Long-term metabolic control in pancreas transplant patients (follow-up 3 to 13 years).
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Adult↗
Biomedical subjects
M Benchaib
Publications and source records attributed to M Benchaib.
Long-term outcome of pancreas transplantation.
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Adult↗
Evaluation of five green fluorescence-emitting streptavidin-conjugated fluorochromes for use in immunofluorescence microscopy.
Fluorescein isothiocyanate (FITC) is largely used in immunofluorescence methods. We propose to analyse the quality of some recent fluorochromes using image analysis. Fluorochromes tested include FITC and dichlorotriazinylaminofluorescein (DTAF), dipyrrometheneboron difluoride (BODIPY), Rhodol Green and cyanine 2. RAMOS cells were immunolabelled against the proliferating cell nuclear antigen (PCNA) revealed by the biotin-streptavidin technique. Slides were mounted in anhydrous glycerol or in buffered glycerol (pH 7.0 or pH 8.5). No antifading medium was added. Cell fluorescence emission intensity and bleaching characteristics were measured. Rhodol Green exhibited the highest fluorescence intensity and the best photobleaching resistance. Although BODIPY also resisted well during the photobleaching assay, its fluorescence intensity was weak. FITC, DTAF and cyanine 2 showed intermediate fluorescence intensity and a fast decay of fluorescence. Among the green emitting fluorochromes tested, Rhodol Green appeared to be the best.
Bacterial Proteins↗
Effect of gastrin-releasing peptide on sperm functions.
Male infertility can be related to defects in motility, capacitation, acrosome reaction, binding and penetration of the zona pellucida. While different in-vitro techniques (such as micromanipulation which is complicated and expensive) are available for the treatment of male infertility, several pharmacological agents have been shown to increase fertilizing capacity under accurate experimental conditions. Gastrin-releasing peptide (GRP, the mammalian homologue of the amphibian skin peptide bombesin) is present in the reproductive tract and expressed by the pregnant ovine endometrium prior to attachment and throughout the pregnancy. A bombesin-like peptide resulting from alternate splicing of the GRP gene in testis has been detected in primates. In this study, we have tested the ability of GRP to enhance human sperm functions such as motility, capacitation, zona binding and acrosome reaction. Analysis of sperm motility was performed with the ATS 20 computer-assisted semen analysis (CASA) system. Zona binding was analysed using intact human unfertilized oocytes and selective labelling of spermatozoa with two fluorochromes. Our results did not show any positive effect of GRP on these parameters under our experimental conditions. However, when GRP at the concentration of 100 nM was added after ionophore treatment, the percentage of reacted cells increased. significantly (P < 0.05) compared with situations where each agent was used alone. This led us to suppose that the role of bombesin in the different stages of fertilization might not exclude other unknown factors.
Acrosome↗
Transvaginal ultrasound studies of vascular and morphological changes in uteri exposed to diethylstilbestrol in utero.
The aim of this prospective study was to establish complementary data of uteri exposed to diethylstilbestrol (DES) in utero for transvaginal analysis and vascularity changes during the menstrual cycle. A total of 28 women with DES-exposed uteri were compared with 60 non-exposed women. Transvaginal ultrasound and colour Doppler imaging were performed on days 5 and 22 of the menstrual cycle. Uteri were measured on sagittal and transverse scans. Uterine length, width, thickness and uterine cavity length and width were measured. Uterine volume and uterine cavity area were calculated. DES-exposed uterine volume was equal to 31.84 +/- 3.37 cm3. The cavity area of DES-exposed uterus was equal to 35.85 +/- 3.93 cm2. Cervix length of DES-exposed uterus was significantly smaller than that of non-exposed uterus. The uterine artery pulsatility index (PI) of DES-exposed uterus was significantly higher than that of normal uterus. Blood flow remained stable throughout the menstrual cycle. The PI of DES-exposed uterus remained stable during the menstrual cycle, as in non-exposed uterus, and it decreased during the luteal phase. This lack of modification in vascularity of DES-exposed uterus may explain miscarriages and obstetric complications such as intrauterine growth retardation or pre-eclampsia. The data may have implications for the assessment of reproductive status and the design of future studies on disorders of implantation in DES-exposed uterus.
Adult↗
n-Butyrate, a cell cycle blocker, inhibits early amplification of duck hepatitis B virus covalently closed circular DNA after in vitro infection of duck hepatocytes.
