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Biomedical subjects

G Levi

Publications and source records attributed to G Levi.

At least 91 records · Page 5Linked to original sources
PubMed1997

Inducible nitric oxide synthase expression in activated rat microglial cultures is downregulated by exogenous prostaglandin E2 and by cyclooxygenase inhibitors.

Prostaglandins and nitric oxide (NO) are among the numerous substances released by activated microglial cells, the brain resident macrophages, and they mediate several important microglial functions. We have previously shown that cyclooxygenase-2 (COX-2) and inducible NO synthase (iNOS), the two key enzymes in prostaglandin and NO synthesis, respectively, are rapidly co-induced in rat neonatal microglial cultures activated by bacterial endotoxin (lipopolysaccharide [LPS]) and that COX-2 expression appears to be under the negative control of endogenous as well as exogenous NO. In this study we show that exogenous prostaglandin E2 (PGE2), which is known to increase cyclic adenosine monophosphate (cAMP) levels in microglial cells, downregulates LPS-induced iNOS expression in a dose-dependent manner. The involvement of cAMP in the PGE2-dependent inhibition of iNOS is supported by several pieces of evidence. First, iNOS expression was also inhibited by agents such as isoproterenol and forskolin, which cause an elevation of cAMP levels, and by dibutyryl cAMP (dbcAMP), a cAMP stable analogue. Second, the inhibitory effect of PGE2 was mimicked by 11-deoxy-16,16-dm PGE2, a selective agonist at the PGE2 receptor subtype EP2, coupled to the activation of adenylyl cyclase, but not by sulprostone, a potent agonist at receptor subtypes EP3 and EP1, associated with an inhibition of adenylyl cyclase activity and intracellular Ca2+ elevation, respectively. Third, the inhibitory effect of PGE2 on NO synthesis was blocked by SQ 22,536, a specific inhibitor of adenylyl cyclase. Interestingly, the abrogation of endogenous prostanoid production by several COX inhibitors caused a reduction of iNOS expression, suggesting a positive modulatory effect of endogenous prostanoids of iNOS expression, as opposed to the inhibitory effect of exogenous PGE2.

Adenine↗
PubMed1997

Depressive disorder in pre-adolescence: comorbidity or different clinical subtypes? (A pharmacological contribution).

The study consisted of evaluating 80 out-patients for DSM-IV diagnoses and giving 75 mg trazodone for four months to children aged 9-13 who fulfilled DSM-IV criteria for Major Depressive Disorder (n = 22); Major Depressive Disorder and Generalized Anxiety Disorder (n = 24), Major Depressive Disorder and Learning Disorder NOS (n = 24); Major Depressive Disorder and Oppositional Defiant Disorder (n = 10). They were followed up weekly by a psychiatrist blind to the original evaluations and to the precise purpose of the study. Trazodone emerges as safe and effective for over 50% of the sample. Those with depression alone and with LD NOS did particularly well, and those with ODD did particularly badly. The presence of psychopathology in the parents was associated with poorer response to trazodone, and life events were also associated with a poor response.

Adolescent↗
PubMed1996

Progressive activation of adult microglial cells in vitro.

Bulk-isolated microglial cells from the adult rat brain grown in N2 medium supplemented with 10% fetal calf serum survived for at least 2 weeks, and their purity was >99% at day 1 and >93% at day 7. The phenotype of freshly plated cells was comparable to that of "resting," ramified microglia in vivo. With time in culture and with different schedules, depending on the parameter considered, microglia acquired antigenic (e.g., positivity for vimentin, ED1, major histocompatibility complex class I antigens, leukocyte common antigen, and to a lesser extent CD4) and functional (e.g., proliferation, phagocytosis) features characteristic of "activated" microglia as described in situ. Production of nitrite and prostaglandin E2 in response to lipopolysaccharide increased greatly with time in culture. Phagocytosis was also accompanied by increased release of nitrite and prostaglandin E2, the latter being more affected than the first by the age of the cultures. The culture system described may be suitable to study the factors that can modulate "activation" of adult microglia.

