RESUMO
To investigate the association between the expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) and the clinicopathological features in lepidic and invasive components of adenocarcinoma of the lung, we performed immunostaining for type IV collagen, various MMPs, and TIMPs in 27 cases of invasive adenocarcinomas and 5 cases of atypical adenomatous hyperplasia of alveolar epithelial cells (AAH). Mean extent of lepidic growth was 61% and the survival was significantly better in cases with 50% or more lepidic component. The preservation of type IV collagen in lepidic areas correlated inversely with lymphatic or vascular invasion (P = 0.02 and 0.002, respectively). Five-year survival was reduced in cases showing destruction of type IV collagen (P = 0.004) or expression of MMP-2 (P = 0.008) in lepidic areas. MMP-2 co-localized with MT-1-MMP (its activating enzyme) and TIMP-2 in neoplastic cells. Reactivity for other MMPs and TIMPs did not correlate with destruction of type IV collagen or prognosis. Type IV collagen was preserved in all cases of AAH. MMP-2, but not MT-1-MMP, was expressed in two of the five cases of AAH. Immunostaining for type IV collagen MMP-2 is useful in evaluating the prognosis of the lung.
Assuntos
Adenocarcinoma Bronquioloalveolar/patologia , Adenoma/patologia , Neoplasias Pulmonares/patologia , Pulmão/patologia , Metaloproteinases da Matriz/biossíntese , Adenocarcinoma Bronquioloalveolar/metabolismo , Idoso , Colágeno Tipo IV/análise , Feminino , Humanos , Hiperplasia , Imuno-Histoquímica , Pulmão/química , Neoplasias Pulmonares/metabolismo , Masculino , Metaloproteinase 2 da Matriz/análise , Metaloproteinases da Matriz Associadas à Membrana , Metaloendopeptidases/análise , Microscopia Confocal , Pessoa de Meia-Idade , Invasividade Neoplásica , Prognóstico , Análise de Sobrevida , Inibidor Tecidual de Metaloproteinase-1/análise , Inibidor Tecidual de Metaloproteinase-2/análiseRESUMO
PURPOSE: To compare the protective effect of dexrazoxane (DRZ) against cardiotoxicity induced by doxorubicin (DXR) and mitoxantrone (MTX). METHODS: Adult male spontaneously hypertensive rats (SHR) were treated with 1 mg/kg DXR (i.v.) or 0.5 mg/kg MTX (i.v.), either alone or 30 min after 25 mg/kg DRZ (i.p.) weekly for up to 12 weeks. Animals treated with DXR alone either died (n = 2) or were killed (n = 3) at a cumulative dose of 10 mg/kg. The severity of cardiac lesions (cytoplasmic vacuolization and myofibrillar loss) were graded semiquantitatively by light microscopy on a scale of 0 to 3. RESULTS: Cardiac lesions were observed in all SHR given DXR or MTX alone, and were attenuated in those given DRZ prior to either DXR (mean lesion scores 2.7 vs 1.5; P < 0.05) or MTX (mean lesion scores 2.0 vs 1.25; P < 0.05). Cardioprotection was also demonstrated by monitoring serum levels of cardiac troponin T (cTnT), which were elevated in all animals receiving DXR or MTX alone. These elevations were attenuated in SHR given the combination of DXR and DRZ (mean values 0.79 ng/ml vs 0.24 ng/ml; P < 0.05) and MTX and DRZ (mean values 0.19 ng/ml vs 0.04 ng/ ml; P < 0.05). Biochemical studies have shown that both DXR and MTX form potentially cardiotoxic complexes with iron. ADR-925 (the hydrolysis product of DRZ) and other chelators (EDTA, diethylenetriaminepentaacetic acid and desferrioxamine) removed Fe(III) from its complex with MTX or DXR. CONCLUSIONS: The present study showed that DRZ significantly attenuates the cardiotoxicity induced by DXR and MTX, and that this protective activity can be assessed by morphological evaluation of cardiac tissues and by monitoring the concentrations of cTnT in serum.
