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Anticuerpos Monoclonales/efectos adversos , Antineoplásicos/efectos adversos , Neoplasias Encefálicas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Índice de Severidad de la Enfermedad , Anciano , Anciano de 80 o más Años , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/secundario , Carcinoma de Pulmón de Células no Pequeñas/inducido químicamente , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Femenino , Humanos , Neoplasias Pulmonares/inducido químicamente , Neoplasias Pulmonares/diagnóstico , Masculino , Persona de Mediana Edad , NivolumabRESUMEN
BACKGROUND: Neutropenia is a common adverse reaction of chemotherapy. We assessed whether chemotherapy-induced neutropenia could be a predictor of survival for patients with non-small-cell lung cancer (NSCLC). METHODS: A total of 387 chemotherapy-naïve patients who received chemotherapy (vinorelbine and gemcitabine followed by docetaxel, or paclitaxel and carboplatin) in a randomised controlled trial were evaluated. The proportional-hazards regression model was used to examine the effects of chemotherapy-induced neutropenia and tumour response on overall survival. Landmark analysis was used to lessen the bias of more severe neutropenia resulting from more treatment cycles allowed by longer survival, whereby patients who died within 126 days of starting chemotherapy were excluded. RESULTS: The adjusted hazard ratios for patients with grade-1 to 2 neutropenia or grade-3 to 4 neutropenia compared with no neutropenia were 0.59 (95% confidence interval (CI), 0.36-0.97) and 0.71 (95% CI, 0.49-1.03), respectively. The hazard ratios did not differ significantly between the patients who developed neutropenia with stable disease (SD), and those who lacked neutropenia with partial response (PR). CONCLUSION: Chemotherapy-induced neutropenia is a predictor of better survival for patients with advanced NSCLC. Prospective randomised trials of early-dose increases guided by chemotherapy-induced toxicities are warranted.
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Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Neutropenia/inducido químicamente , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Femenino , Factor Estimulante de Colonias de Granulocitos/uso terapéutico , Humanos , Incidencia , Neoplasias Pulmonares/mortalidad , Masculino , Persona de Mediana Edad , Estudios Multicéntricos como Asunto , Neutropenia/epidemiología , Pronóstico , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
Asthma is characterised by chronic inflammation of the airways, but the relevance of high-sensitivity assays for C-reactive protein (hs-CRP), which are known to be a sensitive marker of low-grade systemic inflammation, has not been fully studied in asthma. The objective was to examine serum hs-CRP levels in patients with asthma and their relationship to clinical characteristics and degree of airway inflammation. Serum hs-CRP levels were cross-sectionally examined in steroid-naive (n = 22) and steroid-inhaling (n = 23) adult patients with asthma and healthy controls (n = 14). All were nonsmokers. Serum hs-CRP levels were significantly increased in steroid-naive patients (mean+/-sd 1.33+/-1.48 mg.L(-1)) compared with controls (0.21+/-0.30 mg.L(-1)), but not in patients on inhaled corticosteroid. Among steroid-naive patients, serum hs-CRP levels significantly negatively correlated with indices of pulmonary function (forced expiratory volume in one second/forced vital capacity and forced mid-expiratory flow) and positively with sputum eosinophil count. Among patients on inhaled corticosteroid, hs-CRP levels did not correlate with any indices. In conclusion, an increase in serum C-reactive protein levels measured by high-sensitivity assays may be associated with airflow obstruction and airway inflammation, and may serve as a surrogate marker of airway inflammation in asthma.
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Asma/sangre , Asma/fisiopatología , Proteína C-Reactiva/análisis , Adulto , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: The balance between matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) may be critical in extracellular matrix remodelling, a characteristic of asthmatic airways. An excess of TIMP-1 over MMP-9 has been associated with chronic airflow obstruction but the mechanisms underlying this association remain unknown. Recent computed tomographic (CT) studies indicate that airway wall thickening is associated with chronic airflow obstruction. METHODS: Sputum levels of MMP-9, TIMP-1, and their molar ratio were examined in 26 patients with stable asthma and their relationship with pulmonary function and airway wall thickness, assessed by a validated CT technique which measured wall area corrected by body surface area (WA/BSA), the ratio of WA to outer wall area (WA%), and the absolute wall thickness corrected by radicalBSA of a segmental bronchus (T/ radicalBSA), was examined. RESULTS: Sputum MMP-9 levels were inversely correlated with WA% and TIMP-1 levels were positively correlated with WA/BSA and T/ radicalBSA. The MMP-9/TIMP-1 molar ratio was inversely correlated with WA% and T/ radicalBSA and positively correlated with post-bronchodilator values of mid-forced expiratory flow and maximum expiratory flow at the quartile of lung volume. CONCLUSION: Excess TIMP-1 may have a pathogenetic role in airway wall thickening in asthmatic patients which may result in chronic airflow obstruction.
