Search

Resume Introduction La frequence des tests du prostate-specific antigen (PSA) est elevee en France. Cette etude a permis d’estimer leur frequence et celles de biopsies et de cancer de la prostate (CPr) nouvellement diagnostiques selon l’existence d’une hypertrophie benigne (HBP) traitee. Patients et methodes Cette etude observationnelle a concerne les hommes de 40 ans et plus couverts par le regime general (73 % de la population de cet âge). Les informations provenaient du systeme national d’information inter-regimes de l’Assurance maladie (SNIIRAM) qui rassemble de facon individuelle des informations sur les prescriptions et prestations remboursees aux assures des differents regimes et, a l’aide d’un chainage, celles de leurs eventuelles hospitalisations fournies par le programme de medicalisation du systeme d’information (PMSI). Resultats La frequence des hommes sans CPr diagnostique (10,9 millions) avec au moins un dosage du PSA etait tres elevee en 2011 (40 ans et plus : 30 %, 70–74 ans : 56 %, 85 ans et plus : 33 % et hors HBP : 25 %, 41 % et 19 %). Les hommes avec une HBP totalisaient 9 % des hommes de l’etude en 2011, 18 % de ceux avec au moins un test du PSA, 44 % pour la biopsie et 40 % pour un CPr nouvellement diagnostique. En trois ans et hors CPr, 88 % des hommes avec une HBP avaient eu au moins un dosage du PSA et 52 % trois ou plus et, pour ceux sans HBP, 52 % et 15 %. Un an apres un test du PSA, ceux de 55–69 ans avec une HBP avaient plus frequemment une biopsie de la prostate que ceux sans HBP (5,4 % vs 1,8 %) et aussi un CPr nouvellement diagnostique (1,9 % vs 0,9 %). Conclusion En France, les frequences de dosages du PSA sont elevees meme apres exclusion des hommes avec une HBP. Ces derniers avec des frequences plus elevees de dosages du PSA et de biopsies sont plus susceptibles d’avoir un CPr pris en charge. Niveau de preuve 4.

Prostate-specific antigen (PSA) testing is high in France. The aim of this study was to estimate their frequency and those of biopsy and newly diagnosed cancer (PCa) according to the presence or absence of treated benign prostatic hyperplasia (BPH).This study concerned men 40 years and older covered by the main French national health insurance scheme (73 % of all men of this age). Data were collected from the national health insurance information system (SNIIRAM). This database comprehensively records all of the outpatient prescriptions and healthcare services reimbursed. This information are linked to data collected during hospitalisations.The frequency of men without diagnosed PCa (10.9 millions) with at least one PSA test was very high in 2011 (men aged 40 years and older: 30 %, 70-74 years: 56 %, 85 years and older: 33 % and without HBP: 25 %, 41 % and 19 %). Men with treated BPH totalized 9 % of the study population, but 18 % of the men with at least one PSA test, 44 % of those with at least one prostate biopsy and 40 % of those with newly managed PCa. Over a 3-year period, excluding men with PCa, 88 % of men with BPH had at least one PSA test and 52 % had three or more PSA tests versus 52 % and 15 % for men without BPH. One year after PSA testing, men of 55-69 years with BPH more frequently underwent prostate biopsy than those without BPH (5.4 % vs 1.8 %) and presented PCa (1.9 % vs 0.9 %).PSA testing frequencies in France are very high even after exclusion of men with BPH, who can be a group with more frequent managed PCa.4.

This article describes 3 separate methods of predicting maintenance and renewal needs for financial investment in rail infrastructure. The methods include determining and assessing financial ratios; evaluating infrastructure age; and measuring economic and physical life cycles, as well as budget constraints. In addition, the latter method enables quantification of risks that may occur due to underinvestment performance failure.

Cytomegalovirus (CMV) pneumonia is a major cause of illness in immunocompromised patients. The sites of human CMV (HCMV) latency are still not clearly defined. The present study was therefore designed to investigate the hypothesis that alveolar macrophages could constitute such a site. DNA extracted from alveolar cells collected by bronchoalveolar lavage and blood mononuclear cells (BMC) from asymptomatic nonimmunocompromised CMV-seropositive and CMV-seronegative patients were investigated. Controls consisted of DNA from a CMV-infected MRC5 cell line, BMC and alveolar macrophages from patients with acute CMV infection. Polymerase chain reaction (PCR) was designed for detection of a 290-bp fragment of the promoter region of the major immediate early gene of HCMV conserved within the various HCMV strains and without homology with the human genome. The limit of detection of the method was evaluated to be one HCMV viral copy per 10(4) cells. HCMV DNA was detected in BMC or alveolar cells of all patients with acute CMV infection. In contrast, no HCMV DNA was detected in alveolar cells and BMC from nonimmunocompromised asymptomatic HCMV-seropositive patients. In conclusion, in the present experiment, no latent HCMV could be detected in alveolar cells collected in nonimmunocompromised asymptomatic CMV-seropositive patients.

