Search PubMed⌕ Search

Biomedical subjects

J Bonnet

Publications and source records attributed to J Bonnet.

At least 37 records · Page 2Linked to original sources
PubMed2000

Freeze-drying allows double nonradioactive ISH and antigenic labeling.

Because tissue freeze-drying is an excellent way to preserve antigenic conformation, we have tested the feasibility of this technique to reveal nonradioactive in situ hybridization (ISH) of tissue mRNA. We have compared mRNA detection after different methods of tissue preservation, freeze-drying, cryosectioning, and formaldehyde or methanol fixation. Our results show that nonradioactive ISH is more sensitive for tissues preserved by freeze-drying than for other tissue preparations. We have demonstrated that freeze-drying allows combination of ISH and immunohistochemistry for simultaneous detection of mRNA and antigen because with this technique of tissue preservation ISH does not affect the sensitivity or the amount of the detected antigens. This work underscores the fact that tissue freeze-drying is an easy, convenient, and reliable technique for both ISH and immunohistochemistry and achieves excellent structural conditions for nonradioactive detection.

Animals↗
PubMed1999

Trembler as a mouse model of CMT1A?

The Trembler mouse suffers from a dominantly inherited autosomal mutation that results in an abnormal myelination of the peripheral nervous system. Biochemical studies have shown that dysmyelination is the primary event, demyelination being a late-occurring process. The expression of myelin protein genes has been studied. The steady-state levels for PMP22 mRNA represent 10 and 5% of normal values in the nerves of heterozygous and homozygous Trembler, respectively. This is due to a reduced expression of the specific transcript driven by the promoter 1 of the PMP22 gene. Collective results indicate that Trembler dysmyelination is not necessarily the consequence of a large accumulation of the mutated PMP22 protein. Moreover, it appears that the situation in the Trembler is different from that encountered in most CMT1A patients, where an increased PMP22 gene dosage is responsible for the disease. Therefore, the Trembler mutant is perhaps not an ideal model for this human neuropathy.

Animals↗
PubMed1999

IL-2-dependent expression of genes involved in cytoskeleton organization, oncogene regulation, and transcriptional control.

IL-2 induces growth, differentiation, and/or apoptosis of lymphoid cells. To study further the molecular basis of IL-2 function, we used a cDNA subtraction approach involving a cell line grown in IL-2 or IL-4. From the corresponding library, 66 nonredundant sequences were characterized; 16 of them encode identified proteins. The kinetics of in vitro expression of 8 selected sequences, the functions of which could be associated with IL-2-induced T cell activation/differentiation, was investigated using an IL-2-dependent T cell line. IL-2 increased the expression of cytoskeleton proteins (alpha-tubulin), oncogene-regulating proteins (CCCTC-binding factor, Jun inhibitor factor-1), and transcription factors (E2F-4, cyclic AMP-responsive element-binding protein, zhx-1). IL-2 also regulated the expression of genes coding for multifunctional proteins, e.g., beta-catenin and nucleolin. These results were verified using Con A-induced T cell blasts stimulated or not by IL-2. The in vivo expression of four of these genes was also analyzed in spleen and lymph node cells of IL-2-deficient and MRL/lpr mice, which both have high numbers of activated cells, but the latter have intact IL-2 expression. The expression of beta-catenin, CCCTC-binding factor, Jun inhibitor factor-1, and nucleolin was significantly higher in MRL/lpr animals. A similar analysis of thymocytes from IL-2-/- and IL-2+/- mice demonstrated the same expression patterns of the 4 sequences in these strains. The expression of the IL-2-induced genes described herein is similar to the regulatory pattern of IL-2R alpha. Taken together, our data provide additional evidence for the pleiotropic action of IL-2 in the periphery and IL-2 independence of molecular processes involved in thymocyte differentiation.

Animals↗
PubMed1999

Detection of cell and tissue surface antigens using up-converting phosphors: a new reporter technology.

