Can Spirulina maxima reduce the mutagenic potential of sibutramine?

The worldwide obesity pandemic requires the use of anti-obesity drugs. Sibutramine is an anti-obesity drug that has been used worldwide but is indiscriminately consumed in Brazil. Several studies have demonstrated that sibutramine promotes weight loss and weight maintenance, but several side effects have been associated with its systematic consumption. For this reason, sibutramine was withdrawn from the European and American markets, but still remains legal for use in Brazil. Studies have shown that a 5-10% reduction in body weight results in outstanding health benefits for obese patients. However, in order to promote significant weight loss, it is necessary to use sibutramine for at least 2 years. This long-term exposure has carcinogenic potential, as sibutramine causes DNA damage. Thus, this study evaluated the in vivo mutagenic potential of sibutramine alone (5, 7, 10, 15, and 20 mg/kg) and in association with Spirulina maxima (150 and 300 mg/kg), a cyanobacterium with antioxidant potential, using the polychromatic erythrocyte micronucleus test. Our results reinforced the mutagenic potential of sibutramine alone, which showed a time-dependent action. Combinatory treatments with S. maxima were not able to reduce the genotoxicity of sibutramine. These results were confirmed in vitro with the cytokinesis-blocked micronucleus test. In conclusion, our data showed that new alternative anti-obesity treatments are needed since the consumption of sibutramine can increase the risk of cancer in overweight patients.

Mutagenic potential of Cordia ecalyculata alone and in association with Spirulina maxima for their evaluation as candidate anti-obesity drugs.

Obesity is one of the most important nutritional disorders, and can be currently considered as an epidemic. Although there are few weight reduction drugs available on the market, some new drug candidates have been proposed, including Cordia ecalyculata, a Brazilian plant with anorectic properties, and Spirulina maxima, a cyanobacterium with antioxidant and anti-genotoxic activity. In this study, we evaluated the mutagenic potential of C. ecalyculata at doses of 150, 300, and 500 mg/kg alone and in association with S. maxima at doses of 75, 150, and 250 mg/kg, respectively, through an in vivo micronucleus test, using mice of both sexes, and an in vitro micronucleus test and comet assay, using human peripheral blood. For all tests, cyclophosphamide was used as a positive control. The results showed that treatment of 300 mg/kg C. ecalyculata and the combination treatment of 500 mg/kg C. ecalyculata with 250 mg/kg S. maxima resulted in anorectic effects. The mutagenic tests did not reveal any clastogenic or genotoxic activity for any treatment, indicating that these candidates could be marketed as weight-reduction drugs. Moreover, the drugs contain chemo-preventive substances that can protect against tumorigenesis, which has been associated with obesity.

Dynamics of immunosuppression in hamsters with experimental visceral leishmaniasis

Immunosuppression has been reported to occur during active visceral leishmaniasis and some factors such as the cytokine profile may be involved in this process. In the mouse model of cutaneous leishmaniasis using Leishmania (Leishmania) major, the Th1 response is related to protection while the Th2 response is related to disease progression. However, in hamsters, which are considered to be an excellent model for the study of visceral leishmaniasis, this dichotomy is not observed. Using outbred 45- to 60-day-old (140 to 150 g) male hamsters infected intraperitoneally with 2 x 10(7) L. (L.) chagasi amastigotes, we evaluated the immune response of spleen cells and the production of cytokines. We used 3 to 7 hamsters per group evaluated. We detected a preserved response to concanavalin A measured by index of proliferation during all periods of infection studied, while a proliferative response to Leishmania antigen was detected only at 48 and 72 h post-infection. Messenger RNA from cytokines type 1 (IL-2, TNF-α, IFN-γ) and type 2 (IL-4, IL-10 and TGF-β) detected by reverse transcriptase polymerase chain reaction and produced by spleen cells showed no qualitative difference between control non-infected hamsters and infected hamsters during any period of infection evaluated. Cytokines were measured by the DNA band intensity on agarose gel using the Image Lab 1D L340 software with no differences observed. In conclusion, the present results showed an antigen-dependent immunosuppression in hamsters with active visceral leishmaniasis that was not related to the cytokine profile.

Dynamics of immunosuppression in hamsters with experimental visceral leishmaniasis.

