Selective siRNA-mediated suppression of 5-HT1A autoreceptors evokes strong anti-depressant-like effects.

Depression is a major health problem worldwide. Most prescribed anti-depressants, the selective serotonin reuptake inhibitors (SSRI) show limited efficacy and delayed onset of action, partly due to the activation of somatodendritic 5-HT(1A)-autoreceptors by the excess extracellular serotonin (5-HT) produced by SSRI in the raphe nuclei. Likewise, 5-HT(1A) receptor (5-HT(1A)R) gene polymorphisms leading to high 5-HT(1A)-autoreceptor expression increase depression susceptibility and decrease treatment response. In this study, we report on a new treatment strategy based on the administration of small-interfering RNA (siRNA) to acutely suppress 5-HT(1A)-autoreceptor-mediated negative feedback mechanisms. We developed a conjugated siRNA (C-1A-siRNA) by covalently binding siRNA targeting 5-HT(1A) receptor mRNA with the SSRI sertraline in order to concentrate it in serotonin axons, rich in serotonin transporter (SERT) sites. The intracerebroventricular (i.c.v.) infusion of C-1A-siRNA to mice resulted in its selective accumulation in serotonin neurons. This evoked marked anti-depressant-like effects in the forced swim and tail suspension tests, but did not affect anxiety-like behaviors in the elevated plus-maze. In parallel, C-1A-siRNA administration markedly decreased 5-HT(1A)-autoreceptor expression and suppressed 8-OH-DPAT-induced hypothermia (a pre-synaptic 5-HT(1A)R effect in mice) without affecting post-synaptic 5-HT(1A)R expression in hippocampus and prefrontal cortex. Moreover, i.c.v. C-1A-siRNA infusion augmented the increase in extracellular serotonin evoked by fluoxetine in prefrontal cortex to the level seen in 5-HT(1A)R knockout mice. Interestingly, intranasal C-1A-siRNA administration produced the same effects, thus opening the way to the therapeutic use of C-1A-siRNA. Hence, C-1A-siRNA represents a new approach to treat mood disorders as monotherapy or in combination with SSRI.

Sodium tungstate regulates food intake and body weight through activation of the hypothalamic leptin pathway.

AIMS: Sodium tungstate is an anti-obesity drug targeting peripheral tissues. In vivo, sodium tungstate reduces body weight gain and food intake through increasing energy expenditure and lipid oxidation, but it also modulates hypothalamic gene expression when orally administered, raising the possibility of a direct effect of sodium tungstate on the central nervous system. METHODS: Sodium tungstate was administered intraperitoneally (ip) to Wistar rats, and its levels were measured in cerebrospinal fluid through mass spectrometry. Body weight gain and food intake were monitored for 24 h after its administration in the third ventricle. Hypothalamic protein was obtained and subjected to western blot. In vitro, hypothalamic N29/4 cells were treated with 100 µM sodium tungstate or 1 nM leptin, and protein and neural gene expression were analysed. RESULTS: Sodium tungstate crossed the blood-brain barrier, reaching a concentration of 1.31 ± 0.07 mg/l in cerebrospinal fluid 30 min after ip injection. When centrally administered, sodium tungstate decreased body weight gain and food intake and increased the phosphorylation state of the main kinases and proteins involved in leptin signalling. In vitro, sodium tungstate increased the phosphorylation of janus kinase-2 (JAK2) and extracellular signal-regulated kinase-1/2 (ERK1/2), but the activation of each kinase did not depend on each other. It regulated c-myc gene expression through the JAK2/STAT system and c-fos and AgRP (agouti-related peptide) gene expression through the ERK1/2 pathway simultaneously and independently. CONCLUSIONS: Sodium tungstate increased the activity of several kinases involved in the leptin signalling system in an independent way, making it a suitable and promising candidate as a leptin-mimetic compound in order to manage obesity.

Dual effects of sodium tungstate on adipocyte biology: inhibition of adipogenesis and stimulation of cellular oxygen consumption.

