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1.
J Clin Exp Hematop ; 59(1): 1-16, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30918139

RESUMO

The microenvironment influences the behavior of follicular lymphoma (FL) but the specific roles of the immunomodulatory BTLA and TNFRSF14 (HVEM) are unknown. Therefore, we examined their immunohistochemical expression in the intrafollicular, interfollicular and total histological compartments in 106 FL cases (57M/49F; median age 57-years), and in nine relapsed-FL with transformation to DLBCL (tFL). BTLA expression pattern was of follicular T-helper cells (TFH) in the intrafollicular and of T-cells in the interfollicular compartments. The mantle zones were BTLA+ in 35.6% of the cases with similar distribution of IgD. TNFRSF14 expression pattern was of neoplastic B lymphocytes (centroblasts) and "tingible body macrophages". At diagnosis, the averages of total BTLA and TNFRSF14-positive cells were 19.2%±12.4STD (range, 0.6%-58.2%) and 46.7 cells/HPF (1-286.5), respectively. No differences were seen between low-grade vs. high-grade FL but tFL was characterized by low BTLA and high TNFRSF14 expression. High BTLA correlated with good overall survival (OS) (total-BTLA, Hazard Risk=0.479, P=0.022) and with high PD-1 and FOXP3+Tregs. High TNFRSF14 correlated with poor OS and progression-free survival (PFS) (total-TNFRSF14, HR=3.9 and 3.2, respectively, P<0.0001), with unfavorable clinical variables and higher risk of transformation (OR=5.3). Multivariate analysis including BTLA, TNFRSF14 and FLIPI showed that TNFRSF14 and FLIPI maintained prognostic value for OS and TNFRSF14 for PFS. In the GSE16131 FL series, high TNFRSF14 gene expression correlated with worse prognosis and GSEA showed that NFkB pathway was associated with the "High-TNFRSF14/dead-phenotype".In conclusion, the BTLA-TNFRSF14 immune modulation pathway seems to play a role in the pathobiology and prognosis of FL.


Assuntos
Linfoma Folicular/diagnóstico , Linfoma Difuso de Grandes Células B/diagnóstico , Receptores Imunológicos/metabolismo , Membro 14 de Receptores do Fator de Necrose Tumoral/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfócitos B/química , Linfócitos B/patologia , Transformação Celular Neoplásica , Feminino , Humanos , Fatores Imunológicos , Linfoma Folicular/mortalidade , Linfoma Difuso de Grandes Células B/mortalidade , Masculino , Pessoa de Meia-Idade , Prognóstico , Análise de Sobrevida , Linfócitos T/química
2.
BMC Cancer ; 16(1): 805, 2016 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-27756245

RESUMO

BACKGROUND: Pathological stage and grade have limited ability to predict the outcomes of superficial urothelial bladder carcinoma at initial transurethral resection (TUR). AT-motif binding factor 1 (ATBF1) is a tumor suppressive transcription factor that is normally localized to the nucleus but has been detected in the cytoplasm in several cancers. Here, we examined the diagnostic value of the intracellular localization of ATBF1 as a marker for the identification of high risk urothelial bladder carcinoma. METHODS: Seven anti-ATBF1 antibodies were generated to cover the entire ATBF1 sequence. Four human influenza hemagglutinin-derived amino acid sequence-tagged expression vectors with truncated ATBF1 cDNA were constructed to map the functional domains of nuclear localization signals (NLSs) with the consensus sequence KR[X10-12]K. A total of 117 samples from initial TUR of human bladder carcinomas were analyzed. None of the patients had received chemotherapy or radiotherapy before pathological evaluation. RESULTS: ATBF1 nuclear localization was regulated synergistically by three NLSs on ATBF1. The cytoplasmic fragments of ATBF1 lacked NLSs. Patients were divided into two groups according to positive nuclear staining of ATBF1, and significant differences in overall survival (P = 0.021) and intravesical recurrence-free survival (P = 0.013) were detected between ATBF1+ (n = 110) and ATBF1- (n = 7) cases. Multivariate analysis revealed that ATBF1 staining was an independent prognostic factor for intravesical recurrence-free survival after adjusting for cellular grading and pathological staging (P = 0.008). CONCLUSIONS: Cleavage of ATBF1 leads to the cytoplasmic localization of ATBF1 fragments and downregulates nuclear ATBF1. Alterations in the subcellular localization of ATBF1 due to fragmentation of the protein are related to the malignant character of urothelial carcinoma. Pathological evaluation using anti-ATBF1 antibodies enabled the identification of highly malignant cases that had been overlooked at initial TUR. Nuclear localization of ATBF1 indicates better prognosis of urothelial carcinoma.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células de Transição/metabolismo , Proteínas de Homeodomínio/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Western Blotting , Células COS , Carcinoma de Células de Transição/patologia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Progressão da Doença , Feminino , Células HEK293 , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Neoplasias da Bexiga Urinária/patologia
3.
Mol Neurobiol ; 53(8): 5377-83, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-26440668

