RESUMO
BACKGROUND: Pancreatic islet endocrine cell-supporting architectures, including islet encapsulating basement membranes (BMs), extracellular matrix (ECM), and possible cell clusters, are unclear. PROCEDURES: The architectures around islet cell clusters, including BMs, ECM, and pancreatic acinar-like cell clusters, were studied in the non-diabetic state and in the inflamed milieu of fulminant type 1 diabetes in humans. RESULT: Immunohistochemical and electron microscopy analyses demonstrated that human islet cell clusters and acinar-like cell clusters adhere directly to each other with desmosomal structures and coated-pit-like structures between the two cell clusters. The two cell-clusters are encapsulated by a continuous capsule composed of common BMs/ECM. The acinar-like cell clusters have vesicles containing regenerating (REG) Iα protein. The vesicles containing REG Iα protein are directly secreted to islet cells. In the inflamed milieu of fulminant type 1 diabetes, the acinar-like cell clusters over-expressed REG Iα protein. Islet endocrine cells, including beta-cells and non-beta cells, which were packed with the acinar-like cell clusters, show self-replication with a markedly increased number of Ki67-positive cells. CONCLUSION: The acinar-like cell clusters touching islet endocrine cells are distinct, because the cell clusters are packed with pancreatic islet clusters and surrounded by common BMs/ECM. Furthermore, the acinar-like cell clusters express REG Iα protein and secrete directly to neighboring islet endocrine cells in the non-diabetic state, and the cell clusters over-express REG Iα in the inflamed milieu of fulminant type 1 diabetes with marked self-replication of islet cells.
Assuntos
Diabetes Mellitus Tipo 1/patologia , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Litostatina/metabolismo , Pâncreas/patologia , Adolescente , Adulto , Idoso , Diabetes Mellitus Tipo 1/metabolismo , Humanos , Masculino , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Pâncreas/metabolismoRESUMO
A variant located on 14q13.3 nearest to thyroid transcription factor-1 (TTF1) predisposes individuals to thyroid cancer, but whether this variant is related to the RET/PTC rearrangement associated with human papillary thyroid carcinomas (PTCs) is unknown. The aims of this study were to investigate the effects of RET/PTC1 on the expression of thyroid-specific genes in thyrocytes and their relationship with malignant transformation of the thyrocytes. In the absence or presence of TSH, an extracellular signal-regulated kinase was phosphorylated in FRTL5 cells that stably expressed RET/PTC1, and these cells grew independently of TSH. FRTL (RET/PTC1) cells produced 566% more thyroglobulin mRNA and 474% more Na+/I- symporter mRNA than did the control FRTL (pcDNA) cells. FRTL (RET/PTC1) cells expressed 468% more Ttf1 mRNA than did FRTL (pcDNA) cells, but these two cell types did not differ significantly with respect to Pax8 or Ttf2 mRNA levels. When FRTL (RET/PTC1) cells and FRTL (pcDNA), cells were injected into each of nine nude mice, each mouse developed a single tumor at the site of FRTL (RET/PTC1) cell injection; in contrast, tumor formation never occurred at sites of FRTL (cDNA) cells injection. Tumors resulting from FRTL (RET/PTC1) cells retained (125)I-uptake activity; moreover, the cells invaded into surrounding skeletal muscle. When overexpression of Ttf1 in FRTL (RET/PTC1) cells was silenced, the cells completely lost their tumorigenic potential. Exogenous TTF1 cDNA enhanced the tumorigenicity of BHP18-21v cells, human PTC cells that express RET/PTC1, in nude mice. These results indicated that concurrent overexpression of RET/PTC1 and TTF1 confers tumorigenicity to FRTL5 and BHP18-21v cells in nude mice.
Assuntos
Carcinoma Papilar/patologia , Transformação Celular Neoplásica/patologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Tirosina Quinases/metabolismo , Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/patologia , Animais , Western Blotting , Carcinoma Papilar/genética , Carcinoma Papilar/metabolismo , Proliferação de Células , Transformação Celular Neoplásica/genética , Células Cultivadas , Proteínas de Ligação a DNA/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas de Fusão Oncogênica/genética , Fosforilação , Proteínas Tirosina Quinases/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Fatores de TranscriçãoRESUMO
Hypothyroidism in the young leads to irreversible growth failure. hyt/hyt Mice have a nonfunctional TSH receptor (TSHR) and are severely hypothyroid, but growth retardation was not observed in adult mice. We found that epiphysial cartilage as well as cultured chondrocytes expressed functional TSHR at levels comparable to that seen in the thyroid, and that addition of TSH to cultured chondrocytes suppressed expression of chondrocyte differentiation marker genes such as Sox-9 and type IIa collagen. Next, we compared the long bone phenotypes of two distinct mouse models of hypothyroidism: thyroidectomized (THYx) mice and hyt/hyt mice. Although both THYx and hyt/hyt mice were severely hypothyroid and had similar serum Ca(2+) and growth hormone levels, the tibia was shorter and the proliferating and hypertrophic zones in the growth plate was significantly narrower in THYx mice than in hyt/hyt mice. Supplementation of hyt/hyt mice thyroid hormone resulted in a wider growth plate compared with that of wild-type mice. Expressions of chondrocyte differentiation marker genes Sox-9 and type IIa collagen in growth plate from THYx mice were 52 and 60% lower than those of hyt/hyt mice, respectively. High serum TSH causes abnormal skeletal development in young mice with hypothyroidism via suppressive effects on the growth plate.
