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M C Chignol

Publications and source records attributed to M C Chignol.

At least 37 records · Page 2Linked to original sources
PubMed1992

Evaluation of human papillomavirus type 5 on frozen sections of multiple lesions from transplant recipients with in situ hybridization and non-isotopic probes.

Transplant recipients are at high risk to develop multiple cutaneous lesions after grafting. The frequency of the potentially oncogenic human papillomavirus (HPV) type 5 DNA was evaluated in cutaneous lesions taken from sun-exposed areas in transplant recipients (92 lesions and 5 samples from normal skin) and compared with a nontransplanted population (22 lesions and 7 samples from normal skin) using in situ hybridization and biotinylated probes to HPV types 1, 2, 5, 16 and 18. HPV type 5 DNA was identified in 8/92 cutaneous lesions of transplanted recipients: 3 warts, 1 case of seborrheic keratosis, 2 actinic keratoses and 2 keratoacanthomas. HPV type 5 DNA was not detected in 27 malignant tumors (8 basal cell carcinomas and 19 squamous cell carcinomas) from transplant recipients. HPV DNA type 5 was detected in only 1 case of squamous cell carcinoma from the general population. The presence of HPV DNA 5 was confirmed with Southern blotting in 2 out of 6 cases from transplant recipients. The reaction was negative with the squamous cell carcinoma from nontransplant recipients. These data indicate that the presence of HPV DNA type 5 is not very frequent; it can be detected with in situ hybridization and nonisotopic probe, which is easier to handle than Southern blot.

Biopsy↗
PubMed1992

[Human papillomaviruses and cellular oncogenes (c-myc, c-Ha-ras) in cutaneous and mucosal lesions in transplantation recipients].

Transplant recipients develop numerous benign and malignant cutaneous and mucosal lesions in which histological signs of human papillomavirus (HPV) infection are observed. To investigate the role of HPV and c-myc and c-Ha-ras cellular oncogenes' activation in transplanted patients lesions, we used in situ hybridization with biotinylated probes and Southern blot to detect HPV and oncogenes DNA. We analyzed 36 lesions from grafted patients: 11 common warts, 10 actinic keratoses, 13 squamous cell carcinomas and 2 anogenital papillomas. With in situ hybridization, HPV DNA was detected in 14/36 lesions, 10 of which contained several HPV types. Benign and potentially oncogenic HPV types were detected in warts as well as in squamous cell carcinomas. The Southern blot technique confirmed the distribution of HPV types. Group specific viral antigen was detected in 12 lesions, mainly warts. C-Ha-ras oncogene was amplified in 13 lesions and c-myc oncogene in 10 lesions, 9 of which showed both oncogene amplification. The results obtained with in situ hybridization for c-myc gene amplification were confirmed with the Southern blot technique in 11/14 cases. Ras and/or myc amplification was more frequent in squamous cell carcinomas and anogenital papillomas than in warts and actinic keratoses. The amplification was not always linked to the presence of HPV DNA; however, it was more frequent in lesions infected by potentially oncogenic HPV types than in lesions containing only benign HPV types. Myc and p21 oncoproteins were respectively localized in the nucleus and on the membrane of epithelial cells by immunofluorescence. Most lesions showed a good concordance between the detection of oncogene DNA and proteins. Our results suggest than c-Ha-ras and c-myc cellular oncogenes' activation and HPV infection could play a role in the cancerization of cutaneous lesions from transplant recipients.

Genes, myc↗
PubMed1991

Cell fusion of human and mouse cells as a source for new cells retaining human markers. Analysis of DNA content, membrane and cytoplasmic antigen expression.

