Characterization and visualization of [125I] stromal cell-derived factor-1alpha binding to CXCR4 receptors in rat brain and human neuroblastoma cells.

Stromal cell-Derived Factor-1 (SDF-1alpha), binds to the seven-transmembrane G protein-coupled CXCR4 receptor and modulates cell migration, differentiation, and proliferation. CXCR4 has been reported to be expressed in various tissues including brain. Moreover, CXCR4 has recently been shown to be one of the coreceptors for HIV-1 infection which could be implicated in HIV encephalitis. In the present study, the binding properties and autoradiographic distribution of [125I]SDF-1alpha binding to CXCR4 were characterized in the adult rat brain. SDF-1alpha binding and CXCR4 coupling system were also studied in human neuroblastoma cell line SK-N-SH. The binding of [125I]SDF-1alpha on rat brain sections was specific, time-dependent and reversible. The highest densities of CXCR4 were detected in the choroid plexus of the lateral and the dorsal third ventricle. Lower densities of [125I]SDF-1alpha binding sites were observed in various brain regions including cerebral cortex, anterior olfactory nuclei, hippocampal formation, thalamic nuclei, blood vessels and pituitary gland. In the choroid plexus, the IC(50) and K(d) of [125I]SDF-1alpha binding were respectively 0.6 nM and 0. 36 nM. Similar IC(50) values were obtained in other brain structures. A CXCR4 antagonist, bicyclam, competed with SDF-1alpha binding (30% inhibition at 10(-6) M). In SK-N-SH cells, [125I]SDF-1alpha bound to CXCR4 with a K(d) of 5.0 nM and a maximal binding capacity of 460 fmol/mg of protein. SDF-1alpha induced a rapid and transient intracellular calcium increase in SK-N-SH cells. These findings suggest that CXCR4 is highly expressed in some brain structures and have a regulatory role in the nervous system. The significance of this expression in the brain parenchyma and more specifically in the choroid plexus remains to be clarified in the normal as well as in the infected brain.

Activation of receptor gene transcription is required to maintain cell sensitization after agonist exposure. Study on neurotensin receptor.

Neurotensin (NT) acts through specific G protein-coupled receptors to induce effects in the central nervous system and periphery. In this study we have shown that in the human neuroblastoma cell line CHP 212, an NT agonist, JMV 449, induced high affinity neurotensin receptor (NTR) gene activation. 125I-NT binding of cells challenged with JMV 449 rapidly decreased then reappeared and subsequently stabilized at 50% of the control values after 48 h of agonist exposure. These receptors, which reappeared at the cell surface, are as active as those found in control cells as demonstrated by Ca2+ mobilization. Furthermore, the tyrosine hydroxylase (TH) gene, a known NT target gene, remained activated after prolonged NT agonist exposure in this cell line. In the murine neuroblastoma cell line, N1E-115, NT did not stimulate NTR gene activation but induced NTR mRNA destabilization after long term agonist exposure. In this cell line, NT binding dropped to 15% of control values and remained at this value after agonist treatment. The TH expression, which was originally activated upon NT agonist exposure, decreased to control values after prolonged agonist exposure. These observations combined with the data obtained from a complementary study with HT-29 cells (Souazé, F., Rostène, W., and Forgez, P. (1997) J. Biol. Chem. 272, 10087-10094) revealed the crucial role of agonist-induced receptor gene transcription in the maintenance of cell sensitivity. A model for G protein-coupled receptor regulation induced by prolong and intense agonist stimulation is proposed.

Influence of the content in transcription factors on the phenotype of mouse hepatocyte-like cell lines (mhAT).

