Granulocyte macrophage colony stimulating factor improves mucosal repair in a mouse model of acute colitis (P278)

Background and Aims: Granulocyte-macrophage colonystimulating factor (GM-CSF), a cytokine modulating the number and function of innate immune cells, has been shown to provide symptomatic benefit in some patients with Crohn's disease (CD). Since, it becomes widely appreciated that a timely and spatially regulated action of innate immune cells is critical for tissue regeneration, we tested whether GM-CSF therapy may favours intestinal mucosal repair in the acute mouse model of dextran sulfate sodium (DSS)-induced colitis. Methods: Mice treated with GM-CSF or saline were exposed for 7 days to DSS to induce colitis. On day 5, 7 and 10, mice were subjected to colonoscopy or sacrificed for evaluation of inflammatory reaction and mucosal healing. Results: GM-CSF therapy prevented body weight loss, diarrhea, dampened inflammatory reactions and ameliorated mucosal damages. Mucosal repair improvement in GM-CSF-treated mice was observed from day 7 on both by colonoscopy (ulceration score 1.2}0.3 (GM-CSF-treated) vs 3.1}0.5 (untreated), p = 0.01) and histological analysis (percentage of reepithelialized ulcers 55%}4% (GM-CSF-treated) vs 18%}13% (untreated), p = 0.01). GM-CSF therapy can still improve the colitis when hematopoietic, but not non-hematopoietic cells, are responsive to GM-CSF, as shown in WT594;GM-CSFRKO chimeras. Lastly, we observed that GM-CSF-induced promotion of wound healing is associated with a modification of the cellular composition of DSS-induced colonic inflammatory infiltrate, characterized by the reduction of neutrophil numbers and early accumulation of CD11b+Gr1lo myeloid cells. Conclusion: Our study shows that GM-CSF therapy accelerates the complex program leading to tissue repair during acute colitis and suggests that GM-CSF promotion of mucosal repair might contribute to the symptomatic benefits of GM-CSF therapy observed in some CD patients.

Extracellular-signal-regulated kinase 5 modulates the antioxidant response by transcriptionally controlling Sirtuin 1 expression in leukemic cells.

Cancer cell metabolism differs from that of non-transformed cells in the same tissue. This specific metabolism gives tumor cells growing advantages besides the effect in increasing anabolism. One of these advantages is immune evasion mediated by a lower expression of the mayor histocompatibility complex class I molecules. The extracellular-signal-regulated kinase-5 regulates both mayor histocompatibility complex class I expression and metabolic activity. However, the mechanisms underlying are largely unknown. We show here that extracellular-signal-regulated kinase-5 regulates the transcription of the NADH(+)-dependent histone deacetylase silent mating type information regulation 2 homolog 1 (Sirtuin 1) in leukemic Jurkat T cells. This involves the activation of the transcription factor myocyte enhancer factor-2 and its binding to the sirt1 promoter. In addition, extracellular-signal-regulated kinase-5 is required for T cell receptor-induced and oxidative stress-induced full Sirtuin 1 expression. Extracellular-signal-regulated kinase-5 induces the expression of promoters containing the antioxidant response elements through a Sirtuin 1-dependent pathway. On the other hand, down modulation of extracellular-signal-regulated kinase-5 expression impairs the anti-oxidant response. Notably, the extracellular-signal-regulated kinase-5 inhibitor BIX02189 induces apoptosis in acute myeloid leukemia tumor cells without affecting T cells from healthy donors. Our results unveil a new pathway that modulates metabolism in tumor cells. This pathway represents a promising therapeutic target in cancers with deep metabolic layouts such as acute myeloid leukemia.

Mutational screening of splicing factor genes in cases with autosomal dominant retinitis pigmentosa.

PURPOSE: Mutations in genes encoding proteins from the tri-snRNP complex of the spliceosome account for more than 12% of cases of autosomal dominant retinitis pigmentosa (adRP). Although the exact mechanism by which splicing factor defects trigger photoreceptor death is not completely clear, their role in retinitis pigmentosa has been demonstrated by several genetic and functional studies. To test for possible novel associations between splicing factors and adRP, we screened four tri-snRNP splicing factor genes (EFTUD2, PRPF4, NHP2L1, and AAR2) as candidate disease genes. METHODS: We screened up to 303 patients with adRP from Europe and North America who did not carry known RP mutations. Exon-PCR and Sanger methods were used to sequence the NHP2L1 and AAR2 genes, while the sequences of EFTUD2 and PRPF4 were obtained by using long-range PCRs spanning coding and non-coding regions followed by next-generation sequencing. RESULTS: We detected novel missense changes in individual patients in the sequence of the genes PRPF4 and EFTUD2, but the role of these changes in relationship to disease could not be verified. In one other patient we identified a novel nucleotide substitution in the 5' untranslated region (UTR) of NHP2L1, which did not segregate with the disease in the family. CONCLUSIONS: The absence of clearly pathogenic mutations in the candidate genes screened in our cohort suggests that EFTUD2, PRPF4, NHP2L1, and AAR2 are either not involved in adRP or are associated with the disease in rare instances, at least as observed in this study in patients of European and North American origin.

