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Biomedical subjects

A Benoit

Publications and source records attributed to A Benoit.

At least 19 recordsLinked to original sources
PubMed2006

Effects of alveolar macrophage depletion on liposomal vaccine protection against respiratory syncytial virus (RSV).

Little is known about the identities and roles of antigen-presenting cells upon exposure to antigens of respiratory syncytial virus (RSV). Here, we focused on elucidating the importance of alveolar macrophages in conferring protective immunity in mice administered a liposome-encapsulated recombinant fragment of the RSV G protein. Mice were depleted of alveolar macrophages by intranasal inoculation of liposome-encapsulated dichloromethylenediphosphonic acid (DMDP). Mice depleted of alveolar macrophages prior to immunization developed reduced levels of serum RSV-neutralizing antibody and showed dramatically impaired protection against RSV challenge. The severity of interstitial inflammation was also markedly reduced in macrophage-depleted mice. In conclusion, this study demonstrates a pivotal role for alveolar macrophages during exposure to liposome-encapsulated RSV antigen in initiating both protective and histopathological responses against RSV.

Animals↗
PubMed1999

The proapoptotic effect of hepatitis B virus HBx protein correlates with its transactivation activity in stably transfected cell lines.

The role of hepatitis B virus HBx protein in the carcinogenesis associated with chronic viral infection remains ill-defined. Indeed, pleiotropic effects have been ascribed to HBx: in addition to its well-documented ability to indirectly stimulate transcription, the protein has been reported to affect cell growth, signal transduction, DNA repair and apoptosis. In this work, we generated Chang (CCL-13)-derived cell lines constitutively expressing wild type or mutant HBx, as a model of HBx-host cell interaction closer to the chronic infection setting, than the classically used transient expression systems. We document the potentiation by HBx of the apoptotic cell death pathway in the recipient cells. This effect is unlikely to rely on p53 activity since the protein is functionally inactivated in CCL-13. In addition, antioxidants and cyclosporin A failed to reduce the apoptotic response back to the normal level, suggesting that production of reactive oxygen species and calcineurin activation are not directly involved in the proapoptotic effect of HBx. In contrast, our data show that transactivation and stimulation of apoptosis are tightly linked HBx activities. Finally, expression of transactivation-active protein did not result in detectable change in the pattern of MAP kinases phosphorylation nor did it affect the ability of the host cell to repair in vitro irradiated plasmid DNA.

Apoptosis↗
PubMed1997

The comet assay as a repair test for prenatal diagnosis of Xeroderma pigmentosum and trichothiodystrophy.

Xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) are autosomal recessive diseases associated with extreme cutaneous photosensitivity, a defect in nucleotide excision repair (NER), and genetic complexity. Severe prognosis and lack of treatment led families at risk to request genetic counseling. Unscheduled DNA synthesis (UDS) is the classic method for diagnosis and requires 4 to 5 wk before conclusion. The use of the alkaline comet assay (single cell gel electrophoresis assay) is proposed as a simple repair test for earlier prenatal diagnosis. Amniotic or chorionic villus cells in two pregnancies at risk for XP and one for TTD were examined in comparison with skin fibroblasts of family members or with repair-proficient or -deficient control cells. The comet assay and the UDS test were performed in parallel. In repair-proficient cells, DNA strand breaks due to the incision of UV-induced DNA damage result in increased migration of high molecular weight DNA in the comet assay. Fetal cells demonstrate repair capacity similar to that of fibroblasts. In incision repair-deficient XP and TTD cells, after post-UV incubation, migration does not occur and comet moments are reduced. Two fetuses belonging to two XP families responded normally and were diagnosed as unaffected. Fetal cells in a TTD family had reduced comet moments and a low UDS. This fetus was diagnosed and confirmed later as affected. Heterozygotes had normal responses with both assays. The comet assay offers discrimination similar to that of the UDS assay in identifying NER-deficient phenotypes. Practical advantages in view of prenatal diagnosis include the reduced number of cells required, a 24-h delay in obtaining results, and no need for radioactivity.

Cells, Cultured↗
PubMed1996

Recovery of normal DNA repair and mutagenesis in trichothiodystrophy cells after transduction of the XPD human gene.

