RESUMEN
Toll-like receptors (TLRs) are involved in the recognition of bacterial products and thus participate in the induction of the inflammatory cascade. However, much less is known about the evolution of leucocyte TLR expression during human inflammatory stress. We hypothesized that a decrease in leucocyte TLRs could account for the so-called tolerance or hyporesponsiveness state to subsequent stimulation with bacteria-derived products. Because of the profound monocytopenia that ensues after in vivo lipopolysaccharide (LPS) challenge, we also compared monocyte TLR expression using two different techniques of flow cytometric gating. In a first set of experiments, 17 healthy volunteers underwent LPS challenge. Blood was drawn at different time-points and analysed by flow cytometry using light scatter gating and one-colour analysis to assess the expression of the tumour necrosis factor receptor (TNFR) and TLR2 and TLR4 on both monocytes and granulocytes. In a second set of experiments, the assessment of those receptors was made using a more specific gating method that utilized light scatter and CD14 immunofluorescence in a two-colour analysis. This was performed using whole blood drawn from five healthy volunteers and incubated ex vivo for different time periods with or without LPS and in 12 volunteers who underwent LPS challenge in vivo. The pattern of expression for monocyte TNFR was similar for both types of gating. Using only the light scatter gating, an initial drop of TLR 2 and 4 was observed on monocytes. By contrast, when using light scatter x immunofluorescence gating, an up-regulation of these two receptors following both in vivo and in vitro LPS exposure was observed. LPS up-regulates the expression of TLRs on monocytes and granulocytes. Depending upon the methodology utilized, contrasting results were obtained with respect to TLR2 and TLR4 expression. The flow cytometric gating technique used is of importance in determining cellular TLR2 and TLR4 expression, especially in blood samples exhibiting significant monocytopenia.
Asunto(s)
Inflamación/sangre , Leucocitos/metabolismo , Lipopolisacáridos/toxicidad , Glicoproteínas de Membrana/sangre , Receptores de Superficie Celular/sangre , Receptores del Factor de Necrosis Tumoral/sangre , Adolescente , Adulto , Femenino , Citometría de Flujo/métodos , Técnica del Anticuerpo Fluorescente , Granulocitos/metabolismo , Humanos , Masculino , Monocitos/metabolismo , Dispersión de Radiación , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptores Toll-Like , Regulación hacia Arriba/efectos de los fármacosRESUMEN
This study was undertaken to explore the site-specific retention rate and the possibility of shortening fluoride (F) mouthrinsing time of kindergarten children. Fluoride retention after 10-, 20- and 30-second mouthrinsing was determined in 43 kindergarten children aged 4-5 years. Tooth surfaces were sampled by a paper point method. Fluoride concentrations in the salivary film on tooth surfaces increased from primary molars to primary incisors in the maxilla and decreased from primary molars to primary incisors in the mandible. The fluoride solution reached the primary molars even after a 10-second rinse, but F concentrations were higher after 20 s than after 10 s and significantly higher after 30 s than after 10 s. No significant difference was observed between 20 and 30 s. The average total F retained in the mouth was 0.13 mg after 20 s and 0.17 mg after 30 s. It was concluded that 30-second mouthrinsing, which is used extensively in Japanese kindergartens, can be shortened to 20 s.
Asunto(s)
Cariostáticos/administración & dosificación , Antisépticos Bucales/química , Saliva/química , Fluoruro de Sodio/administración & dosificación , Análisis de Varianza , Cariostáticos/análisis , Preescolar , Humanos , Fluoruro de Sodio/análisis , Estadísticas no Paramétricas , Factores de Tiempo , Diente PrimarioRESUMEN
The patient is a 61-year-old woman who had undergone mitral valve replacement with the Starr-Edwards cloth-covered ball valve 31 years ago. She had dyspnea on effort 1 month before admission. The echocardiography revealed mitral and tricuspid regurgitation. Re-replacement of the mitral prosthetic valve with an ATS valve and tricuspid annuloplasty were successfully performed without any complication. The cloth wear of the Starr-Edwards ball valve cage was recognized and no thrombus was found at operation. To our knowledge, there has been no such a reoperative case who underwent so long postoperative period after initial implantation of the Starr-Edwards ball valve in Japan. This experience made us realize again the importance of attention to the complications related to a prosthetic valve like a cloth wear during long-term follow-up for the Starr-Edwards ball valve.
