Immunological fingerprint of 4CMenB recombinant antigens via protein microarray reveals key immunosignatures correlating with bactericidal activity.

Serogroup B meningococcus (MenB) is a leading cause of meningitis and sepsis across the world and vaccination is the most effective way to protect against this disease. 4CMenB is a multi-component vaccine against MenB, which is now licensed for use in subjects >2 months of age in several countries. In this study, we describe the development and use of an ad hoc protein microarray to study the immune response induced by the three major 4CMenB antigenic components (fHbp, NHBA and NadA) in individual sera from vaccinated infants, adolescents and adults. The resulting 4CMenB protein antigen fingerprinting allowed the identification of specific human antibody repertoire correlating with the bactericidal response elicited in each subject. This work represents an example of epitope mapping of the immune response induced by a multicomponent vaccine in different age groups with the identification of protective signatures. It shows the high flexibility of this microarray based methodology in terms of high-throughput information and minimal volume of biological samples needed.

Drug survival and reasons for discontinuation of the first course of biological therapy in 301 juvenile idiopathic arthritis patients.

The objective of this study was to determine long-term effectiveness and safety of 1st biological treatment (BT) in a cohort of 301 juvenile idiopathic arthritis (JIA) patients (pts), non-responders to disease-modifying antirheumatic drugs (DMARDs), in terms of drug survival (continuation rate on therapy) and to identify the baseline predictors of treatment discontinuation. Each JIA pt enrolled in BT is prospectively assessed at the start of treatment and then every 2 months for the evaluation of safety and efficacy according to ACR-Pedi30 criteria. All clinical charts of pts who started a BT between November 1999 and July 2010 have been reviewed. Survival analysis methods suitable for competing risks were used to study time to drug discontinuation due to disease control (defined according to Wallace criteria) or failure [adverse event (AE), lack of efficacy (LaE) or loss of efficacy (LoE) according ACR-Pedi30]. A number of 301 JIA pts, non-responders or intolerant to DMARDs and treated with one or more cycles of BT, were identified. Median disease duration, from onset to the beginning of 1st BT, was 7.8 years (interquartil range 2.21-15.1). In total, there were 294 1st corses with anti-TNF agents, 5 with abatacept and 2 with anakinra. A number of 298 pts were included in the analysis for drug discontinuation (3 pts with no follow-up data after their first dose of BT were excluded). The median follow-up on treatment, before discontinuation due to every cause, was 53.7 months (range 0.45-124.45). One hundred and sixty-five pts discontinued BT: 27 due to disease control, 135 because of failure (78 AEs, 12 LaE and 45 LoE), 3 pts temporarily stopped for pregnancy. Among 135 pts who discontinued for failure, 117 switched to a 2nd BT. Among 27 pts who discontinued due to remission, 13 pts restarted on BT for relapse of disease activity (10 pts restarted with the same BT, 3 switched to a different one). Predictors of discontinuation due to AE were female gender (P=0.01) and longer disease duration (P=0.02). Predictors of discontinuation due to LaE or LoE were systemic onset and polyarthritis FR positive (vs other JIA subtypes) (P<0.05) and use of mAb-anti-TNF (vs sTNFR) (P=0.02). Predictors of discontinuation due to inactive disease were male gender and shorter disease duration (P<0.05). Although only few pts discontinued BT due to a complete and persistent disease control, the majority of them remained on BT for a long time, suggesting that in our cohort of JIA pts, affected by a severe long lasting refractory disease, BT was globally well tolerate and efficacious in controlling the disease.

[Treatment of eating disorders during hospitalization: presentation of a hospital intensive care program in pediatric age]. / Gestione dei disturbi del comportamento alimentare durante il ricovero: presentazione di un programma di terapia intensiva ospedaliera in eta pediatrica.