Assuntos
Antineoplásicos/efeitos adversos , Fármacos Cardiovasculares/farmacologia , Doxorrubicina/efeitos adversos , Mitoxantrona/efeitos adversos , Miocárdio/patologia , Razoxano/farmacologia , Troponina T/sangue , Animais , Biomarcadores/análise , Coração/efeitos dos fármacos , Infusões Intravenosas , Masculino , Ratos , Ratos Endogâmicos SHRRESUMO
PURPOSE: To evaluate the imaging and clinical features of lymphangioleiomyomas and to describe the phenomenon of diurnal variation in the size of lymphangioleiomyomas in patients with lymphangioleiomyomatosis. MATERIALS AND METHODS: One hundred twenty-eight patients with lymphangioleiomyomatosis underwent chest and abdominopelvic computed tomography (CT). Thirteen patients underwent CT in the morning and afternoon of the same day to assess diurnal variation in lymphangioleiomyoma size. RESULTS: Twenty-seven of 128 patients (21%) had 54 lymphangioleiomyomas. The vast majority (96%) of these masses contained material of low attenuation at CT. Associated CT findings included enlarged abdominal lymph nodes, pleural effusions, ascites, and dilatation of the thoracic duct. The prevalence of lymphangioleiomyomas was 15% in patients who had mild pulmonary disease, 19% in patients who had moderate disease, and 26% in patients who had severe disease. Diurnal variation in size of masses was demonstrated in 12 of 13 patients. Seven of the 27 patients who had masses underwent biopsy; all seven were confirmed to have lymphangioleiomyomas. The most common symptoms associated with lymphangioleiomyomas were bloating, abdominal pain, and edema of the lower extremities. The majority of the patients reported worsening of symptoms as the day progressed. CONCLUSION: Lymphangioleiomyomas are common in patients with lymphangioleiomyomatosis. Diurnal variation in size may explain worsening of symptoms during the day.
Assuntos
Neoplasias Abdominais/diagnóstico por imagem , Linfangioleiomiomatose/diagnóstico por imagem , Tomografia Computadorizada por Raios X , Adulto , Idoso , Ritmo Circadiano , Feminino , Humanos , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/patologia , Pessoa de Meia-Idade , Neoplasias Primárias Múltiplas/diagnóstico por imagem , Índice de Gravidade de DoençaRESUMO
The 42 amino acid form of beta amyloid (Abeta42) plays a pivotal role in neurotoxicity and the activation of mononuclear phagocytes in Alzheimer's disease (AD). Our recent study revealed that FPRL1, a G-protein-coupled receptor, mediates the chemotactic and activating effect of Abeta42 on mononuclear phagocytes (monocytes and microglia), suggesting that FPRL1 may be involved in the proinflammatory responses in AD. We investigated the role of FPRL1 in cellular uptake and the subsequent fibrillar formation of Abeta42 by using fluorescence confocal microscopy. We found that upon incubation with macrophages or HEK293 cells genetically engineered to express FPRL1, Abeta42 associated with FPRL1 and the Abeta42/FPRL1 complexes were rapidly internalized into the cytoplasmic compartment. The maximal internalization of Abeta42/FPRL1 complexes occurred by 30 min after incubation. Removal of free Abeta42 from culture supernatants at 30 min resulted in a progressive recycling of FPRL1 to the cell surface and degradation of the internalized Abeta42. However, persistent exposure of the cells to Abeta42 over 24 h resulted in retention of Abeta42/FPRL1 complexes in the cytoplasmic compartment and the formation of Congo red positive fibrils in macrophages but not in HEK 293 cell transfected with FPRL1. These results suggest that besides mediating the proinflammatory activity of Abeta42, FPRL1 is also involved in the internalization of Abeta42, which culminates in the formation of fibrils only in macrophages.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Macrófagos/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptores Imunológicos/fisiologia , Receptores de Lipoxinas , Receptores de Peptídeos/fisiologia , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Colchicina/farmacologia , Relação Dose-Resposta a Droga , Endocitose/efeitos dos fármacos , Humanos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Microscopia Confocal , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Conformação Proteica , Receptores de Formil Peptídeo , Fatores de TempoRESUMO
Lymphangioleiomyomatosis (LAM) is a rare disease, occurring in women, characterized by cystic degeneration of the lungs, abdominal tumors, and proliferation of abnormal smooth muscle cells. Lung function abnormalities consist of impairment of the diffusion capacity (DL(CO)) and airflow obstruction. The objective of this study was to correlate the functional impairment with histologic measures of disease severity to identify predictors of disease outcome. Lung function of 143 patients and lung biopsies of 74 of these patients were reviewed for evidence of airway disease and scoring of disease severity. A positive response to bronchodilators was associated with more severe airflow obstruction, a predominantly solid pattern of LAM lesions in the lung biopsy, and greater rate of decline in expiratory flow. Airway inflammation, present in 61% of the lung specimens, was not associated with reversible airway obstruction and did not correlate with the severity of airflow obstruction. DL(CO) correlated best with the LAM histologic score (LHS), a demonstrated predictor of outcome. We conclude that reversible airway obstruction is found in LAM patients with accelerated loss of lung function and a predominantly solid pattern of LAM lesions. Impairment of DL(CO) correlates with LHS, a predictor of survival and time to lung transplantation.
