Long-term toxicity of intravenous mercury injection.
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Adult↗
Biomedical subjects
A Bernard
Publications and source records attributed to A Bernard.
Selective isolation of transiently transfected cells from a mammalian cell population with vectors expressing a membrane anchored single-chain antibody.
We present here a novel technology for the rapid selection of transiently transfected cells from total populations in culture. This system utilizes recombinant antibody technology to produce a 'molecular hook' by displaying a hapten-binding single-chain antibody (sFv) on the surface of transfected cells. Mammalian cell lines from several origins were transiently transfected with a plasmid (pHook-1) that encodes an sFv fused with a transmembrane anchor and found to express and display the functional hapten-binding sFv on their membranes. Transfected cells were selected from total populations in culture by virtue of their ability to bind to hapten-coated magnetic beads. Some cell lines were able to display sFv sufficient for selection as early as 2 h post-transfection. SK-BR-3 human breast carcinoma cells were co-transfected with pHook-1 and pCR31acZ (expresses beta-galactosidase), selected, and assayed for beta-galactosidase activity. The positive correlation between sFv and beta-galactosidase expression in these cells (95% of selected cells also expressed beta-galactosidase activity) suggests that pHook-1 will be useful in isolating cells co-expressing an exogenous gene of interest. Another vector was constructed in which a gene of interest may be expressed from the same plasmid as the sFv 'hook'. This construct (pHook-2) allows the selection of a homogenous population of cells expressing exogenous genes without co-transfection or the generation of stable transfectants. In experiments where the lacZ gene was co-expressed with the sFv 'hook' from this single plasmid, 100% of 293 human kidney cells and 100% of SK-BR-3 cells selected with antigen-coated magnetic beads stained positively for beta-galactosidase activity. We propose that this system will be a valuable tool for studying the acute and chronic effects of the expression of a variety of wild type and mutant proteins.
Amino Acid Sequence↗
Localization and regulation of the putative membrane fatty-acid transporter (FAT) in the small intestine. Comparison with fatty acid-binding proteins (FABP).
The expression of the putative membrane fatty-acid transporter (FAT) was investigated in the small intestine. The FAT mRNA level was higher in the jejunum than in the duodenum and was lower in the ileum, as observed for cytosolic fatty-acid-binding proteins (FABP) expressed in this tissue. No FAT transcript was found in the stomach or colon. FAT mRNA was constitutively expressed in the epithelial cells located in the upper two thirds of villi, while it was undetectable in the crypt cells and submucosal cells. In jejunal mucosa, immunochemical studies showed that FAT protein was limited to the brush border of enterocytes. No fluorescence was found in the goblet cells. To determine whether FAT responded to changes in fat intake, as reported for FABP, the effect of two high-fat diets, which essentially contained either medium-chain fatty acids or long-chain fatty acids (sunflower-oil diet), was investigated. The sunflower-oil diet greatly increased FAT mRNA abundance throughout the small intestine. In contrast, a weak effect of medium-chain fatty acids was observed only in the jejunum. As found for FABP expression, treatment with the hypolipidemic drug bezafibrate affected FAT expression. These data demonstrate that FAT and FABP are co-expressed in enterocytes, as has been shown in adipocytes, myocytes and mammary cells. The data suggest that these membrane and cytosolic proteins might have complementary functions during dietary-fat absorption.
Animals↗
CD2-induced apoptosis in activated human peripheral T cells: a Fas-independent pathway that requires early protein tyrosine phosphorylation.
Short-term activated peripheral T lymphocytes are susceptible to apoptotic cell death triggered by CD2 mAbs. The aim of this study was to examine whether the CD2-mediated pathway of apoptosis is linked to the Fas death pathway, as this is the case for CD3/TCR-triggered apoptosis in several models of T cells. Using T lymphocytes from patients harboring Fas gene mutations and displaying a profound defect in Fas signaling of cell death, we show that CD2- (but not CD3-) mediated apoptosis still proceeds normally. In normal activated T cells, CD3-mediated apoptosis is prevented by reagents that block the Fas/Fas-ligand interaction, namely soluble M3 (an antagonistic anti-Fas mAb) and soluble human Fas.Fc, a fusion protein able to bind released Fas-ligand. In contrast, CD2 signaling of apoptosis resists these blocking agents. Neither new protein synthesis nor the activation of calcineurin was required for CD2- and Fas-mediated apoptosis, suggesting that latent cytoplasmic "death" molecules were activated upon stimulation of the cells. In both cases, protein tyrosine kinases were transiently activated, as is exemplified by the autophosphorylation and exokinase activity of p56lck, yielding overlapping yet nonidentical profiles of protein tyrosine phosphorylation. Pretreating the cells with herbimycin A, before the addition of the apoptotic stimuli, almost completely inhibited CD2 transmembrane signaling of apoptosis, but left intact Fas-induced apoptosis. Our data suggest that CD2 is a Fas-independent cell death pathway that might contribute directly to the elimination of T cells expanding during an immune reaction.
