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Biomedical subjects

M Vidaud

Publications and source records attributed to M Vidaud.

At least 91 records · Page 5Linked to original sources
PubMed1997

Myotonic dystrophy protein kinase gene expression in skeletal muscle from congenitally affected infants.

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.

Case-Control Studies↗
PubMed1996

Exclusion of allelism of Noonan syndrome and neurofibromatosis-type 1 in a large family with Noonan syndrome-neurofibromatosis association.

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.

Alleles↗
PubMed1996

VJ rearrangements of the TCR gamma locus in peripheral T-cell lymphomas: analysis by polymerase chain reaction and denaturing gradient gel electrophoresis.

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.

Base Sequence↗
PubMed1996

Quantification of apolipoprotein A-I and B messenger RNA in heavy drinkers according to liver disease.

It has previously been shown that, in heavy drinkers, serum apolipoprotein A-I (ApoA-I) levels are closely related to the degree of liver injury: they are at a maximum in patients with steatosis, begin to decrease in patients with fibrosis, and reach a minimum in patients with severe cirrhosis. In contrast with serum ApoA-I variations, serum apolipoprotein B (ApoB) levels are stable. The assessment of messenger RNA (mRNA) levels of ApoA-I and ApoB using a quantitative competitive polymerase chain reaction (PCR) method was performed in 18 alcoholic patients: 8 with normal livers or steatosis (group I), 6 with fibrosis or nonsevere cirrhosis (group II), and 4 with severe cirrhosis (group III). For ApoA-I, group I had higher serum levels (208.4 +/- 37.6 mg/dL mean +/- SE) and mRNA levels (0.51 +/- 0.12) than group II (serum 116 +/- 19 mg/dL, P < .01, mRNA 0.40 +/- 0.11, P < .09) or group III (serum 56.5 +/- 28.6 mg/dL, P < .01, mRNA 0.27 +/- .02, P = .008). For ApoB, the three groups had similar serum ApoB levels: 129.9 +/- 37.7, 121 +/- 51, 120.7 +/- 57.4 mg/dL. Group I presented higher levels of ApoB mRNA than those of group III (0.68 +/- 0.21 vs. 0.41 +/- 0.18, P < .06). There was a significant correlation between serum and mRNA levels of ApoA-I (r = .65, P = .003) but no correlation between serum and mRNA levels of ApoB (r = .19, NS). We suggest that (1) steatosis is associated with increased ApoA-I mRNA; (2) fibrosis is associated with decreased serum ApoA-I, probably caused by a posttranscriptional mechanism; (3) severe alcohol-induced cirrhosis is associated with a nonspecific decrease in ApoA-I and ApoB mRNA; and (4) in contrast to ApoA-I mRNA, the ApoB mRNA level makes a slight contribution to the ApoB serum concentration.

Adult↗
PubMed1996

PCR enzyme-linked immunosorbent assay for diagnosis of leishmaniasis in human immunodeficiency virus-infected patients.

A PCR enzyme-linked immunosorbent assay (ELISA) involving the use of bone marrow aspirates (BMA) and blood samples (BS) for the diagnosis of visceral leishmaniasis (VL) in human immunodeficiency virus-infected patients was developed with primers selected from the sequence of the small-subunit rRNA gene and compared with direct examination and in vitro cultivation. The PCR was optimized for routine diagnosis: processing of samples with lysis of erythrocytes without isolation of leukocytes, enzymatic prevention of contamination, internal control of the reaction, and ELISA testing in a microtitration plate hybridization. Of 79 samples (33 BMA and 46 BS) from 77 patients without VL, all the results were negative. Fifty-three samples (9 BMA and 44 BS) were obtained from 13 patients with VL: 6 samples drawn during anti-Leishmania treatment were negative whatever the technique used, and 47 samples (9 BMA and 38 BS) were positive with at least one technique. The sensitivities were 51% (24 of 47), 81% (38 of 47), and 98% (46 of 47) for direct examination, culture, and PCR, respectively. Thus, PCR ELISA is reliable for diagnosing VL in human immunodeficiency virus-infected patients, and blood sampling should be sufficient for the follow-up.