La toxoplasmose congenitale peut entrainer des sequelles graves qui peuvent s'observer durant la periode neonatale en cas de toxoplasmose severe ou plusieurs annees apres la naissance (chorioretinites). Le diagnostic prenatal de l'infection fœtale est difficile et repose classiquement sur l'echographie (7), l'amniocentese et le prelevement de sang fœtal (2). Obligatoire en France, le depistage des femmes enceintes reste controverse a l'etranger (5). L'introduction du diagnostic prenatal en France a permis de preciser les risques, d'ameliorer le suivi et le traitement in utero, de diminuer l'incidence des toxoplasmoses congenitales severes et enfin de diminuer le nombre d'interruptions de grossesse pour cette pathologie. Considerant le risque d'accidents lies au prelevement de sang fœtal, les delais pour l'obtention des resultats parasitologiques definitifs et la possibilite de diagnostics faussement negatifs (6), une nouvelle strategie a ete definie pour augmenter la securite, la rapidite et la sensibilite du diagnostic prenatal.

To assess the impact of sequential therapy with adefovir dipivoxil (ADV) and pegylated interferon alfa-2a (PEG-IFN) on virological (serum HBV-DNA) and serological (serum HBsAg) response in 20 consecutive HBeAg-negative patients. Patients received ADV for 20 weeks, then ADV and PEG-IFN for 4 weeks and lastly PEG-IFN for 44 weeks. Serum HBV-DNA and HBsAg were assessed at baseline, during therapy (weeks 20, 44 and 68) and follow-up (weeks 92 and 116). Sustained virological response (SVR) was defined as serum HBV-DNA10 000 copies/mL (partial) or70 copies/mL (complete) 24 weeks after stopping treatment. A serological response was defined as a serum HBsAg decrease ≥1 log(10) IU/mL at the end of treatment. Baseline median serum HBV-DNA and HBsAg levels were 7.6 log(10) copies/mL and 3.8 log(10) IU/mL, respectively. Ten patients (50%) achieved SVR, six of them had partial response and four complete response. Four patients (20%) achieved serological response. Complete SVRs showed a major and steep decline in HBsAg level with a median decrease of 0.5, 1.6 and 2.0 log(10) IU/mL at treatment week 20, 44 and 68, respectively. Partial SVRs showed a slight and slow decline in serum HBsAg level (0.1, 0.4, and 0.6 log IU/mL at weeks 20, 44 and 68, respectively). On-treatment serum HBsAg decrease had a high accuracy to predict SVR (AUROC = 0.88). Our results suggest that sequential therapy might be an interesting strategy for HBeAg-negative patients. Serum HBsAg kinetics seem to be an accurate tool to predict SVR. Large clinical trials are needed to explore this strategy with more potent analogues.

Germline loss-of-function mutations in the SPRED1 gene have recently been identified in patients fulfilling the National Institutes of Health (NIH) diagnostic criteria for neurofibromatosis type 1 (NF1) but with no NF1 (neurofibromin 1) mutation found, suggesting a neurofibromatosis type 1-like syndrome.61 index cases with NF1 clinical diagnosis but no identifiable NF1 mutation were screened for SPRED1 mutation.We describe one known SPRED1 mutation (c.190CT leading to p.Arg64Stop) and four novel mutations (c.637CT leading to p.Gln213Stop, c.2TC leading to p.Met1Thr, c.46CT leading to p.Arg16Stop, and c.1048_1060del leading to p.Gly350fs) in five French families. Their NF1-like phenotype was characterised by a high prevalence of café-au-lait spots, freckling, learning disability, and an absence of neurofibromas and Lisch nodules in agreement with the original description. However, we did not observe Noonan-like dysmorphy. It is noteworthy that one patient with the p.Arg16Stop mutation developed a monoblastic acute leukaemia.In our series, SPRED1 mutations occurred with a prevalence of 0.5% in NF1 patients and in 5% of NF1 patients displaying an NF1-like phenotype. SPRED1 mutated patients did not display any specific dermatologic features that were not present in NF1 patients, except for the absence of neurofibromas that seem to be a specific clinical feature of NF1. The exact phenotypic spectrum and the putative complications of this NF1 overlapping syndrome, in particular haematological malignancies, remain to be further characterised. NIH diagnostic criteria for NF1 must be revised in view of this newly characterised Legius syndrome in order to establish a specific genetic counselling.

