RESUMO
Leukoencephalopathies encompass all clinical syndromes that predominantly affect brain white matter. Genetic diagnosis informs clinical management of these patients, but a large part of the genetic contribution to adult leukoencephalopathy remains unresolved. To examine this genetic contribution, we analyzed genomic DNA from 60 Japanese patients with adult leukoencephalopathy of unknown cause by next generation sequencing using a custom-designed gene panel. We selected 55 leukoencephalopathy-related genes for the gene panel. We identified pathogenic mutations in 8 of the 60 adult leukoencephalopathy patients (13.3%): NOTCH3 mutations were detected in 5 patients, and EIF2B2, CSF1R, and POLR3A mutations were found independently in 1 patient each. These results indicate that cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) caused by NOTCH3 mutations is the most frequent adult leukoencephalopathy in our cohort. Moreover, brain imaging analysis indicates that CADASIL patients who do not present typical phenotypes may be underdiagnosed if not examined genetically.
Assuntos
CADASIL/genética , Predisposição Genética para Doença , Leucoencefalopatias/genética , Receptor Notch3/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , CADASIL/diagnóstico por imagem , CADASIL/fisiopatologia , Estudos de Coortes , Fator de Iniciação 2B em Eucariotos/genética , Testes Genéticos , Humanos , Leucoencefalopatias/diagnóstico por imagem , Leucoencefalopatias/fisiopatologia , Imageamento por Ressonância Magnética , Pessoa de Meia-Idade , Mutação , Fenótipo , RNA Polimerase III/genética , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Sequenciamento do ExomaRESUMO
Semaphorin III/collapsin-1 (Sema3A) guides a specific subset of neuronal growth cones as a repulsive molecule. In this study, we have investigated a possible role of non-neuronal Sema3A in lung morphogenesis. Expression of mRNAs of Sema3A and neuropilin-1 (NP-1), a Sema3A receptor, was detected in fetal and adult lungs. Sema3A-immunoreactive cells were found in airway and alveolar epithelial cells of the fetal and adult lungs. Immunoreactivity for NP-1 was seen in fetal and adult alveolar epithelial cells as well as endothelial cells. Immunoreactivity of collapsin response mediator protein CRMP (CRMP-2), an intracellular protein mediating Sema3A signaling, was localized in alveolar epithelial cells, nerve tissue and airway neuroendocrine cells. The expression of CRMP-2 increased during the fetal, neonate and adult periods, and this pattern paralleled that of NP-1. In a two-day culture of lung explants from fetal mouse lung (E11.5), with exogenous Sema3A at a dose comparable to that which induces growth cone collapse of dorsal root ganglia neurons, the number of terminal buds was reduced in a dose-dependent manner when compared with control or untreated lung explants. This decrease was not accompanied with any alteration of the bromodeoxyuridine-positive DNA-synthesizing fraction. A soluble NP-1 lacking the transmembrane and intracellular region, neutralized the inhibitory effect of Sema3A. The fetal lung explants from neuropilin-1 homozygous null mice grew normally in vitro regardless of Sema3A treatment. These results provide evidence that Sema3A inhibits branching morphogenesis in lung bud organ cultures via NP-1 as a receptor or a component of a possible multimeric Sema3A receptor complex.
Assuntos
Glicoproteínas/fisiologia , Pulmão/embriologia , Proteínas do Tecido Nervoso/fisiologia , Animais , Axônios/fisiologia , Western Blotting/métodos , Feminino , Glicoproteínas/genética , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Morfogênese , Proteínas do Tecido Nervoso/genética , Neuropilina-1 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Semaforina-3ARESUMO
We examined the roles of the PI3K-AKT signalling pathway in fetal lung development. By Western blotting, phosphorylated AKT (pAKT) was highly expressed in fetal days 12 and 14 with decreased expression thereafter. By immunohistochemistry, pAKT was expressed mainly in the respiratory epithelium of early fetal days. We examined the effects of fibroblast growth factor 1 (FGF1), PI3K inhibitors (LY294002 and wortmannin), MAPK inhibitor (PD98059) and both of FGF1 and each inhibitor on lung morphogenesis, BrdU incorporation and apoptosis. In the FGF1-treated explants, the number of terminal buds and BrdU-labelled cells increased significantly, while the LY294002-, wortmannin-, PD98059-treated explants demonstrated obvious decreases. The effects by FGF1 were inhibited by LY294002, wortmannin and PD98059. Regardless of the presence of FGF1, the LY294002-, wortmannin- and PD98059-treated explants increased apoptosis revealed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay in the mesenchyme of the explants. At the same time, the effect of LY294002, wortmannin, PD98059 on expression of surfactant apoprotein C (SPC) were also studied. The LY294002 and wortmannin treatments showed decreased expression of SPC. These findings suggest that the PI3K-AKT signalling pathway plays a pivotal role in mouse lung development through various biological processes.