Asunto(s)
Implantación de Prótesis de Válvulas Cardíacas , Insuficiencia de la Válvula Mitral/cirugía , Válvula Mitral/cirugía , Falla de Prótesis , Insuficiencia de la Válvula Tricúspide/cirugía , Femenino , Humanos , Persona de Mediana Edad , Diseño de Prótesis , Reoperación , Factores de Tiempo , Válvula Tricúspide/cirugíaRESUMEN
The effect of daily use of three different dentifrices on glucose retention after glucose mouth rinsing was tested in this study regarding xylitol and fluoride. Six experimental groups used three different dentifrices produced by two different companies: xylitol- and fluoride-containing dentifrice (XF), non-xylitol- and fluoride-containing dentifrice (F), and non-xylitol- and non-fluoride-containing dentifrice (NonX-NonF). Subjects were divided at random and rinsed their mouths for 15s with 20ml of 0.5M glucose solution. Glucose and lactate retention were determined by collecting samples of saliva from the approximal areas of the maxillary and mandibular anterior teeth and using the enzyme membrane test. Samples were collected 0, 1 and 2 months after the start of regular dentifrice use. There were significant differences in glucose retention in relation to the dentifrice used, month of sampling, site of sampling, and time since start of rinsing. Their contribution ratios were 2.0, 4.4, 11.7 and 7.4%, respectively (P<0.01). There were significant differences observed between the XF and NonX-NonF groups, with the XF group presenting lower glucose retention than the NonX-NonF group. The XF group presented lower glucose retention than the F group. The F group showed lower glucose retention than the NonX-NonF group. There were significant differences in lactate retention in relation to the month and site of sampling, and their contribution ratios were 3.3 and 2.8%, respectively (P<0.01). There were, however, no significant differences in glucose and lactate retention in relation to the dentifrice manufacturer. It was concluded that the XF dentifrice was the most effective, and the F dentifrice was more effective in reducing glucose retention than the NonX-NonF dentifrice.
Asunto(s)
Dentífricos , Fluoruros/administración & dosificación , Glucosa/farmacocinética , Saliva/química , Edulcorantes/administración & dosificación , Xilitol/administración & dosificación , Adulto , Análisis de Varianza , Placa Dental/metabolismo , Femenino , Glucosa/análisis , Humanos , Ácido Láctico/análisis , Factores de TiempoRESUMEN
To investigate impairment of cellular immunity in severe acute pancreatitis, alterations of peripheral lymphocytes in acute pancreatitis were examined. In 48 patients with severe acute pancreatitis, the mean peripheral lymphocyte count on admission was 959 + 105/mm3, and it was significantly decreased in the patients with subsequent infection (623 +/- 90/mm3 ) in comparison to those without infection (1084 +/- 135/mm(3)). According to an analysis of lymphocyte subsets, although both B and T lymphocytes were decreased in peripheral circulation in the patients with infection, it was primarily CD8-positive lymphocytes that decreased in these subsets. Cell cycle analysis of lymphocytes collected from these patients indicated that apoptotic changes occurred after 24 hours' incubation in lymphocytes from patients with severe pancreatitis but not in lymphocytes from healthy control subjects. In a rat model of experimental necrotizing pancreatitis, total peripheral lymphocytes and T lymphocytes were significantly decreased 5 hours after induction of pancreatitis. In severe pancreatitis, peripheral lymphocytes are eliminated from systemic circulation possibly as a result of apoptosis. It has been suggested that impairment of cellular immunity due to peripheral lymphocyte apoptosis is linked to the development of subsequent infectious complications in acute pancreatitis.