OBJECTIVE: We have recently shown the in vivo anti-obesity effects of sodium tungstate. In this study, we investigate the in vitro effects of sodium tungstate on adipocyte differentiation and function. METHODS: 3T3-F442A cells were allowed to differentiate in the presence of sodium tungstate, and were analyzed for triglyceride (TG) accumulation, adipocyte differentiation and mitochondrial oxygen consumption. RESULTS: Sodium tungstate treatment of adipose cells decreased TG accumulation and adipocyte differentiation. Expression of key genes for adipocyte function (aP2, ACC, fatty acid synthase (FAS) and lipoprotein lipase (LPL)) and differentiation (CCAAT enhancer-binding protein (C/EBP)alpha and peroxisome proliferator-activated receptor gamma (PPARgamma)) was reduced by sodium tungstate, whereas C/EBPbeta isoform LIP expression level was increased. TG accumulation and changes in C/EBPbeta expression were partially recovered by inactivating the erk1/2 pathway. Finally, tungstate treatment increased the oxygen consumption of adipose cells without changes in the expression of oxidative genes. CONCLUSIONS: Sodium tungstate inhibits adipocyte differentiation by promoting the translation of LIP, a master dominant-negative regulator of this process, and regulates the mitochondrial oxygen consumption of adipose cells. These effects contribute to the anti-obesity activity of sodium tungstate and confirm its potential as a powerful alternative for the treatment of obesity.

Coadministration of coenzyme Q prevents rosiglitazone-induced adipogenesis in ob/ob mice.

OBJECTIVE: The aim of this study is to determine the effect of coenzyme Q (Q) on ob/ob mice treated or not with thiazolidinedione (TZD). DESIGN AND MEASUREMENTS: Ob/ob mice were treated with Q, Rosiglitazone or a combination of both molecules for 13 days; physical and metabolic parameters as well as oral glucose tolerance test were assessed. mRNA expression of genes of energy dissipation and storage were measured by real-time PCR. RESULTS: Q treatment improved some metabolic parameters in ob/ob mice. Surprisingly, cotreatment with Rosiglitazone and Q improved metabolic parameters and prevented TZD increase in body weight and adiposity, mainly by increasing lipid oxidation in adipose tissue, reducing lipid synthesis and balancing adipokine gene expression. CONCLUSIONS: Our finding suggests that Rosiglitazone and coenzyme Q bitherapy could prevent the body weight gain associated with adipogenesis and could improve the clinical use of these compounds.

High Intraspecific Genetic Diversity of Nocardia brasiliensis, a Pathogen Responsible for Cutaneous Nocardiosis Found in France: Phylogenetic Relationships by Using sod and hsp65 Genes.

This study aims at genetic characterization and phylogenetic relationships of Nocardia brasiliensis focusing by using housekeeping rrs, hsp65, and sodA genes. N. brasiliensis is the species responsible for 80% of cases of actinomycetoma, one form of cutaneous nocardiosis which occurs mainly in tropical regions reaching immunocompetent patients in which the disease can lead to amputation. We analyze 36 indigenous cases of N. brasiliensis that happened in France. Phylogenetic analysis targeting rrs gene showed no robustness at phylogenetic nodes level. However, the use of a concatenation of hsp65 and sodA genes showed that the tested strains surprisingly ranked in 3 well-defined genotypes. Genotypes 2 and 3 were phylogenetically closer to each other and both diverged from genotype 1 sustained by a high bootstrap of 81%. This last genotype hosts all the cases of pulmonary forms (3), the sole cerebral form, and almost all the cases of immunocompromised patients (3 out of 4). Moreover, excepting one of them, all the strains belonging to this group present a susceptibility to imipenem which is not the case in the other genotypes that rarely count among them strains being susceptible to this drug. The haplotype diversity (Hd) of hsp65 (0.927) and sodA (0.885) genes was higher than that of rrs (0.824). For this gene, we obtained 16 polymorphic sites whereas, for hsp65 and sodA genes, up to 27 and 29 were identified, respectively. This study reveals that these two genes have an important genetic discriminatory power for the evaluation of the intraspecies genetic variability of N. brasiliensis and they may be useful for identification purposes at species level. This study also reveals the possible existence of a new species harbored by genotype 1.

