Carbamazepine directly inhibits adipocyte differentiation through activation of the ERK 1/2 pathway.

BACKGROUND AND PURPOSE: Carbamazepine (CBZ), known for its anti-epileptic, analgesic and mood-stabilizing properties, is also known to induce weight gain but the pathophysiology of this adverse effect is still largely unknown. We tested the hypothesis that CBZ could have a direct effect on adipocyte development and metabolism. EXPERIMENTAL RESEARCH: We studied the effects of CBZ on morphological biochemical and molecular markers of adipogenesis, using several pre-adipocyte murine cell lines (3T3-L1, 3T3-F442A and T37i cells) and primary cultures of human pre-adipocytes. To delineate the mechanisms underlying the effect of CBZ, clonal expansion of pre-adipocytes, pro-adipogenic transcription factors, glucose uptake and lipolysis were also examined. KEY RESULTS: CBZ strongly inhibited pre-adipocyte differentiation and triglyceride accumulation in a time- and dose-dependent manner in all models. Pleiotropic mechanisms were at the basis of the inhibitory effects of CBZ on adipogenesis and cell lipid accumulation. They included suppression of both clonal expansion and major adipogenic transcription factors such as PPAR-γ and CCAAT/enhancer binding protein-α, activation of basal lipolysis and decrease in insulin-stimulated glucose transport. CONCLUSIONS AND IMPLICATIONS: The effect of CBZ on adipogenesis involves activation of the ERK1/2 pathway. Our results show that CBZ acts directly on pre-adipocytes and adipocytes to alter adipose tissue development and metabolism.

Immunohistochemical and molecular analyses of HER2 status in breast cancers are highly concordant and complementary approaches.

BACKGROUND: Immunohistochemistry (IHC) and fluorescent in situ hybridisation (FISH) are currently the most commonly used methods to assess HER2 status. PCR-based assays allow quantitative determination of HER2 amplification (Q-PCR) or overexpression (Q-RT-PCR), but are not routinely used. We evaluated the relevance of Q-RT-PCR for HER2 status determination. METHODS: We compared IHC and Q-RT-PCR in 466 breast tumours. In discordant or equivocal cases, five additional methods (IHC with two other antibodies, FISH, silver in situ hybridisation (SISH) and Q-PCR) were combined to determine HER2 status. Two cases with HER2 intra-tumour heterogeneity were further explored by allelic profiles analysis and HUMARA clonality determination after microdissection. RESULTS: We observed 97.3% concordance between Q-RT-PCR and non-equivocal IHC. Twelve out of 466 cases (3%) revealed discordances between the two methods. The power of Q-RT-PCR to predict HER2 status (defined by seven methods) was similar to that of IHC. Although rare, some discordances between techniques might be due to HER2 intra-tumour heterogeneity and we report two examples, one tumour containing two distinct clones, another tumour consisting of HER2 amplified and non-amplified subclones. CONCLUSION: Q-RT-PCR and IHC are highly concordant methods for HER2 status assessment, and Q-RT-PCR allows a highly reliable quantitative assessment and could be a useful adjunct to IHC.

Evidence of avian metapneumovirus subtype C infection of wild birds in Georgia, South Carolina, Arkansas and Ohio, USA.

Metapneumoviruses (MPVs) were first reported in avian species (aMPVs) in the late 1970s and in humans in 2001. Although aMPVs have been reported in Europe and Asia for over 20 years, the virus first appeared in the United States in 1996, leaving many to question the origin of the virus and why it proved to be a different subtype from those found elsewhere. To examine the potential role of migratory waterfowl and other wild birds in aMPV spread, our study focused on determining whether populations of wild birds have evidence of aMPV infection. Serum samples from multiple species were initially screened using a blocking enzyme-linked immunosorbent assay. Antibodies to aMPVs were identified in five of the 15 species tested: American coots, American crows, Canada geese, cattle egrets, and rock pigeons. The presence of aMPV-specific antibodies was confirmed with virus neutralization and western blot assays. Oral swabs were collected from wild bird species with the highest percentage of aMPV-seropositive serum samples: the American coots and Canada geese. From these swabs, 17 aMPV-positive samples were identified, 11 from coots and six from geese. Sequence analysis of the matrix, attachment gene and short hydrophobic genes revealed that these viruses belong to subtype C aMPV. The detection of aMPV antibodies and the presence of virus in wild birds in Georgia, South Carolina, Arkansas and Ohio demonstrates that wild birds can serve as a reservoir of subtype C aMPV, and may provide a potential mechanism to spread aMPVs to poultry in other regions of the United States and possibly to other countries in Central and South America.