Assuntos
Lâmina de Crescimento/metabolismo , Hipotireoidismo/metabolismo , Receptores dos Hormônios Tireóideos/metabolismo , Tireotropina/metabolismo , Tíbia/anormalidades , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Condrócitos , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Feminino , Hipotireoidismo/sangue , Hipotireoidismo/genética , Imuno-Histoquímica , Masculino , Camundongos , RNA Mensageiro/química , RNA Mensageiro/genética , Receptores dos Hormônios Tireóideos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Tiroxina/sangue , Tri-Iodotironina/sangueRESUMO
One goal of diabetic regenerative medicine is to instructively convert mature pancreatic exocrine cells into insulin-producing cells. We recently reported that ligand-bound thyroid hormone receptor α (TRα) plays a critical role in expansion of the ß-cell mass during postnatal development. Here, we used an adenovirus vector that expresses TRα driven by the amylase 2 promoter (AdAmy2TRα) to induce the reprogramming of pancreatic acinar cells into insulin-producing cells. Treatment with l-3,5,3-triiodothyronine increases the association of TRα with the p85α subunit of phosphatidylinositol 3-kinase (PI3K), leading to the phosphorylation and activation of Akt and the expression of Pdx1, Ngn3, and MafA in purified acinar cells. Analyses performed with the lectin-associated cell lineage tracing system and the Cre/loxP-based direct cell lineage tracing system indicate that newly synthesized insulin-producing cells originate from elastase-expressing pancreatic acinar cells. Insulin-containing secretory granules were identified in these cells by electron microscopy. The inhibition of p85α expression by siRNA or the inhibition of PI3K by LY294002 prevents the expression of Pdx1, Ngn3, and MafA and the reprogramming to insulin-producing cells. In immunodeficient mice with streptozotocin-induced hyperglycemia, treatment with AdAmy2TRα leads to the reprogramming of pancreatic acinar cells to insulin-producing cells in vivo. Our findings suggest that ligand-bound TRα plays a critical role in ß-cell regeneration during postnatal development via activation of PI3K signaling.
Assuntos
Células Acinares/metabolismo , Desdiferenciação Celular , Células Secretoras de Insulina/metabolismo , Receptores dos Hormônios Tireóideos/biossíntese , Tri-Iodotironina/farmacologia , Células Acinares/citologia , Adenoviridae , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Células Secretoras de Insulina/citologia , Fatores de Transcrição Maf Maior/biossíntese , Fatores de Transcrição Maf Maior/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Camundongos Transgênicos , Morfolinas/farmacologia , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , alfa-Amilases Pancreáticas/genética , alfa-Amilases Pancreáticas/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores dos Hormônios Tireóideos/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Transativadores/biossíntese , Transativadores/genética , Transdução GenéticaRESUMO
We have cloned a thyroid-stimulating hormone receptor (TSHR) cDNA from mouse thyroid glands. The sequence of this cDNA indicated that it encoded a 739 amino acid TSHR splice variant that lacked exon 5 (TSHR739). In thyroid gland samples from adult mice, the amount of TSHR739 mRNA was about 10% of the amount of full-length TSHR (TSHR764) mRNA. A eCFP-tagged TSHR739 integrated into plasma membrane, but lacked TSH binding activity and it did not produce cAMP in response to TSH. However, thyroid-stimulating antibodies from patients with Graves' disease stimulated cAMP production in HEK293 cells that expressed TSHR739. Quantitative PCR revealed that TSHR739 transcript levels were low in the fetal mouse thyroid samples, but TSHR739 transcript levels increased after birth and as the mice grew. We used plasmid injection combined with electroporation into skeletal muscles to immunize BALB/c mice with TSHR739, TSHR764,, or control plasmid; TSHR739 caused goiters, high (125)I uptake activity, thyrotoxicosis, and production of thyroid-stimulating antibodies, but TSHR764, or control did not. These results indicated that immunization with an autologous TSHR antigen, TSHR739, induced Graves'-like disease in mice, and that TSHR739 is a candidate autoantigen in autoimmune thyroid disease.
