Apoptosis as a target for gene therapy in rheumatoid arthritis

Rheumatoid arthritis (RA) is characterized by chronic inflammation of the synovial joints resulting from hyperplasia of synovial fibroblasts and infiltration of lymphocytes, macrophages and plasma cells, all of which manifest signs of activation. All these cells proliferate abnormally, invade bone and cartilage, produce an elevated amount of pro-inflammatory cytokines, metalloproteinases and trigger osteoclast formation and activation. Some of the pathophysiological consequences of the disease may be explained by the inadequate apoptosis, which may promote the survival of autoreactive T cells, macrophages or synovial fibroblasts. Although RA does not result from single genetic mutations, elucidation of the molecular mechanisms implicated in joint destruction has revealed novel targets for gene therapy. Gene transfer strategies include inhibition of pro-inflammatory cytokines, blockade of cartilage-degrading metalloproteinases, inhibition of synovial cell activation and manipulation of the Th1-Th2 cytokine balance. Recent findings have iluminated the idea that induction of apoptosis in the rheumatoid joint can be also used to gain therapeutic advantage in the disease. In the present review we will discuss different strategies used for gene transfer in RA and chronic inflammation. Particularly, we will highlight the importance of programmed cell death as a novel target for gene therapy using endogenous biological mediators, such as galectin-1, a beta-galactoside-binding protein that induces apoptosis of activated T cells and immature thymocytes.

Target-guided synthesis of metal-based phosphatase inhibitors

Report for the scientific sojourn at the Imperial College of London, United Kingdom, from 2007 to 2009. PTEN is a tumour suppressor enzyme that plays important roles in the PI3K pathway which regulates growth, proliferation and survival and is thus related to many human disorders such as diabetes, neurodegenerative diseases, cardiovascular complications and cancer. It is hence of great interest to understand in detail its molecular behaviour and to find small molecules that can switch on/off its activity. For this purpose, metal complexes have been synthesized and preliminary studies in vivo show that all are capable of inhibiting PTEN.

Does history of falling predict length of stay in post-acute geriatric rehabilitation?

Objectives:To investigate the associations between falls before hospital¦admission, falls during hospitalization, and length of stay in elderly¦people admitted to post-acute geriatric rehabilitation. Method: History¦of falling in the previous 12 months before admission was recorded¦among 249 older persons (mean age 82.3±7.4 years, 69.1% women)¦consecutively admitted to post-acute rehabilitation. Data on medical,¦functional and cognitive status were collected upon admission. Falls¦during hospitalization and length of stay were recorded at discharge.¦Results: Overall, 92 (40.4%) patients reported no fall in the 12 months¦before admission; 63(27.6%) reported 1 fall, and 73(32.0%) reported¦multiple falls. Previous falls occurrence (one or more falls) was significantly¦associated with in-stay falls (19.9% of previous fallers fell¦during the stay vs 7.6% in patients without history of falling, P=.01),¦and with a longer length of stay (22.4 ± 10.1 days vs 27.1 ± 14.3 days,¦P=.01). In multivariate robust regression controlling for gender, age,¦functional and cognitive status, history of falling remained significantly¦associated with longer rehabilitation stay (2.8 days more in single fallers,¦p=.05, and 3.3 days more in multiple fallers, p=.0.1, compared to¦non-fallers). Conclusion: History of falling in the 12 months prior to¦post acute geriatric rehabilitation is independently associated with a¦longer rehabilitation length of stay. Previous fallers have also an¦increased risk of falling during rehabilitation stay. This suggests that¦hospital fall prevention measures should particularly target these high¦riskpatients.

Undernutrition in children with profound intellectual and multiple disabilities (PIMD): its prevalence and influence on quality of life.