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Asma/patología , Bronquios/patología , Metaloproteinasa 9 de la Matriz/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Asma/metabolismo , Femenino , Volumen Espiratorio Forzado/fisiología , Humanos , Masculino , Persona de Mediana Edad , Esputo/metabolismo , Tomografía Computarizada por Rayos X/métodos , Capacidad Vital/fisiologíaRESUMEN
Radiation exposure is known to impair healing in irradiated areas. Fibroblasts play a major role in the production and modification of extracellular matrix in wound repair. Since one important aspect of wound repair is the contraction of the wound, this study investigated the effects of radiation on the ability of fibroblasts to mediate collagen gel contraction in an in vitro model of wound retraction. After irradiation, the cells were detached and suspended in a solution of rat tail tendon collagen. Radiation exposure decreased retraction, and this effect was dose dependent. In order to define the mechanism of reduced gel retraction, we investigated alpha2beta1 cell surface integrin and fibronectin, which are thought to mediate contraction, and prostaglandin E2 (PGE2), which is known to inhibit this process. PGE2 release increased dose responsively following radiation. The cyclooxygenase inhibitor indomethacin could partially restore the contractile activity of irradiated fibroblasts. Fibronectin production in gel culture showed a significant decrease. In contrast, there was no decrease in alpha2beta1 integrin expression in radiated cells. In conclusion, radiation decreases fibroblast-mediated gel contraction. Increased PGE2 production and decreased fibronectin production by irradiated fibroblasts may contribute to this effect and may be in part responsible for poor healing of radiated tissue.
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Colágeno Tipo I/metabolismo , Fibroblastos/efectos de la radiación , Rayos gamma , Animales , Dinoprostona/metabolismo , Femenino , Fibroblastos/metabolismo , Fibroblastos/fisiología , Fibronectinas/metabolismo , Humanos , Integrina alfa2beta1/metabolismo , Embarazo , Ratas , Factores de Tiempo , Cicatrización de HeridasRESUMEN
BACKGROUND: Imbalance between proteinases and their inhibitors released from alveolar type II pneumocytes may cause development of inflammatory lung diseases. OBJECTIVES AND METHODS: We examined mRNA expressions of matrix metalloproteinase-3 (MMP-3) and tissue inhibitor of metalloproteinase-3 (TIMP-3) in a cell line (A549) and in primary culture of normal adult human type II pneumocytes using reverse transcription-competitive polymerase chain reaction. RESULTS: Interleukin-1beta (IL-1beta) and transforming growth factor-beta1 (TGF-beta1) increased MMP-3 and TIMP-3 expressions in A549 cells in a time- and concentration-dependent manner. IL-1beta mainly augmented MMP-3 expression, while TGF-beta1 mainly augmented TIMP-3 expression. Dexamethasone attenuated both IL-1beta- and TGF-beta1-stimulated expressions of MMP-3 and TIMP-3. Interleukin-10 had no significant effect. Hepatocyte growth factor alone had no effect on constitutive MMP-3 expression or TIMP-3 expression, but it augmented TGF-beta1-stimulated MMP-3 expression. The constitutive expressions were higher in normal type II pneumocytes than in A549 cells, but the regulations were similar. CONCLUSIONS: These data indicated that the matrix degradation is enhanced by IL-1beta and suppressed by TGF-beta1 via regulations in the balance between MMP-3 and TIMP-3. Further, these regulations were shown to be modulated by glucocorticoids and growth factors.
