Absolute Metabolite Quantification Using Pure Shift NMR: Toward Quantitative Metabolic Profiling of Aqueous Biological Samples.

Accurate quantification of metabolites by nuclear magnetic resonance (NMR) is of prime importance in the field of health sciences for understanding the metabolic pathways of the investigated system, to address the mechanisms of action of diseases, and improving their diagnosis, treatment, and prognosis. Unfortunately, the absolute quantitative analysis of complex samples is still limited by sensitivity and resolution issues that are intrinsic to this technique. Ultrahigh-resolution pure shift methods have especially shown to be suitable for interpreting mixtures of metabolites in biological samples. Here, we introduce a robust analytical protocol based on the use of a pure shift library of calibration reference spectra to fit the fingerprint of each metabolite of interest and determine its concentration. The approach based on the SAPPHIRE pulse sequence enhanced with a block for solvent suppression has been validated through the results of a series of model mixtures, exhibiting excellent trueness (slope values in the range of 0.93-1.02) and linearity (R2 > 0.996) in a total time (a few hours) that is fully compatible with metabolomics studies. Furthermore, we have successfully applied our method to determine the absolute metabolite concentrations in a lymphoma extracellular medium, which improves metabolomic protocols reported to date by providing a quantitative and highly resolved vision of metabolic processes at play.

Biocompatibility of sol-gel hydroxyapatite-titania composite and bilayer coatings.

Titania-Hydroxyapatite (TiO2/HAP) reinforced coatings are proposed to enhance the bioactivity and corrosion resistance of 316L stainless steel (316L SS). Herein, spin- and dip-coating sol-gel processes were investigated to construct two kinds of coatings: TiO2/HAP composite and TiO2/HAP bilayer. Physicochemical characterization highlighted the bioactivity response of the TiO2/HAP composite once incubated in physiological conditions for 7days whereas the TiO2/HAP bilayer showed instability and dissolution. Biological analysis revealed a failure in human stem cells adhesion on TiO2/HAP bilayer whereas on TiO2/HAP composite the presence of polygonal shaped cells, possessing good behaviour attested a good biocompatibility of the composite coating. Finally, TiO2/HAP composite with hardness up to 0.6GPa and elastic modulus up to 18GPa, showed an increased corrosion resistance of 316L SS. In conclusion, the user-friendly sol-gel processes led to bioactive TiO2/HAP composite buildup suitable for biomedical applications.

Unveiling an exceptional zymogen: the single-chain form of tPA is a selective activator of NMDA receptor-dependent signaling and neurotoxicity.

Unlike other serine proteases that are zymogens, the single-chain form of tissue plasminogen activator (sc-tPA) exhibits an intrinsic activity similar to that of its cleaved two-chain form (tc-tPA), especially in the presence of fibrin. In the central nervous system tPA controls brain functions and dysfunctions through its proteolytic activity. We demonstrated here, both in vitro and in vivo, that the intrinsic activity of sc-tPA selectively modulates N-methyl-D-aspartate receptor (NMDAR) signaling as compared with tc-tPA. Thus, sc-tPA enhances NMDAR-mediated calcium influx, Erk(½) activation and neurotoxicity in cultured cortical neurons, excitotoxicity in the striatum and NMDAR-dependent long-term potentiation in the hippocampal CA-1 network. As the first demonstration of a differential function for sc-tPA and tc-tPA, this finding opens a new area of investigations on tPA functions in the absence of its allosteric regulator, fibrin.

NR2D-containing NMDA receptors mediate tissue plasminogen activator-promoted neuronal excitotoxicity.

Although the molecular bases of its actions remain debated, tissue-type plasminogen activator (tPA) is a paradoxical brain protease, as it favours some learning/memory processes, but increases excitotoxic neuronal death. Here, we show that, in cultured cortical neurons, tPA selectively promotes NR2D-containing N-methyl-D-aspartate receptor (NMDAR)-dependent activation. We show that tPA-mediated signalling and neurotoxicity through the NMDAR are blocked by co-application of an NR2D antagonist (phenanthrene derivative (2S(*), 3R(*))-1-(phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid, PPDA) or knockdown of neuronal NR2D expression. In sharp contrast with cortical neurons, hippocampal neurons do not exhibit NR2D both in vitro and in vivo and are consequently resistant to tPA-promoted NMDAR-mediated neurotoxicity. Moreover, we have shown that activation of synaptic NMDAR prevents further tPA-dependent NMDAR-mediated neurotoxicity and sensitivity to PPDA. This study shows that the earlier described pro-neurotoxic effect of tPA is mediated by NR2D-containing NMDAR-dependent extracellular signal-regulated kinase activation, a deleterious effect prevented by synaptic pre-activation.