BACKGROUND: To estimate the prevalence of undernutrition among children with profound intellectual and multiple disabilities (PIMD) and to explore its influence on quality of life. METHODS: Seventy-two children with PIMD (47 male; 25 female; age range 2 to 15 years 4 months; mean age 8.6, SD 3.6) underwent an anthropometric assessment, including body weight, triceps skinfold thickness, segmental measures and recumbent length. Undernutrition was determined using tricipital skinfold percentile and z-scores of weight-for-height and height-for-age. The quality of life of each child was evaluated using the QUALIN questionnaire adapted for profoundly disabled children. RESULTS: Twenty-five children (34.7%) were undernourished and seven (9.7%) were obese. Among undernourished children only eight (32 %) were receiving food supplements and two (8%) had a gastrostomy, of which one was still on a refeeding programme. On multivariate analysis, undernutrition was one of the independent predictors of lower quality of life. CONCLUSION: Undernutrition remains a matter of concern in children with PIMD. There is a need to better train professionals in systematically assessing the nutritional status of profoundly disabled children in order to start nutritional management when necessary.

Assessing multiple sclerosis activity: is the in vitro production of tumor necrosis factor-alpha, interleukins 2, 6, 4, and 10, and immunoglobulin G of value?

Tumor necrosis factor (TNF) alpha, interleukins (IL) 2, 4, 6, and 10, and IgG oligoclonal bands (IgG OB) in vitro production was assessed, after whole-blood stimulation with lipopolysaccharide or concanavalin A, in 61 patients presenting with relapsing-remitting, relapsing-progressive, or chronic progressive multiple sclerosis. Multiple sclerosis patients were receiving no treatment or azathioprine (AZA), cyclosporin, cyclophosphamide, subcutaneous interferon (IFN) beta 1 a, or corticosteroids (CST). Statistical correlations significantly showed that: (a) AZA lowers TNF-alpha (P = 0.002) and increases IL-4 production (P = 0.0024), and IFN-beta 1 a increases TNF-alpha and decreases IL-4 levels; (b) CST has a negative effect on TNF-alpha, IL-6, and IL-4 synthesis; and (c) AZA, IFN-beta 1 a, and CST diminish IgG OB synthesis (P = 0.001). Although our study of the dynamics of TNF-alpha, IL-2, IL-4, IL-6, and IL-10 in vitro production generally found no statistically significant correlations (partly explained by the limited number of values in the various groups), IL-6 was shown to drop during the periods surrounding relapse (P = 0.05) in the absence of treatment, while TNF-alpha (P = 0.04) and IL-6 (P < 0.05) dropped before exacerbation in the presence of AZA. In vitro production of TNF-alpha was closely and positively correlated with that of IL-6, independently of clinical features. The enhanced production of IL-10 detected before or at relapse with AZA and IFN-beta 1 a (trends) may interfere with initiation of the immune reaction and with the development of new CNS lesions. Some discrepancies with previously published results stress the difficulties in studying the state of stimulation of different populations of leukocytes by using a variety of in vitro stimuli and in establishing a correlation between mRNA studies and the amount of final or active protein produced.

Morphological investigation of Toxoplasma gondii in vivo by a multiple beam interference microscope

A recently developed technique, namely multiple beam interference microscopy, has been applied to investigate the morphology of the parasite Toxoplasma gondii for the first time. The interference pattern obtained from the multiple internal reflection of a T. gondii, sandwiched between a glass plate and a cover plate, was focused on the objective of a conventional microscope. Because of the enhance contrast, several details of sub cellular structure and separating compartments are clearly visible. Details reveal the presence of a nucleus, lipid body, dense granule, rhoptry and amylopectin. The wall thickness of the membrane of the lipid body and the amylopectin is of the order of 0.02 µm and can be clearly distinguished with the help of the present technique. The same parasite has also been examined with the help of atomic force microscopy, and because of its thick membrane, the inner structural details were not observed at all. Sub cellular details of T. gondii observed with the present technique have been reported earlier only by low amplification transmission electron microscopy and not by any optical microscopic technique.

Proximale Humerusmehrfragmentfrakturen--Misserfolge nach T-Platten-Osteosynthesen [Proximal humeral multiple fragment fractures--failures after T-plate osteosynthesis].