To determine whether expression of the XPD/ERCC2 repair gene in trichothiodystrophy (TTD) group D cells could restore mutagenesis characteristics of repair-proficient cells, we compared the UV mutagenesis of normal cells, TTD group D cells, and TTD group D cells retrovirally transduced by the wild-type XPD/ERCC2 gene (TTD + ERCC2 cells). We first verified the expression of the XPD protein, correction of UV cell survival, and DNA repair ability of TTD + ERCC2 cells. UV-induced mutations were studied using the pR2 shuttle vector. The addition of the XPD/ERCC2 gene in TTD cells led to a significant but partial decrease of mutation frequency compared with the parental TTD cells. Types of mutations of TTD + ERCC2 cells get closer to those observed in normal cells (ie., a reduction of multiple mutations). New hotspots appeared and some disappeared in the complemented line, suggesting that hotspot distribution is particular to each cell line and cannot be correlated with the repair status of the cells. In conclusion, the expression of the XPD/ERCC2 repair gene completely corrected UV hypersensitivity and almost all types of mutations of TTD group D cells, whereas hypermutagenesis was partially corrected.

Cell Line, Transformed↗
PubMed1996

Cell survival and shuttle vector mutagenesis induced by ultraviolet A and ultraviolet B radiation in a human cell line.

Although it is known that sunlight is carcinogenic,few molecular data are available concerning the mutagenic effects of ultraviolet (UV) B (290-320 nm) and UVA (320-400 nm) radiation in human cells. To analyze the biologic effects of UVA and UVB, we irradiated the 293 human cell line, derived from adenovirus-transformed human embryonic kidney cells, in which we had stably introduced a shuttle vector harboring the lacZ' bacterial gene as the mutagenesis target. Identical cell survival occurred after UVA doses 700-fold higher than UVB. This comparable to the UVA/UVB ratio that reaches the basal cell layer of the skin after sunlight exposure with UVB sunscreen. The frequency of UVA- and UVB- induced mutations increased with the UV dose as cell survival decreased. At cell survival levels greater than 10%, UVA and UVB induced similar frequencies of mutations in the episomal lacZ gene, whereas for cell survival lower than 10%, UVA induced twice as many mutations as UVB. Sequence analysis of 81 independent lacZ mutants (36 UVA- and 45 UVB-induced) revealed specific characteristics for some UV-induced-mutations, particularly for UVB. Mutations at A/T base pairs were induced more frequently by UVA than by UVB. The UVA-induced mutation spectrum that we have observed in human cells may help help to elucidate the mechanism of skin carcinogenesis.

Amino Acid Sequence↗
PubMed1995

Characteristics of UV-induced mutation spectra in human XP-D/ERCC2 gene-mutated xeroderma pigmentosum and trichothiodystrophy cells.

To study the relationships between mutagenesis and carcinogenesis, we compared the mutations and their frequency induced by ultraviolet irradiation at 254 nm (UV-C) in XP-D (GM-08207B/XP6BE), TTD/XP-D (TTD1VI-LAS-KMT11) and wild-type (MRC-5V1) human cells. XP-D and TTD/XP-D cells, mutated in the same XP-D/ERCC2 gene, are deficient in nucleotide excision repair. Whereas XP-D patients develop early skin tumors, TTD patients do not exhibit abnormal levels of cancers. After verification of UV hypersensitivity and DNA repair defect of the immortalized cell lines XP-D and TTD compared with a wild-type cell line, UV-induced mutagenesis was studied with a new shuttle vector pR2, carrying the target lacZ' gene. The UV-mutation frequencies in XP-D and TTD cells were similar and significantly increased compared with normal cells. Sequence analysis of 312 independent mutant plasmids revealed that more rearrangements were induced in TTD cells (16%) than in XP-D (5%) and normal cells (1%), while XP-D cells exhibited a twofold higher rate of tandem mutations compared with TTD and normal cells. In the three cell lines, a predominance of G:C to A:T transitions was found, especially in chiefly on the cytosine at 5'-TC-3' sites. The types of UV-induced point mutations in TTD cells were, however, more similar to those in normal cells than those found in XP-D cells. XP-D mutations were preferentially located in 5'-TCPur-3' sites, while mutations in normal and TTD cells were mostly at 5'-TCC-3' sites. Analysis of mutation spectra revealed differences in the location of the mutational hotspots between the three lines. Although the mutation frequency of the UV-irradiated pR2 vector is much higher in TTD and XP-D cells than in normal cells, the mutation spectrum is closer between TTD and normal cells as compared with XP-D cells. These dissimilarities could contribute to an explanation of some of the differences between the two syndromes.

Amino Acid Sequence↗
PubMed1995

Stable SV40-transformation and characterisation of some DNA repair properties of fibroblasts from a trichothiodystrophy patient.

