RESUMO
Essentials Activated FVII (FVIIa) and FX (FXa) are inhibited by tissue factor pathway inhibitor (TFPI). A monoclonal antibody, mAb2F22, was raised against the N-terminal fragment of TFPI (1-79). mAb2F22 bound exclusively to the K1 domain of TFPI (KD â¼1 nm) and not to the K2 domain. mAb2F22 interfered with inhibition of both FVIIa and FXa activities and restored clot formation. SUMMARY: Background Initiation of coagulation is induced by binding of activated factor VII (FVIIa) to tissue factor (TF) and activation of factor X (FX) in a process regulated by tissue factor pathway inhibitor (TFPI). TFPI contains three Kunitz-type protease inhibitor domains (K1-K3), of which K1 and K2 block the active sites of FVIIa and FXa, respectively. Objective To produce a monoclonal antibody (mAb) directed towards K1, to characterize the binding epitope, and to study its effect on TFPI inhibition. Methods A monoclonal antibody, mAb2F22, was raised against the N-terminal TFPI(1-79) fragment. Binding data were obtained by surface plasmon resonance analysis. The Fab-fragment of mAb2F22, Fab2F22, was expressed and the structure of its complex with TFPI(1-79) determined by X-ray crystallography. Effects of mAb2F22 on TFPI inhibition were measured in buffer- and plasma-based systems. Results mAb2F22 bound exclusively to K1 of TFPI (KD ~1 nm) and not to K2. The crystal structure of Fab2F22/TFPI (1-79) mapped an epitope on K1 including seven residues upstream of the domain. TFPI inhibition of TF/FVIIa amidolytic activity was neutralized by mAb2F22, although the binding epitope on K1 did not include the P1 residue. Binding of mAb2F22 to K1 blocked TFPI inhibition of the FXa amidolytic activity and normalized hemostasis in hemophilia human A-like plasma and whole blood. Conclusion mAb2F22 blocked TFPI inhibition of both FVIIa and FXa activities and mapped a FXa exosite for binding to K1. It reversed TFPI feedback inhibition of TF/FVIIa-induced coagulation and restored clot formation in FVIII-neutralized human plasma and blood.
Assuntos
Anticorpos Monoclonais/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Coagulantes/farmacologia , Fator VIIa/metabolismo , Fator Xa/metabolismo , Hemofilia A/tratamento farmacológico , Lipoproteínas/metabolismo , Fragmentos de Peptídeos/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Sítios de Ligação de Anticorpos , Linhagem Celular , Coagulantes/imunologia , Coagulantes/metabolismo , Cristalografia por Raios X , Epitopos , Fator VIIa/química , Fator Xa/química , Hemofilia A/sangue , Hemofilia A/diagnóstico , Hemofilia A/imunologia , Humanos , Lipoproteínas/química , Lipoproteínas/imunologia , Camundongos , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Interleukin (IL)-20 is a recently discovered cytokine displaying increased levels in psoriatic lesions. Interestingly, IL-20 levels decrease with antipsoriatic treatment, correlating with clinical improvement. However, the role of IL-20 in the aetiology of psoriasis is unknown. OBJECTIVES: In this study, we investigate the effects both of blocking IL-20 signalling in psoriatic plaques and of adding IL-20 to nonlesional psoriasis skin. METHODS: We employed the human skin xenograft transplantation model in which psoriatic plaques and nonlesional keratome skin biopsies obtained from donors with moderate to severe plaque psoriasis were transplanted on to immuno-deficient mice. The transplanted mice were treated with anti-IL-20 antibodies or recombinant human IL-20. RESULTS: We demonstrate that blocking IL-20 signalling with anti-IL-20 antibodies induces psoriasis resolution and inhibits psoriasis induction. We also demonstrate that continuous IL-20 infusion, together with injection of additional nonactivated leucocytes, promotes induction of psoriasis in nonlesional skin from patients with psoriasis. CONCLUSIONS: The results suggest that IL-20 plays a critical role in the induction and maintenance of psoriasis, and IL-20 is suggested as a new possible specific target in psoriasis treatment.