[Milnacipran and venlafaxine at flexible doses (up to 200 mg/d) in the outpatient treatment of adults with moderate-to-severe major depressive disorder: a 24-week randomised, double blind exploratory study]. / Milnacipran et venlafaxine à posologie flexible (jusqu'à 200 mg/j) dans le traitement ambulatoire des épisodes dépressifs majeurs modérés à sévères de l'adulte: étude exploratoire de 24 semaines, randomisée en double insu.

INTRODUCTION: Serotonin (HT) and noradrenaline (NA) reuptake inhibitors (SNRIs) are commonly used as first line treatment of major depressive disorders (MDD). As compared to tricyclic antidepressants, they have proved similar efficacy and better tolerability. Milnacipran (MLN) (Ixel) and venlafaxine (VLF) (Effexor) are two SNRIs pharmacologically differing by their NA/HT ratio of potency: 1:1 and 1:30, respectively. OBJECTIVES: To investigate the efficacy and safety/tolerability of MLN and VLF administered at flexible doses (100, 150 or 200 mg/day) for 24 weeks (including 4 weeks of up-titration) in the outpatient treatment of adults with moderate-to-severe MDD. DESIGN: Multicentre, randomised, double blind, 2-parallel-arm, 24-week exploratory trial conducted in France by 50 psychiatrists. DIAGNOSIS AND MAIN INCLUSION CRITERIA: Male or female outpatients, aged 18 to 70, meeting the DSM-IV-TR and related MINI criteria for recurrent, unipolar, moderate-to-severe MDD, with neither psychotic features nor severe suicidal risk. A Montgomery-Asberg depression rating scale (MADRS) score> or =23 was required at inclusion. TREATMENT SCHEDULE: Patients were randomised to receive either MLN or VLF (1:1 ratio) for 24 weeks in double-blind conditions. Regardless of the treatment received, the following dosing schedule was applied: during the initial 4-week up-titration phase, the dosage was progressively increased from 25 mg/day (qd administration) to 150 mg/day (bid administration). At week 4, the dosage was either maintained at 150 mg/day, or adapted to 100 or 200 mg/day, based on the investigator's clinical judgement. At any time during the 20 following treatment weeks, the dose could be lowered for safety concerns until a minimal threshold of 100 mg/day. From Week 24, the dosage was decreased by 50mg/day every five days. After randomisation, eight assessment visits were organised at 2, 4, 6, 8, 12, 18, 24 weeks, and at study end (after the 5-15 days of down-titration and 10 days free of treatment). Efficacy evaluation ratings included the MADRS and global disease severity (CGI-S) total scores. Rates of MADRS response (reduction of initial score> or =50%) and remission (score< or =10) were calculated at Week 8 and Week 24 in the full analysis set as well as in the subgroups of patients with depressive disorder of severe DSM-IV intensity and with a MINI evaluation of suicidal risk (rated as required 'moderate' at the worst). STATISTICAL ANALYSIS: Standard distribution statistics (including mean and standard deviation [S.D.]) of scores and their changes from baseline, were calculated using the observed-case (OC) approach at all assessment times for the MADRS score, and the last-observation-carried-forward (LOCF) at 8 and 24 weeks for both MADRS and CGI-S scores. MADRS response and remission rates at 8 and 24 weeks were calculated using the LOCF approach by normal approximation of the binomial distribution. Bilateral exploratory statistical tests at 5% significance level were performed for results at 8 and 24 weeks of: (i) MADRS score changes from baseline, based on the score progress at each visit (mixed model for repeated measurements [MMRM]), and (ii) global MADRS response and remission rates (Chi(2)). RESULTS AND PATIENTS: A total of 195 patients were randomly assigned MLN (n=97) or VLF (n=98) and 134 (68.7%: 61.9%/MLN and 75.5%/VLF) completed the trial. At the end of the up-titration, patients received 100 mg/day (11.4%/MLN, 10%/VLF), 150 mg/day (30.4%/MLN, 43.8%/VLF), or 200 mg/day (58.2%/MLN, 46.3%/VLF). Totals of 177 patients (90/MLN and 87/VLF) and 181 patients (90/MLN and 91/VLF) were analysed for efficacy and safety, respectively. Treatment groups were similar for baseline characteristics except a higher proportion of MLN patients with a severe depressive episode (63.3% versus 54%). RESULTS AND EFFICACY: MADRS score (mean [S.D.] initial score: 31 [4.5]) progressively decreased all along the treatment course and similarly in both groups (Week 8-OC : -18.8 [7.7]/MLN and -18.6 [7.3]/VLF, p(MMRM)=0.95 ; Week 24-OC : -23.1 [7.8]/MLN and -22.4 [7.3]/VLF, p(MMRM)=0.37 ). At week 8-LOCF, MADRS response rates were similar in both groups (64.4%/MLN, 65.5%/VLF, p(chi2)=0.88) as well as remission rates (42.2%/MLN, 42.5%/VLF p(chi2)=0.97). At week 24 they remained non clinically and statistically different between groups (response rates: 70%/MLN, 77%/VLF, p(chi2)=0.29; remission rates: 52.2%/MLN, 62.1%/VLF, p(chi2)=0.19). In both "severe depressive episode" and "MINI mild or moderate suicidal risk" subgroups (n=104 and 75, respectively), response and remission rates were non clinically different at both time points, however in the "MINI mild-to-moderate suicidal risk" subgroup, MLN tended to be more rapidly active (remission rate at week 8-LOCF: 44.7%/MLN, 35.1%/VLF). The changes in CGI-S were also indicative of a significant improvement of the global illness severity with both treatments. RESULTS AND SAFETY/TOLERABILITY: The tolerability profile of both drugs was in line with their pharmacological activity. About 70% of patients in both groups experienced at least one adverse event (AE). In both groups, the most common AEs were nausea, dizziness, headache and hyperhidrosis, and, in the male patients, genito-urinary problems: orgasmic disorders (VLF only) and dysuria (MLN only). These AEs were mostly responsible for definitive treatment discontinuation for tolerability concerns. None of the 6 serious adverse events (SAEs) on MLN and 4 of the 8 SAEs on VLF were related to the test drug. CONCLUSION: MLN and VLF at flexible doses up to 200 mg/day globally exhibited similar efficacy and tolerability profiles in the long-term treatment of adults with MDD.