Asunto(s)
Apoptosis/inmunología , Linfopenia/etiología , Pancreatitis/complicaciones , Enfermedad Aguda , Animales , Linfocitos B/inmunología , Linfocitos B/patología , Infecciones Bacterianas/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunidad Celular/inmunología , Recuento de Linfocitos , Linfopenia/inmunología , Masculino , Persona de Mediana Edad , Pancreatitis/inmunología , Pancreatitis Aguda Necrotizante/complicaciones , Pancreatitis Aguda Necrotizante/inmunología , Ratas , Ratas Wistar , Estudios Retrospectivos , Linfocitos T/inmunología , Linfocitos T/patologíaRESUMEN
BACKGROUND: We have reported that pancreatitis-associated ascitic fluid (PAAF) contains a cytotoxic factor(s) inducing apoptosis on renal tubular cells and hepatocytes. It has been suggested that elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) is associated with the development of cell damage and apoptosis. METHODS: To clarify the mechanism of hepatocellular injury in acute pancreatitis, the effect of PAAF on hepatocyte [Ca(2+)](i) was investigated. Primary cultures of rat hepatocytes were loaded with Fura-2/acetoxymethyl, and the changes of [Ca(2+)](i) were measured using spectrofluorometer. RESULTS: The baseline of hepatocyte [Ca(2+)](i) was 172 +/- 17 nM. [Ca(2+)](i) increased from 1 min after the addition of PAAF in a dose-dependent manner. Fractionation of PAAF revealed only one fraction (molecular weight >/= 5 x 10(4)) possessed both [Ca(2+)](i) elevation activity and cytotoxic activity. Neither 8-(N,N-diethyl-amino) octyl-3,4,5-trimethoxybenzoate (TMB-8) nor thapsigargin inhibited the PAAF-evoked [Ca(2+)](i) elevation. Chelation of extracellular Ca(2+) by ethylene glycol bis-(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) prevented the elevation of [Ca(2+)](i), but verapamil did not prevent it. Platelet-activating factor antagonist (TCV-309) blocked the PAAF-elicited [Ca(2+)](i) elevation. Pancreatitis-associated serum also increased hepatocyte [Ca(2+)](i). Moreover, PAAF increased [Ca(2+)](i) on Madin-Darby canine kidney cells in a dose-dependent manner. CONCLUSIONS: These results suggest that the dramatic elevation of hepatocyte [Ca(2+)](i) due to PAAF may be closely related to the hepatocellular injury in severe acute pancreatitis and that platelet-activating factor may play a pivotal role in increasing hepatocyte [Ca(2+)](i). Elevation of [Ca(2+)](i) in various cells may be involved in the mechanism of multiple organ failure in this disease.
Asunto(s)
Líquido Ascítico/química , Calcio/metabolismo , Hígado/metabolismo , Pancreatitis/complicaciones , Enfermedad Aguda , Animales , Células Cultivadas , Perros , Ácido Egtácico/farmacología , Masculino , Insuficiencia Multiorgánica/etiología , Factor de Activación Plaquetaria/fisiología , Ratas , Ratas Wistar , Verapamilo/farmacologíaRESUMEN
A 55 year old male with a history of intermittent claudication presented with an abdominal mass, and was diagnosed by abdominal computed tomography (CT) with an abdominal aortic aneurysm accompanying horseshoe kidney. The horseshoe kidney configuration and governing vessels, urinary duct course, and right common iliac arterial stenosis were shown by methods such as angiogram, spiral CT, and intravenous pyelogram before operation. At the operation, the abdomen was opened by a median incision and, using a staple exclusion technique, the abnormal renal artery was reconstructed using 189 mm knitted Y shaped dacron graft replacement and the great saphenous vein. The isthmus was not resected. There were no post operative complications, nor was there any large decrease in renal function. Good results were obtained, and we herein report our results together with a discussion of the literature.
Asunto(s)
Aneurisma de la Aorta Abdominal/cirugía , Enfermedades Renales/complicaciones , Riñón/anomalías , Suturas , Procedimientos Quirúrgicos Vasculares/instrumentación , Aneurisma de la Aorta Abdominal/complicaciones , Aneurisma de la Aorta Abdominal/diagnóstico por imagen , Aortografía , Implantación de Prótesis Vascular/instrumentación , Humanos , Enfermedades Renales/congénito , Enfermedades Renales/diagnóstico por imagen , Masculino , Persona de Mediana Edad , Tomografía Computarizada por Rayos X , UrografíaRESUMEN
We previously reported that serum hepatocyte growth factor (HGF) levels are elevated in patients with acute pancreatitis and that pancreatitis-associated ascitic fluid (PAAF) contains cytotoxic factor(s) inducing apoptosis on Madin-Darby canine kidney (MDCK) cells. In this study, plasma HGF levels and HGF tissue distribution were investigated in rats with experimental acute pancreatitis, and the effects of HGF on the cytotoxic activity and apoptosis-inducing activity of PAAF also were examined. Plasma HGF levels were elevated in rats with two experimental pancreatitis models of different grades of severity. The degree of its elevation was correlated with the severity and the organ dysfunctions. In rats with severe pancreatitis, HGF protein and messenger RNA (mRNA) levels significantly increased in liver, kidney, and lung, which were injured organs. When anti-HGF neutralizing antibody was administered in severe pancreatitis, liver dysfunction worsened, and apoptotic cells increased in kidney. Recombinant HGF inhibited the cytocidal activity of PAAF on MDCK cells in a dose-dependent manner. Moreover, recombinant HGF prevented the apoptotic cell death (DNA fragmentation, nuclear fragmentation, and caspase-3 activation) induced by PAAF. These results suggest that HGF is produced in injured organs and may function as an organotrophic and antiapoptotic factor against the organ injuries in acute pancreatitis.