Assuntos
Interleucinas/fisiologia , Psoríase/etiologia , Transdução de Sinais/imunologia , Transplante de Pele , Adulto , Idoso , Animais , Especificidade de Anticorpos/imunologia , Proliferação de Células , Humanos , Interleucinas/antagonistas & inibidores , Interleucinas/imunologia , Camundongos , Camundongos SCID , Pessoa de Meia-Idade , Psoríase/tratamento farmacológico , Psoríase/imunologia , Proteínas Recombinantes/imunologia , Indução de Remissão , Transplante HeterólogoRESUMO
BACKGROUND: Interleukin (IL)-20 and IL-19 are recently discovered members of the IL-10 family of cytokines. The skin of transgenic mice overexpressing IL-20 shows histological changes resembling some of those seen in psoriasis, i.e. thickened epidermis, hyperkeratosis and a compact stratum corneum. IL-19 and IL-20, as well as their receptor complexes, IL-20Ralpha/IL-20Rbeta and IL-22Ralpha/IL-20Rbeta, are expressed in human skin. OBJECTIVES: To study the dynamics of IL-19 and IL-20 gene expression as well as the expression of their receptor subunits in psoriatic skin lesions. METHODS: Punch biopsies from patients with plaque-type psoriasis were collected before, during and after 28 days of treatment with either calcipotriol or ciclosporin (CsA). IL-20, IL-19, IL-20Ralpha, IL-20Rbeta and IL-22Ralpha mRNA expression were determined by quantitative reverse transcriptase-polymerase chain reaction. RESULTS: We found IL-19 and IL-20 mRNA expression in lesional psoriatic skin to be strongly upregulated compared with nonlesional psoriatic skin by a factor of 65 and 22, respectively. In contrast to previous reports, IL-20Ralpha and IL-20Rbeta mRNA levels showed a modest but statistically significant decrease in lesional psoriatic skin compared with nonlesional psoriatic skin. During treatment with calcipotriol or CsA, IL-19 and IL-20 mRNA levels decrease in accordance with the clinical improvement of psoriasis. Neither IL-19, IL-20, nor receptor subunit mRNA expression in lesional psoriatic skin reaches the levels of nonlesional skin during this short-term treatment. These findings are in line with the residual disease activity observed at the end of treatment. CONCLUSIONS: The increased IL-19 and IL-20 mRNA expression levels in lesional psoriatic skin suggest that these two cytokines play a role in the pathogenesis of psoriasis. An imbalance in the receptor complexes for IL-19 and IL-20 might contribute to their suspected pathogenic effects.
Assuntos
Interleucina-10/metabolismo , Interleucinas/metabolismo , Psoríase/metabolismo , Adulto , Idoso , Calcitriol/análogos & derivados , Calcitriol/uso terapêutico , Doença Crônica , Ciclosporina/uso terapêutico , Fármacos Dermatológicos/uso terapêutico , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Humanos , Imunossupressores/uso terapêutico , Interleucina-10/genética , Interleucinas/genética , Masculino , Pessoa de Meia-Idade , Psoríase/tratamento farmacológico , RNA Mensageiro/genética , Receptores de Interleucina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Pele/metabolismoRESUMO
BACKGROUND: The trefoil factors (TFF1-3) are cysteine-rich peptides expressed in the gastrointestinal tract where they play a critical role in mucosal protection and repair. The expression is up-regulated at sites of ulceration in various chronic inflammatory diseases. Recently, we presented an ELISA method for measurement of TFF3. The aims of the present study were to develop and evaluate ELISAs for the other two known human trefoil peptides, TFF1 and TFF2, and to carry out a cross-sectional study on serum TFF levels in patients with inflammatory bowel disease (IBD). METHODS: The TFF1-ELISA was based on two polyclonal rabbit antibodies and the TFF2-ELISA on a monoclonal mouse antibody and a polyclonal rabbit antibody. RhTFF1 and 2 were employed to prepare the calibrators. TFF1-3 were assayed in serum from IBD patients (n=41) and controls (n=13). RESULTS: The TFF1- (TFF2-) ELISA had a detection limit of 3 pmol/L (6 pmol/L) and an analytical imprecision (CV(A)) of 7.0-8.8 for mean concentrations of 24-120 pmol/L (6.1-8.0 for mean concentrations of 17-77 pmol/L). The central reference intervals (n=300) were 140-1400 pmol/L (37-190 pmol/L). There was no variation with age and menstrual cycle. Food intake reduced concentrations of TFF1 by approximately 15%, but did not influence concentrations of TFF2. TFF1 and TFF3 were increased in serum from IBD patients. CONCLUSIONS: We have developed assays for measuring TFF1 and TFF2. Finding increased TFF concentrations in serum from IBD patients suggests that measurements of trefoil peptides may be of clinical relevance in IBD.