Assuntos
Desenvolvimento Fetal/fisiologia , Pulmão/embriologia , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Transdução de Sinais , Androstadienos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular , Cromonas/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/biossíntese , Fator 1 de Crescimento de Fibroblastos/farmacologia , Flavonoides/farmacologia , Regulação da Expressão Gênica no Desenvolvimento , Marcação In Situ das Extremidades Cortadas , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos ICR , Morfolinas/farmacologia , Técnicas de Cultura de Órgãos , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt , WortmaninaRESUMO
The pulmonary tumorigenicity of dimethylarsinic acid (DMAA), a main metabolite of inorganic arsenics, was examined in A/J mice fed with drinking water containing DMAA for 25 and 50 weeks. Mice fed with 400 ppm DMAA for 50 weeks produced more pulmonary tumors than untreated mice (mean number per animal 1.36 versus 0.50; P < 0.05). Histological examination revealed that the number of mice which bore adenocarcinomas or papillary adenomas correlated with the concentration of DMAA given (untreated versus 400 ppm; P = 0.002), suggesting that DMAA could promote tumorigenic processes. These results are consistent with the epidemiological studies on the pulmonary carcinogenesis of arsenics and suggest that DMAA alone can act as a carcinogen in mice.
Assuntos
Ácido Cacodílico/toxicidade , Carcinógenos/toxicidade , Neoplasias Pulmonares/induzido quimicamente , Animais , Genes ras , Neoplasias Pulmonares/patologia , Masculino , CamundongosRESUMO
To clarify the process and mechanisms of the development and progression of peripheral lung adenocarcinoma, we investigated the relationships among the patterns of basement membrane (BM), stromal fibrosis, and the expressions of gelatinase A and tissue inhibitor of metalloproteinases-2 (TIMP-2) in 33 lesions of atypical alveolar cell hyperplasia (AAH) and 48 lesions of lung adenocarcinoma, including 24 lesions of bronchioloalveolar carcinoma (BAC). We found that the architecture of alveolar BM was intact in all 33 AAH lesions and 11 nonsclerosing BAC lesions that formed no central scar, suggesting that these lesions are early-stage intraepithelial neoplasia. The preexistent BM of the lung was disrupted, and the BM components around the neoplastic glands were disrupted or absent in the area of the central scar of some sclerosing BAC lesions with collapse fibrosis alone (2 of 4) and in those of all of the adenocarcinoma lesions associated with desmoplastic stromal fibrosis (nine sclerosing BAC and 24 non-BAC tumors). These results suggested that, in lung adenocarcinomas, destruction of the BM was correlated with the formation of a central scar, particularly with desmoplasia. It is likely that adenocarcinomas with a central scar are advanced and invasive cancers potentially having metastatic activity. The expression of gelatinase A and TIMP-2 was associated with central scar formation as well as with destruction of the BM components. Both the neoplastic and stromal cells expressed gelatinase A and TIMP-2 and probably play a role in tumor cell invasion.