Cyclosporin a. Inhibition of experimental autoimmune uveitis in Lewis rats.

Cyclosporin A (CS-A), a selective inhibitor of T lymphocytes, is reported here to prevent S antigen (S-Ag) induced uveitis in Lewis rats. The S-Ag, found in all mammalian retinas, is uveitogenic under experimental conditions and patients with certain uveitic entities demonstrate cell mediated responses to this antigen. Daily treatment with CS-A (10 mg/kg) begun on the same day as S-Ag immunization totally inhibited the development of the uveitis in this experimental autoimmune model. Moreover a greater CS-A dose (40 mg/kg) efficiently prevented the disease process when therapy was started 7 d after S-Ag immunization. Anti-S-Ag antibody titers were observed to be similar in rats either protected or not protected with CS-A. Our data support strongly the need for T cell participation in this disease model. Since ocular inflammatory disease is an important cause of visual impairment, the data further suggest that CS-A may be useful in the treatment of patients with intractable uveitis.

Activators of peroxisome proliferator-activated receptor-alpha induce the expression of the uncoupling protein-3 gene in skeletal muscle: a potential mechanism for the lipid intake-dependent activation of uncoupling protein-3 gene expression at birth.

The recently identified uncoupling protein-3 (UCP-3) gene, predicted to encode a new member of the family of uncoupling proteins, is preferentially expressed in skeletal muscle and has been related to phenotypes of obesity and type 2 diabetes. We have established that during mouse ontogeny, the expression of the UCP-3 gene is switched on in skeletal muscle just after birth. The induction of UCP-3 gene expression is dependent on the initiation of suckling and particularly on lipid intake. Treatment of newborn mice with activators of peroxisome proliferator-activated receptors (PPARs), such as clofibrate, bezafibrate, or (4-chloro-6-(2,3-xylidine)-pirimidinylthio)acetic acid (WY 14,643), mimics the action of food intake on UCP-3 gene expression. The specific ligand of PPAR-alpha WY 14,643 induces UCP-3 gene expression in a time- and dose-dependent manner, whereas the thiazolidinedione BRL 49653, specific for PPAR-gamma, has no effect. These treatments act without altering circulating free fatty acids. During development, skeletal muscle expresses constitutive levels of PPAR-delta mRNA, whereas expression of the PPAR-gamma gene is undetectable. PPAR-alpha gene expression is developmentally regulated in muscle as it is first expressed at birth, just before UCP-3 gene induction occurs. The induction of UCP-3 gene expression by WY 14,643 is impaired in skeletal muscle of premature neonates, which do not express PPAR-alpha. It is proposed that the UCP-3 gene is predominantly regulated in neonatal muscle by PPAR-alpha activation.

Impaired expression of the uncoupling protein-3 gene in skeletal muscle during lactation: fibrates and troglitazone reverse lactation-induced downregulation of the uncoupling protein-3 gene.

