IL-12 family cytokines IL-23 and IL-27 play an important role linking innate and adaptive immunity, and regulating T-cell responses. The production of IL-12, a structurally similar cytokine, is decreased in chronic HIV infection; therefore IL-23 and IL-27 may also be influenced by HIV infection. I hypothesized that HIV inhibits LPS-induced IL-23 and IL-27 production in human MDMs by suppressing the activation of signalling pathways regulating their expression. In vitro HIV-infection of MDMs did not have any effect on basal secretion of IL-23 or IL-27; however, HIV inhibited LPS-induced production of IL-12/23 p40 and IL-23 p19, and IL-27 EBI3 and IL-27 p28 mRNA expression, and IL-23, IL-12/23 p40 and IL-27 secretion. In order to evaluate the molecular mechanisms by which HIV inhibits IL-23 and IL-27 in LPS-stimulated MDMs, the signalling pathways regulating their expression were evaluated. The PI3K, p38 MAPK, and JNK MAPK pathways were found to positively regulate LPS-induced IL-27 secretion. Interestingly, in vitro HIV infection inhibited LPS-induced p38 and JNK MAPK activation in MDMs. In summary, I have shown that HIV inhibits IL-23 and IL-27 production in LPS-stimulated MDMs and that HIV may inhibit LPS-induced IL-27 production through the inhibition of p38 and JNK MAPK activation. It is currently unknown whether PKCs regulate LPS-induced IL-23 or IL-27 in human monocytes/macrophages. I demonstrated that classical PKCs differentially regulate LPS-induced IL-23 and IL-27 secretion within THP-1 cells, primary monocytes, and MDMs. Classical PKCs were found to positively regulate LPS-induced IL-12/23 p40 and IL-27 p28 mRNA expression and IL-12/23 p40, IL-23, and IL-27 secretion in primary human monocytes. Similarly, the classical PKCs were found to positively regulate IL-27 p28 mRNA expression and IL-27 secretion in THP-1 cells. However, classical PKCs did not regulate LPS-induced IL-27 production in MDMs, or LPS-induced IL-23 production in THP-1 cells. Overall, this demonstrates that classical PKCs differentially regulate LPS-induced IL-23 and IL-27 production in different myeloid cells.

The formation of the mRNP complex is a critical component of translational regulation and mRNA decay. Both the 5 ' and 3 ' UTRs of CSF-1 mRNA are involved in post-transcriptional regulation. In CSF-1 mRNA, a small hairpin loop structure is predicted to form at the extreme 5 ' end (2-21 nt) of the 5 ' UTR. Nucleolin binds the hairpin loop structure in the 5 ' UTR of CSF-1 mRNA and enhances translation, while removal of this hairpin loop nucleolin binding element dramatically represses translation. Thus in CSF-1 mRNA, the hairpin loop nucleolin binding element is critical for translational regulation. In addition, nucleolin interacts with the 3 ' UTR of CSF-1 mRNA and facilitates the miRISC formation which results in poly (A) tail shortening. The overexpression of nucleolin increases the association of CSF-1 mRNA containing short poly (A)(n), <= 26, with polyribosomes. Nucleolin both forms an mRNP complex with the eIF4G and CSF-1 mRNA, and is co-localized with the eIF4G in the cytoplasm further supporting nucleolin's role in translational regulation. The distinct foci formation of nucleolin in the cytoplasm of ovarian and breast cancer cells implicates the translational promoting role of nucleolin in these cancers.

Telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase and the limiting factor for the enzyme activity. The expression of TERT and telomerase activity is increased in atherosclerotic plaques. However, the role of TERT dysregulation during atherosclerosis formation remains unknown. The work herein first identified a multi-tiered regulation of TERT expression in smooth muscle cells (SMC) through histone deacetylase (HDAC) inhibition. HDAC inhibition induces TERT transcription and promoter activation. At the protein level in contrast, HDAC inhibition decreases TERT protein abundance through enhanced degradation, which decreases telomerase activity and induces senescence. Furthermore, during vascular remodeling in vivo, TERT protein expression in the neointima is prevented by HDAC inhibition. These data illustrate a differential regulation of TERT transcription and protein stability by HDAC inhibition. TERT is highly expressed in replicating SMC of atherosclerotic and neointimal lesions. Using a model of guidewire-induced arterial injury, neointima formation was reduced in TERT-deficient mice. Studies in SMC isolated from TERT-deficient and TERT overexpressing mice with normal telomere length established that TERT is necessary and sufficient for cell proliferation. TERT deficiency did not induce a senescent phenotype but resulted in G1 arrest albeit hyperphosphorylation of the retinoblastoma protein. This proliferative arrest was associated with stable silencing of the E2F1-dependent S-phase gene expression program which could not be reversed by ectopic overexpression of E2F1. Chromatin immunoprecipitation and accessibility assays revealed that TERT was recruited to E2F1 target sites to increase chromatin accessibility for E2F1 by facilitating the acquisition of permissive histone modifications. These data indicate a mitogenic effect of TERT on SMC growth and neointima formation through epigenetic regulation of proliferative gene expression. Furthermore, TERT expression is induced in activated macrophages during experimental and human atherosclerosis formation. To investigate the role for TERT in lesional macrophages and the subsequent effect on atherosclerosis formation, TERT-deficient mice were crossbred with LDL-receptor-deficient (LDLr-/-) mice to generate first generation G1TERT-/-LDLr-/- offsprings, which were then further intercrossed to obtain third generation G3TERT-/-LDLr-/- mice. G1TERT-/-LDLr-/- mice revealed no telomere shortening while severe telomere attrition was evident in G3TERT-/-LDLr-/- mice. When fed an atherogenic diet, G1TERT-/-LDLr-/- and G3TERT-/-LDLr-/- mice were both protected from atherosclerosis formation compared to their wild-type controls, indicating that genetic TERT-deletion prevents atherosclerosis, and formation of the disease is not affected by telomere attrition. Similarly, atherosclerosis development was decreased in chimeric LDLr-/- mice with TERT deletion in hematopoietic stem cells after bone marrow transplantation. TERT deficiency reduced macrophage accumulation in atherosclerotic lesions and altered chemokine expression, including CXC1/2/3, CCL3, CCL5, CCL21, CCR7, IL-6, and IL-1α. In isolated macrophages, gene ontology (GO) enrichment analysis of silenced inflammatory genes indicated that TERT positively regulates signal transducer and activator of transcription (STAT) cascade, which was confirmed by the decreased tyrosine phosphorylation of STAT3 protein resulting from TERT deletion. These findings indicate genetic TERT deficiency decreases atherosclerosis formation by silencing inflammatory chemokine transcription through inactivation of the STAT3 signaling pathway in activated macrophages. In conclusion, the dysregulation of TERT expression within atherosclerotic plaques plays a causative role for vascular remodeling, including injury-induced neointima formation and hypercholesterolemia-induced atherosclerosis, through inducing SMC proliferation and a pro-inflammatory phenotype in infiltrating macrophages. These findings unveil a mechanism of TERT exacerbating the pathological vascular remodeling, which may provide a novel therapeutic target to combating vascular diseases.

During colonization of the cystic fibrosis airway Pseudomonas aeruginosa converts from non-mucoid to a mucoid phenotype, characterized by the production of the exopolysaccharide alginate. Alginate production has been shown to enhance survival by promoting biofilm formation, evading complement killing, and resisting phagocytosis. The mechanism by which alginate protects P. aeruginosa from phagocytosis is unclear. To investigate the role of alginate in the inhibition of phagocytosis, a human monocytic cell line (THP-1) and a murine alveolar macrophage cell line (MH-S) were used to determine the effects of alginate on macrophage binding, signaling, and phagocytosis. Phagocytosis assays using the mucoid cystic fibrosis clinical isolate FRD1, and its non-mucoid isogenic algD mutant FRD1131, revealed that alginate inhibits opsonic and non-opsonic phagocytosis. The inhibitory effect of alginate production is intrinsic to the bacteria as exogenous alginate was unable to protect non-mucoid FRD1131 from phagocytosis. Decreased binding of FRD1 compared to FRD1131 was also demonstrated by using the actin polymerization inhibitor cytochalasin D to inhibit phagocytosis. Furthermore, studies using blocking antibodies to CD11b and CD14 found that both of these receptors were important for the phagocytosis of FRD, and it is likely that these receptors are blocked by alginate. Alginate production by P. aeruginosa may reduce lipid raft formation, however, it was not found to affect acid sphingomyelinase activity, which is important for ceramide formation within the lipid raft. Decreased binding led to decreased signaling in macrophages demonstrated by reduction in level and alteration in kinetics of phosphorylation of AKT and ERK1/2 kinases. Signaling pathway inhibitors revealed that PI3K, but not MEK, activation was critical for phagocytosis of P. aeruginosa. Despite altered intracellular signaling in murine macrophages, both mucoid and non-mucoid P. aeruginosa induced similar levels of IL-8 and MIP-2 from human and murine macrophages, respectively. By understanding the pathways involved in mediating efficient phagocytosis of clinical isolates, it may be possible to develop a treatment to promote clearance by the resident alveolar macrophages. These experiments may serve as a model to evaluate the effectiveness of such treatments. This approach also provides valuable insight into previously unknown mechanisms of phagocytosis of P. aeruginosa.

