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Biomedical subjects

F Galibert

Publications and source records attributed to F Galibert.

At least 109 records · Page 6Linked to original sources
PubMed1991

The use of labeled fusion protein for detection of B19 parvovirus IgM antibodies by an immunocapture test.

A new anti-B19 IgM ELISA was developed taking advantage of antibody-capture with biotinylated fusion protein as antigen. Specificity was examined using serum IgM antibody positive for rubella, hepatitis B core antigen, cytomegalovirus and Epstein-Barr virus as well as with sera positive for rheumatoid factors or antinuclear antibodies. The specificity was found to be 96%. Of one hundred serum samples compared using the new ELISA or the standard MACRIA tests for the presence of B19 IgM, 88 gave the same results. Fifty-three were negative and 35 were positive. Six sera were ELISA-negative MACRIA-positive, and six MACRIA-negative ELISA-positive. Thus, the ELISA gave 90% agreement with MACRIA. In a clinical study with 725 sera from suspected B19 infections, 161 (22%) were found positive by ELISA. The positive sera were from patients suffering from arthritis (35%), rash (35%), acute or chronic erythroblastopenia (21%), pancytopenia (5%), vascular purpura (2%) and lymphadenopathy (2%). A series of serum specimens obtained from two-B19 infected individuals were also studied. The IgM antibody became undetectable after four months.

Antibodies, Viral↗
PubMed1991

Production of polyclonal antibodies against the S and preS2 regions of woodchuck hepatitis virus: lack of detectable low glycosylated preS2 protein (GP33) in sera from infected animals.

Polyclonal antibodies directed against the preS2 and S domains of the woodchuck hepatitis virus (WHV) envelope proteins were prepared using synthetic peptides and fusion polypeptides as immunogens. They were tested by immunoblotting and immunoprecipitation of infected woodchuck sera and lysates of a eukaryotic cell line expressing WHV envelope proteins. Only one anti-peptide serum directed against the preS2 domain was reactive with WHV envelope proteins, recognizing the preS2 and preS1 proteins by their preS2 epitopes. With recombinant fusion proteins we generated several anti-S sera, which recognized all envelope proteins, and anti-preS2 antisera, which recognized the preS proteins. Results obtained with our antisera showed that sera of infected woodchucks lack the low glycosylated form (GP33) of the preS2 protein, unlike human hepatitis B virus.

Animals↗
PubMed1991

Spectrin Rouen (beta 220-218), a novel shortened beta-chain variant in a kindred with hereditary elliptocytosis. Characterization of the molecular defect as exon skipping due to a splice site mutation.

The molecular defect responsible for the shortened beta-spectrin chain variant, spectrin Rouen, was identified by analysis of cDNA and genomic DNA of affected individuals after amplification by the polymerase chain reaction. Peripheral blood reticulocyte RNA was transcribed into cDNA and amplified using primers corresponding to the 3' end of beta-spectrin cDNA. Agarose gel electrophoresis of cDNA amplification products from affected individuals revealed the expected band of 391 bp as well as a shortened band of 341 bp. Nucleotide sequencing of the shortened cDNA amplification product revealed that the sequences corresponding to the penultimate exon of the beta-spectrin gene (exon Y) were absent. This result was confirmed by hybridization of a Southern blot of amplification products with a labeled probe specific for exon Y. Nucleotide sequencing of the proband's amplified genomic DNA corresponding to this region of the beta-spectrin gene revealed a mutation in the 5' donor consensus splice site of the intron downstream of the Y exon, TGG/GTGAGT to TGG/GTTAGT, in one allele. We postulate that this mutation leads to the splicing out or skipping of exon Y, thus producing a shortened beta-spectrin chain. To our knowledge, this is the first documented example of exon skipping as the cause of a shortened beta-spectrin chain in a case of hereditary elliptocytosis. The exon skip results in the loss of the 17 amino acids of exon Y and creates a frameshift with the synthesis of 33 novel amino acids prior to premature chain termination 14 residues upstream of the normal carboxy terminus of the beta-spectrin chain, giving a mutant beta-spectrin chain that is 31 amino acids shorter than the normal chain.

