is the population attending primary health centers in Andalucía (Spain) in the regional areas of Malaga, Granada, Seville, and Cadiz. Immobilized patients at risk of pressure ulcers will be targeted. The target group will be treated by application of an olive-oil-based formula whereas the control group will be treated by application of HOFA to the control group. The follow-up period will be 16 weeks. The main variable will be the presence of pressure ulcers in the patient. Secondary variables include sociodemographic and clinical information, caregiver information, and whether technical support exists. Statistical analysis will include the Kolmogorov-Smirnov test, symmetry and kurtosis analysis, bivariate analysis using the Student's t and chi-squared tests as well as the Wilcoxon and the Man-Whitney U tests, ANOVA and multivariate logistic regression analysis. DISCUSSION The regular use of olive-oil-based formulas should be effective in preventing pressure ulcers in immobilized patients, thus leading to a more cost-effective product and an alternative treatment. TRIAL REGISTRATION Clinicaltrials.gov identifier: NCT01595347.

Infornut® Process; improves accessibility to diagnosis and nutritional support for the malnourished hospitalized patient; impact on management indicators; two-year assessment.

Introduction: The high prevalence of disease-related hospital malnutrition justifies the need for screening tools and early detection in patients at risk for malnutrition, followed by an assessment targeted towards diagnosis and treatment. At the same time there is clear undercoding of malnutrition diagnoses and the procedures to correct it Objectives: To describe the INFORNUT program/ process and its development as an information system. To quantify performance in its different phases. To cite other tools used as a coding source. To calculate the coding rates for malnutrition diagnoses and related procedures. To show the relationship to Mean Stay, Mortality Rate and Urgent Readmission; as well as to quantify its impact on the hospital Complexity Index and its effect on the justification of Hospitalization Costs. Material and methods: The INFORNUT® process is based on an automated screening program of systematic detection and early identification of malnourished patients on hospital admission, as well as their assessment, diagnoses, documentation and reporting. Of total readmissions with stays longer than three days incurred in 2008 and 2010, we recorded patients who underwent analytical screening with an alert for a medium or high risk of malnutrition, as well as the subgroup of patients in whom we were able to administer the complete INFORNUT® process, generating a report for each.

Gene-lifestyle interaction and type 2 diabetes: the EPIC interact case-cohort study.

BACKGROUND Understanding of the genetic basis of type 2 diabetes (T2D) has progressed rapidly, but the interactions between common genetic variants and lifestyle risk factors have not been systematically investigated in studies with adequate statistical power. Therefore, we aimed to quantify the combined effects of genetic and lifestyle factors on risk of T2D in order to inform strategies for prevention. METHODS AND FINDINGS The InterAct study includes 12,403 incident T2D cases and a representative sub-cohort of 16,154 individuals from a cohort of 340,234 European participants with 3.99 million person-years of follow-up. We studied the combined effects of an additive genetic T2D risk score and modifiable and non-modifiable risk factors using Prentice-weighted Cox regression and random effects meta-analysis methods. The effect of the genetic score was significantly greater in younger individuals (p for interaction  = 1.20×10-4). Relative genetic risk (per standard deviation [4.4 risk alleles]) was also larger in participants who were leaner, both in terms of body mass index (p for interaction  = 1.50×10-3) and waist circumference (p for interaction  = 7.49×10-9). Examination of absolute risks by strata showed the importance of obesity for T2D risk. The 10-y cumulative incidence of T2D rose from 0.25% to 0.89% across extreme quartiles of the genetic score in normal weight individuals, compared to 4.22% to 7.99% in obese individuals. We detected no significant interactions between the genetic score and sex, diabetes family history, physical activity, or dietary habits assessed by a Mediterranean diet score. CONCLUSIONS The relative effect of a T2D genetic risk score is greater in younger and leaner participants. However, this sub-group is at low absolute risk and would not be a logical target for preventive interventions. The high absolute risk associated with obesity at any level of genetic risk highlights the importance of universal rather than targeted approaches to lifestyle intervention.

New insights to occult gastrointestinal bleeding: From pathophysiology to therapeutics.

Obscure gastrointestinal bleeding is still a clinical challenge for gastroenterologists. The recent development of novel technologies for the diagnosis and treatment of different bleeding causes has allowed a better management of patients, but it also determines the need of a deeper comprehension of pathophysiology and the analysis of local expertise in order to develop a rational management algorithm. Obscure gastrointestinal bleeding can be divided in occult, when a positive occult blood fecal test is the main manifestation, and overt, when external sings of bleeding are visible. In this paper we are going to focus on overt gastrointestinal bleeding, describing the physiopathology of the most usual causes, analyzing the diagnostic procedures available, from the most classical to the novel ones, and establishing a standard algorithm which can be adapted depending on the local expertise or availability. Finally, we will review the main therapeutic options for this complex and not so uncommon clinical problem.

Drug assessment by a Pharmacy and Therapeutics committee: from drug selection criteria to use in clinical practice.

BACKGROUND In Spain, hospital medicines are assessed and selected by local Pharmacy and Therapeutics committees (PTCs). Of all the drugs assessed, cancer drugs are particularly important because of their budgetary impact and the sometimes arguable added value with respect to existing alternatives. This study analyzed the PTC drug selection process and the main objective was to evaluate the degree of compliance of prescriptions for oncology drugs with their criteria for use. METHODS This was a retrospective observational study (May 2007 to April 2010) of PTC-assessed drugs. The variables measured to describe the committee's activity were number of drugs assessed per year and number of drugs included in any of these settings: without restrictions, with criteria for use, and not included in formulary. These drugs were also analyzed by therapeutic group. To assess the degree of compliance of prescriptions, a score was calculated to determine whether prescriptions for bevacizumab, cetuximab, trastuzumab, and bortezomib were issued in accordance with PTC drug use criteria. RESULTS The PTC received requests for inclusion of 40 drugs, of which 32 were included in the hospital formulary (80.0%). Criteria for use were established for 28 (87.5%) of the drugs included. In total, 293 patients were treated with the four cancer drugs in eight different therapeutic indications. The average prescription compliance scores were as follows: bevacizumab, 83% for metastatic colorectal cancer, 100% for metastatic breast cancer, and 82.3% for non-small-cell lung cancer; cetuximab, 62.0% for colorectal cancer and 50% for head and neck cancer; trastuzumab, 95.1% for early breast cancer and 82.4% for metastatic breast cancer; and bortezomib, 63.7% for multiple myeloma. CONCLUSION The degree of compliance with criteria for use of cancer drugs was reasonably high. PTC functions need to be changed so that they can carry out more innovative tasks, such as monitoring conditions for drug use.