O estresse é uma realidade na produção avícola mundial. Sabe-se que ambientes estressores prejudicam o bem-estar, os parâmetros produtivos e a imunidade de frangos de corte. Sabe-se, também, que o estresse por calor diminui a atividade de macrófagos em frangos de corte e, que existem, inúmeros estressores ambientais que insidem sobre a produção animal e podem aumentar a susceptibilidade às doenças. A Salmonella spp. é uma das maiores zoonoses do mundo, causando mais de 1 bilhão de casos de infecção. Nesse sentido, o presente trabalho analisa os efeitos do estresse por calor (31±1°C) sobre os índices zootécnicos, a imunidade, a invasão bacteriana e a integridade intestinal em frangos de corte infectados com Salmonella enteritidis; os dados obtidos foram discutidos dentro de uma perspectiva neuroimune. Os frangos foram divididos em quatro grupos: 1) Controle (C); 2) Estresse por Calor a 31±1 °C (HS31°C); 3) Controle infectados com Salmonella enteritidis (Controle Positivo [PC]) e; 4) Estresse por calor a 31±1 °C e infectados com Salmonella (PHS31°C). Nossos resultados mostraram que o estresse por calor em uma situação de infecção experimental por Salmonella enteritidis (grupo PHS31°C) 1) diminuiu os índices zootécnicos; especificamente, diminuiu o ganho de peso, consumo de alimento e a conversão alimentar; 2) diminuiu os níveis plasmáticos de INF-&gamma; e IgA; 3) diminuiu a expressão de, IL-6 e IL-12 em baço e diminui IL1- &beta;, IL-10 e TGF-&beta; em tonsila cecal; 4) diminuiu a expressão de AvBD-4 e AvBD-6 em tonsila cecal e; 5) diminuiu a expressão de TLR-2 em baço e tonsila cecal. Observamos, também, 6) aumento dos níveis séricos de corticosterona nos animais dos grupos HS31°C e PHS31°C e; 7) piora no quadro de enterite produzida pela Salmonella enteritidis, quando os animais foram estressados por calor, caracterizando-se uma enterite moderada ao longo de todo o intestino delgado. Finalmente, 8) observamos que o estresse por calor aumentou a migração de Salmonella enteritidis para baço das aves do grupo PHS31°C, porém esse aumento não foi observado no fígado; observamos, também, presença de Salmonella na medula osssea dos animais estressados e infectado com essa bactéria. Os dados obtidos sugerem que a somatória dos fatores estresse por calor e infecção por Samonella prejudicou os parâmetros produtivos, a integridade intestinal, a imunidade e, em especial a ativação e atividade de macrófagos, possibilitando um aumento da migração de Salmonella enteritidis para o baço e medula óssea dos frangos de corte. Neste sentido, o estresse por calor teria prejudicado a qualidade da barreira imune intestinal, via ativação do eixo HPA e aumento dos níveis de corticosterona, diminuindo a imunidade inata proporcionando a migração das bactérias patogênicas através da mucosa intestinal para o baço e a medula óssea das aves estressadas. / Stress is a reality in the world poultry production. Environmental stressors impair both welfare, performance parameters and immunity in broiler chickens. Heat stress decreases macrophage activity in broiler chickens and many environmental stressors that impact animal production increases animal\'s susceptibility to diseases. Samonella spp is one of the most endemic zoonotic diseases of the world, inducing more than 1 billion infection cases per year. In this way, we studied the effects of 31±1°C heat stress on performance parameters, immunity, bacteria invasion and intestinal integrity in broiler chickens experimentally infected with Salmonella enteritidis; the data were discussed under a neuroimmune perspective. The broiler chickens were divided into four different groups: 1) Control group (C); 2) 31±1 °C heat stressed group (HS31°C); 3) Control group infected with Salmonella enteritidis Positive control (PC) and; 4) 31±1 °C heat stressed and Salmonella enteritidis