Changes in allelic imbalances in locally advanced breast cancers after chemotherapy.

In advanced breast cancers, TP53 mutation is highly predictive of complete response to high-dose epirubicin/cyclophosphamide chemotherapy. In these tumours with an altered control of genomic stability, accumulation of chemotherapy-induced genetic alterations may contribute to cell death and account for complete response. To explore the effects of chemotherapy on stability of the tumour genome, allelic profiles were obtained from microdissected tumour samples before and after chemotherapy in 29 unresponsive breast cancers (9 with TP53 mutation). Ninety-four per cent allelic profiles remained unchanged after treatment. Interestingly, 11 profiles (6%) showed important changes after treatment; allelic imbalances significantly increased (four cases) or decreased (seven cases) after chemotherapy in three distinct experiments, two of which using laser microdissected tumour cells. These genetic changes were not linked to the TP53 status, but one tumour showed complete disappearance of TP53-mutated cells in the residual tumour after treatment. Altogether, these observations carry important implications for the clonal evolution of breast cancers treated with DNA-damaging agents, as they point both to the importance of tumour heterogeneity and chemotherapy-driven selection of subclones.

[Management of intussusception in France in 2004: investigation of the Paediatric Infectious Diseases Group, the French Group of Paediatric Emergency and Reanimation, and the French Society of Paediatric Surgery]. / Modalités de prise en charge des invaginations intestinales aiguës en France en 2004: enquête commune du Groupe de pathologie infectieuse pédiatrique (GPIP), du Groupe francophone d'urgences et de réanimation pédiatrique (GFRUP) et de la Société française de chirurgie pédiatrique (SFCP).

OBJECTIVES: To describe the different pathways of management of intussusception (IS) in infants and children in metropolitan France and to identify paediatric emergency centres that might constitute a surveillance network for IS. MATERIAL AND METHODS: A questionnaire was sent to 273 paediatric emergency centres distributed across France in 2005. Modalities of diagnosis and treatment of IS had to be precised. RESULTS: One hundred and sixty-seven centres (61.2%) responded. The response was given by 131 paediatricians (78.4%) and 36 surgeons (21.6%) working in 38 universitary hospitals (22.7%) and 129 general hospitals (77.2%). The mean number of IS treated in each centre in 2004 was 11+/-13.5 (extr. 0 to 70; median 6). Diagnosis of IS required a collaboration between medical and surgical teams in 51.5% of the centres, but in 40.1% the sole medical team was in charge of the diagnosis. Ultrasonography is used for diagnosis by 98.8% of the centres. Reduction with hydrostatic enema and eventually surgery was performed in the same hospital in 44.3%. Other centres systematically or frequently transferred the patients for reduction, mostly towards universitary hospitals (90%). CONCLUSION: The procedures of IS diagnosis are the same everywhere in France but the pathways of therapeutic management do vary, depending on the availability of surgeons and anaesthetists trained in paediatrics on each site. These disparities will probably change with the implementation of the new plan for sanitary organization in children and adolescents in France. Labellized paediatric emergency centres will gather more surgical patients and could eventually constitute an effective surveillance network for IS.

[Babywalker use (baby-trot, youpala): an unsafe practice]. / Utilisation du trotteur (baby-trot, youpala): une conduite à risque.