Assuntos
Adenocarcinoma/patologia , Membrana Basal/patologia , Gelatinases/metabolismo , Neoplasias Pulmonares/patologia , Metaloendopeptidases/metabolismo , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Adenocarcinoma/enzimologia , Fibrose , Humanos , Hiperplasia , Neoplasias Pulmonares/enzimologia , Metaloproteinase 2 da Matriz , Células Estromais/patologiaRESUMO
Glucose uptake and metabolism are essential for proliferation and survival of cells, and are supposed to be enhanced in actively proliferating cell systems such as embryonic and cancer tissues. Glucose uptake is usually carried out through glucose transporters. In the developing fetal lung, metabolism of glucose is thought to be an important process in cell proliferation, differentiation and maturation. Active glucose uptake could result in accumulation of glycogen in epithelial cells, and utilization of glycogen could be a critical phenomenon for lung epithelial development. In hamsters, although facilitative glucose transporter isoform 1 (GLUT1) and isoform 4 (GLUT4) are not detected in adult lungs, expression of them is detected with immunohistochemical and Western blot analyses in the developing fetal lungs. In human lung carcinomas, GLUT1 expression is seen in most cases of lung carcinoma, and is seen especially frequently in squamous cell carcinoma. GLUT1 expression in adenocarcinoma of the lung is correlated with reduced cell differentiation, larger tumor size and positive lymph node metastasis. A few cases of lung carcinoma show positive staining for GLUT3 and GLUT4. Thus, expression of some facilitative glucose transporter isoforms is detected in developing fetal epithelium and in lung carcinomas. Overexpression of them could enhance uptake of glucose into these cells, and the increased influx of glucose could be involved in active cell proliferation, which is a common character of the developing lung epithelium and carcinoma.
Assuntos
Neoplasias Pulmonares/metabolismo , Pulmão/metabolismo , Proteínas de Transporte de Monossacarídeos/biossíntese , Animais , Cricetinae , Glicogênio/metabolismo , Humanos , Pulmão/embriologiaRESUMO
In order to reveal modulation of the number of pulmonary neuroendocrine cells (PNEC) in interstitial lung diseases and to clarify significance of cell proliferation activity in occurrence of PNEC, we counted airway PNEC of the patients of idiopathic interstitial pneumonia, secondary interstitial pneumonia and control lungs, and compared the number of PNEC with airway Ki-67 labeling. The lung tissue samples were obtained by video-assisted thoracoscopic surgery from 22 patients with usual interstitial pneumonia (UIP), 7 with non-specific interstitial pneumonia (NSIP), 8 with chronic hypersensitivity pneumonia (CHP), 13 with collagen vascular disease (CVD), and were compared with age-matched control lungs. The tissues were immunostained for chromogranin A and for Ki-67. Average incidence of bronchiolar PNEC in normal, UIP, NSIP, CHP, CVD lungs was 0.169%, 0.348%, 0.326%, 0.175% and 0.201%, respectively, and average Ki-67 labeling index in them was 0.241%, 1.186%, 1.605%, 1.058%, and 2.353%, respectively. And, in UIP lungs, PNEC incidence or Ki-67 labeling index was different according to pathological lesions. Thus, PNEC increase in the bronchiole of UIP, and the incidence of PNEC varies according to degree of activity of epithelial cell proliferation probably related to epithelial cell injury. Moreover, enhanced expression of human homolog of achaete-scute complex (hASH1) mRNA in UIP lungs suggests that hASH1 could play roles in the regulation of PNEC.
Assuntos
Doenças Pulmonares Intersticiais/patologia , Pulmão/metabolismo , Pulmão/patologia , Sistemas Neurossecretores/metabolismo , Idoso , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Northern Blotting , Brônquios/patologia , Diferenciação Celular/fisiologia , Cromogranina A , Cromograninas , Proteínas de Ligação a DNA/biossíntese , Feminino , Sequências Hélice-Alça-Hélice , Humanos , Imuno-Histoquímica , Antígeno Ki-67 , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genéticaRESUMO
In this brief review article, we describe how cell fate determination by which the airway epithelial cells become neuroendocrine or non-neuroendocrine is regulated by a network of basic helix-loop-helix transcription (bHLH) factors in a similar manner to neuronal differentiation, and how this system could work to determine cell differentiation of human lung carcinomas. Immunohistochemical studies reveal that mammalina achaete-scute complex homologue (Mash)1 is expressed in pulmonary neuroendocrine cells (PNEC), while hairy and Enhancer of split (Hes)1 is expressed in pulmonary non-neuroendocrine cells (non-PNEC). Studies using gene-deficient mice for the bHLH factors revealed that in Mash1 homozygous null mice no PNEC are detected, while PNEC increase markedly in Hes1 homozygous null mice. These observations suggest that Mash1 is an essential positive factor for neuroendocrine differentiation of lung epithelium, and that Hes1 is one of the repressive factors for neuroendocrine differentiation. Moreover, immunohistochemical studies revealed that Notch receptors are detected in non-PNEC, and thus the Notch signalling pathway could play a role in the determination of airway epithelial cell differentiation. In human lung carcinomas, a similar bHLH network should operate to determine cell differentiation phenotypes. Generally, expression of the human homologue of Mash1 (HASH1) is detected in small cell carcinoma and carcinoids, while Hes1 seems to be expressed mainly in non-small cell carcinoma. Thus, proneuronal bHLH factors may play roles in cell fate determination of the airway epithelial system, and may regulate human airway epithelial cells in diseased conditions.