AIM: The aim of this study was to present a hospital intensive care program for patients affected by a severe eating disorders, with a significant loss of weight (BMI<15 and weight less than 25° centile), severe medical complications (orthostatic hypotension, bradycardia <40 bpm or tachycardia >110 bpm or inability to sustain core body temperature), abnormal laboratory data, especially electrolyte imbalance and refusal to take food and fluids. METHODS: In our study we reported 2 year follow-up of 16 patients treated with the hospital intensive care program between 2007 and 2008 in our department. RESULT: The proposed program was proved an efficient method in a critical phase of the alimentary behavior disorders. It was possible for all the patients to avoid alternative feeding techniques (enteral or parenteral) and to obtain a correct alimentation with a satisfactory improvement of clinical conditions. Eight patients (50%) fully recovered. 5 patients (31.25%) had a significant improvement reaching a BMI>18.5 and one of them had a regular menstrual cycle, too. However in this group of patients a strict modality to alimentation and concern about weight and physical appearance remain. In 3 patients (18.5%) the BMI is still low and amenorrhea persists. CONCLUSIONS: The hospital intensive care program, inspired by the cognitive-behavioral model, through a food rehabilitation and a psychotherapeutic and psychoeducational help, lets the patients and their family understand and modify the dysfunctional patterns, experimenting a right modality to approach alimentation, with a satisfactory improvement in clinical conditions.

Deep sequencing in pre- and clinical vaccine research.

Vaccine research has experienced a quantum leap after the beginning of the genomics era. High-throughput sequencing techniques, unlimited computing resources, as well as new bioinformatic algorithms are now changing the way we perform genomic studies. Whole genome sequencing will soon become the gold standard for phylogenetic and epidemiology studies and is already shedding new light on the dynamics of bacterial evolution. We believe that deep sequencing projects, together with structural studies on vaccine candidates, will allow targeting constant epitopes and avoid vaccine failure due to antigenic variability. Systems biology, which is expected to revolutionize vaccine research and clinical studies, greatly relies on high-throughput technologies such as RNA-seq. Furthermore, genomics is a key element to develop safer vaccines, and the accuracy of deep sequencing will allow monitoring vaccine coverage after their introduction on the market.

EUROPLAN: a project to support the development of national plans on rare diseases in Europe.

BACKGROUND/AIMS: National Plans for Rare Diseases (RDs) are the common denominator of current public health policy concerns on RDs across the EU. With the aim of a better distribution of the available resources, they conjugate the European objective that aims at ensuring that patients with RDs have access to high-quality care - including diagnostics, treatment and rehabilitation - with the national priorities of selecting specific measures for adoption and implementation. METHODS: The European Project for Rare Diseases National Plans Development (EUROPLAN, www.europlanproject.eu) is cofunded by the EU Commission (DG-SANCO) and is coordinated by the Italian National Center for Rare Diseases of the Istituto Superiore di Sanità (ISS). The EUROPLAN goal is to promote the implementation of National Plans or Strategies to tackle RDs and share relevant experiences within countries, linking national efforts, through a common strategy at a European level. In order to fulfill these objectives, EUROPLAN involved health authorities, clinicians, scientists, the European Organisation for Rare Diseases (EURORDIS), and many other patient groups as associated and collaborating partners from several European countries. RESULTS: The project was launched in 2008 and foresaw 2 implementation phases: phase 1 (2008-2011) to build the consensus definition of operational tools (recommendations and indicators), and the ongoing phase 2 (2012-2015), mainly aimed at capacity building with the proactive involvement of multilevel stakeholders. EUROPLAN is facilitating and accelerating the implementation of National Plans in almost all EU and several non-EU Countries. CONCLUSIONS: EUROPLAN is a European and an international process more than a project, and it could be defined as a 'litmus test' demonstrating how the collaboration between institutions and patients' associations can accelerate the process of awareness and development of policies and actions.

A model for the European platform for rare disease registries.

BACKGROUND: The current situation of rare disease (RD) registries is rather heterogeneous, and new ways to support the registration of RD patients are being sought in the European Union (EU) and the US. The project 'Building Consensus and Synergies for the EU Registration of RD Patients', funded by the EU, aimed to define a model platform for EU RD registries. METHODS: A number of surveys and extensive consultations among registry stakeholders have been carried out to study how the platform can best fulfill their needs. RESULTS: This web-based, multidisease and multipurpose platform is intended to provide a number of functions: a metadata and data repository function supporting the planning of research studies and the production of predefined outputs for the funding organizations and the public, provision of tools and resources of use to registries, promotion of registration and networking among patients and professionals. CONCLUSION: Its main impact is expected to be on data and procedures standardization, on the establishment of new registries, on the sustainability of the smaller ones, and on the registration of those RDs for which a dedicated registry is not sustainable, e.g. ultra-rare diseases or diseases for which there is no special research, clinical or economic interest. It will also impact on the production of sounder information on RD and RD-dedicated health systems, by promoting registry data comparability and quality.

