RESUMO
Non-transgenic (wild-type) coho salmon (Oncorhynchus kisutch), growth hormone (GH) transgenic salmon (with highly elevated growth rates), and GH transgenic salmon pair fed a non-transgenic ration level (and thus growing at the non-transgenic rate) were examined for plasma hormone concentrations, and liver, muscle, hypothalamus, telencephalon, and pituitary mRNA levels. GH transgenic salmon exhibited increased plasma GH levels, and enhanced liver, muscle and hypothalamic GH mRNA levels. Insulin-like growth factor-I (IGF-I) in plasma, and growth hormone receptor (GHR) and IGF-I mRNA levels in liver and muscle, were higher in fully fed transgenic than non-transgenic fish. GHR mRNA levels in transgenic fish were unaffected by ration-restriction, whereas plasma GH was increased and plasma IGF-I and liver IGF-I mRNA were decreased to wild-type levels. These data reveal that strong nutritional modulation of IGF-I production remains even in the presence of constitutive ectopic GH expression in these transgenic fish. Liver GHR membrane protein levels were not different from controls, whereas, in muscle, GHR levels were elevated approximately 5-fold in transgenic fish. Paracrine stimulation of IGF-I by ectopic GH production in non-pituitary tissues is suggested by increased basal cartilage sulphation observed in the transgenic salmon. Levels of mRNA for growth hormone-releasing hormone (GHRH) and cholecystokinin (CCK) did not differ between groups. Despite its role in appetite stimulation, neuropeptide Y (NPY) mRNA was not found to be elevated in transgenic groups.
Assuntos
Animais Geneticamente Modificados/genética , Hormônio do Crescimento/genética , Oncorhynchus kisutch/genética , Animais , Animais Geneticamente Modificados/sangue , Animais Geneticamente Modificados/metabolismo , Colecistocinina/genética , Hormônio do Crescimento/sangue , Hormônio do Crescimento/metabolismo , Hormônio Liberador de Hormônio do Crescimento/genética , Hipotálamo/metabolismo , Fator de Crescimento Insulin-Like I/genética , Fígado/metabolismo , Músculos/metabolismo , Neuropeptídeo Y/genética , Oncorhynchus kisutch/sangue , Oncorhynchus kisutch/metabolismo , Hipófise/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores da Somatotropina/genética , Telencéfalo/metabolismoRESUMO
The inflammatory response to ozone in atopic asthma suggests that soluble mediators of inflammation are released in response to oxidant stress. Antioxidants may alleviate additional oxidative stress associated with photochemical oxidant pollution. This study investigates the impact of antioxidant supplementation on the nasal inflammatory response to ozone exposure in atopic asthmatic children. We conducted a randomized trial using a double-blinded design. Children with asthma (n = 117), residents of Mexico City, were given randomly a daily supplement of vitamins (50 mg/day of vitamin E and 250 mg/day of vitamin C) or placebo. Nasal lavages were performed three times during the 4-month follow-up and analysed for content of interleukin-6 (IL-6), IL-8, uric acid and glutathione (GSx). IL-6 levels in the nasal lavage were increased significantly in the placebo group after ozone exposure while no increase was observed in the supplement group. The difference in response to ozone exposure between the two groups was significant (P = 0.02). Results were similar for IL-8, but with no significant difference between the groups (P = 0.12). GSx decreased significantly in both groups. Uric acid decreased slightly in the placebo group. Our data suggest that vitamin C and E supplementation above the minimum dietary requirement in asthmatic children with a low intake of vitamin E might provide some protection against the nasal acute inflammatory response to ozone.