Quantitative modelling of transcription factor binding sites

Abstract One of the most important issues in molecular biology is to understand regulatory mechanisms that control gene expression. Gene expression is often regulated by proteins, called transcription factors which bind to short (5 to 20 base pairs),degenerate segments of DNA. Experimental efforts towards understanding the sequence specificity of transcription factors is laborious and expensive, but can be substantially accelerated with the use of computational predictions. This thesis describes the use of algorithms and resources for transcriptionfactor binding site analysis in addressing quantitative modelling, where probabilitic models are built to represent binding properties of a transcription factor and can be used to find new functional binding sites in genomes. Initially, an open-access database(HTPSELEX) was created, holding high quality binding sequences for two eukaryotic families of transcription factors namely CTF/NF1 and LEFT/TCF. The binding sequences were elucidated using a recently described experimental procedure called HTP-SELEX, that allows generation of large number (> 1000) of binding sites using mass sequencing technology. For each HTP-SELEX experiments we also provide accurate primary experimental information about the protein material used, details of the wet lab protocol, an archive of sequencing trace files, and assembled clone sequences of binding sequences. The database also offers reasonably large SELEX libraries obtained with conventional low-throughput protocols.The database is available at http://wwwisrec.isb-sib.ch/htpselex/ and and ftp://ftp.isrec.isb-sib.ch/pub/databases/htpselex. The Expectation-Maximisation(EM) algorithm is one the frequently used methods to estimate probabilistic models to represent the sequence specificity of transcription factors. We present computer simulations in order to estimate the precision of EM estimated models as a function of data set parameters(like length of initial sequences, number of initial sequences, percentage of nonbinding sequences). We observed a remarkable robustness of the EM algorithm with regard to length of training sequences and the degree of contamination. The HTPSELEX database and the benchmarked results of the EM algorithm formed part of the foundation for the subsequent project, where a statistical framework called hidden Markov model has been developed to represent sequence specificity of the transcription factors CTF/NF1 and LEF1/TCF using the HTP-SELEX experiment data. The hidden Markov model framework is capable of both predicting and classifying CTF/NF1 and LEF1/TCF binding sites. A covariance analysis of the binding sites revealed non-independent base preferences at different nucleotide positions, providing insight into the binding mechanism. We next tested the LEF1/TCF model by computing binding scores for a set of LEF1/TCF binding sequences for which relative affinities were determined experimentally using non-linear regression. The predicted and experimentally determined binding affinities were in good correlation.

Predicting soil erosion using Rusle and NDVI time series from TM Landsat 5

The objective of this work was to evaluate the seasonal variation of soil cover and rainfall erosivity, and their influences on the revised universal soil loss equation (Rusle), in order to estimate watershed soil losses in a temporal scale. Twenty-two TM Landsat 5 images from 1986 to 2009 were used to estimate soil use and management factor (C factor). A corresponding rainfall erosivity factor (R factor) was considered for each image, and the other factors were obtained using the standard Rusle method. Estimated soil losses were grouped into classes and ranged from 0.13 Mg ha-1 on May 24, 2009 (dry season) to 62.0 Mg ha-1 on March 11, 2007 (rainy season). In these dates, maximum losses in the watershed were 2.2 and 781.5 Mg ha-1 , respectively. Mean annual soil loss in the watershed was 109.5 Mg ha-1 , but the central area, with a loss of nearly 300.0 Mg ha-1 , was characterized as a site of high water-erosion risk. The use of C factor obtained from remote sensing data, associated to corresponding R factor, was fundamental to evaluate the soil erosion estimated by the Rusle in different seasons, unlike of other studies which keep these factors constant throughout time.

A functional microsatellite of the macrophage migration inhibitory factor gene associated with meningococcal disease.