Flow cytometry and ultrastructural morphometry were used to study some characteristics of cells obtained by fusion with polyethylene-glycol 4000 between mouse fibroblasts 3T3.4E and normal keratinocytes (3T3.4E x NHK) or hand wart human keratinocytes (3T3.4E x HWK), at late passages. The cell cycle and the expression of human beta 2-microglobulin, human EGF-receptors (EGF-r), vimentin were simultaneously studied by flow cytometry. Epithelial CaSki cells, derived from a human uterine carcinoma, expressing high levels of beta 2-microglobulin, EGF-r and vimentin, were used as a positive control. In mouse fibroblasts 3T3.4E only vimentin was expressed whereas in cells derived from fusion, human beta 2-microglobulin, human EGF-r and vimentin were detected. The cell cycle analysis revealed that the peak position of G0/G1 differed with the cells (channel 11 for 3T3.4E cells, 13 for 3T3.4E x HWK and 15 for 3T3.4E x NHK). The area of the cell compartments from each cell type was also different by quantitative ultrastructural morphometry. The hybrid phenotype was maintained in late passages in cells (3T3.4E x NHK) and (3T3.4E x HWK), as shown by the expression of human antigens, differences in DNA contents and nuclear area. Flow cytometry may be a very accurate and precise tool for studying low antigenic expression. The combination of different methods including analysis of DNA content, antigenic expression and ultrastructural morphometry confirmed that 3T3.4E, 3T3.4E x NHK and 3T3.4E x HWK cells are different cell types. These techniques are complementary to cell phenotype analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗
PubMed1990

Evaluation of mitochondrial content and activity with nonyl-acridine orange and rhodamine 123: flow cytometric analysis and comparison with quantitative morphometry. Comparative analysis by flow cytometry and quantitative morphometry of mitochondrial content and activity.

Mouse fibroblasts 3T3.4E and two cells lines obtained by fusion (3T3.4E cells x normal human keratinocytes), (3T3 x NHK), and (3T3.4E cells x hand wart keratinocytes), (3T3 x HWK), were compared for mitochondrial activity and content between 5 and 20 days of culture, from the 16th to 20th passage, by using Rh 123 and NAO respectively. In 3T3.4E cells both Rh 123 and NAO fluorescence were similar after 5 and 7 days of culture, indicating no modification of mitochondrial activity and content at that time. However, in cells derived from fusion of 3T3 x NHK or 3T3 x HWK, Rh 123 increased from 5 to 20 days whereas NAO fluorescence was maximal at 7 days of culture and then decreased, indicating that their mitochondrial activity differed from that of 3T3.4E cells. No difference was observed between the 16th and 20th passage. Quantitative morphometry and flow cytometry gave good correlations at 7 days of culture for the cell size, estimated either by the cell area or the cell diameter, and for mitochondria content, evaluated either by the number of mitochondria per cell or NAO fluorescence intensity.

Acridine Orange↗
PubMed1990

Study of stringency conditions for human papillomavirus DNA detection on cell lines, frozen and paraffin-embedded tissue sections by in situ hybridization with biotinylated probes.

In situ hybridization was mainly used for typing human papillomavirus (HPV) in paraffin-embedded or frozen sections under stringent conditions (SC). We tested 5 different conditions of stringency with biotinylated HPV 1, 2, 16 and 18 probes on 3 cell lines (Sihà and CaSki with HPV16, HeLa with HPV18) by varying the concentration of formamide in the hybridization mixture and washings in order to determine the stringency conditions to be used to assess the presence of HPV and its typing: A-low stringency, hybridization at 35 degrees C below the melting temperature of DNA (Tm-35 degrees C) and washings without formamide; B-low stringency, hybridization and washings at Tm-35 degrees C; C-medium stringency, hybridization at Tm-35 degrees C and washings at Tm-12 degrees C; D-high stringency, hybridization at Tm-12 degrees C and washing without formamide; E-very high stringency, hybridization and washings at -12 degrees C. This study showed that HPV typing required a high stringency. On the contrary, under non stringent conditions (NSC), each cell line was positive with the heterologous probes. When 3 to 5 stringency conditions were assayed on 4 frozen samples, similar results were obtained. Typing required high stringency conditions whereas NSC allowed HPV detection. Furthermore, this study demonstrated the specificity of the reaction in lesions positive with more than one type. Stringent (Tm-12 degrees C) and non stringent (Tm-35 degrees C) conditions of hybridization were further applied to 57 biopsy sections (17 frozen and 40 paraffin-embedded specimens) from typical wart lesions and lesions suspected of HPV.(ABSTRACT TRUNCATED AT 250 WORDS)

Biopsy↗
PubMed1990

Human papillomavirus infection and filaggrin expression in paraffin-embedded biopsy specimens of extragenital Bowen's disease and genital bowenoid papulosis.