Assuntos
Neoplasias Pulmonares/patologia , Pulmão/patologia , Linfangioleiomiomatose/patologia , Músculo Liso/citologia , Troca Gasosa Pulmonar , Ventilação Pulmonar , Adulto , Idoso , Biópsia , Bronquiolite/patologia , Broncodilatadores/farmacologia , Interpretação Estatística de Dados , Feminino , Seguimentos , Humanos , Pulmão/efeitos dos fármacos , Neoplasias Pulmonares/fisiopatologia , Transplante de Pulmão , Linfangioleiomiomatose/fisiopatologia , Pessoa de Meia-Idade , Análise Multivariada , Músculo Liso/patologia , Prognóstico , Capacidade de Difusão Pulmonar , Ventilação Pulmonar/efeitos dos fármacos , Testes de Função Respiratória , Fumar/efeitos adversos , Fatores de TempoRESUMO
Various types of oculocutaneous albinism (OCA) are associated with reduced pigmentation in the skin, hair, and eyes that results from mutations in genes involved in melanin synthesis. Immortal mouse melanocyte lines (melan-a, melan-b, and melan-c) provide opportune models with which to investigate the etiology of two different types of OCA (types I and III), which arise from mutations in Tyr and Tyrp1, respectively. We compared intracellular processing, sorting, and degradation of tyrosinase and Tyrp1, and the effects on their catalytic function and melanin synthesis, in these wild-type and mutant melanocytes. A mutation in either Tyr or Tyrp1 increased the time of association of tyrosinase and Tyrp1 with calnexin and Bip, which in turn resulted in the retention of these mutant products in the ER. A mutation in either gene selectively enhanced the duration and efficiency of chaperone interactions (even with the wild-type protein in the mutant melanocytes) and markedly slowed their transport to melanosomes. These results show that OCA1 and OCA3 are (in some cases, at least) ER retention diseases wherein a mutation in one melanogenic protein affects the maturation and stability of the other in the melanogenic pathway.
Assuntos
Albinismo Oculocutâneo/etiologia , Retículo Endoplasmático/fisiologia , Proteínas de Choque Térmico , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , Oxirredutases , Albinismo Oculocutâneo/enzimologia , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Calnexina , Proteínas de Transporte/metabolismo , Chaperona BiP do Retículo Endoplasmático , Hexosaminidases/química , Oxirredutases Intramoleculares/metabolismo , Substâncias Macromoleculares , Melaninas/análise , Melanócitos/enzimologia , Melanócitos/metabolismo , Camundongos , Chaperonas Moleculares/metabolismo , Mutação , Células Tumorais CultivadasRESUMO
Multiple factors affect skin pigmentation, including those that regulate melanocyte and/or keratinocyte function. Such factors, particularly those that operate at the level of the melanosome, are relatively well characterized in mice, but the expression and function of structural and enzymatic proteins in melanocytes in human skin are not as well known. Some years ago, we generated peptide-specific antibodies to murine melanosomal proteins that proved to be instrumental in elucidating melanocyte development and differentiation in mice, but cross-reactivity of those antibodies with the corresponding human proteins often was weak or absent. In an effort to characterize the roles of melanosomal proteins in human skin pigmentation, and to understand the underlying mechanism(s) of abnormal skin pigmentation, we have now generated polyclonal antibodies against the human melanocyte-specific markers, tyrosinase, tyrosinase-related protein (TYRP1), Dopachrome tautomerase (DCT) and Pmel17 (SILV, also known as GP100). We used these antibodies to determine the distribution and function of melanosomal proteins in normal human skin (adult and newborn) and in various cutaneous pigmented lesions, such as intradermal nevi, lentigo simplex, solar lentigines and malignant melanomas. We also examined cytokeratin expression in these same samples to assess keratinocyte distribution and function. Immunohistochemical staining reveals distinct patterns of melanocyte distribution and function in normal skin and in various types of cutaneous pigmented lesions. Those differences in the expression patterns of melanocyte markers provide important clues to the roles of melanocytes in normal and in disrupted skin pigmentation.
