Biomedical subjects

G Lizard

Publications and source records attributed to G Lizard.

At least 19 recordsLinked to original sources

PubMed2000

Impairment with various antioxidants of the loss of mitochondrial transmembrane potential and of the cytosolic release of cytochrome c occuring during 7-ketocholesterol-induced apoptosis.

Previous investigations of our laboratory have shown that 7-ketocholesterol was a potent inducer of apoptosis involving a release of cytochrome c into the cytosol, and a lipid peroxidation process that could be the consequence of a production of radical oxygen species. According to these considerations, we asked whether some antioxidants were able to counteract 7-ketocholesterol-induced apoptosis, and whether prevention of cell death was associated with the impairment of mitochondrial events implied in the commitment to apoptosis, i.e., opening of the mitochondrial megachannels leading to the loss of the mitochondrial transmembrane potential (DeltaPsim), and release of cytochrome c from mitochondria into the cytosol. To this end, we studied the effects of glutathione (15 mM), N-acetylcysteine (15 mM), vitamin E (100 microM), vitamin C (50 microM) and melatonin (1 mM) on U937 cells treated with 7-ketocholesterol (40 microg/ml). Only glutathione, N-acetylcysteine, and vitamin E prevented apoptosis measured by the occurrence of cells with condensed and/or fragmented nuclei, as well as the loss of DeltaPsim, and the release of cytochrome c. However, all the antioxidants used were potent inhibitors of the production of O(2)(*) occuring under treatment with 7-ketocholesterol. Collectively, our data demonstrate that impairment of apoptosis by glutathione, N-acetylcysteine, and vitamin E correlates with the prevention of mitochondrial dysfunctions, and they underline that the ability of antioxidants to counteract 7-ketocholesterol-induced apoptosis does not only depend on their capability to inhibit the production of O(2)(*).

Acetylcysteine↗

PubMed2000

Factor analysis of confocal image sequences of human papillomavirus DNA revealed with fast red in cervical tissue sections stained with TOTO-iodide.

OBJECTIVE: To visualize and localize specific DNA sequences by fluorescence in situ hybridization, confocal laser scanning microscopy (CLSM) and factor analysis of biomedical image sequences (FAMIS). STUDY DESIGN: Human papillomavirus (HPV) DNA was identified in cervical tissue sections with biotinylated DNA probes recognizing the whole genome of HPV DNA types 18 and 16, and DNA-DNA hybrids were revealed by streptavidin-alkaline phosphatase and Fast Red (FR). Cell nuclei were counterstained with TOTO-iodide. Image sequences were obtained using successive dynamic or spectral sequences of images on different optical slices from CLSM. The location of fluorescent signals inside tissue preparations was determined by FAMIS and/or selection of filters at emission. Image sequences were summarized into a reduced number of images, called "factor images," and curves, called "factors." Factors estimate spectral patterns and depth emission profiles. Factor images correspond to spatial distributions of the different factors. RESULTS: We distinguished between FR and nucleus staining in HPV DNA hybridization signals by taking into account differences in their spectral patterns and improved visualization by taking into account differences in their focus (depth emission profiles). CONCLUSION: FAMIS, together with CLSM, made possible the detection and characterization of HPV DNA sequences in cells of cervical tissue sections.

Azo Compounds↗

PubMed1999

Impairment of endothelium-dependent arterial relaxation by high-fat feeding in ApoE-deficient mice: toward normalization by human ApoA-I expression.