Human telomerase is a specialized reverse transcriptase that catalyses telomeric repeat addition at the ends of chromosomes. Activation of this enzyme is one of the key steps in cell immortalization and carcinogenesis, and one of its components, hTERT, is considered as the rate-limiting factor. While telomerase activity was found to be prognostically relevant in various cancers, results obtained from renal cell carcinomas (RCC) failed to show any correlation with the usual prognostic factors. The aim of the study was to reassess the role of telomerase and its hTERT component in the biological behaviour of RCC using new quantitative techniques, such as the quantitative evaluation of hTERT mRNA level by a real-time RT-PCR procedure and the measuring of telomerase activity by an ELISA TRAP assay. Since experimental evidence supports a relationship between cell proliferation or c-myc expression and telomerase, the proliferation index and c-myc mRNA levels were also studied. Forty-one RCC (29 conventional renal cell carcinomas (CRCC), 10 papillary RCC and two urothelial carcinomas) were studied. In 73% of cases, normalized hTERT mRNA expression was significantly higher in the tumour sample than in the normal tissue. Telomerase activity was detected in 63% of RCC, while corresponding normal tissue was always negative. Analysis of correlations showed firstly that both telomerase activity and hTERT mRNA level were lower in the group of CRCC versus non-CRCC (TRAP: 0.3+/-0.1 versus 0.6+/-0.2, p0.05; hTERT/PO mRNA: 5+/-3 versus 37+/-8, p0.001, respectively); secondly, that in the group of CRCC, hTERT mRNA expression level was correlated with the stage of the tumour (p=0.01); and thirdly, that no correlation was observed between c-myc mRNA level and hTERT mRNA level. In conclusion, these results support the involvement of telomerase in RCC and the potential interest of hTERT mRNA quantification.

Recent studies performed with null mice suggested a role of either RXR alpha or PPAR gamma in murine placental development. We report here that both PPAR gamma and RXR alpha are strongly expressed in human villous cytotrophoblasts and syncytiotrophoblasts. Moreover, specific ligands for RXRs or PPAR gamma (but not for PPAR alpha or PPAR delta) increase both human CG beta transcript levels and the secretion of human CG and its free beta-subunit. When combined, these ligands have an additive effect on human CG secretion. Pan-RXR and PPAR gamma ligands also have an additive effect on the synthesis of other syncytiotrophoblast hormones such as human placental lactogen, human placental GH, and leptin. Therefore, in human placenta, PPAR gamma/RXR alpha heterodimers are functional units during cytotrophoblast differentiation into the syncytiotrophoblast in vitro. Elements located in the regulatory region of the human CG beta gene (beta 5) were found to bind RXR alpha and PPAR gamma from human cytotrophoblast nuclear extracts, suggesting that PPAR gamma/RXR alpha heterodimers directly regulate human CG beta transcription. Altogether, these data show that PPAR gamma/RXR alpha heterodimers play an important role in human placental development.

Using a protein truncation assay, we have identified a new mutation in the neurofibromatosis type 1 (NF1) gene that causes a severe defect in NF1 pre-mRNA splicing. The mutation, which consists of a G to A transition at position +1 of the 5' splice site of exon 12a, is associated with the loss of both exons 11 and 12a in the NF1 mRNA. Through the use of in vivo and in vitro splicing assays, we show that the mutation inactivates the 5' splice site of exon 12a, and prevents the definition of exon 12a, a process that is normally required to stimulate the weak 3' splice site of exon 12a. Because the 5' splice site mutation weakens the interaction of splicing factors with the 3' splice site of exon 12a, we propose that exon 11/exon 12a splicing is also compromised, leading to the exclusion of both exons 11 and 12a. Our results provide in vivo support for the importance of the exon definition model during NF1 splicing, and suggest that the NF1 region containing exons 11 and 12a plays an important role in the activity of neurofibromin.

Expression of the telomerase catalytic sub-unit (htert) constitutes a key step in the development of human cancer. Although htert regulation is still unclear, several studies suggest that c-myc may activate its expression. Prostate cancer is one of the most common malignancies among men in Western countries. Since de-regulated expression of myc as well as telomerase activation may contribute to the pathogenicity of this cancer, we investigated this pathway in prostate tumorigenesis. For this purpose, myc- and htert-mRNA expression was quantified in 33 sporadic prostate tumors using a real-time quantitative PCR assay based on TaqMan methodology. myc over-expression was observed in 19 (58%) of 33 tumors, whereas telomerase status evaluated by htert expression was observed in 22 (67%). There was no correlation between myc over-expression or htert expression level and tumor stage or Gleason grade. A significant association (p = 0.0024) was found between myc over-expression and elevated htert expression, indicating that the up-regulation of telomerase activity often observed in prostate tumors might be conferred through transactivation of htert by myc. It is likely that the ability of c-myc protein to stimulate expression of htert and thereby enhance telomerase activity represents an important step in prostate tumorigenesis.

The syncytiotrophoblast is the major component of the human placenta as it is involved in the feto-maternal exchanges and the secretion of pregnancy-specific hormones. In normal placenta, the multinucleated syncytiotrophoblast is formed by fusion of mononuclear cytotrophoblast cells. In trisomy 21-affected placenta, we show that it exists a defect (or a delay) in the syncytiotrophoblast formation and a decrease of the production of pregnancy-specific hormones. We show that it is related to the over expression of the SOD-1 located on chromosome 21. These results will be of help for the understanding of maternal hormonal markers of fetal trisomy 21 and the consequences of the placental defects on the fetal development.

