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1.
Lett Appl Microbiol ; 36(4): 208-12, 2003.
Article in English | MEDLINE | ID: mdl-12641712

ABSTRACT

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.


Subject(s)
Bacteria/isolation & purification , Bacteria/pathogenicity , Feces/microbiology , Sewage/microbiology , Sus scrofa/microbiology , Animal Husbandry , Animals , Campylobacter/isolation & purification , Campylobacter/pathogenicity , Drug Resistance, Bacterial , Humans , Ireland , Safety , Salmonella/drug effects , Salmonella/isolation & purification , Salmonella/pathogenicity
2.
Cytokine ; 15(5): 250-60, 2001 Sep 07.
Article in English | MEDLINE | ID: mdl-11594789

ABSTRACT

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.


Subject(s)
Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/therapeutic use , Diabetes Mellitus, Type 1/drug therapy , Interferon-alpha/chemistry , Lupus Erythematosus, Systemic/drug therapy , Amino Acid Sequence , Animals , Biosensing Techniques , Dose-Response Relationship, Drug , Enzyme-Linked Immunosorbent Assay , HeLa Cells , Humans , Immunoglobulin G/chemistry , Interferon-alpha/immunology , Interferon-beta/chemistry , Interferon-beta/immunology , Mice , Models, Molecular , Molecular Sequence Data , Protein Structure, Tertiary , Recombinant Proteins/metabolism
3.
J Biol Chem ; 275(14): 10429-36, 2000 Apr 07.
Article in English | MEDLINE | ID: mdl-10744732

ABSTRACT

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.


Subject(s)
Adrenocorticotropic Hormone/genetics , Gene Expression Regulation/physiology , Leptin/physiology , Obesity/genetics , Pituitary Gland/metabolism , Adipose Tissue/metabolism , Animals , Cells, Cultured , Female , Gene Expression Regulation/drug effects , Hypothalamus/metabolism , Leptin/pharmacology , Liver/metabolism , Mice , Mice, Inbred C57BL , Mice, Obese , Models, Biological , Muscle, Skeletal/metabolism , Obesity/physiopathology , Organ Specificity
4.
J Biol Chem ; 274(47): 33684-90, 1999 Nov 19.
Article in English | MEDLINE | ID: mdl-10559258

ABSTRACT

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.


Subject(s)
Mitogen-Activated Protein Kinase Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-raf/metabolism , Animals , COS Cells , Catalysis , Cell Line , Enzyme Activation , Guanosine Triphosphate/metabolism , Humans , Phosphorylation , Receptor-Interacting Protein Serine-Threonine Kinase 2 , Receptors, Tumor Necrosis Factor/metabolism
6.
J Biol Chem ; 274(24): 16871-5, 1999 Jun 11.
Article in English | MEDLINE | ID: mdl-10358032

ABSTRACT

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.


Subject(s)
Apoptosis , Protein Kinases/metabolism , Amino Acid Sequence , Antigens, CD/metabolism , Caspases/metabolism , Cloning, Molecular , Humans , Molecular Sequence Data , NF-kappa B/metabolism , Protein Binding , Protein Kinases/genetics , Protein Serine-Threonine Kinases , Proteins/metabolism , Receptor-Interacting Protein Serine-Threonine Kinase 2 , Receptor-Interacting Protein Serine-Threonine Kinases , Receptors, Tumor Necrosis Factor/metabolism , Receptors, Tumor Necrosis Factor, Type I , Recombinant Proteins/metabolism , Sequence Analysis , Sequence Homology, Amino Acid , Signal Transduction
7.
J Biol Chem ; 274(15): 10047-52, 1999 Apr 09.
Article in English | MEDLINE | ID: mdl-10187783

ABSTRACT

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.