Assuntos
Especificidade de Anticorpos , Lentigo/patologia , Melanócitos/química , Melanócitos/imunologia , Glicoproteínas de Membrana , Oxirredutases , Pele/química , Adulto , Sequência de Aminoácidos , Animais , Células Cultivadas , Secções Congeladas , Humanos , Imuno-Histoquímica , Recém-Nascido , Oxirredutases Intramoleculares/análise , Oxirredutases Intramoleculares/imunologia , Queratinócitos/química , Queratinócitos/enzimologia , Queratinócitos/imunologia , Melanócitos/enzimologia , Melanoma/patologia , Melanossomas/química , Melanossomas/enzimologia , Melanossomas/imunologia , Dados de Sequência Molecular , Monofenol Mono-Oxigenase/análise , Monofenol Mono-Oxigenase/imunologia , Nevo Intradérmico/patologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Proteínas/análise , Proteínas/imunologia , Coelhos , Pele/citologia , Pele/enzimologia , Neoplasias Cutâneas/patologia , Pigmentação da Pele , Antígeno gp100 de MelanomaRESUMO
BACKGROUND: Insulin-like growth factors (IGF-1 and IGF-2), the IGF-1 receptor (IGF-1R), and IGF-binding proteins (IGFBPs) are involved in normal pulmonary development and in the pathogenesis of smooth muscle cell tumors. METHODS: To evaluate the role of the IGF system in lymphangioleiomyomatosis (LAM), we used immunohistochemical and in situ hybridization techniques to characterize the expression of IGF-1, IGF-2, IGF-1R, and IGFBP-2, -4, -5, and -6 in lung tissue from 18 LAM patients. RESULTS: IGF-1, ICGF-2, IGF-1R, IGFBP-1, IGFBP-2, IGFBP-4, IGFBP-5, and IGFBP-6 were expressed by LAM cells. Reactivity and mRNA expression for IGF-2 were observed in LAM cells and resembled that found in normal smooth muscle cells during pulmonary development as well as in smooth muscle cell tumors. IGFBP-2, IGFBP-4, and IGFBP-6 were associated with spindle-shaped LAM cells, whereas IGFBP-5 was associated mainly with epithelioid LAM cells. CONCLUSIONS: These findings suggest that the IGFBPs modulate the effects of the IGFs on LAM cells. Thus, the patterns of localization and expression of components of the IGF system in LAM strongly suggest that these agents are involved in the proliferation of LAM cells.
Assuntos
Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/análise , Fator de Crescimento Insulin-Like II/análise , Fator de Crescimento Insulin-Like I/análise , Neoplasias Pulmonares/metabolismo , Linfangioleiomiomatose/metabolismo , RNA Mensageiro/análise , Receptor IGF Tipo 1/análise , Adulto , Feminino , Hormônios Esteroides Gonadais/fisiologia , Humanos , Imuno-Histoquímica , Hibridização In Situ , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like II/genética , Pulmão/química , Neoplasias Pulmonares/patologia , Linfangioleiomiomatose/patologia , Pessoa de Meia-Idade , Receptor IGF Tipo 1/genéticaRESUMO
Melanosome biogenesis and function were studied after purification of early stage melanosomes and characterization of specific proteins sorted to that organelle. Melanosomes were isolated from highly pigmented human MNT1 melanoma cells after disruption and initial separation by sucrose density gradient centrifugation. Low-density sucrose fractions were found by electron microscopy to be enriched in stage I and stage II melanosomes, and these fractions were further separated and purified by free flow electrophoresis. Tyrosinase and dopachrome tautomerase (DCT) activities were found exclusively in stage II melanosomes, even though DCT (and to some extent tyrosinase) proteins were sorted to stage I melanosomes. Western immunoblotting revealed that these catalytic proteins, as well as TYRP1, MART1, and GP100, were cleaved and inactivated in stage I melanosomes. Proteolytic cleavage was critical for the refolding of GP100 within the melanosomal milieu, and subsequent reorganization of amorphous stage I melanosomes into fibrillar, ovoid, and highly organized stage II melanosomes appears to stabilize the catalytic functions of melanosomal enzymes and allows melanin biosynthesis to begin. These results provide a better understanding of the structural features seen during melanosome biogenesis, and they yield further clues as to the physiological regulation of pigmentation.
Assuntos
Melanossomas/química , Proteínas de Neoplasias/análise , Oxirredutases , Eletroforese/métodos , Humanos , Oxirredutases Intramoleculares/análise , Melanossomas/ultraestrutura , Glicoproteínas de Membrana/análise , Microscopia Confocal/métodos , Microscopia Imunoeletrônica/métodos , Monofenol Mono-Oxigenase/análise , Proteínas/análise , Células Tumorais Cultivadas , Antígeno gp100 de MelanomaRESUMO
We report 2 patients in whom pulmonary lymphangioleiomyomatosis (LAM) affected the retroperitoneal lymph nodes and was associated with endosalpingiosis. These lesions were large, encapsulated masses with multiple cysts containing chylous fluid. Both were characterized by proliferating LAM cells that formed fascicles separated by slit-like channels. Some cysts were lined by ciliated epithelium resembling that of Fallopian tubes. Other cysts were lined either by flattened endothelial cells or by a mixture of these cells and epithelial cells. Many LAM cells gave a positive reaction with HMB-45 antibody. Most LAM cells in fascicles were reactive for alpha-smooth muscle actin and desmin. In 1 patient, many of the epithelial cells and some of the subjacent LAM cells were positive for estrogen and progesterone receptors. In conclusion, immunostaining with HMB-45 antibody and markers for smooth muscle cells can be helpful in the evaluation of problems in the differential diagnosis of lesions of extrapulmonary LAM, particularly those involving the genital system.