Assuntos
Doença de Graves/etiologia , Receptores da Tireotropina/genética , Receptores da Tireotropina/imunologia , Processamento Alternativo , Sequência de Aminoácidos , Animais , Autoantígenos/genética , Autoantígenos/imunologia , Autoantígenos/metabolismo , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Modelos Animais de Doenças , Feminino , Doença de Graves/genética , Doença de Graves/imunologia , Células HEK293 , Humanos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores da Tireotropina/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismoRESUMO
C.RF-Tshr(hyt/hyt) mice have a mutated thyroid stimulating hormone receptor (P556L-TSHR) and these mice develop severe hypothyroidism. We found that C.RF-Tshr(hyt/wild) heterozygous mice are also in a hypothyroid state. Thyroid glands from C.RF-Tshr(hyt/wild) mice are smaller than those from wild-type mice, and (125)I uptake activities of the former are significantly lower than those in the latter. When TSHR (TSHR(W)) and P556L-TSHR (TSHR(M)) cDNAs were cloned and co-transfected into HEK 293 cells, the cells retained (125)I-TSH binding activity, but cAMP response to TSH was decreased to about 20% of HEK 293 cells transfected with TSHR(W) cDNA. When TSHR(W) and TSHR(M) were tagged with eCFP or eYFP, we observed fluorescence resonance energy transfer (FRET) in HEK 293 cells expressing TSHR(W)-eCFP and TSHR(W)-eYFP in the absence of TSH, but not in the presence of TSH. In contrast, we obtained FRET in HEK 293 cells expressing TSHR(W)-eCFP and TSHR (M)-eYFP, regardless of the presence or absence of TSH. These results suggest that P556L TSHR has a dominant negative effect on TSHR(W) by impairing polymer to monomer dissociation, which decreases TSH responsiveness and induces hypothyroidism in C.RF-Tshr(hyt/wild) mice.
Assuntos
Hipotireoidismo/genética , Mutação , Receptores da Tireotropina/genética , Animais , Sequência de Bases , Western Blotting , AMP Cíclico/metabolismo , Primers do DNA , DNA Complementar , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Camundongos , Reação em Cadeia da Polimerase , Tireotropina/metabolismoRESUMO
To determine the relative importance of TSH in white adipose tissue, we compared the adipose phenotypes of two distinct mouse models of hypothyroidism. These models differed in that the normal reciprocal relationship between thyroid hormone and TSH was intact in one and disrupted in the other. One model, thyroidectomized (THYx) mice, had a 100-fold increase in TSH and a normal TSH receptor (TSHR); in contrast, the other model, hyt/hyt mice, had a 120-fold elevation of TSH but a nonfunctional TSHR. Although both THYx and hyt/hyt mice were in a severe hypothyroid state, the epididymal fat (mg)/body wt (g) (F/B) ratio of THYx mice was much smaller than that of hyt/hyt mice (8.2 ± 0.43 vs. 14.4 ± 0.40, respectively, P < 0.001). The fat cell diameter in THYx mice was also smaller than that in hyt/hyt mice (79 ± 2.8 vs. 105 ± 2.2 µm, respectively, P < 0.001), suggesting that TSH induced lipolysis in adipose tissues. When we transferred a functional mouse TSHR gene and a control plasmid into opposite sides of epididymal fat of hyt/hyt mice by plasmid injection combined with electroporation, fat weight of the TSHR side was decreased to 60% of that of the control side. Messenger RNA levels of hormone-sensitive lipase in epididymal fat containing the transferred TSHR gene were twofold higher than those in tissue from the control side. These results indicated that TSH worked as a lipolytic factor in white adipose tissues, especially in mice in a hypothyroid state.
Assuntos
Tecido Adiposo Branco/metabolismo , Hipotireoidismo/metabolismo , Lipólise/fisiologia , Receptores da Tireotropina/biossíntese , Substituição de Aminoácidos/genética , Substituição de Aminoácidos/fisiologia , Animais , Códon/genética , Eletroporação , Epididimo/metabolismo , Feminino , Técnicas de Transferência de Genes , Lipase/biossíntese , Lipase/genética , Metabolismo dos Lipídeos/genética , Metabolismo dos Lipídeos/fisiologia , Lipólise/genética , Masculino , Camundongos , Camundongos Endogâmicos , Mutação/genética , Mutação/fisiologia , Plasmídeos/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores da Tireotropina/genética , Termogênese/fisiologia , Testes de Função Tireóidea , Glândula Tireoide/fisiologia , Hormônios Tireóideos/sangue , TireoidectomiaRESUMO
Previously, we demonstrated that Runx2 (Cbfa1/AML3), a chondrocyte-specific transcription factor, is expressed in thyroid glands of mice, where it stimulates expression of the thyroglobulin (Tg) gene. Here, we reverse transcribed thyroid transcription factor-1 (TTF-1), Pax-8, Tg, thyroid peroxidase (TPO) and Na(+)/I(-) symporter (NIS) cDNAs from mouse trachea and bronchus RNA samples, but were unable to recover these cDNAs from mouse liver RNA samples. Tg mRNA levels in trachea and bronchus were about 5.1% and 2.1% of those in thyroid glands. ATDC-5 cells, cultured chondrocytes, expressed about 30-fold more Tg mRNA than undifferentiated cells. Gel shift and Tg gene reporter assay revealed that TTF-1 stimulated Tg gene expression in these cells. These results indicate that chondrocytes turn on some aspects of the thyroid gene expression program and that TTF-1 plays important roles in Tg gene expression in chondrocyte.