Amino Acid Sequence↗
PubMed1991

C-myb proto-oncogene: evidence for intermolecular recombination of coding sequences.

We have characterized a novel chicken c-myb exon whose sequences are specifically expressed in thymic cells. In situ hybridization experiments indicate that this thymus-specific coding exon is localized on a small chromosome, distinct from the large acrocentric chromosome 3 on which we recently mapped the bulk of 15 exons, common to the c-myb mRNA species expressed in hematopoietic cells of both B and T lineages. These observations indicate that intermolecular recombination is required for the tissue-specific expression of the c-myb proto-oncogene. We also show that these thymus-specific sequences are conserved in human DNA and lie on chromosome 17q25, whereas the human c-myb locus is localized on chromosome 6q22-23. Sequencing data obtained from genomic DNA and PCR analyses performed with c-myb mRNA species expressed in chicken thymic cells strongly suggest that a repeated decameric sequence plays a key role in the recombination process.

Animals↗
PubMed1991

[Structural characteristics of the human lipoprotein lipase gene].

Lipoprotein lipase plays a major role in the metabolism of circulating triglyceride-rich lipoproteins. In relation with this study, the fundamental results concerning the structure of human LPL gene are first summarized. Sequencing of this gene enabled us to characterize an Alu sequence. Interest of these repetitive sequences is exposed.

Base Sequence↗
PubMed1990

Acute T-cell leukemia/lymphoma mimicking Hodgkin's disease with secondary HTLV I seroconversion.

The authors observed a pleiomorphic lymphoma mimicking Hodgkin's lymphoma in a French Guyana black woman lacking antibodies for human T-cell lymphoma/leukemia virus type I (HTLV I). After two courses of chemotherapy with either mechlorethamine, vincristine, procarbazine, and prednisone (MOPP) or doxorubicin, bleomycin, vincaleukoblastine, and dacarbazine (ABVD), a typical acute T-cell leukemia/lymphoma developed with HTLV I seroconversion. Specific HTLV I DNA sequences were detected using the polymerase chain reaction (PCR) on a lymph node biopsy obtained before chemotherapy. The mechanisms of the seroconversion are discussed.

Adult↗
PubMed1990

Hereditary pyropoikilocytosis and elliptocytosis in a white French family with the spectrin alpha I/74 variant related to a CGT to CAT codon change (Arg to His) at position 22 of the spectrin alpha I domain.

We describe a white French family in which 12 subjects presented with hereditary elliptocytosis (HE) or hereditary pyropoikilocytosis (HPP). Eight of these subjects were shown to be heterozygous for a spectrin (Sp) alpha I/74 variant, as demonstrated by analysis of partial tryptic digestion fragments of spectrin. This abnormal peptide pattern was associated with a decreased ability of Sp dimers to self-associate. In this kindred, in which four generations were available for study, the clinical expression varied from mild HE to HPP with an intermediate status of hemolytic HE. The severity of the disease appeared to be correlated both with the estimated amount of variant Sp (42% to 65%) and the excess of Sp dimers found in the membrane (30% to 51%, with a normal value of 3.7% +/- 1.6%). Reassociation studies using isolated Sp alpha and beta chains from an affected patient and an unaffected control subject showed that the Sp alpha I/74 Kd abnormal tryptic peptide resulted from a defect in the Sp alpha chain. Partial amino acid sequencing showed that the Sp alpha I/74 Kd peptide resulted from cleavage at lysine residue 42 of the Sp alpha I/80 Kd domain. Knowledge of the exon/intron organization of the human alpha Sp gene allowed us to amplify by the polymerase chain reaction the second exon of the alpha Sp gene in total cellular DNA of the HPP proposita. The amplified fragment was subcloned and sequenced. We found a G to A base substitution in the 22nd codon (CAT for CGT), which changes the normal arginine to a histidine. Hybridization of amplified DNAs with allele-specific oligonucleotides corresponding to the normal and mutant sequences confirmed the presence of the mutation in six other HE and HPP members of the family. The identification of this mutation at the DNA level confirmed the transmission of the same molecular defect in Sp through four generations but with different patterns of clinical expression.