BACKGROUND: Atherogenic lipoproteins can impair the endothelium-dependent arterial relaxation, and circumstantial evidence suggests a beneficial role of plasma high density lipoproteins and apolipoprotein (apo) A-I in counteracting the endothelium dysfunction. In the present study, vascular reactivity was determined in control, apoE-deficient mice (apoE-KO mice), and apoE-deficient mice expressing human apoA-I (apoE-KO/HuAITg mice). METHODS AND RESULTS: In the first part of the study, control and apoE-KO mice were fed a low-fat or a high-fat diet for 23 weeks, and the vasoactive responses of isolated thoracic aortic segments to norepinephrine, sodium nitroprusside, and acetylcholine (ACh) were determined. Whereas norepinephrine, sodium nitroprusside, and ACh evoked similar vascular responses in control and apoE-KO mice fed the low-fat diet, high-fat feeding in apoE-KO mice produced a significant 3-fold increase in the mean concentration required to produce a half-maximal relaxing effect (EC(50)) of ACh as compared with control mice. This reflects a weaker sensitivity to ACh of the aortic segments from the apoE-deficient animals. In the second part of the study, the mean EC(50) for ACh after high-fat feeding was found to be 4.4-fold lower in apoE-KO/HuAITg mice than in apoE-KO mice, indicating that the reduced sensitivity to ACh of the thoracic aorta from the apoE-KO mice fed the high-fat diet is improved by the expression of human apoA-I. CONCLUSIONS: The present study demonstrates that the endothelium-dependent arterial relaxation is impaired in apoE-KO mice fed the high-fat diet. The endothelium dysfunction tends to be normalized by human apoA-I expression.

Acetylcholine↗

PubMed1999

Four-dimensional factor analysis of confocal image sequences (4D-FAMIS) to detect and characterize low copy numbers of human papillomavirus DNA by FISH in HeLa and SiHa cells.

Visualization and localization of specific DNA sequences were performed by fluorescence in situ hybridization, confocal laser scanning microscopy (CLSM), and four-dimensional factor analysis of biomedical image sequences (4D-FAMIS). HeLa and SiHa cells containing, respectively 20-50 and 1-2 copies per cell of human papillomavirus (HPV) DNA type 18 and 16 integrated in cellular DNA were used as models. HPV-DNA was identified using DNA probes containing the whole genome of HPV-DNA type 18 or 16, and DNA-DNA hybrids were revealed by alkaline phosphatase and Fast Red. Cell nuclei were counterstained with thiazole orange (TO) or TOTO-iodide. 4D image sequences were obtained using successive dynamic or spectral sequences of images on different optical sections from CLSM. The location of fluorescent signals within the preparations was determined by FAMIS. This original method summarizes image sequences into a reduced number of images called factor images, and curves called factors. Factors estimate different individual physical behaviours in the sequence such as extinction velocity, spectral patterns and depth emission profiles. Factor images correspond to spatial distributions of the different factors. We distinguished between Fast Red and nucleus stainings in HPV-DNA hybridization signals by taking into account differences in their extinction velocities (fluorescence decay rate) or spectral patterns, and in their focus (depth emission profiles). In HeLa cells, factor images showed that Fast-Red-stained targets could be distinguished from nucleus stainings, and were located on different focal planes of the nuclei. In SiHa cells, 4D-FAMIS determined as few as 1-2 copies per cell of HPV-DNA type 16 located in continuous focal planes. Therefore, 4D-FAMIS, together with CLSM, made the detection and characterization of low copy numbers of genes in whole cells possible.

Cell Nucleus↗

PubMed1999

Plasma phospholipid transfer protein prevents vascular endothelium dysfunction by delivering alpha-tocopherol to endothelial cells.

alpha-tocopherol, the most potent antioxidant form of vitamin E, is mainly bound to lipoproteins in plasma and its incorporation into the vascular wall can prevent the endothelium dysfunction at an early stage of atherogenesis. In the present study, the plasma phospholipid transfer protein (PLTP) was shown to promote the net mass transfer of alpha-tocopherol from high density lipoproteins (HDL) and alpha-tocopherol-albumin complexes toward alpha-tocopherol-depleted, oxidized low density lipoproteins (LDL). The facilitated transfer reaction of alpha-tocopherol could be blocked by specific anti-PLTP antibodies. These observations indicate that PLTP may restore the antioxidant potential of plasma LDL at an early stage of the oxidation cascade that subsequently leads to cellular damages. In addition, the present study demonstrated that the PLTP-mediated net mass transfer of alpha-tocopherol can constitute a new mechanism for the incorporation of alpha-tocopherol into the vascular wall in addition to the previously recognized LDL receptor and lipoprotein lipase pathways. In ex vivo studies on rabbit aortic segments, the impairment of the endothelium-dependent arterial relaxation induced by oxidized LDL was found to be counteracted by a pretreatment with purified PLTP and alpha-tocopherol-albumin complexes, and both the maximal response and the sensitivity to acetylcholine were significantly improved. We conclude that PLTP, by supplying oxidized LDL and endothelial cells with alpha-tocopherol through a net mass transfer reaction may play at least two distinct beneficial roles in preventing endothelium damage, i.e., the antioxidant protection of LDL and the preservation of a normal relaxing function of vascular endothelial cells.