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Citocinas/farmacología , Citoplasma/efectos de los fármacos , Citoplasma/fisiología , Pulmón/citología , Metaloproteinasa 3 de la Matriz/efectos de los fármacos , Metaloproteinasa 3 de la Matriz/genética , ARN Mensajero/efectos de los fármacos , ARN Mensajero/fisiología , Inhibidor Tisular de Metaloproteinasa-3/efectos de los fármacos , Inhibidor Tisular de Metaloproteinasa-3/fisiología , Adulto , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Interleucina-1/farmacología , Pulmón/química , Pulmón/enzimología , Inhibidores de Proteasas/farmacología , Valores de Referencia , Factores de Tiempo , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
BACKGROUND AND AIM OF THE WORK: Lipocortin-1 (also known as annexin-1) is upregulated by corticosteroids and plays a prominent part in many of their anti-inflammatory actions. In our previou investigation, lipocortin-1 gene expression in blood monocytes was higher in sarcoidosis patients tha in healthy subjects or IPF patients. In this study, we examined the amounts of lipocortin-1 gene tran scripts [at baseline and after stimulation by synthetic adrenocorticotrophic hormone (ACTH)] in the blood monocytes of sarcoidosis patients, and then we analyzed the relationships between these amount and several clinical indexes. METHODS: Serum and blood monocyte samples were prepared from 21 sar coidosis patients before and after ACTH stimulation. The amounts of lipocortin-1 and beta-actin gene tran scripts obtained from monocytes were quantified by RT-PCR. The patients were prospectively followe for at least 2.5 years. RESULTS: 1. Serum cortisol was increased in all patients after synthetic ACTH in jection. 2. Lipocortin-1 gene transcripts tended to increase in parallel with increases in serum cortisol but the relationship was not significant. 3. The amounts of lipocortin-1 gene transcripts induced by syn thetic ACTH were related to two indexes of disease activity/extension: the presence of parenchymal lesions (p = 0.018), and the presence of extrathoracic lesions (p = 0.043). 4. After 3 years of follow-up the patients with higher basal amounts of lipocortin-1 gene transcripts in monocytes showed improve ment of sACE activity and FEV1/FVC%. CONCLUSION: The amounts of lipocortin-1 gene transcripts induced by synthetic ACTH were related to the presence of parenchymal lesions, while their basa amounts correlated with either improvement of sACE activity or stabilization of FEV1/FVC%.
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Anexina A1/biosíntesis , Monocitos/metabolismo , Sarcoidosis Pulmonar/sangre , Hormona Adrenocorticotrópica , Adulto , Anexina A1/genética , Femenino , Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
It is important to identify in advance patients who will achieve the greatest functional gains from rehabilitation therapy, as specialist rehabilitation resources are still scarce in Japan. The purpose of this study was to determine whether functional score at admission influences the functional change (functional score at discharge minus functional score at admission) after inpatient rehabilitation for first cerebral hemorrhage. One hundred and ninety-three patients with cerebral hemorrhage were enrolled in this study. They were assessed using the Functional Independence Measure (FIM) at admission and discharge and underwent inpatient rehabilitation treatment. Patients were stratified into 3 groups according to their FIM total scores on admission as follows: (1) < or = 36 (severely affected patient group); (2) 37-72 (moderately affected patient group); and (3) >73 (mildly affected patient group). Scheffe's multiple comparison test showed that patients in group 1 were significantly older (mean +/- SD = 63 +/- 10 years) than those in groups 2 (56 +/- 10 years) or 3 (53 +/- 12 years). Patients in group 2 showed significantly greater FIM gain (37 +/- 17) compared with patients in groups 3 (23 +/- 12) or 1 (27 +/- 23). The results suggest that moderately affected patients at admission will show significantly higher functional gain compared with severely or mildly affected patients. Mildly affected patients at admission had a significantly shorter length of hospital stay for rehabilitation than the other groups. There was no significant difference in onset to admission interval between the 3 groups. The functional levels of affected patients on admission, as stratified by the FIM scale, roughly predict the degree of functional gain following rehabilitation in patients with first cerebral hemorrhage. Moderately affected patients will benefit from intensive rehabilitation. This study may be useful in determining how best to prioritize rehabilitation therapy.