Adult↗
PubMed1990

Early and frequent detection of HBxAg and/or anti-HBx in hepatitis B virus infection.

To clarify the significance of the X gene of hepatitis B virus, we have tested for anti-HBx in the serum and HBxAg in the liver at different stages of the natural history of hepatitis B virus infection. Sera were screened by enzyme-linked immunosorbent assay and positive results confirmed by immunoblot. Purified recombinant MS2 Pol-HBx fusion protein was used as target for both assays. Among serial sera of patients with nonfulminant acute hepatitis, 24 of 64 patients (37.5%) were positive for anti-HBx. In fulminant cases, 15 of 36 patients (42%) had anti-HBx. In chronic hepatitis patients with high rates of hepatitis B virus replication, we found a significantly (p less than 0.01) higher prevalence of anti-HBx, 14 of 25 patients (56%), than in those with low replication, 14 of 66 patients (21%), or among asymptomatic HBsAg carrier blood donors (20 of 126 = 16%) without detectable hepatitis B virus replication (p less than 0.0001). The highest prevalence of anti-HBx was found in HBsAg carriers with cirrhosis (41 of 54 patients = 76%) and/or with hepatocellular carcinoma (18 of 33 patients = 54%). The findings suggest that anti-HBx appears as a common and early marker of hepatitis B virus infection, transient in self-limited hepatitis but persisting with progression to chronicity. In chronic hepatitis, the prevalence of anti-HBx correlated with the intensity and duration of hepatitis B virus replication but neither with the severity of the liver disease nor with malignant transformation per se.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗
PubMed1990

Cloning, nucleotide sequence, and expression of the Rhodobacter sphaeroides Y thioredoxin gene.

Synthetic oligodeoxynucleotide probes based on the known amino acid sequence of Rhodobacter sphaeroides Y thioredoxin were used to identify, clone, and sequence the structural gene. The amino acid sequence derived from the DNA sequence of the R. sphaeroides gene was identical to the known amino acid sequence of R. sphaeroides thioredoxin. An NcoI site was created by directed mutagenesis at the beginning of the thioredoxin gene, inducing in the encoded protein the replacement of serine in position 2 by alanine. The 421-base-pair NcoI-PstI restriction fragment obtained was ligated in the pKK233-2 expression vector and the resulting hybrid plasmid was used to transform Escherichia coli strains lacking functional thioredoxin. Transformants that complemented mutations in the trxA gene were identified by increased colony size on rich medium, growth on minimal medium with methionine sulfoxide, and ability to support M13 growth and T7 replication; this latter phenotype implies correct interaction between R. sphaeroides thioredoxin and the product of T7 gene 5. The presence of R. sphaeroides thioredoxin was further confirmed by enzyme assay.

Amino Acid Sequence↗
PubMed1990

A combination of HLA-DQ beta Asp57-negative and HLA DQ alpha Arg52 confers susceptibility to insulin-dependent diabetes mellitus.