Cutaneous Bowen's disease (BD) and genital bowenoid papulosis (BP) are considered as precancerous or cancerous lesions that are sometimes infected with human papillomavirus (HPV). We studied retrospectively paraffin-embedded sections of 11 samples of cutaneous BD and 6 samples of genital BP from the general population for HPV infection and filaggrin expression. Using in situ hybridization with biotinylated probes of HPV types 1, 2, 5, 6, 11, 16, and 18, under stringent and/or non-stringent conditions and a streptavidin-alkaline-phosphatase complex for hybrid detection, HPV DNA was detected in 6/17 cases (5 BD and 1 BP). Positive nuclei were located in intermediate or upper epithelial cell layers. HPV 16 was found in 2 cases of BD but associated either with HPV 2 or 18. Three additional lesions reacted only under non-stringent conditions; HPV could not be typed with the probes used. The positive case of BP reacted with the four probe types 1, 2, 16, 18 and was negative with HPV 6 or 11. Viral antigen was not detected by indirect immunofluorescence with a rabbit antiserum directed to group-specific viral capsid antigen. Differentiation disorders were observed in the intermediate and upper cell layers of these specimens, as shown by a reduced expression of filaggrin/profilaggrin, a marker of terminal differentiation, in extragenital BD (7/11 cases), and an increased expression in genital BP (4/5 cases) although viral DNA was not always detectable. This study shows that in situ hybridization is a valuable technique for HPV DNA detection and its typing in BD and BP lesions on deparaffinized sections. The positive nuclei were located in the cell layers that exhibited abnormal expression of differentiation. There is no relation between the HPV infecting type and the filaggrin expression.

Aged↗
PubMed1990

Detection of human papillomavirus DNA in squamous bronchial metaplasia and squamous cell carcinomas of the lung by in situ hybridization using biotinylated probes in paraffin-embedded specimens.

We examined a series of paraffin-embedded tissue specimens from 10 cases of squamous bronchial metaplasia and 33 cases of squamous cell carcinoma of the lung for histologic characteristics and for the presence and typing of human papillomavirus (HPV) by molecular in situ hybridization with biotinylated probes types 6, 11, 16 and 18 under stringent conditions (temperature, 19 degrees C). Fourteen of these lesions (32.5%) showed typical condylomatous histologic changes. Human papillomavirus DNA was present in seven (16%) specimens. Type 6 HPV DNA was detected in one of the squamous bronchial metaplasia cases. In six of the squamous cell carcinomas cases (18%), HPV DNA was identified (type 18, three cases; type 16, one case; type 11, one case; and type 6, one case); one of the squamous cell carcinoma specimens contained both HPV types 16 and 18. Our data confirm the presence of HPV DNA in squamous metaplastic bronchial mucosa and epidermoid lung carcinoma on paraffin-embedded tissues. This suggests that an HPV infection with benign or potentially oncogenic HPV types could be associated not only with genital tumors, but also with bronchial and lung tumors. The role of HPV DNA in the process of malignancy conversion is not yet known; HPV DNA could possibly be a cocarcinogenic factor. In situ hybridization with biotinylated probes is a useful and appropriate method of retrospective analysis of HPV DNA sequences in routinely paraffin-embedded lesions. It may be used to identify patients at risk of more serious or possibly malignant progression.

Biotin↗
PubMed1990

[Human papillomavirus typing by molecular hybridization in situ with biotinylated probes. Optimization of sample preparation and hybridization time].