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Hemorragia Cerebral/rehabilitación , Rehabilitación de Accidente Cerebrovascular , Adulto , Anciano , Femenino , Indicadores de Salud , Humanos , Japón , Tiempo de Internación , Masculino , Persona de Mediana Edad , Estudios RetrospectivosRESUMEN
The oxidative decomposition of skin lipids by UV exposure and its cell toxicity were studied in vivo and in vitro, using guinea pigs, to investigate the role of oxidative damage in cell membranes and mitochondria in nuclear genome DNA damage resulting in skin cell death by ultraviolet (UV) exposure. Two new methods were developed for this research: selective methylation by trimethylsilyldiazomethane (TMSCHN2) of free fatty acids in crude skin lipids for gas chromatography-mass spectrometry (GC-MS) analysis, and the improvement of the thiobarbituric acid (TBA) reaction for lipid peroxide analysis, which was obtained by adding 0.01% BHT and 1 mM EDTA, an antioxidant additive, into the reaction system described by Ohkawa. The following findings were noted: 1. Using an optical microscope, the infiltration of inflammatory cells such as neutrophils, lymphocytes and monocytes was noted after skin tissue was exposed to UV. 2. The increase of lipid peroxide in exposed areas was small. In the epidermis the increase was about 2 times higher than the non-exposed areas. 3. The fragments of lipid were generated in accordance with the increase of free fatty acids (C16:0, C18:1,:2, C18:0) in the surface of the exposed skin by GC-MS precise assay, and the level of 7-dehydrocholesterol was decreased. 4. The skin homogenate received peroxidation by ultraviolet more easily than living skin, and its peroxidation was inhibited with fat-soluble antioxidative agents such as flavonoids, BHT, BHA, and vitamin E and the metal chelating agent such as Fenton reaction inhibitor as expected; however, it was promoted by water soluble antioxidative agents such as glutathione and vitamin C, which are useful to the human body. 5. Steroids and nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin and aspirin, had no inhibitory effect on lipid peroxidation by ultraviolet as properties of chemicals.
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Peróxidos Lipídicos/metabolismo , Piel/metabolismo , Rayos Ultravioleta/efectos adversos , Animales , Antioxidantes/farmacología , Apoptosis/efectos de la radiación , Ácidos Grasos no Esterificados/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Cobayas , Técnicas In Vitro , Masculino , Piel/citologíaRESUMEN
Injury of the alveolar epithelium by cigarette smoke is presumed to be an important process in the pathogenesis of smoking-related pulmonary diseases. We investigated the cytotoxic effects of cigarette smoke extract (CSE) on an alveolar type II cell-derived cell line (A549). CSE caused apoptosis at concentrations of 5% or less and necrosis at 10% or more. When CSE was exposed to air before application to A549 cells, the cytotoxic effects were attenuated. CSE caused cell death without direct contact with the cells. Acrolein and hydrogen peroxide, two major volatile factors in cigarette smoke, caused cell death in a similar manner. Aldehyde dehydrogenase, a scavenger of aldehydes, and N-acetylcysteine, a scavenger of oxidants and aldehydes, completely inhibited CSE-induced apoptosis. CSE and acrolein increased intracellular oxidant activity. In conclusion, apoptosis of alveolar epithelial cells may be one of the mechanisms of lung injury induced by cigarette smoking. This cytotoxic effect might be due to an interaction between aldehydes and oxidants present in CSE or formed in CSE-exposed cells.
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Nicotiana , Plantas Tóxicas , Alveolos Pulmonares/fisiología , Humo , Aldehídos/farmacología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Humanos , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/metabolismo , Oxidantes/farmacología , Oxidación-Reducción , Alveolos Pulmonares/efectos de los fármacos , Alveolos Pulmonares/metabolismo , Alveolos Pulmonares/patologíaRESUMEN
Bradykinin is a multifunctional mediator of inflammation believed to have a role in asthma, a disorder associated with remodeling of extracellular connective tissue. Using contraction of collagen gels as an in vitro model of wound contraction, we assessed the effects of bradykinin tissue on remodeling. Human fetal lung fibroblasts were embedded in type I collagen gels and cultured for 5 days. After release, the floating gels were cultured in the presence of bradykinin. Bradykinin significantly stimulated contraction in a concentration- and time-dependent manner. Coincubation with phosphoramidon augmented the effect of 10(-9) and 10(-8) M bradykinin. A B2 receptor antagonist attenuated the effect of bradykinin, whereas a B1 receptor antagonist had no effect, suggesting that the effect is mediated by the B2 receptor. An inhibitor of intracellular Ca2+ mobilization abolished the response; addition of EGTA to the culture medium attenuated the contraction of control gels but did not modulate the response to bradykinin. In contrast, the phospholipase C inhibitor U-73122 and the protein kinase C inhibitors staurosporine and GF-109203X attenuated the responses. These data suggest that by augmenting the contractility of fibroblasts, bradykinin may have an important role in remodeling of extracellular matrix that may result in tissue dysfunction in chronic inflammatory diseases, such as asthma.