The syncytiotrophoblast (ST) is one of the major components of the human placenta, as it is involved in feto-maternal exchanges and the secretion of pregnancy-specific hormones. The aim of this study was to elucidate the formation and function of the ST in trisomy 21 (Down's syndrome). We first used the in vitro model of cytotrophoblast differentiation into ST. Cytotrophoblasts were isolated from 15 trisomy 21-affected placentas (12-35 weeks gestation) and 10 gestational age-matched control placentas. In vitro cytotrophoblasts isolated from normal placenta fused to form the ST. This was associated with an increase in transcript levels and in the secretion of hCG, human placental lactogen, placental GH, and leptin. In trisomy 21-affected placentas, we observed a defect (or a delay) in ST formation and a dramatic decrease in the synthesis and secretion of these hormones compared to those in cultured cells isolated from control age-matched placentas. These results were confirmed by a significant (P0.001) decrease in gene expression in total homogenates of trisomy 21-affected placentas compared to controls. These results will be of help in understanding the maternal hormonal markers of fetal trisomy 21 and the consequences of placental defects for fetal development.

The syncytiotrophoblast (ST), which forms the outer layer of the chorionic villi, is the endocrine unit of the human placenta. Bathing in the maternal blood of the intervillous space, the ST secretes its hormonal products directly into the maternal circulation. Leptin is expressed in the ST and is secreted into the maternal circulation. However, its regulation and physiological role during pregnancy remain poorly known. In the present work we used the in vitro model of human cytotrophoblast differentiation into ST to study the effect of physiological and synthetic retinoids on leptin synthesis and secretion. Using specific antibodies we first illustrated by immunocytochemistry the expression of retinoic acid (RA) receptor alpha and retinoid X receptor alpha (RXRalpha) in ST. We then observed that leptin messenger ribonucleic acid and protein expression increased with in vitro ST formation. The 9-cis isomer of RA and the synthetic retinoid specific for RXRs (BMS 649) stimulated leptin messenger ribonucleic acid expression and secretion. In contrast, all-trans-RA and a RA alpha-specific ligand had no effect. These results suggest that retinoids regulate leptin expression and highlight a role for RXRalpha in this process.

A method using multiplex PCR followed by cycle-sequencing has been developed to detect mutations in the FIX gene. The procedure was evaluated in 45 severe or mild haemophilia B patients from 45 unrelated families. At least one deleterious mutation was identified in every haemophiliac demonstrating the efficiency of the method. Furthermore the described procedure offers many advantages compared to other screening detection methods: it is fast (less than 48 h), simple (partly automated) and of relatively low cost (it requires only one PCR).

We took advantage of a recently developed system allowing performance of real-time quantitation of polymerase chain reaction to develop a quantitative method of measurement of PML-RARalpha transcripts which are hallmarks of acute promyelocytic leukemia (APL) with t(15;17) translocation. Indeed, although quantitation of minimal residual disease has proved to be useful in predicting clinical outcome in other leukemias such as chronic myeloid leukemia or acute lymphoblastic leukemia, no quantitative data have been provided in the case of APL. We present here a method for quantitation of the most frequent subtypes of t(15;17) transcripts (namely bcr1 and bcr3). One specific forward primer is used for each subtype in order to keep amplicon length under 200 bp. The expression of PML-RARalpha transcripts is normalized using the housekeeping porphobilinogen deaminase (PBGD) gene. This technique allows detection of 10 copies of PML-RARalpha or PBGD plasmids, and quantitation was efficient up to 100 copies. One t(15;17)-positive NB4 cell could be detected among 106 HL60 cells, although quantitation was efficient up to one cell among 105. Repeatability and reproducibility of the method were satisfying as intra- and inter-assay variation coefficients were not higher than 15%. The efficiency of the method was finally tested in patient samples, showing a decrease of the PML-RARalpha copy number during therapy, and an increase at the time of relapse.

Telomerase is a ribonucleoprotein that synthesizes telomeric DNA on chromosomal ends. While telomerase is undetectable in most normal somatic tissues, telomerase activation has been detected by a polymerase chain reaction (PCR)-based assay (TRAP) in many immortal cell lines and various cancers, including prostate cancers. To investigate the role of telomerase in prostate cancer at the cellular level, the expression of one of the ribonucleoprotein complexes, the RNA component of human telomerase (hTR), was studied in normal, preneoplastic, and cancerous prostate tissues using a non-radioactive in situ hybridization procedure. Nine human prostates resected at the time of radical prostatectomy were studied. In each case, archival paraffin-embedded samples from normal tissue, prostatic intraepithelial neoplasia (PIN) lesions, the putative precancerous lesion, and prostate carcinomas were selected for in situ hybridization. hTR mRNA expression was detected in carcinomatous glands of seven out of the nine cancers (75 per cent). Furthermore, in seven out of the eight cases showing PIN lesions, the epithelial cells of PIN foci also expressed hTR mRNA. By contrast, in normal tissue, epithelial cells were negative, whereas hTR mRNA expression was detected in the basal cells. The detection of hTR mRNA in PIN lesions clearly strengthens the link between PIN and carcinomatous glands and suggests that telomerase expression occurs early in prostate carcinogenesis. Furthermore, this study confirms previous experimental data suggesting that the basal cell layer is the stem cell compartment in prostate.