Dislocated compound fractures of the proximal humerus are often difficult to treat. The choice of treatment influences the final functional result. From 1984-1991 108 patients with dislocated compound fractures of the proximal humerus were operated with a T-plate osteosynthesis, retrospectively examined and classified according to the Neer-Classification. At an average follow up time of 5 years 72 patients had a clinical and radiological examination. 68% of these patients with 3-fragment fractures and 80% with 4-fragment fractures showed a modest to unsatisfactory result caused by fracture biology, imprecise fracture reduction or poor surgical procedure. Incorrect position of T-plates and inadequate material were distinguishable. The T-plate which was widely used in the late eighties for internal fixation has to be considered a failure for these particular types of fractures and should be limited for Collum chirurgicum fractures.

Multiple-scale hydrological and geophysical data integration through non-linear Bayesian sequential simulation

Significant progress has been made with regard to the quantitative integration of geophysical and hydrological data at the local scale. However, extending the corresponding approaches to the scale of a field site represents a major, and as-of-yet largely unresolved, challenge. To address this problem, we have developed downscaling procedure based on a non-linear Bayesian sequential simulation approach. The main objective of this algorithm is to estimate the value of the sparsely sampled hydraulic conductivity at non-sampled locations based on its relation to the electrical conductivity logged at collocated wells and surface resistivity measurements, which are available throughout the studied site. The in situ relationship between the hydraulic and electrical conductivities is described through a non-parametric multivariatekernel density function. Then a stochastic integration of low-resolution, large-scale electrical resistivity tomography (ERT) data in combination with high-resolution, local-scale downhole measurements of the hydraulic and electrical conductivities is applied. The overall viability of this downscaling approach is tested and validated by comparing flow and transport simulation through the original and the upscaled hydraulic conductivity fields. Our results indicate that the proposed procedure allows obtaining remarkably faithful estimates of the regional-scale hydraulic conductivity structure and correspondingly reliable predictions of the transport characteristics over relatively long distances.

Reduction in catheter-related bloodstream infections in critically ill patients through a multiple system intervention.

Els catéters venosos centrals són necessaris per al maneig del pacient crític però poden ser l´origen d´una bacteriemia. Aquest estudi prospectiu de cohort té com a objectiu determinar la utilitat de l´aplicació d´unes mesures bàsiques de prevenció per disminuir la incidència de bacteriemia associada a catéter. Els resultats de l´estudi confirmen que l´aplicació d´aquest sistema d´intervenció múltiple basat en l´evidencia redueix de forma significativa les bacteriemies associades a catéter a la nostra UCI.

Protein-Binding Microarray Analysis of Tumor Suppressor AP2α Target Gene Specificity.

Cheap and massively parallel methods to assess the DNA-binding specificity of transcription factors are actively sought, given their prominent regulatory role in cellular processes and diseases. Here we evaluated the use of protein-binding microarrays (PBM) to probe the association of the tumor suppressor AP2α with 6000 human genomic DNA regulatory sequences. We show that the PBM provides accurate relative binding affinities when compared to quantitative surface plasmon resonance assays. A PBM-based study of human healthy and breast tumor tissue extracts allowed the identification of previously unknown AP2α target genes and it revealed genes whose direct or indirect interactions with AP2α are affected in the diseased tissues. AP2α binding and regulation was confirmed experimentally in human carcinoma cells for novel target genes involved in tumor progression and resistance to chemotherapeutics, providing a molecular interpretation of AP2α role in cancer chemoresistance. Overall, we conclude that this approach provides quantitative and accurate assays of the specificity and activity of tumor suppressor and oncogenic proteins in clinical samples, interfacing genomic and proteomic assays.

Imaging Properties of MS Lesions Correlate with Attention Deficits in Early Multiple Sclerosis