Asunto(s)
Edema/metabolismo , Factor de Crecimiento de Hepatocito/biosíntesis , Insuficiencia Multiorgánica/metabolismo , Pancreatitis/metabolismo , Enfermedad Aguda , Animales , Apoptosis/efectos de los fármacos , Caspasa 3 , Caspasas/análisis , Línea Celular , Ceruletida/toxicidad , Fragmentación del ADN , Ácido Desoxicólico/toxicidad , Perros , Edema/inducido químicamente , Factor de Crecimiento de Hepatocito/sangre , Factor de Crecimiento de Hepatocito/genética , Factor de Crecimiento de Hepatocito/farmacología , Riñón/metabolismo , Riñón/patología , Hígado/metabolismo , Hígado/patología , Pulmón/metabolismo , Pulmón/patología , Masculino , Insuficiencia Multiorgánica/etiología , Pancreatitis/inducido químicamente , Pancreatitis/complicaciones , Pancreatitis Aguda Necrotizante/inducido químicamente , Pancreatitis Aguda Necrotizante/complicaciones , Pancreatitis Aguda Necrotizante/metabolismo , ARN Mensajero/biosíntesis , Ratas , Ratas Wistar , Proteínas Recombinantes de Fusión/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bazo/metabolismo , Bazo/patologíaRESUMEN
A yeast two-hybrid screening for Ras-binding proteins in nematode Caenorhabditis elegans has identified a guanine nucleotide exchange factor (GEF) containing a Ras/Rap1A-associating (RA) domain, termed Ce-RA-GEF. Both Ce-RA-GEF and its human counterpart Hs-RA-GEF possessed a PSD-95/DlgA/ZO-1 (PDZ) domain and a Ras exchanger motif (REM) domain in addition to the RA and GEF domains. They also contained a region homologous to a cyclic nucleotide monophosphate-binding domain, which turned out to be incapable of binding cAMP or cGMP. Although the REM and GEF domains are conserved with other GEFs acting on Ras family small GTP-binding proteins, the RA and PDZ domains are unseen in any of them. Hs-RA-GEF exhibited not only a GTP-dependent binding activity to Rap1A at its RA domain but also an activity to stimulate GDP/GTP exchange of Rap1A both in vitro and in vivo at the segment containing its REM and GEF domains. However, it did not exhibit any binding or GEF activity toward Ras. On the other hand, Ce-RA-GEF associated with and stimulated GDP/GTP exchange of both Ras and Rap1A. These results indicate that Ce-RA-GEF and Hs-RA-GEF define a novel class of Rap1A GEF molecules, which are conserved through evolution.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/química , Factores de Intercambio de Guanina Nucleótido/química , Proteínas del Tejido Nervioso , Proteínas de Unión al GTP rap1/química , Proteínas ras/química , Secuencia de Aminoácidos , Animales , Clonación Molecular , Secuencia Conservada , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Datos de Secuencia Molecular , Unión Proteica , Homología de Secuencia de Aminoácido , Spodoptera , Proteínas de Unión al GTP rap1/metabolismo , Proteínas ras/metabolismoRESUMEN
We conducted mitral valve replacement (MVR) in a patient with mitral regurgitation (MR) complicated with idiopathic thrombocytopenic purpura (ITP). The patient was a 62-year-old male who was diagnosed to have grade IV MR. However, a decrease in platelet count was noted (5.6 x 10(4)/microliter) when he has admitted to this hospital for operation. After detailed examination, he was diagnosed to have ITP. Though mass intravenous infusion of gamma-globulin (400 mg/kg/day, for 5 days) was done before the operation, the treatment was not successful and splenectomy was consequently conducted. In view of the influence of invasive splenectomy, the change in platelet count was carefully observed thereafter. The count subsequently increased to reach a peak (26.9 x 10(4)/microliter) after 2 weeks from the splenectomy. MVR was conducted when the count started to decrease again after the peak. The operation was safely completed without any complication such as hemorrhage during and after the operation. Since gamma-globulin treatment and splenectomy are sometimes ineffective in ITP, it is advisable to wait an operation until the effects of these treatments are clarified.
