RESUMO
AIMS: To examine the prevalence and diversity of bacterial faecal pathogens in unseparated slurry, separated solids and liquid fractions from a commercial pig farm. METHODS: A total of 43 stored slurry specimens originating from a fattening house over the period February-April 2002 were analysed, consisting of unseparated (n = 14) slurry, separated solids (n = 16) and separated liquid (n = 13). Specimens were examined for the presence of five bacterial pathogens including Salmonella spp., Shigella spp., Campylobacter spp., Escherichia coli O157 and Yersinia enterocolitica. Selective enrichment and plating methods were employed for detection of Salmonella spp. and Campylobacter spp. and conventional selective plating techniques for the remaining genera. Antibiogram profiles to 12 antibiotic agents were obtained for all Salmonella isolates obtained. RESULTS: Salmonella spp. were identified in all components of the slurry specimens, whereas Campylobacter spp. was only recovered from the unseparated and separated liquid fractions. In both cases, the separated liquid fraction had the highest prevalence of pathogens and the separated solid fraction had the lowest prevalence. None of the slurry specimens examined were positive for E. coli O157:H7, Shigella spp. or Y. enterocolitica. Twenty-nine isolates of Salmonella were recovered from the slurry specimens, comprising seven serovars, of which Salmonella manhattan was the most prevalent, accounting for over half [15 of 29 (51.7%)] of all Salmonella isolates. Salmonella anatum, Salm. derby, Salm. give, Salm. heidelberg, Salm. simi and Salm. stanley serovars were also recovered. All Salmonella isolates were sensitive to ampicillin, augmentin (amoxicillin/clavulanic acid), chloramphenicol, ciprofloxacin, gentamicin, kanamycin and trimethoprim, but has variable resistance to tetracycline (100%), sulphonamides (84.6%), furazolidone (38.5%), nalidixic acid (15.4%) and streptomycin (15.4%). The majority (57.7%) of isolates displayed antibiotic resistance to at least two antibiotic agents, followed by 34.6% of isolates being resistant to three agents and the remainder (7.7%) being resistant to four antibiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated a marked reduction in the prevalence of Campylobacter and Salmonella in the solids component of separated pig slurry. The adoption of control processes such as aeration of slurry prior to its spread onto agricultural land and newer approaches to pathogen reduction should be investigated, to reduce the transmission of pathogens from pig slurry to the environment.
Assuntos
Bactérias/isolamento & purificação , Bactérias/patogenicidade , Fezes/microbiologia , Esgotos/microbiologia , Sus scrofa/microbiologia , Criação de Animais Domésticos , Animais , Campylobacter/isolamento & purificação , Campylobacter/patogenicidade , Farmacorresistência Bacteriana , Humanos , Irlanda , Segurança , Salmonella/efeitos dos fármacos , Salmonella/isolamento & purificação , Salmonella/patogenicidadeRESUMO
We have developed a panel of murine monoclonal antibodies that recognize human interferon alpha. One of these mononclonal antibodies binds and neutralizes, with high affinity, all of seven tested recombinant human interferon alphas. This mononclonal antibody also neutralizes the interferon activity present in two independent pools of interferon alphas prepared following stimulation of human peripheral blood leukocytes. The complementary determining regions from this murine mononclonal antibody were transferred to a human IgG2 heavy chain and to a human kappa1 light chain. In addition, six (heavy chain) and two (light chain) amino acids were transferred from the framework regions. This generated a humanized mononclonal antibody that retained the specificity of the mouse parent. The humanized anti-interferon alpha antibody is a candidate therapeutic for those diseases, such as insulin-dependent diabetes, systemic lupus erythematosis, psoriasis and Crohn's disease, which are all characterized by pathological expression of interferon alpha.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/uso terapêutico , Diabetes Mellitus Tipo 1/tratamento farmacológico , Interferon-alfa/química , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Sequência de Aminoácidos , Animais , Técnicas Biossensoriais , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Células HeLa , Humanos , Imunoglobulina G/química , Interferon-alfa/imunologia , Interferon beta/química , Interferon beta/imunologia , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismoRESUMO
Absence of the hormone leptin leads to dramatic increases in appetite, food intake, and adiposity. The primary site of action, at least with respect to appetite, is the hypothalamus. Leptin also has significant effects on the function(s) of peripheral organs involved in maintaining body composition. Some of these effects are mediated through direct interaction of leptin with its receptor on the target tissue, and some effects are indirectly mediated through secondary hormonal and neural pathways. Few of the genes that are responsible for regulating body composition and the peripheral effects of leptin are known. We have used a new gene profiling technology to characterize gene expression changes that occur in the pituitary, hypothalamus, fat, muscle, and liver in response to both obesity and treatment with exogenous leptin. These differences were then overlaid to allow the identification of genes that are regulated by obesity and at least partially normalized by leptin treatment. By using this process we have identified five genes (POMC, PC2, prolactin, HSGP25L2G, and one novel) that are both abnormally expressed in the pituitaries of obese mice and are sensitive to the effects of leptin. We also show that adrenocorticotropic hormone appears to be involved in a regulatory loop involving leptin.