In situ hybridization with non radioactive probes is more and more used to detect viral infections, especially human papillomavirus (HPV) infections. The quality of the reaction depends on several factors, such as sample preparation (including fixation and pretreatments). Their importance was evaluated on a model with cell lines including CaSki cells (harboring about 600 copies of HPV 16 DNA per cell) and Hela cells (containing 10-50 copies of HPV 18 DNA per cell). These cell lines were chosen in order to evaluate cytological and histological difficulties. Several parameters were studied: preparation of samples, fixation and hybridization duration. DNAs of biotinylated probes of HPV types 16 and 18 and cells were simultaneously denatured 10 min at 95 degrees C. Hybridization was carried out at 37 degrees C for various periods of time; it was followed by a 3-step reaction for detection of biotinylated DNA-DNA hybrids with immunoenzymatic staining using streptavidin-alkaline phosphatase complex. Typical intranuclear granulations were seen either in cell deposits fixed with acetone, methanol-acetic acid, paraformaldehyde, formaline, Bouin's, Baker's or Carnoy's fixatives; or in cytocentrifuged cells fixed with formaline, Bouin's or Baker's fixatives. The detergent pretreatment was unnecessary. On the contrary, the protease pretreatment was required with formaline, Bouin's or Baker's fixatives. In order to detect constantly HPV 16 in CaSki cells and HPV 18 in HeLa cells, hybridization should be performed for more than 4 h. The sensitivity of the technique could therefore be evaluated to few copies of HPV DNA per cell. This technique is reliable, sensitive and rapid; et can be applied to biopsy specimens fixed with Bouin's or Baker's fixatives and paraffin-embedded; it allows routine detection of HPV infections.

Biotin↗
PubMed1990

Ultrastructure of hybrids derived from electric pulse fusion of human HeLa cells and murine 3T3.4E cells.

Somatic cell hybrids were obtained with electric pulse by fusion of human epithelial HeLa cells derived from uterine carcinoma and mouse fibroblasts 3T3.4E, deficient in thymidine kinase. Hybrids were selected and propagated in HAT medium. After 3 passages the cultures were morphologically homogeneous. They retained an epithelial morphology. Electron microscopic and morphometric studies indicate that these hybrids at the 6th passage differed from parental cells. They exhibited the smallest cellular and cytoplasmic size. The number of mitochondria was similar to that of murine fibroblasts but their size was more homogeneously distributed; the mitochondria matrix was more electron dense than in the parental cells. These data suggest that these hybrids have a different metabolism from that of the parental cells; they contained modified mitochondria.

Animals↗
PubMed1989

A comparison of methods for the detection of human papillomavirus DNA by in situ hybridization with biotinylated probes on human carcinoma cell lines. Application to wart sections.

We compared nine different techniques for the detection of biotinylated DNA-DNA HPV hybrids on HeLa cells with 10-50 copies of HPV 18 DNA per cell. CaSki cells with 600 copies of HPV 16 DNA per cell and tissue sections from frozen or paraffin-embedded biopsy specimens. The cell samples were either cell deposits or cytocentrifuged or cultured slides. In most cases, the samples (cell deposits and tissue sections) were denatured with hybridization mixture prepared under stringent conditions (Tm = -17 degrees C) containing biotinylated DNA probes (cloned HPV types 1, 2, 6, 11, 16 and 18), at 90 degrees C for 10 min. In other cases (cytocentrifuged or cultured cells), the denaturation was performed by HCl hydrolysis and mild heating at 50 degrees C; the probes were denatured separately by heating. All the samples were further incubated overnight at 37 degrees C. For HPV DNA detection, three amplification levels were used on cell deposits. Only the techniques involving a three-step reaction (a rabbit anti-biotin antibody - a biotinylated goat anti-rabbit antibody - a complex of streptavidin-alkaline phosphatase or streptavidin-gold or streptavidin-fluorescein) gave satisfactory results, on both cell lines. With the one step reaction (an avidin-horseradish peroxidase, or streptavidin-alkaline phosphatase or streptavidin-fluorescein complex), no labeling of HeLa cells was observed with any of the HPV probes, including HPV 18. The techniques involving four steps (avidin or streptavidin - anti-avidin goat antibody or anti-streptavidin rabbit antibody - a biotinylated anti-goat (or anti-rabbit) antibody - a complex of avidin-biotin-peroxidase or streptavidin-biotin-alkaline phosphatase or streptavidin-biotin-horseradish peroxidase) resulted in high background on both cell lines. For the reproducible detection of low copy number of HPV DNA (less than 50 copies) such as occur in HeLa cells our data suggested that the three-step technique with the streptavidin-alkaline phosphatase complex was the method of choice. The most intense labeling was always obtained with cell deposits and the technique was successfully applied to frozen and paraffin-embedded tissue sections from typical warts.

