Neutralizing antibodies in Multiple Sclerosis a model-based analysis of Interferon beta signaling pathway in macrophages

Multiple Sclerosis is the most common non-traumatic cause of neurologicaldisability in young people. There is no cure yet, and until recently, few long-termtherapies existed. Interferon beta (IFNβ) was the first treatment, and remains the mostcommonly prescribed. One of the most significant problems of IFNβ therapy is theproduction of drug specific antibodies. Up to 45% of patients develop neutralizingantibodies (NAbs) to IFNβ products. The neutralizing antibody binds to the biologicalagent preventing its interaction with its receptor, inhibiting the biological action of theprotein, which abrogates the clinical efficacy of IFNβ treatment. Interferon-betamediates its response by binding to its high affinity cell surface receptor and initiatingthe JAK/STAT signalling cascade. In this project we have analyzed the IFNβ signalingpathway in macrophages when neutralizing antibodies are present. The response tothis pathway after IFNβ stimulation shows a transient oscillatory rhythm of STAT1phosphorylation, which varies as NAbs concentration increases. To improve ourunderstanding of that behavior, we extended an existing mathematical model based onnonlinear ordinary differential equations of JAK/STAT pathway by including IFN-NAbassociation and IFN-activation receptor. Combining our theoretical model withexperimental data we could study the role of neutralizing antibodies on the molecularresponse and determine its lifetime after cytokine stimulation.

Non-classical karyotypic features in relapsed childhood B-cell precursor acute lymphoblastic leukemia.

Karyotype analysis of acute lymphoblastic leukemia (ALL) at diagnosis has provided valuable prognostic markers for treatment stratification. However, reports of cytogenetic studies of relapsed ALL samples are limited. We compared the karyotypes from 436 nonselected B-cell precursor ALL patients at initial diagnosis and of 76 patients at first relapse. We noticed a relative increase of karyotypes that did not fall into the classic ALL cytogenetic subgroups (high hyperdiploidy, t(12;21), t(9;22), 11q23, t(1;19), <45 chromosomes) in a group of 29 patients at relapse (38%) compared to 130 patients at presentation (30%). Non-classical cytogenetic aberrations in these 29 patients were mostly found on chromosomes 1, 2, 7, 9, 13, 14, and 17. We also describe six rare reciprocal translocations, three of which involved 14q32. The most frequent abnormalities were found in 9p (12/29 cases) and were associated with a marked decrease in the duration of the second remission, but not of the probability of 10-year event-free survival after relapse treatment. From 29 patients with non-classical cytogenetic aberrations, only 8 (28%) had been stratified to a high risk-arm on the first treatment protocol, suggesting that this subgroup might benefit from the identification of new prognostic markers in future studies.

Numerous potentially functional but non-genic conserved sequences on human chromosome 21.

The use of comparative genomics to infer genome function relies on the understanding of how different components of the genome change over evolutionary time. The aim of such comparative analysis is to identify conserved, functionally transcribed sequences such as protein-coding genes and non-coding RNA genes, and other functional sequences such as regulatory regions, as well as other genomic features. Here, we have compared the entire human chromosome 21 with syntenic regions of the mouse genome, and have identified a large number of conserved blocks of unknown function. Although previous studies have made similar observations, it is unknown whether these conserved sequences are genes or not. Here we present an extensive experimental and computational analysis of human chromosome 21 in an effort to assign function to sequences conserved between human chromosome 21 (ref. 8) and the syntenic mouse regions. Our data support the presence of a large number of potentially functional non-genic sequences, probably regulatory and structural. The integration of the properties of the conserved components of human chromosome 21 to the rapidly accumulating functional data for this chromosome will improve considerably our understanding of the role of sequence conservation in mammalian genomes.

Is use of nifurtimox for the treatment of Chagas disease compatible with breast feeding? A population pharmacokinetics analysis.