To characterize nucleotide excision repair properties of cells from trichothiodystrophy (TTD) patients genetically-related to the xeroderma pigmentosum (XP) group D, TTD skin fibroblasts from two unrelated patients (TTD1VI and TTD2VI) belonging to the TTD/XPD group were transformed with a plasmid containing SV40 large T antigen-coding sequences and some DNA repair properties, such as unscheduled DNA synthesis (UDS), UV-survival, in vitro repair synthesis of cell extracts and reactivation of UV-irradiated reporter plasmid were studied. Results showed that: a) both untransformed and transformed TTD cells present a reduced UV-survival, compared to wild-type cells, but at significantly less reduced levels than XP-D cells; b) reduced repair activities were detected in both TTD and XP-D transformed cells by using in vitro cell free extract repair and reactivation of UV-irradiated plasmid procedures, and these relative reduced extents correlated with respective UV-survival; c) surprisingly, near wild-type UDS levels were detected in TTD2VILas transformed cells at different passages after the crisis, suggesting a phenotypic reversion of this transformed cell line; d) fluoro-cytometric analysis of TTD2VILas cells revealed a strong increase of a cell population containing a DNA amount more than twice as high than that of untransformed cells; finally, e) when UDS data were normalized to the DNA content in TTD2VILas cells, it appeared that the repair efficiency was only slightly higher than in untransformed cells. This implies that in transformed cells DNA repair properties should be evaluated, taking into account additional parameters. We obtained an immortalized TTD cell line which maintains DNA repair properties similar to those of parental untransformed cells and may be used to characterize the TTD defect at genetic, molecular and biochemical levels.

Cell Extracts↗
PubMed1995

Development of a new easy complementation assay for DNA repair deficient human syndromes using cloned repair genes.

Nucleotide excision repair (NER)-deficient human cells have been assigned so far to a genetic complementation group by a somatic cell fusion assay and, more recently, by microinjection of cloned DNA repair genes. We describe a new technique, based on the host cell reactivation assay, for the rapid determination of the complementation group of NER-deficient xeroderma pigmentosum (XP), Cockayne's syndrome (CS) and photosensitive trichothiodystrophy (TTD) human cells by cotransfection of a UV-irradiated reporter plasmid with a second vector containing a cloned repair gene. Expression of the reporter gene, either chloramphenicol acetyltransferase (CAT) or luciferase, reflects the DNA repair ability restored by the introduction of the appropriate repair gene. All genetically characterized XP, CS and TTD/XP-D cells tested failed to express the UV-irradiated reporter gene, this reflecting their NER deficiency whereas cotransfection with the repair plasmid expressing a gene specific for the given complementation group increased the enzyme activity to the level reached by normal cells. Selective recovery of both reporter enzyme activities was observed after cotransfection with the XPC gene for the XP17VI cells and with the XPA gene for both XP18VI and XP19VI cells. Using this method, we assigned three new NER-deficient human cells obtained from patients presenting clinical symptoms described as classical XP to either XP group A (XP18VI and XP19VI) and XP group C (XP17VI). Therefore, this technique increases the range of methods now available to determine the complementation group of new NER deficient patients with the advantage, unlike the somatic cell fusion assay or the microinjection procedure, of being simple, rapid, and inexpensive.

Biopsy↗
PubMed1994

Correction by the ERCC2 gene of UV sensitivity and repair deficiency phenotype in a subset of trichothiodystrophy cells.

Trichothiodystrophy (TTD) is a rare genetic disease with heterogeneous clinical features associated with specific deficiencies in nucleotide excision repair. Patients have brittle hair due to a reduced content of cysteine-rich matrix proteins. About 50% of the cases reported in the literature are photosensitive. In these patients an altered cellular response to UV, due to a specific deficiency in nucleotide excision repair, has been observed. The majority of repair-defective TTD patients have been assigned by complementation analysis to group D of xeroderma pigmentosum (XP). Recently, the human excision repair gene ERCC2 has been shown to correct the UV sensitivity of XP-D fibroblasts. In this work we describe the effect of ERCC2 on the DNA repair deficient phenotype of XP-D and on two repair-defective TTD cell strains (TTD1VI and TTD2VI) assigned by complementation analysis to group D of XP. ERCC2 cDNA, cloned into a mammalian expression vector, was introduced into TTD and XP fibroblasts via DNA-mediated transfection or microneedle injection. UV sensitivity and cellular DNA repair properties, including unscheduled DNA synthesis and reactivation of a UV-irradiated plasmid containing the chloramphenicol acetyltransferase reporter gene (pRSVCat), were corrected to wild-type levels in both TTD and XP-D cells. These data show that a functional ERCC2 gene is sufficient to reestablish a wild-type DNA repair phenotype in TTD1VI and TTD2VI cells, confirming the genetic relationship between TTD and XP-D. Furthermore, our findings suggest that mutations at the ERCC2 locus are responsible for causing a similar phenotype in TTD and XP-D cells in response to UV irradiation, but produce quite different clinical symptoms.

