[Pathogenic effect of pandemic influenza virus H1N1 under replication in cultures of human cells].

The propagation of the pandemic influenza virus H1N1 in cultures of bronchial (Calu-3) and intestinal (Caco-2) differentiated epithelial cells of human origin was studied. The canine epithelial cell lines, MDCK-H and MDCK-2, were comparatively tested. The two human cell lines were found to be highly sensitive to the influenza pandemic strains A/Hamburg/05/09 and A/Moscow/501/2011 and maintained their replication without addition of trypsin to culture medium. Virus strains of seasonal influenza H1N1, such as A/Moscow/450/2003, A/Memphis/14/96, and laboratory strain A/PR/8/34, multiplied in these human cells in similar manner. The intracellular cleavage HA0-->HA1+HA2 by the host virus-activating protease (IAP) occurred in both human cell lines under infection with each influenza virus H1N1 including pandemic ones. Comparatively, this cleavage of all influenza H1N1 virus strains appeared to be either undetectable or low-detectible in MDCK-H and MDCK-2, respectively, thereby implying low levels of active IAP in these cells. Multiplication of pandemic and seasonal influenza H1N1 viruses in Calu-3 and Caco-2 cells caused cytopathic effect, which was accompanied with low autophagy and apoptosis events. These data allow recommending human cell lines, Calu-3 and Caco-2, for optimized isolation and passaging of clinical strains of Influenza pandemic viruses H1N1.

Characterization of the neuraminidase of the H1N1/09 pandemic influenza virus.

In this study, we compared properties of the neuraminidase (NA) of the H1N1/2009 pandemic virus (H1N1pdm) and N1 NAs of other influenza viruses. The H1N1pdm NA was more active than NAs of seasonal H1N1 viruses, hydrolyzed Neu5Acα2-3Gal linkage as efficiently as did avian viruses and cleaved Neu5Acα2-6Gal linkage as efficiently as classical swine viruses. To assess the functional balance between heterologous NAs and pandemic virus HA, we generated four recombinant viruses that shared seven genes of A/Hamburg/5/09 and contained the NA gene from representative avian, swine and human viruses. The viruses harboring NA from avian, Eurasian avian-like swine and seasonal human viruses eluted more slowly from red blood cells, were more sensitive to neutralization by human airway mucins, and replicated less efficiently in differentiated human tracheo-bronchial epithelial cultures as compared with the viruses containing the NA of H1N1pdm and the NA of the North American classical swine virus lineage. Our data suggest that functional properties of the NA of H1N1pdm could be closer to those of classical swine viruses than to those of avian, avian-like swine and seasonal human viruses.

[NP gene of pandemic H1N1 virus attenuates virulence of mouse-adapted human influenza virus].

The authors studied a possible role of the caspase cleavage motif located in the nucleoprotein (NP) of pandemic influenza virus H1N1 in the regulation of viral virulence properties. A reverse genetics method was used to obtain chimeric seasonal-like mouse-adapted influenza virus hvA/PE/8/34 (H1N10) carrying either the NP gene of wild type pandemic virus with incomplete caspase motif ETGC or mutated pandemic NP with natural caspase cleavage site of human type ETDG. The wild-type NP gene of the pandemic virus was found to poorly fit to the gene pattern of closely related seasonal-like hvA/PR/8/34 virus (H1N1) and did not rescue mature virus production whereas a mutated NP with human-type caspase cleavage site maintained gene fitness, giving rise to a chimeric virus. The generated chimeric virus hvA/PR/8/34 carrying the mutated pandemic NP successfully replicated in the murine lung, but was attenuated and did not reach the virulence level of seasonal-like mouse-adapted virus hvA/PR/8/34. The findings indicate that the NP caspase cleavage site plays a role in viral adaptation and viral virulence in mammals.

[Multicenter evaluation of h-FABP semi-quantitative assay (Cardio Detect) in central laboratory: the point in acute myocardial infarction diagnosis]. / Evaluation multicentrique du dosage semi-quantitatif de la h-FABP (Cardio Detect) au laboratoire central : intérêt dans le diagnostic de l'infarctus du myocarde.