Carcinoma↗
PubMed1989

Detection of herpes simplex virus DNA in a cutaneous squamous cell carcinoma by in situ hybridization.

A case of a cutaneous squamous cell carcinoma (SCC) occurring at the site of a long-standing recurrent HSV infection is described. No deficit of the cell-mediated immunity was recorded. HSV2 was isolated in several viral cultures. HSV DNA was visualized in the SCC by in situ hybridization with biotinylated probes in paraffin-embedded tissue. The samples were negative for HPV2, 5, 16, and 18 probes. A causal relationship between HSV infection and cutaneous SCC is hypothesized.

Carcinoma, Squamous Cell↗
PubMed1989

Ultrastructural characteristics of hybrids derived from normal and hand wart keratinocytes after serial passages.

Somatic hybrids realized between mouse fibroblasts 3T3.4E and normal human keratinocytes or hand wart keratinocytes were examined from the 6th to the 30th passages by scanning and transmission electron microscopy. Whatever the passage, hybrid cells showed a fibroblastic morphology but, as keratinocytes, they had the capability to stratify. Branched mitochrondria were observed in hybrids whereas normal mitochondria were present in mouse fibroblasts. In human keratinocytes, most of the mitochondria were normal but sometimes few of them were branched. In wart hybrids heterogeneous nucleoli were detected instead of normal nucleoli in normal keratinocyte hybrids, 3T3.4E cells and human keratinocytes.

Animals↗
PubMed1989

Filaggrin expression in cutaneous and mucosal human papillomavirus induced lesions.

A series of 32 human papillomavirus induced lesions derived from epidermis and mucosa was studied for the modulation of filaggrin-profilaggrin (F-PF) expression according to the degree of virus infection as compared to normal skin and mucosa biopsies. This investigation was carried out on frozen sections using indirect immunofluorescence for filaggrin detection and group specific viral antigen and by in situ hybridization with biotinylated probes for viral DNA detection and typing. The 9 cutaneous warts showed an increase of F-PF expression in upper layer cells as compared to normal epidermis, which could be related to the high production of virus (viral antigen and HPV types 1 or 2). The 5 condyloma acuminata displayed also an enhanced expression of these components which was located in several upper layers but virus infection was confirmed in 2 of them with HPV types 6, 11 or 16. The 6 laryngeal papillomas exhibited a granular reactivity pattern for F-PF in suprabasal cell layers with an increase in the upper layers; viral antigen was found in 4 cases and HPV DNA types 6, 11 or 16 were detected in 4 specimens. Conversely among 12 cervical intraepithelial neoplasia, F-PF was expressed only in very superficial layers in few cases, without any correlation with the DNA detection (6, 11 or 16, 18). Taken together these data are suggestive of an intense expression of F-PF in benign lesions which can replicate the virus and a discrete or an absent expression of these components in premalignant or malignant lesions.

Adult↗
PubMed1989

Quantitative studies of keratin expression with the post-embedding immunogold labeling method.

