Development of an ELISA system for tick-borne encephalitis virus infection in rodents

Tick-borne encephalitis (TBE) virus causes severe encephalitis with serious sequelae in humans. An epizootiological survey of wild rodents is effective to detect TBE virus-endemic areas; however, limited serological diagnostic methods are available to detect anti-TBE virus antibodies in wild rodents. In this study, ELISAs for the detection of rodent antibodies against the TBE virus were developed using two recombinant proteins, domain III of the E protein (EdIII) and subviral particles (SPs), as the antigens. As compared with the neutralization test, the ELISA using EdIII had 77.1% sensitivity and 80.0% specificity, and the ELISA using SPs had 91.4% sensitivity and 100% specificity. Furthermore, when the ELISAs were applied to the epizootiological survey in the TBE virus-endemic area, both of the ELISAs was able to detect wild rodents with TBE virus-specific antibodies. This is the first study to show that ELISAs using recombinant antigens can be safe and useful in the detection of TBE virus-infected wild rodents in epizootiological research.     

Apathy/depression, but not subjective fatigue, is related with cognitive dysfunction in patients with multiple sclerosis

Background

Cognitive impairment could affect quality of life for patients with multiple sclerosis (MS), and cognitive function may be correlated with several factors such as depression and fatigue. This study aimed to evaluate cognitive function in Japanese patients with MS and the association between cognitive function and apathy, fatigue, and depression.

Methods

The Brief Repeatable Battery of Neuropsychological tests (BRB-N) was performed in 184 Japanese patients with MS and 163 healthy controls matched for age, gender, and education. The Apathy Scale (AS), Fatigue Questionnaire (FQ), and Beck Depression Inventory Second Edition (BDI-II) were used to evaluate apathy, fatigue, and depression, respectively. Student’s t-test was used to compare MS patients and healthy controls. Correlations between two factors were assessed using the Pearson correlation test, and multiple regression analysis was used to evaluate how much each factor affected the BRB-N score.

Results

In all BRB-N tests, patients with MS scored significantly lower than controls, and the effect size of symbol digit modalities test was the highest among the 9 tests of the BRB-N. Patients with MS had higher AS (p?<?0.001), FQ (p?<?0.0001), and BDI-II (p?<?0.0001) scores than controls. In patients with MS, scores on most of the BRB-N tests correlated with scores on the AS and BDI-II; however, there was little correlation between scores on the BRB-N tests and those on the FQ.

Conclusions

Cognitive function was impaired, particularly information-processing speed, and decreased cognitive function was correlated with apathy and depression in Japanese patients with MS. Despite the association between cognitive variables and depression/apathy, cognitive function was impaired beyond the effect of depression and apathy. However, subjective fatigue is not related with cognitive impairment. Taken together, this suggests that different therapeutic approaches are needed to improve subjective fatigue and cognition, and thereby quality of life, in patients with MS.     

Gating and permeation properties of two types of calcium channels in neuroblastoma cells.