The diagnostic performance of heart-Fatty Acid Binding Protein (h-FABP) (semi-quantitative CardioDetect test) and cardiac troponin I (TnIc) blood assays were compared in one hundred patients presenting with suspicion of acute coronary syndrome. Final patient diagnosis was "acute myocardial infarction" in 36 cases, "non ST myocardial infarction" in 25 cases and "non ischemic pathologies" in 39 cases. h-FABP results were positive in 26 patients, negative in 57 patients and ambiguous in 17 patients, the latter corresponding to the final diagnosis of "acute myocardial infarction" in 5 cases, "non ST myocardial infarction" in 2 cases and "non ischemic pathologies " in 10 cases. At admission, h-FABP and TnIc exhibiteda sensitivity of 54% an 66%, respectively and a specificity of 86% and 95%, respectively. Positive and negative predictive values were 81% and 64% for h-FABP, respectively and 92% and 75% for cTnI, respectively. h-FABP and cTnI demonstrated a similar diagnostic efficiency if admission delay is less than 4 hours after onset of chest pain (area under ROC curve TnIc = 0.767 +/- 0.091 ; area under ROC curve h-FABP = 0.622 +/- 0.109 ; p = 0.144). On the contrary, cTnI assay demonstrated a better efficiency than h-FABP (p< 0.005) for patients admitted in a delay of 4 to 12 hours after the onset of chest pain. If chosen cTnI cut-off corresponded to the recent consensus definition used for monitoring acute coronary syndrome patients, h-FABP semi-quantitative assay realized within central laboratory did not demonstrated a better diagnostic efficiency than cTnI.

Maternal milk as methylmercury source for suckling mice: neurotoxic effects involved with the cerebellar glutamatergic system.

Methylmercury (MeHg) is a highly neurotoxic compound and several studies have reported intoxication signs in children whose mothers were exposed to this environmental toxicant. Although it is well established that the in utero exposure to MeHg causes neurological deficits in animals and humans, there is no evidence of the exclusive contribution of lactational exposure to MeHg as a possible cause of neurotoxicity in the offspring. In this study, we investigated the exclusive contribution of MeHg exposure through maternal milk on biochemical parameters related to the glutamatergic homeostasis (glutamate uptake by slices) and to the oxidative stress (total and nonprotein sulfhydryl groups, nonprotein hydroperoxides, glutathione peroxidase and catalase activities) in the cerebellum of suckling mice (Swiss albino). The same parameters were also evaluated in the cerebellum of mothers. Our results showed, for the first time, that lactational exposure to MeHg caused a high percent of inhibition (50%) on glutamate uptake by cerebellar slices in pups. Contrarily, this effect was not observed in mothers, which were submitted to a direct oral exposure to MeHg (15 mg/l in drinking water). In addition, behavioral/functional changes were observed in the weaning mice exposed to MeHg. It was observed an increase in the levels of nonprotein hydroperoxides in cerebellum, and this increase was negatively correlated to the glutamate uptake by cerebellar slices. This study indicates that (1) the exposure of lactating mice to MeHg causes inhibition of the glutamate uptake by cerebellar slices in the offspring; (2) this inhibitory effect seems to be related to increased levels of hydroperoxide.

Methylmercury increases glutamate release from brain synaptosomes and glutamate uptake by cortical slices from suckling rat pups: modulatory effect of ebselen.

During the early postnatal period the brain is extremely sensitive to external agents. Here, we examined the effect of subcutaneous injections of methylmercury (MeHg; 2 mg/kg) during the suckling period (postnatal days [PND] 3-10, 3-17, or 3-24) on glutamate release from brain synaptosomal preparations and on glutamate uptake by brain cortical slices of rat pups. The possible antagonist effect of ebselen against MeHg effect was also examined at PND 24. MeHg increased the basal (but not K+-stimulated) glutamate release and glutamate uptake at PND 24. A strong tendency of increase in the basal glutamate release from synaptosomes (p= 0.088) was observed at PND 17. Ebselen, which did not affect glutamate release and uptake per se, prevented both effects of MeHg. This study indicates that (1) the effect of MeHg on glutamate release could be involved in its toxicity; (2) the increase in the glutamate uptake could represent a pathophysiological response to MeHg-induced glutamate release; (3) the inhibitory effect of ebselen on MeHg-induced glutamate release could be related to its reported neuroprotective effects.

