Riluzole elevates GLT-1 activity and levels in striatal astrocytes

Drugs which upregulate astrocyte glutamate transport may be useful neuroprotective compounds by preventing excitotoxicity. We set up a new system to identify potential neuroprotective drugs which act through GLT-1. Primary mouse striatal astrocytes grown in the presence of the growth-factor supplement G5 express high levels of the functional glutamate transporter, GLT-1 (also known as EAAT2) as assessed by Western blotting and (3)H-glutamate uptake assay, and levels decline following growth factor withdrawal. The GLT-1 transcriptional enhancer dexamethasone (0.1 or 1muM) was able to prevent loss of GLT-1 levels and activity following growth factor withdrawal. In contrast, ceftriaxone, a compound previously reported to enhance GLT-1 expression, failed to regulate GLT-1 in this system. The neuroprotective compound riluzole (100muM) upregulated GLT-1 levels and activity, through a mechanism that was not dependent on blockade of voltage-sensitive ion channels, since zonasimide (1mM) did not regulate GLT-1. Finally, CDP-choline (10muM-1mM), a compound which promotes association of GLT-1/EAAT2 with lipid rafts was unable to prevent GLT-1 loss under these conditions. This observation extends the known pharmacological actions of riluzole, and suggests that this compound may exert its neuroprotective effects through an astrocyte-dependent mechanism.

Stimulation of the p38 mitogen-activated protein kinase pathway in neonatal rat ventricular myocytes by the G protein-coupled receptor agonists, endothelin-1 and phenylephrine: a role in cardiac myocyte hypertrophy?

We examined the activation of the p38 mitogen-activated protein kinase (p38-MAPK) pathway by the G protein-coupled receptor agonists, endothelin-1 and phenylephrine in primary cultures of cardiac myocytes from neonatal rat hearts. Both agonists increased the phosphorylation (activation) of p38-MAPK by approximately 12-fold. A p38-MAPK substrate, MAPK-activated protein kinase 2 (MAPKAPK2), was activated approximately fourfold and 10 microM SB203580, a p38-MAPK inhibitor, abolished this activation. Phosphorylation of the MAPKAPK2 substrate, heat shock protein 25/27, was also increased. Using selective inhibitors, activation of the p38-MAPK pathway by endothelin-1 was shown to involve protein kinase C but not Gi/Go nor the extracellularly responsive kinase (ERK) pathway. SB203580 failed to inhibit the morphological changes associated with cardiac myocyte hypertrophy induced by endothelin-1 or phenylephrine between 4 and 24 h. However, it decreased the myofibrillar organization and cell profile at 48 h. In contrast, inhibition of the ERK cascade with PD98059 prevented the increase in myofibrillar organization but not cell profile. These data are not consistent with a role for the p38-MAPK pathway in the immediate induction of the morphological changes of hypertrophy but suggest that it may be necessary over a longer period to maintain the response.

Population biology and regeneration of forbs and shrubs after fire in Brazilian Campos grasslands

Fire is an important factor in several ecosystems, affecting plant population biology. Campos grasslands are under constant influence of disturbance, mostly grazing and fire. However, few studies evaluated the effect of fire on plant population biology of grassland species. Therefore, we aim to analyze the effect of fire on the population biology of four species, from different functional groups and regeneration strategies: Chaptalia runcinata (forb, resprouter, absence of belowground organ), Vernonia flexuosa (forb, resprouter, presence of rhizophore), Eupatorium ligulaefolium (shrub, resprouter, presence of xylopodium) and Heterothalamus psiadioides (shrub, obligate seeder). Seven plots were established in different sites in southern Brazil: frequently burned (FB) and excluded from fire since 6 years (E). All plots were subjected to controlled burns during summer. Before experiments, populations were sampled. Further observations were carried out after 90 and after 360 days of fire experiments. In addition, we counted the number of seedlings and resprouters recruited after fire. Heat shock experiments were conducted with two species (H. psiadioides and V. flexuosa), as well as the study of the bud bank of the following species: E. ligulaefolium and V. flexuosa. The obligate seeder species had all individuals killed by fire and established only after 1 year. Resprouters, however, showed new stems immediately after fire. E. ligulaefolium and V. flexuosa showed only vegetative regeneration from belowground organs and more individuals in excluded sites 1 year after the fire. The bud bank of E. ligulaefolium tended to be larger in excluded sites, whilst V. flexuosa showed an opposite result. High temperatures did not enhance nor kill seeds from both studied species. Vegetative regeneration was the most important strategy for all studied species, except for H. psiadioides, the obligate seeder species. Fire thus, plays an important role on population structure and demography, being also important for plant recruitment.