Animals↗

PubMed1999

Characterization and comparison of the mode of cell death, apoptosis versus necrosis, induced by 7beta-hydroxycholesterol and 7-ketocholesterol in the cells of the vascular wall.

Oxidized low density lipoproteins (LDLs) play a central role in atherosclerosis, and their toxicity is due, at least in part, to the formation of oxysterols that have been shown to induce apoptosis in various cell types. As 7beta-hydroxycholesterol and 7-ketocholesterol are the major oxysterols found in oxidized LDLs, we have investigated and compared the mode of cell death, apoptosis versus necrosis, that they induce in the cells of the vascular wall, ie, endothelial cells, smooth muscle cells, and fibroblasts. To this end, human vascular endothelial cells from umbilical cord veins (HUVECs), human artery smooth muscle cells, A7R5 rat smooth muscle cells, MRC5 human fibroblasts, and human fibroblasts isolated from umbilical cord veins were taken at confluence and incubated for 48 hours with 7beta-hydroxycholesterol or 7-ketocholesterol (concentration range, 5 to 80 microg/mL). In all cells, both 7beta-hydroxycholesterol and 7-ketocholesterol exhibited toxic effects characterized by a loss of cell adhesion and an increased permeability to propidium iodide. In oxysterol-treated endothelial and smooth muscle cells, typical features of apoptosis were revealed: condensed and/or fragmented nuclei were detected by fluorescence microscopy after staining with Hoechst 33342, oligonucleosomal DNA fragments were visualized in situ in the cell nuclei by the TdT-mediated dUTP-biotin nick-end labeling (TUNEL) method, and internucleosomal DNA fragmentation was found on agarose gel. In contrast, in oxysterol-treated fibroblasts, fragmented and/or condensed nuclei were never revealed, and no DNA fragmentation was observed either by the TUNEL method or by DNA analysis on agarose gel, indicating that these oxysterols induced necrosis in these cells but not apoptosis. In addition, acetylated Asp-Glu-Val-L-aspartic acid aldehyde (an inhibitor of Asp-Glu-Val-L-aspartic acid-sensitive caspases) prevented 7beta-hydroxycholesterol- and 7-ketocholesterol-induced cell death in HUVECs and smooth muscle cells but not in fibroblasts. Thus, 7beta-hydroxycholesterol and 7-ketocholesterol have dual cytotoxic effects on the cells of the vascular wall by their ability to induce apoptosis in endothelial and smooth muscle cells and necrosis in fibroblasts.

Animals↗

PubMed1998

Different patterns of IL-1beta secretion, adhesion molecule expression and apoptosis induction in human endothelial cells treated with 7alpha-, 7beta-hydroxycholesterol, or 7-ketocholesterol.

Among oxysterols oxidized at C7 (7alpha-, 7beta-hydroxycholesterol, and 7-ketocholesterol), 7beta-hydroxycholesterol and 7-ketocholesterol involved in the cytotoxicity of oxidized low density lipoproteins (LDL) are potent inducers of apoptosis. Here, we asked whether all oxysterols oxidized at C7 were able to trigger apoptosis, to stimulate interleukin (IL)-Ibeta and/or tumor necrosis factor (TNF)-alpha secretion, and to enhance adhesion molecule expression (intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin) on human umbilical venous endothelial cells (HUVECs). Only 7beta-hydroxycholesterol and 7-ketocholesterol were potent inducers of apoptosis and of IL-1beta secretion. TNF-alpha secretion was never detected. Depending on the oxysterol considered, various levels of ICAM-1, VCAM-1 and E-selectin expression were observed. So, oxysterols oxidized at C7 differently injure and activate HUVECs, and the alpha- or beta-hydroxyl radical position plays a key role in apoptosis and IL-1beta secretion.

Apoptosis↗

PubMed1998

Morphological analysis of in situ hybridization signals in cervical intraepithelial neoplasia containing human papillomavirus type 16 or 18: relationship with histological grade and DNA content.