Antibodies, Monoclonal↗
Amniotic fluid for screening of lysosomal storage diseases presenting in utero (mainly as non-immune hydrops fetalis).
Seventy amniotic fluids (AF) were sampled because of abnormal ultrasound findings (mainly non-immune hydrops fetalis (54 cases) or of the presence of vacuolated lymphocytes in fetal blood (3 cases)). They were analysed by a procedure involving AF supernatant analysis (glycosaminoglycans, oligosaccharides, free sialic acid and acid hydrolase activities) and biochemical study of cultured AF cells. Ten cases of lysosomal storage diseases (LSD) were diagnosed. The reported procedure allows an orientating screening within 3 days by analysis of 15 ml of third trimester AF supernatant (except for Gaucher and Niemann-Pick diseases). In some cases, the results allow an LSD diagnosis and a medical abortion without waiting for the formal diagnosis (in cultured AF cells that needs 3 more weeks), considering the poor prognosis of these LSD presenting in utero. Furthermore, the formal assessment of the diagnosis in the cultured fetal cells allows accurate genetic counselling for the couple.
Amniotic Fluid↗
Effect of bile on the intestinal bile-acid binding protein (I-BABP) expression. In vitro and in vivo studies.
Enterocytes actively transport bile acids from the ileal lumen to the portal blood. This physiological process greatly contributes to maintaining the bile acid homeostasis. However, little is known about the molecular mechanisms involved in this transport system. The effect of bile on gene expression of the intestinal bile-acid binding protein (I-BABP) expressed in the enterocytes was studied in vivo, using the by-pass method, and in vitro, using organ culture of ileum explants and Caco-2 cell line. The low cytosolic I-BABP concentration and I-BABP mRNA level found in diverted ileum was totally recovered when bile was added in the ileal lumen. Northern blot analysis of the ileal explants revealed a dose-dependent increase in the I-BABP mRNA in the presence of bile. In Caco-2 cells, the I-BABP transcript was dramatically increased in the presence of human bile while it was undetectable in the control cultures. These data offer the first evidence that biliary components regulate the I-BABP gene expressed in the enterocytes.
Animals↗
[Mucinous pancreatic ductal ectasia: mucus secreting tumor of malignant potential].
We report two cases of mucinous pancreatic ductal ectasia including one which progressed to micro-invasive carcinoma. The frequency of this tumor may be under-estimated (24 cases reported in the literature) because of confusion with pseudocysts or mucinous cystadenoma. The diagnosis is made at retrograde cholangiopancreatography. Endosonography is useful for tumors in the head of the pancreas, the predominant localization. At present, there is no test which can distinguish malignant forms from benign mucinous ductal ectasia. Resection of the tumor and the surrounding pancreatitis is the only curative treatment known. Pancreatoduodenectomy may be discussed in elderly patients with a tumor without signs of malignancy.
Adenocarcinoma, Mucinous↗
Interleukin 6 upregulates TNF-alpha-dependent C3-stimulating activity through enhancement of TNF-alpha specific binding on rat liver epithelial cells.
The authors investigated the role of tumour necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6), two major pro-inflammatory cytokines, in the stimulation of the third component of complement (C3) production by rat liver epithelial cells. Though often considered as the most potent inflammatory cytokine, IL-6 alone displayed no activity, whereas TNF-alpha upregulated C3 production in a dose-dependent manner. However, IL-6 was shown to synergistically stimulate C3 production in the presence of TNF-alpha. To account for this interaction, it was postulated that IL-6 modulates the binding of TNF-alpha on liver target cells. That IL-6 increased the binding of TNF-alpha on the surface of the hepatic cells, whereas TNF-alpha alone downregulated its own specific binding capacity is reported. Furthermore, this upregulatory effect of IL-6 was mainly attributable to an increase in the number of plasma membrane TNF-alpha specific receptors, with little change in their affinity. These results suggest that the synergistic IL-6 activity on C3 production may occur, at least partially, through its capacity to upregulate the number of TNF-alpha receptors on the surface of the rat liver epithelial cells.