The natural history of hepatitis C virus (HCV) infection in human immunodeficiency virus (HIV)-infected patients has never been studied according to the concept of liver fibrosis progression. The aim of this work was to assess the fibrosis progression rate in HIV-HCV coinfected patients and in patients infected by HCV only. A cohort of 122 HIV-HCV coinfected patients was compared with a control group of 122 HIV-negative HCV-infected patients. Groups were matched according to age, sex, daily alcohol consumption, age at HCV infection, and duration and route of HCV infection. The fibrosis progression rate was defined as the ratio between fibrosis stage (METAVIR scoring system) and the HCV duration. The prevalence of extensive liver fibrosis (METAVIR fibrosis scores 2, 3, and 4) and moderate or severe activity were higher in HIV-infected patients (60% and 54%, respectively) than in control patients (47% and 30%, respectively; P.05 and P.001, respectively). The median fibrosis progression rate in coinfected patients and in control patients was 0.153 (95% confidence interval [CI], 0.117-0.181) and 0.106 (95% CI, 0.084-0.125) fibrosis units per year, respectively (P.0001). HIV seropositivity (P.0001), alcohol consumption (50 g/d, P =.0002), age at HCV infection (25 years old, P.0001), and severe immunosuppression (CD4 count/=200 cells/microL, P.0001) were associated with an increase in the fibrosis progression rate. In coinfected patients, alcohol consumption (50 g/d), CD4 count (/=200 cells/microL), and age at HCV infection (25 years old) (P. 0001, respectively) were associated with a higher fibrosis progression rate. HIV seropositivity accelerates HCV-related liver fibrosis progression. In coinfected patients, a low CD4 count, alcohol consumption rate, and age at HCV infection are associated with a higher liver fibrosis progression rate.

Introduction La plupart des modeles animaux d’ischemie-reperfusion retinienne qui ont ete mis au point reposent sur l’injection de solute isotonique dans la chamber anterieure de l’œil ou sur l’occlusion simultanee du nerf optique et des vaisseaux centraux retiniens situes au cœur du nerf optique (artere et veine centrale de la retine). Nous avons mis au point un nouveau modele occlusion vasculaire unilaterale des vaisseaux retiniens sans atteinte concomitante d’origine mecanique des axones des cellules ganglionnaires de la retine. Materiels et Methodes Par occlusion unilaterale de l’artere carotide externe et occlusion transitoire de l’artere pterygopalatine ipsilalaterale, il s’est avere possible chez la souris adulte (Souris avec amaurose Unilaterale (n = 40) et souris Sham (n = 40) d’eviter la reperfusion d’origine controlaterale et d’obtenir une amaurose unilaterale d’origine purement vasculaire. Il est possible de varier la duree d’ischemie et d’etudier le decours de la reperfusion retinienne par diverses methods : electroretinographie et angiographie in vivo puis apres injection de fluoresceine intracardiaque et realisation de retines montees a plat par des methods immunohistochimiques, par des methodes d’etude de l’apoptose et surtout par RTPCR quantitative de certains ARNm, cles des diverses phases de la reperfusion. C’est ainsi qu’ont ete etudies des genes d’expression precoce tels que Zif268, Arc, Jun, les genes codant de tres nombreux recepteurs au glutamate et des genes impliques dans la neurogenese. Les temps etudies ont ete 4 heures, 24 heures, 72 heures, 7 jours et 1 mois avec 8 animaux groupe. Resultats Nous avons pu valider ce modele comme modele d’ischemie-reperfusion oculaire tant chez la souris que chez le rat. Des variations d’expression geniques ont pu etre rattachees a chacun des stades etudies. Par exemple celles des genes COX2, GADD34, HSP70, PAI1. Discussion Nous avons mis au point le premier veritable modele permettant d’etudier l’ischemie-reperfusion retinienne sans interferences de facteurs mecaniques ou autres. Ce modele se prete tout particulierement aux tests precliniques des modulateurs des recepteurs aux glutamates (NMDA, Metabotropiques, AMPA, Kainate). Le glutamate joue en effet un role fondamenteur de neurotransmetteur dans les synapses des circuits retiniens d’une part et dans les phenomenes lies a l’excitotoxicite. Ce modele permettra d’etudier de nouvelles therapies sur des souris KO, Knock in ou transgeniques atteintes d’hypertension arterielles et/ou d’atherosclerose situation frequente retrouvee chez les sujets âges. La thrombolyse peut s’accompagner d’excitotoxicite qui limite ses benefices. Ce modele arrive donc au bon moment pour permettre des avancees therapeutiques. Conclusion Les accidents vasculaires oculaires s’accompagnent trop souvent encore d’echecs therapeutiques du fait du caractere souvent trop tardif de l’examen des patients du a la meconnaissance ou a l’incomprehension des symptomes ou malaises qu’ils ressentent. Ce modele devrait permettre de mieux apprehender le potentiel therapeutique des cellules souches de la retine stimulees par l’ischemie.