Assuntos
Imunoensaio/métodos , Doenças Inflamatórias Intestinais/sangue , Mucinas/sangue , Proteínas Musculares/sangue , Peptídeos/sangue , Proteínas/análise , Adolescente , Adulto , Idoso , Envelhecimento/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Saúde , Humanos , Masculino , Pessoa de Meia-Idade , Mucinas/imunologia , Proteínas Musculares/imunologia , Peptídeos/imunologia , Proteínas/imunologia , Sensibilidade e Especificidade , Fator Trefoil-1 , Fator Trefoil-2 , Fator Trefoil-3 , Proteínas Supressoras de TumorRESUMO
Adipocyte differentiation is co-ordinately regulated by several transcription factors and is accompanied by changes in the expression of a variety of genes. Using mRNA differential display analysis, we have isolated a novel mRNA, DD16, specifically induced during the course of adipocyte differentiation. DD16 mRNAs are present in several tissues, but among the tissues tested, a remarkably higher level of expression was found in white adipose tissue. The DD16 cDNA encoded a polypeptide of 415 amino acids containing a single N-glycosylation site and an N-terminal hydrophobic stretch of 19 amino acids forming a transmembrane segment, indicating that DD16 is a glycosylated membrane-bound protein. Polyclonal antibodies raised against the DD16 peptide detected immunoreactive DD16 in membrane fractions, notably the plasma membrane. Association of DD16 with the plasma membrane was further confirmed by biotinylation studies of cell surface proteins, suggesting that DD16 is an integral plasma membrane protein. Therefore we propose to give DD16 the name APMAP (Adipocyte Plasma Membrane-Associated Protein). Although the biological function of this polypeptide is presently unknown, our data suggest that APMAP may function as a novel protein involved in the cross-talk of mature adipocytes with the environment.
Assuntos
Adipócitos/citologia , Adipócitos/metabolismo , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Células 3T3 , Sequência de Aminoácidos , Animais , Sequência de Bases , Biomarcadores , Diferenciação Celular/fisiologia , Membrana Celular/metabolismo , Clonagem Molecular , DNA Complementar/genética , Glicosilação , Imunoquímica , Masculino , Glicoproteínas de Membrana/química , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
To study the influence of subcellular localization as a determinant of signal transduction specificity, we assessed the effects of wild-type transmembrane and cytoplasmic protein tyrosine phosphatase (PTP) epsilon on tyrosine kinase signalling in baby hamster kidney (BHK) cells overexpressing the insulin receptor (BHK-IR). The efficiency by which differently localized PTPepsilon and PTPalpha variants attenuated insulin-induced cell rounding and detachment was determined in a functional clonal-selection assay and in stable cell lines. Compared with the corresponding receptor-type PTPs, the cytoplasmic PTPs (cytPTPs) were considerably less efficient in generating insulin-resistant clones, and exceptionally high compensatory expression levels were required to counteract phosphotyrosine-based signal transduction. Targeting of cytPTPepsilon to the plasma membrane via the Lck-tyrosine kinase dual acylation motif restored high rescue efficiency and abolished the need for high cytPTPepsilon levels. Consistent with these results, expression levels and subcellular localization of PTPepsilon were also found to determine the phosphorylation level of cellular proteins including focal adhesion kinase (FAK). Furthermore, PTPepsilon stabilized binding of phosphorylated FAK to Src, suggesting this complex as a possible mediator of the PTPepsilon inhibitory response to insulin-induced cell rounding and detachment in BHK-IR cells. Taken together, the present localization-function study indicates that transcriptional control of the subcellular localization of PTPepsilon may provide a molecular mechanism that determines PTPepsilon substrate selectivity and isoform-specific function.