RESUMO

TAR DNA-binding protein 43 (TDP-43) has been identified as a major component of ubiquitin-positive inclusions in the brains and spinal cords of patients with frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U) or amyotrophic lateral sclerosis (ALS). The phosphorylated C-terminal fragment of TDP-43 forms aggregates in the neuronal cytoplasm, possibly resulting in neuronal cell death in patients with FTLD-U or ALS. The inositol pyrophosphate known as diphosphoinositol pentakisphosphate (InsP7) contains highly energetic pyrophosphate bonds. We previously reported that inositol hexakisphosphate kinase type 2 (InsP6K2), which converts inositol hexakisphosphate (InsP6) to InsP7, mediates cell death in mammalian cells. Moreover, InsP6K2 is translocated from the nucleus to the cytosol during apoptosis. In this study, we verified that phosphorylated TDP-43 co-localized and co-bound with InsP6K2 in the cytoplasm of anterior horn cells of the spinal cord. Furthermore, we verified that cell death was augmented in the presence of cytoplasmic TDP-43 aggregations and activated InsP6K2. However, cells with only cytoplasmic TDP-43 aggregation survived because Akt activity increased. In the presence of both TDP-43 aggregation and activated InsP6K2 in the cytoplasm of cells, the expression levels of HSP90 and casein kinase 2 decreased, as the activity of Akt decreased. These conditions may promote cell death. Thus, InsP6K2 could cause neuronal cell death in patients with FTLD-U or ALS. Moreover, InsP6K2 plays an important role in a novel cell death pathway present in FTLD-U and ALS.


Assuntos
Citoplasma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Agregados Proteicos , Caseína Quinase II/metabolismo , Morte Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo
4.
Endocr Pathol ; 24(3): 144-8, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23780556

RESUMO

Succinate dehydrogenase subunit B gene (SDHB) is associated with the development of hereditary paraganglioma (PGL) and pheochromocytoma (PCC). Here we describe a novel germline mutation in SDHB in a 69-year-old Japanese woman with a posterior mediastinal PGL. We summarize the clinical presentation, diagnostic work-up, and pathological features of a patient with a posterior mediastinal PGL and review the pertinent literature. Direct sequencing of SDHB and SDHD was performed. The patient presented with a posterior mediastinal tumor and was normotensive. She underwent abdominal tumor resection at the age of 38 years, but clinical and pathological diagnoses were unknown. She had no family history of hypertension, PGL, or PCC. Imaging studies suggested that the tumor was neurogenic. Endocrinological examinations showed normal plasma catecholamine levels. The tumor was completely removed without metastasis. Pathological findings confirmed PGL. Immunohistochemical staining showed that the tumor cells were positive for chromogranin A, synaptophysin, and CD56, and the Ki67 index was low (<1 %). The patient has not experienced recurrence or metastasis for the last 5 years. DNA sequencing revealed a novel P236S (c.843 C > T) mutation in SDHB. The P236S germline mutation in SDHB was associated with posterior mediastinal PGL. Strict follow-up of the patient is necessary because the SDHB mutation may be related to malignancy.


Assuntos
Mutação em Linhagem Germinativa , Neoplasias do Mediastino/genética , Paraganglioma Extrassuprarrenal/genética , Succinato Desidrogenase/genética , Idoso , Substituição de Aminoácidos , Grupo com Ancestrais do Continente Asiático , Feminino , Humanos , Neoplasias do Mediastino/diagnóstico , Paraganglioma Extrassuprarrenal/diagnóstico , Prolina/genética , Serina/genética
5.
Tokai J Exp Clin Med ; 36(4): 128-33, 2011 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-22167496

RESUMO

A 55-year-old Japanese man was referred to our hospital because of disturbance of consciousness and hyponatremia. He had been aware of general fatigue, nausea, and headache for two weeks. Tests revealed hyponatremia, plasma hypoosmolarity with urine hyperosmolarity, an elevated level of urine sodium excretion, and normal functions of the kidney, adrenal gland, and thyroid. These findings were compatible with syndrome of inappropriate secretion of antidiuretic hormone (SIADH). Head magnetic resonance imaging (MRI) demonstrated a pituitary tumor measuring 20 x 22 x 21 mm that pushed the pituitary stalk upward. Endocrinological evaluations suggested that the pituitary adenoma was non-functional. The pituitary adenoma was surgically removed, and histological examination revealed a biphasic appearance characterized by endocrine cells and a hemangiomatous stroma. After surgery, the patient developed pituitary hypothyroidism, pituitary adrenal insufficiency, and pituitary gonadal failure. Therefore, levothyroxine sodium, 50 µg per day, and hydrocortisone, 10 mg per day, were administered orally. Androgen depot, 250 mg every two months, was also injected intramuscularly. The hyponatremia did not recur, and the patient has done well for the last five years. The pituitary adenoma in this case showed two features: one was the cause of SIADH, and the other was a biphasic histological picture of endocrine cells with a hemangiomatous stroma.