infected group (PHS31°C). We showed the heat stress applied in the course of Salmonella enteritidis infection (PHS31°C group) decreased poultry performance parameters; specifically, it decreased the body weight gain, the feed intake and the food conversion; 2) decresead INF-&gamma; and IgA plasmatic levels; 3) decreased the mRNA expression of IL-6 and IL- 12 in spleen and the mRNA expression of IL1-&beta;, IL-10 and TGF-&beta; in cecal tonsil; 4) decreased the mRNA expression of AvBD-4 and AvBD-6 in cecal tonsil and; 5) decreased the mRNA expression of TLR-2 in spleen and cecal tonsil. We also observed 6) an increase in corticosterone serum levels in the animals of the HS31°C and the PHS31°C groups and, 7) more severe intestinal inflamation produced by Salmonella enteritidis in heat stressed chickens, characterized by a moderate enteritis throughout all the small intestine mucosa (PHS31°C group). Finally, 8) we showed that the heat stress increased splenic Salmonella enteritides invasion in PHS31°C broiler chickens; we also observed the presence of Salmonella in the bone marrow of stressed and infected broiler chickens. These data suggest that heat stress and Salmonella infection working together impair chicken\'s performance parameters, intestinal integrity and immunity (specially the macrophage activity), increasing ate the same time splenic and bone marrow Salmonella enteritidis invasion. Thus, heat stress could have impared the intestinal immunity barrier quality, via HPA axis activation and corticosterone serum levels release, decreasing the inate immunity and, providing pathogenic bacteria migration through the intestinal mucosa for spleen and bone marrow of the heat stressed chickens.

O aumento dos casos de diarreia causados por EPECa evoca a sua capacidade de adaptação e patogenicidade. O objetivo deste estudo foi investigar o comportamento da EPECa na interação com macrófagos (fagocitose e antifagocitose). O estudo da fagocitose das cepas LB7 (O55:H7), LB13 (O111:abH9) e BA487 (O55:H7) em macrófagos J774A1 mostrou sobrevivência intracelular em presença de NO. EPECa impede a maturação do vacúolo parasitóforo. A sobrevivência em macrófago derivado de C3H/HeJ, mutante do tlr4, foi reduzida e em macrófago de C57BL/6 não foi observada. O fator antigagocítico (Fa) secretado pela LB7, já detectado pelo grupo, apresenta natureza peptídica e a sua ação não é específica de EPEC, pois inibe também a fagocitose de Shigella e látex. A secreção do Fa foi avaliada em M9 e DMEM. O produto do fracionamento do sobrenadante do cultivo por SPE apresentou Fa em ambos meios. No entanto, a secreção em M9 é baixa e não foi detectada por HPLC. O Fa do DMEM obtido por HPLC mostrou-se citotóxico. Novos meios de cultivo deverão ser estudados para a identificação do Fa. / The increase in the numbers of diarrhea cases caused by aEPEC denotes its adaptability and pathogenicity. The objective of this study was to investigate the behavior of aEPEC in the interaction with macrophages (phagocytosis and anti-phagocytosis). Strains LB7 (O55:H7), LB13 (O111:abH9) and BA487 (O55:H7) were shown to survive within J774A1 macrophages in the presence of NO. aEPEC prevents maturation of the parasitophorus vacuole. Survival inside C3H/HeJ derived macrophages, mutant for tlr4, decreased and was not observed in C57BL/6 derived macrophages. The anti-phagocytic factor (AF), secreted by LB7 and previously detected by our group, is peptidic and its action is not specific to EPEC as it also inhibits phagocytosis of Shigella and latex. Secretion of AF was evaluated in M9 and DMEM. AF was detected after SPE fractionation of both culture media. However, the secretion in M9 is low and was not detected by HPLC. The DMEM HPLC fraction containing AF was cytotoxic. New culture media will be studied for the identification of AF.

