Biomedical subjects

G Lizard

Publications and source records attributed to G Lizard.

At least 55 records · Page 3Linked to original sources

PubMed1994

Human melanoma cell lines differ in their capacity to release ADP and aggregate platelets.

In this study we have investigated, using three different human melanoma cell lines (M1Do., M3Da., M4Be.). the varying capacity of melanoma cells to induce platelet aggregation in the presence or absence of inhibitors of ADP or thrombin. The expression levels of different integrins (alpha v, beta 3, alpha v beta 3, alpha IIb, alpha v beta 3) were evaluated by immunoprecipitation, binding and flow cytometry studies. The level of ADP in supernatants of melanoma cells were quantified by ADP bioassay and HPLC. Platelets were irreversibly aggregated by M3Da, as shown by electron microscopy, in contrast to M1Do, which induced a slow reversible aggregation. M4Be. did not induce platelet aggregation. In both cases, with M3Da. or M1Do., apyrase but not PPACK inhibited platelet induced aggregation. An anti-alpha v beta 3 monoclonal antibody (LYP18) or polyclonal antibody inhibited platelet aggregation. A similar number of LYP18 molecules bound to the surface of M1Do., M3Da. and M4Be. cell lines. Biological HPLC assays of ADP present in the supernatant of tumour cell lines showed the highest concentration of ADP to be secreted by M3Da., followed by M1Do., and none detected for M4Be. These results show that differences in in vitro aggregating potential of the three human melanoma cell lines are not related to low integrin expression levels but to their ability to generate ADP. Generation of ADP by human melanoma cells may act as important modulator of melanoma-platelet interactions.

Adenosine Diphosphate↗

PubMed1994

Interleukin-4 and interferon-gamma interactions in the induction of intercellular adhesion molecule-1 and major histocompatibility complex class II antigen expression of normal human keratinocytes.

Interleukin-4 (IL-4) that may be produced by T-helper cells in atopic lesions has immunomodulatory activities on skin cells which are poorly known. Our study was aimed at determining whether the cytokine exerts some effects on keratinocyte activation and can either enhance or antagonize interferon-gamma (IFN-gamma)-induced ICAM-1 or HLA-DR antigen expression. Using normal keratinocytes cultured in defined medium and cytofluorography, we showed that treatments of the human cells with the cytokine IL-4 alone had no effect on the induction of ICAM-1 or HLA-DR molecules. However, a transient, but significant enhanced expression of ICAM-1 was observed by the combination of IFN-gamma and IL-4 after 24 h of stimulation, which was followed by a reduction at 48 and 72 h. Conversely, IL-4, when added during the IFN-gamma activation stage, had no effect on MHC class II antigen expression of keratinocytes; however, the cytokine reduced the expression of these antigens when added 24 h before the stimulation by IFN-gamma. These results suggest that IFN-gamma and IL-4 may interact to regulate ICAM-1 and HLA-DR expression on keratinocytes.

Cells, Cultured↗

PubMed1994

Induction of intercellular adhesion molecule 1 expression on normal human keratinocytes by retinoic acid: comparison of cultured keratinocytes and skin explants.

As retinoic acid (RA, all trans) induces irritant reactions when applied topically to skin, we wondered whether it may be involved in the activation state of keratinocytes through ICAM-1 induction. Human skin explants and cultured keratinocytes were treated by RA and/or interferon-gamma (IFN gamma) at different concentrations during 24, 48 and 72 h. ICAM-1 expression was examined and quantified by immunohistochemistry, in situ, on cutaneous frozen sections and in cultured keratinocytes by flow cytometry analysis. Expression of mRNA was checked by Northern blot hybridizations using an oligonucleotide probe. Our results indicate: (1) the absence of spontaneous ICAM-1 expression by normal human keratinocytes both in skin explants and in cultures; (2) an ICAM-1-induced expression after 48 h of treatment by RA concentrations > 10(-6) M; this induction is dose- and time-dependent; in skin explants, ICAM-1 is occasionally observed on foci of epidermal cells; this protein induction is correlated with transcript expression in cultured keratinocytes; and (3) a synergistic effect of RA and IFN gamma (5 IU/ml) on the percentage of ICAM-1-positive cells and the level of expression of the protein. These findings indicate that RA, in a therapeutical range of concentrations can induce or stimulate ICAM-1 expression in normal human keratinocytes. This may in part explain the erythematous reaction observed in vivo after topical applications of RA which may be considered as a mediator of keratinocyte activation.

Adult↗

PubMed1994

[Papillary and cystic tumor of the pancreas. Histological, immunohistochemical and flow cytometric study].