Introduction Women with Chagas disease receiving treatment with nifurtimox are discouraged from breast feeding. Many patients who would receive treatment with nifurtimox live in extreme poverty, have limited access to resources such as clean water and baby formula and may not have safe alternatives to breast milk. Aim We aimed to estimate, using limited available pharmacokinetics data, potential infant exposure to nifurtimox through breast milk. Methods Original nifurtimox plasma concentrations were obtained from published studies. Pharmacokinetic parameters were estimated using non-linear mixed-effect modelling with NONMEM V.VI. A total of 1000 nifurtimox plasma-concentration profiles were simulated and used to calculate the amount of drug that an infant would be exposed to, if breast fed 150 ml/kg/day. Results Breast milk concentrations on the basis of peak plasma levels (1361 ng/ml) and milk-plasma ratio were estimated. We calculated infant nifurtimox exposure of a breastfed infant of a mother treated with this drug to be below 10% of the maternal weight-adjusted dose, even if milk-plasma ratio were overestimated. Simulation led to similar estimates. Discussion Risk for significant infant exposure to nifurtimox through breast milk seems small and below the level of exposure of infants with Chagas disease receiving nifurtimox treatment. This potential degree of exposure may not justify discontinuation of breast feeding.

A simple score for estimating ischemic heart disease in patients with non-traumatic chest pain in primary care

Background: Modelling epidemiological knowledge in validated clinical scores is a practical mean of integrating EBM to usual care. Existing scores about cardiovascular disease have been largely developed in emergency settings, but few in primary care. Such a toll is needed for general practitioners (GP) to evaluate the probability of ischemic heart disease (IHD) in patients with non-traumatic chest pain. Objective: To develop a predictive model to use as a clinical score for detecting IHD in patients with non-traumatic chest-pain in primary care. Methods: A post-hoc secondary analysis on data from an observational study including 672 patients with chest pain of which 85 had IHD diagnosed by their GP during the year following their inclusion. Best subset method was used to select 8 predictive variables from univariate analysis and fitted in a multivariate logistic regression model to define the score. Reliability of the model was assessed using split-group method. Results: Significant predictors were: age (0-3 points), gender (1 point), having at least one cardiovascular risks factor (hypertension, dyslipidemia, diabetes, smoking, family history of CVD; 3 points), personal history of cardiovascular disease (1 point), duration of chest pain from 1 to 60 minutes (2 points), substernal chest pain (1 point), pain increasing with exertion (1 point) and absence of tenderness at palpation (1 point). Area under the ROC curve for the score was of 0.95 (IC95% 0.93; 0.97). Patients were categorised in three groups, low risk of IHD (score under 6; n = 360), moderate risk of IHD (score from 6 to 8; n = 187) and high risk of IHD (score from 9-13; n = 125). Prevalence of IHD in each group was respectively of 0%, 6.7%, 58.5%. Reliability of the model seems satisfactory as the model developed from the derivation set predicted perfectly (p = 0.948) the number of patients in each group in the validation set. Conclusion: This clinical score based only on history and physical exams can be an important tool in the practice of the general physician for the prediction of ischemic heart disease in patients complaining of chest pain. The score below 6 points (in more than half of our population) can avoid demanding complementary exams for selected patients (ECG, laboratory tests) because of the very low risk of IHD. Score above 6 points needs investigation to detect or rule out IHD. Further external validation is required in ambulatory settings.

Genome-wide analysis of Sphingomonas wittichii RW1 behaviour during inoculation and growth in contaminated sand.