AIM: Epidemiological analysis of accidents related to babywalker use admitted to a pediatric emergency department. METHODS: Retrospective, descriptive study of injuries related to babywalkers admitted to the pediatric emergency department between January 1st, 2003 and December 31st, 2005. RESULTS: One hundred and seventy-eight children were admitted due to an accident related to babywalker use. The sex ratio was 1.7 with a male prevalence. Mean age was 11+/-4 months. Seventy-eight percent of babywalker-related injuries were attributable to fall down a flight of stairs. The mean number of steps that a child fell down was 7 (range 1-20 steps). The repartition of accidents was bimodal: during the year, 1 peak in May and 1 in October; during the week: 54% of the cases occurred on Thursday or on the weekend; during the day (1 peak between 10 a.m. and 1 p.m. and 1 peak between 4 p.m. and 7 p.m.). Non-severe head traumas represented the most frequent injury (72%). Twenty-one children were hospitalised for concussion (N=15), cranial fractures (N=3), forearm fracture (N=1), dental subluxation (N=1) and extradural hematoma (N=1). A social problem (families with unsafe domestic practices) was identified in 26 children (15%), 16 of these situations were recognized due to the retrospective character of the study and the analysis of hospital admissions after the first accident. CONCLUSION: Stairway related falls associated with babywalker use and fall down in the stairs are very frequent in children less than 1 year-old. This resulted in babywalkers being prohibited in Canada since 2004. In several countries, advocates are working to ban babywalkers. Active or passive prevention methods have shown their limits. This unsafe and dangerous practice should be banned in France.

[Necrotizing pneumonia and arthritis due to Staphylococcus aureus producing Panton and Valentine leukocidin in a 10-year-old boy]. / Pneumonie nécrosante et ostéoarthrite multifocale à Staphylococcus aureus producteur de la leucocidine de Panton et Valentine chez un garçon de 10 ans.

Staphylococcal necrotizing pneumonia producing the Panton Valentine leukotoxin (PVL) has been described for many years. The french reference center for staphylococcal toxaemia defined it with precision in 1999. A 10-year-old child, died in 36 hours from respiratory distress and shock. Staphylococcal pneumonia was suspected then confirmed: S. Aureus producing PVL was isolated in lung, blood and articulations.

[Severe chloralose intoxication in a toddler]. / Intoxication sévère par le chloralose chez un nourrisson.

UNLABELLED: We report a case of an accidental intoxication in a 20-month-old boy resulting from the ingestion of a rodenticide containing alpha-chloralose. CASE REPORT: Three hours after initial admission to the pediatric emergency department for wheezing bronchitis, this patient was readmitted with a clinical presentation of respiratory insufficiency, a Glasgow coma score of 9 alternating with agitation, areflexia and unilateral mydriasis. Parental interview revealed he had episodes of shaking in the afternoon. Chest x-ray showed thoracic distension. Blood investigations, electrocardiogram, cardiac echography, brain CT scan and CSF were normal. Electroencephalography registered slow delta waves 2-3 cycles/min and an aspect of degraded waves and spikes. The patient was transferred to the intensive care unit where he fully recovered within 48 hours. A second parental interview and clinical presentation confirmed an intoxication with a rodenticide containing alpha-chloralose. The late clinical orientation did not allow us to perform a urinanalysis. DISCUSSION: Clinical association of coma, spontaneous or triggered myoclonias and bronchial hypersecretion are indicative of chloralose intoxication. Presence of specific abnormalities on electroencephalogram and a positive Fujiwara-Ross reaction in an urine sample are additional elements for the diagnosis. The prognosis is usually good after early management which combines gastric lavage, activated charcoal, sedation with benzodiazepines, tracheal intubation and artificial ventilation if required. Severe clinical cases described in voluntary intoxications in adults and teenagers occur very rarely in toddlers.