Assuntos
Proteínas Tirosina Fosfatases/metabolismo , Transdução de Sinais , Animais , Adesão Celular , Divisão Celular , Linhagem Celular , Células Clonais , Cricetinae , Proteína-Tirosina Quinases de Adesão Focal , Insulina/farmacologia , Proteínas de Membrana/metabolismo , Fosforilação , Fosfotirosina/metabolismo , Testes de Precipitina , Isoformas de Proteínas/metabolismo , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Receptor de Insulina/metabolismo , Especificidade por Substrato , TransfecçãoRESUMO
Because cocaine- and amphetamine-regulated transcript (CART) coexists with alpha-melanocyte stimulating hormone (alpha-MSH) in the arcuate nucleus neurons and we have recently demonstrated that alpha-MSH innervates TRH-synthesizing neurons in the hypothalamic paraventricular nucleus (PVN), we raised the possibility that CART may also be contained in fibers that innervate hypophysiotropic thyrotropin-releasing hormone (TRH) neurons and modulate TRH gene expression. Triple-labeling fluorescent in situ hybridization and immunofluorescence were performed to reveal the morphological relationships between pro-TRH mRNA-containing neurons and CART- and alpha-MSH-immunoreactive (IR) axons. CART-IR axons densely innervated the majority of pro-TRH mRNA-containing neurons in all parvocellular subdivisions of the PVN and established asymmetric synaptic specializations with pro-TRH neurons. However, whereas all alpha-MSH-IR axons in the PVN contained CART-IR, only a portion of CART-IR axons in contact with pro-TRH neurons were immunoreactive for alpha-MSH. In the medial and periventricular parvocellular subdivisions of the PVN, CART was co-contained in approximately 80% of pro-TRH neuronal perikarya, whereas colocalization with pro-TRH was found in <10% of the anterior parvocellular subdivision neurons. In addition, >80% of TRH/CART neurons in the periventricular and medial parvocellular subdivisions accumulated Fluoro-Gold after systemic administration, suggesting that CART may serve as a marker for hypophysiotropic TRH neurons. CART prevented fasting-induced suppression of pro-TRH in the PVN when administered intracerebroventricularly and increased the content of TRH in hypothalamic cell cultures. These studies establish an anatomical association between CART and pro-TRH-producing neurons in the PVN and demonstrate that CART has a stimulatory effect on hypophysiotropic TRH neurons by increasing pro-TRH gene expression and the biosynthesis of TRH.
Assuntos
Jejum/metabolismo , Hipotálamo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Precursores de Proteínas/biossíntese , Estilbamidinas , Hormônio Liberador de Tireotropina/biossíntese , Animais , Comportamento Animal/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Contagem de Células , Células Cultivadas , Colchicina/administração & dosagem , Corantes Fluorescentes , Sistema Hipotálamo-Hipofisário/metabolismo , Hipotálamo/citologia , Hipotálamo/efeitos dos fármacos , Injeções Intraventriculares , Masculino , Proteínas do Tecido Nervoso/administração & dosagem , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Núcleo Hipotalâmico Paraventricular/diagnóstico por imagem , Núcleo Hipotalâmico Paraventricular/efeitos dos fármacos , Núcleo Hipotalâmico Paraventricular/metabolismo , Núcleo Hipotalâmico Paraventricular/ultraestrutura , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Precursores de Proteínas/genética , Ácido Pirrolidonocarboxílico/análogos & derivados , RNA Mensageiro/metabolismo , Radiografia , Ratos , Ratos Sprague-Dawley , Glândula Tireoide/metabolismo , Tireotropina/sangue , Hormônio Liberador de Tireotropina/genética , Tiroxina/sangue , alfa-MSH/metabolismoRESUMO
PURPOSE: To investigate whether glycoxidation products and the receptor for advanced glycation end products (RAGE) are present and colocalize in subfoveal membranes of patients with age-related macular degeneration (ARMD). METHODS: Surgically removed subfoveal fibrovascular membranes from 12 patients, 11 related to ARMD and 1 to an idiopathic membrane, were analyzed for the presence of the glycoxidation product N(epsilon)-(carboxymethyl)lysin (CML), one of the receptors for advanced glycation end products, RAGE, and the activation of NFkB, using immunohistochemistry. RESULTS: CML-like immunoreactivity was found in all ARMD specimens examined adjacent or colocalized with RAGE, but not in the idiopathic membrane. RAGE immunoreactive material was found in CD68-positive cells and in the fibrous matrix. CD68-positive cells and surrounding areas stained for p50, the activated form of NFkB. CONCLUSIONS: These results indicate that glycoxidation products are present in subretinal membranes of patients with ARMD. The concomitant expression of RAGE in these membranes and the finding of activated NFkB is suggestive of an implication of glycoxidation product formation in the pathogenesis of the disease.