The efficacy of inoculation of single pure bacterial cultures into complex microbiomes, for example, in order to achieve increased pollutant degradation rates in contaminated material (that is, bioaugmentation), has been frustrated by insufficient knowledge on the behaviour of the inoculated bacteria under the specific abiotic and biotic boundary conditions. Here we present a comprehensive analysis of genome-wide gene expression of the bacterium Sphingomonas wittichii RW1 in contaminated non-sterile sand, compared with regular suspended batch growth in liquid culture. RW1 is a well-known bacterium capable of mineralizing dibenzodioxins and dibenzofurans. We tested the reactions of the cells both during the immediate transition phase from liquid culture to sand with or without dibenzofuran, as well as during growth and stationary phase in sand. Cells during transition show stationary phase characteristics, evidence for stress and for nutrient scavenging, and adjust their primary metabolism if they were not precultured on the same contaminant as found in the soil. Cells growing and surviving in sand degrade dibenzofuran but display a very different transcriptome signature as in liquid or in liquid culture exposed to chemicals inducing drought stress, and we obtain evidence for numerous 'soil-specific' expressed genes. Studies focusing on inoculation efficacy should test behaviour under conditions as closely as possible mimicking the intended microbiome conditions.

Isothermal microcalorimetry for antifungal susceptibility testing of Mucorales, Fusarium spp., and Scedosporium spp.

We evaluated isothermal microcalorimetry for real-time susceptibility testing of non-Aspergillus molds. MIC and minimal effective concentration (MEC) values of Mucorales (n = 4), Fusarium spp. (n = 4), and Scedosporium spp. (n = 4) were determined by microbroth dilution according to the Clinical Laboratory Standard Institute M38-A2 guidelines. Heat production of molds was measured at 37 °C in Sabouraud dextrose broth inoculated with 2.5 × 10(4) spores/mL in the presence of amphotericin B, voriconazole, posaconazole, caspofungin, and anidulafungin. As determined by microcalorimetry, amphotericin B was the most active agent against Mucorales (MHIC 0.06-0.125 μg/mL) and Fusarium spp. (MHIC 1-4 μg/mL), whereas voriconazole was the most active agent against Scedosporium spp. (MHIC 0.25 to 8 μg/mL). The percentage of agreement (within one 2-fold dilution) between the MHIC and MIC (or MEC) was 67%, 92%, 75%, and 83% for amphotericin B, voriconazole, posaconazole, and caspofungin, respectively. Microcalorimetry provides additional information on timing of antifungal activity, enabling further investigation of drug-mold and drug-drug interaction, and optimization of antifungal treatment.

Multiway Array Decomposition Analysis of EEGs in Alzheimer’s Disease

Methods for the extraction of features from physiological datasets are growing needs as clinical investigations of Alzheimer’s disease (AD) in large and heterogeneous population increase. General tools allowing diagnostic regardless of recording sites, such as different hospitals, are essential and if combined to inexpensive non-invasive methods could critically improve mass screening of subjects with AD. In this study, we applied three state of the art multiway array decomposition (MAD) methods to extract features from electroencephalograms (EEGs) of AD patients obtained from multiple sites. In comparison to MAD, spectral-spatial average filter (SSFs) of control and AD subjects were used as well as a common blind source separation method, algorithm for multiple unknown signal extraction (AMUSE). We trained a feed-forward multilayer perceptron (MLP) to validate and optimize AD classification from two independent databases. Using a third EEG dataset, we demonstrated that features extracted from MAD outperformed features obtained from SSFs AMUSE in terms of root mean squared error (RMSE) and reaching up to 100% of accuracy in test condition. We propose that MAD maybe a useful tool to extract features for AD diagnosis offering great generalization across multi-site databases and opening doors to the discovery of new characterization of the disease.

On Automatic Diagnosis of Alzheimer's Disease based on Spontaneous Speech Analysis and Emotional Temperature

Alzheimer's disease is the most prevalent form of progressive degenerative dementia; it has a high socio-economic impact in Western countries. Therefore it is one of the most active research areas today. Alzheimer's is sometimes diagnosed by excluding other dementias, and definitive confirmation is only obtained through a post-mortem study of the brain tissue of the patient. The work presented here is part of a larger study that aims to identify novel technologies and biomarkers for early Alzheimer's disease detection, and it focuses on evaluating the suitability of a new approach for early diagnosis of Alzheimer’s disease by non-invasive methods. The purpose is to examine, in a pilot study, the potential of applying Machine Learning algorithms to speech features obtained from suspected Alzheimer sufferers in order help diagnose this disease and determine its degree of severity. Two human capabilities relevant in communication have been analyzed for feature selection: Spontaneous Speech and Emotional Response. The experimental results obtained were very satisfactory and promising for the early diagnosis and classification of Alzheimer’s disease patients.