Fasting differentially regulates plasma corticosterone-binding globulin, glucocorticoid receptor, and cell cycle in the gastric mucosa of pups and adult rats

Ogias D, de Andrade Sa ER, Kasai A, Moisan M, Alvares EP, Gama P. Fasting differentially regulates plasma corticosterone-binding globulin, glucocorticoid receptor, and cell cycle in the gastric mucosa of pups and adult rats. Am J Physiol Gastrointest Liver Physiol 298: G117-G125, 2010. First published October 15, 2009; doi:10.1152/ajpgi.00245.2009.-The nutritional status influences gastric growth, and interestingly, whereas cell proliferation is stimulated by fasting in suckling rats, it is inhibited in adult animals. Corticosterone takes part in the mechanisms that govern development, and its effects are regulated in particular by corticosterone-binding globulin (CBG) and glucocorticoid receptor (GR). To investigate whether corticosterone activity responds to fasting and how possible changes might control gastric epithelial cell cycle, we evaluated different parameters during the progression of fasting in 18- and 40-day-old rats. Food restriction induced higher corticosterone plasma concentration at both ages, but only in pups did CBG binding increase after short-and long-term treatments. Fasting also increased gastric GR at transcriptional and protein levels, but the effect was more pronounced in 40-day-old animals. Moreover, in pups, GR was observed in the cytoplasm, whereas, in adults, it accumulated in the nucleus after the onset of fasting. Heat shock protein (HSP) 70 and HSP 90 were differentially regulated and might contribute to the stability of GR and to the high cytoplasmic levels in pups and elevated shuttling in adult rats. As for gastric epithelial cell cycle, whereas cyclin D1 and p21 increased during fasting in pups, in adults, cyclin E slowly decreased, concomitant with higher p27. In summary, we demonstrated that corticosterone function is differentially regulated by fasting in 18-and 40-day-old rats, and such variation might attenuate any possible suppressive effects during postnatal development. We suggest that this mechanism could ultimately increase cell proliferation and allow regular gastric growth during adverse nutritional conditions.

The role of glucocorticoid in SIRP alpha and SHP-1 gene expression in AIHA patients

The aim of this work was to evaluate the regulation of SIRP alpha, an inhibitory phagocyte receptor, and the phosphatase SHP-1 in monocytes of patients with autoimmune hemolytic anemia, and the role of dexamethasone on SIRP alpha and SHP-1 gene expression and erythrophagocytosis in vitro. SIRP alpha and SHP-1 expression was higher in monocytes from AIHA patients compared with normal, returning to normal after glucocorticoid therapy. SIRP alpha and SHP-1 mRNA expression was upregulated in healthy monocytes treated with dexamethasone compared with basal; however, the erythrophagocytic ability was not altered. Our results point to a minor role of SIRP alpha and SHP-1 in determining AIHA.

Deficiency of thrombospondin-1 reduces Th17 differentiation and attenuates experimental autoimmune encephalomyelitis

Transforming growth factor beta (TGF-beta) plays a role both in the induction of Treg and in the differentiation of the IL-17-secreting T cells (Th17) which drive inflammation in experimental autoimmune encephalomyelitis (EAE). We investigated the role that thrombospondin-1 (TSP-1) dependent activation of TGF-beta played in the generation of an encephalitic Th17 response in EAE. Upon immunization with myelin oligodendrocyte glycoprotein peptide (MOG(35-55)), TSP-1 deficient (TSP-1(null)) mice and MOG(35-55) TCR transgenic mice that lack of TSP-1 (2D2.TSP-1(null)) exhibited an attenuated form of EAE, and secreted lower levels of IL-17. Adoptive transfer of in vitro-activated 2D2.TSP-1(null) T cells induced a milder form of EAE, independent of TSP-1 expression in the recipient mice. Furthermore, in vitro studies demonstrated that anti-CD3/anti-CD28 pre-activated CD4+ T cells transiently upregulated latent TGF-beta in a TSP-1 dependent way, and such activation of latent TGF-beta was required for the differentiation of Th17 cells. These results demonstrate that TSP-1 participates in the differentiation of Th17 cells through its ability to activate latent TGF-beta, and enhances the inflammatory response in EAE. (C) 2009 Elsevier Ltd. All rights reserved.