Subject(s)
Adaptor Proteins, Signal Transducing , Calcium-Calmodulin-Dependent Protein Kinases/physiology , Expressed Sequence Tags , Growth Substances/physiology , Integrins/physiology , MAP Kinase Kinase Kinase 1 , Mitogen-Activated Protein Kinases , Proteins/genetics , Signal Transduction , Animals , COS Cells , Cloning, Molecular , Crk-Associated Substrate Protein , Electrophoresis, Polyacrylamide Gel , Enzyme Activation , Epidermal Growth Factor/metabolism , ErbB Receptors/metabolism , Guanine Nucleotide Exchange Factors , Insulin/metabolism , JNK Mitogen-Activated Protein Kinases , Molecular Sequence Data , Phosphoproteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Proteins/metabolism , Retinoblastoma-Like Protein p130 , Transcription Factor AP-1/metabolism
8.
N Z Vet J ; 47(2): 77-8, 1999 Apr.
Article in English | MEDLINE | ID: mdl-16032076
9.
Diabetes ; 47(12): 1881-8, 1998 Dec.
Article in English | MEDLINE | ID: mdl-9836519

ABSTRACT

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).


Subject(s)
Glucokinase/genetics , Hyperglycemia/physiopathology , Insulin/metabolism , Animals , Blood Glucose/metabolism , Body Weight , Calcium/metabolism , Dose-Response Relationship, Drug , Glucokinase/drug effects , Glucokinase/metabolism , Glucose/administration & dosage , Glucose/pharmacology , Glucose Tolerance Test , Insulin Secretion , Islets of Langerhans/cytology , Islets of Langerhans/drug effects , Islets of Langerhans/metabolism , Mice , Mutation
10.
Diabetes ; 47(10): 1637-42, 1998 Oct.
Article in English | MEDLINE | ID: mdl-9753304

ABSTRACT

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.


Subject(s)
Diabetic Neuropathies/drug therapy , Diabetic Neuropathies/physiopathology , Nerve Fibers/physiology , Nerve Growth Factors/therapeutic use , Animals , Blood Glucose/metabolism , Diabetic Neuropathies/pathology , Electric Stimulation , Histocompatibility Antigens Class I/genetics , Humans , Insulin/genetics , Islets of Langerhans/pathology , Male , Mice , Mice, Transgenic , Neural Conduction , Recombinant Proteins , Sciatic Nerve/physiopathology
11.
12.
Diabetes ; 45(10): 1336-43, 1996 Oct.
Article in English | MEDLINE | ID: mdl-8826968

ABSTRACT

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.


Subject(s)
Endothelium, Vascular/immunology , HLA-D Antigens/biosynthesis , Histocompatibility Antigens Class I/biosynthesis , Intercellular Adhesion Molecule-1/biosynthesis , Interferon Type I/pharmacology , Islets of Langerhans/immunology , Arteries , Cell Hypoxia , Cells, Cultured , DNA Primers , Endothelium, Vascular/drug effects , HLA-D Antigens/analysis , Humans , Interferon-gamma/pharmacology , Islets of Langerhans/drug effects , Kinetics , Major Histocompatibility Complex/drug effects , Oligonucleotide Probes , Polymerase Chain Reaction , Recombinant Proteins/pharmacology , Tumor Necrosis Factor-alpha/biosynthesis , Umbilical Arteries
13.
J Paediatr Child Health ; 32(5): 378-81, 1996 Oct.
Article in English | MEDLINE | ID: mdl-8933394

ABSTRACT

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.


Subject(s)
Antibodies, Viral/blood , Diphtheria-Tetanus-Pertussis Vaccine/immunology , Analysis of Variance , Australia , Confidence Intervals , Cross-Sectional Studies , Diphtheria/immunology , Diphtheria/prevention & control , Diphtheria-Tetanus-Pertussis Vaccine/standards , Dose-Response Relationship, Drug , Humans , Immunization Schedule , Infant , Sampling Studies , Tetanus/immunology , Tetanus/prevention & control , Whooping Cough/immunology , Whooping Cough/prevention & control
14.
J Orthop Res ; 14(4): 598-604, 1996 Jul.
Article in English | MEDLINE | ID: mdl-8764869

ABSTRACT

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.


Subject(s)
Bone Development/drug effects , Femur/drug effects , Growth Hormone/physiology , Adaptation, Physiological , Animals , Body Weight/physiology , Femur/physiology , Growth Hormone/biosynthesis , Humans , Male , Mice , Mice, Transgenic , Stress, Mechanical
15.
J Immunol ; 157(2): 522-8, 1996 Jul 15.
Article in English | MEDLINE | ID: mdl-8752897

ABSTRACT

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.