Primary Sjögren syndrome: report of a 10 years old girl with local edema and positivity of anti SS-A and anti SS-B autoantibodies.

Sjogren-Larsson syndrome (SLS) is an autoimmune disease, uncommon in childhood. We report a case of SLS in a 10-year-old girl with a history of tumor, calor and rubor in the back of her toes almost every month, which resolved in 4-5 days without therapy. She did not complain of dry mouth or dry eyes. The laboratory findings showed high inflammation markers, rheumatoid factor 128 IU, Waaler-Rose 256 IU, anti nuclear antibody (ANA) 1/640, SSA (anti Sjogren antigen A) and SSB (anti Sjogren antigen B) positive and hypergammaglobulinemia. The Schirmer's test resulted to be pathologic, the ultrasonography images and biopsy of minor salivary glands revealed focal periductal lymphocytic infiltrate and sialoduct ectasia class IV of juvenile Sjogren syndrome. The juvenile Sjogren syndrome is frequently under-diagnosed. Clinical manifestations in children might be different from the adult form, although laboratory findings may be similar to those found in adults.

The sphingosine kinase activator K6PC-5 stimulates C2C12 myoblast differentiation.

Previously, K6PC-5, a synthetic derivative of ceramide, was demonstrated to activate sphingosine kinase (SK)-1 in keratinocytes. In this study its potential biological effect in mouse myoblasts was examined. The obtained results show that K6PC-5 promotes myogenic differentiation by enhancing myogenic marker expression, differentiation index and fusion index. Interestingly, its biological action was prevented by pharmacological inhibition of SK or S1P2 receptor, in full agreement with their recognized role in myoblast differentiation. This is the first evidence that pharmacological activation of SK accelerates myogenesis and suggests that this new therapeutic strategy could be possibly employed in skeletal muscle disorders where muscle regeneration is deficient.

Sphingosine 1-phosphate induces differentiation of adipose tissue-derived mesenchymal stem cells towards smooth muscle cells.

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid which regulates multiple biological parameters in a number of cell types, including stem cells. Here we report, for the first time, that S1P dose-dependently stimulates differentiation of adipose tissue-derived mesenchymal stem cells (ASMC) towards smooth muscle cells. Indeed, S1P not only induced the expression of smooth muscle cell-specific proteins such as alpha-smooth muscle actin (alpha SMA) and transgelin, but also profoundly affected ASMC morphology by enhancing cytoskeletal F-actin assembly, which incorporated alpha SMA. More importantly, S1P challenge was responsible for the functional appearance of Ca(2+) currents, characteristic of differentiated excitable cells such as smooth muscle cells. By employing various agonists and antagonists to inhibit S1P receptor subtypes, S1P(2) turned out to be critical for the pro-differentiating effect of S1P, while S1P(3) appeared to play a secondary role. This study individuates an important role of S1P in AMSC which can be exploited to favour vascular regeneration.

Pleiotropic effects of sphingolipids in skeletal muscle.

Studies of the last two decades have demonstrated that sphingolipids are important signalling molecules exerting key roles in the control of fundamental biological processes including proliferation, differentiation, motility and survival. Here we review the role of bioactive sphingolipids such as ceramide, sphingosine, sphingosine 1-phosphate, ganglioside GM3, in the regulation of skeletal muscle biology. The emerging picture is in favour of a complex role of these molecules, which appear implicated in the activation of muscle resident stem cells, their proliferation and differentiation, finalized at skeletal muscle regeneration. Moreover, they are involved in the regulation of contractile properties, tissue responsiveness to insulin and muscle fiber trophism. Hopefully, this article will provide a framework for future investigation into the field, aimed at establishing whether altered sphingolipid metabolism is implicated in the onset of skeletal muscle diseases and identifying new pharmacological targets for the therapy of multiple illnesses, including muscular dystrophies and diabetes.