[Lethal aortic dissection in a 13-year-old boy with a vascular Ehlers-Danlos syndrome]. / Dissection aortique fatale chez un garçon de 13 ans atteint d'un syndrome d'Ehlers-Danlos vasculaire.

UNLABELLED: Vascular Ehlers-Danlos syndrome(EDS) is at high risk of death by arterial rupture. CASE REPORT: A 13-year-old boy with vascular EDS and aortic dissection was admitted in pediatric emergency care unit. The children died after surgery by massive hemorrhage and prosthetic rupture. DISCUSSION: The precocity of this vascular accident EDS is uncommon. It occurred despite clinical and echocardiographic follow-up. The death of the children confirmed the difficulties in surgery of vascular manifestation, even if appropriate and rapid management was already done. CONCLUSION: Patients with vascular EDS must be identified because of its lethal complications. Friability of the vessels makes surgical treatment difficult. A trial using beta blockers therapy is ongoing.

[Congenital galactosaemia: an unusual presentation]. / La galactosémie congénitale: une révélation singulière.

Congenital galactosaemia reveals usually in the second and third weeks of life with a severe liver dysfunction. We report on a case of congenital galactosaemia with, on the one hand, an early onset liver failure, without any free interval, and on the other hand, an hemophagocytic syndrome as a severe secondary outbreak with pulmonary haemorrhage. Appropriate diet led to normalisation of liver function. Hemophagocytosis, probably linked to an associated Klebsiella Pneumoniae sepsis, had a favourable outcome after antibiotic and corticosteroid therapy.

Long-term effects on tibial growth after intraosseous infusion: a prospective, radiographic analysis.

BACKGROUND: Evaluate, by radiographic analysis, tibial growth after an intraosseous infusion (IOI) in a pediatric population. METHODS: We performed a prospective simple blind study, between January 1, 1994, and July 1, 2001, which included pediatric patients who needed an intraosseous trocar in emergency situations. During the follow-up, roentgenographs were performed. On each radiologic view, different measurements were carried out: anterior and lateral tibial length, anterior and lateral width at 2 diaphyseal levels. We compared the anterior length values to those published in the Anderson et al tables. When only one tibia was punctured, the mean measurements were compared with the control leg measurements using a paired t test. RESULTS: The initial population included 78 patients. Of these 78 subjects, 42 died, 10 families could not be contacted, and one refused to participate. Two children were excluded because they had other conditions that could influence tibial growth. The study included 23 children. The puncture site was the proximal tibia. The mean age was 18.6 months at the time of IOI, the mean time of infusion was 5 hours, and the mean perfused volume was 225 mL. The mean radiologic follow-up time was 29.2 months. When compared with the Anderson et al tables, all the anterior length values were within the 95% confidence interval. For the other measurements, the statistical analysis showed no significant difference between punctured and control legs. CONCLUSION: There is no long-term effect on tibial growth after an IOI when the IO trocar is properly placed.

Effect of mutated TP53 on response of advanced breast cancers to high-dose chemotherapy.

TP53 activation by genotoxic drugs can induce apoptosis or cell-cycle arrest. Thus, whether the gene is mutated or wild type could affect the response of a tumour to chemotherapy. Clinical data are unclear, possibly as a result of heterogeneity of tumours, drugs, methods of assessing response, or TP53 status. We studied 50 non-inflammatory, locally advanced breast cancers that had been treated with high doses of a combination of epirubicin and cyclophosphamide. We noted eight complete responses, which all occurred in the 14 patients with tumours containing mutated TP53 (p<0.0001). In high-grade, advanced breast cancers, inactivation of the TP53 pathway could greatly improve the response to this chemotherapy regimen.

Allelic loss detection in inflammatory breast cancer: improvement with laser microdissection.

