Persistent inhibition of FLIP(L) expression by lentiviral small hairpin RNA delivery restores death-receptor-induced apoptosis in neuroblastoma cells.

Neuroblastoma represents the most common and deadly solid tumour of childhood, which disparate biological and clinical behaviour can be explained by differential regulation of apoptosis. To understand mechanisms underlying death resistance in neuroblastoma cells, we developed small hairpin of RNA produced by lentiviral vectors as tools to selectively interfere with FLIP(L), a major negative regulator of death receptor-induced apoptosis. Such tools revealed highly efficient in interfering with FLIP(L) expression and function as they almost completely repressed endogenous and/or exogenously overexpressed FLIP(L) protein and fully reversed FLIP(L)-mediated TRAIL resistance. Moreover, interference with endogenous FLIP(L) and FLIP(S) significantly restored FasL sensitivity in SH-EP neuroblastoma cell line. These results reveal the ability of lentivirus-mediated shRNAs to specifically and persistently interfere with FLIP expression and support involvement of FLIP in the regulation of death receptor-mediated apoptosis in neuroblastoma cells. Combining such tools with other therapeutic modalities may improve treatment of resistant tumours such as neuroblastoma.

LACK-reactive CD4+ T cells require autocrine IL-2 to mediate susceptibility to Leishmania major.

Mice from most inbred strains are resistant to infection with Leishmania major whereas mice from BALB strains are highly susceptible. Resistance and susceptibility result from the development of Th1 or Th2 cells, respectively. In this report, we document an IL-2 mRNA burst, preceding the reported early IL-4 response, in draining lymph nodes of susceptible mice infected with L. major. Neutralization of IL-2 during the first days of infection redirected Th1 cell maturation and resistance to L. major, through interference with the rapid IL-4 transcription in Leishmania homolog of mammalian RACK1 (LACK)-reactive CD4(+) cells. A burst of IL-2 transcripts also occurred in infected C57BL/6 mice that do not mount an early IL-4 response. However, although the LACK protein induced IL-2 transcripts in susceptible mice, it failed to trigger this response in resistant C57BL/6 mice. Reconstitution experiments using C.B.-17 SCID mice and LACK-reactive CD4(+) T cells from IL-2(-/-) BALB/c mice showed that triggering of the early IL-4 response required autocrine IL-2. Thus, in C57BL/6 mice, the inability of LACK-reactive CD4(+) T cells to express early IL-4 mRNA transcription, important for disease progression, appears due to an incapacity of these cells to produce IL-2.

Cricotracheal resection for pediatric subglottic stenosis.

Until recently, cricotracheal resection (CTR) has not been commonly accepted as a treatment modality for severe subglottic stenosis in the pediatric age group. The reasons have included the risk of a possible dehiscence at the site of the anastomosis, the likelihood of injury to the recurrent laryngeal nerves, and the interference with normal growth of the larynx. Thirty-eight infants and children with a severe subglottic stenosis underwent a partial cricoid resection with primary thyrotracheal anastomosis. Thirty-three patients were tracheotomy-dependent at the time of surgery and 34 were referred cases; 27 were classified as grade III, and 10 as grade IV stenoses according to new Cotton's classification. Nineteen patients were younger than 3 years of age at the time of surgery. The tracheotomy was resected during the surgical procedure in 21 cases. Decannulation was achieved in 36/38 cases after an open procedure. There is one complete restenosis and one good result awaiting decannulation after further surgery for a Pierre Robin syndrome. The authors experienced no lesion of the recurrent laryngeal nerves and no fatality. Thirty-one patients show no exertional dyspnea, three a slight stridor while exercising, and two patients are not decannulated. The postoperative follow-up in longer than 10 years in eight cases. All patients show a normal growth of the larynx and trachea. Compared to laryngotracheoplasties, CTR gives better results for severe subglottic stenosis. This operation should become the treatment of choice for severe (grade III and IV) subglottic stenosis in infants and children.

Acquired epileptic dysgraphia: a longitudinal study.