Assuntos
Linfonodos/patologia , Linfonodos/cirurgia , Linfangioleiomiomatose/patologia , Linfangioleiomiomatose/cirurgia , Espaço Retroperitoneal/patologia , Salpingite/patologia , Adulto , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-IdadeRESUMO
Patients with restrictive cardiomyopathy (RC) have impaired diastolic function, but intact systolic function until later stages of the disease, ultimately leading to heart failure. Primary RC is often sporadic, but also may be inherited in an autosomal dominant fashion, particularly the idiopathic forms. Recently there has been great interest in inherited cardiomyopathy associated with myocyte desmin deposition ('desminopathies'). In some such families, desmin or alpha-B crystallin gene mutation is the underlying cause, and the desmin accumulation affects skeletal muscle as well, usually causing skeletal myopathy. We describe a large family with apparent autosomal dominant inheritance of desmin-associated RC spanning four generations, with the age of onset and severity/rate of progression being highly variable. This family is relatively unique in that there is no symptom-based evidence of skeletal muscle involvement, and the known desminopathy and cardiomyopathy genes/loci have been ruled out. These data support literature suggesting that desmin deposition may be associated with different underlying gene defects, and that a novel desminopathy gene is responsible for the condition in this family.
Assuntos
Cardiomiopatia Restritiva/genética , Desmina/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Northern Blotting , Cardiomiopatia Restritiva/patologia , Criança , Pré-Escolar , Mapeamento Cromossômico , Cristalinas/genética , Análise Mutacional de DNA , Primers do DNA/química , Feminino , Genótipo , Humanos , Técnicas Imunoenzimáticas , Masculino , Repetições de Microssatélites , Pessoa de Meia-Idade , Linhagem , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de RestriçãoRESUMO
Correlations were made between clinical and follow-up data and histopathologic findings in 105 women (mean age +/- standard deviation, 38.3 +/- 9.0 years) with pulmonary lymphangioleiomyomatosis (LAM). The actuarial survival (to pulmonary transplantation or death) of the patients from the time of lung biopsy was 85.1% and 71.0% after 5 and 10 years respectively. The histologic severity of LAM, graded as a LAM histologic score (LHS), was determined on the basis of semiquantitative estimation of the percentage of tissue involvement by the two major features of LAM: the cystic lesions and the infiltration by abnormal smooth muscle cells (LAM cells) in each case: LHS-1, <25%; LHS-2, 25% to 50%; and LHS-3, >50%. Analysis using the Kaplan-Meier method revealed significant differences in survival for patients with LHS-1, -2, and -3 (p = 0.01). The 5-and 10-year survivals were 100% and 100% for LHS-1, 81.2% and 74.4% for LHS-2, and 62.8% and 52.4% for LHS-3. Increased degrees of accumulation of hemosiderin in macrophages also were associated with higher LHS scores (p = 0.029) and a worse prognosis (p = 0.0012). Thus, the current study suggests that the LHS may provide a basis for determining the prognosis of LAM.
Assuntos
Neoplasias Pulmonares/patologia , Linfangiomioma/patologia , Adolescente , Adulto , Idoso , Cistos/patologia , Feminino , Seguimentos , Hemossiderina/metabolismo , Hemossiderose/patologia , Humanos , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/cirurgia , Linfangiomioma/mortalidade , Linfangiomioma/cirurgia , Macrófagos/metabolismo , Macrófagos/patologia , Pessoa de Meia-Idade , Músculo Liso/patologia , Prognóstico , Taxa de SobrevidaRESUMO
The aim of this study was to evaluate the usefulness of determination of telomerase activity and expression of human telomerase RNA component (hTERC) and human telomerase reverse transcriptase (hTERT) for the diagnosis of lung carcinomas. The tissues studied consisted of 115 carcinomas and adjacent nonneoplastic lung, which were removed surgically without previous chemotherapy or radiotherapy. Telomerase activity was determined using a semiquantitative polymerase chain reaction-based telomeric repeat amplification protocol (TRAP) assay. The results obtained were classified into high and low telomerase groups. Localization of expression was examined by using in situ hybridization and immunohistochemistry. The correlation between telomerase activity in lung carcinoma and clinicopathologic features, including prognosis, was investigated. Telomerase activity in lung carcinomas was detected in 107 of 115 (93%) lung carcinomas, but not in any adjacent noncancerous tissues, and was significantly higher in small cell carcinoma than in any other histologic type. This activity also was significantly higher in poorly differentiated than in well-differentiated squamous cell carcinomas and adenocarcinomas. The overall survival rate (P =.020) was significantly lower in the high telomerase group. Messenger RNAs for hTERC and hTERT were mainly detected in the cytoplasm of cancer cells by in situ hybridization, and TERT protein was localized in the nuclei of these cells by immunohistochemical staining. Determinations of telomerase activity by in situ hybridization, immunohistochemistry, and TRAP assay are useful for evaluating the diagnosis and prognosis of lung carcinomas.
