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M Heusterspreute

Publications and source records attributed to M Heusterspreute.

At least 19 recordsLinked to original sources
PubMed2000

Expression of prostate-specific membrane antigen in transitional cell carcinoma of the bladder: prognostic value?

The expression of Prostate-specific membrane antigen (PSMA) mRNA was assessed in the normal bladder urothelium (n = 9), transitional cell carcinoma (TCC) specimens (n = 52), TCC-derived cell lines (n = 3), and preoperative blood samples from TCC patients (n = 27). Specific PSMA mRNA was found in 100% of normal and malignant tissues and two cell lines. PSMA protein was detected in normal (n = 3) and malignant tissues (n = 4). Using a PSMA-specific substrate, PSMA enzymatic activity was found in two bladder cell lines and correlated with immunostaining. Seven of the 27 TCC preoperative blood samples were positive by reverse transcription-PCR. These preliminary results, obtained on a nonrandomized cohort of patients, correlated with tumor invasion (positive RT-PCR: 0% for pT < or = 2 versus 41% for pT > or = 3) and 2-year survival rate (81% in the PSMA-negative group versus 29% in the PSMA-positive group). Although the clinical usefulness of this assay requires confirmation in larger prospective randomized trials, current preliminary results suggest that a blood-borne PSMA mRNA PCR assay may be a useful tool to predict a poor outcome in TCC patients.

Aged↗
PubMed1999

Molecular characterization of femA from Staphylococcus hominis and Staphylococcus saprophyticus, and femA-based discrimination of staphylococcal species.

The femA gene encodes a protein precursor which plays a role in peptidoglycan biosynthesis in Staphylococcus aureus and is also considered as a factor influencing the level of methicillin resistance. A femA homologous gene was recently characterized in S. epidermidis, entailing the possibility of femA phylogenetic conservation in staphylococcal species. Accordingly, we assessed the presence of femA homologous genes in S. hominis and S. saprophyticus. Strategy for identification relied upon alignment of S. aureus and D. epidermidis femA sequences and upon identification of potentially conserved regions. Amplifications of portions of the femA genes were performed under permissive annealing conditions, by using several sets of primers designed to match the consensus regions. DNA sequencing of overlapping PCR fragments led to the characterization of the entire femA genes of S. hominis and S. saprophyticus, and provided more precise information on the femA start codon for all five species. The genomic organization of all these femA genes appeared highly conserved, with alternance of homologous and variable regions. On this basis, a consensus sequence of the femA gene was defined and interspecies variations were exploited to design strategies for staphylococci species-specific identification, including multiplex PCR amplification and a reverse hybridization assay.

Amino Acid Sequence↗
PubMed1999

Peripheral blood stem cell contamination in mantle cell non-Hodgkin lymphoma: the case for purging?

Intensification using peripheral blood stem cells collected after chemotherapy followed by growth factors is being increasingly investigated as an alternative to conventional chemotherapy for mantle cell non-Hodgkin lymphoma. We investigated 14 grades III-IV, t(11;14)-positive cases for contamination of PBSC collected after a polychemotherapy regimen followed by G-CSF. Patients were first treated with a polychemotherapy regimen. There were four CR, seven PR, two refractory and one early death. Seven patients have been transplanted, in whom PBSC were mobilized, using either cyclophosphamide/VP16 or Dexa-BEAM followed by G-CSF. For all patients, whether actually autografted or not, PB cells were tested at the time of regeneration on G-CSF after the first polychemotherapy or after the mobilizing regimen. PCR evaluation of contamination was performed first by a semi-quantitative approach, using serial dilutions of initial DNA, then confirmed using a limiting-dilution analysis. Two patients were not informative (one early death and one without an available molecular marker). PB cells collected at regeneration contained at least one log more lymphoma cells than steady-state blood or marrow, apart from in two cases. Moreover, where a mobilizing treatment diminished tumor burden in the patient, at the same time it increased PB contamination in most cases. We conclude that advanced mantle cell NHL appears to be largely resistant to significant in vivo purging by conventional chemotherapy. Where treatment brings benefits by reducing tumor load, it may at the same time negate it by mobilizing malignant cells into the collections used to intensify. Although the clonogenic potential of this massive infiltration is unknown (only gene marking studies could provide a definitive answer regarding the source of relapses), strategies aimed at reducing the level of contamination in the graft should be considered when designing future protocols.

Adult↗
PubMed1998

Introduction of a normal retinoblastoma (Rb) gene into Rb-deficient lymphoblastoid cells delays tumorigenicity in immunodefective mice.