A male presenting with benign partial epilepsy with rolandic spikes from the age of 7 years was evaluated at age 11 years for worsening of his epilepsy associated with a specific regression of graphomotor skills. A longitudinal study over nearly 2 years showed an improvement in handwriting to an almost normal level under modified antiepileptic therapy. A detailed analysis with a computer-monitored graphics table showed at first a rapid improvement of skills followed by protracted slower progress. We argue that the initial rapid recovery of skills was directly linked to the improvement of his epilepsy. The slower late acquisition of motor programmes that had never been fully established was due to long-standing interference by his epilepsy. The specificity of the deficit within the graphomotor system and its possible neurobiological basis are also discussed. The analytical method and approach used in a single patient might provide an example for other patients in whom epilepsy can interfere in the acquisition, progress, and maintenance of new skills and can be responsible for selective deficits.

Influence of inter-electrode distance, contraction type, and muscle on the relationship between the sEMG power spectrum and contraction force.

INTRODUCTION: Spectral frequencies of the surface electromyogram (sEMG) increase with contraction force, but debate still exists on whether this increase is affected by various methodological and anatomical factors. This study aimed to investigate the influence of inter-electrode distance (IED) and contraction modality (step-wise vs. ramp) on the changes in spectral frequencies with increasing contraction strength for the vastus lateralis (VL) and vastus medialis (VM) muscles. METHODS: Twenty healthy male volunteers were assessed for isometric sEMG activity of the VM and VL, with the knee at 90° flexion. Subjects performed isometric ramp contractions in knee extension (6-s duration) with the force gradually increasing from 0 to 80 % MVC. Also, subjects performed 4-s step-wise isometric contractions at 10, 20, 30, 40, 50, 60, 70, and 80 % MVC. Interference sEMG signals were recorded simultaneously at different IEDs: 10, 20, 30, and 50 mm. The mean (F mean) and median (F median) frequencies and root mean square (RMS) of sEMG signals were calculated. RESULTS: For all IEDs, contraction modalities, and muscles tested, spectral frequencies increased significantly with increasing level of force up to 50-60 % MVC force. Spectral indexes increased systematically as IED was decreased. The sensitivity of spectral frequencies to changes in contraction force was independent of IED. The behaviour of spectral indexes with increasing contraction force was similar for step-wise and ramp contractions. CONCLUSIONS: In the VL and VM muscles, it is highly unlikely that a particular inter-electrode distance or contraction modality could have prevented the observation of the full extent of the increase in spectral frequencies with increasing force level.

Coffee consumption and digestive tract cancers.

The relationship between coffee drinking and the risk of digestive tract neoplasms was analyzed in a case-control study of 50 cases of cancer of the mouth or pharynx, 209 of the esophagus, 397 of the stomach, 455 of the colon, 295 of the rectum, 151 of the liver, 214 of the pancreas, and 1944 control subjects admitted for acute, non-digestive tract disorders. There was no significant or consistent association between coffee and cancers of the mouth or pharynx, esophagus, stomach, liver, or pancreas. In particular, for pancreatic cancer, the multivariate relative risks for the intermediate and upper tertiles were 1.05 and 1.01, respectively. There were significant inverse trends in risk with measures of coffee consumption for colon and rectal cancers, the multivariate relative risks according to tertiles of coffee consumption being 0.86 and 0.64 for colon and 0.97 and 0.66 for rectum. This apparent protection is in agreement with some (but not all) previous epidemiological evidence and finds a possible biological interpretation in terms of interference on bile secretion, causing reduced bile acid and neutral sterol concentrations in the bowel. In conclusion, the results of this study, the major interest of which lies in the opportunity of drawing up an overall pattern of risk for various digestive neoplasms, offer further reassurance as regards the effects of coffee on digestive tract carcinogenesis.

Adaptive filtering methods for identifying cross-frequency couplings in human EEG.

Oscillations have been increasingly recognized as a core property of neural responses that contribute to spontaneous, induced, and evoked activities within and between individual neurons and neural ensembles. They are considered as a prominent mechanism for information processing within and communication between brain areas. More recently, it has been proposed that interactions between periodic components at different frequencies, known as cross-frequency couplings, may support the integration of neuronal oscillations at different temporal and spatial scales. The present study details methods based on an adaptive frequency tracking approach that improve the quantification and statistical analysis of oscillatory components and cross-frequency couplings. This approach allows for time-varying instantaneous frequency, which is particularly important when measuring phase interactions between components. We compared this adaptive approach to traditional band-pass filters in their measurement of phase-amplitude and phase-phase cross-frequency couplings. Evaluations were performed with synthetic signals and EEG data recorded from healthy humans performing an illusory contour discrimination task. First, the synthetic signals in conjunction with Monte Carlo simulations highlighted two desirable features of the proposed algorithm vs. classical filter-bank approaches: resilience to broad-band noise and oscillatory interference. Second, the analyses with real EEG signals revealed statistically more robust effects (i.e. improved sensitivity) when using an adaptive frequency tracking framework, particularly when identifying phase-amplitude couplings. This was further confirmed after generating surrogate signals from the real EEG data. Adaptive frequency tracking appears to improve the measurements of cross-frequency couplings through precise extraction of neuronal oscillations.