Solid tumors are composed not only of tumor cells but also of stromal nonneoplastic cells. In whole tumor samples, stromal cells retaining their alleles may therefore obscure detection of loss of heterozygosity (LOH) in tumor cells. An increasing number of studies have used laser-assisted tissue microdissection to improve LOH detection, but the real gain in sensitivity has been poorly quantified. We studied a group of 16 inflammatory breast carcinomas that were submitted to both standard DNA extraction from frozen whole tumor samples and laser microdissection performed on paraffin-embedded tumor samples. Using PCR with fluorescence-labeled primers, we comparatively analyzed ten polymorphic markers with both sources of DNA. With the LOH detection threshold set at -25%, we showed that 25 LOHs could not be diagnosed with whole tumor samples out of 73 LOHs positively diagnosed in microdissected samples (34%). With the LOH detection threshold set at -50%, the respective figures were 39 LOHs not diagnosed out of 55 LOHs (71%). Measuring the intensity of the allelic decrease, we showed that the mean decrease of the lost allele is -34% with whole tumor samples and -67% with microdissected samples. The increase in sensitivity of LOH detection with microdissection is associated with the density of stromal cells. This strong improvement in LOH detection in this aggressive type of breast cancer indicates that many other molecular studies performed on heterogeneous solid tumors may benefit from a first step of laser microdissection.

Stress-induced aberrant splicing of TSG101: association to high tumor grade and p53 status in breast cancers.

The TSG101 gene, identified through insertional mutagenesis, is localized in a region that exhibits LOH in human cancers, suggesting that TSG101 might be a tumor suppressor gene. Numerous studies have then shown the presence of abnormal transcripts in various tumors which appear to result from aberrant splicing of the gene, rather than from intragenic deletions. Moreover, many studies demonstrated that these aberrantly spliced transcripts were not found in matched normal tissues. We have analysed TSG101 transcripts in 85 breast cancer samples and found that abnormal splicing of the gene is tightly correlated with tumor grade and p53 mutation. In addition, stress induced the appearance of these abnormal transcripts in primary lymphocytes. Hence, TSG101 splicing defects, while unrelated to the oncogenic process per se, could reflect the cellular environment of the tumor cells. The proposed role of stress and hypoxia to select p53 mutant cells could account for the tight association with p53 status.

Dictyostelium discoideum cells shed vesicles with associated DNA and vital stain Hoechst 33342.

Dictyostelium discoideum cells are highly resistant to xenobiotics. We previously observed that these primitive eukaryotic cells contain a 170-kDa P-glycoprotein, mediating multidrug resistance in mammalian cells, but nonfunctional in Dictyostelium cells. We show here that D. discoideum cells vitally stained with the DNA-specific dye, Hoechst 33342, release fluorescent material in their culture medium. Electron microscopy and lipid analysis demonstrate the vesicular nature of this material. Moreover, nucleic acids associate with these extracellular vesicles independently of Hoechst vital staining. The main vesicular DNA component exhibits a size > 21 kb. Shedding of microvesicles during cell growth is not concomitant with programmed cell death. We propose that these extracellular vesicles are involved in a new cellular resistance mechanism against xenobiotics. Furthermore, since the association of DNA with vesicles occurs in physiological growth conditions and independently of vital staining, the new shedding process might be involved in a more general intercellular mechanism.

In adrenocortical tissue, annexins II and VI are attached to clathrin coated vesicles in a calcium-independent manner.

We have previously characterized three populations of clathrin coated vesicles (CCVs) isolated from bovine adrenocortical tissue and designated them as large, medium and small coated vesicles, i.e., LCV, MCV and SCV, respectively. Here, we show that annexins II and VI, two of the annexins involved in membrane traffic, are present in the three populations of CCVs but with different distributions between coat proteins (CP) and lipidic vesicle membrane. Annexin VI is only associated with the membrane, whatever the CCV population. In contrast, annexin II is differently distributed between coat and membrane, depending on the CCV population. Both annexins are bound to membranes in a calcium-independent manner and solubilization studies in Triton X114 (TX114) suggest that they interact poorly with lipids by hydrophobic interactions. Ligand blotting experiments show that both annexins bind to CCV proteins: annexin II to a 200-kDa component in all CCVs and annexin VI to a 100-kDa component in LCV and SCV identified as dynamin, a GTPase essential for endocytic CCV pinching off. Dynamin is tightly associated to annexin VI only in LCVs, the endocytic [transferrin (Tf) positive] vesicles. Our data suggest that annexins II and VI could define specific protein-lipid interaction microdomains that could play a role in the different functions of the CCVs.