Among 345 lesions histologically defined as cervical intraepithelial neoplasia (CIN) examined by in situ hybridization (ISH) for the presence of DNA from human papillomavirus (HPV) types 6/11, 16, 18, 31, 33, and 51, a group of 69 lesions (41 low grade and 28 high grade) containing HPV 16 or 18 was further characterized with the following criteria: DNA ploidy and morphological patterns of ISH spots, i.e., punctate or diffuse throughout the nuclei corresponding to integrated or episomal state of HPV DNA, respectively. The highest percentage of aneuploid lesions, the highest diploid index values, and the highest proportion of CIN with punctate ISH signals were associated with high-grade lesions. In addition, punctate ISH signals were also most frequently found in aneuploid CIN. These results underline that punctate ISH signals considered as integrated HPV DNA were preferentially associated with aneuploid and high-grade lesions, and lead to suggest that this later criteria could be used to predict the evolution of a lesion towards malignancy.

Adolescent↗

PubMed1998

Detection of low copy numbers of HPV DNA by fluorescent in situ hybridization combined with confocal microscopy as an alternative to in situ polymerase chain reaction.

In genital lesions infected by human papillomavirus (HPV), histological criteria and HPV DNA typing are of prognostic value. Therefore, non-radioactive methods such as in situ hybridization are used extensively since they preserve the histological organization of the tissue, and allow the detection and characterization of HPV DNA. However, the sensitivity of these methods is often limited to detection of low copy numbers of HPV DNA in isolated cells or in tissue sections, and therefore alternative techniques have been explored. In the present study, 1-2 copies of HPV DNA were visualized in SiHa cells either by in situ amplification of nucleic acid sequences with the polymerase chain reaction (PCR) or by fluorescent in situ hybridization (FISH) associated with observation by laser scanning confocal microscopy (LSCM). The latter procedure was evaluated for use on histological tissue sections to identify low copy numbers of HPV DNA. Genital lesions which were negative by enzymatic in situ hybridization and FISH but histologically suspected of HPV infection were investigated, and intense signals were obtained both with in situ PCR and with the combined use of FISH and LSCM. Therefore, the combination of FISH with LSCM examination may be as valuable as in situ PCR to detect viral genes present in small amounts in isolated cells and in tissue sections.

Condylomata Acuminata↗

PubMed1998

Glutathione is implied in the control of 7-ketocholesterol-induced apoptosis, which is associated with radical oxygen species production.

In a number of experimental systems, inhibition of apoptosis by antioxidants has led to the production of radical oxygen species (ROS) in certain apoptotic forms of cell death. Since antioxidant therapies can reduce vascular dysfunctions in hypercholesterolemic patients who frequently have increased plasma levels of oxysterols constituting potent inducers of apoptosis, we speculate that oxysterol-induced apoptosis could involve oxidative stress. Here, we tested the protective effects of the aminothiols glutathione (GSH) and N-acetylcysteine (NAC), which are two potent antioxidants, on apoptosis induced by 7-ketocholesterol in U937 cells, and we present evidence indicating that oxidative processes are involved in 7-ketocholesterol-induced cell death. Thus, GSH and NAC prevented phenomenona linked to apoptosis such as reduction of cell growth, increase cellular permeability to propidium iodide, and occurrence of nuclear condensation and/or fragmentation, and they delayed internucleosomal DNA fragmentation. In addition, cell treatment with GSH impaired cytochrome c release into the cytosol and degradation of caspase-8 occurring during cell death. During 7-ketocholesterol-induced apoptosis, we also observed a rapid decrease in cellular GSH content, oxidation of polyunsaturated fatty acids, and a production of ROS by flow cytometry with the use of the dye 2', 7'-dichlorofluorescin-diacetate; both phenomena were inhibited by GSH. Prevention of cell death by GSH and NAC does not seem to be a general rule since these antioxidants impaired etoposide (but not cycloheximide) -induced apoptosis. Taken together, our data demonstrate that GSH is implied in the control of 7-ketocholesterol-induced apoptosis associated with the production of ROS.

Acetylcysteine↗

PubMed1998

Inhibition by cholesterol oxides of NO release from human vascular endothelial cells.

