RESUMEN
BACKGROUND: Acute pancreatitis (AP) is an acute inflammatory process of the pancreas characterized by self-digestion of pancreatic tissue, which can trigger a systemic inflammatory response. Venous thrombosis, resulting from a hypercoagulable state, is a vascular complication of AP. AP complicated by pulmonary embolism (PE) is very rare, and the combined use of extracorporeal membrane oxygenation (ECMO) with a vascular interventional procedure for AP complicated by PE is even rarer. CASE SUMMARY: A 32-year-old man with a history of obesity developed rapidly worsening AP secondary to hypertriglyceridemia. During treatment, the patient developed chest tightness, shortness of breath, and cardiac arrest. Computed tomography (CT) scans of his upper abdomen were consistent with pancreatitis. PE was identified by chest CT angiography involving the right main pulmonary artery and multiple lobar pulmonary arteries. The patient's D-dimer level was significantly elevated (> 20 mg/L). The patient received high-frequency oxygen inhalation, continuous renal replacement therapies, anti-infective therapy, inhibition of pancreatic secretion, emergent endotracheal intubation, and advanced cardiac life support with cardiopulmonary resuscitation. Following both ECMO and a vascular interventional procedure, the patient recovered and was discharged. CONCLUSION: PE is a rare but potentially lethal complication of AP. The early diagnosis of PE is important because an accurate diagnosis and timely interventional procedures can reduce mortality. The combined use of ECMO with a vascular interventional procedure for AP complicated by PE can be considered a feasible treatment method. A collaborative effort between multiple teams is also vital.
RESUMEN
Genetic diversity of Pneumocystis jirovecii isolates based on internal transcribed spacer (ITS) of the nuclear rRNA locus has previously been reported. The information about ITS genotype and epidemiology of this organism in Chinese human immunodeficiency virus-infected patients has not been available. In this study, 12 bronchoalveolar lavage fluid specimens obtained from HIV-infected patients were analyzed by PCR followed by cloning, sequencing and typing. Three ITS1 genotypes (E, B and 'H') and four ITS2 genotypes (b, g, i and r) as previously reported were identified, the most common of which were E, b and i. Five ITS haplotypes (Eg, Eb, Bi, Er and 'H'r) and 19 new combination types were also identified with the most common types being Eg (four of 12 patients, 10 of 60 clones), Eb (three of 12 patients, 11 of 60 clones) and Bi (three of 12 patients, 10 of 60 clones). Nine patients were found to be co-infected with more than one ITS genotype of P. jirovecii. The prevalence of ITS genotypes in HIV patients from one Chinese hospital did not seem to be significantly different when compared to reports from other countries.
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Infecciones Oportunistas Relacionadas con el SIDA/microbiología , Variación Genética , Pneumocystis carinii/genética , Neumonía por Pneumocystis/microbiología , Infecciones Oportunistas Relacionadas con el SIDA/epidemiología , Adulto , Secuencia de Bases , Líquido del Lavado Bronquioalveolar/microbiología , China , Coinfección , ADN de Hongos/genética , ADN Espaciador Ribosómico/química , ADN Espaciador Ribosómico/genética , Femenino , Genotipo , Técnicas de Genotipaje , Humanos , Huésped Inmunocomprometido , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Tipificación Molecular , Técnicas de Tipificación Micológica , Pneumocystis carinii/clasificación , Pneumocystis carinii/aislamiento & purificación , Neumonía por Pneumocystis/epidemiología , Prevalencia , ARN Ribosómico 5.8S/genética , Análisis de Secuencia de ADNRESUMEN
OBJECTIVE: To identify the protein-protein interaction between aldolase and actin of Toxoplasma gondii by GST pull-down. METHODS: The aldolase and actin genes were obtained from cDNA library by PCR amplification, and subcloned respectively into pGEX-4T-1 and pET30a. The fusion protein GST-Aldolase and Actin-His6 were expressed in E. coli upon induction by 1 mmol/L IPTG and then purified with affinity chromatography. Fifteen rats were immunized intradermally with 200 microg Actin-His6 protein per rat at first time to produce the polyclonal antibodies. Then 100 microg Actin-His6 protein per rat on the 2nd-4th immunizations. Rats were immunized for 4 times with 7 days interval. The serum of rats was collected from heart at the fifth day after the final immunization. Glutathione sepharose beads were incubated with GST-Aldolase protein, then incubated with Actin-His6, and bound proteins were eluted using sample buffer. Eluants were resolved by SDS-PAGE and Western blotting. RESULTS: The aldolase and actin genes were obtained, and the recombinant plasmid aldolase/pGEX-4T-1, actin/pET30a were successfully constructed. Protein GST-Aldolase and Actin-His6 were expressed and purified in vitro. Serum samples were prepared from rats immunized with protein Actin-His6, and polyclonal antibody was purified with affinity chromatography. SDS-PAGE and Western blotting analysis of products from GST pull-down experiment showed that the protein bands on NC membrane were specifically recognized by anti-Aldolase-His6 and anti-Actin-His6 antibody. CONCLUSION: Aldolase interacts with Actin of Toxoplasma gondii.