The authors are reporting a case of papillary and cystic neoplasm of the pancreas. This rare malignant epithelial neoplasm occurs mostly in young women and has a more favorable prognosis than the adenocarcinoma. Radiologic investigations showed a well demarcated, heterogeneous, hypovascular mass. The histological study showed the proliferation of epithelial cells in a papillary pattern. The immunochemistry was negative for markers of adenocarcinoma but positive for vimentin, alpha-1-antitrypsin, alpha-1-antichymotrypsin and neuron specific enolase. Receptors of progesterone were also detected. By flow cytometric analysis, the tumours was hypoploid. The histogenesis of these tumours remained controversed. Ultrastructural details of the neoplastic cells suggest that these tumours originate from small duct cells; detection in several cases of markers for endocrine neoplasm suggests that they originate from primitive multipotential cells, that may show exocrine or endocrine differentiation.

Adult↗

PubMed1994

[Value of rhodamine 123 in the detection of minor amounts of multidrug resistant cells].

The neoplasias resistance to chemotherapy is mainly due to multidrug resistance phenomenon (MDR) mediated by an ATP-dependent efflux pump called P-glycoprotein. This function can be reversed by many multidrug reversing agents so numerous chemotherapy regimens have been initiated in malignancies. To make sure the success of these protocols it is necessary to detect as soon and as certain as possible the presence of resistant cells among malignant population. We have chosen to evaluate functional test using rhodamine 123 in this aim in view. We have made various mixtures of resistant ans sensitive cells of three cell lines. Rhodamine 123 allows to detect 1% of resistant cells among sensitive cells. Influence of dead cells on the interpretation is discussed.

Antimetabolites, Antineoplastic↗

PubMed1994

[Detection of human papillomavirus DNA by in situ hybridization and gene amplification].

In situ hybridization with non radioactive probes is attractive for human papillomaviruses (HPV) detection. Its sensitivity has been greatly improved by using different hybridization conditions, techniques for revealing the DNA-DNA hybrids and method of observation and various cell lines derived from human uterine carcinomas (CaSki, SiHa and HeLa cells) which contain 500-600 copies of HPV DNA, 1-2 copies of HPV 16 and 20-50 copies of HPV DNA 18, respectively. In situ gene amplification increased the detection of HPV DNA since hybridization spots were visible in SiHa cells on slides; a specific signal was observed in HeLa cells in suspensions examined by flow cytometry. Confocal microscopy is an alternative method to in situ gene amplification since viral DNA is detectable in SiHa cells with or without gene amplification. Thus, the techniques used in this study are potentially useful for research of single cellular genes.

Carcinoma↗

PubMed1994

Improvements in visualisation and localisation of human papillomavirus DNA in CaSki cells by fluorescence in situ hybridization, laser scanning confocal microscopy and three-dimensional image reconstruction.

The visual interpretation and localisation of specific DNA sequences in three dimensions in cell nuclei was investigated by fluorescence in situ hybridization (FISH) and laser scanning confocal microscopy (LSCM) using CaSki cells containing 600 copies per cell of human papillomavirus (HPV) DNA type 16 integrated in cellular DNA. Biotinylated DNA probes were used and DNA-DNA hybrids were revealed by a three-step reaction involving a rabbit anti-biotin antibody, a biotinylated goat anti-rabbit antibody and a streptavidin-fluorescein isothiocyanate complex. The DNA from cell nuclei was counterstained with propidium iodide. With standard fluorescence microscopy, some dense fluorescent spots were seen in the cell nuclei. Similarly, with LSCM, some hybridization spots were observed in the cell nuclei but they were at different levels of the nuclei as shown by successive nuclear sections taken along the z axis. The visualisation of multiple hybridization spots confirmed the presence of multiple integration sites of HPV 16 DNA in CaSki cells. Association of LSCM with three-dimensional reconstructions lead to spatial images of hybridization spots obtained by stacking (x,y) images from consecutive confocal planes. Rotation of the reconstructed cell nuclei around the y axis makes it possible to distinguish closely adjacent spots. The combination of these techniques improves the detection of hybridization spots and may be of interest to further determine whether the HPV DNA is episomal or integrated in infected cells.

DNA, Viral↗

PubMed1993

Detection of human papillomavirus DNA in CaSki and HeLa cells by fluorescent in situ hybridization. Analysis by flow cytometry and confocal laser scanning microscopy.