Molecular Analysis of the PTEN Gene in a Choroidal Schwannoma in the Context of a Hamartomatous Syndrome

Purpose: To investigate the molecular involvement of PTEN, a tumor suppressor gene, in a case of cellular pigmented choroidal Schwannoma in a patient with hamartomatous syndrome due to heterozygous PTEN germline mutation. Methods: Histopathological, immunohistochemical, and electron microscopy analyses were performed by standard procedures. Paraffin-embedded samples of normal and tumor eye tissues were collected and DNA was extracted. A 145 bp region flanking the heterozygous c.406T>C mutation in exon 5 of PTEN was amplified by PCR and sequenced. To evaluate the allelic status of PTEN in the tumor sample, we cloned different PCR products in E. coli using a TA cloning procedure. Results: Histopathology demonstrated a posterior choroidal mass measuring 1.3 x 1.6 x 1.4 cm. The tumor was composed by fascicles of spindle cells with wavy cytoplasm. No Verrocay bodies could be identified. Scattered histiocytes with clear cytoplasm were present. By immunohistochemistry, the cells were expressing S100 and focally Melan A proteins. Pericellular type IV collagen could be demonstrated. Interlacing cytoplasmic processes covered by thick basement membrane could be found by electron microscopy as well as few premelanosomes. Moderate PTEN expression by immunohistochemistry was identified in some cells. As expected, the germline mutation could be detected by DNA sequencing in both the paraffin-embedded normal and tumor eye tissues. Analysis of 33 E. coli colonies bearing clones from the tumor eye tissue DNA surprisingly revealed that most of them contained the PTEN wild-type allele (29 vs. 4, Fisher's test p-value = 0.002). Conclusions: This is the first reported case of choroidal cellular Schwannoma arising in the context of a PTEN hamartomatous syndrome. Allelic analysis of PTEN in the tumor suggests a statistically-significant partial loss of heterozygozity in favor of the wild-type allele. Our findings are in clear contrast with what is usually observed in cancer tissues, for which mutated alleles of tumor suppressor genes are usually brought to homozygosity. Similar results were previously reported in human non-Hodgkin's lymphomas, displaying an overexpression of the wild-type form of the tumor suppressor gene p53. We are in the process of investigating additional DNA derived from other fresh and paraffin-embedded tissues from the patient, in order to gain insights on the molecular bases of PTEN involvement in this rare choroidal Schwannoma.

Are myotonic dystrophy and peripheral neuropathy associated? : morphological, morphometric and electrophysiological analysis in DM1 transgenic mice

