Pitfalls in target mRNA quantification for real-time quantitative RT-PCR in overload-induced skeletal muscle hypertrophy.

Quantifying target mRNA using real-time quantitative reverse transcription-polymerase chain reaction requires an accurate normalization method. Determination of normalization factors (NFs) based on validated reference genes according to their relative stability is currently the best standard method in most usual situations. This method controls for technical errors, but its physiological relevance requires constant NF values for a fixed weight of tissue. In the functional overload model, the increase in the total RNA concentration must be considered in determining the NF values. Here, we pointed out a limitation of the classical geNorm-derived normalization. geNorm software selected reference genes despite that the NF values extensively varied under experiment. Only the NF values calculated from four intentionally selected genes were constant between groups. However, a normalization based on these genes is questionable. Indeed, three out of four genes belong to the same functional class (negative regulator of muscle mass), and their use is physiological nonsense in a hypertrophic model. Thus, we proposed guidelines for optimizing target mRNA normalization and quantification, useful in models of muscle mass modulation. In our study, the normalization method by multiple reference genes was not appropriate to compare target mRNA levels between overloaded and control muscles. A solution should be to use an absolute quantification of target mRNAs per unit weight of tissue, without any internal normalization. Even if the technical variations will stay present as a part of the intergroup variations, leading to less statistical power, we consider this method acceptable because it will not generate misleading results.

Quantification of low-expressed mRNA using 5' LNA-containing real-time PCR primers.

Real-time RT-PCR is the most sensitive and accurate method for mRNA quantification. Using specific recombinant DNA as a template, real-time PCR allows accurate quantification within a 7-log range and increased sensitivity below 10 copies. However, when using RT-PCR to quantify mRNA in biological samples, a stochastic off-targeted amplification can occur. Classical adjustments of assay parameters have minimal effects on such amplification. This undesirable amplification appears mostly to be dependent on specific to non-specific target ratio rather than on the absolute quantity of the specific target. This drawback, which decreases assay reliability, mostly appears when quantifying low-expressed transcript in a whole organ. An original primer design using properties of LNA allows to block off-target amplification. 5'-LNA substitution strengthens 5'-hybridization. Consequently on-target hybridization is stabilized and the probability for the off-target to lead to amplification is decreased.

Musclin gene expression is strongly related to fast-glycolytic phenotype.

Musclin has been described as a muscle-derived secretory peptide, responsive to insulin in vivo, and inducing insulin resistance in vitro. Because muscle fibers display very different metabolic properties and insulin sensitivity, we tested the hypothesis that musclin expression could depend on myofiber type. Musclin mRNA was detected at high level in fast gastrocnemius and plantaris muscles, but only as traces in soleus, a slow-twitch muscle. A single fiber analysis showed that musclin was produced by muscle fibers themselves, almost exclusively type IIb fibers. Slow to fast transition of soleus phenotype after hindlimb suspension increased musclin mRNA levels, whereas fast to slow transition of plantaris phenotype after functional overload decreased musclin mRNA levels. This clearly suggests that musclin transcription is strongly related to fast-glycolytic phenotype. We conclude that musclin is produced by myocytes in a highly fiber-type specific manner and that physiological changes in type IIb MHC lead to coordinated musclin expression.

Quantification by real-time PCR of developmental and adult myosin mRNA in rat muscles.

A real-time RT-PCR assay using newly designed primers was developed to analyze developmental and adult MHC mRNA expression both in skeletal muscles and single fibers. Only 4 ng of total RNA was necessary for the analysis of the relative mRNA expression of MHC genes. Different validation steps were realized concerning both specificity and sensitivity of each primer set, and linearity and efficiency of each real-time PCR amplification. Then, quantification of MHC mRNA in neonatal and adult muscles as well as in single fibers was done by the deltaC(T) method, with CycA gene as the reference gene. Due to a higher sensitivity than that of a competitive PCR method, we demonstrated that this assay is suitable to study very low level of MHC mRNA expression as developmental MHC in adult muscle and to quantify mRNA from very small samples.