Assuntos
Cartilagem/metabolismo , Condrócitos/metabolismo , Expressão Gênica , Glândula Tireoide/metabolismo , Animais , Células Cultivadas , DNA Complementar/genética , Iodeto Peroxidase/genética , Camundongos , Proteínas Nucleares/genética , Fatores de Transcrição Box Pareados/genética , Simportadores/genética , Tireoglobulina/genética , Fator Nuclear 1 de Tireoide , Fatores de Transcrição/genéticaRESUMO
OBJECTIVE: The contribution of innate immunity responsible for aggressive ß-cell destruction in human fulminant type 1 diabetes is unclear. RESEARCH DESIGN AND METHODS: Islet cell expression of Toll-like receptors (TLRs), cytoplasmic retinoic acid-inducible gene I (RIG-I)-like receptors, downstream innate immune markers, adaptive immune mediators, and apoptotic markers was studied in three autopsied pancreata obtained 2 to 5 days after onset of fulminant type 1 diabetes. RESULTS: RIG-I was strongly expressed in ß-cells in all three pancreata infected with enterovirus. Melanoma differentiation-associated gene-5 was hyperexpressed in islet cells, including ß- and α-cells. TLR3 and TLR4 were expressed in mononuclear cells that infiltrated islets. Interferon (IFN)-α and IFN-ß were strongly expressed in islet cells. Major histocompatibility complex (MHC)-class I, IFN-γ, interleukin-18, and CXC motif ligand 10 were expressed and colocalized in affected islets. CD11c+ MHC-class II+ dendritic cells and macrophage subsets infiltrated most islets and showed remarkable features of phagocytosis of islet cell debris. CD4+ forkhead box P3+ regulatory T cells were not observed in and around the affected islets. Mononuclear cells expressed the Fas ligand and infiltrated most Fas-expressing islets. Retinoic acid-receptor responder 3 and activated caspases 8, 9, and 3 were preferentially expressed in ß-cells. Serum levels of IFN-γ were markedly increased in patients with fulminant type 1 diabetes. CONCLUSIONS: These findings demonstrate the presence of specific innate immune responses to enterovirus infection connected with enhanced adoptive immune pathways responsible for aggressive ß-cell toxicity in fulminant type 1 diabetes.
Assuntos
Imunidade Adaptativa/imunologia , RNA Helicases DEAD-box/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Imunidade Inata/imunologia , Células Secretoras de Insulina/metabolismo , Adolescente , Adulto , Idoso , Análise de Variância , Morte Celular/imunologia , Proteína DEAD-box 58 , Diabetes Mellitus Tipo 1/imunologia , Infecções por Enterovirus/imunologia , Infecções por Enterovirus/metabolismo , Feminino , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Imuno-Histoquímica , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/virologia , Helicase IFIH1 Induzida por Interferon , Interferon beta/imunologia , Interferon beta/metabolismo , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-18/imunologia , Interleucina-18/metabolismo , Masculino , Pessoa de Meia-Idade , Receptores Imunológicos , Receptores Toll-Like/imunologia , Receptores Toll-Like/metabolismoRESUMO
Failure of the functional pancreatic beta-cell mass to expand in response to increased metabolic demand is a hallmark of type 2 diabetes. Lineage tracing studies indicate that replication of existing beta-cells is important for beta-cell proliferation in adult animals. In rat pancreatic beta-cell lines (RIN5F), treatment with 100 nM thyroid hormone (triiodothyronine, T(3)) enhances cell proliferation. This result suggests that T(3) is required for beta-cell proliferation or replication. To identify the role of thyroid hormone receptor alpha (TR(alpha)) in the processes of beta-cell growth and cell cycle regulation, we constructed a recombinant adenovirus vector, AdTR(alpha). Infection with AdTR(alpha) to RIN5F cells increased the expression of cyclin D1 mRNA and protein. Overexpression of the cyclin D1 protein in AdTR(alpha)-infected cells led to activation of the cyclin D1/cyclin-dependent kinase/retinoblastoma protein/E2F pathway, along with cell cycle progression and cell proliferation following treatment with 100 nM T(3). Conversely, lowering cellular cyclin D1 by small interfering RNA knockdown in AdTR(alpha)-infected cells led to down-regulation of the cyclin D1/CDK/Rb/E2F pathway and inhibited cell proliferation. Furthermore, in immunodeficient mice with streptozotocin-induced diabetes, intrapancreatic injection of AdTR(alpha) led to the restoration of islet function and to an increase in the beta-cell mass. These results support the hypothesis that liganded TR(alpha) plays a critical role in beta-cell replication and in expansion of the beta-cell mass during postnatal development. Thus, liganded TR(alpha) may be a target for therapeutic strategies that can induce the expansion and regeneration of beta-cells.