[Belated diagnosis of medullar compression in a case of post-polio syndrome]. / Découverte tardive dune compression médullaire à loccasion de la prise en charge dun syndrome post-polio.

The physiatrist observes about his practice individuals with sequela of old poliomyelitics. A part of them have unusual fatigue and muscular pains and weakness. The hypothesis of an evolution of neuro-biological mechanism suggested by few authors isn't, actually, demonstrated. More probably, the modifications of lesional and, functional changes with disability observed are the consequence of elderly effects and decreasing of physical activites. We report a case of spinal cord compression by intramedullar tumor, associated with a post-polio syndrome.

Cytokine gene expression in lymph node and spleen of sheep in response to Salmonella infection by two serotypes displaying different host specificity.

In order to investigate the determinism of the host specificity and to better understand the host resistance mechanisms, infections of sheep were performed with either S. abortusovis, serotype specific for ovine species, or with S. dublin, serotype adapted to cattle and accidentally transmissible to human. Following a subcutaneous challenge, S. dublin disseminated more rapidly towards lymphoid tissues than S. abortusovis. However, S. abortusovis tended to persist in spleen more efficiently than S. dublin. Using a quantitative RT-PCR method, the expression level of ovine cytokines genes was measured in the draining lymph node and in the spleen, in the course of infection. Inflammatory cytokine response was characterised by an early and strong increase of IL-1beta and TNFalpha mRNA in both lymphoid organs following S. dublin infection, while S. abortusovis challenge only induced IL-1beta mRNA increase in the spleen at day 3 post-inoculation. Likewise, S. dublin infection provoked a marked increase of IL-12 mRNA and a slight up-regulation of IFNgamma gene transcription in the local lymphoid site, in contrast to S. abortusovis infection. Elsewhere, both serotypes induced a strong and early IL-10 mRNA production and had no effect on IL-4 gene expression. Finally, taken together, these data suggest that the intensity of inflammatory and anti-infectious cytokine responses, but not the type 2 cytokine response, is serotype-dependent. They also suggest that the host-specific serotype, by limiting the host cytokine-mediated defence, could favour its persistence within lymphoid organs.