The expression of uncoupling protein (UCP)-3 mRNA in skeletal muscle is dramatically reduced during lactation in mice. The reduction in UCP-3 mRNA levels lowers the amount of the UCP-3 protein in skeletal muscle mitochondria during lactation. Spontaneous or abrupt weaning reverses the downregulation of the UCP-3 mRNA but not the reduction in UCP-3 protein levels. In lactating and virgin mice, however, fasting increases UCP-3 mRNA levels. Changes in UCP-3 mRNA occur in parallel with modifications in the levels of free fatty acids, which are reduced in lactation and are upregulated due to weaning or fasting. Modifications in the energy nutritional stress of lactating dams achieved by manipulating litter sizes do not influence UCP-3 mRNA levels in skeletal muscle. Conversely, when mice are fed a high-fat diet after parturition, the downregulation of UCP-3 mRNA and UCP-3 protein levels due to lactation is partially reversed, as is the reduction in serum free fatty acid levels. Treatment of lactating mice with a single injection of bezafibrate, an activator of the peroxisome proliferator-activated receptor (PPAR), raises UCP-3 mRNA in skeletal muscle to levels similar to those in virgin mice. 4-chloro-6-[(2,3-xylidine)-pirimidinylthio] acetic acid (WY-14,643), a specific ligand of the PPAR-alpha subtype, causes the most dramatic increase in UCP-3 mRNA, whereas troglitazone, a specific activator of PPAR-gamma, also significantly increases UCP-3 mRNA abundance in skeletal muscle of lactating mice. However, in virgin mice, bezafibrate and WY-14,643 do not significantly affect UCP-3 mRNA expression, whereas troglitazone is at least as effective as it is in lactating dams. It is proposed that the UCP-3 gene is regulated in skeletal muscle during lactation in response to changes in circulating free fatty acids by mechanisms involving activation of PPARs. The impaired expression of the UCP-3 gene is consistent with the involvement of UCP-3 gene regulation in the reduction of the use of fatty acids as fuel by the skeletal muscle and in impaired adaptative thermogenesis, both of which are major metabolic adaptations that occur during lactation.

9-cis retinoic acid induces the expression of the uncoupling protein-2 gene in brown adipocytes.

The expression of uncoupling protein-2 (UCP2) mRNA is up-regulated during the differentiation of brown adipocytes in primary culture. When differentiation of brown adipocytes is impaired, UCP2 mRNA expression is down-regulated. 9-cis Retinoic acid causes a dose-dependent induction of UCP2 mRNA levels in brown adipocytes, whereas all-trans retinoic acid has no effect. Specific agonists of retinoid X receptors (RXR) induce UCP2 mRNA expression, whereas specific activators of retinoic acid receptors do not. 9-cis Retinoic acid, acting through RXR receptors, is identified as a major regulator of the expression of the UCP2 gene in the brown fat cell.

Uncoupling protein-3 gene expression in skeletal muscle during development is regulated by nutritional factors that alter circulating non-esterified fatty acids.

Uncoupling protein-3 gene expression in skeletal muscle is up-regulated during postnatal development of mice. A high-carbohydrate diet at weaning induces a decrease in uncoupling protein-3 mRNA levels that does not occur when mice were weaned onto a high-fat diet. Uncoupling protein-3 mRNA levels do not increase in response to fasting in young pups. Only after day 15 of life, when fasting increases serum non-esterified fatty acids, uncoupling protein-3 mRNA is up-regulated by starvation. Over-nutrition or under-nutrition during lactation increases or decreases, respectively, uncoupling protein-3 mRNA expression in skeletal muscle. Regulation of uncoupling protein-3 gene expression in skeletal muscle during development is mediated by ontogenic and nutritional factors determining changes in circulating non-esterified fatty acids.

Inverse distribution of uncoupling proteins expression and oxidative capacity in mature adipocytes and stromal-vascular fractions of rat white and brown adipose tissues.

To investigate relationships between the uncoupling protein (UCP) family and oxidative metabolism in fat pads, we measured the cytochrome oxidase activity, used as an index of oxidative capacity, and the mRNA content encoding UCP1, UCP2 and UCP3. Most oxidative potential was found in the stromal-vascular fraction (SVF) of brown fat and in mature adipocytes of white fat (inguinal and periovarian). Considering the whole fat pads, the oxidative potential observed in mature white adipocytes fraction was not negligible compared with that of brown adipocytes fraction. UCP1 and UCP3 were expressed exclusively in mature brown adipocytes. Whatever the deposit, UCP2 mRNA was mainly localized in the SVF. These results indicate that, in fat, high oxidative potential is not necessarily linked to high UCPs transcripts content and point out the oxidative capacity of SVF from brown fat.