Recent studies have demonstrated that, unlike cholesterol, cholesterol oxidized at position 7 can reduce the maximal endothelium-dependent relaxation of isolated rabbit aortas (Circulation. 1997;95:723-731). The aim of the current study was to determine whether cholesterol oxides reduce the release of nitric oxide (NO) from human umbilical vein endothelial cells (HUVECs). The amount of NO released by histamine-stimulated HUVECs was determined by differential pulse amperometry using a nickel porphyrin- and Nafion-coated carbon microfiber electrode. The effects of cholesterol (preserved from oxidation by butylated hydroxytoluene), 7-ketocholesterol, 7beta-hydroxycholesterol, 5alpha,6alpha-epoxycholesterol, 19-hydroxycholesterol (60 microg/mL), and alpha-lysophosphatidylcholine (10 microg/mL) were compared. Pretreatment of HUVECs with cholesterol, 5alpha,6alpha-epoxycholesterol, or 19-hydroxycholesterol did not alter histamine-activated NO production. In contrast, pretreatment with 7-ketocholesterol or 7beta-hydroxycholesterol significantly decreased NO release. The inhibitory effect of 7-ketocholesterol was time and dose dependent and was maintained in the presence of L-arginine. In the absence of serum, lysophosphatidylcholine also reduced NO production. In ionomycin-stimulated cells, pretreatment with 7-ketocholesterol did not inhibit NO release. These results demonstrate that cholesterol derivatives oxidized at the 7 position, the main products of low density lipoprotein oxidation, reduce histamine-activated NO release in HUVECs. Such an inhibitory effect of cholesterol oxides may account, at least in part, for the ability of oxidized low density lipoprotein to reduce the endothelium-dependent relaxation of arteries.

Arginine↗

PubMed1997

Induction of apoptosis and of interleukin-1beta secretion by 7beta-hydroxycholesterol and 7-ketocholesterol: partial inhibition by Bcl-2 overexpression.

The oxysterols, 7beta-hydroxycholesterol and 7-ketocholesterol, are involved in the cytotoxicity of oxidized LDL. To elucidate their molecular mechanisms, the human promonocytic leukemia cells U937 and U4 were used. U4 cells overexpressing Bcl-2 were obtained by transfection of U937 cells. 7Beta-hydroxycholesterol and 7-ketocholesterol induced nuclear condensation and/or fragmentation, internucleosomal DNA fragmentation, and IL-1beta secretion, which were partially inhibited by Bcl-2 overexpression. These findings underline that these oxysterols could constitute major risk factors in atherosclerosis by their cytotoxicity and their ability to induce IL-1beta release which might favor the recruitment of immunocompetent cells in the atherosclerotic plaque.

Apoptosis↗

PubMed1997

Loss of heterozygosity at the TP53 gene: independent occurrence from genetic instability events in node-negative breast cancer.

TP53 abnormalities have been reported as an early event in the process of cellular transformation of human breast cancers, and involved in mammary-tumor evolution, from in situ to invasive disease. In this study, node-negative (N-) tumors were examined for TP53 allelic loss in relation to different genetic instability events, including allelic loss at chromosome 17p13.3 and c-H-ras-1 loci, as well as alteration of the c-myc and c-erbB-2/neu oncogenes. TP53 allelic loss was analyzed to determine whether such an abnormality was the more important, among other genetic events, in the N- tumors, whether it appeared independently of these genetic events, and whether accumulation of genetic events arises in this group of breast tumors. Clinicopathological parameters were also examined. Loss of heterozygosity (LOH) at the TP53 gene appears the most frequent alteration detected (26% vs. 13%, 8%, 9% and 3% for LOH at D17S30 and c-H-ras-1 loci, and amplification of c-myc and c-erbB-2/neu respectively). There was no association between LOH at the TP53 locus and other genetic events. Among clinicopathological parameters, significant associations were observed only with estrogen-receptor-negative tumors (p = 0.05). Our results demonstrate that LOH at TP53 arises more frequently in the N- breast cancer, thus supporting earlier findings suggesting that TP53 abnormality has a role early in the pathogenesis of breast lesions. Moreover, the data indicate that accumulation of many genetic events occurs at a low level in N- breast tumors, and that TP53 abnormality occurs independently of these genetic events.