Immunosuppression has been reported to occur during active visceral leishmaniasis and some factors such as the cytokine profile may be involved in this process. In the mouse model of cutaneous leishmaniasis using Leishmania (Leishmania) major, the Th1 response is related to protection while the Th2 response is related to disease progression. However, in hamsters, which are considered to be an excellent model for the study of visceral leishmaniasis, this dichotomy is not observed. Using outbred 45- to 60-day-old (140 to 150 g) male hamsters infected intraperitoneally with 2 x 10(7) L. (L.) chagasi amastigotes, we evaluated the immune response of spleen cells and the production of cytokines. We used 3 to 7 hamsters per group evaluated. We detected a preserved response to concanavalin A measured by index of proliferation during all periods of infection studied, while a proliferative response to Leishmania antigen was detected only at 48 and 72 h post-infection. Messenger RNA from cytokines type 1 (IL-2, TNF-α, IFN-γ) and type 2 (IL-4, IL-10 and TGF-ß) detected by reverse transcriptase polymerase chain reaction and produced by spleen cells showed no qualitative difference between control non-infected hamsters and infected hamsters during any period of infection evaluated. Cytokines were measured by the DNA band intensity on agarose gel using the Image Lab 1D L340 software with no differences observed. In conclusion, the present results showed an antigen-dependent immunosuppression in hamsters with active visceral leishmaniasis that was not related to the cytokine profile.

DNA extraction from hair shafts of wild Brazilian felids and canids.

Wild felids and canids are usually the main predators in the food chains where they dwell and are almost invisible to behavior and ecology researchers. Due to their grooming behavior, they tend to swallow shed hair, which shows up in the feces. DNA found in hair shafts can be used in molecular studies that can unravel, for instance, genetic variability, reproductive mode and family structure, and in some species, it is even possible to estimate migration and dispersion rates in given populations. First, however, DNA must be extracted from hair. We extracted successfully and dependably hair shaft DNA from eight wild Brazilian felids, ocelot, margay, oncilla, Geoffroy's cat, pampas cat, jaguarundi, puma, and jaguar, as well as the domestic cat and from three wild Brazilian canids, maned wolf, crab-eating fox, and hoary fox, as well as the domestic dog. Hair samples came mostly from feces collected at the São Paulo Zoo and were also gathered from non-sedated pet or from recently dead wild animals and were also collected from museum specimens. Fractions of hair samples were stained before DNA extraction, while most samples were not. Our extraction protocol is based on a feather DNA extraction technique, based in the phenol:chloroform:isoamyl alcohol general method, with proteinase K as digestive enzyme.

Genetic diversity of microsatellite loci in Leopardus pardalis, Leopardus wiedii and Leopardus tigrinus.

The microsatellite loci FCA045, FCA077, FCA008, and FCA096 are highly variable molecular markers which were used to determine the genetic diversity in 148 captive Leopardus sp. The PCR-amplified products of microsatellite loci were characterized in ABI Prism 310 Genetic Analyzer. Allele numbers, heterozygosity, polymorphism information content, exclusive allele number, and shared alleles were calculated. Sixty-five alleles were found and their sizes ranged from 116 to 216 bp in four microsatellite loci. The heterozygosity ranged from 0.36 to 0.81 in Leopardus pardalis, 0.57 to 0.67 in L. tigrinus and 0.80 to 0.92 in L. wiedii. The polymorphism information content was from 0.80 to 0.88 in L. pardalis, 0.76 to 0.88 in L. tigrinus and 0.77 to 0.90 in L. wiedii. The margay (L. wiedii) showed the highest index of polymorphism among the three species in this study. These results imply that microsatellite DNA markers can help in the study of the genetic diversity of Leopardus specimens.