Animals↗
Intestinal lymph absorption of butter, corn oil, cod liver oil, menhaden oil, and eicosapentaenoic and docosahexaenoic acid ethyl esters in rats.
Adult male rats were surgically given a drainage catheter in the main mesenteric lymph duct. After an overnight fast, five groups of rats received intragastrically, in one bolus, butter, corn oil (CO), cod liver oil (CLO), menhaden oil (MO), or ethyl esters of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids (K80). Intestinal lymph was collected in these conscious animals, each hour during the first 6 h and in a single sample for the next 18 h. The absorption peak appeared earlier after MO and CO than after CLO administration. The quantities of triglycerides recovered during the first 6 h were significantly lower after butter (91 mg) and K80 (54 mg) administration than for the other three oils. No difference was observed between the vegetable oil and the marine oils (CO = 173 mg, CLO = 148 mg, MO = 180 mg). The total triglyceride recovered in 24 h was highest after CLO (410 mg) and lowest with K80 (146 mg). An increase in the weight percentage of some characteristic fatty acids of the lipid mixtures was observed: oleic acid for butter, oleic and linoleic acids for CO, EPA and DHA for CLO, MO, and K80. Chylomicrons were the largest with CO, more numerous and smaller with CLO, and the smallest with K80. Results obtained illustrated the relation between gastrointestinal hydrolysis, enterocyte biochemical events, and lymph triglyceride absorption profiles as related to the composition and distribution of triglyceride fatty acids.
Animals↗
Resection of pulmonary nodules using video-assisted thoracic surgery. The Thorax Group.
BACKGROUND: The aim of this study was to assess the experience with video-assisted thoracic surgery for the resection of pulmonary nodules. METHODS: This voluntary registry (20 centers) included 388 patients with either benign (n = 171) or malignant (n = 217) pulmonary nodules. Pulmonary nodules were located using computed tomography scan-guided injection of methylene blue (59 patients) and hook wire technique (17 patients). RESULTS: Video-assisted thoracic surgery was converted into thoracotomy in 67 patients (17%) because of technical-emergency in 4, inability to complete resection in 33, and the need to perform lobectomy for cancer through thoracotomy in 30. In other patients, video-assisted thoracic surgery allowed wedge resection in 300 cases and lobectomy in 21 cases. No intraoperative and two postoperative deaths (0.56%) occurred. The complication rate was 8% (n = 31). Mean durations of chest tube placement and hospital stay were 3.3 days (range, 1 to 20 days) and 6 days (range, 1 to 25 days), respectively. Video-assisted thoracic surgery was judged by the surgeon to be a diagnostic procedure 226 times (58%) and a therapeutic procedure 162 times (42%). CONCLUSION: Video-assisted thoracic surgery appears to be safe and remains mainly a diagnostic procedure for malignant tumors.
Adolescent↗
Selective induction of cytokines in mouse brain infected with canine distemper virus: structural, cellular and temporal expression.
We have previously shown that, in experimentally inoculated mice, canine distemper virus (CDV), a neurotropic virus, selectively infects certain brain structures (hypothalamus, hippocampus, monoaminergic nuclei, etc). Here we demonstrate that tumor necrosis factor (TNF)-alpha, interleukin (IL)-1 beta and IL-6 transcripts are selectively expressed in these CDV-targeted structures, except in the dentate gyrus, where cytokines are induced without prior CDV replication. The time-course of TNF-alpha expression vs. viral replication in the hypothalamus was different from that in hippocampus. In addition, we show that a substantial number of neurons express TNF-alpha and IL-6. These findings provide new insights into the possible participation of cytokines in the neurological disorders triggered by CDV infection.
Animals↗
Cytokines are increased in the rat hippocampus after serotonergic neuron degeneration and upregulate the expression of GDH, an enzyme involved in glutamate detoxification.
The degeneration of serotonergic neurons increases the expression of glutamate dehydrogenase (GDH) in hippocampal astrocytes. This process was demonstrated to be independent of the serotonin level. At the same time, upregulation of tumor necrosis factor (TNF) alpha and interleukin (IL)-1 alpha mRNA were observed, whereas levels of transforming growth factor (TGF) beta 1 mRNA remained unchanged. The level of GDH mRNA was increased in primary cultures of hippocampal astrocytes treated with TNF alpha and IL-1 alpha suggesting that these cytokines act on the GDH metabolism. TNF alpha and IL-1 alpha induced an increase in GDH promoter activity in C8S (an astrocytic cell line) transfected with constructs containing 5' flanking genomic sequences of GDH driving the expression of a reporter gene. These observations suggest that cytokines may be signals that upregulate the astrocytic GDH expression in response to the degeneration of serotonergic terminals in the hippocampus.