A novel luminescent reporter for the sensitive detection of antigens in tissue sections or on cell membranes is described. It consists of submicron-size phosphor crystals (0.2-0.4 microm), which are surface labeled with avidin or antibodies and capable of binding specifically to antigens on intact cells or in tissue sections. These phosphor reporters exhibit two-photon, anti-Stokes luminescence by up-converting infrared to visible light and are named Up-converting Phosphor Technology (UPT). They typically consist of yttriumoxysulfides doped with two different lanthanides exhibiting photostable, strong emission in the visible (blue, green, and red) upon excitation in the infrared. This report describes the conjugation of phosphor particles to NeutrAvidin with the subsequent use of this conjugate in a model system consisting of prostate-specific antigen in tissue sections and the CD4 membrane antigen on human lymphocytes. An epi-illumination fluorescence microscope was adapted to provide near-IR excitation using a xenon lamp for visualization of the visible emission. Advantages of UPT are (i) permanent, strong, anti-Stokes emission of discrete wavelengths; (ii) unmatched contrast in biological specimens due to the absence of autofluorescence upon excitation with IR light; (iii) simultaneous detection of multiple target analytes; and (iv) low-cost microscope modifications. The new methodology has not only high potential value in diagnostic pathology as described here, but may offer advantages for the detection of proteins or nucleic acids when applied to molecular biology, genomic research, virology, and microbiology.

Animals↗
PubMed1999

Vascular cell adhesion molecule-1 gene expression during human smooth muscle cell differentiation is independent of NF-kappaB activation.

Vascular cell adhesion molecule-1 (VCAM-1) gene expression in cytokine-activated cells depends on two kappaB elements. Since VCAM-1 expression appears developmentally regulated and cytokine-inducible in smooth muscle cells (SMCs), we have studied the role of NF-kappaB in differentiated SMC VCAM-1 expression. Confluent SMCs were cultured either in a serum-free medium in order to induce differentiation, or in medium with serum, stimulated or not by tumor necrosis factor alpha (TNF-alpha). The expression of smooth muscle myosin heavy chain, a SMC marker, and VCAM-1 was induced concomitantly in serum-free medium, whereas only VCAM-1 expression was induced by cytokine-treatment. We showed that the p50 and p65 subunits of NF-kappaB were localized in the cytoplasm of differentiating SMCs, whereas they were translocated into the nucleus of TNF-alpha-activated SMCs. Electrophoretic mobility shift assays with VCAM-1 gene kappaB elements failed to detect any induction of DNA-protein complex with nuclear extracts of differentiating SMCs in contrast to the cytokine-activated SMC nuclear extracts. Furthermore, VCAM-1 mRNA induction was inhibited in TNF-alpha-stimulated SMCs, but not in differentiating SMCs, by pyrrolidine dithiocarbamate, an inhibitor of NF-kappaB protein activation. Taken together, these findings suggest that in contrast to TNF-alpha activation, NF-kappaB is not involved in VCAM-1 gene expression during SMC differentiation.

Base Sequence↗
PubMed1999

Immunohistological and ultrastructural analysis of the intimal thickening in coarctation of human aorta.

OBJECTIVES: The histological nature and characteristics of aortic coarctation are not clearly defined, the aim of this study is to analyse intimal thickening in aortic coarctation. METHODS: In order to characterize the components of intimal thickening in coarctation, narrowed segments of aorta obtained after surgery from ten children were examined immunocytochemically and by electron microscopy. RESULTS: Histological analysis of aortic coarctation demonstrated a widened subendothelial region with separation of endothelial cells from the internal elastic lamina. Masson's trichrome staining showed a marked increase in extracellular matrix and cell numbers in the intimal thickening compared with normal aorta. Cellular component analysis demonstrated invagination of the intima by smooth muscle actin-positive cells, with a fragmentation of the internal elastic lamina. No proliferating smooth muscle and inflammatory cells were identified in the intima. In order to characterize the smooth muscle cell phenotypes, various smooth muscle cell markers were sought using specific monoclonal antibodies: alpha-smooth muscle actin, smooth muscle-myosin heavy chain, heavy caldesmon, desmin. In moderate coarcted aorta, at least two distinct smooth muscle phenotypes were identified. In the juxtamedial part of the intima smooth muscle, cells were differentiated and expressed all smooth muscle markers; in the subendothelial part of the intimal thickening, the majority of smooth muscle cells expressed only alpha-smooth muscle actin and appeared dedifferentiated. In regions of marked stenosis, a strong expression of smooth muscle-myosin heavy chain, and heavy caldesmon in the intimal thickening pointed to the presence of redifferentiated smooth muscle cells, not still expressing desmin. Electron microscopic examination also revealed a variety of smooth muscle cell phenotypes in the intimal thickening. In the superficial layer, smooth muscle cells appeared to be in the synthetic state, while in the deeper part, both synthetic and contractile components were identified. CONCLUSIONS: These observations indicated that human coarctation was characterized by intimal recruitment of non-proliferating smooth muscle cells with dedifferentiated phenotype. However, the presence of smooth muscle cells with an intermediate phenotype in the narrowest part of the coarctation suggest that the redifferentiation process could participate in the pathogenesis of aortic coarctation.