Impact of hepatitis C treatment has never taken into account the dynamics of fibrosis progression. This study assessed the impact of interferon on liver fibrosis progression in patients with chronic hepatitis C according to 3-month aminotransferase activity response.We recruited 287 patients, 185 treated and 102 control, with paired biopsy specimens. Before follow-up, the fibrosis progression rate per year was estimated as the ratio between fibrosis stage in METAVIR units (1 U, 1 stage; 4 U, cirrhosis) and the duration of infection. During follow-up, fibrosis progression was assessed by the observed difference between stages divided by duration between biopsies.The median fibrosis progression rate in treated patients decreased compared with the rate before treatment from 0.103 F METAVIR U/yr (95% confidence interval [CI], 0.087-0.120) to 0.000 (95% CI, 0.000-0.000; P/= 0.0001). Among 91 treated responders, fibrosis stage worsened in 19 (22%), compared with 21 (22%) of 94 treated nonresponders and 57 of 102 controls (56%; P/= 0.0001 compared with treated patients), and improved in 26 (29%), 17 (18%), and 8 (8%; P = 0.0002 compared with 29% and P = 0.03 compared with 18%), respectively. These observed differences persisted after genotype, viremia, sex, age at infection, duration of infection, and alcohol consumption were taken into account.Interferon treatment changes the natural fibrosis progression rate in patients with chronic hepatitis C independently of genotype and early response.

Polypeptide growth factors play an important role in the regulation of human embryonic development. Insulin-like 4 gene (INSL4) is a member of the insulin family, which includes insulin, IGF-I, IGF-II, relaxin, and INSL3. Using RT-PCR, we previously found abundant INSL4 mRNA in the human placenta. In this study, we examined the chronology and spatial expression of this gene in sections of human placenta and conceptus by means of in situ hybridization. Expression of the IGF-II gene was studied as a positive control. INSL4 distribution was tissue- and cell-specific. Indeed, INSL4 mRNA was most abundant in syncytiotrophoblast cells. In fetal tissues, INSL4 mRNA was identified in the perichondrium of all four limbs, vertebrae, and ribs. Moreover, INSL4 mRNA was abundant in interbone ligaments. These findings indicate that the INSL4 gene may play an important role in trophoblast development and regulation of bone formation. IGF-II mRNA, in agreement with the literature, are mainly located in the mesodermal core in the villous trophoblast and in most embryonic tissues.

Neurofibromatosis type 1 (NF1), a genetic disorder with neuroectodermal involvement, demonstrates phenotypic overlap in some patients with Noonan syndrome (NS), ultimately resulting in the so-called neurofibromatosis-Noonan syndrome (NF-NS). A strong association of the two phenotypic traits was recently illustrated by a four-generation family, although NF1 and NS were eventually demonstrated to segregate independently on the basis of polymorphic DNA markers [Bahuau et al., 1996: Am J Med Genet 66:347-355]. Identification of the causal NF1 mutation seemed a prerequisite to further dissecting this singular familial association. Using the protein truncation assay, a nonsense mutation (C2446T--R816X) of the neurofibromin gene was evidenced. This mutation occurred on a CpG dinucleotide within exon 16 and 5' to the GAP domain-specifying region of the gene. R816X creates a recognition site for endonuclease HphI, absent in 2 individuals with NS only. Screening 184 unrelated NF1 patients, three novel occurrences of the mutation were found in individuals diagnosed with classical NF1. Based on the assumption of genotype-phenotype correlation in these individuals, clinical and molecular analyses of this four-generation family demonstrated that the NF-NS phenotype was additive, being the result of both classical NF1 and NS. This particular observation also suggests the presence of an NS locus on 17q, which might be of interest for further linkage studies.

Identification of mutations responsible for human monogenic hereditary diseases has lately undergone rapid development. It is now possible to establish the mutation spectrum for a given genetic disease and international databases are now available on the Internet. New mutational mechanisms have been identified:transposition, inversion, trinucleotide repeat expansion. At the disease level, knowledge of molecular defects has allowed to better delimit mechanisms of gain or loss of function, to understand dominant or recessive mode of inheritance, to explain anticipation phenomena, to identify founder effect amid a selected population, to establish, in some privileged cases, phenotype-genotype correlations and above all to elicit allelic and genetic heterogeneity hitherto unsuspected. Identification of deleterious mutations is also the basis of diagnosis at the gene level which today has a growing impact on genetic counselling and prenatal diagnosis.

Myotonic dystrophy (DM) is an autosomal dominant neuromuscular disorder characterized by marked variability of its clinical manifestations. The mutational basis of DM is an unstable (CTG)n trinucleotide repeat in the 3' untranslated region of the myotonic dystrophy protein kinase gene (DMPK). We used quantitative RT-PCR to determine DMPK mRNA levels in muscular biopsies from three congenitally affected (CDM) and two control infants. The CDM infants had increased DMPK mRNA levels, which were not correlated to increased expression of the mutant allele. This increase may be the consequence of a maturational muscular arrest, which may maintain an elevated level of DMPK mRNA until birth.