Animals↗
Extracellular matrix-remodeling metalloproteinases and infection of the central nervous system with retrovirus human T-lymphotropic virus type I (HTLV-I).
Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are involved in physiological processes and contribute to the phenotype of several pathological conditions associated with uncontrolled tissue degradation. In the central nervous system (CNS), MMPs are thought to play a role in cell migration and synaptic plasticity. We have investigated the expression, regulation and possible role of MMPs and TIMPs during infection of glial cells with human T-lymphotropic virus type I (HTLV-I), the causative agent of a progressive chronic myelopathy, TSP/HAM. The major alteration consists in a high increase in MMP-9 secretion and TIMP-2 mRNA expression. Cytokines TNF alpha and IL1 alpha, induced in glial cells during HTLV-I infection, promote the upregulation of MMP-9. In addition, cerebrospinal fluid from TSP/HAM patients contain high MMP-9 level. The exact role of dysregulated MMPs/TIMPs in the pathogenesis of TSP/HAM is not known; however, functions of these proteases in physiological processes should provide valuable clues. MMPs can affect the blood-brain barrier and the intercellular connectivity by degrading the extracellular matrix of endothelial and neural cells. They can be involved in autoimmunity by generating preformed specific peptides from myelin components. Finally, they can direct and prolong TNF activity in the CNS by converting its inactive precursor into active molecules.
Central Nervous System↗
[Digestive manifestations in hemolytic uremic syndrome in children].
BACKGROUND: Gastro-intestinal manifestations are relatively frequent during the course of hemolytic uremic syndrome (HUS), some of them requiring special supportive care. This work was aimed at retrospectively studing gastrointestinal manifestations of HUS and determining their place in the prognosis. PATIENTS: Thirty-seven children aged 4 months to 11 years (22 girls and 15 boys) were included in the study. RESULTS: All children but one had gastrointestinal prodromes. During the course of HUS, various manifestations were seen: bloody diarrhea in 32% of patients, ileo-ileal intussusception in 3%, rectal prolapse in 8% and hepatic cytolysis in 38%. Seven patients with bloody diarrhea had a complicated course, lethal in one. Comparison between these seven children and the 30 others revealed some indicators of severe gut involvement: female sex, short duration of gastrointestinal prodromes, hemorrhagic colitis with rectal prolapse, high WBC count, high neutrophils count and less important degree of anemia at admission. Severity of the gastrointestinal lesions was correlated with that of the outcome of the renal disease. CONCLUSION: Gastrointestinal tract is frequently affected in HUS and severe complications can appear, potentially leading to death. Total parenteral nutrition could prevent occurrence of gastrointestinal complications. Severe gastrointestinal lesions are associated with a poor renal outcome.
Child↗
Low molecular weight proteinuria in human immunodeficiency virus-infected patients.
To determine whether human immunodeficiency virus (HIV) infection is associated with incipient tubular or glomerular defects, we determined the urinary excretion of four low molecular weight proteins (LMWP); beta2-microglobulin (U-beta2-m), cystatin C (U-cyst C), Clara cell protein (U-CC16), and retinol-binding protein (U-RBP), the markers of tubular dysfunction, the excretion of albumin (U-Alb), a marker of glomerular defect, and the excretion of N-acetyl-beta-D-glucosaminidase (U-NAG), a marker of structural damage of the proximal tubular epithelium. Their determinants have been assessed by stepwise regression analysis using as possible predictors age, sex, serum-beta2-m (S-beta2-m), CD4 lymphocyte count, or HIV infection stage and therapy. The study involved 76 HIV-infected patients without renal disease, 56 with S-beta2-m < 5 mg/L (Group B1), 20 with S-beta2-m > or = 5 mg/L (Group B2), and 30 HIV-negative controls. Fourteen patients (18.4%) had no abnormal urinary protein loss, and 62 (81.6%) had elevated urinary excretion of at least one protein (Alb, LMWP, or NAG). A single urinary protein was abnormal in 21 patients (U-beta2-m, n = 9; U-RBP, n = 2; U-CC16, n = 4; and U-Alb, n = 6). At least two LMWP were abnormal without increased U-Alb in 23 patients (12 with increased and 11 with normal U-NAG). Ten patients had an increased urinary excretion of at least one LMWP together with U-Alb (5 with increased and 5 with normal U-NAG). An increased urinary excretion of all proteins was observed in the last 8 patients. The average urinary excretion of all proteins (except cyst C) was significantly higher in HIV than in the control group. As expected, U-beta2-m and the prevalence of abnormal U-beta2-m values were higher in the B2 than in the B1 group (P = 0.0001), whereas the average urinary excretion and the prevalence of elevated values of Alb, LMWP (except beta2-m) or NAG were the same in both HIV groups. By stepwise regression analysis, age emerged as a significant determinant of urinary excretion of beta2-m and CC16, whereas male sex was associated with increased U-CC16. S-beta2-m, CD4-lymphocyte count, or HIV infection stage emerged as significant determinants only for U-beta2-m as a consequence of a close correlation between S-beta2-m and either HIV infection stage (r = -0.52, P = 0.0001), or CD4 count (r = -0.45, P = 0.0002). Over 80% of HIV-infected patients without overt renal disease have evidence of glomerular permeability defects or tubular dysfunction, whatever the stage of the disease. U-Alb, RBP, and CC16 appear as the most sensitive and reliable early markers of these abnormalities. Their cause and prognostic value remain to be determined.
