Morphology and myofiber composition of skeletal musculature of the forelimb in young and aged wild type and myostatin null mice

Most current research into therapeutic approaches to muscle diseases involves the use of the mouse as an experimental model. Furthermore, a major strategy to alleviate myopathic symptoms through enhancing muscle growth and regeneration is to inhibit the action of myostatin (Mstn), a transforming growth factor-beta (TGF-beta) family member that inhibits muscle growth. Presently, however, no study has expanded the morphological analysis of mouse skeletal muscle beyond a few individual muscles of the distal hindlimb, through which broad conclusions have been based. Therefore, we have initially undertaken an expansive analysis of the skeletal musculature of the mouse forelimb and highlighted the species-specific differences between equivalent muscles of the rat, another prominently used experimental model. Subsequently, we examined the musculature of the forelimb in both young and old adult wild-type (mstn(+/+)) and myostatin null (mstn(-/-)) mice and assessed the potential beneficial and detrimental effects of myostatin deletion on muscle morphology and composition during the aging process. We showed that: (1) the forelimb muscles of the mouse display a more glycolytic phenotype than those of the rat; (2) in the absence of myostatin, the induced myofiber hyperplasia, hypertrophy, and glycolytic conversion all occur in a muscle-specific manner; and, importantly, (3) the loss of myostatin significantly alters the dynamics of postnatal muscle growth and impairs age-related oxidative myofiber conversion.

Opposite effects of fasting on TGF-beta 3 and T beta RI distribution in the gastric mucosa of suckling and early weanling rats

Objective: Our aim was to evaluate the effects of a dietary regimen (suckling or early weaning) and feeding status (fed or fasted) on the distribution of transforming growth factor-beta 3 (TGF-beta 3) and TGF receptor-I (T beta RI) in the gastric epithelium of pups Methods: Wistar rats were used At 15 d, half of the pups were separated from dams and fed with hydrated powered chow On day 17, suckling and early weanling rats were subjected to fasting (17 h). Four different conditions were established. suckling fed and fasted and early weanling fed and fasted At 18 d stomachs were collected under anesthesia and were fixed in 4% formaldehyde for immunohistochemistry The number of immunostained epithelial cells per microscopic field was determined for TGF-beta 3 and T beta RI in longitudinal sections from the gastric mucosa Results: We found that during suckling, fasting reduced the number of immunolabeled cells per field of both molecules when compared with the fed group (P < 0.05), whereas in early weaning, food restriction increased TGF-beta 3 and T beta RI distributions (P < 0.05) We also observed that TGF-beta 3 and T beta RI were more concentrated in parietal cells in the upper gland in suckling pups, whereas after early weaning these were displaced to parietal and chief cells at the bottom of the gland Conclusion: Suckling and early weaning directly influence TGF-beta 3 and T beta RI distributions in the gastric epithelium in response to fasting, such that early weaning anticipates the effects observed in adult rats. Furthermore, the differential concentrations of TGF-beta 3 and T beta RI indicate that they might be important for cell proliferation events in growth control (C) 2010 Elsevier Inc. All rights reserved

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.

Comportamento de células pulpares humanas expostas ao TGFβ1 a ao aFGF em cultura

O propósito do presente estudo foi avaliar o comportamento de células pulpares humanas expostas ao TGFβ1 e ao aFGF, em cultura, nas seguintes concentrações: TGFβ1 a 1ng/mL, TGFβ1 a 5ng/mL, TGFβ1 a 1ng/mL + aFGF a 5ng/mL, TGFβ1 a 5ng/mL + aFGF a 5ng/mL e aFGF a 5ng/mL. Foi avaliada a morfologia celular, a atividade da fosfatase alcalina, através de ensaio com pNPP como substrato e a expressão das proteínas osteocalcina, sialoproteína óssea e sialofosfoproteína de dentina, através de RT-PCR. Após quatro dias, verificou-se que a média do número de nucléolos no grupo tratado com TGFβ1 a 1ng/mL foi significativamente maior que no grupo tratado com aFGF a 5ng/mL. A média da atividade da fosfatase alcalina no grupo tratado com TGFβ1 a 1ng/mL foi significativamente maior que no grupo tratado com TGFβ1 a 5ng/mL + aFGF a 5ng/mL. Foi observada a expressão de osteocalcina em todas as células pulpares humanas que proliferaram em cultura. Entretanto, no grupo em que foi utilizado o aFGF a 5ng/mL houve diminuição da expressão da osteocalcina. A exposição dos fatores não induziu a expressão de componentes da matriz de dentina tais como BSP e DSPP. Sugere-se que as células expostas ao TGFβ1 1ng/mL foram estimuladas, apresentando uma maior atividade celular e as células expostas ao aFGF 5ng/mL foram inibidas, apresentando uma menor atividade celular.