Assuntos
Células Secretoras de Insulina/citologia , Receptores alfa dos Hormônios Tireóideos/fisiologia , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Proliferação de Células , Ciclina D1/metabolismo , Regulação da Expressão Gênica , Humanos , Ligantes , Camundongos , Camundongos SCID , Modelos Biológicos , RNA Interferente Pequeno/metabolismo , Proteína do Retinoblastoma/metabolismo , Receptores alfa dos Hormônios Tireóideos/metabolismoRESUMO
We recently reported on the overexpression of Runx2 (Cbfa1/AML3), an osteoblast-specific transcription factor, in human papillary thyroid cancer tissues. We report here that normal thyrocytes also express Runx2 and that Runx2(+/-) mice are in a hypothyroid state. To clarify the mechanism, we studied the effects of small interfering RNA-mediated silencing of Runx2 on thyroid-specific gene expression in FRTL-5 cells. Lowering the levels of Runx2 had no effect on the amount of Na(+)/I(-) symporter mRNA but markedly decreased the amount of thyroglobulin (Tg) mRNA. A Runx2 binding consensus sequence is present on the Tg gene promoter, and gel-shift assay revealed that Runx2 binds to this region. Reporter assay showed that deletion of the region or introduction of a mutation into the binding site significantly impairs promoter function. These results indicate that Runx2 deficiency in mice causes decreased Tg expression and a novel type of hypothyroidism.
Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/deficiência , Hipotireoidismo/metabolismo , Tireoglobulina/metabolismo , Animais , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Inativação Gênica , Hipotireoidismo/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Regiões Promotoras Genéticas/genética , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Tireoglobulina/genética , Testes de Função Tireóidea , Glândula Tireoide/crescimento & desenvolvimento , Glândula Tireoide/patologia , Glândula Tireoide/fisiopatologiaRESUMO
OBJECTIVE: Fulminant type 1 diabetes is characterized by the rapid onset of severe hyperglycemia and ketoacidosis, with subsequent poor prognosis of diabetes complications. Causative mechanisms for accelerated beta-cell failure are unclear. RESEARCH DESIGN AND METHODS: Subjects comprised three autopsied patients who died from diabetic ketoacidosis within 2-5 days after onset of fulminant type 1 diabetes. We examined islet cell status, including the presence of enterovirus and chemokine/cytokine/major histocompatibility complex (MHC) expressions in the pancreata using immunohistochemical analyses and RT-PCR. RESULTS: Immunohistochemical analysis revealed the presence of enterovirus-capsid protein in all three affected pancreata. Extensive infiltration of CXCR3 receptor-bearing T-cells and macrophages into islets was observed. Dendritic cells were stained in and around the islets. Specifically, interferon-gamma and CXC chemokine ligand 10 (CXCL10) were strongly coexpressed in all subtypes of islet cells, including beta-cells and alpha-cells. No CXCL10 was expressed in exocrine pancreas. Serum levels of CXCL10 were increased. Expression of MHC class II and hyperexpression of MHC class I was observed in some islet cells. CONCLUSIONS: These results strongly suggest the presence of a circuit for the destruction of beta-cells in fulminant type 1 diabetes. Enterovirus infection of the pancreas initiates coexpression of interferon-gamma and CXCL10 in beta-cells. CXCL10 secreted from beta-cells activates and attracts autoreactive T-cells and macrophages to the islets via CXCR3. These infiltrating autoreactive T-cells and macrophages release inflammatory cytokines including interferon-gamma in the islets, not only damaging beta-cells but also accelerating CXCL10 generation in residual beta-cells and thus further activating cell-mediated autoimmunity until all beta-cells have been destroyed.