Assuntos
Poluentes Atmosféricos/toxicidade , Antioxidantes/administração & dosagem , Ácido Ascórbico/administração & dosagem , Asma/imunologia , Suplementos Nutricionais , Ozônio/imunologia , Vitamina E/administração & dosagem , Asma/dietoterapia , Criança , Método Duplo-Cego , Exposição Ambiental/efeitos adversos , Feminino , Glutationa/análise , Humanos , Interleucina-6/análise , Interleucina-8/análise , Masculino , Cavidade Nasal/imunologia , Estresse Oxidativo/imunologia , Ozônio/toxicidade , Ácido Úrico/análise , alfa-Tocoferol/sangueRESUMO
To determine whether antioxidants can influence human susceptibility to ozone (O(3))-induced changes in lung function and airway inflammation, we placed 31 healthy nonsmoking adults (18 to 35 yr old) on a diet low in ascorbate for 3 wk. At 1 wk, subjects were exposed to filtered air for 2 h while exercising (20 L/min/m(2)), and then underwent bronchoalveolar lavage (BAL) and were randomly assigned to receive either a placebo or 250 mg of vitamin C, 50 IU of alpha-tocopherol, and 12 oz of vegetable cocktail daily for 2 wk. Subjects were then exposed to 0.4 ppm O(3) for 2 h and underwent a second BAL. On the day of the O(3) exposure, supplemented subjects were found to have significantly increased levels of plasma ascorbate, tocopherols, and carotenoids as compared with those of the placebo group. Pulmonary function testing showed that O(3)-induced reductions in FEV(1) and FVC were 30% and 24% smaller, respectively, in the supplemented cohort. In contrast, the inflammatory response to O(3) inhalation, as represented by the percent neutrophils and the concentration of interleukin-6 recovered in the BAL fluid at 1 h after O(3) exposure was not different for the two groups. These data suggest that dietary antioxidants protect against O(3)-induced pulmonary function decrements in humans.
Assuntos
Antioxidantes/uso terapêutico , Pneumopatias/induzido quimicamente , Pneumopatias/prevenção & controle , Ozônio/efeitos adversos , Adulto , Feminino , Humanos , MasculinoRESUMO
Fish losses from infectious diseases are a significant problem in aquaculture worldwide. Therefore, we investigated the ability of cationic antimicrobial peptides to protect against infection caused by the fish pathogen Vibrio anguillarum. To identify effective peptides for fish, the MICs of certain antimicrobial peptides against fish pathogens were determined in vitro. Two of the most effective antimicrobial peptides, CEME, a cecropin-melittin hybrid peptide, and pleurocidin amide, a C-terminally amidated form of the natural flounder peptide, were selected for in vivo studies. A single intraperitoneal injection of CEME did not affect mortality rates in juvenile coho salmon infected with V. anguillarum, the causative agent of vibriosis. Therefore, the peptides were delivered continuously using miniosmotic pumps placed in the peritoneal cavity. Twelve days after pump implantation, the fish received intraperitoneal injections of V. anguillarum at a dose that would kill 50 to 90% of the population. Fish receiving 200 microg of CEME per day survived longer and had significantly lower accumulated mortalities (13%) than the control groups (50 to 58%). Fish receiving pleurocidin amide at 250 microg per day also survived longer and had significantly lower accumulated mortalities (5%) than the control groups (67 to 75%). This clearly shows the potential for antimicrobial peptides to protect fish against infections and indicates that the strategy of overexpressing the peptides in transgenic fish may provide a method of decreasing bacterial disease problems.
Assuntos
Antibacterianos/uso terapêutico , Doenças dos Peixes/prevenção & controle , Oncorhynchus kisutch , Peptídeos/uso terapêutico , Vibrioses/veterinária , Vibrio/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Doenças dos Peixes/microbiologia , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/farmacologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Vibrioses/prevenção & controleRESUMO
A potent interleukin-6 (IL-6) antagonist (Sant 5), which binds tightly to the IL-6alpha receptor but has impaired gp130 heterodimerization, has been developed recently by site-directed mutagenesis of human IL-6. We report here that Sant 5 inhibits IL-6-stimulated osteoclast-like multinucleated cell (MNC) formation in human marrow cultures but also inhibits the stimulatory effects of IL-1 or tumor necrosis factor alpha (TNF-alpha in these cultures. We further show that a neutralizing antibody to IL-6 also inhibits the stimulatory effects of IL-1 or TNF-alpha in these cultures. In contrast, Sant 5 had no effect on parathyroid hormone related protein (PTHrP) or 1,25-dihydroxyvitamin D3 stimulated MNC formation in human marrow cultures. Transfection of a human marrow stromal cell line, which normally induces osteoclast formation through production of IL-6, with the Sant 5 cDNA driven by a cytomegalovirus (CMV) promoter blocked the capacity of these cells to stimulate osteoclast-like cell formation. These Sant 5 transfected cells and conditioned media from these cells also inhibited the stimulatory effects of the parent cell line on MNC formation. These data suggest that IL-6 mediates the effects of IL-1 and TNF on human osteoclast formation, but in contrast to murine systems, does not mediate the effects of PTHrP. These data further demonstrate that stromal cells transfected with the Sant 5 cDNA can constitutively produce high levels of the IL-6 antagonist and inhibit osteoclast formation in vitro.