Assuntos
Produtos Finais de Glicação Avançada/metabolismo , Lisina/análogos & derivados , Degeneração Macular/metabolismo , Proteínas de Membrana/metabolismo , Receptores Imunológicos/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Membrana Epirretiniana/metabolismo , Feminino , Humanos , Técnicas Imunoenzimáticas , Lisina/metabolismo , Masculino , NF-kappa B/metabolismo , Receptor para Produtos Finais de Glicação Avançada , Neovascularização Retiniana/metabolismoRESUMO
AIMS/HYPOTHESIS: Glycated proteins, formed by reaction of glucose and protein, react further yielding numerous, mostly undefined advanced glycation end products (AGE). The recently characterized imidazolone-type AGE (AG-1) is non-oxidatively formed involving 3-deoxyglucosone whereas some AGEs, particularly N(epsilon)-(carboxymethyl)lysine (CML), are formed only in the presence of oxygen. METHODS: To study the possible contribution of oxidative and non-oxidative AGE formation in the development of diabetic retinopathy antibodies directed against CML-type and imidazolone-type AGEs were characterized by dot blot analysis and used to localize these well-characterized epitops in the retinas from diabetic rats (early course) and from human Type I (insulin-dependent) diabetes mellitus with laser-treated proliferative diabetic retinopathy (late course). RESULTS: In non-diabetic rats CML was moderately positive in neuroglial and vascular structures of non-diabetic rat retinas and increased strongly in diabetic retinas. Anti-imidiazolone antibody staining was strongly positive only in diabetic capillaries. Advanced human diabetic retinopathy showed strong CML-immunolabelling of the entire retina whereas control samples showed moderate staining of neuroglial structures only with the polyclonal CML-antibody. Anti-imidiazolone antibody staining was faint in the inner retina of control sections but were strong throughout the entire diabetic retina. Immunolabelling for the AGE-receptor was congruent with a marker of Müller cells. CONCLUSION/INTERPRETATION: Our data indicate that the oxidatively formed CML is present in non-diabetic retinas as a regular constituent but increases in diabetes both in neuroglial and vascular components. Imidazolone-type AGE are restricted to microvessels and spread during later stages over the entire retina, co-localizing with the expression of AGE-receptor.
Assuntos
Retinopatia Diabética/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Retinopatia Diabética/patologia , Humanos , Imuno-Histoquímica , Proteínas de Membrana/metabolismo , Ratos , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/metabolismo , Retina/metabolismoRESUMO
The novel neuropeptide cocaine-amphetamine-regulated transcript (CART) is expressed in several hypothalamic regions and has recently been shown to be involved in the central control of food intake. To characterize the hypothalamic CART neurons and understand the physiological functions they might serve, we undertook an in situ hybridization and immunohistochemical study to examine distribution and neurochemical phenotype of these neurons. In situ hybridization studies showed abundant CART mRNA in the periventricular nucleus (PeV), the paraventricular nucleus of the hypothalamus (PVN), the supraoptic nucleus (SON), the arcuate nucleus (Arc), the zona incerta, and the lateral hypothalamic area. The distribution of CART-immunoreactive neurons as revealed by a monoclonal antibody raised against CART(41-89) displayed complete overlap with CART mRNA. Double immunohistochemistry showed co-existence of CART immunoreactivity (CART-IR) and somatostatin in some neurons of the PeV. In the magnocellular division of the PVN as well as the SON, CART-IR was demonstrated in both oxytocinergic and vasopressinergic perikarya. In the medial parvicellular region of the PVN a few CART-IR neurons co-localized galanin, but none was found to co-localize corticotropin-releasing hormone. In the Arc, almost all pro-opiomelanocortinergic neurons were shown to contain CART, whereas no co-localization of CART with NPY was found. In the lateral hypothalamic area nearly all CART neurons were found to contain melanin-concentrating hormone. The present data support a role for CART in neuroendocrine regulation. Most interestingly, CART is co-stored with neurotransmitters having both positive (melanin-concentrating hormone) as well as a negative (pro-opiomelanocortin) effect on food intake and energy balance.
Assuntos
Hipotálamo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Hipotálamo/anatomia & histologia , Imuno-Histoquímica , Hibridização In Situ , Masculino , Neuropeptídeos/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos WistarRESUMO
AIMS/HYPOTHESIS: Previous studies in our laboratory have shown that the vascular changes in diabetes include hypertrophy of the mesenteric vasculature and that this process can be attenuated by the inhibition of advanced glycation with aminoguanidine. Since aminoguanidine can also act as an inhibitor of nitric oxide synthase, the effect of a novel inhibitor of advanced glycation end-products, formation that does not inhibit nitric oxide synthase, known as 2,3 diaminophenazine (2,3 DAP) was evaluated. METHODS: Initially, in vitro assessment of the ability of 2,3 diaminophenazine to inhibit formation of advanced glycation products was performed. Subsequently, in vivo studies evaluating 2,3 diaminophenazine and aminoguanidine were carried out. Animals were followed for 3 weeks after induction of diabetes and randomised to no treatment, aminoguanidine or 2,3 diaminophenazine. Mesenteric vessels were weighed and advanced glycation end-products were measured by radioimmunoassay in vessel and kidney homogenates. In addition, these products were assessed in mesenteric vessels by immunohistochemistry. RESULTS: When compared with control animals, diabetes was associated with an increase in mesenteric vascular weight. Treatment of diabetic rats with aminoguanidine or 2,3 diaminophenazine resulted in attenuation of vascular hypertrophy. Both aminoguanidine and 2,3 diaminophenazine reduced the formation of advanced glycation end-products as measured by radioimmunoassay and as assessed immunohistochemically in these vessels. This reduction was also observed in the kidney. CONCLUSION/INTERPRETATION: These data support the concept that the effects of aminoguanidine in reducing diabetes associated vascular hypertrophy are via inhibition of advanced glycation end-products dependent pathways.