Assuntos
Adenoma/complicações , Hemangioma/complicações , Síndrome de Secreção Inadequada de HAD/etiologia , Neoplasias Hipofisárias/complicações , Adenoma/patologia , Adenoma/cirurgia , Terapia Combinada , Hemangioma/patologia , Humanos , Hipotireoidismo/etiologia , Masculino , Pessoa de Meia-Idade , Neoplasias Hipofisárias/patologia , Neoplasias Hipofisárias/cirurgia , Resultado do Tratamento
6.
Pathobiology ; 78(5): 239-52, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21849805

RESUMO

OBJECTIVE: To evaluate the role of matrix metalloproteinase (MMP)-13 gene expression in the early phase of recovery from liver fibrosis/cirrhosis. METHODS: Liver fibrosis was induced in male Wistar rats by administration of carbon tetrachloride (CCl(4)) for 10 weeks. Recombinant adenovirus-mediated human MMP-13 gene transfer (RAdMMP-13) was performed via the femoral vein on day 3 after the last CCl(4) injection. The role of MMP-13 in stably expressing cell lines was also analyzed. RESULTS: Fibrous deposition in the liver was decreased in RAdMMP-13-injected rats by day 3 after gene transfer compared with empty vector RAd66-injected rats. Furthermore, MMP-2 and MMP-9 enzymatic activity was markedly enhanced in the liver of RAdMMP-13 injected rats. Hepatocyte growth factor (HGF) induction was also increased in RAdMMP-13 injected rats. In established stable HT-1080 cells transfected with MMP-13, HGF-α expression and MMP-2 and MMP-9 enzymatic activity were increased. The conversion of precursor HGF into mature HGF was also increased in the MMP-13 expressing cell lines. CONCLUSION: Forced MMP-13 expression effectively accelerated recovery from liver cirrhosis via the effects of MMP-13-mediated HGF, MMP-2, and MMP-9 expression, which induced the degradation of collagen fibers and promoted hepatic regeneration.


Assuntos
Cirrose Hepática Experimental/metabolismo , Metaloproteinase 13 da Matriz/metabolismo , Animais , Western Blotting , Regulação Enzimológica da Expressão Gênica , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Imuno-Histoquímica , Cirrose Hepática Experimental/genética , Masculino , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção
7.
Med Mol Morphol ; 44(2): 63-70, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21717308

RESUMO

Combined in situ hybridization (ISH) and immunohistochemistry (IHC) under electron microscopy (EM-ISH & IHC) has sufficient ultrastructural resolution to provide two-dimensional images of subcellular localization of pituitary hormone and its mRNA in a pituitary cell. The advantages of semiconductor nanocrystals (Quantum dots; Qdots) and confocal laser scanning microscopy (CLSM) enable us to obtain three-dimensional images of the subcellular localization of pituitary hormone and its mRNA. Both EM-ISH & IHC and ISH & IHC using Qdots and CLSM are useful for understanding the relationship between protein and mRNA simultaneously in two or three dimensions. CLSM observation of rab3B and SNARE proteins such as SNAP-25 and syntaxin revealed that both rab3B and SNARE system proteins play an important role and work together as the exocytotic machinery in anterior pituitary cells. Another important issue is the intracellular transport and secretion of pituitary hormone. An experimental pituitary cell line, the GH3 cell, in which growth hormone (GH) is linked to enhanced yellow fluorescein protein (EYFP), has been developed. This stable GH3 cell secretes GH linked to EYFP upon being stimulated by Ca(2+) influx or Ca(2+) release from storage. This GH3 cell is useful for real-time visualization of the intracellular transport and secretion of GH. These three methods enable us to visualize consecutively the processes of transcription, translation, transport, and secretion of pituitary hormone.


Assuntos
Hormônio do Crescimento , Imagem Tridimensional/métodos , Proteínas Qa-SNARE , RNA Mensageiro/metabolismo , Proteína 25 Associada a Sinaptossoma , Proteínas rab3 de Ligação ao GTP , Animais , Proteínas de Bactérias , Transporte Biológico/fisiologia , Linhagem Celular , Exocitose/fisiologia , Hormônio do Crescimento/metabolismo , Humanos , Imuno-Histoquímica/métodos , Hibridização In Situ/métodos , Proteínas Luminescentes , Microscopia Confocal/métodos , Microscopia Eletrônica/métodos , Hipófise/metabolismo , Hipófise/ultraestrutura , Proteínas Qa-SNARE/metabolismo , Proteínas Qa-SNARE/ultraestrutura , Pontos Quânticos , Ratos , Proteína 25 Associada a Sinaptossoma/metabolismo , Proteína 25 Associada a Sinaptossoma/ultraestrutura , Proteínas rab3 de Ligação ao GTP/metabolismo , Proteínas rab3 de Ligação ao GTP/ultraestrutura
8.
J Biol Chem ; 286(30): 26680-6, 2011 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-21652713

RESUMO

Inositol pyrophosphate diphosphoinositol pentakisphosphate is ubiquitously present in mammalian cells and contains highly energetic pyrophosphate bonds. We have previously reported that inositol hexakisphosphate kinase type 2 (InsP(6)K2), which converts inositol hexakisphosphate to inositol pyrophosphate diphosphoinositol pentakisphosphate, mediates apoptotic cell death via its translocation from the nucleus to the cytoplasm. Here, we report that InsP(6)K2 is localized mainly in the cytoplasm of lymphoblast cells from patients with Huntington disease (HD), whereas this enzyme is localized in the nucleus in control lymphoblast cells. The large number of autophagosomes detected in HD lymphoblast cells is consistent with the down-regulation of Akt in response to InsP(6)K2 activation. Consistent with these observations, the overexpression of InsP(6)Ks leads to the depletion of Akt phosphorylation and the induction of cell death. These results suggest that InsP(6)K2 activation is associated with the pathogenesis of HD.