Macrophage migration inhibitory factor (MIF) is an abundantly expressed proinflammatory cytokine playing a critical role in innate immunity and sepsis and other inflammatory diseases. We examined whether functional MIF gene polymorphisms (-794 CATT(5-8) microsatellite and -173 G/C SNP) were associated with the occurrence and outcome of meningococcal disease in children. The CATT(5) allele was associated with the probability of death predicted by the Pediatric Index of Mortality 2 (P=0.001), which increased in correlation with the CATT(5) copy number (P=0.04). The CATT(5) allele, but not the -173 G/C alleles, was also associated with the actual mortality from meningoccal sepsis [OR 2.72 (1.2-6.4), P=0.02]. A family-based association test (i.e., transmission disequilibrium test) performed in 240 trios with 1 afflicted offspring indicated that CATT(5) was a protective allele (P=0.02) for the occurrence of meningococcal disease. At baseline and after stimulation with Neisseria meningitidis in THP-1 monocytic cells or in a whole-blood assay, CATT(5) was found to be a low-expression MIF allele (P=0.005 and P=0.04 for transcriptional activity; P=0.09 and P=0.09 for MIF production). Taken together, these data suggest that polymorphisms of the MIF gene affecting MIF expression are associated with the occurrence, severity, and outcome of meningococcal disease in children.

Treatment intensification and risk factor control: toward more clinically relevant quality measures.

BACKGROUND: Intensification of pharmacotherapy in persons with poorly controlled chronic conditions has been proposed as a clinically meaningful process measure of quality. OBJECTIVE: To validate measures of treatment intensification by evaluating their associations with subsequent control in hypertension, hyperlipidemia, and diabetes mellitus across 35 medical facility populations in Kaiser Permanente, Northern California. DESIGN: Hierarchical analyses of associations of improvements in facility-level treatment intensification rates from 2001 to 2003 with patient-level risk factor levels at the end of 2003. PATIENTS: Members (515,072 and 626,130; age >20 years) with hypertension, hyperlipidemia, and/or diabetes mellitus in 2001 and 2003, respectively. MEASUREMENTS: Treatment intensification for each risk factor defined as an increase in number of drug classes prescribed, of dosage for at least 1 drug, or switching to a drug from another class within 3 months of observed poor risk factor control. RESULTS: Facility-level improvements in treatment intensification rates between 2001 and 2003 were strongly associated with greater likelihood of being in control at the end of 2003 (P < or = 0.05 for each risk factor) after adjustment for patient- and facility-level covariates. Compared with facility rankings based solely on control, addition of percentages of poorly controlled patients who received treatment intensification changed 2003 rankings substantially: 14%, 51%, and 29% of the facilities changed ranks by 5 or more positions for hypertension, hyperlipidemia, and diabetes, respectively. CONCLUSIONS: Treatment intensification is tightly linked to improved control. Thus, it deserves consideration as a process measure for motivating quality improvement and possibly for measuring clinical performance.

Tumor-necrosis factor can enhance radio-antibody uptake in human colon carcinoma xenografts by increasing vascular permeability.

Marked differences in the tumor uptake of a 125I-labeled monoclonal antibody (MAb) directed against carcinoembryonic antigen (CEA) were observed in 4 serially transplanted human colorectal carcinomas in nude mice. A comparative study showed that elevated values of measurable tumor vascular parameters, such as permeability, blood flow and blood volume, correlated better with high MAb tumor uptake than the concentration of target antigen in the tumor. In an attempt to modify the vascular parameters and to determine if this could increase antibody uptake by the tumor, rhTNF alpha (TNF) was injected i.t. or i.v. and antibody localization experiments were performed immediately thereafter. Results showed that the permeability of the tumor vessels increased 8 to 10 fold 1 hr after i.t. injection of TNF as compared to control tumors injected with saline. Tumor uptake of 125I-labeled anti-CEA MAb, was 3 times higher 2 hr after i.v. injection and still 27% higher 22 hr later, as compared to results from controls. Intravenous injection of TNF simultaneously with the 125I-labeled anti-CEA MAb also resulted in a 2-fold increase in tumor uptake 4 hr after injection, but the increase was no longer significant 24 hr after injection. Interestingly after i.v. injection of TNF, the MAb concentration in the blood and other normal tissues, such as liver, kidneys, lungs and heart was decreased, resulting in significantly higher ratios of tumor to normal tissue. Taken together the results demonstrate that injection of TNF can increase tumor vascular permeability and improve radio-antibody uptake. This raises the possibility of increasing the radiation dose delivered by antibody to the tumor in the course of radioimmunotherapy.

Risk factor impact on blood flow velocities and clinical outcomes of stented cervical and intracranial stenoses: preliminary observations.