Assuntos
Células da Medula Óssea/efeitos dos fármacos , Calcitriol/farmacologia , Interleucina-1/farmacologia , Interleucina-6/antagonistas & inibidores , Osteoclastos/efeitos dos fármacos , Proteínas/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Anticorpos Monoclonais , Células Cultivadas , Técnicas de Cocultura , Citocinas/biossíntese , Citomegalovirus/genética , DNA Complementar/genética , Células Gigantes/efeitos dos fármacos , Humanos , Interleucina-6/análise , Proteína Relacionada ao Hormônio Paratireóideo , Regiões Promotoras Genéticas , Células Estromais/efeitos dos fármacos , TransfecçãoRESUMO
Autocrine products of osteoclasts such as interleukin-6 may play an important role in normal osteoclast formation and activity. To identify novel stimulatory factors for osteoclasts, we have prepared a mammalian cDNA expression library generated from highly purified human osteoclast-like multinucleated cells (MNC) formed in long term bone marrow cultures and screened this library for autocrine factors that enhance MNC formation. A candidate clone which stimulated MNC formation was isolated. Sequence analysis showed that this cDNA encoded annexin II (AXII). Purified recombinant AXII significantly increased MNC formation in human bone marrow cultures in the absence of 1,25-(OH)2 vitamin D3 and enhanced MNC formation in mouse bone marrow cultures treated with 10(-9) M 1,25-(OH)2 vitamin D3. The enhanced MNC formation in murine marrow cultures resulted in increased bone resorption. Treatment of fetal rat long bones with AXII and 1,25-(OH)2 vitamin D3 significantly increased bone resorption compared to 1,25-(OH)2 vitamin D3 alone. Reverse transcriptase polymerase chain reaction analysis demonstrated that AXII mRNA was expressed at high levels in RNA isolated from highly purified giant cells from osteoclastomas, human osteoclast-like MNC, and pagetic bone. Western blot analysis of conditioned media collected from human marrow cultures showed that AXII was present in the media. Furthermore, approximately 50% of total AXII produced by cells transfected with AXII cDNA was present in the conditioned media. These data suggest that the AXII is an autocrine factor that enhances osteoclast formation and bone resorption and demonstrate a previous unknown function for AXII.
Assuntos
Anexina A2/genética , Reabsorção Óssea , Osteoclastos/citologia , Animais , Anexina A2/fisiologia , Calcitriol/farmacologia , Células Cultivadas , Clonagem Molecular , Meios de Cultivo Condicionados , DNA Complementar , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Cultura de Órgãos , Osteoclastos/efeitos dos fármacos , Ratos , Proteínas Recombinantes/farmacologia , TransfecçãoRESUMO
Clotting factors synthesized by monocytes and macrophages may initiate coagulation reactions during inflammation. Functional vitamin K-dependent coagulation factors have been found to be associated with human monocytes/macrophages, but there are no reports identifying mRNA coding for vitamin K-dependent proteins in these cells. In the present studies, factor VII mRNA was found in total RNA extracted from freshly isolated human alveolar macrophages using hybridization with a complementary DNA probe. On the other hand, vitamin K-dependent carboxylase activity which is required for postribosomal modification of the protein, was not detectable in the macrophages before or after culture, and human blood mononuclear leukocytes also lacked this enzyme activity. Control human and rat hepatoma cells exhibited high levels of carboxylase activity within the same experiments. Using sensitive kinetic assays, no increase in factor VII activity was detected during culture of alveolar macrophages under conditions promoting 1.78 +/- .24 (n = 8) fold increases of tissue factor activity. These findings with freshly isolated cells demonstrate that alveolar macrophages synthesize factor VII mRNA in vivo. However, the mRNA was found in the absence of evidence for gamma-carboxylase activity or processing of the factor into a functional clotting enzyme. The results imply that functional expression of any synthesized coagulation factor VII in alveolar macrophages may be limited or prevented due to a cellular deficiency at the level of postribosomal processing.