Assuntos
Apoptose , Núcleo Celular/enzimologia , Doença de Huntington/enzimologia , Linfócitos/enzimologia , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Núcleo Celular/ultraestrutura , Citoplasma/enzimologia , Citoplasma/ultraestrutura , Ativação Enzimática/genética , Células HEK293 , Humanos , Doença de Huntington/genética , Doença de Huntington/patologia , Linfócitos/ultraestrutura , Fosforilação/genética , Fosfotransferases (Aceptor do Grupo Fosfato)/genética , Ácido Fítico/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo
9.
Molecules ; 16(5): 3618-35, 2011 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-21540793

RESUMO

In situ hybridization (ISH) at the electron microscopic (EM) level is essential for elucidating the intracellular distribution and role of mRNA in protein synthesis. EM-ISH is considered to be an important tool for clarifying the intracellular localization of mRNA and the exact site of pituitary hormone synthesis on the rough endoplasmic reticulum. A combined ISH and immunohistochemistry (IHC) under EM (EM-ISH&IHC) approach has sufficient ultrastructural resolution, and provides two-dimensional images of the subcellular localization of pituitary hormone and its mRNA in a pituitary cell. The advantages of semiconductor nanocrystals (quantum dots, Qdots) and confocal laser scanning microscopy (CLSM) enable us to obtain three-dimensional images of the subcellular localization of pituitary hormone and its mRNA. Both EM-ISH&IHC and ISH & IHC using Qdots and CLSM are useful for understanding the relationships between protein and mRNA simultaneously in two or three dimensions. CLSM observation of rab3B and SNARE proteins such as SNAP-25 and syntaxin has revealed that both rab3B and SNARE system proteins play important roles and work together as the exocytotic machinery in anterior pituitary cells. Another important issue is the intracellular transport and secretion of pituitary hormone. We have developed an experimental pituitary cell line, GH3 cell, which has growth hormone (GH) linked to enhanced yellow fluorescein protein (EYFP). This stable GH3 cell secretes GH linked to EYFP upon stimulation by Ca²+ influx or Ca²+ release from storage. This GH3 cell line is useful for the real-time visualization of the intracellular transport and secretion of GH. These three methods from conventional immunohistochemistry and fluorescein imaging allow us to consecutively visualize the process of transcription, translation, transport and secretion of anterior pituitary hormone.


Assuntos
Imuno-Histoquímica/métodos , Hipófise/citologia , Hipófise/metabolismo , Animais , Hormônio do Crescimento/genética , Hormônio do Crescimento/metabolismo , Humanos , Hibridização In Situ/métodos , Microscopia Eletrônica
10.
Circ Res ; 109(1): 20-37, 2011 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-21566217

RESUMO

The precise and conceptual insight of circulating endothelial progenitor cell (EPC) kinetics is hampered by the absence of an assay system capable of evaluating the EPC differentiation cascade. An assay system for EPC colony formation was developed to delineate circulating EPC differentiation. EPC colony-forming assay using semisolid medium and single or bulk CD133(+) cells from umbilical cord blood exhibited the formation of two types of attaching cell colonies made of small or large cells featuring endothelial lineage potential and properties, termed small EPC colony-forming units and large EPC colony-forming units, respectively. In vitro and in vivo assays of each EPC colony-forming unit cell revealed a differentiation hierarchy from small EPC to large EPC colonies, indicating a primitive EPC stage with highly proliferative activity and a definitive EPC stage with vasculogenic properties, respectively. Experimental comparison with a conventional EPC culture assay system disclosed EPC colony-forming unit cells differentiate into noncolony-forming early EPC. The fate analysis of single CD133(+) cells into the endothelial and hematopoietic lineage was achieved by combining this assay system with a hematopoietic progenitor assay and demonstrated the development of colony-forming EPC and hematopoietic progenitor cells from a single hematopoietic stem cell. EPC colony-forming assay permits the determination of circulating EPC kinetics from single or bulk cells, based on the evaluation of hierarchical EPC colony formation. This assay further enables a proper exploration of possible links between the origin of EPC and hematopoietic stem cells, representing a novel and powerful tool to investigate the molecular signaling pathways involved in EPC biology.