OBJECTIVES: The role of angioplasty/stenting procedures, neurointerventionist experience, vascular risk factors, medical treatment and blood flow velocities were analysed to identify possible causes of intra-stent restenosis (ISR) following stenting of cervical and/or intracranial arteries, assuming progressive atherosclerosis to be the shared mechanism in both territories. Patients. 26 cerebrovascular patients subjected to stenting of severe (Ͱ5;85%) symptomatic or asymptomatic carotid stenoses or moderate-to-severe (Ͱ5;50%) intracranial or vertebral stenoses were included. METHODS: Clinical, radiological and ultrasonographic follow-up data were analysed retrospectively. RESULTS: Overall, stenting of the internal carotid artery (ICA) induced significant reductions in peak systolic velocities at 2 years (96±31cm/s vs. 358.2±24.9cm/s at baseline). The procedure-related ischemic complications rate was 7.4% (one hemispheric stroke and one TIA). The rate of ISR≤50% was 8% in the ICA at 2 years; was 50% in the common carotid artery (CCA) at 1 year, with concomitant distal ICA stenosis in 75% of CCA stenting, but all ISR were asymptomatic. Patients with ISR of the ICA were significantly younger (56.8±4.5 vs. 71.3±3.6 years, P=0.042) and had significantly more risk factors (5.5±0.9 vs. 3±0.3, P=0.012). No ISRͰ5;70% was detected. CONCLUSIONS: ISR is relatively infrequent and, when present, it is mild and asymptomatic. Restenosis is more frequent in younger patients and those with several risk factors, and it may also be related to stenting of previous carotid endarterectomy.

Relaciones entre la estructura del 16PF-5 y el modelo de cinco grandes factores de personalidad

Este estudio fue diseñado para evaluar las relaciones del 16PF-5 y el modelo de Cinco Grandes factores de personalidad evaluado a partir de un listado de adjetivos unipolares propuestos por Goldberg (1992). Mediante sucesivos análisis factoriales se obtuvo un listado de 40 adjetivos, 8 para cada uno de los Cinco Grandes, que presentaron una estructura robusta y una consistencia interna aceptable. Al comparar los cinco factores del 16PF-5 con los Cinco Grandes, mediante análisis correlacionales y factoriales, se observa que Surgencia (Extraversión), Neuroticismo (Ansiedad) y Responsabilidad (Auto-Control) son dimensiones muy parecidas en ambos instrumentos y coincidentes con los resultados obtenidos por otros autores entre el NEO-PI, NEO-PI-R y el 16PF. Las dimensiones de Extraversión e Independencia del 16PF-5 están muy relacionadas entre sí, en cambio Independencia y Dureza tienden a relacionar más con el factor de Intelecto de Goldberg. Se estudian tres estructuras factoriales de 3, 4 y 5 factores a partir de los cinco factores de segundo orden de Cattell y los cinco factores de Goldberg. Se discute sobre la equivalencia de ambas estructuras.

Replicabilidad de los factores de segundo orden del 16PF-5 en muestras americanas y españolas

El objetivo de este estudio fue replicar las estructuras de 5 y 6 factores de segundo orden del 16PF-5. Para la estructura de 5 factores se toma como referencia teórica la estructura obtenida por Russell y Karol (1995), y para la estructura de 6 factores (incluyendo un factor adicional de Razonamiento) la obtenida en muestras americanas por Cattell y Cattell (1995). Se utilizan tres procedimientos para el estudio de la replicabilidad, a) análisis factorial exploratorio, b) análisis de la estructura ortogonal Procrustes, y c) análisis de los índices de congruencia entre las tres matrices factoriales. Las matrices factoriales obtenidas en el presente estudio son similares a las informadas en los estudios de referencia, aunque la solución Procrustes se revela ligeramente más parecida. Los índices de congruencia en ge- neral son aceptables por lo que se concluye que el 16PF-5 demuestra buena replicabilidad.

Sensitivity analysis, dominant factors, and robustness of the ECETOC TRA v3, Stoffenmanager 4.5, and ART 1.5 occupational exposure models

Occupational exposure modeling is widely used in the context of the E.U. regulation on the registration, evaluation, authorization, and restriction of chemicals (REACH). First tier tools, such as European Centre for Ecotoxicology and TOxicology of Chemicals (ECETOC) targeted risk assessment (TRA) or Stoffenmanager, are used to screen a wide range of substances. Those of concern are investigated further using second tier tools, e.g., Advanced REACH Tool (ART). Local sensitivity analysis (SA) methods are used here to determine dominant factors for three models commonly used within the REACH framework: ECETOC TRA v3, Stoffenmanager 4.5, and ART 1.5. Based on the results of the SA, the robustness of the models is assessed. For ECETOC, the process category (PROC) is the most important factor. A failure to identify the correct PROC has severe consequences for the exposure estimate. Stoffenmanager is the most balanced model and decision making uncertainties in one modifying factor are less severe in Stoffenmanager. ART requires a careful evaluation of the decisions in the source compartment since it constitutes ∼75% of the total exposure range, which corresponds to an exposure estimate of 20-22 orders of magnitude. Our results indicate that there is a trade off between accuracy and precision of the models. Previous studies suggested that ART may lead to more accurate results in well-documented exposure situations. However, the choice of the adequate model should ultimately be determined by the quality of the available exposure data: if the practitioner is uncertain concerning two or more decisions in the entry parameters, Stoffenmanager may be more robust than ART.