Animals↗
PubMed1996

Selective enhancement by serum factors of cyclic AMP accumulation in rat microglial cultures.

Using purified microglial cultures obtained from the neonatal rat brain we found that media containing fetal calf serum (as well as human, horse and goat sera) enhanced by about 3-fold the accumulation of cyclic AMP induced by the beta-adrenergic agonist isoproterenol and did not affect in a significant way that induced by the direct adenylyl cyclase stimulator forskolin. The effect of fetal calf serum was (i) dose dependent, and statistically significant also at serum concentrations below 1%; (ii) rapidly lost (half life of about 15 min) when the serum-containing medium was exposed to microglia, astrocytes or neuroblastoma cells; (iii) present also when cyclic AMP accumulation was enhanced by prostaglandin E2 or by cholera toxin; (iv) absent on basal cyclic AMP levels. When media containing fetal calf serum or the other mammalian sera mentioned above were tested on astrocyte cultures, an inhibitory, rather than enhancing activity on cyclic AMP levels was observed, indicating that the facilitatory factor(s) present in serum acts specifically on microglial cells. Moreover, in astrocytes the effect of serum was identical when tested on basal and on isoproterenol or forskolin-stimulated cyclic AMP levels. Thus, the mechanism of cyclic AMP inhibition in astrocytes is unrelated to the mechanism of activation in microglia. Our observations suggest that serum contains factor(s), promptly cleared by different cell types. Such factors may interact with so far unidentified microglial receptors responsible for a facilitation of G protein-mediated activation of adenylyl cyclase. Regulation of the cyclic AMP cascade at this step has not been described previously, and may be important for the modulation of microglial functions controlled by the cyclic nucleotide.

Adrenergic beta-Agonists↗
PubMed1996

Cytokine-regulated expression of platelet-derived growth factor gene and protein in cultured human astrocytes.

To elucidate mechanisms regulating the production of platelet-derived growth factor (PDGF) in the CNS, we analyzed the influence of a panel of cytokines on PDGF mRNA and protein levels in astrocyte-enriched cultures from the human embryonic brain and spinal cord. Using a specific ELISA, PDGF, AB protein was detected in serum-free astrocyte supernatants and its levels were significantly increased after treatment of the cultures with transforming growth factor-beta 1 (TGF-beta 1) or tumor necrosis factor-alpha (TNF-alpha); the largest increase was detected after combined treatment with the two cytokines. Interleukin-1beta (IL-1beta) by itself had little or no effect but synergized with TGF-beta 1 in enhancing PDGF AB production. Supernatants from human astrocyte cultures stimulated the proliferation of rat oligodendrocyte progenitors, and most of the mitogenic activity could be accounted for by PDGF. By northern blot analysis, both PDGF A- and PDGF B- chains mRNAs were detected in untreated astrocytes. PDGF B-chain mRNA levels were increased by TGF-beta 1, TNF-alpha, TNF-alpha/TGF-beta, or IL-1-beta/TGF-beta 1, whereas PDGF A-chain mRNA levels were not consistently affected by cytokine treatments. These in vitro data indicate that TGF-beta 1, TNF-alpha, and IL-1 beta are able to stimulate astrocyte PDGF production. This cytokine network could play a role in CNS development and repair after injury or inflammation.

Astrocytes↗
PubMed1996

Interferon-gamma and nitric oxide down-regulate lipopolysaccharide-induced prostanoid production in cultured rat microglial cells by inhibiting cyclooxygenase-2 expression.