Assuntos
Ensaio de Unidades Formadoras de Colônias/métodos , Células Endoteliais/citologia , Células-Tronco/citologia , Antígeno AC133 , Adulto , Animais , Antígenos CD/análise , Diferenciação Celular , Células Cultivadas , Glicoproteínas/análise , Células-Tronco Hematopoéticas/citologia , Humanos , Receptores de Lipopolissacarídeos/análise , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos/análise , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/farmacologia
11.
Infect Immun ; 79(1): 512-7, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20956568

RESUMO

A combinatorial immunoglobulin gene library was constructed from lymphocytes in peripheral blood of a patient with toxoplasmosis and screened for production of human monoclonal antibody Fab fragments to recombinant surface antigen 1 (SAG1) of Toxoplasma gondii. Two Fab clones, Tox203 and Tox1403, which consisted of a common heavy chain and different light chains, showed positive staining on the entire surface of tachyzoites in confocal microscopy. Sequence analysis of the heavy-chain gene revealed that the closest germ line V segments were VH3-23. The germ line D segment was D1-7, and the closest germ line J segment was JH4. In the light-chain genes, the closest germ line V segment was Vκ1-17 with the Jκ1 or Jκ4 segments. The dissociation constants of these Fab fragments with recombinant SAG1 were 3.09 × 10(-9) M for Tox203 and 2.01 × 10(-8) M for Tox1403, indicating that the affinity of Tox203 was 7 times higher than that of Tox1403. Preincubation of T. gondii tachyzoites with Tox203 significantly inhibited their attachment to cultured MDBK cells. Passive immunization of mice with Tox203 also significantly reduced mortality after challenge with T. gondii tachyzoites. This is the first report of bacterial expression of human monoclonal antibody Fab fragments to SAG1 of T. gondii. These results also demonstrate that human Fab fragments to SAG1 might be applicable for immunoprophylaxis of toxoplasmosis.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Antígenos de Superfície/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Toxoplasma/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários , Antígenos de Protozoários/imunologia , Clonagem Molecular , Feminino , Biblioteca Gênica , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , Imunoglobulinas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Dados de Sequência Molecular , Toxoplasmose Animal/imunologia , Toxoplasmose Animal/prevenção & controle
12.
Glycoconj J ; 27(7-9): 661-72, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21046235

RESUMO

The expressions of heparan sulfate glycosaminoglycans (HSGAGs) in breast carcinoma specimens from 60 patients were immunohistochemically investigated using monoclonal antibodies (mAbs) that recognized different epitopes of the glycan structure. Cytoplasmic expression of GlcA-GlcNH(3)(+) on HSGAG was detected in carcinomas at high frequency (58.3%) using mAb JM403, whereas it was almost undetectable in normal breast ducts. This cytoplasmic expression was confirmed using confocal laser scanning microscopy. The expression of JM403 antigen in invasive carcinomas significantly correlated with nuclear atypia score (p = 0.0004), mitotic counts score (p = 0.0018), nuclear grade (p = 0.0061) and the incidence of metastasis to axillary lymph nodes (p = 0.0061). Furthermore, its expression was significantly correlated with the Ki67-labeling index in 55 invasive carcinomas (p < 0.05) as well as in 26 non-invasive carcinomas (5 non-invasive carcinomas and 21 non-invasive carcinomas that were observed in individual invasive carcinomas) (p < 0.005). Interestingly, the JM403 antigen GlcA-GlcNH(3)(+) was also expressed in the cytoplasm of normal crypt epithelial cells where Ki67 protein was expressed in the cell nuclei in the proliferative compartment of the human small intestines. To date, HSGAGs have generally been found to exist on cell surface membranes and in extracellular matrices as components of HS proteoglycans, and the negatively-charged sulfated domains on HSGAGs are considered to be important for their functions. However, our present findings indicate that the cytoplasmic expression of the JM403 antigen GlcA-GlcNH(3)(+) on positively charged, non-sulfated HSGAG may be involved in cell proliferation and associated with increased degrees of malignancy. The unordinary carbohydrate antigen of GlcA-GlcNH(3)(+) on HSGAGs recognized by mAb JM403 may represent a novel proliferative biomarker for highly malignant mammary carcinomas.


Assuntos
Antígenos de Neoplasias/biossíntese , Biomarcadores Tumorais/biossíntese , Neoplasias da Mama/metabolismo , Citoplasma/metabolismo , Proteoglicanas de Heparan Sulfato/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/patologia , Neoplasias da Mama/ultraestrutura , Proliferação de Células , Feminino , Humanos , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Antígeno Ki-67/biossíntese , Glândulas Mamárias Humanas/metabolismo , Pessoa de Meia-Idade , Receptor ErbB-2/biossíntese , Receptores Estrogênicos/biossíntese , Receptores de Progesterona/biossíntese
13.
Endocrine ; 38(1): 18-23, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20960097