Assuntos
Células Epiteliais/patologia , Sequências Hélice-Alça-Hélice/genética , Neoplasias Pulmonares/patologia , Pulmão/fisiologia , Neurônios/fisiologia , Sistemas Neurossecretores/fisiologia , Fatores de Transcrição/fisiologia , Animais , Diferenciação Celular/fisiologia , Humanos , Pulmão/citologia , Pulmão/embriologia , Pulmão/patologia , Neoplasias Pulmonares/genética , Neurônios/patologia , Sistemas Neurossecretores/citologia , Sistemas Neurossecretores/embriologia , Sistemas Neurossecretores/patologiaRESUMO
The expression of gap junction protein was examined immunohistochemically using affinity-purified antibody against rat liver gap junction protein, connexin 32 (Cx32), in the kidneys of fetal (gestation days 13-16) and adult Syrian golden hamsters. Phalloidin histochemical staining, PNA- and RCA I-lectin staining, NCAM immunostaining, and alkaline phosphatase and Na(+)-K(+)-ATPase enzyme-histochemical staining were performed in combination with Cx32 immunostaining. The kidney sections were observed with a confocal scanning laser microscope. By gestation day 13, Cx32 immunoreactivity was observed in the differentiating tubules. The Cx32 staining was localized on the lateral cell membrane of the cells lining the developing proximal tubules, while the S-shaped bodies, developing distal tubules, and collecting tubules showed no positive immunostaining. As the kidney developed, the density of Cx32 immunoreactivity increased. As the gap junction provides pathways for cell-cell communication, the development of Cx32 expression may imply that this structure plays an important role in renal tubule development. Confocal scanning laser microscopy provided a clear image of the fluorescence-labeled cell structures, free from out-of-focus blur. Using the same sections, stereoscopic images were easily reconstructed from serial optical sections, and were helpful in understanding the spatial distribution of Cx32 expression in the developing fetal proximal tubules.
Assuntos
Conexinas/metabolismo , Desenvolvimento Embrionário e Fetal , Junções Comunicantes/metabolismo , Rim/metabolismo , Mesocricetus/embriologia , Animais , Cricetinae , Histocitoquímica , Imuno-Histoquímica , Rim/embriologia , Mesocricetus/metabolismo , Ratos , Proteína beta-1 de Junções ComunicantesRESUMO
Excessive stimulation of insulin secretion may be one cause of the beta-cell dysfunction induced by hyperglycemia. We investigated a possible link between the prior endogenous hypersecretion of insulin and this dysfunction by performing a 7-day glucose infusion (50% wt/vol, 1.2 ml/h) on ventromedial hypothalamic VMH-lesioned hyperinsulinemic rats. Intravenous glucose tolerance tests (i.v.GTT 1.0 g/kg) revealed that a 3-day glucose infusion enhanced the insulin responses in both the sham- and VMH-lesioned rats compared with saline infusions. A similar 7-day glucose infusion enhanced the insulin response to glucose in sham-lesioned rats but not in VMH-lesioned rats. Batch-incubation of islets isolated from sham-lesioned rats showed an enhanced insulin response to glucose after 7 days of glucose treatment compared with the saline infusions. Conversely, the glucose infusion in VMH-lesioned rats markedly suppressed the in vitro insulin response. In sham- and VMH-lesioned rats, similar islet insulin contents were produced by saline and glucose treatments. Electron microscopy revealed that glucose infusions impaired the granule-releasing function of the beta-cells in VMH-lesioned rats, while insulin synthesis was accelerated in either group. These findings support the notion that excessive secretion is partly responsible for the beta-cell dysfunction induced by hyperglycemia without signs of exhaustion.