Assuntos
Hormônio Adrenocorticotrópico/genética , Regulação da Expressão Gênica/fisiologia , Leptina/fisiologia , Obesidade/genética , Hipófise/metabolismo , Tecido Adiposo/metabolismo , Animais , Células Cultivadas , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hipotálamo/metabolismo , Leptina/farmacologia , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Modelos Biológicos , Músculo Esquelético/metabolismo , Obesidade/fisiopatologia , Especificidade de ÓrgãosRESUMO
RIP2 is a serine-threonine kinase associated with the tumor necrosis factor (TNF) receptor complex and is implicated in the activation of NF-kappaB and cell death in mammalian cells. However, the function of its kinase domain is still enigmatic as it is not required in engaging these responses. Here we show that RIP2 activates the extracellular signal-regulated kinase (ERK) pathway and that the kinase activity of RIP2 appears to be important in this process. RIP2 activates AP-1 and serum response element regulated expression by inducing the activation of the Elk1 transcription factor. RIP2 directly phosphorylates and activates ERK2 in vivo and in vitro. RIP2 in turn is activated through its interaction with Ras-activated Raf1. Kinase-defective point and deletion variants of RIP2 also significantly blocked the activation of ERK2 by TNFalpha but not epidermal growth factor. These results describe a novel pathway of ERK activation and the first catalytic function ascribed to any of the RIP-like kinases associated with the TNF receptor superfamily.
Assuntos
Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Animais , Células COS , Catálise , Linhagem Celular , Ativação Enzimática , Guanosina Trifosfato/metabolismo , Humanos , Fosforilação , Proteína Serina-Treonina Quinase 2 de Interação com Receptor , Receptores do Fator de Necrose Tumoral/metabolismoRESUMO
RIP3 is a novel gene product containing a N-terminal kinase domain that shares extensive homology with the corresponding domain in RIP (receptor-interacting protein) and RIP2. Unlike RIP, which has a C-terminal death domain, and RIP2, which has a C-terminal caspase activation and recruitment domain, RIP3 has a unique C terminus. RIP3 binds RIP through its unique C-terminal segment and by virtue of this interaction is recruited to the tumor necrosis factor (TNF) receptor-1 signaling complex. Previous studies have shown that RIP mediates TNF-induced activation of the anti-apoptotic NF-kappaB pathway. RIP3, however, attenuates both RIP and TNF receptor-1-induced NF-kappaB activation. Overexpression studies revealed RIP3 to be a potent inducer of apoptosis, capable of selectively binding to large prodomain initiator caspases.