We have used purified microglial cultures obtained from neonatal rat brains to study some aspects of the regulation of prostanoid production in these cells. We found that nitric oxide, which is released into the culture medium along with prostanoids when the cells are exposed to lipo-polysaccharide (1-100 ng/ml), can down-regulate prostanoid biosynthesis. Indeed, the abrogation of endogenous nitric oxide production, obtained by depleting the medium of the precursor L-arginine or by blocking the activity of nitric oxide synthase by the specific inhibitor NG-monomethyl-L-arginine, led to a remarkable increase in lipopolysaccharide-induced prostanoid release. Moreover, the nitric oxide-generating compound 3-morpholinosydnonimine caused a substantial reduction of prostanoid formation, in the absence of endogenous nitric oxide, suggesting that both endogenous and exogenous nitric oxide may inhibit the induced prostanoid production. We also found that interferon-gamma potentiated the effect of lipopolysaccharide on nitrite accumulation as previously described by others and depressed the lipopolysaccharide-evoked production of prostaglandin E2, prostaglandin D2, and thromboxane. It is interesting that the inhibitory effect of interferon-gamma on prostanoid production did not appear to depend on the potentiation of NO release, as it was present also when the endogenous synthesis of nitric oxide was abrogated. Additional experiments showed that interferon-gamma and nitric oxide act mainly by down-regulating the lipopolysaccharide-induced enzymatic activity and expression (analyzed by western blot) of cyclooxygenase-2. Our data indicate that microglial prostanoid biosynthesis induced by proinflammatory stimuli, such as lipopolysaccharide, is tightly regulated by nitric oxide. Interferon-gamma appears to affect the balance between these local mediators by favoring nitric oxide production and inhibiting the prostanoid cascade and may thus contribute to the modulation of inflammation, local immune reactivity, and neuronal damage.

Animals↗
PubMed1996

Reversible inhibitory effects of interferon-gamma and tumour necrosis factor-alpha on oligodendroglial lineage cell proliferation and differentiation in vitro.

We have investigated the effects of the two prominent inflammatory cytokines, interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha), on oligodendroglial lineage cell development and survival. Purified oligodendrocytes and oligodendrocyte precursors obtained from neonatal rat brain primary cultures were subcultured in a defined, serum-free medium and exposed to IFN-gamma (1-100 U/ml, TNF-alpha (25-100 ng/ml) or both (100 U/ml and 50 ng/ml respectively) from day 1 to day 3 or from day 3 to day 6. While cell survival was not affected in any of the conditions tested, IFN-gamma dose-dependently inhibited [3H]thymidine or bromodeoxyuridine incorporation (by up to 50%) and the reduction of the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT; by up to 33%). TNF-alpha synergized with IFN-gamma, but was ineffective by itself. Moreover, IFN-gamma totally antagonized the induction by basic fibroblast growth factor and platelet-derived growth factor of the proliferation of the oligodendroglial lineage cell population under study. IFN-gamma also blocked the differentiation of oligodendrocyte precursors, as evidenced by cell morphology, immunostaining for early and late differentiation markers (galactocerebroside and myelin basic protein respectively) and activity of ceramide galactosyl transferase. Again, the effect of IFN-gamma was potentiated by TNF-alpha, which was ineffective when tested alone. The inhibitory activity of IFN-gamma was rapidly reversible: 3 days after removal of the cytokine, administered from day 1 to day 3, complete recovery of cll proliferation and differentiation could be documented. The cytokine-induced arrest in the expression of differentiation antigens was accompanied by perturbations in the expression of the corresponding mRNAs, revealed by a semiquantitative reverse transcription-polymerase chain reaction method. In particular, the message for myelin basic protein (and, in the case of treatment from days 3 to 6, also that for myelin associated glycoprotein) was decreased in cultures exposed to IFN-gamma, and further depressed in cultures treated with IFN-gamma and TNF-alpha, while TNF-alpha alone was ineffective. The above observations may help explain the role of IFN-gamma and TNF-alpha in the pathogenesis of inflammatory demyelinating diseases, in which increases in the levels of these substances have been described. In particular, in the case of multiple sclerosis, our results may bear on the problem of defective remyelination and are consistent with the frequent relapsing-remitting course of the disease.