CaSki and HeLa cell lines, isolated from human uterine carcinomas and containing integrated human papillomavirus (HPV) DNA type 16 and 18, respectively were used to evaluate the sensitivity of HPV-DNA detection on suspended cells by fluorescent in situ hybridization using flow cytometry and on corresponding cell deposits using confocal laser scanning microscopy (CLSM). HPV DNAs were detected in cell suspensions with biotinylated DNA probes and revealed with a three-step technique: a rabbit antibiotin antibody, a biotinylated goat anti-rabbit antibody and a streptavidin-fluorescein isothiocyanate complex. By flow cytometry, HPV DNA was detectable only in CaSki cells which contained about 600 copies of HPV DNA per cell. In HeLa cells, with only 20-50 copies of HPV DNA, flow cytometry could not detect HPV DNA, whereas CLSM permitted visualization of fluorescent labelling of HPV DNA hybrids. Furthermore, CLSM showed good preservation of cellular morphology and the nucleus was clearly recognizable after fluorescent in situ hybridization and counterstaining with propidium iodide. Moreover, this examination confirmed that the fluorescent foci were specifically confined to the cell nuclei.

DNA, Viral↗

PubMed1993

An improved assay for the detection of Toxoplasma gondii antibodies in human serum by flow cytometry.

The diagnosis of toxoplasmosis relies primarily on the demonstration of specific antibodies to Toxoplasma gondii. We describe a flow cytometry method for the determination of antibodies to whole fixed tachyzoites by indirect immunofluorescence. Fixed tachyzoites in suspension have characteristic light scattering properties. The amount of IgM, IgG, and IgA antibodies from patients' sera bound to the tachyzoites can be estimated from the mean fluorescence intensities observed using class-specific conjugates for different Ig heavy chains. Appropriate serum dilutions for the estimation of specific IgG titres and for the discrimination between sera positive and negative for IgA and IgM antibodies were established using 40 random sera from pregnant women. The method proved to be quantitative and highly sensitive as compared with currently used assays. Coefficients of variation between series ranged from 6.4% to 12.2% and could be controlled by the inclusion of positive and negative standard sera in the test series. The method might represent a major improvement in cost effectiveness as compared with routine immunofluorescence assay by fluorescent microscopy when the volume of testing is sufficient. It can be fully automated, and its large scale application is currently under investigation.

Animals↗

PubMed1993

Inhibition of human T cell response to staphylococcal enterotoxin B by prior ligation of surface CD4 molecules.

The mechanisms whereby anti-CD4 monoclonal antibodies can block human peripheral blood lymphocyte response to staphylococcal enterotoxin B (SEB) were investigated. Preincubation of peripheral blood mononuclear cells (PBMC) with anti-CD4 mAbs resulted in a profound inhibition of SEB-induced DNA synthesis, while simultaneous addition of antibody and superantigen did not reproducibly decrease the proliferative response. Inhibition was achieved at a very low antibody concentration (0.1 microgram/ml). It was not increased by cross-linking of anti-CD4 mAb nor mediated by Fc-dependent signals as F(ab')2 antibody fragments were as effective as intact antibodies. Inhibition of proliferation was associated with a profound diminution of IL-2 and IFN-gamma secretion, CD25 (the alpha chain of IL-2 receptor) expression, and blast transformation. Stimulation by SEB after prior ligation of surface CD4 proteins by antibodies was associated with an increased percentage of lymphocytes with chromatin condensation and nuclear fragmentation. It was concluded that stimulation of mature peripheral T cells by SEB through T cell receptors induces an apoptotic signal providing that a small proportion of surface CD4 molecules has interacted with antibodies or F(ab')2 fragments before stimulation by SEB, while simultaneous addition of SEB and anti-CD4 mAb does not prevent the development of a complete activation program in this system. Possible implications of these observations regarding selective clonal deletion of autoreactive T cells by administration of anti-CD4 mAbs in patients with auto-immune diseases are discussed.

Antibodies, Monoclonal↗

PubMed1993

Response of a multidrug-resistant human small-cell lung cancer xenograft to chemotherapy.