Effect of the mouse Nramp1 genotype on the expression of IFN-gamma gene in early response to Salmonella infection.

The early interferon-gamma (IFN-gamma) response after Salmonella infection was compared in resistant and susceptible congenic mice strains differing by the allele of Nramp1. IFN-gamma gene expression in vivo was studied by quantitative reverse transcription-PCR performed from the spleen of mice challenged by Salmonella either in multiplication or in resting phase. The spleen colonisation by Salmonella in vivo was investigated and was shown to be identical in a genetically defined host, whatever the initial phase of the bacteria. However, the bacterial load of the spleen was significantly reduced in Nramp1(r) (resistant) mice compared to Nramp1(s) (susceptible) mice. The background level of IFN-gamma mRNA was higher in the spleen of resistant mice than in the susceptible mice, before infection. Interestingly, the early upregulation of IFN-gamma gene transcription, which was observed after infection with Salmonella, was reproducibly delayed in susceptible mice compared to resistant mice. Finally, the kinetics of the host IFN-gamma response seems to be Nramp1 dependent. Resistant mice present the advantage of being more prompt to express this anti-infectious cytokine gene than susceptible mice in response to Salmonella infection.

Quantification of ovine cytokine gene expression by a competitive RT-PCR method.

A quantitative competitive RT-PCR method was developed in order to measure IL-1beta, IL-4, IL-12, IFNgamma, TNFalpha and G(3)PDH mRNA from samples of ovine tissue such as lymph node or spleen. The main advantage of the method relies on the use, for each target sequence, of an internal competitor construct similar to the relevant target, but 4-bp different in size. This competitive strategy is validated by the equivalence of the amplification process, observed separately between competitor DNA and target DNA species. Furthermore, the copy number of each cytokine cDNA is normalized to a fixed copy number of G(3)PDH cDNA. The cDNA level of this constitutive gene was effectively shown to remain constant whatever the tissue studied and independently of the experimental conditions used. The accurate and reproducible data obtained permit the application of this quantitative RT-PCR method to measure the sheep cytokine response to Salmonella infection. Early induction of IFNgamma mRNA was observed in the draining lymph node 1 day after infection. At the same time, a strong increase of IL-1beta mRNA was observed in local and systemic lymphoid organs, suggesting the initiation of the inflammatory response. Finally, the overall results demonstrate the efficiency of the method and its suitability for further studies of the immune response in the ovine species.

Long-term effects of glycylglycine peritoneal dialysis solution with neutral pH on peritoneum in rats.

This study was designed to test the morphological and functional effects of neutral, bicarbonate-based peritoneal dialysis solution containing glycylglycine on the peritoneum of chronically dialyzed rats. Peritoneal dialysis catheters were implanted in 36 rats. The animals were dialyzed twice daily for 4 weeks with a solution containing bicarbonate (35 mmol/L), glycylglycine (10 mmol/L), and 4% of anhydrous glucose (pH 7.35) (group 1; n = 18) or with lactate-based standard 4.25% Dianeal (pH 5.3 (group 2; n = 18). At the beginning of the study, reabsorption of glucose was slower in group 1 (p < 0.02); at the same time, the hyaluronic acid level in the effluent was higher in this group (p < 0.05). However, towards the end of the study these differences disappeared. After 4 weeks of dialysis in rats exposed to bicarbonate-based solution only, the transperitoneal loss of proteins was slower. In morphological studies of the parietal peritoneum, we detected no statistically significant differences between control nondialyzed rats and those exposed to tested solutions. In a biopsy of visceral peritoneum a tendency was observed for increased thickness of peritoneum in rats dialyzed with both tested peritoneal dialysis solutions when compared to control animals. In conclusion, neutral pH glycylglycine peritoneal dialysis solutions seem to be more biocompatible than standard dialysis solutions.