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Bradiquinina/farmacología , Colágeno/efectos de los fármacos , Colágeno/fisiología , Fibroblastos/fisiología , Animales , Bradiquinina/antagonistas & inhibidores , Células Cultivadas , Geles , Humanos , Concentración Osmolar , Inhibidores de Proteasas/farmacología , Ratas , Transducción de Señal/fisiología , Factores de TiempoRESUMEN
Like bacteria and many fungi, the pathogenic fungus Candida albicans can utilize GlcNAc as a carbon source for growth. A cluster of six genes was identified in the C. albicans genome. One of the genes in the cluster was CaNAG1, which is responsible for GlcN6P deaminase and is therefore essential for GlcNAc-dependent growth. The other five genes were designated CaNAG2, CaNAG3, CaNAG4, CaNAG5 and CaNAG6. The mRNA levels of CaNAG1, CaNAG2 and CaNAG5 were significantly induced by GlcNAc, whereas those of CaNAG3, CaNAG4 and CaNAG6 were not. Neither CaNAG2 nor CaNAG5 was essential for growth, but disruption of CaNAG2 or CaNAG5 greatly retarded the growth of cells using GlcNAc as the sole carbon source. Although no homolog of CaNAG2 or CaNAG5 was found in the Saccharomyces cerevisiae genome, CaNag2p displayed sequence similarities to Escherichia coli nagA, and CaNag5p is homologous to a wide variety of hexose kinases. When expressed as a fusion protein with glutathione S-transferase (GST), CaNag5p produced GlcNAc-P from GlcNAc in the presence of ATP, whereas GST alone did not. Furthermore, the recombinant GST-CaNag2p fusion protein converted GlcNAcP, which was produced by CaNag5p, into GlcNP. These results clearly demonstrate that CaNAG2 and CaNAG5 encode GlcNAcP deacetylase and GlcNAc kinase, respectively. CaNag5p recognized glucose and mannose as substrates, whereas the recently identified human GlcNAc kinase was specific to GlcNAc. Deletion of CaNAG2 or CaNAG5 markedly, and that of CaNAG1 moderately, attenuated the virulence of C. albicans in a mouse systemic infection model. Thus, it appears that GlcNAc metabolism of C. albicans is closely associated with its virulence.
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Amidohidrolasas/genética , Candida albicans/enzimología , Candida albicans/genética , Proteínas Fúngicas , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Animales , Candida albicans/patogenicidad , Carbono/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/metabolismo , Eliminación de Gen , Glutatión Transferasa/metabolismo , Masculino , Proteínas de la Membrana/genética , Ratones , Modelos Genéticos , Datos de Secuencia Molecular , Familia de Multigenes , Mutagénesis Sitio-Dirigida , Plásmidos , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Especificidad por Sustrato , Factores de TiempoRESUMEN
OBJECTIVE: To determine whether the admission functional score influences the functional change after stroke rehabilitation. DESIGN: Two hundred forty-three patients who had received the Functional Independence Measure (FIM) assessment at admission and at discharge were enrolled in the study. The patients were stratified into three groups according to their FIM total scores at admission, i.e., < or =36, 37 to 72, and > or =73. RESULTS: The Scheffé's multiple comparison test showed that patients with FIM total scores of > or =73 at admission were significantly younger (58 +/- 11 [SD] yr) than those who had scores of 37 to 72 (64 +/- 11 yr) or < or =36 (66 +/- 12 yr). Patients with FIM total scores of 37 to 72 at admission showed significantly higher FIM gain (37 +/- 15) compared with those patients who had scores of > or =73 (20 +/- 10) or < or =36 (29 +/- 23). CONCLUSION: The functional levels of affected patients at admission stratified by the FIM scale roughly predict the degree of functional gain after rehabilitation in survivors with a first episode of ischemic stroke. Moderately affected patients will benefit from intensive rehabilitation. These findings may be useful for rehabilitation triage.
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Indicadores de Salud , Rehabilitación de Accidente Cerebrovascular , Anciano , Femenino , Hospitalización , Humanos , Masculino , Persona de Mediana Edad , PronósticoRESUMEN
OBJECTIVES: To reveal increased lipid peroxidation in diabetics by quantification of cholesterol oxidation products (COPs) not only in plasma, but also in erythrocytes. DESIGN AND METHODS: We quantified 7-ketocholesterol (7-kCho) by gas chromatography-mass spectrometry as a surrogate measure for COPs. These assays were performed on both plasma and erythrocytes in 20 control subjects and 20 treated patients with relatively poorly controlled Type 2 diabetes. RESULTS: Both plasma and erythrocyte 7-kCho levels in diabetics were significantly higher than those in control subjects. Although neither plasma nor erythrocyte 7-kCho levels were associated with markers for glucose tolerance in diabetics, a negative correlation of serum HDL-cholesterol levels with erythrocyte, but not plasma, 7-kCho levels was found. CONCLUSION: Increased oxidative stress in diabetics affects oxidation of cholesterol. Assays of COPs not only in plasma, but also in erythrocytes, may yield complementary information in lipid peroxidation.