Assuntos
Apoptose , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Antígenos CD/metabolismo , Caspases/metabolismo , Clonagem Molecular , Humanos , Dados de Sequência Molecular , NF-kappa B/metabolismo , Ligação Proteica , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases , Proteínas/metabolismo , Proteína Serina-Treonina Quinase 2 de Interação com Receptor , Proteína Serina-Treonina Quinases de Interação com Receptores , Receptores do Fator de Necrose Tumoral/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral , Proteínas Recombinantes/metabolismo , Análise de Sequência , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
As part of a program to further understand the mechanism by which extracellular signals are coordinated and cell-specific outcomes are generated, we have cloned a novel class of related adaptor molecules (NSP1, NSP2, and NSP3) and have characterized in more detail one of the members, NSP1. NSP1 has an Shc-related SH2 domain and a putative proline/serine-rich SH3 interaction domain. Treatment of cells with epidermal growth factor or insulin leads to NSP1 phosphorylation and increased association with a hypophosphorylated adaptor protein, p130(Cas). In contrast, cell contact with fibronectin results in Cas phosphorylation and a transient dissociation of NSP1 from p130(Cas). Increased expression of NSP1 in 293 cells induces activation of JNK1, but not of ERK2. Consistent with this observation, NSP1 increases the activity of an AP-1-containing promoter. Thus, we have described a novel family of adaptor proteins, one of which may be involved in the process by which receptor tyrosine kinase and integrin receptors control the c-Jun N-terminal kinase/stress-activated protein kinase pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Etiquetas de Sequências Expressas , Substâncias de Crescimento/fisiologia , Integrinas/fisiologia , MAP Quinase Quinase Quinase 1 , Proteínas Quinases Ativadas por Mitógeno , Proteínas/genética , Transdução de Sinais , Animais , Células COS , Clonagem Molecular , Proteína Substrato Associada a Crk , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Fatores de Troca do Nucleotídeo Guanina , Insulina/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno , Dados de Sequência Molecular , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/metabolismo , Proteína p130 Retinoblastoma-Like , Fator de Transcrição AP-1/metabolismoRESUMO
The present study was undertaken to test the hypothesis that exposure to high glucose concentrations enhances insulin secretion in pancreatic islets from glucokinase-deficient mice. Insulin secretion and intracellular calcium ([Ca2+]i) were measured as the glucose concentration was increased from 2 to 26 mmol/l in islets from heterozygous glucokinase (GK)-deficient mice (GK+/-) and their wild-type littermates (GK+/+). Results obtained in islets incubated in 11.6 or 30 mmol/l glucose for 48-96 h were compared. GK+/- islets that had been incubated in 30 mmol/l glucose showed improved although not normal insulin secretory and [Ca2+]i responses to the standard glucose challenge as well as an enhanced ability to sense small amplitude glucose oscillations. These effects were associated with increased glucokinase activity and protein. In contrast, exposure of GK+/+ islets to 30 mmol/l glucose increased their basal insulin secretion but reduced their incremental secretory responses to glucose and their ability to detect small amplitude glucose oscillations. Thus exposure of GK+/- islets to 30 mmol/l glucose for 48-96 h enhanced their ability to sense and respond to a glucose stimulus, whereas similar exposure of GK+/+ islets induced evidence of beta-cell dysfunction. These findings provide a mechanistic framework for understanding why glucokinase diabetes results in mild hyperglycemia that tends not to increase over time. In addition, the absence of one allele of the glucokinase gene appears to protect against glucose-induced beta-cell dysfunction (glucose toxicity).