Assuntos
Adenocarcinoma/enzimologia , Neoplasias Pulmonares/enzimologia , RNA Mensageiro/metabolismo , RNA , Telomerase/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/mortalidade , Adenocarcinoma/secundário , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Proteínas de Ligação a DNA , Feminino , Humanos , Imuno-Histoquímica , Hibridização In Situ , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Linfonodos/patologia , Metástase Linfática/genética , Metástase Linfática/patologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Estadiamento de Neoplasias , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Telomerase/genéticaRESUMO
Prion diseases are transmissible and fatal neurodegenerative disorders which involve infiltration and activation of mononuclear phagocytes at the brain lesions. A 20-aa acid fragment of the human cellular prion protein, PrP(106-126), was reported to mimic the biological activity of the pathologic isoform of prion and activates mononuclear phagocytes. The cell surface receptor(s) mediating the activity of PrP(106-126) is unknown. In this study, we show that PrP(106-126) is chemotactic for human monocytes through the use of a G protein-coupled receptor formyl peptide receptor-like 1 (FPRL1), which has been reported to interact with a diverse array of exogenous or endogenous ligands. Upon stimulation by PrP(106-126), FPRL1 underwent a rapid internalization and, furthermore, PrP(106-126) enhanced monocyte production of proinflammatory cytokines, which was inhibited by pertussis toxin. Thus, FPRL1 may act as a "pattern recognition" receptor that interacts with multiple pathologic agents and may be involved in the proinflammatory process of prion diseases.
Assuntos
Fatores Quimiotáticos/fisiologia , Quimiotaxia de Leucócito/imunologia , Proteínas de Ligação ao GTP/metabolismo , N-Formilmetionina Leucil-Fenilalanina/metabolismo , Fragmentos de Peptídeos/agonistas , Fragmentos de Peptídeos/toxicidade , Príons/agonistas , Príons/toxicidade , Receptores Imunológicos/fisiologia , Receptores de Lipoxinas , Receptores de Peptídeos/fisiologia , Animais , Sinalização do Cálcio/imunologia , Linhagem Celular , Fatores Quimiotáticos/toxicidade , Citocinas/biossíntese , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Ligantes , Microglia/imunologia , Microglia/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Fragmentos de Peptídeos/fisiologia , Príons/fisiologia , Ratos , Receptores de Formil Peptídeo , Receptores Imunológicos/metabolismo , Receptores de Peptídeos/metabolismo , Células Tumorais CultivadasRESUMO
A human cDNA encoding an 841-aa guanine nucleotide-exchange protein (GEP) for ADP-ribosylation factors (ARFs), named ARF-GEP(100), which contains a Sec7 domain, a pleckstrin homology (PH)-like domain, and an incomplete IQ-motif, was identified. On Northern blot analysis of human tissues, a approximately 8-kb mRNA that hybridized with an ARF-GEP(100) cDNA was abundant in peripheral blood leukocytes, brain, and spleen. ARF-GEP(100) accelerated [(35)S]GTPgammaS binding to ARF1 (class I) and ARF5 (class II) 2- to 3-fold, and to ARF6 (class III) ca. 12-fold. The ARF-GEP(100) Sec7 domain contains Asp(543) and Met(555), corresponding to residues associated with sensitivity to the inhibitory effect of the fungal metabolite brefeldin A (BFA) in yeast Sec7, but also Phe(535) and Ala(536), associated with BFA-insensitivity. The PH-like domain differs greatly from those of other ARF GEPs in regions involved in phospholipid binding. Consistent with its structure, ARF-GEP(100) activity was not affected by BFA or phospholipids. After subcellular fractionation of cultured T98G human glioblastoma cells, ARF6 was almost entirely in the crude membrane fraction, whereas ARF-GEP(100), a 100-kDa protein detected with antipeptide antibodies, was cytosolic. On immunofluorescence microscopy, both proteins had a punctate pattern of distribution throughout the cells, with apparent colocalization only in peripheral areas. The coarse punctate distribution of EEA-1 in regions nearer the nucleus appeared to coincide with that of ARF-GEP(100) in those areas. No similar coincidence of ARF-GEP(100) with AP-1, AP-2, catenin, LAMP-1, or 58K was observed. The new human BFA-insensitive GEP may function with ARF6 in specific endocytic processes.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fator 6 de Ribosilação do ADP , Sequência de Aminoácidos , Animais , Sequência de Bases , Cálcio/metabolismo , Calmodulina/metabolismo , Linhagem Celular , Primers do DNA , DNA Complementar , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Microscopia de Fluorescência , Dados de Sequência Molecular , Fosfolipídeos/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Spodoptera , Frações Subcelulares/metabolismoRESUMO