During chronic hepadnavirus infection, virus persistence depends on the regulation of the pool of covalently closed circular DNA (cccDNA), which is the template for transcription of viral RNA species. The development of in vitro infection of duck hepatocyte primary cultures by duck hepatitis B virus (DHBV) provides a unique opportunity to study the regulation of cccDNA synthesis. After DHBV in vitro infection, cccDNA is detected 1 day later and is amplified to a high copy number after 1 week in culture. We studied whether this amplification occurs during cell cycle progression of duckling hepatocytes. By using [3H]thymidine incorporation, we found that hepatocytes obtained from 3-week-old ducklings spontaneously entered the S phase of the cell cycle when cultured in serum-free medium without added growth factors. Bromodeoxyuridine labeling confirmed that cellular DNA synthesis took place in more than 50% of parenchymal cells. Cytofluorometry analysis revealed the presence of asynchronous populations and polyploidization processes. The addition of a cell cycle blocker, n-butyrate, completely inhibited [3H]thymidine incorporation and blocked duckling hepatocytes in the G1 phase of the cell cycle. Simultaneously, butyrate inhibited cccDNA amplification and allowed the establishment of DHBV infection, as demonstrated by the detection of a basal level of cccDNA in treated hepatocytes. Both effects were reversible since active cell DNA synthesis was restored and cccDNA accumulated after drug withdrawal.
Animals↗
Methods for cell proliferation analysis by fluorescent image cytometry.
Methods were developed for multimodal microscopic image analysis in order to identify and analyze one cell type under various microscopic conditions. Our purpose was to quantify both total DNA content using propidium iodide (PI) stain and S-phase fraction using the bromodeoxyuridine (BrdUrd) incorporation technique in cell population subsets. The model chosen was plasma cells in bone marrow triply labelled with fluorescein isothiocyanate (FITC) for intracytoplasmic immunoglobulins, with amino-methylcoumarin-acetate (AMCA) for BrdUrd, and with PI for DNA. Image analysis included three phases. First, plasma cells were recognized on FITC images, and the centroid positions were stored. Second, plasma cell nuclei were geodesically reconstructed from these stored positions using PI images in which DNA content was measured, and the nuclear mask outlines were stored. Third, BrdUrd incorporation level of plasma cells was measured on AMCA images inside PI nuclei masks and stored. Image DNA vs. BrdUrd scatterplots were obtained for cells selected according to the expression of intracytoplasmic immunoglobulin. Thus, both ploidy and proliferation could be independently evaluated on a subset of the cellular population.
Animals↗
Cdk1 is a marker of proliferation in human lymphoid cells.
To better understand the relationship between the proliferation of human lymphoid cells and the expression of cdk1, a catalytic subunit of the histone H1 kinase (H1K), we examined its mRNA and protein content in 3 B-cell lines: Ramos, Reh-6 and IARC 963. Cells were elutriated according to their position in the cell cycle. Cell fractions were analyzed for cdk1 mRNA and protein cellular content by Northern blot and immunoblot, respectively, as well as for H1K activity. Both mRNA and protein amounts and H1K activity varied according to cell cycle phase, the lowest values being observed in G1-enriched fractions. For comparison, elutriated fractions were also tested for the expression of cdk2 and cdk4 proteins. Both showed some variations among fractions, but they were less clear than those of cdk1. We also tested 29 samples of lymphoid neoplastic and non-neoplastic tissues for proliferative activity (percentage of S and G2/M cells estimated by flow cytometry) and expression of cdk1, cdk2 and cdk4 proteins. We found a significant correlation between the percentage of cells in S or S + G2/M phases and cdk1 protein content but not cdk2 or cdk4 content. We conclude that cdk1 expression in human lymphoid cells varies during the cell cycle at both mRNA and protein levels.
Biomarkers↗
Expression of cell cycle regulatory proteins in chronic lymphocytic leukemias. Comparison with non-Hodgkin's lymphomas and non-neoplastic lymphoid tissue.
The expression of certain cell cycle regulatory proteins: cdk1, cdk2, cdk4, cyclin A, cyclin B, cyclin E, Bcl2 and PCNA was examined in peripheral blood lymphocytes (PBL) from 25 cases of chronic lymphocytic leukemias (CLL) in order to analyze a possible cell cycle involvement of CLL lymphocytes. For comparison, we also studied the expression of these proteins in: 23 samples of non-Hodgkin's lymphoma (NHL) tissue of different histological types, 10 samples of non-neoplastic lymphoid tissue (NLT), non-stimulated PBL (NS-PBL) and PHA-stimulated PBL (PHA-PBL) from three healthy donors. Samples were lysed and proteins were resolved on polyacrylamide gel followed by Western blot. The expression of cdk4 and cyclin E, both known to act in early cell cycle stage, was approximately on the same level in all groups of lymphoid pathology examined. In particular, we found that that 19 out of 24 CLL cases were cyclin E positive and all but one were cdk4 positive, ie they expressed these markers over twice the level of non-stimulated healthy PBL. The cdk1 expression was above the level seen in NS-PBL in 14 (56%) cases, but the average expression was significantly lower than in the other tissues examined, including low-grade lymphomas. Cdk2 expression was comparable in CLL and in low malignancy grade NHL, but weaker than in other NHL and in NLT. Cyclins A and B, normally observed in advanced cell cycle phases, were not seen in any CLL case. The presence of cdk4 and cyclin E in the blood cells of the majority of CLL cases studied, as well as cdk1 and cdk2 in some cases, indicate that the CLL cells are not quiescent, but are blocked in an early stage of the G1 cell cycle phase, and/or that the expression of these proteins is pathologically deregulated.