RESUMO

Nuclear genes succinate dehydrogenase B subunit and succinate dehydrogenase D subunit, which encode two mitochondrial complex II subunits, are associated with the development of familial paraganglioma (PGL). Succinate dehydrogenase B subunit gene mutation is highly associated with extraadrenal PGL and subsequent distant metastasis. We describe the case of a 29-year-old Japanese man with a 3-year history of hypertension, headache, and palpitation. Endocrinological examinations showed that the patient had elevated levels of catecholamines, and imaging studies revealed a right paraaortic PGL without distant metastases. The PGL was surgically removed. Genetic analysis of the patient showed a heterozygous thymine deletion at position 470 (c.470delT) in exon 5 of the succinate dehydrogenase B subunit gene complementary DNA. This thymine deletion changed TTG (leucine) to TGA (stop codon) at codon 157 (L157X). It remains unclear whether this mutation was associated with PGL malignancy because the patient has had no metastases for the past 3 years. It has been recently reported that L157X is associated with malignant paraaortic PGL. Thus, strict follow-up is required because this succinate dehydrogenase B subunit gene's nonsense mutation (L157X) may be related to the malignancy.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Códon sem Sentido , Paraganglioma/genética , Neoplasias do Sistema Nervoso Periférico/genética , Succinato Desidrogenase/genética , Adulto , Aorta , Mutação em Linhagem Germinativa , Humanos , Masculino , Paraganglioma/diagnóstico por imagem , Paraganglioma/cirurgia , Neoplasias do Sistema Nervoso Periférico/diagnóstico por imagem , Neoplasias do Sistema Nervoso Periférico/cirurgia , Tomografia Computadorizada de Emissão de Fóton Único
14.
Int J Biochem Cell Biol ; 42(12): 2065-71, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20883817

RESUMO

We and other authors have previously reported that increasing cellular diphosphoinositol pentakisphosphate (InsP(7)) levels increases cell sensitivity to cell death. In the present study, we elucidated the relationship between inositol hexakisphosphate kinases (InsP(6)Ks), which form InsP(7), and autophagy using InsP(6)Ks overexpression and disruption systems. A large number of autophagosomes were induced in cells transfected with InsP(6)Ks, as revealed by the conversion of LC3-I to LC3-II, which was examined using immunoblotting, immunocytochemistry, and immuno-electron microscopy for LC3; consequently, the rate of cell death was higher among these cells than among cells transfected with a control vector, as shown using propidium iodide staining. However, the reduction of InsP(6)Ks levels using RNAi suppressed the formation of autophagosomes. Moreover, the number of autophagosomes and the rate of cell death were significantly higher among cells transfected with InsP(6)Ks subjected to staurosporine-induced stress than among cells transfected with InsP(6)Ks subjected to normal conditions. The cell death induced by InsP(6)Ks was not completely suppressed by z-VAD-fmk, a pan-caspase inhibitor. The phosphorylation of mammalian target of rapamycin (mTOR) was also depressed in cells overexpressing InsP(6)Ks, suggesting that the mTOR pathway regulates autophagosomes generated by InsP(6)Ks. These findings imply that InsP(6)Ks promote autophagy and induce caspase-independent cell death. This phenomenon opens a new pathway of autophagy via InsP(6)Ks.


Assuntos
Autofagia/fisiologia , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Morte Celular/fisiologia , Sobrevivência Celular , Células HEK293 , Células HeLa , Humanos , Imuno-Histoquímica , Fosfatos de Inositol/metabolismo , Fosforilação , Fosfotransferases (Aceptor do Grupo Fosfato)/genética , Transfecção
15.
Int J Oncol ; 37(5): 1077-83, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20878055

RESUMO

The somatostatin analogue (SA) Octreotide has been used as a therapeutic reagent for somatostatin receptor type 2a (SSTR2a)-positive cancers. The purpose of this study is to detect SSTR2a in human prostate carcinomas and to elucidate the effects of SA on SSTR2a-positive prostate carcinoma cells to determine the potential of this drug as a new therapeutic method for advanced prostate carcinoma. Immunohistochemical study of SSTR2a was performed on 95 prostate carcinoma cases, and the results showed expression of SSTR2a in 14 of the 95 cases (14.74%); the histological grade (Gleason) and capsular invasion of the prostate carcinoma were directly related to SSTR2a expression. Among the ten cases of lymph node metastasis, SSTR2a expression was markedly higher. In vitro studies were performed using SSTR2a-positive prostate cancer cells, DU145 and PC3. Migration and invasion abilities of DU145 and PC3 cells were inhibited by SA in a dose-dependent manner. This inhibition was reversed by Rho-kinase inhibitor Y-27632. Morphological changes of the prostate cancer cells treated with SA and Y27632 corroborate the migration and invasion assays, although SA had no effect on proliferation of DU145 and PC3 cells. In conclusion, the somatostatin analogue may be beneficial for patients with advanced prostate carcinoma or to protect from distal metastasis if they are positive for SSTR2a.