Citocinas e proteínas de fase aguda do soro como marcadores de regressão da resposta inflamatória ao tratamento da tuberculose pulmonar

OBJETIVO: Analisar o padrão de citocinas pró- e antiinflamatórias e da resposta de fase aguda (RFA) como marcadores de resposta ao tratamento da tuberculose pulmonar. MÉTODOS: Determinação dos níveis de interferon-gama (IFN-γ), tumor necrosis factor-alpha (TNF-α, fator de necrose tumoral-alfa), interleucina-10 (IL-10) e transforming growth factor-beta (TGF-β, fator transformador de crescimento-beta), pelo método ELISA, em sobrenadante de cultura de células mononucleares do sangue periférico e monócitos, assim como dos níveis de proteínas totais, albumina, globulinas, alfa-1-glicoproteína ácida (AGA), proteína C reativa (PCR) e velocidade de hemossedimentação (VHS) em 28 doentes com tuberculose pulmonar, em três tempos: antes (T0), aos três meses (T3) e aos seis meses (T6) de tratamento, em relação aos controles saudáveis, em um único tempo. RESULTADOS: Os pacientes apresentaram valores maiores de citocinas e RFA que os controles em T0, com diminuição em T3 e diminuição (TNF-α, IL-10, TGF-β, AGA e VHS) ou normalização (IFN-γ e PCR) em T6. CONCLUSÕES: PCR, AGA e VHS são possíveis marcadores para auxiliar no diagnóstico de tuberculose pulmonar e na indicação de tratamento de indivíduos com baciloscopia negativa; PCR (T0 > T3 > T6 = referência) pode também ser marcador de resposta ao tratamento. Antes do tratamento, o perfil Th0 (IFN-γ, IL-10, TNF-α e TGF-β), indutor de e protetor contra inflamação, prevaleceu nos pacientes; em T6, prevaleceu o perfil Th2 (IL-10, TNF-α e TGF-β), protetor contra efeito nocivo pró-inflamatório do TNF-α ainda presente. O comportamento do IFN-γ (T0 > T3 > T6 = controle) sugere sua utilização como marcador de resposta ao tratamento.

Infiltrating CD8(+) T lymphocytes, natural killer cells, and expression of IL-10 and TGF-beta 1 in chemically induced neoplasms in male Wistar rats

The present study aimed to estimate the number of CD8(+) T and natural killer (NK) infiltrating cells and the expression of interleukin-10 (IL-10) and transforming growth factor beta 1 (TGF-beta1) in chemically induced neoplasms in an initiation-promotion bioassay for carcinogenesis. Male Wistar rats were treated with N-nitrosodiethylamine, N-methyl-N-nitrosourea, N-butyl-N-(4-hydroxybutyl) nitrosamine, dihydroxy-di-N-propylnitrosamine, and 1,2-dimethylhydrazine for 4 weeks. Two groups were subsequently exposed through diet to phenobarbital (0.05%) or 2-acetylaminofluorene (0.01%) for 25 weeks. An untreated group was used as a control. Immune cells and cytokines were immunohistochemically evaluated in neoplasms and in surrounding normal tissues at the liver, kidneys, lung, and small and large intestines. When compared to the respective normal tissues, an increased number of NK cells was verified infiltrating the colon, lung, and kidney neoplasms, while the number of CD8+ T cells decreased in the intestine and lung neoplasms. Expression of IL-10 was found mainly in kidney tumors. TGF-beta1 was expressed mainly in the liver and kidneys tumors. The results indicate that the differential occurrence of immune cells between neoplastic and normal tissues could be dependent upon tumor microenvironment.