Impaired expression of the inducible cAMP early repressor accounts for sustained adipose CREB activity in obesity.

OBJECTIVEIncrease in adipose cAMP response binding protein (CREB) activity promotes adipocyte dysfunction and systemic insulin resistance in obese mice. This is achieved by increasing the expression of activating transcription factor 3 (ATF3). In this study we investigated whether impaired expression of the inducible cAMP early repressor (ICER), a transcriptional antagonist of CREB, is responsible for the increased CREB activity in adipocytes of obese mice and humans.RESEARCH DESIGN AND METHODSTotal RNA and nuclear proteins were prepared from visceral adipose tissue (VAT) of human nonobese or obese subjects, and white adipose tissue (WAT) of C57Bl6-Rj mice that were fed with normal or high-fat diet for 16 weeks. The expression of genes was monitored by real-time PCR, Western blotting, and electromobility shift assays. RNA interference was used to silence the expression of Icer.RESULTSThe expression of Icer/ICER was reduced in VAT and WAT of obese humans and mice, respectively. Diminution of Icer/ICER was restricted to adipocytes and was accompanied by a rise of Atf3/ATF3 and diminution of Adipoq/ADIPOQ and Glut4/GLUT4. Silencing the expression of Icer in 3T3-L1 adipocytes mimicked the results observed in human and mice cells and hampered glucose uptake, thus confirming the requirement of Icer for appropriate adipocyte function.CONCLUSIONSImpaired expression of ICER contributes to elevation in CREB target genes and, therefore, to the development of insulin resistance in obesity.

Augmented epithelial multidrug resistance-associated protein 4 expression in peritoneal endometriosis: regulation by lipoxin A(4).

OBJECTIVE: To compare the expression of the prostaglandin (PG) E(2) transporter multidrug resistance-associated protein 4 (MRP4) in eutopic and ectopic endometrial tissue from endometriosis patients with that of control subjects and to examine whether MRP4 is regulated by the antiinflammatory lipid lipoxin A(4) (LXA(4)) in endometriotic epithelial cells. DESIGN: Molecular analysis in human samples and a cell line. SETTING: Two university hospitals and a private clinic. PATIENT(S): A total of 59 endometriosis patients and 32 age- and body mass index-matched control subjects undergoing laparoscopy or hysterectomy. INTERVENTION(S): Normal, eutopic, and ectopic endometrial biopsies as well as peritoneal fluid were obtained during surgery performed during the proliferative phase of the menstrual cycle. 12Z endometriotic epithelial cells were used for in vitro mechanistic studies. MAIN OUTCOME MEASURE(S): Tissue MRP4 mRNA levels were quantified by quantitative reverse-transcription polymerase chain reaction (qRT-PCR), and localization was analyzed with the use of immunohistochemistry. Cellular MRP4 mRNA and protein were quantified by qRT-PCR and Western blot, respectively. PGE(2) was measured in peritoneal fluid and cell supernatants using an enzyme immunoassay (EIA). RESULT(S): MRP4 was expressed in eutopic and ectopic endometrium, where it was overexpressed in peritoneal lesions and localized in the cytoplasm of glandular epithelial cells. LXA(4) attenuated MRP4 mRNA and protein levels in endometriotic epithelial cells in a dose-dependent manner, while not affecting the expression of enzymes involved in PGE(2) metabolism. Investigations employing receptor antagonists and small interfering RNA revealed that this occurred through estrogen receptor α. Accordingly, LXA(4) treatment inhibited extracellular PGE(2) release. CONCLUSION(S): We report for the first time that MRP4 is expressed in human endometrium, elevated in peritoneal endometriosis, and modulated by LXA(4) in endometriotic epithelial cells.

Tissue-specific and SRSF1-dependent splicing of fibronectin, a matrix protein that controls host cell invasion.