[Longitudinal division of the penis caused by an indwelling urinary catheter in a paraplegic patient]. / Section longitudinale du pénis secondaire au port de la sonde urinaire à demeure chez un paraplégique.

The authors report a rare case of longitudinal section of the penis, due to an urethral catheter. The patient was paraplegic and schizophrenic. They found only six such cases in the literature. They stress the risk of such complications in psychiatric patients.

[Complications of cervical spine manipulation. A case of "locked-in syndrome"]. / Complications de la manipulation cervicale. Une observation de "locked-in syndrome".

The current craze for cervical spine manipulation is due to its rapid and even dramatic results and to the ineffectiveness of many treatments prescribed by physicians. Complications of thoracic or lumbar spine manipulation are very rarely reported, but this is not the case with the cervical spine. We present the case of a woman who suffered a severely disabling complication caused by an "osteopathic" manoeuvre. This technique should be rejected as it is dangerous and as harmless methods can be used with good results in common cervicalgia, even if it is very severe.

[Pharmacokinetic interaction of cefixime and 2 antacids. Preliminary results]. / Etude de l'interaction pharmacocinétique entre le céfixime et deux antiacides. Résultats préliminaires.

A crossover study was undertaken in 8 healthy volunteers to evaluate the pharmacokinetics of cefixime administered orally alone or combined with an antacid. Each subject received successively: cefixime alone, Maalox followed 30 min later by cefixime, then Maalox followed 4 hours later by cefixime and finally Alka-Seltzer followed 30 min later by cefixime. Sixteen blood samples were drawn from 0 to 24 hours after oral administration, and plasma cefixime parameters were determined, using a HPLC assay. Four parameters were used to detect a possible interaction: Cmax, Tmax, AUCO-N and AUCO-alpha. No significant difference was observed between the four parameters. Thus, poor absorption of cefixime when combined with an antacid can be ruled out.

Maffucci's syndrome.

We report a case of Maffucci's syndrome, an infrequent tumoral process of unknown etiology. Its bizarre aspects make it conspicuously different from other cases in Peru. The number, size, and location of the tumors and the dystrophic changes after a prolonged evolution led to severe incapacity. The amputations performed as surgical treatment paradoxically contributed to improvement of the patient's condition.

[Rapid disappearance of calcification in a case of multiple tendinous calcifications (author's transl)]. / Une calcification disparaît: un cas de maladie des calcifications tendineuses multiples.

The authors describe a case of multiple tendinous calcification which was of unusual interest because of its localization (left gluteus medius tendon), the associated fever, its rapid favourable evolution (72 hours), and the disappearance of radiological signs of calcification.

Modification by prostaglandin E 2 (PGE 2 ) of the response of guinea-pig isolated vasa deferentia and atria to adrenergic stimuli.

1. Prostaglandin E(2) (PGE(2)) exerted positive cardiostimulant effects on isolated guinea-pig atria. The response was not altered by treatment of the animal with reserpine or by addition of propranolol to the organ bath. These results suggest that the cardiostimulatory actions of PGE(2) are not mediated through the release of catecholamines or stimulation of adrenoceptors.2. On the electrically driven atria, PGE(2) consistently exerted a cardiostimulant action which was not appreciably altered by changes in calcium ion in the bathing medium. PGE(2) showed no effect on the transport of calcium by the fragments of heart sarcoplasmic reticulum.3. PGE(2) reduced the responses to both noradrenaline and tyramine in the isolated atria. The shifted dose-response curve was not parallel to the original.4. PGE(2) increased the contractor response of the isolated vas deferens to nerve stimulation or to direct electrical stimulation.5. PGE(2) antagonized the increase caused by noradrenaline in contractor response of isolated vas deferens to direct electrical stimulation, whereas it affected the potentiation by noradrenaline differently when the vas deferens was contracting in response to nerve stimulation. In low concentration it inhibited and in large concentrations, it slightly enhanced the potentiation by catecholamine.6. It is concluded that PGE(2) has actions on multiple sites. It has post-junctional as well as pre-junctional effects on adrenergic neurones.