Assuntos
Diabetes Mellitus Experimental/complicações , Angiopatias Diabéticas/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Fenazinas/farmacologia , Animais , Diabetes Mellitus Experimental/metabolismo , Indução Enzimática , Hipertrofia/prevenção & controle , Masculino , Artérias Mesentéricas/patologia , Veias Mesentéricas/patologia , Camundongos , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase Tipo II , Coelhos , Radioimunoensaio , Ratos , Ratos Sprague-DawleyRESUMO
The hypothalamic satiety peptide CART (cocaine and amphetamine regulated transcript) is expressed at high levels in anorectic rat glucagonomas but not in hypoglycemic insulinomas. However, a non-anorectic metastasis derived from the glucagonoma retained high CART expression levels and produced circulating CART levels comparable to that of the anorectic tumors. Moreover, distinct glucagonoma lines derived by stable HES-1 transfection of the insulinoma caused severe anorexia but retained low circulating levels of CART comparable to that of insulinoma bearing or control rats. Islet tumor associated anorexia and circulating CART levels are thus not correlated, and in line with this peripheral administration of CART (5-50 mg/kg) produced no effect on feeding behavior. In the rat two alternatively spliced forms of CART mRNA exist and quantitative PCR revealed expression of both forms in the hypothalamus, in the different islet tumors, and in the islets of Langerhans. Immunocytochemistry as well as in situ hybridization localized CART expression to the somatostatin producing islet D cell. A potential endocrine/paracrine role of islet CART remains to be clarified.
Assuntos
Adenoma de Células das Ilhotas Pancreáticas/sangue , Adenoma de Células das Ilhotas Pancreáticas/genética , Anorexia/sangue , Anorexia/genética , Ilhotas Pancreáticas/metabolismo , Proteínas do Tecido Nervoso/sangue , Proteínas do Tecido Nervoso/genética , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/genética , Animais , Sequência de Bases , Sondas de DNA/genética , Ingestão de Alimentos/efeitos dos fármacos , Feminino , Expressão Gênica , Glucagonoma/sangue , Glucagonoma/genética , Imuno-Histoquímica , Hibridização In Situ , Insulinoma/sangue , Insulinoma/genética , Proteínas do Tecido Nervoso/farmacologia , RNA Mensageiro/genética , RNA Neoplásico/genética , Ratos , Células Secretoras de Somatostatina/metabolismoRESUMO
Cocaine- and amphetamine-regulated transcript (CART) is a recently discovered hypothalamic peptide regulated by leptin and with a potent appetite-suppressing activity. In the rat, the CART gene encodes a peptide of 116 amino acid residues (or a splice variant 13 residues longer). The predicted signal sequence is 27 amino acid residues, resulting in a prohormone of 89 residues. The CART prohormone contains several potential posttranslational processing sites in the form of mono- and dibasic sequences. In the present study we have purified CART peptides from extracts of adrenal gland, hypothalamus, nucleus accumbens, and pituitary gland (anterior and neurointermediate lobe) of the rat and determined the peptide structures by using microsequencing and mass spectrometry. In none of the tissues examined the long splice variant was found. From the adrenal gland, the CART(1-89) and CART(10-89) peptides were isolated, in contrast to the hypothalamus and nucleus accumbens, from which the shorter form peptides CART(42-89) and CART(49-89) were purified. From the anterior lobe of the pituitary gland, CART(42-89) was isolated, in contrast to the neurointermediate lobe, which contains only CART(49-89). This tissue-specific processing indicates that CART peptides may have different biological functions in the periphery and in the central nervous system.