Genetic diversity of microsatellite loci in Leopardus pardalis, Leopardus wiedii and Leopardus tigrinus

The microsatellite loci FCA045, FCA077, FCA008, and FCA096 are highly variable molecular markers which were used to determine the genetic diversity in 148 captive Leopardus sp. The PCR-amplified products of microsatellite loci were characterized in ABI Prism 310 Genetic Analyzer. Allele numbers, heterozygosity, polymorphism information content, exclusive allele number, and shared alleles were calculated. Sixty-five alleles were found and their sizes ranged from 116 to 216 bp in four microsatellite loci. The heterozygosity ranged from 0.36 to 0.81 in Leopardus pardalis, 0.57 to 0.67 in L. tigrinus and 0.80 to 0.92 in L. wiedii. The polymorphism information content was from 0.80 to 0.88 in L. pardalis, 0.76 to 0.88 in L. tigrinus and 0.77 to 0.90 in L. wiedii. The margay (L. wiedii) showed the highest index of polymorphism among the three species in this study. These results imply that microsatellite DNA markers can help in the study of the genetic diversity of Leopardus specimens.

B and T cell responses elicited by monoclonal anti-idiotypic antibody (Ab2beta) mimicking gp43 from Paracoccidioides brasiliensis.

Paracoccidioidomycosis is a systemic mycosis endemic in Latin America, with a high prevalence in Brazil, Argentina, Colombia and Venezuela. The aetiological agent of disease is the thermal dimorphic fungus, Paracoccidioides brasiliensis. A glycoprotein of 43 kD (gp43) is the major antigen of P. brasiliensis. Antibodies directed to this antigen are detected in the sera of all patients with paracoccidioidomycosis (PCM). Recently, it has been shown that mice immunized with anti-gp43 monoclonal antibodies (MAbs) (Ab1), induce the idiotypic cascade in the gp43 system, which produced both, anti-Id antibodies (Ab2) and anti-anti-Id antibodies (Ab3). To further characterize the idiotypic cascade modulation in mice immunized with anti-gp43 MAb 17c, hybridomas were produced. Ab2 MAbs named 7.B12 inhibited (>95%) the binding of gp43 to MAb 17c (Ab1), suggesting that this anti-Id MAb bind to the idiotope, thus fulfilling the internal image criteria. To elucidate whether Ab2 MAb could act as antigen in serological assays, instead of gp43, sera from PCM patients were tested. Using an ELISA test, it was observed that antibodies from patients and not normal serum bound to Ab2. However, the ELISA test using Ab2 bound to the solid phase made possible to serologically monitor the patients after antifungal therapy, showing an equivalent curve when compared with ELISA test employing purified gp43. Our results also showed that, when mice were immunized with Ab2beta and their cells were exposed to gp43 in vitro, a T cell proliferation response was observed.

Effect of non-peptide corticotropin-releasing factor receptor type 1 antagonist on adrenocorticotropic hormone release and interleukin-1 receptors followed by stress.

Previous studies demonstrated that ether-laparotomy significantly increased iodine-125-labeled interleukin-1alpha ([125I]IL-1alpha) binding in the mouse anterior pituitary at 2 h after the onset of stress. Corticotropin-releasing factor (CRF) receptor antagonist, D-Phe CRF (12-41), abolished ether-laparotomy-induced increase in [125I]IL-1alpha binding in the pituitary, showing that CRF plays a pivotal role in the regulation of IL-1 receptors under stress conditions. In an attempt to define the effect of CRA 1000 (2-(N-(2-methylthio-4-isopropylphenyl)-N-ethylamino-4-(4-(3-fluorophenyl)-1,2,3,6-tetrahydropyridin-1-yl)-6-methylpyrimidine), a non-peptide CRF receptor type 1 antagonist on the regulation of hypothalamic-pituitary-adrenal (HPA) axis and IL-1 receptors in the mouse, we measured plasma adrenocorticotropic hormone (ACTH) and corticosterone levels, [125I]IL-1alpha binding and the expression of transcripts for type 1 IL-1 receptor (IL-1R1 mRNA) in the pituitary at 2 h after endotoxin lipopolysaccharide (LPS) treatment or ether-laparotomy stress with or without CRA 1000 pretreatment. A single injection of LPS dramatically increased plasma ACTH and corticosterone levels compared with saline injection. In contrast, plasma ACTH levels were significantly attenuated in response to one LPS injection following oral CRA 1000 pretreatment. LPS-induced plasma corticosterone levels tended to be lower after CRA 1000 pretreatment but it did not reach statistical significance. Ether-laparotomy stress significantly increased plasma ACTH and corticosterone levels at 2 h after the onset of stress and CRA 1000 pretreatment did not affect the peak ACTH and corticosterone levels following stress. Ether-laparotomy stress resulted in a robust increase in [125I]IL-1alpha binding and IL-1R1 mRNA levels in the pituitary. CRA 1000 pretreatment significantly decreased ether-laparotomy stress-induced IL-1R1 mRNA levels but did not affect [125I]IL-1alpha binding. Pretreatment with CRA 1000 without stress significantly increased [125I]IL-1alpha binding and IL-1R1 mRNA levels compared with those in vehicle pretreatment. These data demonstrate differential effects of CRA 1000 in HPA axis following endotoxin and ether-laparotomy stress and complex interactions between CRF and IL-1 receptors during stress.