Assuntos
Glucoquinase/genética , Hiperglicemia/fisiopatologia , Insulina/metabolismo , Animais , Glicemia/metabolismo , Peso Corporal , Cálcio/metabolismo , Relação Dose-Resposta a Droga , Glucoquinase/efeitos dos fármacos , Glucoquinase/metabolismo , Glucose/administração & dosagem , Glucose/farmacologia , Teste de Tolerância a Glucose , Secreção de Insulina , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Camundongos , MutaçãoRESUMO
Mice (Ins.Dd1) with hypoinsulinemic diabetes were created by increased expression of syngeneic major histocompatibility complex (MHC) class I protein in pancreatic beta-cells. The diabetic state was characterized in these mice by high glucose concentrations and islet pathology. To determine whether a neuropathy would develop, motor and sensory conduction velocities (CV) were determined in the sciatic nerves of 2-, 4-, and 7-month-old control and diabetic littermate male mice. Recording bipolar electrodes were placed in the plantar muscles of the hind foot of anesthetized (ketamine/xylazine) mice. Bipolar stimulating electrodes were positioned near the sciatic nerve at the sciatic notch or near the tibial nerve at the ankle. Motor CV from alpha-motor fibers and sensory CV from proprioceptive Aalpha nerves were measured and expressed as meters per second (m/s). Group data are reported as mean +/- SE and compared by analysis of variance. The CVs from nondiabetic mice (controls) were not different across the three ages and averaged 41.3 +/- 1.7 m/s for motor and 38.7 +/- 1.7 m/s for sensory. The motor CVs from diabetic mice at 2 and 4 months were similar to controls. Sensory CVs were unchanged at 2 months but were lower at 4 months (18.9 +/- 2.4 m/s). Both sensory (23.9 +/- 2.1 m/s) and motor (18.9 +/- 1.8 m/s) CVs were significantly reduced at 7 months, which is indicative of a polyneuropathy. NGF has well-known trophic effects on sympathetic and small sensory neurons. To determine whether NGF could influence this neuropathy, 6-month-old control and diabetic mice were divided into the following groups: 1) control + vehicle, 2) diabetic + vehicle, and 3) diabetic + NGF (1 mg/kg, 3x week, s.c.). After 1 month of treatment, motor and sensory CVs were determined. In some mice, the branches of the sciatic nerve were exposed and in situ recordings from the sural nerve were performed to determine compound C-fiber CV, integral, and amplitude. Sensory CV, determined via Hoffmann's reflex (H-reflex) (A-fiber), was decreased in diabetic compared with control animals as expected (P < 0.05), and NGF did not alter this parameter. Continuing diabetes reduced the amplitude (0.9 +/- 0.2 vs. 3.2 +/- 0.7 mV x 10(-2); P < 0.05) and integral (6.9 +/- 1.9 mV/ms vs. 18.8 +/- 4.4 mV/ms; P < 0.05) of the C-fiber response versus control, suggesting fiber loss. NGF treatment normalized C-fiber amplitude (2.9 +/- 0.8 mV x 10(-2)) and integral (21.2 +/- 6.5 mV/ms) in animals with established diabetes, with no effect on blood glucose. The C-fiber CV was similar in all groups, indicating that the animals had some normally conducting small fiber sensory nerves. These studies characterized a motor and sensory polyneuropathy in transgenic diabetic mice and are the first to demonstrate directly that NGF treatment can protect or restore abnormal sensory C-fiber function.
Assuntos
Neuropatias Diabéticas/tratamento farmacológico , Neuropatias Diabéticas/fisiopatologia , Fibras Nervosas/fisiologia , Fatores de Crescimento Neural/uso terapêutico , Animais , Glicemia/metabolismo , Neuropatias Diabéticas/patologia , Estimulação Elétrica , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Insulina/genética , Ilhotas Pancreáticas/patologia , Masculino , Camundongos , Camundongos Transgênicos , Condução Nervosa , Proteínas Recombinantes , Nervo Isquiático/fisiopatologiaAssuntos
Liderança , Cultura Organizacional , Anedotas como Assunto , Ética Institucional , Ética Médica , História , Humanos , Mentores , Princípios Morais , Estados UnidosRESUMO
The ability of interferon-alpha (IFN-alpha) to induce the adhesion molecules that characterize the islets of patients with type I diabetes has been investigated. We have found that all tested recombinant IFN-as will induce major histocompatibility complex (MHC) class I on arterial endothelial cells. Some but not all IFN-as will induce intercellular adhesion molecule-1 (ICAM-1). However, there is only a transient and modest increase in VCAM on arterial endothelial cells. IFN-alpha has very little effect on endothelial MHC class II expression but will induce these proteins on monocytes. Thus, there is a close concordance between the biological actions of IFN-alpha and the appearance of those adhesion molecules induced in the islets of patients with type I diabetes. IFN-alpha is also produced in normal human islets during short-term cultures, probably as a result of the ischemia present at the center of the islet. This induction of IFN-alpha by hypoxia may explain the previously reported spontaneous induction of ICAM-1 in human islets and may also be a contributing factor to the failure of islet grafts.