AIDS-Associated Nephropathy↗
Partition of oleic acid between the lymph and portal blood in rats having a diverted bile-pancreatic duct.
The present study examines the suggestion that in the absence of adequate bile and pancreatic juice, which support the absorption from the gut of long-chain fatty acids into lymph, the fatty acids are absorbed directly into the portal blood. Oleic acid (18:1) partitioning between lymph and portal blood was investigated in intact and bile- and pancreatic juice-diverted rats. In a first set of experiments, 18:1 absorption from the gut into lymph and blood was studied by continuous recovery of the mesenteric lymph for 6 h and mesenteric portal venous blood for 1 h. In a second set of experiments, esterification processes were investigated by study of the mucosal distribution of labelled lipids and by mono- and diacylglycerol acyltransferase (EC 2.3.1.22 and EC 2.3.1.20 respectively) specific activities. In the bile- and pancreatic juice-diverted rats the absorption of labelled 18:1 into lymph was significantly reduced during the first 3 h of intraluminal infusion of this substrate. In such rats a compensatory absorption of labelled 18:1 into mesenteric portal blood was not observed. At 6 h after micellar lipid-mixture infusion, the overload of lipids both in free form and as triacylglycerols persisting in the mucosa paralleled the lower acyltransferase specific activities observed in bile- and pancreatic juice-diverted rats. These studies demonstrate the absence of a previously proposed compensatory absorption of 18:1 into portal blood when absorption into lymph is impaired by an inadequate supply of bile and pancreatic juice.
Animals↗
Cellular and molecular aspects of fat metabolism in the small intestine.
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Animals↗
Endocytosis of GPI-anchored proteins in human lymphocytes: role of glycolipid-based domains, actin cytoskeleton, and protein kinases.
GPI-anchored surface proteins mediate many important functions, including transport, signal transduction, adhesion, and protection against complement. They cluster into glycolipid-based membrane domains and caveolae, plasmalemmal vesicles involved in the transcytosis and endocytosis of these surface proteins. However, in lymphocytes, neither the characteristic flask shaped caveolae nor caveolin, a transmembrane protein typical of caveolae, have been observed. Here, we show that the GPI-anchored CD59 molecule on Jurkat T cells is internalized after cross-linking, a process inhibited by nystatin, a sterol chelating agent. Clustered CD59 molecules mostly accumulate in non-coated invaginations of the lymphocyte membrane before endocytosis, in marked contrast with the pattern of CD3-TCR internalization. Cytochalasin H blocked CD59 internalization in lymphocytes, but neither CD3 internalization nor transferrin uptake. Confocal microscopy analysis of F-actin distribution within lymphocytes showed that CD59 clusters were associated with patches of polymerized actin. Also, we found that internalization of CD59 was prevented by the protein kinase C inhibitor staurosporine and by the protein kinase A activator forskolin. Thus, in lymphocytes, as in other cell types, glycolipid-based domains provide sites of integration of signaling pathways involved in GPI-anchored protein endocytosis. This process, which is regulated by both protein kinase C and A activity, is tightly controlled by the dynamic organization of actin cytoskeleton, and may be critical for polarized contacts of circulating cells.
Actins↗