Performance-enhancing effects of CRF-BP ligand inhibitors.

Intracerebroventricular (i.c.v.) administration of corticotropin-releasing factor (CRF) peptide fragments with low affinity for CRF receptors reportedly improves cognitive performance without producing anxiety. These compounds are hypothesized to act by displacing endogenous peptide from the CRF-binding protein (CRF-BP). To test this hypothesis, the present study determined whether the performance-enhancing potency of CRF fragments was related to their affinity for the CRF-BP. Rank ordering of the optimal doses of these compounds for facilitating spatial navigation corresponded to their affinity for the CRF-BP. i.c.v. pretreatment with performance-enhancing doses of r/h CRF(1-41)-OH (5 micrograms) or r/h CRF(6-33) (25 micrograms) did not increase emotionality. These findings replicate the dissociability of the cognition- and anxiety-related effects of CRF-related compounds and suggest that CRF fragments facilitate performance via the CRF-BP.

Effect of orchidectomy on the age-related modulation of IL-1beta and IL-1 receptors following lipopolysaccharide treatment in the mouse.

OBJECTIVE: To compare the effect of orchidectomy (ODX) in 7- and 24-week-old C57BL/6 mice on the age-related responses of the cytokine interleukin (IL)-1beta and its receptor to intraperitoneal injection of the bacterial endotoxin, lipopolysaccharide (LPS). METHODS: We measured IL-1beta concentrations in the plasma, hippocampus, hypothalamus and adrenal gland using ELISA and iodine-125-labeled recombinant human IL-1alpha ([(125)I]IL-1alpha) binding in the hippocampus following the intraperitoneal administration of saline or LPS. RESULTS: There were no significant differences in the concentrations of IL-1beta and its receptors in the brain and peripheral tissues between sham-operated and ODX mice in both age groups injected with saline. LPS induced significantly higher IL-1beta production in the plasma and hippocampus in sham-operated 24-week-old mice than in 7-week-old mice. Coincident with the heightened IL-1beta response to LPS, hippocampal [(125)I]IL-1alpha binding was lower in 24-week-old mice than in 7-week-old mice after LPS injection in the sham-operated group. The age-related differences in the IL-1beta concentrations in the plasma and hippocampus and [(125)I]IL-1alpha binding in the hippocampus in response to LPS administration were abolished by ODX. Although LPS dramatically increased IL-1beta levels in the hypothalamus, no significant age-related differences in IL-1beta concentrations were seen, and ODX did not affect IL-1beta levels. In contrast, there were no significant differences between saline- and LPS-injected 7-week-old mice in relation to concentrations in the adrenal gland. Moreover, although the adrenal IL-1beta concentrations in 24-week-old mice were significantly higher than those in 7-week-old mice, ODX did not abolish these age-related differences in concentrations in the adrenal gland. CONCLUSIONS: Our data suggest the involvement of testosterone (or gonadal product) in plasma and hippocampal IL-1beta regulation in relation to age, and demonstrate the importance of the gonadal development in mediating the effects of infectious challenge on the brain and immune function.

Nephroblastoma overexpressed gene (NOV) codes for a growth factor that induces protein tyrosine phosphorylation.