A five-generation family is here reported in which several members developed malignant melanoma, dysplastic naevi, astrocytoma in all grades, benign or malignant schwannoma, neurofibroma, or meningioma in a single instance. Significant cosegregation of skin and nervous tumours, preclusion of allelism to type 1 neurofibromatosis and phenotypic departure from known syndromes of hereditary proneness to cancer make one suggest an original familial predisposition to both malignant melanoma and central/peripheral nervous tumours.

A large four-generation family with Noonan syndrome (NS) and neurofibromatosis-type 1 (NF1) was studied for clinical association between the two diseases and for linkage analysis with polymorphic DNA markers of the NF1 region in 17q11.2. Nonrandom segregation between NS and NF1 phenotypes was observed. Neurofibromatosis was tightly linked to NF1 markers, whereas Noonan syndrome was found not be allelic to NF1. These results suggest that two mutations at two independent but closely linked loci are the cause of neurofibromatosis-Noonan syndrome (NF-NS) association in this family.

Apolipoprotein A-I, a protein produced mainly by hepatocytes, is of major importance in prevention of atherosclerosis. Its serum level varies according to the degree of liver fibrosis and the mechanism of this regulation is unknown. The aim of this study was to investigate the role of extracellular matrix in the regulation of apolipoprotein A-I by the liver. Primary mouse hepatocytes were cultured on different extracellular matrix components. The apolipoprotein A-I mRNA level was quantified in these different culture conditions by a sensitive quantitative RT-PCR procedure and compared according to the extracellular matrix component used as substrate. A significant decrease in the apolipoprotein A-I mRNA level was observed when cells were plated on fibronectin by comparison with cells cultured on all other components. Potential binding of apolipoprotein A-I to the different matrix components was also studied in vitro. We demonstrated that apolipoprotein A-I significantly bound to fibronectin in a concentration-dependent, saturable and specific manner. Thus, fibronectin, a major liver extracellular matrix component, can interact with apolipoprotein A-I both by downregulating its mRNA level in liver cells and by binding this molecule after its secretion in the extracellular space. Since fibronectin is the first matrix component to be produced in excess and deposited in liver fibrosis, it could be involved in the decrease in serum apolipoprotein A-I in alcoholic patients with liver fibrosis and cirrhosis.

Using Southern blotting for the diagnosis of clonality in peripheral T-cell lymphomas (PTCLs), analysis of the T-cell receptor (TCR) gamma gene rearrangement was shown to be more informative than that of the TCR beta gene rearrangement. In order to amplify every VJ gamma rearrangement, a polymerase chain reaction (PCR) procedure using newly designed GC-clamp primers has been developed. All primers can be mixed in a single multiplex PCR. PCR products are analysed by denaturing gradient gel electrophoresis (DGGE), providing tumour-specific imprints inasmuch as the procedure characterizes N sequence polymorphism at the VJ junctions. In a series of 30 PTCL cases, the PCR procedure demonstrated 27 cases to be clonally rearranged and failed in three cases. PCR was more accurate than Southern blotting, showing 47 rearranged gamma alleles, four of which were undetectable on the Southern blot. When lymphomas were studied at different sites and at relapse, the DGGE pattern remained unchanged. In PTCL, the proposed PCR is helpful for the diagnosis and staging of the disease and should improve the follow-up monitoring. The undetectability of clonal rearrangements in a few cases is discussed in the light of concepts of lymphomagenesis and T-cell differentiation.

Increased serum levels of human chorionic gonadotropin beta subunit (hCG beta) were described previously in patients with bladder cancer. To obtain insight into such production of hCG beta, the expression of hCG beta 7, 8, 5, and 3 genes in bladder carcinomas and normal urothelia was investigated by reverse transcription PCR. Surprisingly, hCG beta mRNAs were detected in both normal urothelial and carcinomatous cells. However, tumor progression was characterized by different patterns of transcription of the hCG beta genes; the beta 7 gene was the only gene transcribed in normal urothelia and Ta tumors included in this study, whereas in addition to beta 7, genes beta 5, 8, and 3 were transcribed in T1 to T4 tumors. Moreover, transcription levels of the latter three genes increased with the stage of the disease. These observations showed that dramatic modifications in the expression of hCG beta genes accompany progression of bladder carcinomas.

Alterations in exons 5-8 of the p53 gene have been found in the vast majority of human tumors. Although some specific codons have been revealed as preferential mutational sites in defined tumor types, mutations are generally so scattered within this region that various screening methods for their detection have been widely used. However, the capacity of these techniques to detect exhaustively all mutations has not been evaluated. In this report, conditions for analysing exons 5-8 of the p53 gene by denaturing gradient gel electrophoresis (DGGE) have been elaborated using the Melt87 and SQHTX computer programs. The procedure requires five different amplifications. The screening of 90 colorectal tumors revealed 56 amplification products demonstrating abnormal DGGE behavior. Direct sequencing of these amplification products after asymmetric polymerase chain reaction (PCR) showed, besides polymorphism, 49 somatically mutated DNA samples (54.4%) corresponding to 38 different p53 variants. Moreover, 25 additional p53 variants identified in other tumor types were also detectable with our conditions. Thus, altogether the procedure has been successfully used in the detection of 63 different p53 variants. The experimental differences in migration between the wild-type homoduplex DNA molecule and the heteroduplex(es) containing one mismatch were systematically compared with those calculated by the SQHTX program. The computer program was found to be reliably predictive. This DGGE procedure appears thus to be effective and reliable for detecting mutations in exons 5-8 of the p53 gene.