Assuntos
Proteínas do Tecido Nervoso/biossíntese , Processamento de Proteína Pós-Traducional , Animais , Cromatografia Líquida de Alta Pressão , Feminino , Especificidade de Órgãos , Ratos , Ratos WistarRESUMO
To investigate whether type 1 diabetes in man is associated with a preferential Th1/Th2 response, and whether autoantibodies to one of the main autoantigens would reflect such a response, we characterized the immunoglobulin isotype profile to the 65-kDa isoform of glutamic acid decarboxylase (GAD65) in siblings to IDDM patients. Samples obtained from affected subjects before and at clinical onset of IDDM, from unaffected individuals at high risk and at low risk and from healthy controls were studied. The immunoglobulin isotype profile in the siblings at low risk reflected a more immature, i.e., IgM and Th2 like, i.e., IgE response compared to the progressors and siblings at high risk, with significantly higher median levels of IgM and IgE. The rank order of anti-GAD65 immunoglobulin isotypes was similar in the siblings before and at clinical onset of IDDM, IgG1 > IgG4 > IgM > IgE > IgA > IgG3 > IgG2, but markedly different in the individuals at low risk, IgG1 > IgM > IgE > IgG4 > IgG3 > IgA > IgG2. Based on these observations, we suggest that progression to clinical onset of IDDM is associated with a maturation and a decrease in the Th2 immune response against GAD65; findings which could have implications for future intervention and prediction strategies.
Assuntos
Autoanticorpos/sangue , Diabetes Mellitus Tipo 1/etiologia , Diabetes Mellitus Tipo 1/imunologia , Glutamato Descarboxilase/imunologia , Isotipos de Imunoglobulinas/sangue , Adolescente , Autoantígenos , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Finlândia , Humanos , Imunoglobulina E/sangue , Imunoglobulina M/sangue , Masculino , Fatores de Risco , Células Th1/imunologia , Células Th2/imunologiaRESUMO
OBJECTIVE: To investigate whether children and adolescents with type 1 diabetes have increased serum levels of the glycoxidation product Nepsilon-(carboxymethyl)lysine (CML) at an early stage of the disease. RESEARCH DESIGN AND METHODS: The serum levels of CML in 38 patients with type 1 diabetes aged 14+/-3.2 (mean+/-SD) years were compared with those in 26 control subjects aged 16+/-1.7 years. The mean duration of diabetes was 5+/-4.7 years, ranging from 0.5 to 15 years. The mean levels of HbA1c were 10.3+/-2.5% in the patient group. The serum levels of CML were measured using a monoclonal anti-CML antibody in a fluoremetric immunoassay. Serum protein levels of advanced glycation end products (AGEs) were assayed using a polyclonal antibody from rabbit immunized with AGE-RNase (pAGE). RESULTS: The serum levels of CML and pAGE were significantly increased in the patient group versus the control group: 1.08 (0.45-2.97) U/ml CML (median 10-90 percentiles) vs. 0.70 (0.36-1.79) U/ml CML, P < 0.03, and 6.6 (5.1-9.9) U/ml pAGE vs. 5.5 (3.7-8.2) U/ml AGEs, P < 0.01. A significant relationship between CML and pAGE was found in the IDDM group, r = 0.76, P < 0.001. The CML levels were not associated with the HbAlc levels (n = 23, r = -0.02, NS), cholesterol levels (n = 21, r = 0.07, NS), age, sex, or diabetes duration. CONCLUSIONS: Serum levels of CML are increased in patients with type 1 diabetes. This increase precedes the development of micro- and macrovascular complications.
Assuntos
Diabetes Mellitus Tipo 1/sangue , Produtos Finais de Glicação Avançada/sangue , Lisina/análogos & derivados , Adolescente , Animais , Anticorpos Monoclonais , Criança , Ensaio de Imunoadsorção Enzimática , Feminino , Hemoglobinas Glicadas/análise , Humanos , Lisina/sangue , Masculino , Coelhos , Sensibilidade e EspecificidadeRESUMO
Glutamic acid decarboxylase (GAD) is an important autoantigen in insulin-dependent diabetes mellitus (IDDM), but little is known about its regulation and function in islet cells. We investigated the effects of the GABA-transaminase inhibitor gamma-vinyl-GABA (GVG) on GAD expression in rat islets and brain in vitro and in vivo. In islets incubated in high glucose culture medium there was an increase in GAD activity, GAD65 and GAD67 protein levels compared to low-glucose conditions; however, even in high glucose, GVG still significantly suppressed GAD activity and GAD67 expression. Our observations suggest that glucose and GVG act on GAD in islets through different mechanisms. Quantitative immunohistochemistry of pancreatic sections from rats treated with GVG in vivo using novel monoclonal antibodies specific for GAD65 and GAD67, showed a decrease in GAD67 expression (p < 0.005) relative to untreated rats. The effects of GVG on rat pancreatic islets were very similar to those observed in brain of rats treated with GVG in vivo. In homogenates of cerebral tissue from GVG treated rats containing both membrane-bound and soluble protein GAD67 levels were significantly decreased while GAD65 levels were not significantly changed compared to untreated rats. In contrast, in homogenates of cerebral tissues containing only soluble cytosolic protein, GVG-treatment was also significantly found to decrease GAD65 levels. Taken together, these results suggest that GVG potentially could be of use to decrease GAD expression in islet cells and consequently to deviate/inhibit the autoimmune response against the beta cells seen in IDDM.