Nocardia brasiliensis: from microbe to human and experimental infections.

Nocardia brasiliensis is a Gram-positive bacterium that lives as a saprophyte in soil. In this article the physical properties, chemical composition and taxonomic position of this species is reviewed. Human infections and an experimental model of actinomycetoma in BALB/c mice as well as the host-immune response is described.

Opposite regulation of PPAR-alpha and -gamma gene expression by both their ligands and retinoic acid in brown adipocytes.

The peroxisome proliferator-activated receptors (PPARs) are lipid-activated transcription factors involved in the regulation of lipid metabolism and adipocyte differentiation. Little is known, however, about the control of the expression of the genes encoding each of all three receptor subtypes: alpha, delta, and gamma. We have addressed this question in the brown adipocyte, the only cell type that co-expresses high levels of the three PPAR subtypes. Differentiation of brown adipocytes is associated with enhanced expression of PPAR genes. However, whereas PPARgamma and PPARdelta genes are already expressed in preadipocytes, the mRNA for PPARalpha appears suddenly in association with the acquisition of the terminally differentiated phenotype. Both retinoic acid isomers and PPAR agonists, specific for either PPARalpha or PPARgamma, regulate expression of each PPAR subtype gene in the opposite way: they up-regulate PPARalpha and down-regulate PPARgamma. The effects on PPARalpha mRNA are independent of protein synthesis, whereas inhibition of PPARgamma mRNA expression depends on protein synthesis, except when its specific ligand prostaglandin J2 is used. Our results indicate a strictly opposite autoregulation of PPAR subtypes, which supports specific physiological roles for them in controlling brown fat differentiation and thermogenic activity.

Modulation of experimental autoimmune uveitis with cyclosporin A.

Cyclosporin A has been shown to be an effective inhibitor of T cell-mediated diseases. We show here that cyclosporin A was capable of totally preventing the clinical appearance of experimental autoimmune uveitis in Lewis rats, even when administered on an every-other-day schedule (10 mg/kg) or when begun seven days after immunization (40 mg/kg). At lower doses of the drug, a modulation of the disease was seen with evidence of a more chronic, granulomatous process. A long-lasting unresponsive state to the immunizing antigen was not uniformly induced with cyclosporin A if therapy was begun seven days after S antigen immunization. Because of cyclosporin A's effective control of this experimental model that is induced by an antigen to which certain patients with uveitis demonstrate cell-mediated immune responses, cyclosporin A may be an effective mode of therapy for T cell-mediated intraocular inflammatory disease.

Elastoidin turn-over during tail fin regeneration in teleosts. A morphometric and radioautographic study.

During teleostean fin regeneration the actinotrichia, rods of a collagen-like protein, the elastoidin, are immersed in the blastema, maintaining their apical position. In this epimorphic event the latter fact might be achieved by either a cellular carriage or a continuous turn-over of these hyperpolimerized fibrils. By means of a 3H-proline pulse and radioautographic chase experiment of the isolated actinotrichia we have found a turn-over of collagen within the structure. From these and additional morphometric results, we present in this work an operational hypothesis of how gradually differentiating blastemal cells and an appropriately shaped basal lamina, can control the number and distribution of actinotrichia which might be under the balanced control of their synthesis and degradation.

[Immunoelectroblotting in Mexican subjects at high risk for infection with human immunodeficiency virus (HIV)]. / La inmunoelectrotransferencia en sujetos mexicanos de alto riesgo para la infección por el virus de la inmunodeficiencia humana (VIH).

Sera from 124 persons in high risk groups were analyzed including homosexuals, blood recipients, and spouses or siblings from AIDS patients. In this study, 118 individuals had a positive ELISA for anti-HIV antibodies. Six persons had a complete immunodeficiency syndrome and a negative ELISA test. In the Western blot, 111 sera were positive, four negative, and nine scored indeterminate; four of the latter converted to positive when retested three months later. Antibodies present in the positive sera were directed against the HIV gp 41 kD in 100% of the cases and against the gp 120 kD in 82%. Frequency of recognition of p55 kD was 96% but p18 kD was only 42%.