The gating and permeation properties of two types of calcium channels were studied in the neuroblastoma cell line N1E-115. Calcium channel currents as carried by Ba2+ (50 mM) were recorded using the whole-cell variation of the patch electrode voltage-clamp technique. The two types of calcium channels showed similar membrane potential dependence with respect to the steady-state activation and inactivation gating properties. However, the properties of the long-lasting type II channels were shifted approximately 30 mV in the depolarizing direction compared with those of the transient type I channels. Activation of type I channels developed with a sigmoidal time course which was described by m2 kinetics, whereas the activation of type II channels was described by a single exponential function. Tail current upon repolarization followed an exponential decay in either type of calcium channels. In comparison to type I channels, the activation process of type II channels was shifted approximately 30 mV in the positive direction, while the deactivation process showed a 60 mV shift in the positive direction. The rate constants of activation obtained from the activation and deactivation processes indicated that under comparable membrane potential conditions, type II channels close 2.4 times faster than type I channels upon repolarization. When external 50 mM Ba2+ was replaced with Ca2+ or Sr2+ on the equimolar basis, the amplitudes of transient and long-lasting currents were altered without a significant change in their time courses. The ion permeability ratios determined from the maximum amplitude of the inward current were as follows: Ba2+ (1.0) = Sr2+ (1.0) greater than Ca2+ (0.7) for type I channels, and Ba2+ (1.0) greater than Sr2+ (0.7) greater than Ca2+ (0.3) for type II channels. Replacement of Ba2+ with Ca2+ caused a 10-12 mV positive shift in the current-voltage relation for type II channels. However, the shift for type I channels was much less. This suggests that negative surface charges are present around type II channels. After correction for the surface charge effect on the ion permeation, there was no significant difference between the permeability ratios of these cations for the two channel types. It was concluded that the two types of calcium channels have many common properties in their gating and permeation mechanisms despite their differential voltage sensitivity and ion selectivity.     

Bromocriptine Mesylate Attenuates Amyotrophic Lateral Sclerosis: A Phase 2a,Randomized, Double-Blind,Placebo-Controlled Research in Japanese Patients

Objective

Bromocriptine mesylate (BRC), a dopamine D2 receptor agonist has been shown to confer neuroprotection, sustained motor function and slowed disease progression in mouse models of amyotrophic lateral sclerosis (ALS) Here we report a first in human trial in ALS.

Design

A multicenter, Riluzole add-on, randomized, double-blind, placebo controlled 102-week extension BRC clinical trial.

Methods

The trial was conducted between January 2009 and March 2012 on 36 Japanese ALS patients. A 12-week treatment with Riluzole observational period was followed by combined treatment (Riluzole + BRC; n = 29 or Riluzole + placebo; n = 7). The dosing commenced at 1.25 mg/day increasing in steps at two weeks intervals to a maximum of 15 mg/day. The efficacy of BRC was evaluated by comparing BRC and placebo groups upon completion of stepwise dosing at 14 weeks 2 points (1^st endpoint) and upon completion or discontinuation of the study (2^nd endpoint) of the dosing.

Results

Statistics analyses revealed a marginal BRC treatment efficacy with P≦20%to placebo by 1^st and 2^nd endpoint analysis. In the 1^st endpoint analysis, BRC group was significantly effective on the scores of ALSAQ40-communicaton (P = 1.2%), eating and drinking (P = 2.2%), ALSFRS-R total (P = 17.6%), grip strength (P = 19.8%) compared to the placebo group. In the 2^nd endpoint analysis, differences between the scores of Limb Norris Scale (P = 18.3%), ALSAQ40-communication (P = 11.9%), eating and drinking (P = 13.6%), and neck forward-bent test (P = 15.4%) of BRC group were detected between the two groups. There was no significant difference between the treatment groups for adverse events or serious drug reactions incidence.

Conclusions

BRC sustains motoneuronal function at least in part through BRC treatment. Further analysis involving a Phase 2b or 3 clinical trial is required but BRC currently shows promise for ALS treatment.

Trial Registration

UMIN Clinical Trials UMIN000008527     

Modification of the effects of naloxone in morphine-dependent mice

Mice were rendered dependent on morphine by mixing morphine with their food (2 mg/g) for three days. Increasing doses of naloxone precipitated dose-dependent withdrawal reactions such as weight loss and jumping. These withdrawal reactions were antagonized by morphine pretreatment. Effects of morphine, such as increased locomotor activity, inhibition of intestinal transport, and analgesia were antagonized by naloxone in both non-dependent and dependent subjects. The antagonist actions of naloxone were increased in dependent subjects; lower doses of naloxone were sufficient to antagonize effects of morphine. The present results confirm earlier studies indicating that precipitation of withdrawal can be antagonized by morphine pretreatment suggesting that withdrawal reactions are due to actions of naloxone at the same receptor at which opioid agonists act. The increased antagonist potency of naloxone in dependent subjects extends earlier results obtained with analgesic effects to several other agonist effects of morphine and is consistent with the interpretation that exposure to an opioid agonist induces a change in the conformation of opioid receptors.     