Abstract: Myotonic dystrophy (DM1), also known as Steinert disease, is an inherited autosomal dominant disease. It is characterized by myotonia, muscular weakness and atrophy, but DM1 may have manifestations in other organs such as eyes, heart, gonads, gastrointestinal and respiratory tracts, as well as brain. In 1992, it was demonstrated that this complex disease results from the expansion of CTG repeats in the 3' untranslated region of the DM protein kinase (DMPK) gene on chromosome 19. The size of the inherited expansion is critically linked to the severity of the disease and the age of onset. Although several electrophysiological and histological studies have been carried out to verify the possible involvement of peripheral nerve abnormality with DM1, the results have not been univocal. Therefore, at present the possible association between peripheral neuropatliy and DM1 remains debated. Recently, transgenic mice have been generated, that carry the human genomic DM1 region with 300 CTG repeats, and display the human DMl phenotype. The generation of these DM1 transgenic mice provides a useful tool to investigate the type and incidence of structural abnormalities in the peripheral nervous system associated with DM1 disease. By using the DM1 transgenic mice, we investigated the presence/absence of the three major peripheral neuropathies: axonal degeneration, axonal demyelination and neuronopathy. The morphological and morphometric analysis of sciatic, sural and phrenic nerves demonstrated the absence of axonal degeneration or demyelination. The morphometric analysis also ruled out any loss in the numbers of sensory or motor neurons in lumbar dorsal root ganglia and lumbar spinal cord enlargement respectively. Moreover, the éxamination of serial hind limb muscle sections from DMl mice showed a normal intramuscular axonal arborization as well as the absence of changes in the number and structure of endplates. Finally, the electrophysiological tests performed in DM1 transgenic mice showed that the compound muscle axon potentials (CMAPs) elicited in the hind limb digits in response to a stimulation of the sciatic nerve with anear-nerve electrode were similar to thosé obtained in wild type mice. On the basis of all our results, we hypothesized that 300 CTG repeats are not sufficient to induce disorder in the peripheral nervous system of this DM1 transgenic mouse model. Résumé La dystrophie myotonique (DM1), connue aussi sous le nom de maladie de Steinert, est une maladie héréditaire autosornale dominante. Elle est caractérisée par une myotonie, une faiblesse et une atrophie musculaires, mais peut aussi se manifester dans d'autres organes tels que les yeux, les voies digestive et respiratoire, ou le cerveau. En 1992, il a été montré que cette maladie complexe résultait de l'expansion d'une répétition de CTG dans une partie non traduite en 3' du gène codant pour la protéine kinase DM (DMPK), sur le chromosome 19. La taille de l'expansion héritée est étroitement liée à la sévérité et l'âge d'apparition de DM1. Bien que plusieurs études électrophysiologiques et histologiques aient été menées, pour juger d'une implication possible d'anomalies au niveau du système nerveux périphérique dans la DM1, les résultats n'ont jusqu'ici pas été univoques. Aujourd'hui, la question d'une neuropathie associée avec la DM1 reste donc controversée. Des souris transgéniques ont été élaborées, qui portent la séquence DM1 du génome humain avec 300 répétitions CTG et expriment le phénotype des patients DM1: Ces souris transgéniques DMl procurent un outil précieux pour l'étude du type et de l'incidence d'éventuelles anomalies du système nerveux périphérique dans la DM1. En utilisant ces souris transgéniques DM1, nous avons étudié la présence ou l'absence des trois principaux types de neuropathies périphériques: la dégénération axonale, la démyélinisation axonale et la neuronopathie. Les études morphologiques et morphométrique des nerfs sciatiques, suraux et phréniques ont montré l'absence de dégénération axonale ou de démyélinisation. L'analyse du nombre de cellules neuronales n'a pas dévoilé de diminution des nombres de neurones sensitifs dans les ganglions des racines dorsales lombaires ou de neurones moteurs dans la moëlle épinière lombaire des souris transgéniques DMl. De plus, l'examen de coupes sériées de muscle des membres postérieurs de souris DM1 a montré une arborisation axonale intramusculaire normale, de même que l'absence d'irrégularité dans le nombre ou la structure des plaques motrices. Enfin, les tests électrophysiologiques effectués sur les souris DMl ont montré que les potentiels d'action de la composante musculaire (CMAPs) évoqués dans les doigts des membres postérieurs, en réponse à une stimulation du nerf sciatique à l'aide d'une électrode paranerveuse, étaient identiques à ceux observées chez les souris sauvages. Sur la base de l'ensemble de ces résultats, nous avons émis l'hypothèse que 300 répétitions CTG ne sont pas suffisantes pour induire d'altérations dans le système nerveux périphérique du modèle de souris transgéniques DM 1.