RESUMO
OBJECTIVE: A chronic decrease in neuromuscular activity results in atrophy and capillary regression in skeletal muscles. The purposes of this study were to determine the effects of Enterococcus faecium strain R30 (R30) administration on (i) the hemodynamics of the rat soleus muscle, and (ii) the capillary regression normally associated with HU. METHODS: Experiment 1: The VRBC was measured for up to 1 hour after administration of R30 with or without the ß-blocker propranolol. Experiment 2: R30 was administered daily to control and HU rats for 2 weeks. Mean capillary luminal diameter, volume, and the levels of eNOS and VEGF protein were measured. RESULTS: Experiment 1: VRBC was faster 20, 40, and 60 minutes after than before the administration of R30: This effect was suppressed by propranolol administration. Experiment 2: R30 administration during HU increased capillary luminal diameter and volume and eNOS and VEGF protein levels in the soleus of HU rats. CONCLUSIONS: The results suggest that R30 increases VRBC in the soleus muscle via muscle sympathetic nerve activity (Experiment 1) and that R30 supplementation lessens the capillary regression normally associated with HU via the eNOS/VEGF pathway (Experiment 2).
Assuntos
Velocidade do Fluxo Sanguíneo , Capilares/ultraestrutura , Enterococcus faecium/fisiologia , Eritrócitos/fisiologia , Óxido Nítrico Sintase Tipo III/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Capilares/metabolismo , Elevação dos Membros Posteriores , Músculo Esquelético/irrigação sanguínea , Ratos , Transdução de SinaisRESUMO
Leukemia-inhibitory factor (LIF) is indispensable for maintaining the undifferentiated state when propagating mouse embryonic stem (ES) cells. We previously cloned chicken LIF (chLIF) cDNA and demonstrated that it maintained chicken ES cell cultures in an undifferentiated state. Here, we developed two monoclonal antibodies, HUL-1 and HUL-2, against chLIF, which specifically recognized recombinant chLIF (rchLIF) produced by Escherichia coli and Chinese hamster ovary K1 cells, in enzyme-linked immunosorbent assays and Western blot analysis. In addition, HUL-2 inhibited the phosphorylation of signal transducer and activator of transcription 3 by rchLIF in chicken blastodermal cells (CBCs), but not that of mitogen-activated protein kinase kinase. Furthermore, the addition of HUL-2 to CBC cultures resulted in embryoid bodies forming earlier than in normal cultures. These results indicated that HUL-2 recognized not only rchLIF but also native chLIF, and suggested that CBCs in culture produce LIF, which functions in autocrine signaling.
Assuntos
Anticorpos Monoclonais/imunologia , Blastoderma/metabolismo , Interleucina-6/metabolismo , Animais , Blastoderma/citologia , Diferenciação Celular , Células Cultivadas , Embrião de Galinha , Cricetinae , Cricetulus , Escherichia coli/metabolismo , Interleucina-6/imunologia , Fator Inibidor de Leucemia , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Fosforilação , Proteínas Recombinantes/imunologia , Fator de Transcrição STAT3/metabolismo , Transdução de SinaisRESUMO
Mouse embryonic stem (ES) cells can be maintained in an undifferentiated state in the presence of leukemia inhibitory factor (LIF), a member of the interleukin-6 cytokine family. In other mammals, this is not possible with LIF alone. Chicken ES-like cells (blastodermal cells) have only been cultured with mouse LIF because chicken LIF was not available. However the culture system is imperfect and chicken ES-like cells equivalent to mouse ES cells were not observed. In the present study, we cloned the cDNA-encoding chicken LIF using mRNA subtraction and RACE methodology. The chicken LIF cDNA encodes a protein with approximately 40% sequence identity to mouse LIF. It has 211 amino acids including a putative N-terminal signal peptide of 24 residues. Chicken blastodermal cells were cultured in the presence of bacterially expressed chicken LIF or mouse LIF. The expression of alkaline phosphatase and embryonal carcinoma cell monoclonal antibody-1 and stage-specific embryonic antigen-1 and the activation of STAT3 were examined, all of which are indices of the undifferentiated state. Exposure in the blastodermal cells to recombinant chicken LIF but not to mouse LIF maintained the expression of these various markers. After 9 days of incubation, the blastodermal cells formed cystic embryoid bodies in the presence of mouse LIF but not in the presence of recombinant chicken LIF. We conclude that chicken LIF is able to maintain chicken ES cell cultures in the undifferentiated state.
Assuntos
Técnicas de Cultura de Células/métodos , Embrião de Mamíferos/citologia , Embrião não Mamífero , Interleucina-6/fisiologia , Células-Tronco/citologia , Fosfatase Alcalina/química , Fosfatase Alcalina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Blastocisto/metabolismo , Western Blotting , Carcinoma Embrionário/química , Diferenciação Celular , Galinhas , Clonagem Molecular , DNA Complementar/metabolismo , Escherichia coli/metabolismo , Humanos , Interleucina-6/metabolismo , Fator Inibidor de Leucemia , Sistema de Sinalização das MAP Quinases , Camundongos , Microscopia de Fluorescência , Dados de Sequência Molecular , Fosforilação , Sinais Direcionadores de Proteínas , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Fatores de TempoRESUMO
BACKGROUND: The house dust mite Dermatophagoides farinae is the most common aeroallergen causing human allergic asthma. Previously, we demonstrated that a high-molecular-weight allergenic fraction (HM1), which was abundant in D. farinae extracts, induced a proliferative response of T cells from healthy donors. The induction was mediated through the activation of macrophages without MHC class II restriction. In this study, we investigate whether HM1 influences the development of airway inflammation in murine models of asthma. METHODS: BALB/c mice were injected twice intraperitoneally with D. farinae fecal extract (Dff) at an interval of 5 days. They were exposed daily to aerosolized antigen (group 1: Dff, group 2: HM1, group 3: HM1-depleted Dff and group 4: PBS) for 10 days. The effect of HM1 on their airway inflammation was evaluated by measuring acetylcholine-induced airway hyperresponsiveness and inflammatory cell infiltration in lung tissue. RESULTS: The inhalation of the whole fecal extract or the HM1 fraction induced airway hyperresponsiveness which was detectable after 24 h and was maintained for as long as 120 h. The inhalation of extract depleted of the HM1 fraction induced hyperresponsiveness measured at 24 h but this was not maintained for 120 h. Macrophage infiltration was significantly prolonged in mice inhaling the whole extract and the HM1 fraction compared to the HM1-depleted extract. CONCLUSION: The inhalation of the high-molecular-weight HM1 fraction of D. farinae prolonged airway hyperresponsiveness and macrophage inflammation in a mouse model of hypersensitivity. The results indicate that the HM1 fraction which can induce T cell proliferation through macrophage activation may play a role in the duration of airway responsiveness.