Small-cell lung carcinomas (SCLC) are highly responsive to various chemotherapies. However only a minority of patients benefit from long survival. SCLC patients treated at the Institut Gustave Roussy received a combined chemotherapy (CCAV) including cisplatin, cyclophosphamide (Cpa), Adriamycin (doxorubicin; Adm) and vepeside (VP16). We report here the intrinsic sensitivity of a small-cell lung carcinoma, designated SCLC-6, grafted in nude mice. This xenografted tumour was derived from an untreated patient. The CCAV regimen given to the patient donor of the tumour sample resulted in a complete response followed by recurrence and death, 8 months after the initial cure. The expression of P-glycoprotein encoded by the MDR1 gene was detected with the C219 antibody on the membrane of SCLC-6 tumour cells. When given to SCLC-6-tumour-bearing nude mice, CCAV induced a strong inhibition of tumour growth (84% of growth inhibition, 20 days after start of the treatment), but no cure. Intensification of CCAV doses did not improve the response. The efficacy of individual agents of the CCAV, given at maximal tolerated doses was analysed. Only cisplatin (10 mg/kg) and Cpa (3 x 50 mg/kg) inhibited SCLC-6 growth (79% and 100% inhibition respectively), VP16 (3 x 24 mg/kg) was poorly efficient (42%) and Adm (10 mg/kg) not at all. Two-drug combinations such as cisplatin plus VP16 or cisplatin plus Cpa inhibited tumour growth (81% and 70%, respectively). Curiously, the efficacy of Cpa, given in combination with cisplatin was less than that of Cpa alone. Repeated treatments with CCAV administered to mice at each in vivo passage of the tumour induced a loss of chemosensitivity, which was observed until the ninth passage. An improvement of the therapeutic response was obtained by adding a headline reverser of multi-drug resistance, verapamil (25 mg/kg), to CCAV (81% versus 63% inhibition). MDR1-related resistance appeared to play a role in the failure of SCLC-6 chemotherapy; frequent recurrences after treatment with cisplatin and Cpa, two drugs that are not recognized by the P-glycoprotein, indicated that other modes of resistance were simultaneously active.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

[Cancers of the kidney: multiple drug resistance].

The treatment of kidney cancers raise difficult problems. Usual treatments combining surgery, radiation therapy, chemotherapy and hormonotherapy are poorly effective. The immunotherapy gave only an objective response rate of 25% in metastatic renal cell carcinomas. Among cellular resistance mechanisms of renal carcinoma, the multi-drug resistance (MDR) phenotype and the glutathione redox cycle have been studied. The MDR is related to overexpression of a 170 kDa membrane glycoprotein, the so-called P glycoprotein (Pgp). This protein is a pump able to extrude from cytoplasm drugs with various structures and mechanisms. The Pgp is encoded by the MDR1 gene expression is very low in most of the normal tissues, except in colon, liver and kidney. In these tissues, the Pgp would ensure a detoxifying function related to cellular toxic compounds, elimination or transfer of molecules synthesized by the cells. According to the intrinsic resistance of renal carcinoma, MDR1 gene expression has been determined. Approximately, 80% of fresh kidney cancer before chemotherapy express resistance phenotype. Reversal compounds specifically inhibiting Pgp were found, such as verapamil, cyclosporin or quinidine. Unfortunately, the two first compounds give cardiac toxicity and immunosuppression, respectively. So far, clinical trials have not been demonstrating, but no biological resistance measures were determined, in parallel. According to the multifactorial resistance the association of reversals to chemotherapy should be introduced in clinical trials, in correlating clinical response to biological mechanisms of resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

[Value of cytological study by flow cytometry or analysis of image as a complement to macroscopic study by cytoscopy. Apropos of therapeutic follow-up of a case of tumor of the bladder].

The case described corresponds to a grade G2 urothelial tumor with stage pTa extension (according to the World Health Organisation classification). After transurethral resection of the tumor and treatment by Bacillus Calmette Guerin (BCG), the efficacy of treatment was evaluated by cystoscopy, standard cytology, flow cytometry or image analysis. According to these various methods it has been shown that a normal cystoscopy may or may not be associated with aneuploidy revealed by flow cytometry. Such a case clearly illustrates the value of combining macroscopic examination and a cytologic analysis in particular by flow cytometry in order to increase the accuracy of diagnosis and to evaluate without ambiguity the efficacy of treatment.

BCG Vaccine↗

PubMed1992

Doxorubicin-loaded nanospheres bypass tumor cell multidrug resistance.