Production of pro- and anti-inflammatory cytokines by monocytes from patients with paracoccidioidomycosis

Monocytes and macrophages can produce a large repertoire of cytokines and participate in the pathogenesis of granulomatous diseases. We investigated the production of pro- and anti-inflammatory cytokines by monocytes from patients with active paracoccidioidomycosis. Peripheral blood monocytes from 37 patients and 29 healthy controls were cultivated with or without 10 mug/ml of lipopolysaccharide (LPS) for 18 h at 37 degreesC, and the cytokine levels were determined in the culture supernatants by enzyme immunoassay. The results showed that the endogenous levels of tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1beta), IL-6, IL-8, IL-10 and transforming growth factor beta detected in the supernatant of patient monocytes cultivated without stimulus were significantly higher than those produced by healthy controls. These data demonstrated that monocytes from patients with active paracoccidioidomycosis produce high levels of cytokines with both inflammatory and anti-inflammatory activities. However, patient monocytes produced significantly lower TNF-alpha and IL-6 levels in response to LPS when compared to normal subjects, suggesting an impairment in their capacity to produce these cytokines after LPS stimulation. Concentrations of IL-1beta, IL-8 and IL-10 in cultures stimulated with LPS were higher in patients than in controls. These results suggest that an imbalance in the production of pro- and anti-inflammatory cytokines might be associated with the pathogenesis of paracoccidioidomycosis. (C) 2003 Editions scientifiques et medicales Elsevier SAS. All rights reserved.

Ultrastructural and immunocytochemical evaluation of the effects of extracorporeal shock wave treatment in the hind limbs of horses with experimentally induced suspensory ligament desmitis

Objective-To evaluate the effects of extracorporeal shock wave therapy (ESWT) on affected ligaments in the hind limbs of horses with experimentally induced suspensory ligament desmitis by use of ultrasonographic, ultrastructural, and immunocytochemical techniques.Animals-10 horses.Procedure-Suspensory ligament desmitis was induced in both hind limbs of each horse by use of 2 collagenase injections (administered 2 weeks apart) in each suspensory ligament. Two weeks after the second injection, the right hind limb of each horse was treated with ESWT (3 treatments at 3-week intervals)- the left hind limb was not treated (control limb). Periodically during the study, the healing process was monitored ultrasonographically and the proportions of ligaments affected with lesions were assessed. Four weeks after the last ESWT treatment, biopsy specimens were collected from all ligaments for ultrastructural evaluation and immunocytochemical analysis of transforming growth factor beta-1.Results-The difference in the proportion of the lesion-affected ligament between ESWT-treated and control limbs was significant (P < 0.05) from 3 weeks after the second ESWT treatment to the end of the study. Compared with control ligaments, ESWT-treated ligaments had more small, newly formed collagen fibrils and greater expression of transforming growth factor beta-1 4 weeks after the last ESWT treatment was administered.Conclusions and Clinical Relevance-Results have indicated that ESWT appears to facilitate the healing process in horses with experimentally induced hind limb suspensory ligament desmitis.

Cardiac benefits of exercise training in aging spontaneously hypertensive rats

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

Effects of long-term cyclosporin therapy on gingiva of rats--analysis by stereological and biochemical estimation.

Cyclosporin A (CsA) is used as an immunosuppressive agent and its prominent side effect is the induction of gingival overgrowth, which remains a significant problem. The risk factors appraised include the duration of treatment. However, there are no stereological and biochemical studies exploring the effects of long-term CsA therapy on gingival tissue. The purpose of the present study was to investigate the level of TGF-beta1 in saliva and describe the densities of fibroblasts and collagen fibers in the gingival tissue of rats treated with CsA for long periods. Rats were treated for 60, 120, 180 and 240 days with a daily subcutaneous injection of 10 mg/kg of body weight of CsA. At the end of the experimental periods, saliva was collected for the determination of TGF-beta1 levels. After histological processing, the oral epithelium and the connective tissue area were measured as well as the volume densities of fibroblasts (Vf) and collagen fibers (Vcf). After 60 and 120 days of CsA treatment, there was a significant increase in Vf and Vcf as well as a significant increase in TGF-beta1 levels. After 180 and 240 days, reduction in the gingival overgrowth associated with significant decreases in the level of TGF-beta1, and also decreased Vf and Vcf, were observed. The data presented here suggest that after long-term therapy, a decrease in TGF-beta1 levels occurs, which might contribute to an increase in the proteolytic activity of fibroblasts in the gingiva, favoring the normality of extracellular matrix synthesis.