We have described new well-differentiated mouse hepatocyte-like cell lines (mhAT) derived from transgenic mice expressing simian virus 40 large T antigen under the control of antithrombin III gene promoter (Exp. Cell Res. (1992) 200, 175-185). In an attempt to understand the phenotypic variations of the different cell lines, we analyzed their content in liver-specific transcription factors at the level of both the proteins, by gel shift analysis, and the mRNA, by quantitative reverse-transcription PCR. Moreover, the transactivating ability of endogenous HNF1 alpha and C/EBP alpha was also evaluated by measuring the activity of transfected synthetic promoters consisting of DNA element homopolymers upstream of a TATA box. High levels of HNF1, HNF3, and HNF4 transcription factors were maintained in mhAT cells. In contrast, C/EBP alpha was much more variable between the different cell lines and was less abundant than it was in vivo, in the liver. We investigated the influence of HNF1 alpha and C/EBP alpha on the activity of transfected liver-specific promoters. HNF1 alpha was not limiting for the activity of transfected liver-type pyruvate kinase and albumin promoters. In contrast, the activity of the albumin promoter in the different lines was clearly dependent on the C/EBP alpha content, which seems, therefore, to be an essential factor modulating the expression of this gene in HNF1 alpha-containing cells. This work shows that the correlations between promoter activities and transacting factor contents in a panel of well-differentiated cultured cells can be used to determine the respective role of transcription factors on the strength of some promoters.

Gene expression in hepatocyte-like lines established by targeted carcinogenesis in transgenic mice.

New hepatocyte-like cell lines (mhAT) were derived from the liver of a transgenic mouse expressing SV40 early genes under the direction of the liver-specific antithrombin III gene promoter (ATIII-TSV40). Their differentiated phenotypes were improved and stabilized by the use of liver-specific growth media (arginine-free, glucose-free, or low-fructose/glucose-free medium). The best differentiated lines display a very high level of albumin, transferrin, and L-type pyruvate kinase (L-PK) gene expression that is comparable to that observed in the mouse liver. Abundance of the aldolase B and phosphoenolpyruvate carboxykinase (PEPCK) transcripts varied from 5 to 35% of the in vivo concentrations while abundance of the alpha-fetoprotein and phenylalanine hydroxylase transcripts remained very low. Hormonal (cAMP and insulin) and nutritional (glucose) gene controls of PEPCK and L-PK were, at least partially, conserved. mhAT cells are readily transfectable by the calcium phosphate coprecipitation technique and exhibit a liver-specific pattern of expression of exogenous genes. Thus, mhAT cells seem suitable for the analysis of the regulatory regions involved in the tissue-specific transcription of genes. This work demonstrates, therefore, the great efficiency of targeted carcinogenesis in transgenic mice to create new differentiated cell lines. The availability of various lines of liver-specific cells with different phenotypes will constitute useful tools to establish correlations between expression of trans-acting factors and control of the phenotype.

Expression of the rat aldolase B gene: a liver-specific proximal promoter and an intronic activator.

The nature and location of the cis-acting DNA sequences regulating expression of the rat aldolase B gene has been investigated. Two liver-specific DNAse I hypersensitive sites were detected, one located just upstream from the cap site, the second in the middle of the first, 4.8-kbp-long, intron. A fragment of 190 bp 5' to the cap site behaved as a tissue-specific but weak core promoter: it directed a detectable reporter gene expression in the Hep G2 cells and hepatocytes, but not in fibroblasts. The tissue-specific expression was stimulated at least 16 fold when constructs contained the entire first intron. The intronic activating sequences could be ascribed to an inner 2 kbp fragment in which the downstream liver-specific DNAse I hypersensitive site was located.

Transfection of hepatic genes into adult rat hepatocytes in primary culture and their tissue-specific expression.

We describe in this paper a method for studying transient gene expression in a primary culture of adult rat hepatocytes. After isolation by collagenase perfusion, hepatocytes in a monolayer were transfected with foreign DNA by the calcium phosphate precipitation technique during the first 24 hours after plating. When they were transfected with a plasmid containing the gene for chloramphenicol acetyltransferase driven by the early promoter of simian virus 40, hepatocytes reproducibly expressed high levels of chloramphenicol acetyltransferase (CAT); this transient expression was much higher than that obtained with the rat hepatoma cell line H4II. Different medium conditions have been tested; an optimal level of CAT activity can be obtained using a serum-free, hormonally defined medium. Using these techniques, we have investigated the expression of liver-specific genes transferred into hepatocytes. We show that the L-pyruvate kinase promoter is active in these hepatocytes while it is silent in fibroblasts. Moreover, the use of serum-free medium may allow investigation of the role of hormones and nutrients in cells which respond normally to these effectors.