Actins↗
PubMed1999

Familial dilated cardiomyopathy: clinical features in French families.

UNLABELLED: The aims of the study were to analyze the clinical features, the penetrance and the mode of inheritance of 13 French families with dilated cardiomyopathy using diagnostic criteria recently established by a European collaboration. METHODS: Screening consisted of physical examination, ECG and Echo of all the probands first degree relatives (n = 118). Using major Echo criteria [ejection fraction (EF) < 45% or FS < 25% and left ventricular diameter (LVD) > 117% of the predictive value], or combined minor Echo/ECG criteria, relatives were classified as affected, unknown or healthy. RESULTS: (1) Adult affected relatives (n = 31) were identified with major Echo criteria in 74% of cases, and with combined minor Echo/ECG criteria in 26% of cases. (2) In the unknown relatives (n = 21), the most common abnormality was an isolated left ventricular dilation (67%). (3) Mode of inheritance was autosomal dominant (AD) in 11 families and possibly autosomal recessive in two. (4) In AD families, the penetrance was incomplete in adults (72%), age-related (O.R.: 1.3 per 10 years; 95% CI 1.03-1.56) and sex-related [greater in men (87%) than in women (61%), actuarial survival curve: P<0.002]. (5) Mortality related to end stage heart failure was 2.2 times as high as mortality related to sudden death (11% vs. 5%). CONCLUSIONS: (1) In the absence of a specific phenotype of FDC, the characterization of relatives appears more accurate when minor criteria were added. (2) Since high mortality (16%) and incomplete penetrance frequently give rise to small nuclei of clinically affected and alive relatives per family, the accurate model of penetrance that we proposed might be helpful in the future to enhance the statistical power of linkage analysis in this disease.

Adult↗
PubMed1999

Evidence of early subchondral bone changes in the meniscectomized guinea pig. A densitometric study using dual-energy X-ray absorptiometry subregional analysis.

OBJECTIVE: Subchondral bone changes are thought to be an important aetiological element in the pathogenesis of osteoarthritis (OA). To confirm this hypothesis in the meniscectomized (MNX) guinea pig model, bone densitometry was performed in the subchondral bone of the distal femur. METHODS: MNX and sham-operated (SH) guinea pigs were studied 1 and 3 months after partial meniscectomy at the medial side of the left knee. Bone mineral density was measured at the lateral (BMD-L) and medial (BMD-M) sides of the distal femur using dual-energy X-ray absorptiometry (DXA). BMD-M was then compared to the bone volume evaluated by histomorphometry at the medial epiphyseal part of the proximal tibia (BV-M). RESULTS: One month after operation, in MNX animals left femur BMD-M was significantly lower than in the contralateral femur (-9%, P< 0.01) and than in the left femur of SH (-11%, P< 0.01). By contrast, 3 months after meniscectomy BMD-M was higher in the femur than in the contralateral femur (+4%, P< 0.05); BV-M tended to be higher on the left than on the right side (+4%, P< 0.06), and was significantly correlated with BMD-M at the 2 grouped time points: r=0.74 (P< 0.001). CONCLUSIONS: These data emphasize the usefulness of DXA as a simple tool to assess subchondral bone changes at the OA-affected side of the femur and reveal typical variations of bone metabolism in the initiation of OA pathology in the MNX guinea pig: early bone loss at the subchondral level followed by increased bone density.

Absorptiometry, Photon↗
PubMed1999

MRI dosimetry: a fast quantitative MRI method to determine 3D absorbed dose distributions.

RATIONALE AND OBJECTIVES: Magnetic resonance imaging (MRI) techniques seem to be very promising for 3D dosimetry studies, but long imaging acquisition time limits their use. A new fast T1 mapping protocol, easy to implement on a conventional MR imager, has been used to determine dose distributions on Fricke gels. METHODS: The method has been tested on manganese chloride (MnCl2) doped ferrous gelatin gels. The T1 measuring times range from 1 minute 40 seconds to 3 minutes 30 seconds for a 256x256 matrix image. RESULTS: The two- and three-dimensional profiles agree with those obtained with conventional dosimetry techniques (ion chambers). The precision and the spatial resolution principally depend on the signal-to-noise ratio of the used imaging RF coil. For example, for a surface coil, the accuracy is about 2.5% with a 1.56 mm spatial resolution. CONCLUSION: These preliminary results support the feasibility of the proposed technique for accurate MRI dosimetry studies and also have potential for various clinical quantitative MRI applications.