AIDS-Related Opportunistic Infections↗
PubMed1996

Histological features predictive of liver fibrosis in chronic hepatitis C infection.

AIMS: To assess which pathological features are associated with a sensitive marker of liver fibrogenesis and thus of the potential development of fibrosis in hepatitis C. METHODS: The degree of liver fibrogenesis was evaluated by quantification of type I collagen mRNA and transforming growth factor (TGF) beta 1 mRNA (a major profibrogenic cytokine) in liver biopsy specimens from 28 patients with chronic hepatitis C and five controls, using a quantitative reverse transcription polymerase chain reaction (RT-PCR) assay. Results of mRNA quantification were correlated with histological lesions scored semiquantitatively in the same specimens. RESULTS: Type I collagen mRNA was more strongly expressed in patients than in controls and correlated with the degree of fibrosis, but not with any of the necro-inflammatory lesions (portal inflammation, piecemeal necrosis, and lobular necrosis). TGF beta 1 mRNA concentration was higher in patients than in controls and correlated with histological grade of activity and lobular necrosis. This result was confirmed by in situ hybridisation experiments which showed that TGF beta 1 mRNA was mainly expressed in areas of focal lobular necrosis in chronic hepatitis C. CONCLUSION: This study shows that fibrosis, rather than necro-inflammatory lesions or activity scores, is associated with fibrogenesis and thus with potential aggravation of the fibrous deposit in chronic hepatitis C. Lobular necrosis is an important predictor of prognosis in chronic hepatitis C, as shown by its strong association with TGF beta 1 mRNA expression.

Adult↗
PubMed1996

Role of liver extracellular matrix in transcriptional and post-transcriptional regulation of apolipoprotein A-I by hepatocytes.

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.

Animals↗
PubMed1995

Factor VIII gene inversions in severe hemophilia A: results of an international consortium study.

Twenty-two molecular diagnostic laboratories from 14 countries participated in a consortium study to estimate the impact of Factor VIII gene inversions in severe hemophilia A. A total of 2,093 patients with severe hemophilia A were studied; of those, 740 (35%) had a type 1 (distal) factor VIII inversion, and 140 (7%) showed a type 2 (proximal) inversion. In 25 cases, the molecular analysis showed additional abnormal or polymorphic patterns. Ninety-eight percent of 532 mothers of patients with inversions were carriers of the abnormal factor VIII gene; when only mothers of nonfamilial cases were studied, 9 de novo inversions in maternal germ cells were observed among 225 cases (approximately 1 de novo maternal origin of the inversion in 25 mothers of sporadic cases). When the maternal grandparental origin was examined, the inversions occurred de novo in male germ cells in 69 cases and female germ cells in 1 case. The presence of factor VIII inversions is not a major predisposing factor for the development of factor VIII inhibitors; however, slightly more patients with severe hemophilia A and factor VIII inversions develop inhibitors (130 of 642 [20%]) than patients with severe hemophilia A without inversions (131 of 821 [16%]).

Blotting, Southern↗
PubMed1995

Expression of human chorionic gonadotropin beta subunit genes in superficial and invasive bladder carcinomas.

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.

Base Sequence↗
PubMed1995

Detection of Aspergillus species DNA in bronchoalveolar lavage samples by competitive PCR.

A competitive PCR assay involving the use of bronchoalveolar lavage (BAL) samples for the diagnosis of invasive pulmonary aspergillosis (IPA) was developed. For this purpose, a 1-kb mitochondrial DNA fragment of Aspergillus fumigatus was sequenced. The primers used allowed amplification of A. fumigatus, A. flavus, A. terreus, and A. niger DNAs but not DNAs of other fungi and yeasts. BAL samples from 55 consecutively enrolled patients were tested. Three samples were excluded because of failure of correct amplification of the internal competitive control. Of 28 immunocompromised patients, 6 were PCR positive; 3 died of IPA and their BAL cultures yielded A. fumigatus; and 3 were culture negative and did not develop IPA. Of 15 human immunodeficiency virus-positive patients and 9 immunocompetent patients, 5 and 4, respectively, were both PCR positive and culture negative, and none developed aspergillosis. Thus, PCR confirmed IPA in three patients but gave positive results for 25% (12 of 49) of the patients who did not develop aspergillosis. The predictive value of PCR-positive results seems low for patients at risk for aspergillosis. Moreover, the risk of contamination of reaction buffers or biological samples with Aspergillus conidia seems high and has to be weighed in regard to the potential diagnostic benefit of PCR testing as a routine procedure.