Introduction: Cognitive impairment affects 40-65% of multiple sclerosis (MS) patients, often since early stages of the disease (relapsing remitting MS, RRMS). Frequently affected functions are memory, attention or executive abilities but the most sensitive measure of cognitive deficits in early MS is the information processing speed (Amato, 2008). MRI has been extensively exploited to investigate the substrate of cognitive dysfunction in MS but the underlying physiopathological mechanisms remain unclear. White matter lesion load, whole-brain atrophy and cortical lesions' number play a role but correlations are in some cases modest (Rovaris, 2006; Calabrese, 2009). In this study, we aimed at characterizing and correlating the T1 relaxation times of cortical and sub-cortical lesions with cognitive deficits detected by neuropsychological tests in a group of very early RR MS patients. Methods: Ten female patients with very early RRMS (age: 31.6 ±4.7y; disease duration: 3.8 ±1.9y; EDSS disability score: 1.8 ±0.4) and 10 age- and gender-matched healthy volunteers (mean age: 31.2 ±5.8y) were included in the study. All participants underwent the following neuropsychological tests: Rao's Brief Repeatable Battery of Neuropsychological tests (BRB-N), Stockings of Cambridge, Trail Making Test (TMT, part A and B), Boston Naming Test, Hooper Visual Organization Test and copy of the Rey-Osterrieth Complex Figure. Within 2 weeks from neuropsychological assessment, participants underwent brain MRI at 3T (Magnetom Trio a Tim System, Siemens, Germany) using a 32-channel head coil. The imaging protocol included 3D sequences with 1x1x1.2 mm3 resolution and 256x256x160 matrix, except for axial 2D-FLAIR: -DIR (T2-weighted, suppressing both WM and CSF; Pouwels, 2006) -MPRAGE (T1-weighted; Mugler, 1991) -MP2RAGE (T1-weighted with T1 maps; Marques, 2010) -FLAIR SPACE (only for patient 4-10, T2-weighted; Mugler, 2001) -2D Axial FLAIR (0.9x0.9x2.5 mm3, 256x256x44 matrix). Lesions were identified by one experienced neurologist and radiologist using all contrasts, manually contoured and assigned to regional locations (cortical or sub-cortical). Lesion number, volume and T1 relaxation time were calculated for lesions in each contrast and in a merged mask representing the union of the lesions from all contrasts. T1 relaxation times of lesions were normalized with the mean T1 value in corresponding control regions of the healthy subjects. Statistical analysis was performed using GraphPad InStat software. Cognitive scores were compared between patients and controls with paired t-tests; p values ≤ 0.05 were considered significant. Spearmann correlation tests were performed between the cognitive tests, which differed significantly between patients and controls, and lesions' i) number ii) volume iii) T1 relaxation time iv) disease duration and v) years of study. Results: Cortical and sub-cortical lesions count, T1 values and volume are reported in Table 1 (A and B). All early RRMS patients showed cortical lesions (CLs) and the majority consisted of CLs type I (lesions with a cortical component extending to the sub-cortical tissue). The rest of cortical lesions were characterized as type II (intra-cortical lesions). No type III/IV lesions (large sub-pial lesions) were detected. RRMS patients were slightly less educated (13.5±2.5y vs. 16.3±1.8y of study, p=0.02) than the controls. Signs of cortical dysfunction (i.e. impaired learning, language, visuo-spatial skills or gnosis) were rare in all patients. However, patients showed on average lower scores on measures of visual attention and information processing speed (TMT-part A: p=0.01; TMT-part B: p=0.006; PASAT-included in the BRB-N: p=0.04). The T1 relaxation values of CLs type I negatively correlated with the TMT-part A score (r=0.78, p<0.01). The correlations of TMT-part B score and PASAT score with T1 relaxation time of lesions as well and the correlation between TMT-part A, TMT-part B and PASAT score with lesions' i) number ii) volume iii) disease duration and iv) years of study did not reach significance. In order to preclude possible influences from partial volume effects on the T1 values, the correlation between lesion volume and T1 value of CLs type I was calculated; no correlation was found, suggesting that partial volume effects did not affect the statistics. Conclusions: The present pilot study reports for the first time the presence and the T1 characteristics at 3 T of cortical lesions in very early RRMS (< 6 y disease duration). It also shows that CLS type I represents the most frequent cortical lesion type in this cohort of RRMS patients. In addition, it reveals a negative correlation between the attentional test TMT-part A and the T1 properties of cortical lesions type I. In other words, lower attention deficits are concomitant with longer T1-relaxation time in cortical lesions. In respect to this last finding, it could be speculated that long relaxation time correspond to a certain degree of tissue loss that is enough to stimulate compensatory mechanisms. This hypothesis is in line with previous fMRI studies showing functional compensatory mechanisms to help maintaining normal or sub-normal attention performances in RR MS patients (Penner, 2003).