[Definitions and recommendations for studying the delay of antidepressant action. Part 3: methodological aspects]. / Définitions et recommandations pour l'étude du délai d'action des antidépresseurs.

AIMS: To provide a journal publishing all literature relevant to the time of response to antidepressants, in order to find out any existing consensus which might bring in fine recommendations for a thorough study of the concept. Three sections corresponding to 3 articles surveyed by the Encephale deal with the pharmacological, clinical, and metho-dological aspects. METHOD: A group of experts (see conclusion) was trained on the initiative of the authors, following a think-talk at a meeting of scientific learned societies (FUAG and AFPB in particular). Dr Sophie Banzet, from the Boehringher-Ingelheim Laboratories, attended the meeting as a partner supporting this project. The article tries to give the most faithfull coverage of the main topics discussed by the group of experts, allowing each author to highlight his own contribution after the rereading of his text by the two other authors of the group. Are also included in the analysis the major studies published in the literature at the date when the present article was submitted. RESULTS AND CONCLUSION: There are no unequivocal criteria to assess or identify the time of response to antidepressants on a pharmacological level. In fact, the criteria depend on the symptomatic impact of the antidepressant while depression cannot be defined as a single-symptom pathology. The criteria retained for assessment depend on the chosen definition which is: either quantitative (patients profiles global method), or qualitative (sympto-matic approach). One should discriminate between the assessment of the time of response and the stage proving the efficacy of the antidepressant. The threshold proposed for efficacy is based on the values of the thresholds used in clinical tests and in literature, as well as those commonly accepted by the various drug agencies. Choosing the variable for the response to antidepressants must take into account the popu-lation of patients responding to treatment. A consensus on the choice of comparators can be observed in studies on the time of response to antidepressants: using a placebo is necessary. The active reference product depends on the population concerned. The statistical methods which have been used, in particular the survival analysis methods are very useful indeed but they entails loss of information and must be completed by tests which, though simpler, allow simpler conclusions (such as c(2)). Lastly, the experimental level, which aims to assess the time of response, remains by far the most difficult part. It is still unsolved and the study aiming to prove efficacy. We have not included the considerations on this particular point as this would require an article in its own right. The cognitive symptoms belonging to the DSM IV diagnosis criteria in major depressive periods can help to build new tools.

[Management of American cutaneous leishmaniasis. Outcome apropos of 326 cases treated with high-dose pentamidine isethionate]. / Thérapeutique des leishmanioses tégumentaires du Nouveau Monde. Expérience à propos de 326 cas traités par iséthionate de pentamidine à fortes doses.

The purpose of this retrospective analysis was to evaluate the outcome of pentamidine isethionate treatment (4 mg/kg of Pentamidine by the intramuscular route on Days 1 and 3) of cutaneous leishmaniasis in 326 cases that occurred during an outbreak among French military personnel in French Guyana from 1998 to 1999. A great difference was found between the 205 patients treated in French Guyana (series G) and 32 patients treated at the Laveran Military Hospital in Marseille, France (series L). Failure rate, i.e. 25% in series L versus 5% in series G, was significantly correlated with the delay to treatment which was much longer in series L. Extensive rhabdomyolysis was observed in all cases tested: this side-effect has not been reported. Based on these findings and a review of the literature on pentamidine isethionate, the authors recommend prompt treatment using lower doses. Other treatment alternatives for American cutaneous leishmaniasis are also presented including two of the latest developments in the field, i.e., oral treatment using miltefosine and topical treatment using agents such as paromomycine.

[Physical activity in the heat: physiology of hydration recommendations]. / L'activité physique à la chaleur: de la physiologie aux recommandations d'apport hydrique.