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Diabetes Mellitus Tipo 2/sangre , Eritrocitos/metabolismo , Cetocolesteroles/sangre , Estrés Oxidativo , Plasma/metabolismo , Anciano , Calibración , Estudios de Casos y Controles , Femenino , Cromatografía de Gases y Espectrometría de Masas , Humanos , Masculino , Persona de Mediana Edad , Oxígeno/metabolismo , Factores de Riesgo , Factores Sexuales , Factores de TiempoRESUMEN
The aim of this study was to compare the function of lung fibroblasts obtained from surgically biopsied specimens of patients with idiopathic pulmonary fibrosis/usual interstitial pneumonia (UIP; n = 5), nonspecific interstitial pneumonia (NSIP; n = 5), and normal parts of surgically resected lungs (control; n = 5). The results showed that (1) fibroblasts obtained from UIP showed increased contractility compared with those obtained from NSIP or controls (UIP, 72.7 +/- 6.21%; NSIP, 32.8 +/- 5.46; controls, 28.5 +/- 3.51, p < 0.01 in UIP versus NSIP or control); (2) this increase in contractility was consistent with enhanced F-actin content in fibroblasts; (3) conditioned media from UIP fibroblast cultures enhanced control fibroblast contractility, whereas those obtained from NSIP or controls did not; (4) the 180 and 25 kD products representing the contractility in conditioned media were identified as fibronectin (ED-A domain) and TGF-beta1 by immunoblots, respectively; (5) the UIP-conditioned media contained higher amounts of fibronectin or TGF-beta 1 (fibronectin: UIP 289 +/- 47.1 ng/ml, NSIP 121 +/- 23.0, control 118 +/- 16.0; TGF-beta1: UIP 798 +/- 119 pg/ml, NSIP 246 +/- 69.1, control 247 +/- 53.6, p < 0.01 in UIP versus NSIP or control); () the contractility positively correlated with the amount of either fibronectin (r = 0.867, p < 0.001, n = 15) or TGF-beta 1 (r = 0.939, p < 0.001, n = 15), respectively. Thus, UIP fibroblasts showed greater contractility than did NSIP fibroblasts and up-regulated control fibroblasts.
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Fibroblastos/fisiología , Enfermedades Pulmonares Intersticiales/fisiopatología , Actinas/análisis , Adenocarcinoma/patología , Análisis de Varianza , Biopsia , Células Cultivadas , Medio de Cultivo Libre de Suero , Femenino , Fibroblastos/química , Geles , Hamartoma/patología , Humanos , Pulmón/patología , Enfermedades Pulmonares/patología , Enfermedades Pulmonares Intersticiales/metabolismo , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Factor de Crecimiento Transformador beta/análisis , Factor de Crecimiento Transformador beta1RESUMEN
Conventional tools for elucidating gene function are relatively scarce in Candida albicans, the most prevalent human fungal pathogen. To this end, we developed a convenient system to control gene expression in C. albicans by the tetracycline-regulatable (TR) promoters. When the sea pansy Renilla reniformis luciferase gene (RLUC1) was placed under the control of this system, doxycycline (DOX) inhibited the luciferase activity almost completely. In the absence of DOX, the RLUC1 gene was induced to express luciferase at a level 400- to 1,000-fold higher than that in the presence of DOX. The same results were obtained in hypha-forming cells. The replacement of N-myristoyltransferase or translation elongation factor 3 promoters with TR promoters conferred a DOX-dependent growth defect in culture media. Furthermore, all the mice infected with these mutants, which are still virulent, survived following DOX administration. Consistently, we observed that the number of these mutant cells recovered from the mouse kidneys was significantly reduced following DOX administration. Thus, this system is useful for investigating gene functions, since this system is able to function in both in vitro and in vivo settings.