Lesion Recognition in Multiple Sclerosis: A Sequence Comparison and Quantification Study at 3T

Introduction Lesion detection in multiple sclerosis (MS) is an essential part of its clinical diagnosis. In addition, radiological characterisation of MS lesions is an important research field that aims at distinguishing different MS types, monitoring drug response and prognosis. To date, various MR protocols have been proposed to obtain optimal lesion contrast for early and comprehensive diagnosis of the MS disease. In this study, we compare the sensitivity of five different MR contrasts for lesion detection: (i) the DIR sequence (Double Inversion Recovery, [4]), (ii) the Dark-fluid SPACE acquisition schemes, a 3D variant of a 2D FLAIR sequence [1], (iii) the MP2RAGE [2], an MP-RAGE variant that provides homogeneous T1 contrast and quantitative T1-values, and the sequences currently used for clinical MS diagnosis (2D FLAIR, MP-RAGE). Furthermore, we investigate the T1 relaxation times of cortical and sub-cortical regions in the brain hemispheres and the cerebellum at 3T. Methods 10 early-stage female MS patients (age: 31.64.7y; disease duration: 3.81.9y; disability score, EDSS: 1.80.4) and 10 healthy controls (age and gender-matched: 31.25.8y) were included in the study after obtaining informed written consent according to the local ethic protocol. All experiments were performed at 3T (Magnetom Trio a Tim System, Siemens, Germany) using a 32-channel head coil [5]. The imaging protocol included the following sequences, (all except for axial FLAIR 2D with 1x1x1.2 mm3 voxel and 256x256x160 matrix): DIR (TI1/TI2/TR XX/3652/10000 ms, iPAT=2, TA 12:02 min), MP-RAGE (TI/TR 900/2300 ms, iPAT=3, TA 3:47 min); MP2RAGE (TI1/TI2/TR 700/2500/5000 ms, iPAT=3, TA 8:22 min, cf. [2]); 3D FLAIR SPACE (only for patient 4-6, TI/TR 1800/5000 ms, iPAT=2, TA=5;52 min, cf. [1]); Axial FLAIR (0.9x0.9x2.5 mm3, 256x256x44 matrix, TI/TR 2500/9000 ms, iPAT=2, TA 4:05 min). Lesions were identified by two experienced neurologist and radiologist, manually contoured and assigned to regional locations (s. table 1). Regional lesion masks (RLM) from each contrast were compared for number and volumes of lesions. In addition, RLM were merged in a single "master" mask, which represented the sum of the lesions of all contrasts. T1 values were derived for each location from this mask for patients 5-10 (3D FLAIR contrast was missing for patient 1-4). Results & Discussion The DIR sequence appears the most sensitive for total lesions count, followed by the MP2RAGE (table 1). The 3D FLAIR SPACE sequence turns out to be more sensitive than the 2D FLAIR, presumably due to reduced partial volume effects. Looking for sub-cortical hemispheric lesions, the DIR contrast appears to be equally sensitive to the MP2RAGE and SPACE, but most sensitive for cerebellar MS plaques. The DIR sequence is also the one that reveals cortical hemispheric lesions best. T1 relaxation times at 3T in the WM and GM of the hemispheres and the cerebellum, as obtained with the MP2RAGE sequence, are shown in table 2. Extending previous studies, we confirm overall longer T1-values in lesion tissue and higher standard deviations compared to the non-lesion tissue and control tissue in healthy controls. We hypothesize a biological (different degree of axonal loss and demyelination) rather than technical origin. Conclusion In this study, we applied 5 MR contrasts including two novel sequences to investigate the contrast of highest sensitivity for early MS diagnosis. In addition, we characterized for the first time the T1 relaxation time in cortical and sub-cortical regions of the hemispheres and the cerebellum. Results are in agreement with previous publications and meaningful biological interpretation of the data.