Family and population studies indicate that predisposition to insulin-dependent (type I) diabetes mellitus (IDDM) is polygenic. It has been shown that the absence of the aspartic acid in position 57 (Asp57) of the DQ beta chain is positively correlated to IDDM. However, Asp57-negative haplotypes do not always confer susceptibility and conversely, some Asp57-positive haplotypes seem to be disease associated. It has been suggested that other HLA class II sequences, probably belonging to the HLA DQA1 gene, confer susceptibility to IDDM. This report, based on extensive oligonucleotide dot blot hybridization of PCR-amplified DQA1 and DQB1 genes, reinforces the importance of the Asp57-negative DQ beta chain, but also introduces the possibility that a DQ alpha chain bearing an arginine in position 52 (Arg52) confers susceptibility to IDDM. A molecular model of susceptibility to IDDM is proposed. This model strongly suggests that the disease susceptibility correlates quantitatively with the expression at the cell surface of a heterodimer, composed of a DQ alpha-chain bearing an Arg52 and a DQ beta chain lacking an Asp57. In view of the respective positions of the two residues and their charge, we might anticipate that both residues DQ beta Asp57 and DQ alpha Arg52 are critical for modulation of susceptibility, presumably via viral-antigenic peptide and/or autoantigen presentation.

Base Sequence↗
PubMed1990

Use of oligonucleotide probes directed against T cell antigen receptor gamma delta variable-(diversity)-joining junctional sequences as a general method for detecting minimal residual disease in acute lymphoblastic leukemias.

To provide a sensitive and generally applicable method to detect clonal cells in acute lymphoblastic leukemias (ALL), we have designed a new strategy based on the polymerase chain reaction (PCR) amplification of the T cell receptor gamma delta gene rearrangements found in most T and B lineage ALLs. PCR allows rapid sequencing of variable-(diversity)-joining (V-[D]-J) junctions from tumor DNA and construction of anti-junctional oligonucleotides (AJOs) used as probes to detect clonal cells in the same patient. We have defined oligonucleotides suitable for all T cell receptor (TCR) rearrangements involving functional V gamma segments. Oligonucleotides corresponding to preferential TCR delta rearrangements in T and B lineage ALLs were also used. By analysis of the nucleotide sequence of 52 V gamma-V gamma junctions from 30 cases of B and T ALLs, we demonstrate that V-J junctional sequences are clone specific in both lineages and at all stages of differentiation examined despite the frequent presence of the recently described P nucleotides. Experiments performed with TCR gamma delta AJOs on DNA from tumor cells and polyclonal T cells show that AJOs can be used to differentiate clonal cells from polyclonal T cells, distinguish between different T cell clones, and detect residual clonal populations at 10(-4)/10(-5) dilution. AJOs were also used to detect residual disease in samples from patients in clinical and morphological complete remission. Finally, rearrangement patterns were studied by classical Southern analysis in selected cases at both presentation and subsequent relapse showing absence of clonal evolution in most cases. V-(D)-J nucleotide sequences of rearrangements with an identical pattern of rearrangement at presentation and relapse were identical in all cases analyzed. We therefore describe a new, specific, and clinically useful strategy for the detection of minor clonal populations applicable in the majority of cases of ALL.

Alleles↗
PubMed1990

HLA DQ alpha/beta molecule associated with the susceptibility to insulin-dependent diabetes mellitus. A model for HLA/autoimmune disease association.

We have proposed a molecular model of susceptibility to Insulin-Dependent Diabetes Mellitus (IDDM) based on the proportional expression at the cell surface of the following susceptible (S-S) HLA-DQ heterodimere composed of a chain DQ alpha Arg 52 positive (S) and a chain HLA-DQ beta Asp 57 negative (S). All other DQ alpha/beta chains associations are considered as protective molecules. This model allows a predictive scale of risk for the disease.

Alleles↗
PubMed1990

Development of an IgM antibody capture test using labelled fusion protein as antigen for diagnosis of B19 human parvovirus infections.