Animals↗
PubMed1996

Defective bone formation in Krox-20 mutant mice.

Endochondral ossification is the prevalent mode of vertebrate skeleton formation; it starts during embryogenesis when cartilage models of long bones develop central regions of hypertrophy which are replaced by bony trabeculae and bone marrow. Although several transcription factors have been implicated in pattern formation in the limbs and axial skeleton, little is known about the transcriptional regulations involved in bone formation. We have created a null allele in the mouse Krox-20 gene, which encodes a zinc finger transcription factor, by in frame insertion of the E. coli lacZ gene and shown that hindbrain segmentation and peripheral nerve myelination are affected in Krox-20-/- embryos. We report here that Krox-20 is also activated in a subpopulation of growth plate hypertrophic chondrocytes and in differentiating osteoblasts and that its disruption severely affects endochondral ossification. Krox-20-/- mice develop skeletal abnormalities including a reduced length and thickness of newly formed bones, a drastic reduction of calcified trabeculae and severe porosity. The periosteal component to bone formation and calcification does not appear to be affected in the homozygous mutant suggesting that the major role for Krox-20 is to be found in the control of the hypertrophic chondrocyte-osteoblast interactions leading to endosteal bone formation.

Animals↗
PubMed1996

Cell adhesion-dependent regulation of cell growth during sea urchin development.

In the sea urchin embryo there are at least two cell adhesion molecules related to mammalian cadherins, one of them, similar to E-cadherin, is expressed in embryos at very early developmental stages, the second appears at the blastula stage (G. Ghersi et al. Mech. Dev. 41, 47-55 (1993)). We show here that when sea urchin embryos are treated with monovalent fragments of antibodies directed against the extracellular domain of these molecules, the decompaction of the embryo is accompanied by a sharp reduction of the rate of cell division. Treatment of the embryos with Fab fragments inhibits thymidine incorporation, but does not affect thymidine uptake or amino acid incorporation. After the first day of development treated embryos have 10 times less blastomeres than normal; later, however, they resume development and give eventually rise to normal-looking plutei. Analysis of putative second messengers shows that treatment of the embryos with anti-cadherin Fabs leads to a decreased tyrosine phosphorylation of the two cadherins and of two cadherin-associated proteins and to a doubling of the intracellular concentration of cAMP. These results are discussed in view of the importance of cell adhesion signals for cell growth control.

Animals↗
PubMed1995

Ion channels in rat microglia and their different sensitivity to lipopolysaccharide and interferon-gamma.

In order to study the voltage-dependent ion channels in microglia, and their possible modulation by pro-inflammatory substances like lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) we employed the patch-clamp technique on purified rat microglial cell subcultures grown for 1 - 5 days in control condition or after a 24 hour treatment with those agents. Regardless of the culture condition, almost 100% of the cells presented inward-rectifying (IR) K+ currents identified by the following features: (a) extracellular K(+)-dependence of Vrev and whole-cell conductance; (b) inward-rectifying property; (c) channel blocking mechanism by Cs+; and (d) single channel conductance of 27 pS. A 'n' type outward-rectifying (OR) K+ current was present in 30% of the cells during the first 2 days of subcultivation. Its occurrence was strongly dependent on the preparation, varying from 0% to almost 80%, and it decreased to 13% of the cells after three days in culture. It showed the following features: (i) threshold of activation close to -30 mV; (ii) sigmoid current onset; (iii) voltage-dependent kinetics; and (iv) sensitivity to 4-aminopyridine (4-AP) and tetraethylammonium (TEA). Furthermore, we detected two ion currents not previously described in microglia: (i) a slowly activating outward current which appeared at potentials more positive than +20 mV and with a reversal potential close to 0 mV, tentatively identified as a proton current; and (ii) a Cl- conductance identified in ion substitution experiments as the current sensitive to the Cl- channel blocker SITS. The two agents, LPS (20 - 2,000 ng/ml) and IFN-gamma (10 - 100 u/ml), shared the following effects: (a) enhancement of membrane capacitance, and (b) increase of OR current amplitude and frequency of occurrence. Moreover, IFN-gamma was also able to increase IR current density, especially in cells with ameboid morphology, while LPS was ineffective. We conclude that the voltage-dependent ion channel pattern of microglia is more complex than previously thought and that activating agents such as LPS and IFN-gamma share some electrophysiological effects, but differ in others.