Inactivation of the Rb susceptibility gene occurs in various human cancers and has been associated with tumorigenicity. Rb gene is inactivated in 30% of acute leukaemias. The effect of Rb protein expression was assessed in the lymphoblastoid cell line IM-9 defective for Rb protein, after stable transfection with a wild-type Rb gene. The Rb transgene was under the control of the MoMuLv-LTR. Protein expression by the transduced cells was confirmed by Western blot and flow cytometry analyses. Compared to the parental cell line, growth rate remained unchanged in the Rb transfected clones. In SCID mice however, tumor formation originating from these clones was delayed. The current data suggest therefore that, in this Rb-defective haematopoietic cell line, Rb expression correlates with reduced tumorigenicity but not with reduced growth rate.

Animals↗
PubMed1998

The prothrombin gene G20210A variant in an unselected thromboembolic population. A Belgian prospective clinical study.

The presence of the 20210A allele of the prothrombin gene has recently been shown to be a risk factor of venous thromboembolism, probably mediated through increased prothrombin levels. The aim of the study was to determine the frequency of the prothrombin 20210A allele in 193 consecutive unselected patients with venous thromboembolism and 100 healthy controls and to analyze the clinical profile associated with this new inherited thrombophilic factor. In agreement with previous reports, we found a frequency of 7.3% of heterozygous carriers of the 20210A allele among patients and 1% among controls. We confirm that plasma prothrombin levels are more elevated in the individuals bearing the prothrombin 20210A allele compared with those who do not. We did not find any relationship between the presence of the prothrombin 20210A allele and either a family history of thromboembolism, the rate of recurrences or the age at disease onset. However, the co-inheritance in the same individual of both prothrombin 20210A allele and factor V Leiden was associated with a significantly lower age at disease onset suggesting a synergistic contribution of both abnormalities.

Adult↗
PubMed1998

Expression of prostate-specific antigen and prostate-specific membrane antigen transcripts in blood cells: implications for the detection of hematogenous prostate cells and standardization.

Circulating prostate cells can be detected in cancer patients by using reverse transcriptase-PCR (RT-PCR) assay for prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSM) mRNA. A quality-control study involving a conventional RT-PCR assay was performed and, surprisingly, detected both transcripts in many negative control cell lines and in normal blood samples. The existence of an illegitimate transcription of the PSA and PSM genes was evidenced by sequence analysis of several PSM and PSA-PCR products. Sequencing indeed demonstrated the presence of a PSA or PSM polymorphism in some but not all the cell lines and patient samples, as well as a heterozygous mutation (G to A; Asp to Asn) in the Jurkat cell line. Moreover, the amount of PSA transcript in MCF-7, a PSA-negative breast line, increased after incubation with cycloheximide. Interestingly, the frequency of positivity was as high as 12% in male samples if only tested once, but dropped to 3% upon multiple testing of the same cDNA. This highlights the stochastic effects in RT-PCR results at high sensitivity, hence the importance of repetitive testing in clinical samples. Decreasing the number of cycles avoided the amplification of illegitimate transcripts but also affected the limit of detection, as evidenced with PSA and PSM cDNA containing plasmids, mixing of LNCap with normal blood samples, and the PSA-PSM-negative K562 cell line. The current data raise the need for a multicentric standardization of the RT-PCR methodology used to amplify PSA and PSM transcripts.

Adult↗
PubMed1997

Prevalence of activated protein C resistance and analysis of clinical profile in thromboembolic patients. A Belgian prospective study.

OBJECTIVES: To assess the prevalence of activated protein C resistance (APC-R) among healthy subjects and thromboembolic patients and to determine the clinical characteristics associated with APC-R. DESIGN: A prospective study. SETTING: One academic medical centre. SUBJECTS: 91 health controls and 126 thromboembolic patients. MEASUREMENTS: Patients and control were genotyped for the factor V Leiden (VaQ506) mutation. The anticoagulant response of the patient's plasma to activated protein C was also determined. RESULTS: The frequency of APC-R was 3.3% among healthy control subjects and 22% among thrombotic patients of whom 18% were heterozygous and 4% were homozygous. The mean age at the first thrombotic event and the severity of thrombotic disease including the proportion of proximal deep vein thrombosis and the frequency of lung embolism were identical among APC-R positive and negative patients. A family history of thromboembolic disease was elicited more frequently in APC-R positive than in APC-R negative patients (57% vs. 22%, P < 0.001). The recurrence rate was higher for APCR-R positive patients (57% vs. 34%, P < 0.05). The percentage of cases with a factor predisposing to thrombosis was very similar in APC-R positive (57%) and negative (68%) patients. CONCLUSIONS: A familial history of thromboembolic disease and recurrences are significantly more frequent among APC-R positive than APC-R negative patients.