Assuntos
Glucose/farmacologia , Hipotálamo Médio/fisiopatologia , Insulina/metabolismo , Obesidade/fisiopatologia , Animais , Glicemia/análise , Feminino , Teste de Tolerância a Glucose , Técnicas In Vitro , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/ultraestrutura , Microscopia Eletrônica , Obesidade/patologia , Ratos , Ratos Sprague-DawleyRESUMO
The subcellular localization of five isoforms of facilitated-diffusion glucose transporters (GLUTs), from GLUT1 to GLUT5, in rat pancreatic islets was studied by immunohistochemistry using rabbit polyclonal antisera against mouse or rat GLUT peptides. Animals were perfusion-fixed with phosphate-buffered 4% paraformaldehyde and the pancreases were removed. Some specimens were embedded in paraffin, serially sectioned, and immunostained for glucagon, insulin, somatostatin, and the GLUTs for light microscopic observation. Others were prepared for immunoelectron microscopy by the post-embedding method. By these methods, GLUT2 immunostaining was observed on the lateral membranes of pancreatic beta-cells, whereas GLUT3 immunoreaction was predominantly localized in the cytoplasm of beta-cells and was not found in alpha-cells. In contrast, GLUT5 immunostaining was preferentially localized in the cytoplasm of alpha-cells compared to that of beta-cells. However, GLUT1 and GLUT4 were either barely or not at all detectable in any cells. These results suggest that rat islets take up glucose by at least three different processes and that blood glucose levels could be modulated differentially by: a high Km glucose transporter, GLUT2, in beta-cells; by a low Km glucose transporter, GLUT3, in beta-cells; and by a low Km glucose transporter, GLUT5, in alpha-cells.
Assuntos
Ilhotas Pancreáticas/química , Proteínas de Transporte de Monossacarídeos/análise , Proteínas Musculares , Proteínas do Tecido Nervoso , Sequência de Aminoácidos , Animais , Glucagon/análise , Transportador de Glucose Tipo 1 , Transportador de Glucose Tipo 2 , Transportador de Glucose Tipo 3 , Transportador de Glucose Tipo 4 , Transportador de Glucose Tipo 5 , Imuno-Histoquímica , Insulina/análise , Ilhotas Pancreáticas/ultraestrutura , Masculino , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Ratos , Ratos Sprague-Dawley , Organismos Livres de Patógenos EspecíficosRESUMO
We report a fatal case of death due to unusual aspiration of sardine fry in an elderly Japanese man with lung cancer. The cause of death was sudden respiratory arrest while eating. Autopsy revealed peculiar materials with cell nests and pigmented particles, together with striated muscle and skin, in the ectatic bronchioles of the left lower lobe. Serial histologic sections suggested that the structures observed were the eyeballs of small animals that appeared to have been inhaled. The patient had habitually eaten sardine fry and rice gruel, which were also detected in the gastric contents. Therefore, the eyes were considered to be those of the fry, which is a popular food item in Japan. This was confirmed by histologic examination of fry that were obtained commercially.
Assuntos
Produtos Pesqueiros , Corpos Estranhos/complicações , Neoplasias Pulmonares/complicações , Pneumonia Aspirativa , Idoso , Causas de Morte , Evolução Fatal , Medicina Legal , Humanos , Larva , MasculinoAssuntos
Hepatite/etiologia , Sífilis/complicações , Dor Abdominal/etiologia , Doença Aguda , Adulto , Colangite/etiologia , Humanos , Masculino , RecidivaRESUMO
In order to ascertain that alpha-subunit of guanine nucleotide-binding protein Go (Go alpha)-positive cells in the lung epithelia are pulmonary neuroendocrine cells (PNECs), we carried out an immunohistochemical study in young adult and fetal lungs of rodents and in cultured fetal lung explants. Serial sections showed that Go alpha-positive cells were immunostained for calcitonin gene-related peptide and serotonin in young adult mouse, rat, and hamster lungs and that these cells are, therefore, PNECs. In the fetal lungs of hamster and mouse, Go alpha-positive PNECs appeared in the epithelium of the lobar bronchus by gestational day 13 in hamster and by day 15.5 in mouse, and they increased with a proximal-to-distal wave during the late fetal period. Explants of immature lung from the fetal hamster on gestational day 11 were cultured. After 2 days of culture, Go alpha-positive PNEC clusters appeared in the main and lobar bronchi and many PNEC clusters were seen after 4 days of culture. To determine the functional significance of Go in the development of the fetal lung, pertussis toxin, a Go inhibitor, was added to the medium, and changes in branching morphogenesis and PNEC development were studied. Although branching morphogenesis was not disturbed by pertussis toxin, the toxin treatment induced large PNEC clusters in the cultured lung explant. In summary, we showed that Go alpha is a neuroendocrine marker for PNECs and that Go alpha-positive cells appear along with development of PNECs in fetal hamster lung in vivo and in vitro. The functional significance of Go in the development of fetal lung is obscure, but signals mediated through this GTP-binding protein could be related to some functions of PNECs.