Animals↗
PubMed1995

Interferon-gamma and lipopolysaccharide reduce cAMP responses in cultured glial cells: reversal by a type IV phosphodiesterase inhibitor.

The aim of the present study was to determine whether two classical macrophage activators, bacterial lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) could affect the accumulation of the second messenger cAMP in cultured rat microglia and astrocytes. Purified microglia and astrocyte secondary cultures obtained from the neonatal rat were grown for 3 days in basal medium Eagle (BME) + 10% fetal calf serum (FCS). Exposure of microglia to LPS resulted into a dose- and time-dependent decrease in the accumulation of cAMP induced by receptor-mediated (isoproterenol or prostaglandin E2) or direct (forskolin) activation of adenylate cyclase. The inhibitory effect of LPS was rapid (a 10 min preincubation was sufficient to approach a maximal effect), occurred at low doses (IC50 = 1.2 ng/ml), and was not abrogated by pertussis toxin. A selective inhibitor of type IV phosphodiesterase (rolipram, 100 nM) prevented the effect of LPS on cAMP accumulation, while inhibitors of other forms of phosphodiesterase were unable to do so. IFN-gamma (100 u/ml) also caused a depression of the evoked cAMP accumulation in microglia after a 10 min preincubation, and its effect was prevented by rolipram, as in the case of LPS. Astrocytes differed from microglia in that LPS (1-100 ng/ml) did not inhibit the accumulation of cAMP induced by either isoproterenol or forskolin; on the other hand, IFN-gamma did have an inhibitory effect (though less pronounced than in microglia) that could be prevented by rolipram.(ABSTRACT TRUNCATED AT 250 WORDS)

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1995

Induction of prostanoid biosynthesis by bacterial lipopolysaccharide and isoproterenol in rat microglial cultures.

We have used purified microglial cultures obtained from neonatal rat cerebral cortex to investigate the ability of microglia to release prostanoids after exposure to bacterial lipopolysaccharide, a classic macrophage activator. Release of prostaglandin E2, prostaglandin D2, and thromboxane A2 was low in basal conditions and increased in a dose- and time-dependent way upon lipopolysaccharide treatment (1-100 ng/ml), by a mechanism requiring de novo protein synthesis. When compared with astrocytes, microglial cells appeared to respond more effectively to lipopolysaccharide, being able to release prostanoids after exposure to a 100-fold lower concentration of lipopolysaccharide. In addition to prostanoids, we also measured the release of leukotriene B4; although lipopolysaccharide failed to stimulate leukotriene B4 release by microglial cells, it doubled the basal production in astrocytes. Lipopolysaccharide enhanced the release of preloaded [3H]arachidonic acid from microglial membrane phospholipids by a mechanism inhibited by the protein synthesis inhibitor cycloheximide, which suggests that the increased availability of arachidonic acid contributed to the enhanced prostanoid production. Lipopolysaccharide, however, also stimulated prostanoid synthesis by inducing cyclooxygenase activity, as shown by determining the activity of newly synthesized enzyme after inactivating the endogenous enzyme with aspirin and by assessing the level of the inducible form of cyclooxygenase by western blot analysis. Among the mechanisms potentially involved in the regulation of microglial prostanoid production, we studied the effect of beta-adrenergic receptor activation. The beta-agonist isoproterenol was inactive by itself but doubled the effect of lipopolysaccharide.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

The costimulatory molecule B7 is expressed on human microglia in culture and in multiple sclerosis acute lesions.