Assuntos
Encéfalo/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Glucose/farmacologia , Glutamato Descarboxilase/biossíntese , Glutamato Descarboxilase/efeitos dos fármacos , Ilhotas Pancreáticas/efeitos dos fármacos , Ácido gama-Aminobutírico/análogos & derivados , Animais , Especificidade de Anticorpos , Encéfalo/enzimologia , Células Cultivadas , Indução Enzimática , Glutamato Descarboxilase/imunologia , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/enzimologia , Isoenzimas/biossíntese , Isoenzimas/efeitos dos fármacos , Isoenzimas/imunologia , Masculino , Ratos , Ratos Wistar , Vigabatrina , Ácido gama-Aminobutírico/farmacologiaRESUMO
The mammalian hypothalamus strongly influences ingestive behaviour through several different signalling molecules and receptor systems. Here we show that CART (cocaine- and amphetamine-regulated transcript), a brain-located peptide, is a satiety factor and is closely associated with the actions of two important regulators of food intake, leptin and neuropeptide Y. Food-deprived animals show a pronounced decrease in expression of CART messenger RNA in the arcuate nucleus. In animal models of obesity with disrupted leptin signalling, CART mRNA is almost absent from the arcuate nucleus. Peripheral administration of leptin to obese mice stimulates CART mRNA expression. When injected intracerebroventricularly into rats, recombinant CART peptide inhibits both normal and starvation-induced feeding, and completely blocks the feeding response induced by neuropeptide Y. An antiserum against CART increases feeding in normal rats, indicating that CART may be an endogenous inhibitor of food intake in normal animals.
Assuntos
Anorexia/metabolismo , Hipotálamo/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Proteínas/fisiologia , Animais , Núcleo Arqueado do Hipotálamo/metabolismo , Clonagem Molecular , Escherichia coli , Jejum , Comportamento Alimentar/fisiologia , Regulação da Expressão Gênica , Hipotálamo/metabolismo , Técnicas Imunoenzimáticas , Leptina , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neuropeptídeo Y/biossíntese , Neuropeptídeo Y/fisiologia , Obesidade/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Ratos Zucker , Proteínas Recombinantes de Fusão , Transdução de SinaisRESUMO
The genetic and immunological basis of respiratory anaphylaxis was studied in two strains of guinea pigs selectively bred for either high or low respiratory anaphylactic response to inhalation of ovalbumin. Individuals of the parental strains, F1-hybrids and backcross offspring were examined for class II MHC type and asthmatic response to ovalbumin. Analysis of the results revealed an association of asthma phenotype and MHC class II type. Apart from the gene(s) in the MHC several other genes seem to be involved in the control of guinea pig asthma.
Assuntos
Asma/imunologia , Cobaias/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Anafilaxia/genética , Anafilaxia/imunologia , Animais , Anticorpos/imunologia , Asma/genética , Genes MHC da Classe II , Cobaias/genética , Hibridização Genética , Complexo Principal de Histocompatibilidade , Ovalbumina/imunologiaRESUMO
The serological typing of determinants within the guinea pig histocompatibility complex (GPLA-complex) has, until now, largely been carried out by the 51Cr-release technique. As target cells, a mixture of B and T cells from peripheral blood and lymph nodes were used. However, for the detection of Ia-determinants, which predominantly are expressed on the surface of B cells, the use of lymphocyte suspensions with a relatively low content of B cells can cause some evaluation problems. We have developed a simple method for obtaining a relatively pure suspension of B cells, making use of the ability of guinea pig T cells to form rosettes with rabbit red blood cells and the possibility of removing these by sedimentation on lymphoflot. By employing these purified B cells, an improved serological detection of Ia-determinants of the guinea pig histocompatibility complex is made possible.