Assuntos
Proteínas de Ligação ao GTP/análise , Pulmão/química , Animais , Cricetinae , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP , Pulmão/citologia , Pulmão/embriologia , Mesocricetus , Camundongos , Camundongos Endogâmicos A , Mucosa/química , Mucosa/citologia , Mucosa/embriologia , Sistemas Neurossecretores/química , Sistemas Neurossecretores/citologia , Sistemas Neurossecretores/embriologia , Coelhos , Ratos , Ratos Sprague-DawleyRESUMO
To elucidate the role of p53 and apoptosis in the pathogenesis of lung injury, we examined histological changes, expressions of p53 and p21waf1/cip1 (p21), apoptosis, DNA double strand breaks, cell kinetics, and DNA synthesis in C57/BL6 mice (p53+/+) and mice deficient for p53 (p53-/-) at 2 hours to 7 days after a single intravenous administration of bleomycin. We also compared these parameters between the lung cells and small intestinal epithelial cells to explore potential differences in their response to DNA damage. Bleomycin induced p21 expression in a p53-dependent manner in p53+/+ mice but neither p53 nor p21 expression in p53-/- mice. In the lung of both groups of mice, focal inflammation followed by fibrosis was observed, but there was no evidence of apoptosis. Cells with DNA breaks and those undergoing DNA synthesis were unequivocally increased, but the cycling cell fraction remained unchanged, suggesting that the DNA synthesis detected in the lung reflected unscheduled DNA synthesis for repair of damaged DNA. DNA breaks and unscheduled DNA synthesis were prolonged in p53-/- mice compared to p53+/+ mice. By contrast, in the small intestine, marked cell cycle arrest and extensive apoptosis were evoked in the cycling crypt cells of both groups of mice, but these changes were milder and DNA breaks remained detectable for a longer time in p53-/- mice than in p53+/+ mice. Among the resting enterocytes in the villi, apoptosis was observed almost equally in both groups, but repair of DNA breaks was significantly delayed in the p53-/- mice. These observations imply that apoptosis is mediated largely by the p53-dependent pathway in the crypts but exclusively by the p53-independent pathway in the villi, that this pathway is particularly important in DNA repair in the villi, and that despite this difference in the significance of apoptosis, p53 plays an important role in DNA repair in both the crypts and villi. Our results suggest that the lung cells and small intestinal cells respond to the bleomycin treatment in different ways in terms of the induction of apoptosis and that p53 carries out an essential role in the early response to and repair of DNA damage by a non-apoptotic mechanism which appears to be crucial in the noncycling lung cells and enterocytes. Importantly, the p53-p21 pathway and apoptosis are unlikely to be essential for bleomycin-induced tissue injury in the lung.
Assuntos
Bleomicina/toxicidade , Dano ao DNA , Intestino Delgado/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Proteína Supressora de Tumor p53/fisiologia , Animais , Apoptose , Western Blotting , Morte Celular , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/biossíntese , DNA/biossíntese , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Intestino Delgado/patologia , Antígeno Ki-67/metabolismo , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Organismos Livres de Patógenos Específicos , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genéticaRESUMO
Fetal hamster lung explant was cultured in serum-free medium on gestational Day 11-2 days before the appearance of pulmonary neuroendocrine cells (PNEC)--and the development and differentiation of PNEC from immature fetal lung epithelium was examined through immunostaining for neural cell adhesion molecule (NCAM) to establish an in vitro system to study the mechanisms involved. PNEC were present in the main bronchus after 2 days of culture. Thereafter, NCAM-positive clusters of PNEC increased and were distributed from the large bronchus to the terminal bronchiole with a proximal-to-distal wave. To elucidate the role of NCAM in the fetal development of PNEC, whole fetal lung was cultured on gestational Day 11 with an anti-NCAM antibody. This antibody slightly inhibited the growth and branching morphogenesis of the lung and disturbed the formation of PNEC clusters. NCAM may function to form clusters of PNEC known as neuroepithelial bodies. We cultured fetal lung epithelial explant at gestational Day 11 after removing mesenchyme, including nerve tissue, with dispase digestion. Immunohistochemical staining for NCAM revealed that PNEC were induced in cultured fetal epithelium without mesenchymal tissue, but basement membrane Matrigel was necessary to maintain cultured epithelium. In conclusion, PNEC derive from immature airway epithelial cells. This organ culture system, therefore, is a useful experimental model and should facilitate further investigations of the development and differentiation of PNEC. Mesenchymal and neural tissues are not always necessary for the development of PNEC, but matrix substance and/or growth factors may be required to induce or maintain PNEC.