Assuntos
Quimiocina CXCL10/genética , Diabetes Mellitus Tipo 1/patologia , Infecções por Enterovirus/complicações , Células Secretoras de Insulina/patologia , Receptores CXCR3/genética , Adulto , Idoso , Autopsia , Proteínas do Capsídeo/genética , Quimiocina CXCL10/sangue , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/imunologia , Cetoacidose Diabética/genética , Cetoacidose Diabética/patologia , Infecções por Enterovirus/sangue , Infecções por Enterovirus/imunologia , Evolução Fatal , Feminino , Antígenos HLA-D/genética , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Masculino , Pessoa de Meia-Idade , RNA Viral/genética , RNA Viral/isolamento & purificaçãoRESUMO
We immunized AKR/N mice with bovine thyroglobulin (Tg) once every 2 weeks and monitored their time-dependent changes in (125)I uptake activity in the thyroid glands. After 3 months, anti-Tg antibody was positive in all sera from the immunized mice. Serum free tri-iodothyronine (T(3)) and free thyroxine (T(4)) levels in the immunized mice (n=6) were significantly higher than those in the saline injected (control) mice (n=6). Neck counts as well as scintigraphy of the thyroid glands revealed that iodide uptake activity of the immunized mice was not suppressed, but was instead higher than that of the control mice. Two of the six immunized mice showed extremely high iodide uptake activity. The thyroid glands of these two mice were diffusely enlarged and the height of the epithelial cells was also increased. In addition, two mice with high iodide uptake activity produced a high titer of thyroid-stimulating antibody. Additional experiments showed that 4 out of 11 AKR/N mice and 3 out of 10 C57BL6 mice immunized with Tg had high serum free T(3)/free T(4) levels, high (125)I uptake activity of the thyroid, and positive thyroid-stimulating antibody activity. Diffuse goiter, thyrotoxicosis, high iodide uptake activity, and positive thyroid-stimulating antibody are the characteristics of Graves' disease. Thus, these mice exhibit the symptoms of Graves' disease. These results suggest that immunization with Tg induces Graves'-like disease in mice and that our methods will provide a new animal model of Graves' disease.
Assuntos
Doença de Graves/imunologia , Imunização , Tireoglobulina/imunologia , Animais , Autoanticorpos/sangue , Bovinos , Modelos Animais de Doenças , Feminino , Doença de Graves/sangue , Doença de Graves/patologia , Imunoglobulinas Estimuladoras da Glândula Tireoide/sangue , Radioisótopos do Iodo/farmacocinética , Camundongos , Camundongos Endogâmicos AKR , Camundongos Endogâmicos C57BL , Cintilografia , Glândula Tireoide/diagnóstico por imagem , Glândula Tireoide/metabolismo , Glândula Tireoide/patologia , Tiroxina/sangue , Tri-Iodotironina/sangueRESUMO
To search autoantigens in autoimmune pancreatitis (AIP), we have screened the human pancreas cDNA library with a patient's serum and obtained 10 positive clones. Seven out of 10 clones were amylase alpha-2A, the autoantibody to which was specifically detected in sera from patients with AIP and fulminant type 1 diabetes (FT1DM) [T. Endo, S. Takizawa, S. Tanaka, M. Takahashi, H. Fujii, T. Kamisawa, T. Kobayashi, Amylase alpha-2A autoantibodies: novel marker of autoimmune pancreatitis and fulminant type 1 diabetes mellitus, Diabetes 58 (2009) 732-737]. Sequencing of 1 out of remaining 3 positive clones revealed that it was identical to heat shock protein 10 (HSP 10) cDNA. Using a recombinant HSP 10, we have developed enzyme-linked immunosorbent assay (ELISA) system for detecting autoantibodies against HSP 10. We found that autoantibody against HSP 10 was also produced with high frequency in sera from patients with AIP (92%) and FT1DM (81%), but not in chronic alcoholic pancreatitis (8%) or healthy volunteers (1.4%). These results suggest that an autoantibody against HSP 10 is also a new diagnostic marker for both AIP and FT1DM.
Assuntos
Autoantígenos/imunologia , Doenças Autoimunes/imunologia , Chaperonina 10/imunologia , Diabetes Mellitus Tipo 1/imunologia , Pancreatite/imunologia , Adolescente , Adulto , Idoso , Autoantígenos/análise , Autoantígenos/genética , Doenças Autoimunes/sangue , Chaperonina 10/análise , Chaperonina 10/genética , Diabetes Mellitus Tipo 1/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Biblioteca Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Pancreatite/sangue , Análise de Sequência de DNA , Adulto JovemRESUMO
OBJECTIVE: The pathogenesis of autoimmune pancreatitis (AIP) and fulminant type 1 diabetes remains unclear, although it is known that immune-mediated processes severely compromise the endocrine and exocrine functions in both diseases. RESEARCH DESIGN AND METHODS: We have screened a lambdaTriplEx2 human pancreas cDNA library with serum from a patient with AIP and obtained positive clones. Sequence analysis revealed that 7 of 10 clones were identical to human amylase alpha-2A. Using a recombinant COOH-terminal amylase alpha-2A protein, we developed an enzyme-linked immunosorbent assay system to detect autoantibodies against human amylase alpha-2A. RESULTS: All 15 serum samples from patients with AIP recognized the recombinant protein, whereas sera from 25 patients with chronic alcoholic pancreatitis and sera from 25 patients with a pancreas tumor did not. Interestingly, 88% (15/17) of patients with fulminant type 1 diabetes were positive for an autoantibody against amylase alpha-2A. These antibodies were detected in 21% of patients with acute-onset type 1 diabetes (9 of 42) and 6% of type 2 diabetic patients (4 of 67). CONCLUSIONS: These results suggest that an autoantibody against amylase alpha-2A is a novel diagnostic marker for both AIP and fulminant type 1 diabetes and that, clinically and immunologically, AIP and fulminant type 1 diabetes are closely related.