Asunto(s)
Implantación de Prótesis de Válvulas Cardíacas , Insuficiencia de la Válvula Mitral/cirugía , Púrpura Trombocitopénica Idiopática/complicaciones , Humanos , Masculino , Persona de Mediana Edad , EsplenectomíaRESUMEN
Ras proteins are conserved from yeasts to mammals and implicated in regulation of the actin cytoskeleton. The flightless-1 (fli-1) gene of Drosophila melanogaster and its homologs in Caenorhabditis elegans and humans encode proteins (FLI-1) comprising a fusion of a leucine-rich repeats (LRRs) domain and a gelsolin-like domain. This LRRs domain is highly homologous to those of three proteins involved in Ras-mediated signaling; Saccharomyces cerevisiae adenylyl cyclase, C. elegans SUR-8, and mammalian RSP-1. Here we report that the LRRs domain of C. elegans FLI-1 (Ce-FLI-1) associates directly with Ras (Kd = 11 nM) and, when overexpressed, suppresses the heat shock sensitive phenotype of yeast cells bearing the activated RAS2 gene (RAS2(Val-19)). Further, the gelsolin-like domain of Ce-FLI-1 is shown to possess a Ca2+-independent G-actin-binding activity as well as F-actin-binding and -severing activities. FLI-1 may be involved in regulation of the actin cytoskeleton through Ras.
Asunto(s)
Caenorhabditis elegans/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Drosophila , Gelsolina/química , Proteínas del Helminto/metabolismo , Proteínas de Insectos/metabolismo , Leucina/química , Proteína Oncogénica p21(ras)/metabolismo , Actinas/metabolismo , Actinas/ultraestructura , Adenilil Ciclasas/metabolismo , Animales , Unión Competitiva , Caenorhabditis elegans/química , Caenorhabditis elegans/genética , Calcio/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/genética , Relación Dosis-Respuesta a Droga , Ácido Egtácico/farmacología , Activación Enzimática/efectos de los fármacos , Gelsolina/genética , Respuesta al Choque Térmico , Proteínas del Helminto/química , Proteínas del Helminto/genética , Humanos , Proteínas de Insectos/química , Proteínas de Insectos/genética , Leucina/genética , Proteína Oncogénica p21(ras)/antagonistas & inhibidores , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Unión Proteica/efectos de los fármacos , Biosíntesis de Proteínas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Mammalian Ras proteins regulate multiple effectors including Raf, Ral guanine nucleotide dissociation stimulator (RalGDS), and phosphoinositide 3-kinase. In the nematode Caenorhabditis elegans, LIN-45 Raf has been identified by genetic analyses as an effector of LET-60 Ras. To search for other effectors in C. elegans, we performed a yeast two-hybrid screening for LET-60-binding proteins. The screening identified two cDNA clones encoding a phosphoinositide-specific phospholipase C (PI-PLC) with a predicted molecular mass of 210 kDa, designated PLC210. PLC210 possesses two additional functional domains unseen in any known PI-PLCs. One is the C-terminal Ras-associating domain bearing a structural homology with those of RalGDS and AF-6. This domain, which could be narrowed down to 100 amino acid residues, associated in vitro with human Ha-Ras in a GTP-dependent manner and competed with yeast adenylyl cyclase for binding Ha-Ras. The binding was abolished by specific mutations within the effector region of Ha-Ras. The other functional domain is the N-terminal CDC25-like domain, which possesses a structural homology to guanine nucleotide exchange proteins for Ras. These results strongly suggest that PLC210 belongs to a novel class of PI-PLC, which is a putative effector of Ras.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/enzimología , Fosfolipasas de Tipo C/metabolismo , Proteínas ras/metabolismo , Secuencia de Aminoácidos , Animales , Clonación Molecular , ADN Complementario/genética , Guanosina Trifosfato/farmacología , Humanos , Datos de Secuencia Molecular , Mutagénesis , Fosfatidilinositol Diacilglicerol-Liasa , Fosfoinositido Fosfolipasa C , Unión Proteica/efectos de los fármacos , Homología de Secuencia de Aminoácido , Transducción de Señal , Fosfolipasas de Tipo C/genética , Proteínas ras/genéticaRESUMEN
Mammalian Ras proteins associate with multiple effectors, including Raf, Ral guanine nucleotide dissociation stimulator, phosphoinositide 3-kinase and AF-6. In the nematode Caenorhabditis elegans, LIN-45/Raf has been identified genetically as an effector of LET-60/Ras. To search for other effectors in C. elegans, we carried out a yeast two-hybrid screening for LET-60-associating proteins. The screening identified a novel protein, designated Ce-AF-6, which exhibited a strong structural homology with human AF-6, rat Afadin and Drosophila melanogaster Canoe and possessed both the Ras-associating (RA) domain and the PSD-95/DlgA/ZO-1 (PDZ) domain. Ce-AF-6 associated with human Ha-Ras in a GTP-dependent manner, with an efficiency comparable to that of human Raf-1 Ras-binding domain. When the effects of mutations of the Ras effector region residues were examined for associations with various effectors, Ce-AF-6 was found to possess a distinct and the most rigorous requirement for the effector region residues. These results strongly suggest that Ce-AF-6 is a putative effector of Ras that possesses a distinct recognition mechanism for association with Ras.