Physical exercise in the heat causes severe disturbances in homeostasis. The need for evaporative thermolysis is increased due to the combination of endogenous and exogenous heat production. Despite a marked increase in cardiac output, muscles and skin must compete for sufficient blood flow. In addition progressive dehydration can impair the ability of the cardiocirculatory to adjust adequately. The most serious risk associated with exercise in a hot environment is heat stroke. Although deleterious effects of dehydration occur only if large amounts of water and electrolytes are lost without being replaced, even moderate fluid depletion can reduce both physical and cognitive performance. Another mechanism by which heat exposure directly affects performance involves core temperature elevation which can induce profound changes in muscular activity and energy consumption, thereby accelerating exhaustion. Prevention of deleterious effects on health and performance requires an effective rehydration strategy to maintain body fluid balance. This strategy must optimize all three potentially limiting factors for fluid replacement, i.e., fluid intake, gastric emptying, and intestinal absorption. Practical guidelines are given to answer the questions of when, what and how much to drink.

[Clinical overview of New World tegumentary leishmaniasis]. / Panorama clinique des leishmanioses tégumentaires du Nouveau Monde.

This richly illustrated article (80 color photographs) based on the authors' experience in French Guyana documents the clinical diversity of American tegumentary leishmaniasis. Main highlights include the often outstanding aspect of lesions, the high frequency of forms not associated with ulceration or scab formation that must be recognized to achieve diagnosis in travellers returning from endemic zones, and the special prognosis of clinical forms associated with intradermic, lymphatic or hematogenous spread. The article also reviews an original diagnostic method based on culture of cutaneous biopsy specimens on specific nutrient mediums that provides isolates in a high percentage of cases (80%) and thus allows identification of offending parasite.

[Epidemiologic surveillance of cutaneous leishmaniasis in Guiana. Summary of military data collected over 10 years]. / Surveillance épidémiologique des leishmanioses tégumentaires en Guyane. Synthése de données militaires sur 10 ans.

This report describes the results of epidemiological surveillance of cutaneous leishmaniasis in French military personnel in French Guiana. Data was collected regarding microscopic diagnosis, clinical manifestations, and lesion location as well as compliance with vector control measures. Year-to-year variations in the incidence in the general population have been attributed to changes in climatic conditions. Monitoring incidence and density curves, correlation of findings with local epidemiological data, and analysis of the most recent epidemic in 1998/99 (326 cases, attack rate 3.2% men years) highlight the importance of behavioral factors. The proportion of total cases involving military personnel varied widely from 20 to 85%. Investigation consistently showed that failure to apply elementary protective measures against sandfly bites was the most determinant factor in this proportion. Strict compliance with these measures appears to reduce the risk of infection considerably.

[Epidemics of cutaneous leishmaniasis in military personnel working in French Guiana]. / Epidémies de leishmanioses cutanées chez les militaires en opération en Guyane française.

Cutaneous leishmaniasis transmitted by phlebotomine sandflies is endemic in the rain forests of French Guyana. The 3rd Régiment Etranger d'Infanterie, based in Kourou carries out numerous operations in the Amazonian areas. In 1998 two outbreaks of cutaneous leishmaniasis occurred: one during an exercise at the training center in the equatorial forest of Regina (10 patients) and the other during a mission in Saint Elie (21 patients). Clinical findings were variable and diagnosis was confirmed by skin smear tests. Patients were treated by two intramuscular injections of pentmidine isethionate (Pentacarinate). Recurrence was observed in two patients who were retreated using the same agent. Persistent lesions were treated by intralesional injection of meglumine antimoniate (Glucantime). Both outbreaks were characterized by high attack rates (91 p. 100 and 84 p. 100) and were facilitated by non-observance of standard procedures because of training or operational requirements at the beginning of the leishmaniasis season. Strict planning of activities, wearing protective clothing, deployment of insecticide treated bed nets, and of candles rather than electrical lamps for lighting are key preventive measures. Greater emphasis is needed on the use of insect repellents.