Assuntos
Autoanticorpos/sangue , Diabetes Mellitus Tipo 1/imunologia , Ilhotas Pancreáticas/imunologia , alfa-Amilases Pancreáticas/imunologia , Pancreatite/imunologia , Idoso , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Doença Crônica , Clonagem Molecular , Primers do DNA , Diabetes Mellitus Tipo 1/enzimologia , Ensaio de Imunoadsorção Enzimática , Humanos , Inflamação/imunologia , Masculino , Ductos Pancreáticos/imunologia , Ductos Pancreáticos/patologia , alfa-Amilases Pancreáticas/genética , Reação em Cadeia da Polimerase , Proteínas Recombinantes/imunologiaRESUMO
A high proportion of patients with autoimmune pancreatitis (AIP) have diabetes. The decreased ß-cell function in active AIP, which leads to diabetes, can sometimes be reversed by corticosteroid treatment. However, the immunological mechanisms causing this ß-cell dysfunction are largely unclear. Our recent studies on AIP complicated with diabetes, and data from other animal models of AIP, suggest the presence of distinct mechanisms responsible for ß-cell damage in AIP. The presence of immunological cross-reactivity against antigens that are localized both in exocrine pancreatic tissue and ß-cells may explain the concomitant occurrence of pancreatitis and ß-cell damage in AIP.
RESUMO
CONTEXT: Histopathological analysis has demonstrated lymphocytic infiltration in both the endocrine and the exocrine pancreas in some patients with type 1 diabetes and non-alcoholic chronic pancreatitis, suggesting an immune-mediated mechanism which affects both diabetes mellitus and chronic pancreatitis. OBJECTIVE: The examination of exocrine pancreatic humoral markers in Caucasian patients with respect to the interactions between exocrine and endocrine pancreatic diseases. PATIENTS: One hundred and thirty-six European Caucasian subjects subdivided into three groups: type 1 diabetes (n=48); non-alcoholic chronic pancreatitis (n=48); controls (n=40). MAIN OUTCOME MEASURE: Autoantibodies against carbonic anhydrase II (CAIIAb) and lactoferrin (LACAb) (both of which are exocrine pancreatic antigens) were analyzed by enzyme-linked immunosorbent assay. RESULTS: No positivity for CAIIAb and LACAb were found in the controls. Patients with type 1 diabetes had a significantly higher prevalence of CAIIAb (25.0%) than the controls while the prevalence of LACAb (8.3%) was not significantly higher than the controls. The prevalence of CAIIAb (12.5%) and LACAb (20.8%) in the patients with non-alcoholic chronic pancreatitis was significantly higher than that in the controls. A significantly higher prevalence of CAIIAb and/or LACAb was found in patients with type 1 diabetes (29.2%) and non-alcoholic chronic pancreatitis (22.9%) compared to that in the controls (0%). There was a significant association between CAIIAb and LACAb titers both in patients with type 1 diabetes (P=0.042) and in patients with non-alcoholic chronic pancreatitis (P<0.001). CONCLUSION: We have clearly demonstrated that some European Caucasian patients with type 1 diabetes and non-alcoholic chronic pancreatitis have autoantibodies against the exocrine pancreatic antigens CAIIAb and LACAb.
Assuntos
Autoanticorpos/sangue , Autoantígenos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Pâncreas Exócrino/imunologia , Pancreatite Crônica/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anidrase Carbônica II/imunologia , Criança , Pré-Escolar , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/complicações , Ensaio de Imunoadsorção Enzimática , Europa (Continente) , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Glutamato Descarboxilase/imunologia , Humanos , Lactoferrina/imunologia , Pessoa de Meia-Idade , Pancreatite Crônica/sangue , Pancreatite Crônica/complicações , Estudos Prospectivos , Radioimunoensaio , População Branca , Adulto JovemRESUMO
C.RF- Tshr(hyt/hyt) mice have a mutated thyroid-stimulating hormone receptor (TSHR), and, without thyroid hormone supplementation, these mice develop severe hypothyroidism. When hypothyroid Tshr(hyt/hyt) mice were exposed to cold (4 degrees C), rectal temperature rapidly dropped to 23.9 +/- 0.40 degrees C at 90 min, whereas the wild-type mice temperatures were 37.0 +/- 0.15 degrees C. When we carried out functional rat TSHR gene transfer in the brown adipose tissues by plasmid injection combined with electroporation, there was no effect on the serum levels of thyroxine, although rectal temperature of the mice transfected with pcDNA3.1/Zeo-rat TSHR 90 min after cold exposure remained at 34.6 +/- 0.34 degrees C, which was significantly higher than that of Tshr(hyt/hyt) mice. Transfection of TSHR cDNA increased mRNA and protein levels of uncoupling protein-1 (UCP-1) in brown adipose tissues, and the weight ratio of brown adipose tissue to overall body weight also increased. Exogenous thyroid hormone supplementation to Tshr(hyt/hyt) mice restored rectal temperature 90 min after exposure to cold (36.8 +/- 0.10 degrees C). These results indicate that not only thyroid hormone but also thyroid-stimulating hormone (TSH)/TSHR are involved in the expression mechanism of UCP-1 in mouse brown adipose tissue. TSH stimulates thermogenesis and functions to protect a further decrease in body temperature in the hypothyroid state.