NOV (nephroblastoma overexpressed gene) is a member of the CCN (connective tissue growth factor [CTGF], Cyr61/Cef10, NOV) family of proteins. These proteins are cysteine-rich and are noted for having growth-regulatory functions. We have isolated the rat NOV gene, and the DNA sequence shares 90% identity with the mouse and 80% identity with the human sequences. The rat NOV gene was expressed in all rat tissues examined, including brain, lung, heart, kidney, liver, spleen, thymus and skeletal muscle. Higher levels of rat NOV mRNA were seen in the brain, lung and skeletal muscle compared to the other tissues. Examination of NOV expression in various human cell lines revealed that NOV was expressed in U87, 293, T98G, SK-N-MC and Hs683 but not in HepG2, HL60, THP1 and Jurkat. The human NOV gene was transfected into 293 cells and the expressed protein purified. When 3T3 fibroblasts were treated with this recombinant NOV protein, a dose-dependent increase in proliferation was observed. Analysis of tyrosine-phosphorylated proteins revealed that when 3T3 cells were treated with NOV, a 221 kDa protein was phosphorylated. These data suggest that NOV can act as a growth factor for some cells and binds to a specific receptor that leads to the phosphorylation of a 221 kDa protein.

Differential effects of one and repeated endotoxin treatment on pituitary- adrenocortical hormones in the mouse: role of interleukin-1 and tumor necrosis factor-alpha.

The role of endogenous interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) in modulating the hypothalamic-pituitary-adrenal (HPA) axis response was examined in male C57BL/6 mice injected with endotoxin (lipopolysaccharide, LPS) or saline at 24-hour intervals for 4 or 8 consecutive days. The mice were divided into four groups: (1) LPS injections for 4 or 8 days and LPS injection on day 5 or 9, respectively (LPS-LPS); (2) LPS injections for 4 or 8 days and saline injection on day 5 or 9, respectively (LPS-saline); (3) saline injections for 4 or 8 days and LPS injection on day 5 or 9, respectively (saline-LPS), and (4) saline injections for 4 or 8 days and saline injection on day 5 or 9 (saline-saline). The mice were sacrificed by decapitation 2 h after the last injection and plasma levels of hormones and cytokines and tissue levels of IL-1beta were measured. Plasma adrenocorticotropin (ACTH) levels were significantly attenuated in the LPS-LPS group compared with the dramatic increases in the saline-LPS group following 4 or 8 days of endotoxin treatment. Plasma corticosterone concentrations were comparable in the LPS-LPS group after 4 days' treatment, but significantly lower following 8 days of treatment when compared with saline-LPS group. Repeated endotoxin treatment followed by a single saline injection (LPS-saline) did not alter the levels of IL-1beta in plasma or any of the tissues examined. IL-1beta levels in the hippocampus, hypothalamus, adrenal gland and plasma were elevated to comparable levels in the saline-LPS and LPS-LPS groups after 4 days of treatment. In contrast to the plasma IL-1beta response, TNFalpha levels were dramatically increased in the saline-LPS group but not in the LPS-LPS group following the 4-day treatment regimen. Increases in IL-1beta concentrations were seen in all tissues following one endotoxin challenge in the saline-LPS group following the 8-day treatment regimen, while increases were significantly attenuated in the hypothalamus, adrenal gland and plasma in LPS-LPS for 8 days. The sustained increases in tissue levels of IL-1beta following 4-day endotoxin treatment appears to have functional consequences since [125I]IL-1alpha binding was significantly decreased in the LPS-saline group compared with the saline-saline group. Furthermore, [125I]IL-1alpha binding was markedly reduced in the LPS-LPS group compared with the saline-LPS group. There was a significant positive correlation between plasma ACTH and IL-1beta after a single and repeated LPS treatment for 4 days, while a significant correlation was seen between plasma ACTH and TNFalpha following one but not repeated LPS treatment. These data demonstrate a differential regulation of IL-1beta and TNFalpha by repeated endotoxin treatment and suggest that while TNFalpha may be important modulating the attenuated pituitary adrenocortical response following the 4-day endotoxin treatment, IL-1beta appears to be the primary regulator of the response following the 8-day endotoxin treatment in the regulation of the HPA axis.

Chemokines and chemokine receptors in the CNS: a possible role in neuroinflammation and patterning.