Gels↗
PubMed1999

Phosphorescent platinum/palladium coproporphyrins for time-resolved luminescence microscopy.

Streptavidin and antibodies were labeled with phosphorescent platinum and palladium coproporphyrin. The optimal conjugates were selected on the basis of spectroscopic analysis (molar extinction coefficient, quantum yield, lifetime) and using ELISA assays to determine the retention of biological activity and immunospecificity. They were subsequently tested for the detection of prostate-specific antigen, glucagon, human androgen receptor, p53, and glutathione transferase in strongly autofluorescent tissues. Furthermore, platinum and palladium coproporphyrin-labeled dUTPs were synthesized for the enzymatic labeling of DNA probes. Porphyrin-labeled DNA probes and porphyrin-labeled streptavidin conjugates were evaluated for DNA in situ hybridization on metaphase spreads, using direct and indirect methods, respectively. The developed in situ detection technology is shown to be applicable not only in mammals but also in plants. A modular- based time-resolved microscope was constructed and used for the evaluation of porphyrin-stained samples. The time-resolved module was found suitable for detection of antigens and DNA targets in an autofluorescent environment. Higher image contrasts were generally obtained in comparison with conventional detection systems (e.g., fourfold improvement in detection of glutathione transferase).

Acyltransferases↗
PubMed1999

The farnesyltransferase inhibitor FTI-277 suppresses the 24-kDa FGF2-induced radioresistance in HeLa cells expressing wild-type RAS.

In this paper, we describe the effect of the inhibitor of farnesyltransferase (FTI-277) on radioresistance induced by the 24-kDa isoform of FGF2 in human cells expressing wild-type RAS. Treatment with FTI-277 (20 microM) for 48 h prior to irradiation led to a significant decrease in survival of radioresistant cells expressing the 24-kDa isoform (HeLa 3A) but had no effect on the survival of control cells (HeLa PINA). The radiosensitizing effect of FTI-277 is accompanied by a stimulation of postmitotic cell death in HeLa 3A cells and by a reduction in G(2)/M-phase arrest in both cell types. These results clearly demonstrate that at least one farnesylated protein is involved in the regulation of the radioresistance induced by the 24-kDa isoform of FGF2. Furthermore, the radiation-induced G(2)/M-phase arrest is also under the control of farnesylated protein. This work also demonstrates that FTase inhibitors may be effective radiosensitizers of certain human tumors with wild-type RAS.

Alkyl and Aryl Transferases↗
PubMed1999

[Coronary atherectomy. An essential tool for specific indications].

Technological advances in the manufacturing of stents have extended the indications of angioplasty and considerably reduced the immediate complications, death and myocardial infarction. Nevertheless, intra-stent restenosis remains a problem and some complex lesions are still inaccessible. Atherectomy has not been shown to be effective in limiting restenosis but it has a primordial role in the treatment of lesions of bifurcation and could improve long-term results as a complement of angioplasty and stenting. Rotational atherectomy is still useful, even essential, for lesions which cannot be passed with the balloon and for calcified plaques of atheroma. A possible new indication may be the treatment of intra-stent stenosis. The indications of directional atherectomy are more limited, mainly non-calcified ostial stenosis and of bifurcations of large arteries. The association with stenting has given encouraging results which require confirmation. These techniques have a place in the in the angioplasty physician's arsenal even though they are reserved for specific anatomical situations.

Angioplasty, Balloon, Coronary↗
PubMed1998

Purification of guinea pig YKL40 and modulation of its secretion by cultured articular chondrocytes.