Are FPCIs a source of increased risk for children? Results of a multicenter, experimental study comparing children's behaviour with FPCIs and toys.

INTRODUCTION: Food Products Containing Inedibles (FPCIs) are believed to represent a source of higher choking risk in children. The aim of this study was to set up a controlled study, conducted on children aged 3-6 in a laboratory setting, in order to understand their behavior when interacting with FPCIs (with reference to mouthing activities, double nature recognition, and toy assembling ability). METHOD: The experimental phase was divided into two sessions: a FPCI session and a Toy session, to which 247 children were randomly assigned. During these sessions children were observed in order to catch their mouthing activity according to the two types of objects available to them (FPCIs and Toys). RESULTS: This study shows that: (a) children's behavior with respect to toys contained in FPCIs and toys presented alone is not significantly different; (b) children's ability to distinguish between the edible and non-edible part of the FPCI was very high; and (c) mouthing episodes of the inedible parts were negligible and comparable between FPCIs and toys presented alone. This strongly suggests that, with respect to choking risk, FPCIs are not per se distinguishable from toys containing small parts. IMPACT ON INDUSTRY: Restrictions on the sale of FPCIs with small toys exist in the U.S. market. In Europe, FPCIs are allowed to be on sale, under the condition that, in case, they will follow the general regulatory requirements of small toys packaged and sold alone. In this case, they must provide age warnings and labels. Our findings do not justify the different attention that toys in FPCIs are at times afforded by regulators when compared to "stand alone" toys.

Dynamics and configurational entropy in the Lewis-Wahnström model for supercooled orthoterphenyl.

We study thermodynamic and dynamic properties of a rigid model of the fragile glass-forming liquid orthoterphenyl. This model, introduced by Lewis and Wahnström in 1993, collapses each phenyl ring to a single interaction site; the intermolecular site-site interactions are described by the Lennard-Jones potential whose parameters have been selected to reproduce some bulk properties of the orthoterphenyl molecule. A system of N=343 molecules is considered in a wide range of densities and temperatures, reaching simulation times up to 1 micros. Such long trajectories allow us to equilibrate the system at temperatures below the mode coupling temperature T(c) at which the diffusion constant reaches values of order 10(-10) cm(2)/s and thereby to sample in a significant way the potential energy landscape in the entire temperature range. Working within the inherent structures thermodynamic formalism, we present results for the temperature and density dependence of the number, depth and shape of the basins of the potential energy surface. We evaluate the total entropy of the system by thermodynamic integration from the ideal-noninteracting-gas state and the vibrational entropy approximating the basin free energy with the free energy of 6N-3 harmonic oscillators. We evaluate the configurational part of the entropy as a difference between these two contributions. We study the connection between thermodynamical and dynamical properties of the system. We confirm that the temperature dependence of the configurational entropy and of the diffusion constant, as well as the inverse of the characteristic structural relaxation time, are strongly connected in supercooled states; we demonstrate that this connection is well represented by the Adam-Gibbs relation, stating a linear relation between logD and the quantity 1/TS(c). This relation is found to hold both above and below the critical temperature T(c)-as previously found in the case of silica-supporting the hypothesis that a connection exists between the number of basins and the connectivity properties of the potential energy surface.

Relationship of visceral fat distribution to angiographically assessed coronary artery disease: results in subjects with or without diabetes or impaired glucose tolerance.