A new anti-B19 IgM ELISA was developed using the antibody-capture principle. Biotinylated fusion protein was used as antigen. The specificity of the test was analysed using sera IgM positive to rubella, hepatitis B core antigen, cytomegalovirus and Epstein Barr virus as well with sera positive for rheumatoid factors, antinuclear antibodies and with sera from normal blood donors. The specificity of the test was 96.18%. One hundred serum samples were tested by the new ELISA and the standard MACRIA tests for the presence of B19 IgM. Eighty-eight sera gave the same results with both tests. Fifty-three were negative and 35 were positive. Six sera were ELISA negative MACRIA positive and six MACRIA negative ELISA positive. In a clinical study with 725 sera from suspected B19 infections, 161 (22%) were recorded as positive by the ELISA test. The positive sera were from patients suffering from arthritis (35%), rash (35%), acute or chronic erythroblastopenia (21%), pancytopenia (5%), vascular purpura (2%) and lymphadenopathy (2%).

Capsid↗
PubMed1990

Typing for HLA DQ using oligonucleotidic probes.

We have typed 25 homozygous B lymphoblastoid cell lines, defined as reference panel during the Xth International Histocompatibility Workshop, using PCR (Polymerase Chain Reaction) and oligonucleotidic probes recognizing sequences of DQA and DQB genes. The polymorphism of 8 HLA-DQA1, and 12 DQB1 alleles is reported and the advantages of this technique are compared to that of other typing methods, currently used.

Cell Line↗
PubMed1990

Diagnosis of HTLV-I infected seronegative neurological patients by polymerase chain reaction amplification in Martinique.

HTLV-I is a type C human retrovirus, endemic in Japan, central Africa, South America and the Caribbean, which causes adult T cell leukemia/lymphoma and chronic progressive paraparesis. Some neurological patients with chronic progressive myelopathies, but seronegative for HTLV-I have been reported in Martinique. We performed polymerase chain reaction (PCR), to look for the presence of HTLV-I genomic sequences in those patients. Two primers were chosen for gag and tax genes. Liquid hybridization was performed on the amplified products using an internal 32p labelled oligonucleotide as a probe. The hybridized material was run on a 6% polyacrylamide gel. Forty-three of forty-four seropositive subjects analysed as "positive controls" were positive for gag, but only 16/29 for tax. Twenty HTLV-I seronegative neurological patients with a symptomatology suggesting HAM/TSP were tested: 18 were found positive for at least one of the 2 oligonucleotide pairs. Two "negative controls": Moya-Moya and vascular brain failure were found negative. 8/9 subjects with uninterpretable WB were also studied by PCR, and 8 were found positive for at least one oligo pair. Our conclusion is that PCR methodology is able to detect genetic information related to HTLV-I in a number of cases where classical serology is negative.

Blotting, Western↗
PubMed1989

Analysis of junctional diversity in the preferential V delta 1-J delta 1 rearrangement of fresh T-acute lymphoblastic leukemia cells by in vitro gene amplification and direct sequencing.

To define the junctional diversity of T-cell antigen receptor delta gene rearrangements in fresh T-acute lymphoblastic cells and to correlate cell phenotype with the coding potential of rearrangements, we determined the junctional nucleotide sequences of 13 T-cell antigen receptor delta gene rearrangements involving the preferentially rearranged V (V delta 1) and J (J delta 1) segments using in vitro gene amplification and direct sequencing. We showed that, as in gamma delta+ cell lines, extensive junctional diversity exists in these clones and that this diversity is due both to random nucleotide deletions/additions and to the use of at least two D delta segments. We also showed that a high percentage of these rearrangements are potentially translatable (7:13) and that such functional rearrangements occur in both surface CD3+ and CD3- cells. Comparison of alpha beta versus gamma delta surface expression demonstrates that all CD3+ T acute lymphoblastic leukemias with a functional V delta 1-J delta 1 rearrangement express a surface gamma delta receptor and are recognized by the anti-delta monoclonal antibody delta TCS1, whereas a control CD3+ gamma delta+ leukemic case that had not undergone V delta 1 rearrangement was delta TCS1-. In addition, expression of this monoclonal antibody is not restricted by V gamma or C gamma usage or by the covalent or noncovalent link between gamma and delta chains.

Amino Acid Sequence↗