Chemokines constitute a growing family of structurally and functionally related small (8-10 kDa) proteins associated with inflammatory-cell recruitment in host defence. In addition to their well-established role in the immune system, recent data suggest their involvement in the maintenance of CNS homeostasis, in neuronal patterning during ontogeny and as potential mediators of neuroinflammation, playing an essential role in leukocyte infiltration into the brain. Chemokines and their G protein-coupled receptors are constitutively expressed at low-to-negligible levels in various cell types in the brain. Their expression is rapidly induced by various neuroinflammatory stimuli, implicating them in various neurological disorders such as trauma, stroke and Alzheimer's disease, in tumour induction and in neuroimmune diseases such as multiple sclerosis or acquired immunodeficiency syndrome (AIDS). Here, F. Mennicken, R. Maki, E. B. De Souza and R. Quirion briefly summarize recent exciting findings in the field.

Non-peptide corticotropin-releasing hormone antagonists: syntheses and structure-activity relationships of 2-anilinopyrimidines and -triazines.

Screening of our chemical library using a rat corticotropin-releasing hormone (CRH) receptor assay led to the discovery that 2-anilinopyrimidine 15-1 weakly displaced [125I]-0-Tyr-oCRH from rat frontal cortex homogenates when compared to the known peptide antagonist alpha-helical CRH(9-41) (Ki = 5700 nM vs 1 nM). Furthermore, 15-1 weakly inhibited CRH-stimulated adenylate cyclase activity in the same tissue, but it was less potent than alpha-helical CRH(9-41) (IC50 = 20 000 nM vs 250 nM). Systematic structure-activity relationship studies, using the cloned human CRH1 receptor assay, defined the pharmacophore for optimal binding to hCRH1 receptors. Several high-affinity 2-anilinopyrimidines and -triazines were discovered, some of which had superior pharmacokinetic profiles in the rat. This paper describes the structure-activity studies which improved hCRH1 receptor binding affinity and pharmacokinetic parameters in the rat. Compound 28-17 (mean hCRH1 Ki = 32 nM) had a significantly improved pharmacokinetic profile in the rat (19% oral bioavailability at 30 mg/kg) as well as in the dog (20% oral bioavailability at 5 mg/kg) relative to the early lead structures.

Corticotropin-releasing factor antagonists, binding-protein and receptors: implications for central nervous system disorders.

Corticotrophin-releasing factor (CRF; interchangeable with corticotrophin-releasing hormone, CRH) is a neurohormone family of peptides which implements endocrine, physiological and behavioural responses to stressor exposure. Built-in biological diversity and selectivity of CRF system function is provided by multiple endogenous ligands and receptors which are heterogeneously distributed in both brain and peripheral tissues across species. At present, there are at least five distinct targets for CRF with unique cDNA sequences, pharmacology and localization. These fall into three distinct classes, encoded by three different genes and have been termed the CRF1 and CRF2 receptors and the CRF-binding protein. Significant gains in knowledge about the physiological role of CRF binding sites in brain have emerged recently due to the proliferation of novel, high-affinity, receptor-selective pharmacological tools as well as multiple knock-out and knock-in mutant mouse models. These results support a role for CRF binding sites in co-ordinating stress reactivity, emotionality and energy balance over the life-span of the organism.

Characterization of [125I-Tyr0]-corticotropin releasing factor (CRF) binding to the CRF binding protein using a scintillation proximity assay.

We describe the characterization of high affinity [125I-Tyr0]-human CRF binding to purified recombinant human CRF-binding protein (CRF-BP) using a scintillation proximity assay (SPA). For this stable nonseparation technique developed in 96 well microtiter plates, biotinylated CRF-BP is captured by streptavidin-coated SPA beads for the detection of bound [125I-Tyr0]-CRF. Unbound [125I-Tyr0]-CRF represented little or no signal in the assay. Total binding observed was greater than 5000 cpm with a nonspecific signal of < 100 cpm determined in the presence of excess unlabeled human CRF. A comparison of the SPA method with a charcoal precipitation method confirmed that the biotinylation procedure did not adversely affect affinity of the CRF-BP for [125I-Tyr0]-CRF. Saturation binding analysis yielded an apparent equilibrium dissociation constant (Kd) of 208 +/- 5.0 pM (+/- S.D., n = 3). An inhibition constant (Ki) for unlabeled CRF was calculated to be 0.22 +/- 0.03 nM (+/- S.D., n = 8) and a pharmacological profile for eight CRF-related neuropeptides gave a rank potency similar to previously reported results. Finally, the assay variability was assessed with intra- and inter-plate coefficients of variation which were less than 5% each.