[Evaluation of prognosis after Q wave myocardial infarction. Comparison of invasive and noninvasive diagnostic strategies]. / Evaluation du pronostic après infarctus du myocarde avec onde Q. Comparaison de stratégies diagnostiques non invasives et invasives.

The predictive value of several diagnostic strategies after myocardial infarction was assessed in 178 patients (mean age 55 +/- 9 years) treated medically after a primary Q wave myocardial infarction. Within 6 weeks of onset of symptoms the authors performed exercise stress test coupled with Thallium 201 scintigraphy, isotopic left ventriculography and conventional coronary angiography with ventriculography. The average left ventricular ejection fraction was 45 +/- 12%. Two non-invasive diagnostic strategies with and without results of scintigraphy and two invasive strategies with and without ventricular volumes were studied. The average follow-up period was 58 +/- 22 months. Sixteen cardiac deaths occurred. Multivariate Cox analysis showed that, in contrast to left ventricular volumes, coronary angiography did not provide additional prognostic value compared with the non-invasive model with Thallium scintigraphy and did not appear to be essential in terms of predictive value in this population. Moreover, the size of reversible defect on Thallium scintigraphy was an independent predictive factor of cardiac death and provided additional and independent prognostic information in the non-invasive and invasive strategies. Therefore, the reduction of residual ischaemia by coronary revascularisation could improve the long-term prognosis after myocardial infarction.

[Alteration of sinus variability after cardiac surgery]. / Altération de la variabilité sinusale après chirurgie cardiaque.

UNLABELLED: Sinus tachycardia is frequent after cardiac surgery and this tachycardia is probably due to changes of the autonomic nervous system. The objective of this study was to evaluate the changes possibly induced by cardiac surgery, by studying sinus variability (SV) during a 24-hour Holter monitoring. The examination was performed in 28 patients who had undergone cardiac surgery 1 to 6 weeks previously. These patients had no alteration of left ventricular function, or any causes likely to modify SV and they had a normal postoperative course. Their results were compared to those of 4 subjects developing a postoperative complication (1 case of ventricular tachycardia and 3 cases of resuscitated cardiac arrest). The results were also compared to those of 24 age-matched adult controls without heart disease (control group). The study of SV included temporal and spectral analysis of SV with measurement of the standard deviation of normal RR intervals (SD), mean heart rate (HR), percentage of RR intervals differing by more than 50 m/sec from the adjacent interval (pNN50), coefficient of variability (CV) (SD/RR), square root of the differences between successive RR (rMSSD), spectral properties of low frequencies (LF) and high frequencies (HF) and the fractionated spectral property (LF/HF). RESULTS: (see tables, page 151 and 152). An alteration of SV was therefore observed in the surgical group, and lasted 4 to 6 months after surgery. No difference was observed between subjects without cardiac events and those presenting a cardiac event. IN CONCLUSION: cardiac surgery decreases all parameters of SV during the first few postoperative months. Certain unexpected cardiac accidents during this period could be explained by these changes.

Mechanism of QRS electrical alternans.

In patients with latent dual atrioventricular nodal pathways a 2:1 ventriculoatrial block often occurs during ventricular pacing and is generally associated with the concomitant appearance of QRS alternans. This type of QRS alternans is related to retrograde conduction, and a concealed retrograde conduction in the His Purkinje system could explain the QRS alternans. A case that confirms the hypothesis that electrical alternans is secondary to a 2:1 block in the activation of some part of the ventricles is reported.

[Heart rate response to ventricular stimulation]. / Réponse de la fréquence cardiaque à la stimulation ventriculaire.