Adult↗

PubMed1997

Laser scanning confocal microscopy and factor analysis of biomedical image sequences (FAMIS) to detect and characterise HPV DNA sequences by FISH in HeLa cells.

Visualisation and localisation of specific DNA sequences were performed by fluorescence in situ hybridisation (FISH), confocal laser scanning microscopy (CLSM), and factor analysis of biomedical image sequences (FAMIS). HeLa cells containing 10-50 copies per cell of human papillomavirus (HPV) DNA type 18 integrated in cellular DNA were used as a model. HPV-DNA was identified by DNA probes and DNA-DNA hybrids were revealed by alkaline phosphatase and Fast Red (FR) TR salt/naphtol-MX phosphate. Cell nuclei were counterstained with thiazole orange (TO). FAMIS summarises image sequences into a reduced number of images called factor images and curves called factors. Factor images correspond to spatial distributions of the different factors. Factors estimate different individual physical behaviours in the sequence (extinction velocity, spectral emission, depth emission profiles). We verified that HPV-DNA hybridisation signals are specific to the spectrum of FR, and distinguished between FR and TO. The latter result was found by taking into account differences in their extinction velocities. The focus of CLSM was improved on 3D image sequences, and the location of fluorescent signals inside the preparations was determined. Factor images showed that FR stained targets were located on different focal planes at the periphery of the nuclei.

DNA, Viral↗

PubMed1997

Inhibitors of arterial relaxation among components of human oxidized low-density lipoproteins. Cholesterol derivatives oxidized in position 7 are potent inhibitors of endothelium-dependent relaxation.

BACKGROUND: Oxidized low-density lipoproteins (LDLs) are known to impair arterial relaxation. The aim of the present study was to identify the components of oxidized LDL that may account for inhibition of endothelium-dependent relaxation. METHODS AND RESULTS: LDLs from 12 healthy subjects were either maintained at 4 degrees C (native LDL) or incubated at 37 degrees C in the presence of copper sulfate (oxidized LDL). Unlike pretreatment with native LDL, pretreatment with oxidized LDL reduced significantly the acetylcholine-mediated relaxation of rabbit aortic segments compared with control segments incubated in Krebs' buffer (maximal relaxation [Emax], 72.0 +/- 6.7% versus 94.1 +/- 0.8%, respectively, P < .01; negative logarithm of the concentration required to produce a half-maximal relaxing effect [pD2], 6.6 +/- 0.1 versus 7.2 +/- 0.1, respectively, P < .001). The absolute difference between Emax values obtained with oxidized and native LDL (delta Emax) correlated significantly with the formation of 7 ketocholesterol, 7 alpha-hydroxycholesterol, and 7 beta-hydroxy-cholesterol. In contrast, delta Emax did not correlate with the amount of lipoperoxides or lysophosphatidylcholine formed, and the difference of pD2 values measured with oxidized and native LDL (delta pD2) did not correlate significantly with any of the oxidation-derived LDL compounds. When added individually, 7-ketocholesterol and 7 beta-hydroxycholesterol reduced Emax values but not pD2 values in a time- and concentration-dependent manner. CONCLUSIONS: Cholesterol derivatives in oxidized LDL can reduce maximal arterial relaxation through a specific effect on vascular endothelial cells.

Acetylcholine↗

PubMed1997

Induction of similar features of apoptosis in human and bovine vascular endothelial cells treated by 7-ketocholesterol.