AIDS-Related Opportunistic Infections↗
PubMed1995

Quantitative competitive PCR with bronchoalveolar lavage fluid for diagnosis of toxoplasmosis in AIDS patients.

Of 121 AIDS patients, 12 (10%) had Toxoplasma gondii DNA detected by competitive PCR in their bronchoalveolar lavage samples. Quantitation of the PCR results showed a correlation between the parasite burden and the serum lactic dehydrogenase titer. Quantitative competitive PCR could be useful to assess the diagnosis and the severity of pulmonary toxoplasmosis.

AIDS-Related Opportunistic Infections↗
PubMed1995

Late toxoplasmosis evidenced by PCR in a marrow transplant recipient.

We report a case of disseminated toxoplasmosis occurring 12 months after allogeneic BMT. The patient was seropositive for Toxoplasma gondii, and the donor was seronegative, so the patient was given anti-Toxoplasma prophylaxis. One year after BMT, he developed fever and muscle pain without other clinical symptoms. PCR amplification for T. gondii performed on blood was positive. Toxoplasma were found in bronchoalveolar lavage by PCR and in the marrow by special stains. With treatment, the PCR signal disappeared in 3 days while clinical symptoms resolved over 15 days. This case emphasizes the possibility of late toxoplasmosis after BMT despite prophylatic treatment, and the value of PCR in making the diagnosis.

Adult↗
PubMed1994

Prenatal diagnosis of congenital toxoplasmosis with a polymerase-chain-reaction test on amniotic fluid.

BACKGROUND: Congenital infection with Toxoplasma gondii can produce serious sequelae. However, there is little consensus about screening during pregnancy, and the tests used to establish a prenatal diagnosis of toxoplasmosis are complex and slow. We evaluated a simpler approach that is based on a polymerase-chain-reaction (PCR) test. METHODS: Prenatal diagnostic tests, including ultrasonography, amniocentesis, and fetal-blood sampling, were performed in 2632 women with T. gondii infection acquired during pregnancy. In 339 consecutive women, a competitive PCR test for T. gondii was performed on amniotic fluid, and its results were compared with those of conventional diagnostic tests. The PCR test targets the B1 gene of T. gondii, uses an internal control, and can be completed in a day. Positive tests were confirmed by serologic testing of newborns or by autopsy in terminated pregnancies. RESULTS: Overall, the risk of fetal infection was 7.4 percent, but it increased sharply with gestational age. Congenital infection was demonstrated in 34 of 339 fetuses by conventional methods, and the PCR test was positive in all 34. In three other fetuses, only the PCR test gave positive results, and follow-up testing confirmed the presence of congenital toxoplasmosis. The PCR test gave one false negative result but no false positive results. The PCR test performed better than conventional parasitologic methods (sensitivity, 97.4 vs. 89.5 percent; negative predictive value, 99.7 vs. 98.7 percent). CONCLUSIONS: For the prenatal diagnosis of congenital T. gondii infection, an approach based on a PCR test performed on amniotic fluid is rapid, safe, and accurate.

Amniotic Fluid↗
PubMed1994

Evaluation of human cytomegalovirus latency in alveolar macrophages.

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.

Adult↗
PubMed1994

Chemiluminescent detection of amplified pseudorabies virus gp50 DNA with immobilized probes on microtiter wells.

A highly specific and sensitive PCR assay for the envelope glycoprotein gp50 has been developed for the detection of PRV-DNA sequences. Primer pairs from PRV gp50 gene were used with the enzyme uracil N-glycosylase and dUTP instead dTTP to prevent contamination due to PCR product carry-over. Biotinylated PCR products were captured in microtiter wells by specific oligonucleotide covalently linked to the polystyrene wells. After recognition of the biotinylated PCR product with a streptavidin phosphatase conjugate, a chemiluminescent detection was realised. Different factors influencing the binding, the hybridization and the detection efficiency have been tested.

Base Sequence↗