We describe a direct method for detection of the Spanish and Sicilian (delta beta)zero-thalassemias. This method is based on the use of the deletion-junction sequences to design specific PCR primers. It permits a rapid screening of heterozygotes in populations at risk and provides a useful tool for early prenatal diagnosis of these forms of thalassemia.

Genomic DNA from 170 unrelated hemophilia A patients was examined for gene defects in the coding region of the Factor VIII gene. Exons 18, 22-24 and 26 contain a CGA codon for arginine within the recognition sequence for the restriction enzyme Taq I. These five sites were amplified by the polymerase chain reaction and tested for abnormal Taq I restriction patterns. In five cases, the enzyme Taq I failed to digest the amplified fragments. Direct sequencing of the amplified products demonstrated a C to T transition in the coding strand of exons 18, 22 and 24 in three severe hemophilia A patients resulting in TGA termination codons. Two patients showed G to A transition in exons 24 and 26 reflecting a C to T transition in the non-coding strand substituting a glutamine for an arginine. Three deletions involving exon 26 and one exons 23-26 were found in severe hemophiliac patients. In contrast, exons 23 and 24 failed to amplify in one patient with a moderate form of the disease suggesting an in-frame splicing of exons 22 and 25. Exon 8 and the 3' end of exon 14 were analyzed by denaturing gradient gel electrophoresis (DGGE). Two patients with a moderate form of the disease demonstrated an abnormal electrophoretic pattern in exon 8 and sequencing demonstrated missense mutations at codon 372 for arginine within a thrombin activation site. One missense mutation was a C to T transition substituting cysteine for arginine and the other was an infrequent G to C transversion at an adjacent nucleotide changing the same arginine to proline.(ABSTRACT TRUNCATED AT 250 WORDS)

A directed-search strategy for point mutations in the factor VIII gene causing hemophilia A was used to screen eight potentially hypermutable CpG dinucleotides occurring at sites deemed to be of functional importance. Polymerase chain reaction-amplified DNA samples from 793 unrelated individuals with hemophilia A were screened by discriminant oligonucleotide hybridization. Point mutations were identified in 16 patients that were consistent with a model of 5-methylcytosine (5mC) deamination. Four new examples of recurrent mutation were demonstrated at the following codons: 336 (CGA----TGA), 372 (CGC----TGC), 372 (CGC----CAC), and 1689 (CGC----TGC). These are functionally important cleavage sites for either activated protein C or thrombin. Further novel C----T transitions were identified in the remaining arginine codons screened (-5, 427, 583, 795, and 1696), resulting in the creation of TGA termination codons. Differences in mutation frequency were found both within and between the CpG sites and between ethnic groups. These differences are assumed to be due to differences in the level of cytosine methylation at these sites, although direct evidence for this inference is lacking.

The polymerase chain reaction (PCR) allows the detection of minimal amounts of nucleic sequences and has been successfully used to test for the chronic myeloid leukemia-specific bcr/abl transcripts. We studied blood samples from 17 patients who had undergone allogeneic bone marrow transplantation for CML, using a modified polymerase chain reaction-based assay for the detection of leukemic mRNA. This nested PCR technique was found to be highly sensitive, detecting the chimeric bcr/abl transcript in 16 of 17 patients including several long-term survivors. Cytogenetic techniques failed to detect Ph mitoses. The clinical significance of the persisting bcr/abl transcript for long periods following BMT is poorly understood and remains to be elucidated by further studies.

Objectifs pedagogiques Comprendre les enjeux professionnels des trois partenaires « Medecins-Cadres-Administratifs ». Connaitre et mesurer les zones d’intervention de chacun dans un fonctionnement collegial et efficace. Prevoir les etapes de developpement et d’evolution du fonctionnement du pole. Messages a retenir Analyse de la situation : etat des lieux du Pole. Regles communes : indicateurs d’activite et de qualite. Collaboration et force de proposition vis-a-vis du coordonnateur au niveau strategique et organisationnel. Animation equipe d’encadrement.

Using very specific vasopressin (VP) analogues, the human platelet VP receptor was characterized as a V1a rather than a V1b receptor, on the basis of the effect of the analogues on shape-change and aggregation. The platelet VP binding sites appeared to be subject to homologous down-regulation by plasma VP, in view of the inverse correlation found between the maximal capacity of binding of tritiated VP to platelets and the immunoreactive VP concentration in poor platelet plasma from the same individual. Aggregating effect of VP on human platelets was potentiated by both ADP and epinephrine. In addition, VP was able to release serotonin from human platelets, but only at high concentration.