Autolytic Mycobacterium leprae Hsp65 fragments may act as biological markers for autoimmune diseases

Investigating the proteolytic activity of the recombinant Mycobacterium leprae Heat Shock Protein of 65 kDa (rHsp65), chaperonin 2 (cpn2), we observed that it displays high instability. The fragmentation process starts at the C-terminus followed by progressive degradation of the N-terminus, which leads to a stable fragment comprising the middle region of the molecule. Urea was able to prevent autolysis, probably due to its denaturing action, while EDTA increased degradation levels indicating the need for metal ions. Peptides originated from autolysis were purified and analyzed by mass spectrometry, generating a continuous map. Since the bacteria and mammalian Hsp60 are known to be targets of the immune response and have been implicated in autoimmune diseases and chronic inflammation, the in vivo effect of rHsp65 peptides was evaluated in the spontaneous Systemic Lupus Erythematosus (SLE) model developed by the (NZB/NZW)F(1) mouse hybrids, and their individual anti-rHsp65 IgG2a/IgG1 antibody titer ratio was determined. The results showed orientation toward a T(H)1 responsiveness, and the treatment with the rHsp65 peptides diminished the environmental variance of the survival time of treated animals. These results outline the fact that environmental factors may also act through the modified stability expression of Heat Shock Proteins intervening during autoimmune processes. (C) 2011 Elsevier Ltd. All rights reserved.

Interleukin-10 attenuates vascular responses to endothelin-1 via effects on ERK1/2-dependent pathway

Giachini FR, Zemse SM, Carneiro FS, Lima VV, Carneiro ZN, Callera GE, Ergul A, Webb RC, Tostes RC. Interleukin-10 attenuates vascular responses to endothelin-1 via effects on ERK1/2-dependent pathway. Am J Physiol Heart Circ Physiol 296: H489-H496, 2009. First published December 12, 2008; doi:10.1152/ajpheart.00251.2008.-Interleukin-10 (IL-10) is an anti-inflammatory cytokine with protective actions on the vasculature. On the other hand, endothelin ( ET)-1 has potent vasoconstrictor, mitogenic, and proinflammatory activities, which have been implicated in the pathophysiology of a number of cardiovascular diseases. We hypothesized that, in a condition where ET-1 expression is upregulated, i.e., on infusion of TNF-alpha, IL-10 confers vascular protection from ET-1-induced injury. Aortic rings and first-order mesenteric arteries from male C57BL/6 (WT) and IL-10-knockout (IL-10(-/-)) mice were treated with human recombinant TNF-alpha (220 ng.kg(-1).day(-1)) or vehicle (saline) for 14 days. TNF-alpha infusion significantly increased blood pressure in IL-10(-/-), but not WT, mice. TNF-alpha augmented vascular ET-1 mRNA expression in arteries from WT and IL-10(-/-) mice. ET type A (ETA) receptor expression was increased in arteries from IL-10(-/-) mice, and TNF-alpha infusion did not change vascular ETA receptor expression in control or IL-10(-/-) mice. Aorta and mesenteric arteries from TNF-alpha-infused IL-10(-/-) mice displayed increased contractile responses to ET-1, but not the ET type B receptor agonist IRL-1620. The ETA receptor antagonist atrasentan completely abolished responses to ET-1 in aorta and mesenteric vessels, whereas the ERK1/2 inhibitor PD-98059 abrogated increased contractions to ET-1 in arteries from TNF-alpha-infused IL-10(-/-) mice. Infusion of TNF-alpha, as well as knockdown of IL-10 (IL-10(-/-)), induced an increase in total and phosphorylated ERK1/2. These data demonstrate that IL-10 counteracts ET(A)-mediated vascular responses to ET-1, as well as activation of the ERK1/2 pathway.

TLR2 and TLR4 expression after kidney ischemia and reperfusion injury in mice treated with FTY720