Assuntos
Adenocarcinoma/patologia , Antineoplásicos Hormonais/farmacologia , Octreotida/farmacologia , Neoplasias da Próstata/patologia , Receptores de Somatostatina/biossíntese , Adenocarcinoma/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Metástase Linfática/patologia , Masculino , Invasividade Neoplásica/patologia , Estadiamento de Neoplasias , Neoplasias da Próstata/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Somatostatina
16.
Acta Histochem Cytochem ; 43(2): 33-44, 2010 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-20514290

RESUMO

Estrogen-induced pituitary prolactin-producing tumors (PRLoma) in F344 rats express a high level of vascular endothelial growth factor (VEGF) associated with marked angiogenesis and angiectasis. To investigate whether tumor development in E2-induced PRLoma is inhibited by anti-VEGF monoclonal antibody (G6-31), we evaluated tumor growth and observed the vascular structures. With simultaneous treatment with G6-31 for the latter three weeks of the 13-week period of E2 stimulation (E2+G6-31 group), the following inhibitory effects on the PRLoma were observed in the E2+G6-31 group as compared with the E2-only group. In the E2+G6-31 group, a tendency to reduction in pituitary weight was observed and significant differences were observed as (1) reductions in the Ki-67-positive anterior cells, (2) increases in TUNEL-positive anterior cells, and (3) repair of the microvessel count by CD34-immunohistochemistry. The characteristic "blood lakes" in PRLomas were improved and replaced by repaired microvascular structures on 3D observation using confocal laser scanning microscope. These inhibitory effects due to anti-VEGF antibody might be related to the autocrine/paracrine action of VEGF on the tumor cells, because VEGF and its receptor are co-expressed on the tumor cells. Thus, our results demonstrate that anti-VEGF antibody exerted inhibitory effects on pituitary tumorigenesis in well-established E2 induced PRLomas.

17.
Endocr J ; 57(1): 31-8, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-19834252

RESUMO

Small-cell carcinoma (SCC) of neuroendocrine type is an uncommon tumor of the endometrium. No previous report has documented Cushing's syndrome due to ectopic ACTH production by SCC of the endometrium. We describe a 56-year-old Japanese woman with SCC of the endometrium and multiple lung metastases presenting as Cushing's syndrome. The patient was referred to our hospital because of general fatigue with facial and leg edema, and multiple nodular lesions in the bilateral lungs on chest X-ray examination. A physical examination revealed that the patient had moon face, buffalo hump, and truncal obesity. Endocrinological examinations confirmed ACTH-dependent Cushing's syndrome. Thoracic computed tomography imaging showed multiple nodular lesions in the bilateral lungs. Abdominal magnetic resonance imaging suggested a malignant tumor of the uterus. The patient received a lung tumor biopsy and surgical hysterectomy. The endometrial carcinoma was histologically a SCC admixed with endometrioid adenocarcinoma. The SCC of the endometrium showed immunoreactivity for pro-opiomelanocortin, ACTH, and vimentin, but not for thyroid transcription factor-1. The lung biopsy specimen had the same features. These findings indicated that the SCC originated from the endometrium, and the ectopic ACTH-producing tumor caused Cushing's syndrome. This study provides the evidence that SCC of endometrial origin was an ectopic ACTH-producing tumor causing Cushing's syndrome.


Assuntos
Carcinoma de Células Pequenas/complicações , Síndrome de Cushing/etiologia , Neoplasias do Endométrio/complicações , Carcinoma de Células Pequenas/diagnóstico , Carcinoma de Células Pequenas/cirurgia , Síndrome de Cushing/diagnóstico , Diagnóstico Diferencial , Neoplasias do Endométrio/diagnóstico , Neoplasias do Endométrio/cirurgia , Evolução Fatal , Feminino , Humanos , Histerectomia , Imuno-Histoquímica , Imagem por Ressonância Magnética , Pessoa de Meia-Idade , Tomografia Computadorizada por Raios X
18.
Eur Urol ; 55(2): 441-9, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18395325

RESUMO

BACKGROUND: Metastin, the final peptide of the KiSS-1 gene, has been proposed to suppress cell motility. OBJECTIVE: This study investigated whether renal cell carcinoma (RCC) tissue expresses metastin or its receptor, and clarified whether metastin can suppress migration and/or invasion and/or proliferation of RCC cells in vitro. DESIGN, SETTING, AND PARTICIPANTS: Twenty-five RCC samples were submitted. Fresh RCC tissues were prepared for real-time RT-PCR, and formalin-fixed and paraffin-embedded tissues blocks were examined by immunohistochemistry. RCC cell lines Caki-1 and ACHN were supplied for cell migration, invasion, and proliferation assays. MEASUREMENTS: Real-time RT-PCR was performed by using Taq Man gene expression system. ENVISION system was used in immunohistochemistry. Wound-healing assay and matrigel assays were used to identify migration and invasion abilities of RCC cell lines. Cell Counting Kit-8 was applied to measure the cell proliferation. Cell morphology was examined under a META system. Statistical analysis was performed with SPSS15.0J. RESULTS AND LIMITATIONS: In twenty-five RCC samples, the mRNA level of metastin receptor was identified to be significantly higher than non-neoplastic renal cortex by real-time RT-PCR (p=0.011). Immunohistochemical study also detected metastin receptor protein in all RCC tumors. In vitro, this study showed that metastin inhibited migration and invasion of Caki-1 and ACHN cells. In contrast, it had no effects on cell proliferation. Metastin (10 micromol/l) induced excessive formation of focal adhesions and stress fibers in Caki-1 and ACHN cells; this phenomenon was inhibited by pretreating pharmacological Rho-kinase inhibitor (Y-27632) to those cells. CONCLUSION: This is the first report regarding overexpression of the metastin receptor hOT7T175 in human RCC. We demonstrate that metastin can inhibit migration and invasion of the RCC cell line, which is regulated by a Rho-kinase inhibitor. Metastin and its receptor are therefore probable targets for suppressing RCC.