Assuntos
Endotélio Vascular/imunologia , Antígenos HLA-D/biossíntese , Antígenos de Histocompatibilidade Classe I/biossíntese , Molécula 1 de Adesão Intercelular/biossíntese , Interferon Tipo I/farmacologia , Ilhotas Pancreáticas/imunologia , Artérias , Hipóxia Celular , Células Cultivadas , Primers do DNA , Endotélio Vascular/efeitos dos fármacos , Antígenos HLA-D/análise , Humanos , Interferon gama/farmacologia , Ilhotas Pancreáticas/efeitos dos fármacos , Cinética , Complexo Principal de Histocompatibilidade/efeitos dos fármacos , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase , Proteínas Recombinantes/farmacologia , Fator de Necrose Tumoral alfa/biossíntese , Artérias UmbilicaisRESUMO
OBJECTIVE: To determine antibody levels to the Australian manufactured combined diphtheria, tetanus and pertussis (DTP) vaccine (Triple Antigen, CSL Ltd) in infants before and after their primary immunization course. METHODOLOGY: Serosurvey (antibody prevalence study) in two groups: infants aged 5-9 weeks who had not received any immunizations (n = 25), and infants aged 7-10 months who had received two (n = 25) or three immunizations (n = 57) with DTP, sampled from infants attending the Royal Children's Hospital, Melbourne, either as inpatients or outpatients between February and April 1993. The immunization history for each infant was determined from hospital records, the parent-held child health record, or the local council or family doctor who immunized the infant. RESULTS: Enzyme immunoassay (EIA) of antibodies to diphtheria and tetanus showed all infants to have adequate protective levels after two or three vaccinations (> or = 0.01 IU/mL). All subjects who had received all three DTP vaccinations had detectable antibody to at least one pertussis antigen. Antibodies to the pertussis antigens filamentous haemagglutinin and pertussigen (pertussis toxin) were comparable to levels determined for whole cell pertussis vaccines used elsewhere in the world. EIA-determined antibodies to pertussis agglutinogen type 2 and agglutinogen type 3 showed substantially higher geometric mean titres when results for pre-immunization and post-immunization subjects were compared. CONCLUSIONS: These data show that the Australian manufactured DTP vaccine has immunogenic properties similar to those of vaccines used elsewhere, and that antibody concentrations following immunization are at levels consistent with efficacy.
Assuntos
Anticorpos Antivirais/sangue , Vacina contra Difteria, Tétano e Coqueluche/imunologia , Análise de Variância , Austrália , Intervalos de Confiança , Estudos Transversais , Difteria/imunologia , Difteria/prevenção & controle , Vacina contra Difteria, Tétano e Coqueluche/normas , Relação Dose-Resposta a Droga , Humanos , Esquemas de Imunização , Lactente , Estudos de Amostragem , Tétano/imunologia , Tétano/prevenção & controle , Coqueluche/imunologia , Coqueluche/prevenção & controleRESUMO
Insulin-dependent diabetes is an autoimmune disease characterized by the loss of the insulin-producing beta cells and the appearance of autoreactive (anti-islet) T cells. The mechanism by which these autoimmune T cells become activated has not been resolved. We demonstrate that the expression of IFN-alpha by the pancreatic beta cells leads to the development of CD4+ T cells that proliferate in the presence of islet Ags. These autoreactive T cells have a Th1 phenotype and are able to lyse islet cells, possibly through an indirect, cytokine-mediated mechanism. We also demonstrate that the pancreatic infiltrating leukocytes in the transgenic mice express increased levels of 87.2 and ICAM-1 and that IFN-alpha can directly induce these two costimulatory molecules on nontransgenic splenic APCs. Treatment of the transgenic mice with Abs against these costimulatory molecules demonstrated that B7.2 is essential for the induction of autoreactive T cells, whereas ICAM-1 contributes to but is not essential for the formation of these autoreactive cells. As B7.2 and ICAM-1 are able to synergize to provide costimulatory signals to naive T cells under conditions of limiting Ag, we propose that IFN-alpha expression normally contributes to the development of an anti-viral cellular immune response, but that uncontrolled expression of this cytokine will induce autoimmunity.