Animals↗
PubMed1991

[QT prolongation and circulatory arrest after an injection of erythromycin in a newborn infant].

A 8 day-old full-term newborn showed severe cardiac disturbances after intravenous injection of erythromycin. The neonate, suspected of having Chlamydia pneumonitis because of tachypnea and rhinitis, had been given 5 injections of erythromycin without clinical effect. Pallor, vomiting and bradycardia developed a few minutes after the 6th injection, and ECG showed ventricular arrhythmia, prolonged QT interval and an atrioventricular block. The infant died in intensive care unit. This case and the analysis of other published cases of cardiac disturbances following the parenteral use of erythromycin, indicate the potential arrhythmogenic risk of this drug. It is suggested that newborns treated with erythromycin should be monitored by ECG.

Erythromycin↗
PubMed1990

Constraints in simian virus 40 (SV40) encapsidation, as determined by SV40-based shuttle viruses.

Simian virus 40 (SV40)-based shuttle vectors, containing the SV40 late genes, can be packaged as infectious pseudovirions. In terms of their function as bacterial plasmids, modifications in the overall size of these plasmids can be tolerated within a very wide range, which has allowed us to determine the requirements for SV40 encapsidation, free of the more stringent limitations of SV40 virus. Monkey COS7 cells were transfected with over- and undersized SV40-based shuttle virus plasmids and their progeny have been analysed to follow the stability and evolution of these genomes. Two of the three plasmids analysed undergo recombination, generating molecules with sizes of between 4.0 and to 4.8 kb which were selected after multiple lytic cycles. This size range may correspond to the DNA lengths preferentially packaged in SV40 capsids. The structure of the rearranged plasmids indicates that there is a strong selective pressure for genomes that retain the functions necessary for replication and virus production. Depending on the parent DNA, two main classes of rearrangements were generated: duplications in tandem with the SV40 origin of replication and deletions. Both classes are probably a result of selective size and replicative advantages, which are then biologically amplified during plasmid transmission as virus particles.

Animals↗
PubMed1989

Ornithine carbamoyltransferase deficiency. A new variant with subnormal enzyme activity.

A new ornithine carbamoyltransferase variant is described with a nearly normal specific enzymatic activity. This new variant is characterized by a ten-fold increased Km value for L-ornithine (3.75 vs. 0.33 mmol/l in control) and by a normal Km value for carbamoylphosphate (0.42 vs. 0.29 mmol/l). Its pH optimum is shifted towards the alkaline side. This enzyme is highly unstable at 55 degrees C.

Amino Acids↗
PubMed1989

Respective roles of pyrimidine dimer and pyrimidine (6-4) pyrimidone photoproducts in UV mutagenesis of simian virus 40 DNA in mammalian cells.

UV light induces DNA lesions which are mutagenic in mammalian cells. We used simian virus 40 tsB201 (unable to produce viral capsid at the restrictive temperature of 41 degrees C because of a point mutation in the VP1 gene) to analyze the mutagenic potency of the two major UV-induced lesions, pyrimidine dimers (Py-Py) and pyrimidine (6-4) pyrimidones [Py(6-4)Py], which are formed on the same nucleotide sites. The mutagenesis criterion was the reversion toward a wild-type growth phenotype. After UV irradiation (mainly at 254 nm), part of the DNA was treated with the photoreactivating enzyme of Escherichia coli, which monomerizes Py-Py but does not modify the Py(6-4)Py photoproduct. Higher survival and lower mutation frequency rates for the photoreactivated DNA indicated that the two lesions were lethal and mutagenic. The VP1 gene of some mutants was entirely sequenced. The mutation spectra showed that the two lesions did not induce the same mutation hot spots, although some sites were common to both. The induced mutation hot spots were not only correlated with lesion hot spots but seemed partially directed by local DNA structures.

Animals↗