We have demonstrated that in vitro resistance of tumor cells to doxorubicin (Dox) can be fully circumvented by using doxorubicin-loaded nanospheres (Dox-NS), consisting of biodegradable polyisohexylcyanoacrylate polymers of 300 nm diameter and containing 2.83 mg of Dox per 31.5 mg of polymer. Five different multidrug-resistant cell lines, characterized by mdr1 amplification, were used in this study: Dox-R-MCF7, a human breast adenocarcinoma; SKVBL1, a human ovarian adenocarcinoma; K562-R, a human erythroleukemia; and two murine lines: P388-Adr-R, a monocytic leukemia of DBA2 mouse, and LR73MDR, a Chinese hamster ovarian cell line. These lines were 38.7, 210, 232, 143 and 20 times more resistant than their corresponding sensitive counterparts, respectively. Using Dox-NS, we obtained complete reversion of drug resistance in vitro, i.e. cell growth inhibition comparable with that obtained with sensitive cells exposed to free Dox. In vivo, we significantly prolonged the survival of DBA2 mice which had previously received P388-Adr-R cells by i.p. injections of Dox-NS, while free Dox injection was ineffective toward this rapidly growing tumor. (Prolongation of survival time: 115% vs 167% after Dox vs Dox-NS treatment, respectively.) Using the MCF7 cell line and its resistant variant, we studied the intracellular concentration and the cytoplasmic and nuclear distribution of Dox by laser microspectrofluorometry (LMSF). In sensitive cells, we observed a similar accumulation and distribution of Dox whatever the form of Dox delivery, i.e. whether free or carried by nanospheres. Analysis by LMSF showed that 99% of intranuclear Dox was bound to DNA after treatment with both forms of Dox. Of Dox, 81 and 83% were found in the intranuclear compartment of sensitive cells incubated with free Dox and Dox-NS, respectively. Resistant cells incubated with Dox-NS accumulated the same amount of Dox as sensitive cells incubated with free Dox or with Dox-NS. Dox, when loaded in nanospheres, bypasses the efflux mechanism responsible for multidrug resistance. LMSF analysis showed that Dox, transported and released by nanospheres, interacts with DNA identically in sensitive and resistant cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1992

Abnormal expression of neurofilament proteins in Ewing's sarcoma cell cultures.

A neural origin of Ewing's sarcoma (ES) has often been suggested and we have demonstrated neurofilament protein expression in ES cells. However, only the 200-kD subunit has been revealed in all of the ES cells analyzed. The 160- and 68-kD subunits were always absent. For these reasons, we have attempted to induce neural differentiation in 3 ES cell lines with different types of inducers: tetradecanoylphorbol-13-acetate (TPA) retinoic acid and nerve growth factor. When the cell lines were cultured for 7 days with TPA (10(-9) M) or retinoic acid (10(-7) M), only the 68-kD neurofilament subunit was slightly induced. No inducation was obtained when nerve growth factor was used, even at a 21-day culture. These results are in agreement with the putative neural origin of ES and may indicate an abnormal expression of neurofilament proteins in this tumor.

Cell Division↗

PubMed1992

Characterization of seven kidney tumors by flow cytometry: analysis of cell cycle, DNA content and P-glycoprotein expression.

Seven kidney tumors obtained from patients aged from 5 to 76 years were analyzed by flow cytometry for cell cycle, DNA content and P-glycoprotein expression involved in multidrug resistance. The DNA index seems to be an important criterion since all the tumors were aneuploid. In a case of clear cell carcinoma, two aneuploid clones were identified. In 5 cases of kidney tumors a high proportion of cells in proliferation (S + (G2 + M)) was observed; it was comprised between 13 and 33%. As for P-glycoprotein it was detected only in few tumor cells (5-15%) respectively in a case of clear cell carcinoma and in a case of Wilms' tumor.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

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↗

PubMed1991

Cytotoxic activity of lymphocytes infiltrating progressive and regressive tumor variants from a rat colonic cancer.

Two tumor cell variants (PROb and REGb) isolated from a single chemically-induced rat colon adenocarcinoma were previously shown to differ in their tumorigenicity. When injected into syngeneic BDIX rats, PROb cells induce progressive tumors whereas REGb cells give rise to tumors which always regress. PROb and REGb variants also differ in their capacity to induce an immune response in the syngeneic host. Regression of REGb tumors could have been mediated by cytotoxic effector cells acting at the tumor site. To test this hypothesis, the cytolytic activity of non-adherent lymphoid cells isolated from PROb and REGb tumors and from the spleen of the same animals were compared. The same number of infiltrating lymphocytes was recovered per gram of PROb or REGb tumor. The cytolytic activity of tumor infiltrating lymphocytes, as that of spleen lymphocytes, was predominantly non specific, as demonstrated by their ability to kill YAC-1 cells, an NK-sensitive cell line. PROb cells were relatively resistant to the cytotoxic activity of spleen or tumor infiltrating lymphocytes. In the regressing REGb tumors, the cytotoxic activity of tumor infiltrating lymphocytes to homologous cells or to YAC-1 cells was low and significantly inferior to that of the corresponding spleen lymphocytes. These results suggest that the cytotoxic activity of lymphocytes was impaired at the local, intratumoral level, even in spontaneously regressing tumors.

Adenocarcinoma↗