Lack of an association between candidate gene loci and idiopathic generalized epilepsy in Kuwaiti Arab children

Summary Idiopathic generalized epilepsies (IGEs) are the most common types of epilepsy in childhood and adolescence. A variety of data suggest that IGEs have a predominant genetic etiology. Recently, a number of gene mutations have been found to be associated with various types of epilepsy in mainly the Caucasian populations. The objective of this study was to investigate the association of three different candidate genes with IGE in Kuwaiti Arab children. This study includes 123 Kuwaiti patients with a confirmed diagnosis of epilepsy. Most of the patients have had a diagnostic EEG with generalized spike-wave discharges (GSWs). All patients were evaluated by using a validated seizure questionnaire. The clinical type of epilepsy was determined by a trained neurologist/pediatrician. The study also include 100 controls, the control subjects were children which did not have any history of neurological disorders. Blood samples were collected from all patients and control subjects after taking informed consent. DNA was isolated and analyzed by molecular methods. A FokI polymorphism in neuronal nicotinic acetylcholine receptor alpha-4 subunit (CHRNA4) gene was detected by PCR-RFLP method. A missense mutation (Ser248Phe) in CHRNA4 gene was analyzed by PCR-RFLP using HpaII. A C121W mutation in sodium-channel beta-1 subunit (SCN1B) gene was screened by a PCR-RFLP method using HinPI. A 2-bp deletion in Cystatin B gene was detected by PCR-RFLP using XcmI. The incidence of three FokI polymorphism genotypes in Kuwaiti IGE patients was 1,1 (85%), 1,2 (14%) and 2,2 (1%) respectively. The missense mutation Ser248Phe of CHRNA4 gene was not detected at all in Kuwaiti IGE patients. The C387G transversion resulting in C121W change in third exon of the SCN1B gene was detected in 3/123 patients (2%). The patients carrying this mutation also exhibited febrile seizures. The incidence of 2 bp deletion in the cystatin B gene was found to be 4% (5/123 IGE patients). The data obtained from molecular analysis show a lack of association between three candidate genes and clinical expression of IGE in Kuwaiti Arab children. This is completely different from the findings reported from Caucasian populations of France, Australia and USA in which case a strong association has been reported between IGE and these genes. To whom corresspondence should be addressed. Tel: +965-5319486; Fax: +965-5338940; E-mail: haider@hsc.edu.kw     

Neutrophils exhibit distinct phenotypes toward chitosans with different degrees of deacetylation: implications for cartilage repair

Introduction  

Osteoarthritis is characterized by the progressive destruction of cartilage in the articular joints. Novel therapies that promote resurfacing of exposed bone in focal areas are of interest in osteoarthritis because they may delay the progression of this disabling disease in patients who develop focal lesions. Recently, the addition of 80% deacetylated chitosan to cartilage microfractures was shown to promote the regeneration of hyaline cartilage. The molecular mechanisms by which chitosan promotes cartilage regeneration remain unknown. Because neutrophils are transiently recruited to the microfracture site, the effect of 80% deacetylated chitosan on the function of neutrophils was investigated. Most studies on neutrophils use preparations of chitosan with an uncertain degree of deacetylation. For therapeutic purposes, it is of interest to determine whether the degree of deacetylation influences the response of neutrophils to chitosan. The effect of 95% deacetylated chitosan on the function of neutrophils was therefore also investigated and compared with that of 80% deacetylated chitosan.     

Biodegradable chitosan particles induce chemokine release and negligible arginase-1 activity compared to IL-4 in murine bone marrow-derived macrophages

Alternatively activated macrophages have been implicated in the therapeutic activity of biodegradable chitosan on wound healing, however, the mechanisms of phenotypic differentiation are still unclear.In vitro, macrophages stimulated with high doses of chitosan (?500 μg/mL) were reported to produce low-level markers associated with alternative activation (arginase-1) as well as classical activation (nitric oxide), and to undergo apoptosis. In this study, we tested the hypothesis that 40 kDa biodegradable chitosan (5–500 μg/mL) is sufficient to polarize mouse bone marrow-derived macrophages (BMDM) in vitro to an alternatively activated phenotype. Control cultures were stimulated with IL-4 (alternative activation), IFN-γ/LPS (classical activation), 1 μm diameter latex beads (phagocytosis), or left untreated. After 48 h of in vitro exposure, BMDM phagocytosed fluorescent chitosan particles or latex beads, and remained viable and metabolically active, although some cells detached with increasing chitosan and latex bead dosage. Arginase-1 was over 100-fold more strongly induced by IL-4 than by chitosan, which induced only sporadic and weak arginase-1 activity over untreated BMDM, and no nitric oxide. IFN-γ/LPS stimulated nitric oxide production and arginase-1 activity and high concentrations of inflammatory cytokines (IL-6, IL-1β, TNF-α, MIP-1α/MIP-1β), while latex beads stimulated nitric oxide and not arginase-1 activity. Chitosan or latex bead exposure, but not IL-4, tended to promote the release of several chemokines (MIP-1α/β, GM-CSF, RANTES, IL-1β), while all treatments promoted MCP-1 release. These data show that chitosan phagocytosis is not sufficient to polarize BMDM to the alternative or the classical pathway, suggesting that biodegradable chitosan elicits alternatively activated macrophages in vivo through indirect mechanisms.     