Adolescent↗
PubMed1997

Beta-thalassaemia in indigenous Belgians: an update.

Beta-thalassaemia, a widespread autosomal recessive disorder, occurs sporadically in Northern and Western European countries. Molecular analysis of the beta-globin gene has been carried out in 30 members of 15 unrelated indigenous Belgian families which presented with non sideropenic hypochromic and microcytic anaemia. For all of them, extensive search failed to find an ancestor at risk for the disease. The beta-globin genes were first screened for frequent beta-thalassemic mutations by dot-blot hybridization with specific radiolabeled oligonucleotide probes. Direct automated fluorescence-based DNA sequencing and, in one case, Southern blotting were also used. All the 30 patients were found to be heterozygous for a beta-thalassemic mutation. Eight different mutations were identified. Among these, four are commonly found in the Mediterraneans: codon 8 (-AA), IVS-I-1 (G-->A), IVS-1-6 (T-->C) and codon 39 (C-->T); three have occasionally been reported: initiation codon (T-->C) and codon 35 (C-->A) and a rare deletion of 12.6 kb which removes all the beta-globin gene and its flanking regions. A new mutation, a -CC deletion at codon 38/39 was found in one family. These results both at the biological and molecular level show that beta-thalassaemia exist in indigenous Belgian families with no known ancestor a risk for the disease. They also show that clinicians and biologists should keep in mind the existence of beta-thalassaemia in indigenous Belgian families when investigating hypochromic and microcytic anaemia in patients whom the past familial history does not evocate a risk for the disease.

Adenine↗
PubMed1996

Beta-thalassaemia in indigenous Belgian families: identification of a novel mutation.

The molecular basis of beta-thalassemia was investigated at the DNA level in 28 Belgians from 14 unrelated families. All the patients were heterozygous for beta-thalassaemia. Seven different mutations were identified using a combination of dot-blot hybridization with allele-specific oligonucleotide probes and direct automated fluorescence-based DNA sequencing. Among these mutations, four are commonly found in the Mediterraneans - codon 8 (-AA), IVS-I-1 (G --> A), IVS-I-6 (T --> C) and codon 39 (C --> T)-and two have occasionally been reported-initiation codon (T --> C) and codon 35 (C --> A). The last mutation, a -CC deletion at codons 38/39, appears to be a novel mutation and can routinely be investigated by AvaII restriction on amplified DNA. We report our findings, discuss the diversity of the mutations found in Belgium and show the usefulness of direct DNA sequencing in a population in which the molecular defects of beta-thalassaemia have yet to be characterized and in which screening is hampered by the wide range of potential mutations.

Belgium↗
PubMed1995

HLA class II genotyping: two assay systems compared.

In the last few years, a variety of DNA-based human leukocyte antigen (HLA) typing methods have emerged, revealing the extreme polymorphism of HLA genes. This polymorphism makes it difficult for a clinical laboratory to establish the best HLA typing strategy. In this study we have compared two techniques for performing HLA-DRB typing: a commercial rapid assay based on the polymerase chain reaction (PCR) followed by reverse dot-blot hybridization of the PCR products (the Inno-LiPA assay), and a method based on PCR followed by restriction fragment length polymorphism analysis. We found that both methods provide reliable results with a high rate of concordance (97%) and that Inno-LiPA is convenient for large-scale routine typing. However, if a high-resolution allelic typing is required, each method lacks accuracy but using them in association improves the accuracy of the results.

Alleles↗
PubMed1995

An anti-HIV-1 gag protein rat monoclonal antibody library.

A set of 18 rat monoclonal antibodies (MAbs) directed against human immunodeficiency virus type 1 (HIV-1) gag proteins was derived from 4 independent fusion protocols. The epitopes recognized were delineated using a random fragment expression library representing the whole HIV-1IIIB genome. This panel of rat MAbs was used to analyze the antigenicities of the HIV-1 CAp24 major core protein and the HIV-1 NCp7 nucleocapsid protein. As a result, a limited set of antigenic domains as defined, 3 on CAp24 between amino acids (aa) 195 and 268, 323 and 329, 329 and 352, and one on NCp7 (aa 382-392). Only 4 mouse anti-CAp24 MAbs appeared to recognize the COOH-terminal domain (aa 329-352) defined by the majority of our MAbs. The rat anti-CAp24 (Q1B10) and the rat anti-NCp7 (I5B11) MAbs described here, defined two newly described epitopes, aa 323-329 and aa 382-392.

Animals↗
PubMed1993

Isolation of a C-type retrovirus from an HIV infected cell line.