Cholesterol oxides have numerous cytotoxic effects and those oxidized in the C7 position have been shown to induce apoptosis in bovine aortic endothelial cells (BAEC). The aim of the present study was to determine whether apoptosis also occurs in human vascular endothelial cells (HUVEC) treated with 7-ketocholesterol. To this end, cultured BAEC and HUVEC were incubated for 48 h with 7-ketocholesterol (concentration range 5-80 micrograms/ml) and the characteristics of cell death were assessed by various methods: counting of adherent and non-adherent cells; analysis of DNA fragmentation pattern; and morphological study by light, fluorescence, and electron microscopy. The 7-ketocholesterol treatment was accompanied by a decrease in the number of adherent cells and an increase in the number of non-adherent cells. Apoptotic cells, recognized by fragmented and/or condensed nuclei after staining with Hoechst 33342 or Giemsa, were mainly detected among non-adherent cells, and agarose gel electrophoresis revealed a typical internucleosomal DNA fragmentation among 7-ketocholesterol-treated cells. The DNA fragmentation was no longer detected when HUVEC and BAEC were simultaneously incubated with 0.5 mmol/l zinc chloride, which is known to inhibit Ca2+/Mg(2+)-dependent endonucleases. Finally, the ultrastructural abnormalities observed by electron microscopy in both 7-ketocholesterol-treated HUVEC and BAEC were remarkably similar and were mainly characterized by condensed chromatin, altered mitochondria, disturbed organization of the cytoskeleton, and vacuoles containing myelin figures and/or cell debris; apoptotic bodies were also frequently detected. It is concluded that 7-ketocholesterol constitutes a potent inducer of apoptosis in endothelial vascular cells of both bovine and human origin, suggesting that cholesterol oxides may be involved in the early steps of the atherosclerotic process in humans.

Animals↗

PubMed1997

Detection of human papillomavirus DNA in genital lesions by enzymatic in situ hybridization with Fast Red and laser scanning confocal microscopy.

Human papillomavirus (HPV) infection with potentially oncogenic types 16 or 18 is common in genital lesions especially in uterine carcinomas. In such lesions, in situ hybridization with non-radioactive probes is a powerful tool for the histopathologist to detect and type HPV DNA either on cell deposits or on tissue sections. The use of an immunohistochemical method involving alkaline phosphatase and Fast Red TR salt/naphthol AS-MX phosphate is proposed for use with conventional bright-field or fluorescence microscopy as well as by laser scanning confocal microscopy. The alkaline phosphatase-Fast Red reaction has the advantage of producing a red precipitate that permits the detection of in situ hybridization signals by bright-field microscopy, and of obtaining a strong red fluorescence characterized by a lack of bleaching when excited by a green light. Therefore, the alkaline phosphatase-Fast Red reaction is well adapted for observations by fluorescence and confocal microscopy, the latter method allowing the detection, in tissue sections of cervical intraepithelial lesions, of small punctate and large diffuse hybridization signals, considered as integrated and episomal states of HPV DNA respectively. The combination of in situ hybridization with the alkaline phosphatase-Fast Red reaction and confocal microscopy is particularly convincing when hybridization signals are of small size and/or of low fluorescence intensity, especially if they are present in various focal planes; in such conditions, infected cells are easily detected by three-dimensional reconstruction. Therefore, this combination is a suitable method for identifying and characterizing HPV DNA in cells and tissue sections.

Coloring Agents↗

PubMed1997

Comparative evaluation of the antiproliferative effect of cyclosporin A and gamma-interferon on normal and HPV-transformed keratinocytes by cell counting, MTT assay and tritiated thymidine incorporation.

We compared three techniques, the MTT tetrazolium assay, cell counting, and tritiated thymidine ([3H]TdR) incorporation assay to measure the antiproliferative effect of cyclosporin A (CsA) and interferon-gamma (IFN-gamma) on normal human skin keratinocyte cultures (NHK) used at the second passage and human papilomavirus type 16- and 18-transformed cell lines (EK16 and EK18) exposed continuously to the drugs for 3 days. The three techniques showed that under CsA (0.5 and 8 micrograms/ml) and IFN-gamma (5 and 160 U/ml) treatments the cells remained viable and that the growth of keratinocytes was inhibited. For IFN-gamma, the MTT colorimetric assay consistently underestimated its growth inhibitory activity as compared to cell counting or [3H]TdR incorporation, whatever the cells used. For high doses of CsA, MTT and cell counting gave similar percentages, of inhibitory activity whatever the cells; MTT underestimated this activity as compared to [3H]TdR incorporation only in NHK and EK18 cells, whereas similar results were obtained with EK16 cells. In conclusion, this investigations shows that MTT sensitivity differed with the drug and also according to the keratinocyte cultures. The MTT test is clearly not appropriate for study of IFN-gamma treatment whatever the keratinocytes used. Such discrepancies indicate that the MTT test should be done with care on cultures to measure the effects of drugs on cell growth; the growth inhibition should be carefully considered and it would be best if two different methods were used.

Adult↗