Cyclosporin but not tacrolimus significantly increases salivary cytokine contents in rats

Background: Cyclosporin (CsA) and tacrolimus (FK-506) are immunosuppressive drugs that specifically inhibit T-cell activation via calcineurin inhibition. Gingival overgrowth is a common side effect following the administration of CsA. The severity of gingival overgrowth seen in patients taking FK-506 is less than that observed with CsA. Little is known about the involvement of saliva in drug-induced gingival overgrowth. The purpose of this study was to investigate the salivary contents of tumor growth factor β1 (TGF-β1), epidermal growth factor (EGF), and interleukin-6 (IL-6) as well as the hystometry of gingival tissue obtained from rats treated with either FK-506 or CsA. Methods: For 30 or 60 days rats received daily subcutaneous injection doses of either CsA or FK-506 (10 mg/kg). The concentrations of TGF-β1, EGF, and IL-6 in saliva were determined by enzyme-linked immunosorbent assay, and after histological processing, the oral epithelium and connective tissue were assessed at the region of the lower first molars. Results: The levels of TGF-β1, EGF, and IL-6 in saliva were not significantly altered by any of the treatments after 30 days. After 60 days of treatment with CsA, gingival overgrowth and significant increase in salivary TGF-β1, EGF, and IL-6 concentrations were observed; no statistically significant changes were induced by FK-506. Conclusion: Within the limits of this experimental study, it can be concluded that CsA, but not FK-506, induced gingival overgrowth associated with an increase of the salivary levels of the cytokines TGF-β1, EGF, and IL-6.

Pro- and anti-inflammatory cytokines produced by human monocytes challenged in vitro with Paracoccidioides brasiliensis

Monocytes and macrophages play a central role in innate and adaptive immune response against systemic fungal infections. Imbalances in suppressor or stimulatory cytokine secretion caused by these cells may influence disease development, microorganism death, and the nature of the adaptive immune response. This study analyzed the monocyte cytokine profiles of healthy individuals challenged with high and low virulent strains of P. brasiliensis and mRNA cytokine expression kinetics by reverse transcription polymerase chain reaction (RT-PCR). Peripheral blood monocytes from healthy volunteers were cultured in vitro with and without virulent (Pb18) or low virulence (Pb265) strains from P. brasiliensis viable yeast cells. Interleukin-1 beta (IL-1β), IL-6, IL-8, IL-10, tumor necrosis factor-alpha (TNF-α), and transforming growth factor-beta (TGF-β1) were measured in culture supernatants by enzyme immunoassay (ELISA), and mRNA cytokine expression was determined by RT-PCR at 0, 4, 8, 12, 18 and 48 hr. Both P. brasiliensis strains induced monocyte production of IL-1β, IL-6, IL-10 and TNF-α. Pb18 induced higher levels of IL-1β, IL-6, and IL-10 than Pb265. IL-8 and TGF-β1 levels were not significantly different from those cultured without stimulus. The mRNA cytokine expression was similar to supernatant cytokines measured by ELISA. In vitro monocyte challenge with virulent P. brasiliensis strain induces earlier and higher levels of pro- and anti-inflammatory cytokines than low virulence strain.

Silibinin attenuates oxidative metabolism and cytokine production by monocytes from preeclamptic women.

Silibinin is a polyphenolic plant flavonoid with anti-inflammatory properties. The present study investigated the effect of silibinin on oxidative metabolism and cytokine production - tumor necrosis factor-alpha (TNF-α), interleukin (IL)12, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, IL-10, and transforming growth factor beta (TGF-β1) - by peripheral blood monocytes (PBM) from preeclamptic pregnant women. It is a case-controlled study involving women with preeclampsia (PE, n = 30) compared with normotensive pregnant (NT, n = 30) and with non-pregnant (NP, n = 30) women. Monocytes were obtained and cultured with or without silibinin (5 μM or 50 μM) for 18 h. Superoxide anion (O2-) and hydrogen peroxide (H2O2) release were determined by specific assays, and cytokine levels were determined by immunoenzymatic assays (ELISA). Monocytes from preeclamptic women cultured without stimulus released higher levels of O22, H2O2 and TNF-α, and lower levels of IL-10 and TGF-β1 than did monocytes from NT and NP women. Treatment in vitro with silibinin significantly inhibited spontaneous O2- and H2O2 release and TNF-α production by monocytes from preeclamptic women. The main effect of silibinin was obtained at 50 μM concentration. Thus, silibinin exerts anti-oxidative and anti-inflammatory effects on monocytes from preeclamptic pregnant women by inhibiting the in vitro endogenous release of reactive oxygen species and TNF-α production.

Influência da amoxicilina na virulência do Helicobacter pylori

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Cellular and Molecular Tissue Response to Triple Antibiotic Intracanal Dressing

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