Cell invasion targets specific tissues in physiological placental implantation and pathological metastasis, which raises questions about how this process is controlled. We compare dermis and endometrium capacities to support trophoblast invasion, using matching sets of human primary fibroblasts in a coculture assay with human placental explants. Substituting endometrium, the natural trophoblast target, with dermis dramatically reduces trophoblast interstitial invasion. Our data reveal that endometrium expresses a higher rate of the fibronectin (FN) extra type III domain A+ (EDA+) splicing isoform, which displays stronger matrix incorporation capacity. We demonstrate that the high FN content of the endometrium matrix, and not specifically the EDA domain, supports trophoblast invasion by showing that forced incorporation of plasma FN (EDA-) promotes efficient trophoblast invasion. We further show that the serine/arginine-rich protein serine/arginine-rich splicing factor 1 (SRSF1) is more highly expressed in endometrium and, using RNA interference, that it is involved in the higher EDA exon inclusion rate in endometrium. Our data therefore show a mechanism by which tissues can be distinguished, for their capacity to support invasion, by their different rates of EDA inclusion, linked to their SRSF1 protein levels. In the broader context of cancer pathology, the results suggest that SRSF1 might play a central role not only in the tumor cells, but also in the surrounding stroma.

Basal and stimulated lactate fluxes in primary cultures of astrocytes are differentially controlled by distinct proteins.

Lactate release by astrocytes is postulated to be of importance for neuroenergetics but its regulation is poorly understood. Basigin, a chaperone protein for specific monocarboxylate transporters (MCTs), represents a putatively important regulatory element for lactate fluxes. Indeed, basigin knockdown by RNA interference in primary cultures of astrocytes partially reduced both proton-driven lactate influx and efflux. But more strikingly, enhancement of lactate efflux induced by glutamate was prevented while the effect of sodium azide was significantly reduced by treatment of cultured astrocytes with anti-basigin small interfering RNA. Enhancement of glucose utilization was unaffected under the same conditions. Basal lactate uptake and release were significantly reduced by MCT1 knockdown, even more so than with basigin knockdown, whereas glutamate-driven or sodium azide-induced enhancement of lactate release was not inhibited by either MCT1, 2, or 4 small interfering RNAs. In conclusion, MCT1 plays a pivotal role in the control of basal proton-driven lactate flux in astrocytes while basigin is only partly involved, most likely via its interaction with MCT1. In contrast, basigin appears to critically regulate the enhancement of lactate release caused by glutamate (or sodium azide) but via an effect on another unidentified transporter at least present in astrocytes in vitro.

The category 'natural' in the discourse of couples who used medically assisted procreation

As a constantly evolving set of complex biotechnologies, medically assisted procreation (MAP) jeopardises a category that seems to be taken for granted: that of 'natural'. What is 'natural' or not when MAP is used to procreate? What are the boundaries between a 'natural' and a 'non-natural' fertilisation? Drawing upon a dialogical approach to language and cognition, our study examined the semantic field of the category 'natural' as expressed in interviews between a psychiatrist and seven couples who resorted to MAP and had to decide whether to keep their frozen pre-embryonic cells (zygotes) for further procreation or to allow them be destroyed. We examined how these couples evoked the category 'natural' and showed that in their argumentation, the category 'natural' encompassed a wide variety of phenomena, which shifted the boundaries between the 'natural' and 'non-natural'. In so doing, the couples 'renaturalised' MAP, normalized it, moved the boundaries between what is legitimate or not, and showed their accountability. Hence, reference to the category 'natural' seemed to act both as an argumentative and a psychological resource in the elaboration of the person's experience in resorting to MAP.

Dialogism and dialogicality in the study of the self

This article stems from the statement that dialogical approaches to a study of the self face a double challenge: that of developing a conception of the self that both avoids social reductionism and accounts for the stability of the self. In discussing this double challenge, we identify three much debated issues: (a) To what does the notion of "Alter" exactly refer? (b) How could we conceptualize the fact that Subject-Alter interactions are not only interpersonal but entail larger social entities, in particular institutions? (c)What importance should we attach to the materiality of objects? We discuss these three questions from two standpoints - that of linguistics and that of psychology - and illustrate our theoretical proposals with an analysis of an excerpt taken from a focus-group discussion. In conclusion, we argue that the dialogism of discourse provides us with some clues about the dialogicality of the mind, whereas the latter invites us to develop a theory showing the importance of interactions in the construction of the self, to pay more attention to the transpersonal dimension of the social, and to consider that the material world contributes to the construction of the self.