Assuntos
Tecido Adiposo Marrom/fisiologia , Receptores da Tireotropina/fisiologia , Termogênese/fisiologia , Animais , Northern Blotting , Temperatura Corporal/fisiologia , Peso Corporal/fisiologia , Temperatura Baixa , Imuno-Histoquímica , Canais Iônicos/biossíntese , Canais Iônicos/genética , Canais Iônicos/fisiologia , Camundongos , Camundongos Mutantes , Proteínas Mitocondriais/biossíntese , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/fisiologia , Tamanho do Órgão/fisiologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores da Tireotropina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Organismos Livres de Patógenos Específicos , Tiroxina/sangue , Transfecção , Proteína Desacopladora 1RESUMO
CONTEXT: Development of calcifying foci is a common finding in human thyroid papillary carcinoma, but its mechanisms remain unknown. OBJECTIVE: We therefore investigated whether osteocalcin and/or Cbfa-1 genes are expressed in malignant thyroid epithelial cells. We also studied the effects of Cbfa-1 on the expression of osteoblast-specific and thyrotropin receptor genes in thyrocytes. RESULTS: The human thyroid papillary carcinoma cell line BHP18-21 expresses bone-type osteocalcin mRNA at higher levels than in MG63 osteosarcoma cells. Northern blot analysis and EMSA using nuclear extracts from BHP18-21 cells and FRTL-5 cells demonstrated that cells of thyroid epithelial origin expressed Cbfa-1/Runx2, the main transcription factor for the expression of osteocalcin. When we transfected pcDNA3.1-human Cbfa-1 into FRTL-5 cells, Cbfa-1 increased the gene expression of alkaline phosphatase, type I collagen, and osteocalcin but suppressed the expression of thyrotropin receptor. We then stained the calcified regions of human papillary thyroid carcinoma tissues with antiosteocalcin antibody and found that malignant cells, as well as follicular epithelial cells, were immunopositive for osteocalcin. Northern blot analysis revealed that the Cbfa-1/Runx2 gene was strongly expressed in tissues from four cases of surgically resected papillary carcinoma. CONCLUSIONS: Thyrocytes share characteristics with osteoblasts. Cbfa-1 may play a role in calcification processes in human thyroid papillary carcinoma tissues.
Assuntos
Carcinoma Papilar/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/fisiologia , Neoplasias da Glândula Tireoide/genética , Animais , Anticorpos Monoclonais/farmacologia , Calcinose/genética , Calcinose/metabolismo , Carcinoma Papilar/metabolismo , Linhagem Celular , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Células Epiteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Osteocalcina/genética , Osteocalcina/imunologia , Osteocalcina/metabolismo , Ratos , Neoplasias da Glândula Tireoide/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
A new insulin sensitivity index was devised on the basis of an autoregressive model and its validity was investigated. Using data from the 75-g oral glucose tolerance test (OGTT), 115 subjects were divided into 3 groups: 40 with normal glucose tolerance, 34 with impaired glucose tolerance, and 41 with type 2 diabetes mellitus. The new insulin sensitivity index: oral glucose insulin sensitivity index (GSI) was calculated from five sets of plasma glucose and insulin levels obtained at 0, 30, 60, 90 and 120 min during OGTT using a formula based on an autoregressive model. Forty-three of the 115 subjects were examined for insulin sensitivity index (ISI) by euglycemic hyperinsulinemic clamp. GSI decreased in the order of normal glucose tolerance group>impaired glucose tolerance group>diabetic group. There was a significant correlation between GSI and the ISI derived from euglycemic hyperinsulinemic clamp study data in all 43 subjects who underwent both tests (r=0.72; P<0.0001). The ISI calculated by previous methods poorly correlated with the ISIs obtained by euglycemic hyperinsulinemic clamp study. In conclusion, this new insulin sensitivity index based on the data obtained from OGTT using an autoregressive model is comparable to an insulin sensitivity index by euglycemic hyperinsulinemic clamp technique and may be superior to previous indexes that have been devised to determine insulin sensitivity from OGTT data.