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Candida albicans/genética , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Regiones Promotoras Genéticas , Tetraciclina/farmacología , Animales , Doxiciclina/farmacología , Masculino , Ratones , Transactivadores/fisiologíaRESUMEN
In Saccharomyces cerevisiae, phosphoacetylglucosamine mutase is encoded by an essential gene called AGM1. The human AGM1 cDNA (HsAGM1) and the Candida albicans AGM1 gene (CaAGM1) were functionally cloned and characterized by using an S. cerevisiae strain in which the endogenous phosphoacetylglucosamine mutase was depleted. When expressed in Escherichia coli as fusion proteins with glutathione S-transferase, both HsAgm1 and CaAgm1 proteins displayed phosphoacetylglucosamine mutase activities, demonstrating that they indeed specify phosphoacetylglucosamine mutase. Sequence comparison of HsAgm1p with several hexose-phosphate mutases yielded three domains that are highly conserved among phosphoacetylglucosamine mutases and phosphoglucomutases of divergent organisms. Mutations of the conserved amino acids found in these domains, which were designated region I, II, and III, respectively, demonstrated that alanine substitutions for Ser(64) and His(65) in region I, and for Asp(276), Asp(278), and Arg(281) in region II of HsAgm1p severely diminished the enzyme activity and the ability to rescue the S. cerevisiae agm1Delta null mutant. Conservative mutations of His(65) and Asp(276) restored detectable activities, whereas those of Ser(64), Asp(278), and Arg(281) did not. These results indicate that Ser(64), Asp(278), and Arg(281) of HsAgm1p are residues essential for the catalysis. Because Ser(64) corresponds to the phosphorylating serine in the E. coli phosphoglucosamine mutase, it is likely that the activation of HsAgm1p also requires phosphorylation on Ser(64). Furthermore, alanine substitution for Arg(496) in region III significantly increased the K(m) value for N-acetylglucosamine-6-phosphate, demonstrating that Arg(496) serves as a binding site for N-acetylglucosamine-6-phosphate.
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Fosfotransferasas (Fosfomutasas)/genética , Fosfotransferasas (Fosfomutasas)/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , Candida albicans/enzimología , Candida albicans/genética , Catálisis , Clonación Molecular , Análisis Mutacional de ADN , ADN Complementario/análisis , Biblioteca Genómica , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Homología de Secuencia de AminoácidoRESUMEN
Human bronchial epithelial cells are involved in airway immune mechanisms through secretion of cytokines and through cell-cell contacts with immunocompetent cells. The aim of our study was to assess the ability of interferon (IFN) alpha and gamma alone and in combination to modulate human bronchial epithelial cell (HBECs) release of the inflammatory cytokines IL-8 and IL-6 and fibronectin and to induce the surface expression of HLA-DR and ICAM-1 molecules involved in immune interactions with other cells. HBECs spontaneously secreted a limited amount of IL-8, which was significantly increased by IFN gamma. IFN alpha inhibited IFN gamma stimulated IL-8 secretion in a concentration-dependent manner. Further, IFN gamma induced IL-6 and fibronectin secretion, and this was also inhibited by IFN alpha. The expression of HLA-DR antigens was significantly increased by IFN gamma and partially inhibited by co-stimulation with IFN alpha. In contrast, IFN gamma also induced ICAM-1 expression by HBECs but co-stimulation with IFN alpha had no significant effect on the expression of this surface antigen. IFN alpha modulation of HBEC functions does not seem to be restricted to IFN gamma stimulation since either stimulatory or inhibitory effects of INF alpha on IL-8 production have been found in pilot experiments using IL-1 beta, TNF alpha, and TGF beta as stimuli. In summary, IFN-gamma induces a number of responses in HBECs including increased secretion of IL-6, IL-8 and fibronectin and increased expression of HLA-DR and ICAM-1. IFN alpha can inhibit all these except expression of ICAM-1 which is unaffected. IFN alpha can also interact with other inflammatory cytokines, but whether the effects are inhibitory or augmentive depends on the cytokines.