Assuntos
Antígenos CD/biossíntese , Autoimunidade/efeitos dos fármacos , Molécula 1 de Adesão Intercelular/biossíntese , Interferon-alfa/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Glicoproteínas de Membrana/biossíntese , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Animais , Antígenos CD/efeitos dos fármacos , Autoimunidade/imunologia , Antígeno B7-2 , Diabetes Mellitus Tipo 1/imunologia , Molécula 1 de Adesão Intercelular/efeitos dos fármacos , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/metabolismo , Glicoproteínas de Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
The effect of local production of human growth hormone on murine cortical bone was investigated using a transgenic mouse model. Femora and humeri from human growth hormone transgenic mice and littermate control mice were obtained, and the geometrical, biomechanical, compositional, and histomorphometric properties of all specimens were determined. The goals were to investigate the effects of local expression of human growth hormone on skeletal integrity, including the functional geometry of long bone and its related structural and mechanical behavior, as well as tissue composition and integrity. As expected, local production of human growth hormone by osteoblasts indeed resulted in longer femora with significantly greater mid-diaphyseal cross-sectional geometry in the transgenic mice (16% increase in cross-sectional area and 29% increase in bending moments of inertia). However, the significant increase in geometry was not associated with a proportional increase in bending stiffness and other structural properties, which suggested that the mechanical properties of the cortical bone tissue may have been inferior. Microspecimen bending tests verified this prediction, given that transgenic cortical bone tissue had significantly lower apparent elastic modulus and ultimate strength (52 and 68%, respectively, of control values). These defects in the whole bone structural and tissue mechanical properties of transgenic specimens were associated with a smaller fraction of ash, larger fractions of woven bone and cartilage islands, and greater porosity in the mid-diaphyseal cortices. These results suggest that local production of human growth hormone by osteoblasts is indeed anabolic for bone, but at the expense of bone tissue integrity.
Assuntos
Desenvolvimento Ósseo/efeitos dos fármacos , Fêmur/efeitos dos fármacos , Hormônio do Crescimento/fisiologia , Adaptação Fisiológica , Animais , Peso Corporal/fisiologia , Fêmur/fisiologia , Hormônio do Crescimento/biossíntese , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Estresse MecânicoRESUMO
Cytokines, particularly interferons, may participate in the development of type I diabetes. This involvement could be from direct cytotoxic actions of the interferons on the pancreatic beta-cells or from an indirect influence on the number, activity, or type of inflammatory cells that invade the islets in type I diabetes. To examine directly the role of interferon (IFN)-gamma in a mouse model of type I diabetes, we have introduced an inactivating mutation in the IFN-gamma gene (ifg) into NOD mice. The genetic absence of IFN-gamma does not prevent either insulitis or diabetes in the NOD mice, but it does increase the time to onset. Although it might have been predicted that the absence of IFN-gamma in these mice would lead to an increase in expression of Th2 T-helper cell-related cytokines, we found instead a profound decrease in the expression of two of the characteristic Th2 cytokines, interleukin (IL)-4 and IL-10. We also demonstrate that the splenocytes taken from IFN-gamma-deficient diabetic mice are fully capable of transferring diabetes to naive recipients.
Assuntos
Citocinas/biossíntese , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Interferon gama/genética , Ilhotas Pancreáticas/imunologia , Envelhecimento/fisiologia , Animais , Diabetes Mellitus Tipo 1/patologia , Feminino , Deleção de Genes , Expressão Gênica , Genótipo , Imunoterapia Adotiva , Interferon gama/deficiência , Ilhotas Pancreáticas/crescimento & desenvolvimento , Ilhotas Pancreáticas/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Mutantes , Reação em Cadeia da PolimeraseRESUMO
The secretion of insulin is controlled by the rate of glucose metabolism in the pancreatic beta cells. As phosphorylation by glucokinase (GLK) appears to be the rate-limiting step for glucose catabolism in beta cells, this enzyme may be the glucose sensor. To test this possibility and to resolve the relative roles of liver and beta cell GLK in maintaining glucose levels, we have generated mice completely deficient in GLK and transgenic mice in which GLK is expressed only in beta cells. In mice with only one GLK allele, blood glucose levels are elevated and insulin secretion is reduced. GLK-deficient mice die perinatally with severe hyperglycemia. Expression of GLK in beta cells in the absence of expression in the liver is sufficient for survival. These mice demonstrate the critical need for beta cell GLK in maintaining normal glucose levels and provide a novel model for one form of noninsulin-dependent diabetes.