Reaction of the hydrated electron with histone H1 and related compounds studied by e.s.r. and spin-trapping

The reactions of the hydrated electron with histone H1, protamine and related compounds (poly-L-lysine, poly-L-arginine and poly-D,L-alanine) were investigated by the spin-trapping technique. In order to identify the radical structure of the spin-adducts originating from macromolecules, the usual spin-trapping technique was developed as follows: N2-saturated aqueous solutions of proteins containing sodium formate were X-irradiated (4.5 kGy) in the presence of 2-methyl-2-nitrosopropane (MNP) as a spin-trap. The side-products due to the self trapping of MNP radicals were then removed from the spin-adducts of the proteins by a Sephadex G-25 column. Finally the spin-adducts were enzymatically digested to transform the broad e.s.r. signals due to slow tumbling of nitroxyl radicals to identifiable ones. The e.s.r. spectra obtained for all samples showed that the deaminated radical, R--CH--CO--NH--(R:amino acid side chain), was produced. Furthermore, polyacrylamide gel electrophoresis of the irradiated protamine and histone H1 indicated reduction of molecular size. These results confirm that hydrated electrons react with proteins and induce the deamination reaction which leads to main-chain scission.     

Chemical synthesis and hepatic biotransformation of 3 alpha,7 alpha-dihydroxy-7 beta-methyl-24-nor-5 beta-cholan-23-oic acid, a 7-methyl derivative of norchenodeoxycholic acid: studies in the hamster.

A new bile acid analogue, 3 alpha,7 alpha-dihydroxy-7 beta-methyl-24-nor-5 beta-cholan-23-oic acid (7-Me-norCDCA) was synthesized from the methyl ester of norursodeoxycholic acid, and its hepatic biotransformation was defined in the hamster. To synthesize 7-Me-norCDCA, the 3 alpha-hydroxyl group of methyl norursodeoxycholate was protected as the hemisuccinate, and the 7 beta-hydroxyl group was oxidized with CrO3 to form the 7-ketone. A Grigard reaction with methyl magnesium iodide followed by alkaline hydrolysis gave 7-Me-norCDCA (greater than 70% yield). The structure of the new compound was confirmed by proton magnetic resonance and mass spectrometry. After intraduodenal administration of the 14C-labeled compound into the anesthetized biliary fistula hamster, it was rapidly and efficiently secreted into the bile; 80% of radioactivity was recovered in 2 h. After intravenous infusion, the compound was efficiently extracted by the liver and secreted into the bile (greater than 75% in 3 h). Most (93%) of the biliary radioactivity was present in biotransformation products. The major biotransformation product (48.7 +/- 6.0%) was a new compound, assigned the structure of 3 alpha,5 beta,7 alpha- trihydroxy-7 beta-methyl-24-nor-5 beta-cholan-23-oic acid (5 beta-hydroxy-7- Me-norCDCA). In addition, conjugates of 7-Me-norCDCA with taurine (13.7 +/- 5.0%), sulfate (10.3 +/- 3.0%), or glucuronide (5.1 +/- 1.7%) were formed. 7-Me-norCDCA was strongly choleretic in the hamster; during its intravenous infusion, bile flow increased 2 to 3 times above the basal level, and the calculated choleretic activity of the compound (and its metabolic products) was much greater than that of many natural bile acids, indicating that the compound induced hypercholeresis. It is concluded that the biotransformation and physiological properties of 7-Me-norCDCA closely resemble those of norCDCA. Based on previous studies, the major biological effect of the 7-methyl group in 7-Me-norCDCA is to prevent its bacterial 7-dehydroxylation in the distal intestine.