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Bronquios/efectos de los fármacos , Citocinas/biosíntesis , Interferón-alfa/farmacología , Interferón gamma/farmacología , Bronquios/citología , Bronquios/inmunología , Células Cultivadas , Células Epiteliales/efectos de los fármacos , Células Epiteliales/inmunología , Fibronectinas/biosíntesis , Antígenos HLA-DR/biosíntesis , Humanos , Molécula 1 de Adhesión Intercelular/biosíntesis , Interleucina-6/biosíntesis , Interleucina-8/biosíntesis , FenotipoRESUMEN
BACKGROUND: Various factors such as serum angiotensin-converting enzyme (sACE) activity, bronchoalveolar lavage (BAL) fluid lymphocyte percent, CD4/CD8 ratio, and shadows on chest radiograph have been identified as indexes of disease activity in patients with sarcoidosis. However, it remains to be confirmed whether these factors can predict clinical outcomes. OBJECTIVE: To examine whether the interleukin-1 receptor antagonist (IL-1ra)/IL-1 beta ratio can predict the clinical course, we prospectively followed the clinical courses of 30 patients with pulmonary sarcoidosis 4 years after measurement of immunoreactive amounts of IL-1ra or IL-1 beta in the culture supernatants obtained from BAL fluid macrophages. METHODS: Immunoreactive amounts of IL-1ra or IL-1 beta were measured using ELISA. Changes in pulmonary function, sACE activity, and shadows on chest radiographs during observation periods were evaluated as markers of changes in disease activity. RESULTS: We found that the patients whose shadows on chest radiographs showed improvement had a higher molar IL-1ra/IL-1 beta ratio than the patients whose shadows persistently remained 4 years after BAL examination (p < 0.05). The molar ratio was found to be positively correlated with improvement of percent vital capacity (p < 0.05) and negatively correlated with the ratio of sACE activity at the time of the last observation to sACE activity at the time of BAL (sACE(LAST)/sACE(BAL), p < 0.01). The sACE(LAST)/sACE(BAL) ratio was significantly lower in patients whose shadows on chest radiographs decreased than in those whose shadows remained unchanged (p < 0.005). CONCLUSION: The IL-1ra/IL-1 beta ratio in the BAL fluid macrophage culture supernatants in patients with pulmonary sarcoidosis could be a useful marker in predicting the persistence of granulomatous lesions (chronicity).
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Receptores de Interleucina-1/metabolismo , Sarcoidosis Pulmonar/metabolismo , Sarcoidosis Pulmonar/fisiopatología , Sialoglicoproteínas/metabolismo , Adulto , Líquido del Lavado Bronquioalveolar/citología , Células Cultivadas , Femenino , Humanos , Proteína Antagonista del Receptor de Interleucina 1 , Pulmón/fisiopatología , Macrófagos/metabolismo , Masculino , Concentración Osmolar , Peptidil-Dipeptidasa A/sangre , Pronóstico , Radiografía Torácica , Sarcoidosis Pulmonar/diagnóstico por imagen , Sarcoidosis Pulmonar/patología , Factores de TiempoRESUMEN
OBJECTIVE: Nilvadipine, a calcium antagonist, has been shown to have antioxidant activity in vitro, but its effect on in vivo oxidation has not been assessed. The aim of this study was to investigate the antioxidant effect of this agent in vivo. The ratios of 7-keto cholestadien to cholesterol are believed to be an available marker of lipid peroxidation. Using these ratios, we have assessed the antioxidant effect of nilvadipine on low-density lipoprotein (LDL) in hypertensive patients with high risk of atherosclerosis. The risk factors of atherosclerosis may involve oxidation of LDL. METHODS: Fifteen healthy subjects (seven females and eight males aged 35-72 years, mean +/- SD = 55.3 +/-13.8 years) and fifteen hypertensive patients (seven females and eight males aged 45-80 years, mean +/- SD = 60.2 +/- 11.8 years) were recruited. Patients were treated orally with nilvadipine (4 mg b.i.d.) for 4 weeks. Cholesterol oxidation levels of LDL in patients before and after nilvadipine therapy and healthy subjects were studied. RESULTS: The ratios of 7-keto cholestadien to cholesterol in LDL of hypertensive patients before and 4 weeks after nilvadipine treatment and in healthy subjects were 6.5 +/- 1.6% (mean +/- SD), 3.8 +/- 1.2%, and 0.2 +/- 0.1%, respectively. There were significantly (P < 0.001) increased levels of cholesterol oxidation in LDL in patients with hypertension both before and after nilvadipine treatment compared with healthy subjects. However, there was a significantly (P < 0.001) decreased level of cholesterol oxidation in LDL in patients after nilvadipine treatment compared with patients before nilvadipine treatment. CONCLUSION: Our data showed that nilvadipine may protect LDL cholesterol from in vivo oxidation in hypertensive patients with high risk of atherosclerosis.