Assuntos
Diabetes Mellitus Tipo 2/genética , Modelos Animais de Doenças , Glucoquinase/fisiologia , Glucose/metabolismo , Hiperglicemia/genética , Ilhotas Pancreáticas/enzimologia , Alelos , Animais , Glicemia/análise , Diabetes Mellitus Tipo 2/enzimologia , Indução Enzimática , Feminino , Genes Letais , Glucoquinase/deficiência , Glucoquinase/genética , Homeostase , Hiperglicemia/enzimologia , Insulina/metabolismo , Secreção de Insulina , Fígado/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Camundongos TransgênicosRESUMO
Individuals with insulin resistance show increased levels of PC-1 expression in skeletal muscle and fibroblasts, and in transfected cell lines that overexpress PC-1 there is a reduction in the insulin-stimulated insulin receptor tyrosine phosphorylation. As PC-1 is a type II transmembrane protein with extracellular phosphodiesterase and pyrophosphatase activity, increased expression of PC-1 at the cell surface will decrease extracellular adenosine triphosphate levels and increase extracellular adenosine levels. Consequently it is possible that PC-1-mediated insulin resistance could be caused either by a decrease in adenosine triphosphate or an indirect increase in adenosine levels. We have tested this hypothesis and find that the PC-1-mediated inhibition of insulin-stimulated insulin receptor autophosphorylation is not altered by agents that alter the level or action of adenosine. Further, a mutated PC-1 with a single amino acid change that abolishes the phosphodiesterase and pyrophosphatase activities is still able to inhibit insulin-stimulated insulin receptor phosphorylation. The results of these experiments indicate that the phosphodiesterase activity of PC-1 is not involved in the inhibition of insulin receptor autophosphorylation.
Assuntos
Trifosfato de Adenosina/metabolismo , Adenosina/metabolismo , Insulina/farmacologia , Glicoproteínas de Membrana/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Pirofosfatases/metabolismo , Receptor de Insulina/metabolismo , Proteínas Repressoras/isolamento & purificação , Sequência de Bases , Humanos , Técnicas In Vitro , Resistência à Insulina , Fatores de Transcrição Kruppel-Like , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos/química , Fosfodiesterase I , Fosforilação , Antagonistas de Receptores Purinérgicos P1 , Receptor de Insulina/antagonistas & inibidores , Receptores Purinérgicos P1/fisiologia , Proteínas Repressoras/genética , Transdução de Sinais , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
We have used a reverse transcriptase-polymerase chain reaction (RT-PCR) protocol to examine the expression of cytokines in the pancreases and islets of patients with type I diabetes. We detect a significant increase in the level of expression of interferon (IFN)-alpha in the pancreases of the diabetic patients as compared with the control pancreases. In contrast, IFN-beta was detected at comparable levels in both groups, while IFN-gamma was detected in three of four control pancreases and one of four pancreases from the diabetic individuals. The IFN-alpha cDNAs generated by the RT-PCR were cloned and sequenced to determine which alpha-subtypes were being expressed. We found that the repertoire of subtypes was quite limited in any one individual (diabetic or not), although each individual was different with respect to the pattern of subtypes expressed. We also examined these pancreases for the expression of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1 beta, IL-2, IL-4, and IL-6. We found no detectable expression of TNF-alpha or IL-2 in any pancreases, and the expression of the other cytokines was variable, with no pattern emerging from the comparison of the diabetic and nondiabetic individuals. We conclude that, of the cytokines examined, only IFN-alpha was significantly increased in the diabetic patients, a result that is consistent with the possibility that this cytokine is directly involved in the development of type I diabetes.