B7 is a costimulatory molecule which is expressed on antigen-presenting cells and which plays a pivotal role in T cell activation and proliferation. To elucidate mechanisms regulating intracerebral immune responses, expression of B7 was examined in cultured microglial cells and in brain tissue from control and multiple sclerosis patients. Using immunocytochemical and polymerase chain reaction techniques, we show that B7 was expressed in cultured microglial cells from the human embryonic brain. Microglia also bound the soluble form of the B7 receptor CTLA-4 (CTLA-4-Ig). B7 gene expression and binding of anti-B7 antibodies and CTLA-4-Ig increased after treatment with interferon-gamma. B7 was not inducible in human astrocytes. Human microglia expressed other costimulatory molecules, such as intercellular adhesion molecule-1, LFA-1 and LFA-3. In sections of multiple sclerosis brains, B7 immunoreactivity was detected on activated microglia and infiltrating macrophages within active lesions. In chronic lesions, only perivascular cells were stained. B7 immunoreactivity was undetectable in sections from Alzheimer's disease or normal brain tissue. These data suggest that B7 may be involved in T cell activation and lesion development in multiple sclerosis and that the regulated expression of B7 on microglia may contribute to the local stimulation of T cell proliferation and effector functions.

Acute Disease↗
PubMed1995

Medical students' contribution to the development of a smoke-free hospital policy in a university medical centre: a relevant learning experience.

In a research methodology course, second-year medical students conducted a survey on 'Enforcing a Smoking Ban in the Soroka Medical Center: a Survey of Hospital Employees on Facilitating Factors and Obstacles'. They defined the study objectives and design, developed the study instrument, carried out the survey, coded and entered the data into mainframe computers, analysed the computer output, and prepared oral and written reports. The aims of the project were twofold: to survey employees' attitudes to a hospital smoking ban and to train medical students in the planning and conduct of a research project on public health or preventive medicine. Twelve students conducted a cross-sectional survey of 208 hospital employees (10% of the hospital staff). Employees were surveyed regarding smoking status, interest in quitting smoking, knowledge of the law banning smoking in public places, knowledge of the health effects of passive smoking, attitudes towards a hospital smoking ban and potential obstacles to its implementation. The students rated the course as excellent. They gained important research skills, as well as practical medical and public health experience through active participation in the design and execution of a study project with public health implications. At the first meeting of the hospital committee appointed to enforce a smoke-free hospital, the students' findings were reported in full, and their recommendations have guided policy decisions.

Health Policy↗
PubMed1995

Nucleolar localisation of three Hox homeoproteins.

Homeoproteins encoded by genes of the Hox family are nuclear proteins believed to act as transcription factors and to participate in the determination of the body plan. Here we show that in several vertebrate cells, they exhibit a subnuclear localisation associated with the nucleolus. We used monoclonal antibodies to study the distribution of three homeoproteins, namely HOXB7, HOXC6 and HOXD4. The immunoreactivity to antibodies against HOXC6 protein in Xenopus laevis embryonic tissues is restricted to one or two spots within the nucleus; this distribution partially overlaps that of fibrillarin, a protein of the fibrillar zone of the nucleoli. Indirect immunofluorescence analysis of the distribution of HOXB7 protein in 3T3 cells, and of HOXD4 protein in human neuroblastoma and Raji lymphoma cell lines and activated lymphocytes, results invariably in a nucleolar localisation. Purified nucleoli from stimulated T lymphocytes, and Raji cells contain an activity capable of binding, in a gel retardation assay, to an oligonucleotide specifically recognised by the HOXD4 homeoprotein. This activity is specifically removed by anti-HOXD4 antibodies and is found associated in southwestern blots with a single band with an apparent M(r) of 30,000, corresponding to that of recombinant HOXD4. The functional significance of the nucleolar localisation of Hox proteins remains to be determined.

Animals↗