BACKGROUND AND AIM: To evaluate the relationship between the degree of coronary artery disease (CAD) and the amount of visceral fat deposition in a mixed population of CAD patients with or without diabetes or impaired glucose tolerance (IGT), and with different body weights. METHODS AND RESULTS: A total of 55 patients undergoing coronary angiography (43 men and 12 women with a mean age of 58.9 +/- 1.1 years, range 37-70, and a mean body mass index [BMI] of 27.9 +/- 0.4, range 21.3-38.7) were studied in order to establish whether the coronary damage exclusively depends on intra-abdominal adipose tissue per se, or may be influenced by the coexistence of diabetes or IGT. Twenty-one subjects were non-diabetic, 13 had type 2 diabetes, and 21 IGT. Hypertension was found in 47% and dyslipidemia in 55%; 69% were smokers. The angiographic evaluation of CAD was made using the method of Gensini, and computed tomography (CT) was used to estimate the amount of visceral adipose tissue (VAT) based on a single scan at L4 level. Clinical, anthropometric, biochemical and hormonal variables, as well as smoking and alcohol consumption were determined. In the study population as a whole, the coronary score did not correlate with VAT, but only with smoking. However, both univariate and multivariate regression analysis showed that CAD significantly correlated with VAT in the non-diabetic patients, particularly in those with VAT of > 130 cm2. This correlation did not appear in the diabetic or IGT patients, nor when the group of patients with VAT > 130 cm2 was extended to include diabetic or IGT patients. No relationship was found between CAD and BMI or the other considered variables. CONCLUSIONS: In a mixed population of CAD patients with or without diabetes, CAD correlates with VAT only in the absence of diabetes or IGT, and especially when VAT exceeds 130 cm2 at an L4 CT scan, regardless of weight or obesity. Diabetes or IGT therefore seem to contribute towards the development of CAD regardless of the amount of VAT.

Dual regulation of sphingosine 1-phosphate-induced phospholipase D activity through RhoA and protein kinase C-alpha in C2C12 myoblasts.

Previous studies showed that in C2C12 cells, phospholipase D (PLD) and its known regulators, RhoA and protein kinase Calpha (PKCalpha), were downstream effectors in sphingosine 1-phosphate (SPP) signalling. Moreover, the role of PKC for SPP-mediated PLD activation and the requirement of PKCalpha for RhoA translocation were reported. The present results demonstrated that inactivation of RhoA, by overexpression of RhoGDP dissociation inhibitor (RhoGDI) as well as treatment with C3 exotoxin, attenuated SPP-stimulated PLD activity, supporting the involvement of RhoA in the stimulation of PLD activity by the bioactive lipid in C2C12 myoblasts. In addition, the effect of PKCalpha inhibitor Gö6976 on the SPP-induced PLD activation in myoblasts, where RhoA function was inactivated, was consistent with a dual regulation of the enzyme through RhoA and PKCalpha. Interestingly, the subcellular distribution of PLD isoforms, RhoA and PKCalpha, in SPP-stimulated cells supported the view that the functional relationship between the two PLD regulators, demonstrated to occur in SPP signalling, represents a novel mechanism of regulation of specifically localized PLD.

[Oxygen therapy in the long term treatment of Meniere's disease]. / L'ossigenoterapia nel trattamento a lungo termine della malattia di Ménière.

Endolymphatic hydrops is the hystopathological substrate characteristic of Ménière's disease. Besides the classical treatment with diuretics and/or osmotic drugs for some time, now treatment in a "pressure chamber" (OTI) has also been applied. The oxygen administered in the hyperbaric chamber can reduce the hydrops both by increasing the hydrostatic pressure and by mechanically stimulating the flow of endolymph toward the duct and endolymphatic sac. In addition, an increase is seen in the amount of O2 dissolved in the labyrinthine fluids and this contributes to recovering cell metabolism and restoring normal cochlear electrophysiological functions. Between 1992 and 1996 40 patients with monolateral Ménière's disease were studied: 15 underwent oxygen therapy at a constant pressure (2.2 ATA) (HOT), 25 with a continuous variation in pressure (from 1.7 to 2.2 ATA) (Alternobaric therapy, AOT). During the acute phase the patients underwent daily OTI treatment for 15 days in a row. The maintenance treatment called for one treatment cycle (one session a day for 5 days in a row) a month for 1 year, followed by for one treatment cycle (one session a day for 5 days in a row) every three months during the 2nd, 3rd and 4th years. The controls consisted of a group of 18 patients treated with 10% glycerol i.v. (during the acute phase) and betahystine (8 mg x 3/die) between episodes. A comparison was made of the average hearing threshold for the frequencies 500-3000 Hz (PTA), how frequently episodes of dizziness arose and extent of hearing loss in the three groups after the initial 15 days of treatment and at the end of the 4-year follow-up, in compliance with the criteria laid down by the Committee on Hearing and Equilibrium in 1995. At the end of the first 15 days of treatment, there were no statistically significant differences between the three groups. At the end of the follow-up, on the other hand, hyperbaric treatment, and in particular alternobaric therapy, enabled a significant reduction in the episodes of dizziness as compared to the control group. PTA and deafness also improved significantly in the patients who had undergone hyperbaric treatment. The results of the present work show that HOT, and in particular AOT, offer a valid alternative to drugs in the treatment of Ménière's disease.