A diabetes tipo 1 (DT1) está associada `a inflamação estéril (IE) e maior susceptibilidade a sepse. A sepse induz a síndrome da resposta inflamatória sistêmica (SIRS) e a inflamação pulmonar aguda (ALI). O leucotrieno (LT) B4 produzido condições inflamatórias induz a expressão de MyD88 em macrófagos (MA). Hipotetizamos que a DT1 induz a síntese de LTB4 promovendo a IE e isto contribui para SIRS, susceptibilidade a sepse e ALI. Os diabéticos apresentaram níveis elevados de LTB4 e IL-1b no soro e seu MA expressaram mais MyD88/STAT-1. A expressão de STAT-1 foi induzida por c-Jun de forma dependente de LTB4. O tratamento com insulina restaurou os níveis de LTB4 e STAT-1/MyD88 e a inibição de LTB4 restaurou os níveis de MyD88 e IL-1b. Na sepse, a inibição de 5LO prolongou a sobrevida dos diabéticos e diminuiu a SIRS menos IL-1b e IL-10 no soro e TNF-a e IL-1b na cavidade peritoneal. O pulmão dos diabéticos apresentaram ALI menos intensa que se correlacionou com um altos níveis de SOCS-1, baixos níveis de MyD88 e falha na ativação de NFkB nos macrófagos alveolares. / Type 1 diabetes (T1D) is associated with sterile inflammation (SI) and increased sepsis susceptibility. Sepsis induces Systemic Inflammatory Response Syndrome (SIRS) and Acute Lung Injury (ALI). Leukotriene (LT) B4 is produced in inflammatory conditions and induces MyD88 expression in macrophages (MA). We hypothesized that T1D induce LB4 that promotes SI contributing to SIRS, sepsis susceptibility and ALI. Diabetics presented higher levels of LTB4 and e IL-1b in the serum and MA expressed more MyD88/STAT-1. STAT-1 expression was induced by c-Jun on LTB4 dependent manner. Insulin treatment restored LTB4 and STAT-1/MyD88 levels and inhibition of LTB4 restored MyD88 and IL-1b levels. During sepsis, 5LO inhibition increased diabetics survival and inhibited SIRS- lower levels of IL-1b and IL-10 in the serum and TNF-a and IL-1b in the peritoneal cavity. Lungs from diabetics presented milder ALI that correlated with high levels of SOCS-1, low levels of MyD88 and impaired NFkB activation in alveolar macrophages.

Os piretróides sintéticos, em especial os do tipo II, como a cialotrina, são extensivamente utilizados para o controle de uma ampla variedade de ectoparasitas que acometem os animais de produção. Entretanto, no Brasil e em outros países, sua utilização vai além da saúde animal, sendo utilizados também em saúde pública, no controle de diversos vetores, como é o caso do vetor da dengue, dentre outros. Visto que a cialotrina modifica a atividade de macrófagos peritoneais, o objetivo deste trabalho foi investigar os prováveis mecanismos através dos quais este piretróide modifica a atividade destas células. Os presentes resultados, analisados em seu conjunto, mostram de maneira inequívoca que a cialotrina tem um efeito direto e/ou indireto sobre a atividade de macrófagos peritoneais. Especificamente, observou-se neste trabalho que o praguicida causou em ratos: 1 ? marcação fos positiva em neurônios do núcleo paraventricular do hipotálamo (NPH), após a dose de 3,0 mg/kg/dia; 2 - diminuição do percentual e intensidade de fagocitose de macrófagos peritoneais ativados e avaliados por citometria de fluxo; 3 - diminuição dose-dependente da produção de nitrito (NO2); 4 ? diminuição do percentual e intensidade de fagocitose de macrófagos peritoneais ativados, em ratos adrenalectomizados e/ou tratados com metirapona (inibidor da síntese de corticosterona) e RU 486 (antagonista de receptores glicocorticóides) com a finalidade de modular os níveis de glicocorticóides, e tratados com 3,0 mg/kg/dia de cialotrina; 5 ? aumento dos níveis de noradrenalina hipotalâmica em animais tratados com a dose de 3,0mg/kg/dia de cialotrina; 6 - diminuição do percentual e intensidade de fagocitose, bem como diminuição da produção de nitrito de macrófagos peritoneais ativados, em ratos simpatectomizados químicamente com 6-OHDA; 7 - diminuição dose dependente do percentual e intensidade de fagocitose, bem como da produção de nitrito de macrófagos peritoneais ativados e tratados in vitro com 10 e 100 nM de