Immunoreactivity of the 56.5 KD acidic (type I) keratin was localized ultrastructurally and quantified in normal human epidermis using the specific monoclonal antibody KL1 and post-embedding immunogold labeling. The protein was detected in keratin intermediate filament bundles of all suprabasal keratinocytes. Keratohyalin granules and desmosomal plaques were labeled only on the periphery, in regions where keratin filaments penetrate these structures. The 56.5 KD keratin immunoreactivity increased from the first suprabasal layer onwards and reached its maximum in the outmost spinous layer. A subsequent abrupt decrease of the specific immunogold labeling was observed in the granular layer. This low reactivity, which persisted also in the horny layer, may be partially explained by either protein degradation or masking of the antigenic sites by a filament-aggregating material occurring at these stages of keratinocyte terminal differentiation. Statistical comparison of the quantitative results obtained in various cell and tissue compartments revealed no significant differences between the background labeling levels observed in the basal layer of epidermis with KL1, a control monoclonal antibody, or the immunogold conjugate alone. Our results confirm the specificity of 56.5 KD keratin for terminally differentiating suprabasal keratinocytes and demonstrate the importance of appropriate control studies when a post-embedding immunogold labeling method is employed.

Adult↗
PubMed1986

HB8: a monoclonal antibody recognizing elastic fibre microfibrils.

We report an ultrastructural study of the reactivity of an IgG monoclonal antibody, which reacts with human dermal elastic fibres. On electron microscopy, immunogold labelling demonstrated the reactivity of HB8 with the microfibrillar component of the fibres. In contrast, the electron-dense granular component which contains elastin was unlabelled with HB8. A fine network of microfibrils from the level of the oxytalan fibres to the large elastic fibres of the deep dermis was labelled with gold particles. No reactivity was detected on collagen fibres. These results demonstrate that HB8 reacts with the microfibrillar glycoproteins of the elastic fibres in normal skin. The precise biochemical nature of the antigens identified by HB8 is still unknown.

Animals↗
PubMedDate not supplied

The up-regulation of vascular endothelial growth factor in mutated Ha-ras HaCaT cell lines is reduced by a farnesyl transferase inhibitor.

The induction of tumor angiogenesis is mediated in particular by an increased production of VEGF. As ras oncogene is implicated in tumorigenesis, the inhibition of farnesyl transferase activity has recently been developed. The purpose of this study was to evaluate whether expression of mutated Ha-ras oncogene is associated with an altered expression of VEGF in an in vitro model of human skin carcinogenesis and to appreciate the effect of a new farnesyl transferase inhibitor on this VEGF expression. The amounts of VEGF secreted by an HaCaT cell line and two cell clones (metastatic or not) obtained after mutated c-Ha-ras transfection were compared. Our findings showed that the release of VEGF is greater for HaCaT-ras than for HaCaT cells and could be down-regulated using a protein farnesyl transferase inhibitor, in a reversible and dose-dependent manner. These results confirm that the Ha-ras oncogene can contribute to tumor development and progression of epidermal tumors through neoangiogenesis and that farnesyl transferase inhibitors as anticancer drugs may be efficient for the reduction of skin tumor growth.

Alkyl and Aryl Transferases↗
PubMedDate not supplied

Papilloma viruses, warts, carcinoma and Langerhans cells.

In human papillomavirus (HPV) infections, Langerhans cells (LC) are essential in the control of viral infection. The evolution of HPV-derived lesions in the normal population and in graft patients is drastically different, since a high proportion of papillomas progress towards malignancy in transplant recipients. We analyzed the distribution of markers of LC and T lymphocytes, the level of keratinocyte activation and the prevalence of HPV in a series of epithelial lesions obtained from the normal population and from graft patients. The local immune response of warts, condyloma acuminata, Bowen, basal and squamous cell carcinomas (SCC) showed a moderate to intense inflammatory reaction of HLA-DR positive cells, the intensity of the immune reaction being correlated with the degree of malignancy. In the normal population, CD4-positive cells were mainly overexpressed in the dermal infiltrate of condyloma and malignant lesions, whereas in grafted patients such infiltrates were CD4- and CD8-positive without significant predominance of a single T cell subset. The epidermis of most lesions was characterized by a reduced number of CD1a-positive LC with an altered morphology. This was concomitant with the decrease or loss of beta 2-microglobulin by epithelial cells. HLA-DR antigen was sometimes expressed by keratinocytes in genital lesions and SCC from the normal population but has not been detected in immunosuppressed patients. Whereas in the normal population HPV infection was only detected in benign papillomas, both benign and oncogenic HPV DNA may be present in carcinomas from graft patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