A study by electron microscopy of HUT 78 cells infected with the ARV-2 strain of HIV-1 revealed, in addition to virions having the characteristic morphology of a lentivirus, the presence of numerous C-type particles, suggesting that the analysed specimen might in fact contain two different viruses. In order to further investigate this observation, the cell culture supernatant was filtered and mixed at serial dilutions with uninfected HUT 78 cells. In this way, it was possible to obtain cells producing only virions with C-type morphology and a Mn++ dependent reverse transcriptase activity which in a sucrose gradient was found to peak at a buoyant density of 1.16 g/cm3. The RNA purified from the culture medium was not detected by reference DNA probes for either HIV-1 and -2 or HTLV-I and -II. In an indirect immunofluorescence assay, sera from patients seropositive for these human retroviruses failed to recognize any antigen in the cells producing only the C-type particles. Using the same technique, plasma samples from 100 blood donors also gave negative results. The presence of this retrovirus could be the result of previous laboratory contamination but the possibility that it is indeed a human virus has to be considered.

Cell Line↗
PubMed1990

Isolation of HIV-1 from seropositive people living in Cotonou, Benin.

Benin is located in West Africa and is situated between HIV-2 and HIV-1-endemic zones. The first cases of HIV-1 infection in Benin were reported in 1987. Since then, AIDS cases have been diagnosed there and the number of known HIV-seropositive people has rapidly increased. Blood samples were collected from 14 seropositive and 11 seronegative patients living in the main city, Cotonou, and their peripheral blood mononuclear cells were cultured. In seven of the seropositive cases, a retrovirus was detected by measurement of Mg2(+)-dependent reverse transcriptase activity and electron microscopy. HIV-1 antigen assay and genomic analysis indicated that the isolated viruses belong to the first serotype. In each positive case, an HIV-1 DNA probe hybridized to the RNA extracted from the virus and six isolates were found positive by the polymerase chain reaction using HIV-1-specific primers.

Base Sequence↗
PubMed1987

The first-step transfer-DNA injection-stop signal of bacteriophage T5.

Bacteriophage T5 is different from most phages in that its DNA is injected in two steps during infection. The region containing the injection stop signal (iss) has been cloned and sequenced and found to contain numerous large repeats and inverted repeats which may be part of the iss. The most impressive of these are the 31-bp repeat units (rb) which are present three times in 99 bp. The rb repeats, themselves, contain inverted repeats so that mutually exclusive stem-and-loop structures may potentially form, not only within the repeats, but also between them. Another pair of repeats (21 bp each) contains two sequences resembling DnaA protein-binding sites. The region sequenced also contains one of the T5 site-specific strand interruptions and this was found to lie at the base of a perfect 9-bp palindrome.

Base Composition↗
PubMed1987

A 'phase-shift' fusion system for the regulation of foreign gene expression by lambda repressor in gram-negative bacteria.

A 'phase-shift' translation fusion vector was constructed in which mutually compatible restriction sites BamHI, BclI and BglII are positioned in such a manner that the cut point is in a different reading frame, immediately following the ATG start codon and ribosome-binding site of the lambda cro gene. The lambda cro gene is expressed from promoter pR and controlled by a thermosensitive (cI857) lambda repressor. The usefulness of the expression vector was demonstrated using a galK gene lacking the ATG start codon and fusing this to the pR promoter and ATG start codon of the lambda cro gene, resulting in cI857-regulated expression of galactokinase. The vector is of general use for foreign gene expression in Escherichia coli when the target gene has a compatible cohesive end (5'-GATC-3') at the N terminus (provided, for example, by a BamHI linker). The lambda cI857-pR-cro-galK cassette was cloned into pJRD215, a wide-host-range plasmid and transferred by conjugation to a variety of Gram-negative bacteria. In all cases, thermosensitive regulation of galactokinase could be demonstrated, though the levels of induction varied considerably. These results show that the powerful lambda pR promoter and the efficient lambda repressor can be used to regulate expression of foreign genes in Gram-negative organisms other than E. coli.

Bacteriophage lambda↗
PubMed1987

Vectors with restriction site banks. V. pJRD215, a wide-host-range cosmid vector with multiple cloning sites.

The construction of a new wide-host-range, restriction-site bank, cosmid-cloning vehicle (pJRD215) is described. The wide-host-range properties and the ability to be transferred by conjugation, extend genetic engineering to those Gram-negative species that cannot be transformed. The vector permits the cloning of genes from Gram-negative bacteria using a complementation screening procedure in a mutant host. This procedure is simplified by the possibility of construction of a cosmid gene bank so that only a few hundred clones need to be screened. Subsequent subcloning of the gene of interest is facilitated by the presence of at least 23 unique cloning sites.

Base Sequence↗