Syntax and the brain: disentangling grammar by selective anomalies.

Many paradigms employed so far with functional imaging in language studies do not allow a clear differentiation of the semantic, morphological, and syntactic components, as traditionally defined within linguistic theory. In fact, many studies simply consider the brain's response to lists of unrelated words, rather than to syntactic structures, or do not neutralize the confounding effect of the semantic component. In the present PET experiment, we isolated the functional correlates of morphological and syntactic processing. The neutralization of the access to the lexical-semantic component was achieved by requiring the detection of anomalies in written sentences consisting of pseudowords. In both syntactic and morphosyntactic processing, the involvement of a selective deep component of Broca's area and of a right inferior frontal region was detected. In addition, within this system, the left caudate nucleus and insula were activated only during syntactic processing, indicating their role in syntactic computation. These findings provide original in vivo evidence that these brain structures, whose individual contribution has been highlighted by clinical studies, constitute a neural network selectively engaged in morphological and syntactic computation.

Permissive role of protein kinase C alpha but not protein kinase C delta in sphingosine 1-phosphate-induced Rho A activation in C2C12 myoblasts.

Rho GTPases participate in various important signaling pathways and have been implicated in myogenic differentiation. Here the first evidence is provided that in C2C12 myoblasts sphingosine 1-phosphate (SPP) rapidly and transiently induced membrane association of Rho A in a pertussis toxin-insensitive manner. The bioactive lipid preferentially relocalized the GTPase to Golgi-enriched membrane. Translocation of Rho A was abolished by inhibition or down-regulation of protein kinase C (PKC). Notably, treatment with Gö6976, an inhibitor of conventional PKCs, which selectively blocked PKC alpha in these cells, prevented SPP-induced Rho A translocation. Conversely rottlerin, a selective inhibitor of PKC delta, was without effect, demonstrating that SPP signaling to Rho A involves PKC alpha but not PKC delta activation. This novel functional relationship between the two proteins may have a role in SPP-mediated regulation of downstream effectors.

Role of unstable directions in the equilibrium and aging dynamics of supercooled liquids

The connectivity of the potential energy landscape in supercooled atomic liquids is investigated through a calculation of the instantaneous normal modes spectrum and a detailed analysis of the unstable directions in configuration space. We confirm the hypothesis that the mode-coupling critical temperature is the T at which the dynamics crosses over from free to activated exploration of configuration space. We also observe changes in the local connectivity of configuration space sampled during aging, following a temperature jump from a liquid to a glassy state.

Receptor-activated phospholipase D is present in caveolin-3-enriched light membranes of C2C12 myotubes.

Caveolin-3 (cav-3) is a key structural component of caveolar membrane in skeletal muscle. Cav-3-enriched light membrane (CELM) fractions obtained from C2C12 myotubes contain phospholipase D1 (PLD1) and its major regulators, RhoA and protein kinase Calpha (PKCalpha). All these proteins were found bound to cav-3. An in vivo assay of PLD activity, which allows to localize the reaction product in CELMs, indicated that the enzyme associated to this membrane microdomain was active. Moreover, bradykinin (BK), thrombin and phorbol 12-myristate 13-acetate induced rapid stimulation of PLD activity in CELMs. The cav-3-PLD1 complex was not affected by BK treatment, whereas the agonist induced a marked increase of RhoA association with cav-3. Furthermore, BK-induced PLD activation in CELMs was dependent, at least in part, on PKCalpha.