Early adversity and 5-HTT-BDNF genes: new evidences of gene-environment interactions on depressive symptoms in a general population

Adverse childhood experiences have been described as one of the major environmental risk factors for depressive disorder. Likewise, the deleterious impact of early traumatic experiences on depression seems to be moderated by individual genetic variability. Serotonin transporter (5-HTT) and the Brain-Derived Neurotrophic Factor (BDNF) seem to modulate the effect of childhood adversity on adult depression, although inconsistencies across studies have been found. Moreover, the GxE interaction concerning the different types of childhood adversity remains poorly understood. The aim of this study is to analyse the putative interaction between the 5-HTT gene (5-HTTLPR polymorphism), BDNF gene (Val66Met polymorphism) and childhood adversity in accounting for adult depressive symptoms.

Rainfall is a risk factor for sporadic cases of Legionella pneumophila Pneumonia

It is not known whether rainfall increases the risk of sporadic cases of Legionella pneumonia. We sought to test this hypothesis in a prospective observational cohort study of non-immunosuppressed adults hospitalized for community-acquired pneumonia (1995-2011). Cases with Legionella pneumonia were compared with those with non-Legionella pneumonia. Using daily rainfall data obtained from the regional meteorological service we examined patterns of rainfall over the days prior to admission in each study group. Of 4168 patients, 231 (5.5%) had Legionella pneumonia. The diagnosis was based on one or more of the following: sputum (41 cases), antigenuria (206) and serology (98). Daily rainfall average was 0.556 liters/m2 in the Legionella pneumonia group vs. 0.328 liters/m2 for non-Legionella pneumonia cases (p = 0.04). A ROC curve was plotted to compare the incidence of Legionella pneumonia and the weighted median rainfall. The cut-off point was 0.42 (AUC 0.54). Patients who were admitted to hospital with a prior weighted median rainfall higher than 0.42 were more likely to have Legionella pneumonia (OR 1.35; 95% CI 1.02-1.78; p = .03). Spearman Rho correlations revealed a relationship between Legionella pneumonia and rainfall average during each two-week reporting period (0.14; p = 0.003). No relationship was found between rainfall average and non-Legionella pneumonia cases (−0.06; p = 0.24). As a conclusion, rainfall is a significant risk factor for sporadic Legionella pneumonia. Physicians should carefully consider Legionella pneumonia when selecting diagnostic tests and antimicrobial therapy for patients presenting with CAP after periods of rainfall.

Hepatocyte nuclear factor 4alpha transactivates the mitochondrial alanine aminotransferase gene in the kidney of Sparus aurata

Alanine aminotransferase (ALT) plays an important role in amino acid metabolism and gluconeogenesis. The preference of carnivorous fish for protein amino acids instead of carbohydrates as a source of energy lead us to study the transcriptional regulation of the mitochondrial ALT (mALT) gene and to characterize the enzyme kinetics and modulation of mALT expression in the kidney of gilthead sea bream (Sparus aurata) under different nutritional and hormonal conditions. 5′-Deletion analysis of mALT promoter in transiently transfected HEK293 cells, site-directed mutagenesis and electrophoretic mobility shift assays allowed us to identify HNF4α as a new factor involved in the transcriptional regulation of mALT expression. Quantitative RT-PCR assays showed that starvation and the administration of streptozotocin (STZ) decreased HNF4α levels in the kidney of S. aurata, leading to the downregulation of mALT transcription. Analysis of the tissue distribution showed that kidney, liver, and intestine were the tissues with higher mALT and HNF4α expression. Kinetic analysis indicates that mALT enzyme is more efficient in catalyzing the conversion of L-alanine to pyruvate than the reverse reaction. From these results, we conclude that HNF4α transactivates the mALT promoter and that the low levels of mALT expression found in the kidney of starved and STZ-treated fish result from a decreased expression of HNF4α. Our findings suggest that the mALT isoenzyme plays a major role in oxidazing dietary amino acids, and points to ALT as a target for a biotechnological action to spare protein and optimize the use of dietary nutrients for fish culture.