Analysis of heart rate variability (HRV) by Holter monitoring is the method of choice for assessing the cardiac autonomic regulation. Rapid ventricular stimulation also provokes changes in the autonomic nervous system tone. The aim of this study was to compare time and frequency domain analysis of HRV (Elatec version 3.02) with variations of HR observed after incremental ventricular stimulation to 200/min in 130 patients. In 80 patients, ventricular stimulation provoked an initial acceleration in HR followed by a slowing with a variation of over 10%. In the other 50 patients, these variations were not observed. Holter analysis of HRV showed concordance between the two methods. In time domain analysis, the standard deviation of normal R-R intervals, the coefficient of variability [(CV = SD/mean RR) and percentage of adjacent RR intervals with a difference of more than 50 msec (pNN 50) were significantly reduced in the abnormal group, the respective values in the normal and abnormal groups being: SD 122 vs 72 msec; CV 15 versus 9% and pNN50 9 versus 5%)]. In frequency domain analysis, there was a reduction of low and high frequency spectra and of the ratio of low/high frequencies in abnormal subjects. The authors conclude that the disappearance of HR changes after ventricular stimulation is correlated to the absence of HRV on Holter recording. This simple test may be performed systematically during electrophysiological investigations. The measurements are reproductible and the results are not affected by arrhythmias or technical problems of quality recording by the Holter method which may affect analysis of HRV.

A closed flow chamber for long-term multichannel recording and optical monitoring.

An autoclavable chamber and associated medium circulation system has been constructed to provide a stable environment for mammalian cultures for long periods of time. The chamber was specifically designed for (a) multichannel electrophysiological recording from monolayer networks with photoetched multielectrode matrices, (b) for microscope observations of networks in the living state and (c) for the manipulation of such networks with laser cell surgery. The chamber components can be assembled under sterile conditions in less than 30 s to minimize pH and osmolarity stress to the monolayer cultures. An open chamber version provides a constant medium bath for pharmacological studies. The closed chamber version, attached to a 10-ml medium reservoir and a peristaltic pump, has so far provided constant conditions for continual recording over an 8-day period. Flow characteristics within the closed chamber, optical properties, pH maintenance, and schemes for drug addition are described.

Stimulation of monolayer networks in culture through thin-film indium-tin oxide recording electrodes.

Monolayer networks, obtained from murine spinal cord tissue and grown on a matrix of 64 photo-etched, indium-tin oxide (ITO) microelectrodes, can be electrically stimulated through such thin-film recording electrodes. Multichannel coordinated network activity can be evoked and spontaneous network activity can be modified by generation of additional, multichannel bursting. Although single pulses through 1 electrode may trigger network responses, networks generally react best to short trains of pulses. Response thresholds approximate standard physiological strength/duration relationships. Repetitive stimulation trains often generate network activity patterns akin to epileptiform activity. The ITO conductors remain stable for recording under warm saline for long periods of time (maximum test period: 8 months). However, most electrodes show a reduction in impedance after several thousand stimulus pulses. Electrode breakdown in the form of ITO oxidation and loss of light transmittance occurs before hydrolysis is observed but requires a combination of voltage levels and pulse lengths beyond that needed for effective network stimulation.

The effect of phenobarbital on the transcriptional activity of liver.

The effect of phenobarbital on the transcriptional activity of liver was studied by measuring the synthesis of RNA by suspensions of hepatocytes isolated from rats treated with phenobarbital for various time periods. The absolute rates of RNA synthesis by isolated hepatocytes were determined by measuring the incorporation of [3H]orotic acid into RNA as UMP and the specific radioactivity of the UTP pool. The specific radioactivity of the UTP extracted from hepatocytes isolated from phenobarbital-treated rats was consistently lower than that of the UTP pool of hepatocytes from untreated rats. Phenobarbital treatment increased the rate of RNA synthesis 10-fold over that observed for hepatocytes from untreated rats. The maximum rate of RNA synthesis was observed 16-18 h after phenobarbital administration. Phenobarbital treatment also affected the nuclear-cytoplasmic transport of RNA by isolated hepatocytes. Immediately after phenobarbital treatment, the transport of RNA decreased; however, 24 h after phenobarbital administration, the transport of RNA was increased 4-fold. An increase in the synthesis of RNA in vivo by liver was found 18 h after phenobarbital treatment, and the incubation of suspensions of hepatocytes with various concentrations of phenobarbital increased RNA synthesis significantly.