Specific lipid protein interactions characterize 3 populations of clathrin coated vesicles involved in the LDL receptor traffic.

We have previously isolated 3 different populations of clathrin coated vesicles (CCV) involved in the LDL-receptor traffic in bovine adrenal cortex. We now show that each CCV type contains the transferrin-R and the CI-MPR, therefore, they provide a good model for studying the membrane organization that may govern their targeting in one of the biosynthetic, endocytic and/or recycling pathways. Transferrin--prototype of recylcing ligand--, and alpha adaptin, dynamin and the 110 kDa phosphatidylinositol-3-kinase subunit--of the trafficking machinery--were mainly detected in only 2 of the vesicle populations which could be involved in the endocytic/recycling pathway. The third population which contained larger amounts of gamma adaptin and do not carry transferrin could be involved in the biosynthetic pathway. The vesicle lipid pattern and the saturation of their fatty acyl chains were analyzed and confirmed these results. The nature of the interactions between vesicle components was then determined using several classes of detergents. Only non ionic ones could solubilize the LDL-R in a complex with either alpha or gamma adaptin. In contrast, they dissociated clathrin or beta-beta' adaptins. Taken together these results prompt us to suggest an integrated model for targeting in membrane traffic. Besides specific targeting signals carried by cargo proteins and recognized by proteins from the coat and the cytosolic trafficking machinery, lipids would play a key modulatory role. At each step in the membrane traffic, the proteins which carry multiple targeting signals would interact transiently with a specific set of lipids. This would result in the exposure of the appropriate targeting signals which could now become recognized by the proper targeting machinery.

Musculo-articular modelling of the triceps brachii.

With the aim of proposing modelling equations for the length, force and shortening velocity of the triceps brachii, an X-ray and biomechanical study was carried out on the elbow joint. Two series of three upper right limbs removed from cadavers were used. It allowed us to situate the articular rotational axis of the elbow and to modelise the lever arm of the muscle group of the elbow extensors. Eight superimposed X-ray films with opaque markers at different articular angles show that the rotational axis of the elbow is different from the epitroch-lea-epicondyle axis, commonly localised by palpation and used as such in functional and biomechanical explorations. This rotational axis of the elbow, situated in the trochlea, is fixed at the centre of the articular range and moves noticeably to both extremities of this range. Biomechanical study, based on measurement of internal and external torques established using an overload, allowed us to modelize the lever arm of the triceps brachii between 0 and 2.44 radians, according to length of the ulna. The applications concerning length, force and shortening velocity of the triceps brachii are described. This method of modelling is applicable to other joints and muscle groups.

Subjective estimates of sleep differ from polysomnographic measurements in obstructive sleep apnea patients.

It is well established that, as a group, insomnia patients overestimate sleep onset latency (SOL) and underestimate total sleep time (TST) when compared to objective polysomnographic (PSG) findings. Whether a similar phenomenon occurs with other sleep disorders is not fully established. We compared the PSG sleep of 84 patients with suspected sleep apnea (SA) to their subjective experience of sleep reported on a sleep diary the morning after PSG testing. Both patients with SA (SA+) and those without (SA-) tended to overestimate SOL, but the SA+ group (n = 50) made larger overestimations (p < 0.02). The SA+ and SA- groups also differed in their accuracy at estimating TST, with SA+ patients underestimating TST (p < 0.05). These findings support the premise that marked discrepancies between subjective and PSG-determined sleep may not be limited to insomnia, but present in other sleep disorders as well, and should be appreciated by practitioners when obtaining sleep histories.