[Antigens of the human immunodeficiency virus in serum of high risk people in the city of Monterrey]. / Antígenos del virus de inmunodeficiencia humana en el suero de personas del grupo de alto riesgo de la ciudad de Monterrey.

One hundred forty human sera distributed in 2 groups were analyzed. In group I, 50 serum samples from healthy individuals that did not belong to the high-risk acquired immune deficiency syndrome (AIDS) population were included. In group II, there were 90 individuals, most of whom were apparently healthy but were at high risk of getting AIDS through their life styles or by transfusion. Of the 90 persons, 5 had a clinical picture of AIDS. All sera were analyzed by the enzyme - linked immunosorbent assay (ELISA) for the anti VIH antibodies. The positive cases were confirmed by the Western blot assay. In all samples the presence of the human immune deficiency virus antigens was sought. The results showed that the 50 healthy individuals (control group) were negative for both HIV antigens and antibodies. Of the 90 sera for the high-risk group, 50 were negative for antibodies, and 2 of them (4%) were positive for HIV antigens. Forty sera were positive for anti HIV antibody and among them, 5 patients were diagnosed as AIDS, and showed positive for antigen and antibody. The other 35 patients were all positive for HIV antibody and in 8 of them HIV antigen was also present.

[Antibodies against human T-cell lymphotropic viruses in subjects at high risk for HIV in Monterrey]. / Anticuerpos anti-virus linfotrópicos de células T humanas en sujetos de alto riesgo para la infección por VIH en Monterrey.

HIV and HTLV-1 are retrovirus that can produce human disease. It is known that HTLV-1 is associated to the adult T cell leukemia and to the spastic tropical paraparesis. AIDS is now a pandemic infection and HTLV-1 has a high endemicity in the Caribbean region and Japan, whereas the south of the United States has a low endemicity. In Mexico there is little information on HTLV-1 incidence. In the present work we looked for anti HTLV-1 antibodies in one hundred persons that belong to the high risks AIDS population in the city of Monterrey, Mexico. We found that 93 sera were positive for anti HIV antibodies in a ELISA test and seven were negative. All 93 sera were also positive in the Western Blot assay. In the confirmatory test two out of the seven negative sera were classified as indeterminate and five as negative. We also included in this study 50 sera from healthy control volunteers that did not belong to the high risk AIDS population and resulted negative in the HIV and HTLV-1 test. Anti HTLV-1 antibodies were determined by using an agglutination test with gelatin particles covered with HTLV-1 and confirmed by a Western Blot assay. We found that only three sera resulted positive in this agglutination test, but were negative by the Western Blot technique.

[Anti-Nocardia brasiliensis antibodies in patients with actinomycetoma and their clinical usefulness]. / Anticuerpos anti-Nocardia brasiliensis en pacientes con actinomicetoma y su utilidad clínica.

Anti-Nocardia brasiliensis antibodies quantification and its clinical utility was confirmed in this study. A protein cellular extract from a N. brasiliensis strain named HUJEG-1 and registered at the ATCC # 700358 was used in a western blot assay to identify the immunodominant antigens. The protein P24 was selected to set up an ELISA test because it exhibit no cross-reaction with sera from tuberculosis and leprosy patients. A purified protease was also used as antigen in the ELISA test to compare its utility. Sera from N. brasiliensis mycetoma persons gave absorbance values above 0.3 when the disease was active using the P24 as antigen, these values decreased after patients completed their medical treatment. Anti-protease antibodies showed great variation and absorbance values similar to the healthy controls. We confirmed the clinical usefulness of the ELISA test both in serodiagnosis and in assessing the response to medical treatment. This is the first sensitive and specific serologic test for routine clinical laboratory.