Subject(s)
Antigens, CD/biosynthesis , Autoimmunity/drug effects , Intercellular Adhesion Molecule-1/biosynthesis , Interferon-alpha/pharmacology , Lymphocyte Activation/drug effects , Membrane Glycoproteins/biosynthesis , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism , Animals , Antigens, CD/drug effects , Autoimmunity/immunology , B7-2 Antigen , Diabetes Mellitus, Type 1/immunology , Intercellular Adhesion Molecule-1/drug effects , Islets of Langerhans/drug effects , Islets of Langerhans/immunology , Islets of Langerhans/metabolism , Membrane Glycoproteins/drug effects , Mice , Mice, Inbred C57BL , Mice, Transgenic
16.
Diabetes ; 45(6): 812-7, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8635658

ABSTRACT

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.


Subject(s)
Cytokines/biosynthesis , Diabetes Mellitus, Type 1/genetics , Diabetes Mellitus, Type 1/immunology , Interferon-gamma/genetics , Islets of Langerhans/immunology , Aging/physiology , Animals , Diabetes Mellitus, Type 1/pathology , Female , Gene Deletion , Gene Expression , Genotype , Immunotherapy, Adoptive , Interferon-gamma/deficiency , Islets of Langerhans/growth & development , Islets of Langerhans/pathology , Mice , Mice, Inbred NOD , Mice, Mutant Strains , Polymerase Chain Reaction
17.
Cell ; 83(1): 69-78, 1995 Oct 06.
Article in English | MEDLINE | ID: mdl-7553875

ABSTRACT

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.


Subject(s)
Diabetes Mellitus, Type 2/genetics , Disease Models, Animal , Glucokinase/physiology , Glucose/metabolism , Hyperglycemia/genetics , Islets of Langerhans/enzymology , Alleles , Animals , Blood Glucose/analysis , Diabetes Mellitus, Type 2/enzymology , Enzyme Induction , Female , Genes, Lethal , Glucokinase/deficiency , Glucokinase/genetics , Homeostasis , Hyperglycemia/enzymology , Insulin/metabolism , Insulin Secretion , Liver/enzymology , Male , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Knockout , Mice, Transgenic
18.
J Biol Chem ; 270(38): 22085-8, 1995 Sep 22.
Article in English | MEDLINE | ID: mdl-7673181

ABSTRACT

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.


Subject(s)
Adenosine Triphosphate/metabolism , Adenosine/metabolism , Insulin/pharmacology , Membrane Glycoproteins/metabolism , Phosphoric Diester Hydrolases/metabolism , Pyrophosphatases/metabolism , Receptor, Insulin/metabolism , Repressor Proteins/isolation & purification , Base Sequence , Humans , In Vitro Techniques , Insulin Resistance , Kruppel-Like Transcription Factors , Molecular Sequence Data , Mutagenesis, Site-Directed , Oligodeoxyribonucleotides/chemistry , Phosphodiesterase I , Phosphorylation , Purinergic P1 Receptor Antagonists , Receptor, Insulin/antagonists & inhibitors , Receptors, Purinergic P1/physiology , Repressor Proteins/genetics , Signal Transduction , Structure-Activity Relationship , Tumor Cells, Cultured
19.
Diabetes ; 44(6): 658-64, 1995 Jun.
Article in English | MEDLINE | ID: mdl-7540571

ABSTRACT

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.


Subject(s)
Diabetes Mellitus, Type 1/metabolism , Interferons/biosynthesis , Islets of Langerhans/metabolism , Adolescent , Adult , Base Sequence , Child , Child, Preschool , DNA Primers , Female , Humans , Infant , Interferon-alpha/biosynthesis , Interferon-beta/biosynthesis , Interferon-gamma/biosynthesis , Interleukin-1/biosynthesis , Interleukin-2/biosynthesis , Interleukin-4/biosynthesis , Interleukin-6/biosynthesis , Male , Molecular Sequence Data , Polymerase Chain Reaction , Tumor Necrosis Factor-alpha/biosynthesis
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