Ischemia and reperfusion injury (IR) is an antigen independent inflammatory process that causes tissue damage. After IR, kidneys up-regulate leukocyte adhesion molecules and toll-like receptors (TLRs). Moreover, injured kidneys can also secrete factors (i.e. heat shock protein) which bind to TLRs and trigger intracellular events culminating with the increase in the gene expression of inflammatory cytokines. FTY720 is an immunomodulatory compound and protects at least in part kidneys submitted to IR. The mechanisms associated with FTY720`s beneficial effects on kidneys after IR remain elusive. We investigated whether FTY720 administration in mice submitted to kidney IR is associated with modulation of TLR2 and TLR4 expression. C57BL/6 mice submitted to 30 min of renal pedicles clamp were evaluated for serum parameters (creatinine, urea and nitric oxide), kidney histology, spleen and kidney infiltrating cells expression of TLR2 and TLR4, resident kidney cells expression of TLR2 and TLR4 and IL-6 protein expression in kidney. FTY720-treated mice presented decrease in serum creatinine, urea and nitric oxide, diminished expression of TLR2 and TLR4 both in spleen and kidney infiltrating cells, and reduced kidney IL-6 protein expression in comparison with IR non-treated mice. However, acute tubular necrosis was present both in IR non-treated and IR + FTY720-treated groups. Also, FTY720 did not prevent TLR2 and TLR4 expression in kidney resident cells. In conclusion, FTY720 can promote kidney function recovery after IR by reducing the inflammatory process. Further studies are needed in order to establish whether TLR2 and TLR4 down regulation should be therapeutically addressed as protective targets of renal function and structure after IR. (C) 2011 Elsevier B.V. All rights reserved.

MuRF1 is a muscle fiber-type II associated factor and together with MuRF2 regulates type-II fiber trophicity and maintenance

MuRF1 is a member of the RBCC (RING, B-box, coiled-coil) superfamily that has been proposed to act as an atrogin during muscle wasting. Here, we show that MuRF1 is preferentially induced in type-II muscle fibers after denervation. Fourteen days after denervation, MuRF1 protein was further elevated but remained preferentially expressed in type-II muscle fibers. Consistent with a fiber-type dependent function of MuRF1, the tibialis anterior muscle (rich in type-II muscle fibers) was considerably more protected in MuRF1-KO mice from muscle wasting when compared to soleus muscle with mixed fiber-types. We also determined fiber-type distributions in MuRF1/MuRF2 double-deficient KO (dKO) mice, because MuRF2 is a close homolog of MuRF1. MuRF1/MuRF2 dKO mice showed a profound loss of type-II fibers in soleus muscle. As a potential mechanism we identified the interaction of MuRF1/MuRF2 with myozenin-1, a calcineurin/NFAT regulator and a factor required for maintenance of type-II muscle fibers. MuRF1/MuRF2 dKO mice had lost myozenin-1 expression in tibialis anterior muscle, implicating MuRF1/MuRF2 as regulators of the calcineurin/NFAT pathway. In summary, our data suggest that expression of MuRF1 is required for remodeling of type-II fibers under pathophysiological stress states, whereas MuRF1 and MuRF2 together are required for maintenance of type-II fibers, possibly via the regulation of myozenin-1. (C) 2010 Elsevier Inc. All rights reserved.

Transcriptome analysis of Taenia solium cysticerci using Open Reading Frame ESTs (ORESTES)

Background: Human infection by the pork tapeworm Taenia solium affects more than 50 million people worldwide, particularly in underdeveloped and developing countries. Cysticercosis which arises from larval encystation can be life threatening and difficult to treat. Here, we investigate for the first time the transcriptome of the clinically relevant cysticerci larval form. Results: Using Expressed Sequence Tags (ESTs) produced by the ORESTES method, a total of 1,520 high quality ESTs were generated from 20 ORESTES cDNA mini-libraries and its analysis revealed fragments of genes with promising applications including 51 ESTs matching antigens previously described in other species, as well as 113 sequences representing proteins with potential extracellular localization, with obvious applications for immune-diagnosis or vaccine development. Conclusion: The set of sequences described here will contribute to deciphering the expression profile of this important parasite and will be informative for the genome assembly and annotation, as well as for studies of intra- and inter-specific sequence variability. Genes of interest for developing new diagnostic and therapeutic tools are described and discussed.

Expressão gênica dos receptores toll-símele e sua relação com citocinas potencialmente envolvidas em lesão de reperfusão durante a fase inicial do transplante pulmonar em humanos