Testing for anaplastic lymphoma kinase rearrangement to target crizotinib therapy: oncology, pathology and health economic perspectives.

Crizotinib is a first-in-class oral anaplastic lymphoma kinase (ALK) inhibitor targeting ALK-rearranged non-small-cell lung cancer. The therapy was approved by the US FDA in August 2011 and received conditional marketing approval by the European Commission in October 2012 for advanced non-small-cell lung cancer. A break-apart FISH-based assay was jointly approved with crizotinib by the FDA. This assay and an immunohistochemistry assay that uses a D5F3 rabbit monoclonal primary antibody were also approved for marketing in Europe in October 2012. While ALK rearrangement has relatively low prevalence, a clinical benefit is exhibited in more than 85% of patients with median progression-free survival of 8-10 months. In this article, the authors summarize the therapy and alternative test strategies for identifying patients who are likely to respond to therapy, including key issues for effective and efficient testing. The key economic considerations regarding the joint companion diagnostic and therapy are also presented. Given the observed clinical benefit and relatively high cost of crizotinib therapy, companion diagnostics should be evaluated relative to response to therapy versus correlation alone whenever possible, and both high inter-rater reliability and external quality assessment programs are warranted.

Astrocyte dense-core vesicle gliosecretion is governed by rest/nrsf

Astrocytes are the brain non-nerve cells competent for the expression of clear and dense-core vesicles (DCVs) and for their regulated exocytosis. This process, called gliosecretion, nearly resembles the neurosecretion occurring in neurons and neurosecretory cells. REST/NRSF is a transcription repressor known to orchestrate nerve-cell differentiation, governing the expression of hundreds of neuron-specific genes through their repression in the non-nerve and their fine modulation in the nerve cells. Our previous studies in neurosecretory rat PC12 cells identified REST as the critical factor for the expression not only of individual genes, but also of the whole neurosecretory process via multiple, direct and indirect mechanisms (D'Alessandro et al., J. Neurochem., 2008; Klajn et al., J. Neurosci., 2009). Therefore we wondered whether gliosecretion was governed by REST. We investigated rat astrocyte primary cultures: they exhibited high REST, which directly represses the transcription of at least one target gene, and expressed neither DCVs nor their markers (granins, peptides, membrane proteins). Transfection of a dominant-negative construct of REST (REST/ DBD-GFP) induced the appearance of DCVs filled with secretogranin2 and NPY that are distinct from other intracellular organelles. TIRF analysis of astrocytes co-transfected with REST/DBD-GFP and NPY-mRFP constructs revealed NPY-mRFP-positive DCVs undergoing Ca2þ-dependent exocytosis, largely prevented by BoNT/B. Immunohistochemistry of the I-II layers of the human temporal brain cortex showed all neurons and microglia exhibiting the expected inappreciable and high levels of REST, respectively. In contrast astrocyte RESTwas variable, going from inappreciable to high, accompanied by variable expression of DCVs. In this work it has been demonstrated that astrocyte DCV expression and gliosecretion are governed by REST (Prada et al., 2011 in press). The variable in situ REST levels may contribute to the well known structural/functional heterogeneity of astrocytes and this new observation might be of great interest for the understanding of both astrocyte physiology and pathology.

Altruism across disciplines: one word, multiple meanings

Altruism is a deep and complex phenomenon that is analysed by scholars of various disciplines, including psychology, philosophy, biology, evolutionary anthropology and experimental economics. Much confusion arises in current literature because the term altruism covers variable concepts and processes across disciplines. Here we investigate the sense given to altruism when used in different fields and argumentative contexts. We argue that four distinct but related concepts need to be distinguished: (a) psychological altruism, the genuine motivation to improve others' interests and welfare; (b) reproductive altruism, which involves increasing others' chances of survival and reproduction at the actor's expense; (c) behavioural altruism, which involves bearing some cost in the interest of others; and (d) preference altruism, which is a preference for others' interests. We show how this conceptual clarification permits the identification of overstated claims that stem from an imprecise use of terminology. Distinguishing these four types of altruism will help to solve rhetorical conflicts that currently undermine the interdisciplinary debate about human altruism.