Progranulin directly binds to the CRD2 and CRD3 of TNFR extracellular domains

We previously reported that PGRN directly bound to TNF receptors (TNFR) in vitro and in chondrocytes (Tang, et al., Science, 2011). Here we report that PGRN also associated with TNFR in splenocytes, and inhibited the binding of TNFα to immune cells. Proper folding of PGRN is essential for its binding to TNFR, as DTT treatment abolished its binding to TNFR. In contrast, the binding of PGRN to Sortilin was enhanced by DTT. Protein interaction assays with mutants of the TNFR extracellular domain demonstrated that CRD2 and CRD3 of TNFR are important for the interaction with PGRN, similar to the binding to TNFα. Taken together, these findings provide the molecular basis underlying PGRN/TNFR interaction and PGRN-mediated anti-inflammatory activity in various autoimmune diseases and conditions.     

Potent in vitro methicillin-resistant Staphylococcus aureus activity of 2-(1H-indol-3-yl)quinoline derivatives

A novel structural class of antibacterials, 2-(1H-indol-3-yl)quinolines, effective against methicillin-resistant Staphylococcus aureus (MRSA), was discovered from a combinatorial library. A structure-activity relationship (SAR) study was conducted to determine the pharmacophore and increase the potency of these compounds. Compounds were prepared that had minimum inhibitory concentrations (MICs) < 1.0 microg/mL against MRSA and retained activity against two strains of glycopeptide intermediate-resistant S. aureus (GISA).     

At-line monitoring of bioreactor protein production by Surface Plasmon Resonance

An innovative and automated method for the at-line monitoring of secreted protein was developed by harnessing a Surface Plasmon Resonance-based biosensor to a bioreactor. The proof of concept was performed by following at-line the relative concentration of a secreted protein produced by transient transfection of mammalian cells in a bioreactor. Our results suggest that our approach can be readily applied to the at-line determination of both protein concentration and bioactivity. Our experimental setup and strategy can thus satisfy the needs related to the development of novel bioprocess control protocols in the context of the new process analytical technology that arises in the biopharmaceutical industry.     

At-line quantification of bioactive antibody in bioreactor by surface plasmon resonance using epitope detection

We report an innovative at-line method to monitor concentration of bioactive antibody (i.e., antibody with conserved antigen-binding activity) secreted during bioreactor culture by the use of surface plasmon resonance (SPR)-based biosensor technology. In a first series of experiments, conditions for SPR-based measurements were validated off-line by monitoring bioactive antibody concentration in conditioned medium from 500-ml baffled flask hybridoma cell cultures. A fully automated experimental setup in which the SPR-based biosensor was harnessed to a bioreactor was then used at-line to monitor the concentration of bioactive antibody produced in a 3.5-L bioreactor. Quantitative SPR measurements performed both at-line and off-line were in excellent agreement with quantitative Western blotting followed by densitometry analyses. Thus, our experimental study confirms that SPR biosensors can be applied to at-line quantification of correctly folded proteins that are secreted by cells cultured in a bioreactor. Our experimental approach represents a novel and robust analytical strategy to be applied to the control and optimization of the production of bioactive secreted proteins.     

Potent in vitro methicillin-resistant Staphylococcus aureus activity of 2-(1H-indol-3-yl)tetrahydroquinoline derivatives.

Novel antibacterials agents, 2-(1H-indol-3-yl)tetrahydroquinolines, were prepared using hetero Diels-Alder chemistry and found to be effective in vitro against methicillin-resistant Staphylococcus aureus (MRSA). A structure-activity relationship (SAR) study was conducted to determine the important features of this series and to increase the potency of these compounds. Compounds were prepared that had minimum inhibitory concentrations (MIC's) < 1.0 microg/mL against MRSA, but had no activity versus vancomycin-resistant Enterococcus (VRE).     

Precise derivatization of structurally distinct chitosans with rhodamine B isothiocyanate

Work to date shows that structurally distinct chitosans have reacted inefficiently and unpredictably with fluorescein isothiocyanate (FITC) in an acid–methanol solvent that maintains both chitosan and fluorophore solubility. Since isothiocyanate preferentially reacts with neutral amine groups, and chitosan solubility typically depends upon a minimal degree of protonation, we tested the hypothesis that precise derivatization of chitosan with rhodamine isothiocyanate (RITC) can be achieved by controlling the reaction time and the degree of protonation. Addition of 50% v/v methanol reduced the chitosan degree of protonation in acetic acid but not HCl solutions. At various degrees of protonation, chitosan reacted inefficiently with RITC as previously observed with FITC. Nevertheless, precise derivatization was achieved by allowing the reaction to proceed overnight at a given degree of protonation (p < 0.0001, n = 18) and fixed initial fluorophore concentration. A reproducible 2% to 4% fraction of neutral amines reacted with RITC in proportion to the initial fluorophore concentration (p < 0.005). Using our optimized protocol, chitosans with different degree of deacetylation and molecular weight were derivatized to either 1% or 0.5% mol/mol RITC/chitosan-monomer with a precision of 0.1% mol/mol. The average molecular weight of fluorescent RITC-chitosan was similar to the unlabeled parent chitosan. Precise molar derivatization of structurally distinct chitosans with RITC can be achieved by controlling chitosan degree of protonation, initial fluorophore concentration, and reaction time.