cialotrina. No entanto, não observamos: 1 ? alterações na produção de nitrito realizada por macrófagos peritoneais ativados, em ratos adrenalectomizados e/ou tratados com metirapona e RU 486; 2 - alterações na viabilidade celular induzida pelo tratamento in vitro com a cialotrina na concentração de 10 e 100 nM e 3 ? alterações nos efeitos da cialotrina sobre a atividade de macrófagos tratados in vitro com os ligantes de receptores benzodiazepínicos periféricos. Em conjunto, os presentes dados mostram que a cialotrina interfere com a atividade de macrófagos por atuar indiretamente, através da ativação do eixo Hipotálamo-Hipófise-Adrenal (HHA), e/ou diretamente sobre os mesmos modulando sua atividade. É muito provável que o efeito resultante do tratamento in vivo com este praguicida esteja ligado à somatória destas ações / Synthetic pyrethroids, particularly those of type II, such as cyhalothrin, are extensively used in agriculture for the control of a broad range of ectoparasites in farm animals. However, in Brazil and some other countries, these pyrethroids have also been used in public health, for the control of insects that are known to be vectors of diseases such as dengue. Since it has been suggested that cyhalothrin alters activity of peritoneal macrophages, the objective of our study was to investigate the putative mechanisms for the changes induced by pyrethroid in these cells. The results presented here show, in an unequivocal manner, that cyhalothrin has a direct or indirect (or both) effect on the activity of peritoneal macrophages. We specifically observed in this work that this pesticide induced in rats: 1- Fos-positive immunostaining in neurons of the paraventricular nucleus of the hypothalamus (NPH), after 3.0 mg/kg/day; 2 ? a reduction in the percentage and intensity of phagocytosis by activated peritoneal macrophages, evaluated by flow cytometry; 3 ? a dose-dependent reduction in nitrite production (NO2); 4 ? a reduction in the percentage and intensity of phagocytosis by activated peritoneal macrophage from adrenalectomized rats treated or not with metirapone (inhibitor of corticosterone synthesis) or RU 486 (antagonist of glicocorticoids receptors) with the propose of modulating the levels of glicocorticoids, and treated with 3.0 mg/kg/day of cyhalothrin; 5 ? an increase in the hypothalamic levels of noradrenaline in rats treated with 3.0 mg/kg/day of cyhalothrin; 6 ? a reduction in the percentage and intensity of phagocytosis and also a decrease in the production of nitrite by activated peritoneal macrophages, after chemical sympatectomy with 6-OHDA; 7 ? a dose-dependent reduction of the percentage and intensity of phagocytosis, and also a decrement in nitrite production by activated peritoneal macrophages treated in vitro with 10 and 100 nM of cyhalothrin. However, we found no differences on: 1 ? nitrite production by activated peritoneal macrophages after adrenalectomy, treated or not with metirapone or RU 486; 2 ?cell viability of peritoneal macrophages treated in vitro with 10 and 100 nM of cyhalothrin, and 3 ? the effects of cyhalothrin on macrophage activity after in vitro treatment with peripheral benzodiazepine receptor ligands. Altogether, the present results show that cyhalothrin interferes with the activity of peritoneal macrophages by acting indirectly, via activation of the hypothalamus-pituitary-adrenal axis, or directly on these cells, altering their activity. As a matter of fact, it is quite possible that the results of in vivo cyhalothrin treatment on macrophage activity would be related to the combined effect of these direct and indirect influences