Assuntos
Pulmão/citologia , Pulmão/embriologia , Sistemas Neurossecretores/citologia , Sistemas Neurossecretores/embriologia , Animais , Anticorpos/farmacologia , Diferenciação Celular/imunologia , Separação Celular , Cricetinae , Células Epiteliais/citologia , Células Epiteliais/imunologia , Feminino , Pulmão/imunologia , Masculino , Moléculas de Adesão de Célula Nervosa/imunologia , Sistemas Neurossecretores/imunologia , Técnicas de Cultura de ÓrgãosRESUMO
We have examined the distribution of calcium-binding proteins (CaBPs) in adult and fetal lungs of Syrian golden hamsters (Mesocricetus auratus) using immunostaining with confocal laser microscopy and electron microscopy. Single and grouped (neuroepithelial body; NEB) endocrine cells were distributed from bronchi to alveolar ducts in the adult lung. Serial frozen sections immunostained for CaBPs in combination with immunostaining for endocrine markers such as calcitonin gene-related peptide, serotonin, PGP9.5, and synaptophysin revealed that positive immunostaining for calbindin-D28K (CB-D28K) was seen in single endocrine cells and NEBs. However, other so-called EF-hand family CaBPs, parvalbumin and calretinin, were not detected. Electron microscopically, positive immunoreaction for CB-D28K was mainly in the organelle-free cytoplasmic matrix of endocrine cells, and partly in nuclei and associated with secretory granules and endoplasmic reticulum. In fetal developing lungs, endocrine cells appeared first on gestational day 13, and they were positive for all the endocrine markers used. However, pulmonary endocrine cells were positively immunostained for CB-D28K from gestational days 15 and 16 onward. In summary, our observations suggest that CB-D28K is a useful marker for endocrine cells of the lung, and CB-D28K could function as a mediator of endocrine stimulation or calcium homeostasis in pulmonary endocrine cells.
Assuntos
Sistema Endócrino/química , Pulmão/química , Proteína G de Ligação ao Cálcio S100/análise , Animais , Brônquios/química , Calbindinas , Cricetinae , Sistema Endócrino/citologia , Imuno-Histoquímica , Pulmão/citologia , Masculino , Mesocricetus , Microscopia Confocal , Microscopia Eletrônica , Alvéolos Pulmonares/químicaRESUMO
We immunostained mouse lung tumors using a mouse monoclonal antibody against recombinant Ki-67 antigen (clone; MIB 5) to establish an MIB 5 immunostaining method and to determine the extent of MIB 5 labeling to monitor cell proliferation activity in mouse lung tumors. A/J mice, treated with 4-nitroquinoline 1-oxide, were killed after 18 months. One hour before killing, bromodeoxyuridine (BrdU) was injected intraperitoneally. Lung tissues including tumors were fixed with phosphate-buffered 4% paraformaldehyde and embedded in paraffin. For MIB 5 immunostaining, two antigen-retrieval buffers, citrate buffer pH 6 and TRIS-HCl buffer pH 9.5 containing 5% urea, were tested, and constant and reproducible staining was obtained only with the TRIS-HCI buffer. The mean values of the MIB 5-positive cell index (PCI), the BrdU labeling index (LI), and the mitotic cell count for adenocarcinomas were 4.6%, 2.3%, and 7/mm2, and those for adenomas were 1.2%, 0.7%, and 1.3/mm2, respectively. Each of these values was significantly higher for adenocarcinomas than for adenomas. A close correlation was seen between the MIB 5 PCI and the BrdU LI for adenocarcinomas and adenomas and between the MIB 5 PCI and the mitotic cell count in adenocarcinomas. Thus, MIB 5 immunostaining is a useful method for assessing the proliferative activity of mouse tumor tissues.