Assuntos
Carcinoma de Células Renais/patologia , Neoplasias Renais/patologia , RNA Mensageiro/genética , Receptores Acoplados a Proteínas-G/genética , Proteínas Supressoras de Tumor/uso terapêutico , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/cirurgia , Adesão Celular , Divisão Celular , Linhagem Celular Tumoral , Movimento Celular , Primers do DNA , Feminino , Genes Supressores de Tumor , Humanos , Imuno-Histoquímica , Neoplasias Renais/genética , Neoplasias Renais/cirurgia , Kisspeptinas , Masculino , Invasividade Neoplásica , Receptores de Kisspeptina-1 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Supressoras de Tumor/genética
19.
Infect Immun ; 77(1): 549-56, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19001071

RESUMO

Four fully human monoclonal antibodies (MAbs) to Entamoeba histolytica intermediate subunit lectin (Igl) were prepared in XenoMouse mice, which are transgenic mice expressing human immunoglobulin loci. Examination of the reactivities of these MAbs to recombinant Igl1 and Igl2 of E. histolytica showed that XEhI-20 {immunoglobulin G2(kappa) [IgG2(kappa)]} and XEhI-28 [IgG2(kappa)] were specific to Igl1, XEhI-B5 [IgG2(kappa)] was specific to Igl2, and XEhI-H2 [IgM(kappa)] was reactive with both Igls. Gene analyses revealed that the V(H) and V(L) germ lines were VH3-48 and L2 for XEhI-20, VH3-21 and L2 for XEhI-28, VH3-33 and B3 for XEhI-B5, and VH4-4 and A19 for XEhI-H2, respectively. Flow cytometry analyses showed that the epitopes recognized by all of these MAbs were located on the surfaces of living trophozoites. Confocal microscopy demonstrated that most Igl1 and Igl2 proteins were colocalized on the surface and in the cytoplasm, but different localization patterns in intracellular vacuoles were also present. The preincubation of trophozoites with XEhI-20, XEhI-B5, and XEhI-H2 caused significant inhibition of the adherence of trophozoites to Chinese hamster ovary cells, whereas preincubation with XEhI-28 did not do so. XEhI-20, XEhI-B5, and XEhI-H2 were injected intraperitoneally into hamsters 24 h prior to intrahepatic challenge with E. histolytica trophozoites. One week later, the mean abscess size in groups injected with one of the three MAbs was significantly smaller than that in controls injected with polyclonal IgG or IgM isolated from healthy humans. These results demonstrate that human MAbs to Igls may be applicable for immunoprophylaxis of amebiasis.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antiprotozoários/imunologia , Anticorpos Antiprotozoários/isolamento & purificação , Entamoeba histolytica/imunologia , Lectinas/imunologia , Proteínas de Protozoários/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Antiprotozoários/uso terapêutico , Células CHO , Adesão Celular , Membrana Celular/química , Cricetinae , Cricetulus , Citoplasma/química , Citometria de Fluxo , Imunização Passiva , Imunoglobulina G/imunologia , Região Variável de Imunoglobulina/genética , Abscesso Hepático/prevenção & controle , Masculino , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Dados de Sequência Molecular , Alinhamento de Sequência , Análise de Sequência de DNA , Vacúolos/química
20.
Acta Histochem Cytochem ; 41(2): 15-21, 2008 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-18493590

RESUMO

The aim of this study was to evaluate the immunohistochemical expression of MUC2, MUC5AC, MUC6, and CD10 in ovarian mucinous adenoma (MA), mucinous borderline tumor (MB), and mucinous adenocarcinoma (MC), and to analyze the relationship between prognosis and these expressions. The expression of MUC2, MUC5AC, MUC6, and CD10 was evaluated by immunohistochemical analysis in 29 cases of MA, 29 cases of MB, and 26 cases of MC and scored based on the percentage of positive cells. Moreover, the ovarian mucinous tumors were classified into 4 phenotypes based on the staining patterns: intestinal, gastrointestinal, gastric, and unclassified patterns. The gastrointestinal pattern and the expression of MUC2 and CD10 increased from MA to MC. Conversely, the gastric pattern and MUC5AC expression decreased from MA to MC. Low MUC2 expression in MC was correlated with a better long-term survival rate. MUC2 expression in MC may be a useful predictor of the clinical outcome. The expression patterns of MUC2, MUC5AC, MUC6, and CD10 indicated that intestinal metaplasia may arise from the gastric-like epithelium in MA and that a close association exists between carcinogenesis and intestinal metaplasia in major ovarian mucinous tumors.

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