Adult↗
Quantitative immunochemistry of endothelial cells in cutaneous tissue.
In vivo endothelial cell exploration in human pathology has been limited by the lack of a suitable procedure for quantification of endothelial molecule expression. The objective of the present study was the development of a precise, simple and rapid image analysis method to quantify dermis endothelial cells and associated molecules. Cutaneous tissue sections were labelled by immunoperoxidase procedure using a monoclonal anti-von Willebrand factor (vWF) antibody, which identifies endothelial cells. The image analysis programme was tested to quantify final reaction deposit (DAB) with reference to different anti-vWF antibody concentrations. The entire biopsy section was acquired, field by field. Analysis was restricted to three dermis zones geodesically defined from epidermis. Optical density and area measurements were performed in whole area and in positive areas. This work demonstrated the feasibility of endothelial cell study assisted by image analysis. Surface labelled by anti-vWF antibody at saturating concentration may be considered as an endothelial reference surface, and thus may be useful for future quantification of other endothelial molecules.
Antibodies, Monoclonal↗
Choice of fixation and denaturation for the triple labelling of intra-cytoplasmic antigen, bromodeoxyuridine and DNA. Application to bone marrow plasma cells.
A triple staining method of intra-cytoplasmic antigen, bromodeoxyuridine (BrdU), and DNA for fluorescence image analysis is described. Several kinds of fixation and DNA denaturation methods were tested to obtain a technique suitable for heterogeneous tissues. The model chosen was the analysis of plasma cells in bone marrow. The fluorochromes used were fluorescein isothiocyanate (FITC) for intra-cytoplasmic antigens (light chain immunoglobulins), aminomethylcoumarin acetic acid (AMCA) for BrdU, and propidium iodide (PI) for DNA. The quality of the staining was analysed according to: (1) cell morphology with a good preservation of the chromatin structure, (2) intensity of light chains and of BrdU labelling, and (3) the quality of DNA staining judged from a DNA histogram. For most of the analysed tissues, fixation with methanol followed by 0.5% paraformaldehyde and denaturation by an NaOH concentration adapted to the tissue gave good results. However, in our model fixation by methanol, followed by methanol/acetic acid and denaturation of DNA by 0.03 N NaOH was the solely satisfactory technique. A good correlation (P < 0.001) was found with the plasma cell BrdU labelling index obtained with our reference immuno-enzymatic technique. Quantification of DNA content showed a satisfactory G1 peak coefficient of variation (CV) in diploid cells and a 4C to 2C ratio equal to 2. With this technique, the nuclear and cytoplasmic structures of both myeloid cells and plasma cells were well preserved, while their sensitivity to DNA denaturation was quite different.
Antigens↗
Fluorescence image analysis of plasma cells in monoclonal gammopathies.
The DNA content of plasma cells was estimated by fluorescence image analysis in monoclonal gammopathies. Image analysis provides the possibility of measuring the bone marrow plasma cell population separately from the other myeloid cells. Plasma cells were labeled, identified and then relocated for propidium iodide intensity measurements. Patients with DNA malignancy grade > 0.20 or with DNA index > 1.15 had a poorer survival rate than did patients with a low DNA malignancy grade or DNA index. DNA malignancy grade is based on the variance of DNA values around the normal diploid peak, as proposed by Böcking. Of all the biologic and clinical parameters investigated, the serum beta 2 microglobulin level was the most important prognostic factor for survival in the multivariate regression analysis, and the DNA entropy index provided the most significant additional information.
Aged↗
Applications of laser scanning confocal microscopy to the observation of human oocytes and embryos.
Laser scanning confocal microscopy (LSCM) is a powerful and useful tool in developmental biology. The diverse applications of LSCM in biomedical field has led to advances in the microscopes themselves and the synthesis of novel specific probes for the observation of biological structures and the hypothesis of physiological process. LSCM was used to visualize the cellular actin cortex together with the chromatin in human arrested preimplantation embryos and in unfertilized oocytes obtained by in vitro fertilization. In embryos, we observed a majority of multinucleated blastomeres and some with fragmented nuclei or anucleated. LSCM also showed nuclei displaying the chromatin condensation and fragmentation patterns suggesting the phenomenon of apoptosis. Moreover, in arrested fragmented embryos of poor morphology, unequal sized blastomeres often showed cellular fragments without nuclei, compatible with aspects of apoptotic bodies.
Actins↗