Assuntos
Adenocarcinoma/metabolismo , Adenoma/metabolismo , Antígeno Ki-67/metabolismo , Neoplasias Pulmonares/metabolismo , 4-Nitroquinolina-1-Óxido/toxicidade , Adenocarcinoma/induzido quimicamente , Adenocarcinoma/patologia , Adenoma/induzido quimicamente , Adenoma/patologia , Animais , Anticorpos Monoclonais , Bromodesoxiuridina/metabolismo , Carcinógenos/toxicidade , Contagem de Células , Divisão Celular/efeitos dos fármacos , Técnicas Imunoenzimáticas , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos A , Proteínas Recombinantes/metabolismoRESUMO
To clarify the mechanisms that regulate neuroendocrine differentiation of fetal lung epithelia, we have studied the expression of the mammalian homologs of achaete-scute complex (Mash1) (Ascl1 - Mouse Genome Informatics); hairy and enhancer of split1 (Hes1); and the expression of Notch/Notch-ligand system in the fetal and adult mouse lungs, and in the lungs of Mash1- or Hes1-deficient mice. Immunohistochemical studies revealed that Mash1-positive cells seemed to belong to pulmonary neuroendocrine cells (PNEC) and their precursors. In mice deficient for Mash1, no PNEC were detected. Hes1-positive cells belong to non-neuroendocrine cells. In the mice deficient in Hes1, in which Mash1 mRNA was upregulated, PNEC appeared precociously, and the number of PNEC was markedly increased. NeuroD (Neurod1 - Mouse Genome Informatics) expression in the lung was detected in the adult, and was enhanced in the fetal lungs of Hes1-null mice. Expression of Notch1, Notch2, Notch3 and Notch4 mRNAs in the mouse lung increased with age, and Notch1 mRNA was expressed in a Hes1-dependent manner. Notch1, Notch2 and Notch3 were immunohistochemically detected in non-neuroendocrine cells. Moreover, analyses of the lungs from the gene-targeted mice suggested that expression of Delta-like 1 (Dll1 - Mouse Genome Informatics) mRNA depends on Mash1. Thus, the neuroendocrine differentiation depends on basic helix-loop-helix factors, and Notch/Notch-ligand pathways may be involved in determining the cell differentiation fate in fetal airway epithelium.
Assuntos
Diferenciação Celular , Proteínas de Drosophila , Células Epiteliais/citologia , Sequências Hélice-Alça-Hélice , Pulmão/citologia , Pulmão/embriologia , Fatores de Transcrição/metabolismo , Envelhecimento/genética , Envelhecimento/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Constituição Corporal , Contagem de Células , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/metabolismo , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Imuno-Histoquímica , Ligantes , Pulmão/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Notch , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de Transcrição HES-1 , Fatores de Transcrição/deficiência , Fatores de Transcrição/genéticaRESUMO
We examined the hypothesis that superoxide mediates infiltration of neutrophils to the airways through nuclear factor (NF)-kappaB and interleukin-8 (IL-8) after acute exposure to cigarette smoke (CS) in vivo. Male Hartley strain guinea pigs were exposed to air or 20 puffs of CS and killed 5 h after the exposure. The differential cell count of bronchoalveolar lavage fluid and specific myeloperoxidase enzyme assay demonstrated that acute exposure to CS caused neutrophil accumulation to the airways and parenchyma, respectively. Acute exposure to CS increased DNA-binding activity of NF-kappaB in the lung. Acute exposure to CS also increased IL-8 messenger RNA (mRNA) expression in the lung. Pretreatment of guinea pigs with recombinant human superoxide dismutase (rhSOD) aerosols reduced the CS-induced neutrophil accumulation to the airways. Both activation of NF-kappaB and increased IL-8 mRNA expression were also inhibited by the pretreatment of rhSOD aerosols. Strong immunoreactivities for p65 and p50 were detected in the nuclei of alveolar macrophages after acute exposure to CS. The signal for IL-8 mRNA expression was demonstrated in the alveolar space after acute exposure to CS. Neither significant immunoreactivities for p65 and p50 nor IL-8 mRNA signals were observed in airway epithelium. These observations suggest that acute exposure to CS initiates superoxide-dependent mechanism that, through NF-kappaB activation and IL-8 mRNA expression, produces infiltration of neutrophils to the airways in vivo. It was also suggested that the alveolar macrophage is one potential source of NF-kappaB activation and IL-8 mRNA expression after acute exposure to CS.