No intuito de avaliar o papel do receptor b2-adrenérgico no processo de regeneração muscular, os músculos tibialis anterior de camundongos knockout para o adrenoceptor b2 (b2KO) foram criolesados e analisados após 1, 3, 10 e 21 dias. Análises de aspectos morfológicos e contráteis, atuação de macrófagos M1 e M2, conteúdo de AMPc e ativação de elementos da via de sinalização TGF-b/smad foram realizadas. Os músculos em regeneração dos animais b2KO apresentaram redução do calibre das fibras musculares, redução na função contrátil em 10 dias após criolesão, atenuado aumento do conteúdo de AMPc nos músculos em 10 dias após criolesão, aumento da inflamação e do número de macrófagos nos músculos em regeneração em 3 e 10 dias após lesão, predominância de macrófagos M1, diminuição da ativação de TbR-I e smad2/3 e da expressão de smad4 em 3 dias após lesão, e aumento na expressão de akirina1 em 10 dias após lesão. Nossos resultados sugerem que o adrenoceptor b2 contribui para a regulação das fases iniciais da regeneração muscular. / In this study, we investigated the role of the b2-adrenoceptor in skeletal muscle regeneration. Tibialis anterior muscles from b2-adrenoceptor knockout (b2KO) mice were cryolesioned and analysed after 1, 3, 10, and 21 days. Analysis of structural and contractile aspects, M1 and M2 macrophage profile, cAMP content, and activation of TGF-b/smad signalling elements. Regenerating muscles from b2KO mice showed diminished calibre of regenerating myofibres, decreased muscle contractile function at 10 days when compared with those from wild-type, attenuated augment in cAMP content in muscles at 10 days post-injury, increase in inflammatory process and in the number of macrophages at 3 and 10 days, prevalence of M1 macrophage phenotype, reduction in TbR-I and smad2/3 activation, and in the smad4 expression at 3 days, and an increase in akirin1 expression at 10 days in muscles from b2KO mice when compared to those from wild-type. Our data suggest that the b2-adrenoceptor contributes to the control of the initial stages of muscle regeneration.

Murine Mikrogliakulturen wurden mit Astrozyten-konditioniertem Medium (ACM) in einen deaktivierten Zustand überführt. Dies wurde anhand morphologischer (Grad der Ramifizierung) und immunologischer (Expression von Adhäsionsmolekülen) Parameter verifiziert. Durch den Einsatz von Makrophagen-koloniestimulierenden Faktor (M-CSF), Granulozyten/Makrophagen-koloniestimulierenden Faktor (GM-CSF), transformierenden Wachstumsfaktor beta (TGF-beta) und den gegen sie gerichteten Antikörpern wurde gezeigt, daß alle untersuchten Zytokine in unterschiedlichem Maße an der Deaktivierung der Mikrogliazellen durch ACM beteiligt sind. Außerdem wurde nach Stimulation mit ACM an murinen Mikrogliazellen eine transiente Hochregulation eines Kaliumauswärtsstromes beobachtet Das Auftreten dieses Kalium-stromes nach Inkubation der Mikrogliazellen mit ACM konnte auf die Wirkung von TGF-beta, welches im ACM enthalten ist, zurückgeführt werden. Der durch ACM in deaktivierter Mikroglia induzierte Kaliumkanal entsprach in seinen kinetischen und pharma-kologischen Eigenschaften am ehesten dem klonierten Kanal Kv1.3. Die Kv1.3 Expression durch TGF-beta oder ACM war durch den unspezifischen Proteinkinaseinhibitor H7 unterdrückbar. Diese Ergebnisse zeigen, daß die Expression des Kv1.3 Kanals nicht, wie bisher angenommen, ein Indikator für aktivierte Mikroglia ist. / Murine microglial cultures were deactivated with astrocyte-conditioned medium (ACM). The deactivation process was verified measuring morphological (ramification index) and immunological (expression level of adhesion molecules) parameters. By using macrophage-colony stimulating factor (M-CSF), granulocyte/macrophage-colony stimulating factor (GM-CSF), transforming growth factor beta (TGF-beta) and their corresponding antibodies it was shown, that to a different extent all of these cytokines influence the deactivation process of microglial cells by ACM. ACM treatment of microglial cultures also lead to a transient upregulation of a delayed potassium outward current. This upregulation was due to the impact of TGF-beta contained in ACM. The ACM induced potassium channel resembled in its kinetic and pharmacological properties the cloned Kv1.3 channel. Expression of Kv1.3 in microglial cells by TGF-beta or ACM was inhibited by the unspecific protein kinase inhibitor H7. These results show, that expression of Kv1.3 channels is not a special feature of activated microglia, which has been proposed in recent publications.