BACKGROUND: Pathogens infecting the arterial wall with resultant inflammation may contribute to atherogenesis. Human coronary artery smooth muscle cells (SMCs) infected with human cytomegalovirus (CMV) demonstrate a rapid increase in reactive oxygen species (ROSs), with activation of genes involved in viral replication and inflammation. Because estrogen appears to have antioxidant properties, we wished to determine whether this hormone attenuates SMC responses to CMV infection. METHODS AND RESULTS: Using confocal microscopy and an intracellular fluorescent dye activated by ROSs, we found that 17beta-estradiol (0.1 to 10 nmol/L) and its stereoisomer 17alpha-estradiol (which has low affinity for the estrogen receptor) dose-dependently inhibited ROS generation in CMV-infected SMCs. These effects were not blocked by the estrogen receptor inhibitor ICI 182,780. 3-Methoxyestrone, which lacks the phenolic hydroxyl group, did not interfere with ROS generation. We found that 17beta-estradiol and 17alpha-estradiol, but not 3-methoxyestrone, prevented binding of nuclear factor (NF)-kappaB to DNA. Furthermore, in SMCs transfected with the reporter constructs 3XkappaB-CAT, MIEP-CAT, or ICAM-CAT, cotransfection with a CMV-IE72 expression plasmid caused promoter and CAT activation. Treatment with 17beta-estradiol and 17alpha-estradiol, but not 3-methoxyestrone, inhibited CAT activity and, in CMV-infected SMCs, prevented IE72 and ICAM-1 protein expression and cytopathic effects. CONCLUSIONS: These findings indicate that estrogen molecules with an A-ring hydroxyl group have estrogen receptor-independent anti-CMV effects at physiological concentrations by inhibiting ROS generation, NF-kappaB activation, NF-kappaB-dependent transcription, and viral replication. To the extent that chronic infection of the vascular wall with CMV contributes to atherogenesis, these antioxidant actions of estrogen may be of therapeutic importance.
Assuntos
Antioxidantes/farmacologia , Vasos Coronários/efeitos dos fármacos , Citomegalovirus/efeitos dos fármacos , Estrogênios/farmacologia , Regulação Viral da Expressão Gênica/genética , Músculo Liso Vascular/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Proteínas Virais , Adulto , Células Cultivadas , Vasos Coronários/patologia , Vasos Coronários/fisiologia , Citomegalovirus/genética , Citomegalovirus/fisiologia , Feminino , Fluorescência , Humanos , Proteínas Imediatamente Precoces/antagonistas & inibidores , Proteínas Imediatamente Precoces/biossíntese , Molécula 1 de Adesão Intercelular/biossíntese , Molécula 1 de Adesão Intercelular/genética , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiologia , NF-kappa B/metabolismo , Tamoxifeno/farmacologia , Transcrição Gênica/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: Little is known of the morphology of the pneumocytes lining the parenchymal cysts in lymphangioleiomyomatosis (LAM). OBJECTIVE: To evaluate the structural characteristics of the alveolar epithelial cells in LAM. METHODS: Immunohistochemical and electron microscopic studies were performed on lung tissue from 22 women with pulmonary LAM. RESULTS: Epithelial cells that reacted with PE-10 (a mouse monoclonal antibody that recognizes the surfactant apoprotein A in type II pneumocytes) and TTF-1 (an antibody that identifies nuclear transcription factor found in type II pneumocytes) were the predominant cell type lining the surfaces of lesions of LAM and normal areas of lung. Scanning and transmission electron microscopic studies confirmed that these cells were type II pneumocytes as demonstrated by (1) apical microvilli, (2) electron-dense lamellar bodies, and (3) cytoplasmic projections that extended from the basal surfaces into the underlying connective tissue, where they made extensive contact with interstitial connective tissue cells. A few cells had morphologic characteristics of type I pneumocytes, including large flat surfaces lacking microvilli. Cells that appeared intermediate between type I and type II pneumocytes were observed occasionally. CONCLUSIONS: These observations and the reactivity of these cells with antibody to proliferating cell nuclear antigen demonstrate that extensive hyperplasia of type II pneumocytes is a major characteristic of LAM.