The aim of this study was to purify, characterize, and study the regulation at the chondrocyte level of the guinea pig (gp) homologue of human (R) YKL40, a putative marker of arthritic disorders. Studying YKL40 in guinea pigs is of particular interest, as age-related osteoarthritis develops in this species spontaneously. Both N-terminal sequencing and total amino acid composition of gpYKL40 purified from the secretion medium of cultured articular chondrocytes indicate a high degree of identity with hYKL40. gpYKL40 was found to contain complex N-linked carbohydrate, as demonstrated by N-glycosidase F and endoglycosidase F digestion. Isoelectric focusing demonstrated the presence of a major band at pI 6.7. The secretion of gpYKL40 by confluent articular chondrocytes in the extracellular medium was studied by immunoblotting. gpYKL40 was released by chondrocytes continuously over a 7 day period and did not appear to be degraded by proteinases, as its signal intensity in cell-free medium at 37 degrees C did not decrease with time. Thus, gpYKL40 displays high stability and accumulates in extracellular medium without reaching a steady-state level. Among the main factors known to regulate cartilage metabolism, IL-1beta, TNF-alpha, bFGF, or 1,25(OH)2D3 did not alter the basal level of gpYKL40, and retinoic acid had a slight inhibitory effect; TGF-beta and IGF-I and -II dose-dependently and inversely modulated this basal level. TGF-beta at 5 ng/ml decreased extracellular gpYKL40 2.9-fold, whereas IGF-I and IGF-II at 50 ng/ml increased extracellular gpYKL40 3.6- and 3.4-fold, respectively. The present biochemical and biological findings give new insights for studying the function of YKL40 in cartilage.

Adipokines↗
PubMed1998

Lack of ceramide generation in TF-1 human myeloid leukemic cells resistant to ionizing radiation.

The mechanism(s) by which ionizing radiation (IR) induces cell death is of fundamental importance in understanding cell sensitivity and resistance. Here we evaluated the response to IR of two subclones of the autonomous human erythromyeloblastic cell line TF-1: TF-1-34 (which expresses CD34) and TF-1-33 (which lacks CD34). In clonogenic assays, TF-1-34 cells were found to be relatively less sensitive to IR compared to TF-1-33 cells based on the D0 determination (3.01 vs 1.56 Gy). Furthermore, after IR at 12 Gy, TF-1-33 cell viability decreased by approximately 50% within 24 h, whereas TF-1-34 cell growth was unaffected during this time. Gradual loss of TF-1-34 cell viability was observed only after 48 h. Morphological and molecular analysis revealed that TF-1-33 cells died of apoptosis, and TF-1-34 cells of delayed reproductive cell death. While IR produced comparable amounts of DNA double strand breaks (DSB) in both cell lines, TF-1-34 retained DSB much longer than TF-1-33 suggesting that radioresistance and the defective apoptotic response of TF-1-34 cells was not related to a higher DNA repair capacity. However, the lack of an apoptotic response in TF-1-34 was correlated to the absence of a sphingomyelin (SM)-ceramide (CER) signaling pathway. Indeed, IR triggered in TF-1-33 cells but not in TF-1-34, SM hydrolysis (25% at 12 Gy) and CER generation (>50%) through the activation of neutral but not acid sphingomyelinase. Synthetic cell permeate CER induced apoptosis in both TF-1-33 and TF-1-34 cells. This study indicates that alterations of the SM-CER signaling pathway can significantly influence the cell death process as well as the survival of acute myeloid leukemia cells after IR exposure.

Antimetabolites↗
PubMed1998

Granulocytic sarcoma of the jejunum: a rare cause of small bowel obstruction.

We report the case of a 40-yr-old man presenting with symptoms of small bowel obstruction. Small bowel x-rays revealed a stricture of the mid-jejunum. Push enteroscopy found a polypoid mass at 1 meter of the ligament of Treitz. Histopathological examination of the biopsy and surgical specimens showed a diffuse infiltrate of the mucosa made of medium to large cells, which were stained on immunohistochemistry by the leucocyte marker CD45 and the histiocyte/monocyte marker CD68 but were negative for the B and T cell markers. Cytological examination of the ascitic fluid revealed many myelobasts with cytoplasmic Auer rods and positive myeloperoxidase staining. There was no evidence of blood or bone marrow involvement suggestive of acute leukemia or myeloproliferative disorders. These findings were consistent with the diagnosis of preleukemic granulocytic sarcoma (or chloroma). Chemotherapy led to complete remission, but 21 months later the patient developed an acute myeloid leukemia. He died from aspergillus pneumonitis, 10 months after bone marrow allograft. Preleukemic granulocytic sarcoma of the small bowel is a rare condition and its diagnosis is usually not easy, requiring histochemical or immunohistochemical studies. Most cases have progressed to acute myeloid leukemia.

Adult↗