Introdução: Imunidade inata é a primeira linha de defesa do hospedeiro contra microorganismos invasores, a qual é mediada por moléculas específicas que reconhecem patógenos, chamadas receptores toll-símile (TLRs). Os TLRs são também capazes de reconhecer ligantes endógenos, tais como conteúdos de células necróticas e proteínas de choque térmico (HSP), resultando na produção de citocinas e ativação do sistema imune adquirido. A função exata dos TLRs ainda é pouco entendida em transplante de órgãos. No entanto, tem sido sugerido que eles podem estar envolvidos na rejeição aguda ou crônica e atuar na resposta do enxerto a lesão por isquemia e reperfusão. Objetivo: Examinar as alterações na expressão gênica dos TLRs durante a fase inicial do transplante pulmonar em humanos e sua relação com citocinas potencialmente envolvidas na lesão por isquemia e reperfusão em transplante de órgãos. Métodos: Foram analisadas biópsias pulmonares de 14 pacientes submetidos a transplante pulmonar (LTx). Estas amostras foram coletadas no final do período de isquemia fria (TIF, n=14), no final do período de isquemia quente (TIQ, n=13),1 hora (n=12) e 2 horas (n=8) após a reperfusão do enxerto. RNA total foi isolado a partir de tecido pulmonar e os níveis de RNA mensageiro (mRNA) dos TLRs (1-10) bem como citocinas (IL-8, IL-6, IL-10, IFN-γ, IL-1β) e proteína de choque térmico 70 (HSP70) foram medidos por reação em cadeia pela polimerase em tempo real. Resultados: Foi detectada a expressão de mRNA de todos TLRs em tecido pulmonar. Nas amostras no TIF, os níveis de mRNA dos TLRs apresentaram-se com diferentes expressões gênicas. Os níveis de expressão dos TLRs, com exceção para o TLR3, estavam altamente correlacionados entre si no TIF e com os níveis de mRNA de IFN-γ, IL-10 e IL-1β e menos significativamente com os níveis de IL-6 e IL-8. Houve diminuição dos níveis de mRNA na grande maioria dos TLRs após reperfusão, o que foi diferente para a maioria das citocinas e HSP70, que apresentaram tendência a aumentar após transplante. A expressão gênica de TLR4 apresentou-se correlacionada com os níveis de IL-8 e IL-1β antes e após transplante (P<0.05). Pulmões de doadores que foram intubados por períodos acima de 72 horas (n=5) apresentaram níveis mais elevados de TLR2 e TLR10 (P<0.05). Conclusão: Pela primeira vez, foi demonstrado que a expressão dos TLRs altera-se durante o período de isquemia e reperfusão em transplante pulmonar em humanos. O tempo de intubação dos doadores pulmonares pode influenciar a expressão de receptores Toll-símile específicos. A correlação entre TLR4 e IL-8/IL-1β sugere que os TLRs pulmonares podem ter alguma função na resposta precoce do enxerto.

Estudo do papel da proteína multifuncional APE1/Ref-1 sobre a resposta inflamatória na meningite bacteriana

Despite advances in antibiotic therapy, bacterial meningitis (BM) remains with high mortality and morbidity rates in worldwide. One important mechanism associated to sequels during disease is the intense inflammatory response which promotes an oxidative burst and release of reactive oxygen species, consequently leading to cell death. Activation of DNA repair enzymes during oxidative stress has been demonstrated in several neurological disorders. APE1/Ref-1 is a multifunctional protein involved in DNA repair and plays a redox function on transcription factors such as NFkB and AP-1.The aim of this study was assess the role of APE1/Ref-1 on inflammatory response and the possibility of its modulation to reduce the sequels of the disease. Firstly it was performed an assay to measure cytokine in cerebrospinal fluid of patients with BM due to Streptococcus pneumoniae and Neisseriae meningitides. Further, a cellular model of inflammation was used to observe the effect of the inhibition of the endonuclease and redox activity of APE1/Ref-1 on cytokine levels. Additionally, APE1/Ref-1 expression in cortex and hippocampus of rat with MB after vitamin B6 treatment was evaluated. Altogether, results showed a similar profile of cytokines in the cerebrospinal fluid of patients from both pathogens, although IFNy showed higher expression in patients with BM caused by S. pneumoniae. On the other hand, inhibitors of APE1/Ref-1 reduced cytokine levels, mainly TNF-α. Reduction of oxidative stress markers was also observed after introduction of inhibitors in the LPS-stimulated cell. In the animal model, BM increased the expression of the protein APE1/Ref-1, while vitamin B6 promoted reduction. Thereby, this data rise important factors to be considered in pathogenesis of BM, e.g., IFNy can be used as prognostic factor during corticosteroid therapy, APE1/Ref-1 can be an important target to modulate the level of inflammation and VIII oxidative stress, and vitamin B6 seems modulates several proteins related to cell death. So, this study highlights a new understanding on the role of APE1/Ref-1 on the inflammation and the oxidative stress during inflammation condition

Characterization of heat waves affecting mortality rates of broilers between 29 days and market age

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)