Assuntos
Neoplasias Pulmonares/patologia , Pulmão/patologia , Linfangioleiomiomatose/patologia , Adulto , Apolipoproteínas A/análise , Feminino , Humanos , Hiperplasia , Pulmão/química , Pulmão/ultraestrutura , Neoplasias Pulmonares/metabolismo , Linfangioleiomiomatose/metabolismo , Metaloproteinase 1 da Matriz/análise , Microscopia Confocal , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Pessoa de Meia-Idade , Proteínas Nucleares/análise , Antígeno Nuclear de Célula em Proliferação/análise , Fator Nuclear 1 de Tireoide , Fatores de Transcrição/análiseRESUMO
Desmin myopathy is a hereditary or sporadic cardiac and skeletal myopathy characterised by intracytoplasmic accumulation of desmin reactive deposits in muscle cells. We have characterised novel splice site mutations in the gene desmin resulting in deletion of the entire exon 3 during the pre-mRNA splicing. Sequencing of cDNA and genomic DNA identified a heterozygous de novo A to G change at the +3 position of the splice donor site of intron 3 (IVS3+3A-->G) in a patient with sporadic skeletal and cardiac myopathy. A G to A transition at the highly conserved -1 nucleotide position of intron 2 affecting the splice acceptor site (IVS2-1G-->A) was found in an unrelated patient with a similar phenotype. Expression of genomic DNA fragments carrying the IVS3+3A-->G and IVS2-1G-->A mutations confirmed that these mutations cause exon 3 deletion. Aberrant splicing leads to an in frame deletion of 32 complete codons and is predicted to result in mutant desmin lacking 32 amino acids from the 1B segment of the alpha helical rod. Functional analysis of the mutant desmin in SW13 (vim-) cells showed aggregation of abnormal coarse clumps of desmin positive material dispersed throughout the cytoplasm. This is the first report on the pathogenic potentials of splice site mutations in the desmin gene.
Assuntos
Desmina/genética , Doenças Musculares/genética , Splicing de RNA/genética , Adulto , Sequência de Bases , DNA/química , DNA/genética , Análise Mutacional de DNA , DNA Complementar/química , DNA Complementar/genética , Saúde da Família , Feminino , Haplótipos , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Músculo Esquelético/patologia , Doenças Musculares/patologia , Mutação , Miocárdio/patologia , Linhagem , Polimorfismo de Nucleotídeo ÚnicoRESUMO
We describe the lesions of extrapulmonary lymphangioleiomyomatosis (LAM) affecting the lymph nodes of the mediastinum and retroperitoneum in 22 women (mean age +/- SD, 42.4+/-10.5 years). In most of these patients, the diagnosis of extrapulmonary LAM preceded that of pulmonary LAM, usually by 1 to 2 years. Eleven patients had distinct symptoms, including chylous pleural effusion and/or ascites, abdominal pain, and palpable abdominal masses. In the other 11 patients, the masses caused no symptoms. Well-circumscribed, encapsulated masses, measuring up to 20 cm in size, occurred in the mediastinum in 2 patients, the upper retroperitoneum in 15, extensive areas of the retroperitoneum in 2, and the pelvis in 3. The masses exceeding 3 cm in diameter contained large, multiple cysts filled with yellow-tan chylous fluid. Histologically, the masses were characterized by a proliferation of smooth muscle cells (LAM cells) arranged in fascicular, trabecular, and papillary patterns, which were associated with slit-like vascular channels. The LAM cells varied from small, spindle-shaped cells to large epithelioid cells. Immunohistochemical studies showed a strong reactivity of most LAM cells for alpha-smooth muscle actin and smooth muscle myosin heavy chain and a weak to moderate reactivity of a lesser number of cells for desmin and nonmuscle myosin heavy chain II-B. A reaction for HMB-45 and estrogen and progesterone receptors was observed mainly in epithelioid LAM cells. These patterns of reactivity are similar to those observed in pulmonary LAM. However, the chylous cysts are not a feature of pulmonary LAM and are thought to result from obstruction of lymphatics.