Retrovirus antigens recognized by cytolytic T lymphocytes activate tumor rejection in vivo.

We have initiated the molecular definition of the antigens recognized by Gross MuLV-specific cytolytic T lymphocytes on the surface of Gross MuLV-induced tumor cells. A panel of target cells was obtained by the double transfection and expression of a retrovirus gene and a foreign H-2 gene in recipient mouse fibroblasts. Our results show that class I H-2 transplantation antigens have a directive influence in determining the antigenicity of proteins encoded by the gag and env MuLV genes. Genes not linked to H-2 influence the intensity and the specificity of the cytolytic T lymphocyte response to Gross MuLV-induced tumors. Finally, MuLV-induced antigens expressed by transfected fibroblasts induce tumor immunity and lead to accelerated tumor rejection in vivo.

Molecular definition of retrovirus-induced antigens recognized by tumour-specific H-2-restricted cytolytic T lymphocytes.

The antigenic specificities of H-2-restricted, tumour-specific cytolytic T lymphocytes (CTL) were studied at the molecular level using CTL from BALB.B and C57BL/6 (H-2b) mice sensitized to an H-2b Gross murine leukaemia virus (MuLV)-induced tumour. Target cells were produced by the double transfection of mouse L cells (H-2k) with the cloned H-2Kb or H-2Db gene and retroviral DNA derived from a molecular clone of Akv MuLV (closely related to Gross MuLV). Doubly transfected L cells which express either H-2Kb or H-2Db antigen and retroviral antigens are lysed in a virus-specific manner by Gross MuLV-immune CTL. The existence of two independent Gross MuLV-immune CTL subpopulations, one restricted by H-2Kb and the other by H-2Db, is thus confirmed. Gross MuLV-immune CTL from both BALB.B and C57BL/6 mice killed L cells that express Akv MuLV gag gene products and H-2Kb or H-2Db antigen. In contrast, only CTL from C57BL/6 mice killed L cells that express Akv MuLV env gene products and H-2Kb or H-2Db. This indicates that specific recognition of MuLV-induced antigens by CTL can be selective and varies according to the origin of the CTL.

[Tuberculosis in France. Current epidemiologic status and evolution over the last 10 years]. / La tuberculose en France. Situation épidémiologique actuelle et évolution au cours des 10 dernières années.

By evaluating the statistics of the cause of death, the current reports of antituberculous clinics and the information gathered from the tuberculosis registers of certain departments, an epidemiological survey in France shows that there were 2,048 deaths due to tuberculosis in 1981 (3.8 per 100,000). The level is steadily falling; these were 8.2/100,000 in 1970. It is the same for morbidity, the incidence of all forms of tuberculosis was 54 0/0000 in 1972 and 26.9 in 1980. In the register in the Bas-Rhin the level fell from 70.5 to 30.4, in the Rhône from 41.6 to 14.4 and in the Hautes Pyrénées from 22.2 (in 1973) to 15.3. In the Bas-Rhin the prevalence has fallen steadily: 233.3 in 1972 to 97.7 in 1979. Foreigners are five times more affected than the French by respiratory forms and 8 times more for extra-respiratory tuberculosis. Those who came from Black Africa are the most affected.

[Production of monoclonal antibodies against human cytomegalovirus]. / Production d'anticorps monoclonaux contre le cytomégalovirus humain.

Cell fusion of Sp2/0, a murine myeloma derived non-secreting variant, with splenic lymphocytes from Mice immunized against human cytomegalovirus, has originated two stable hybridomas producing monoclonal antibodies (IgG) which react specifically with antigens that appear early in infectious cycle and remain localized in the cell nucleus.