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Actinas/metabolismo , Fructosa-Bifosfato Aldolasa/metabolismo , Proteínas Protozoarias/metabolismo , Toxoplasma/metabolismo , Animales , Masculino , Mapeo de Interacción de Proteínas , Ratas , Ratas Sprague-Dawley , Toxoplasma/inmunologíaRESUMEN
Pneumocystis jirovecii dihydropteroate synthase (DHPS) gene mutations are associated with failure of sulfur prophylaxis of Pneumocystis pneumonia. Here the P. jirovecii DHPS gene was amplified from bronchoalveolar fluid of 10 AIDS patients in China. No DHPS gene mutations were observed. The results suggest that the prevalence of DHPS mutations in China may be low.
Asunto(s)
Infecciones Oportunistas Relacionadas con el SIDA/microbiología , Dihidropteroato Sintasa/genética , Mutación , Pneumocystis carinii/genética , Neumonía por Pneumocystis/microbiología , Infecciones Oportunistas Relacionadas con el SIDA/tratamiento farmacológico , Adulto , Anciano , Antifúngicos/uso terapéutico , Líquido del Lavado Bronquioalveolar/microbiología , China , Femenino , Genes Fúngicos , Humanos , Masculino , Persona de Mediana Edad , Pneumocystis carinii/enzimología , Pneumocystis carinii/aislamiento & purificación , Neumonía por Pneumocystis/complicaciones , Neumonía por Pneumocystis/tratamiento farmacológico , Combinación Trimetoprim y Sulfametoxazol/uso terapéuticoRESUMEN
OBJECTIVE: To explore the anti-tumor immune responses of dendritic cells (DCs) loaded with intact wild-type p53 to mice challenged with tumor cells expressing p53 genes with mutations at different sites. METHODS: Ad-p53-DC immunization function was assessed by the expression of surface molecules and allogeneic MLR. DCs derived from bone marrow were transduced with adenovirus or a human wild-type p53 containing recombinant adenovirus (Ad-DC and Ad-p53-DC) and immunized C57BL/6 mice. Splenocytes were separated and cell cytotoxicity was measured against tumor cells expressing mutant p53 (MethA, D459 and P815) in a standard 6-h(51)Cr-release assay. Effector and target cells were incubated in the presence of anti-CD(4) or anti-CD(8) antibody. Ad-p53-DC was immunized in control Ad-DC before or after mice were challenged with either D459 tumor or with MethA sarcoma cells to observe whether immune response would provide tumor protection. RESULTS: Immunization with Ad-p53-DC developed significantly higher substantial CTL responses against Ad-p53-P815, D459 and MethA cells (effectors: target cells = 50:1), (27.8 +/- 3.4)%, (23.5 +/- 2.7)%, (58.3 +/- 9.2)% than with Ad-DC (9.3 +/- 1.8)%, (4.6 +/- 1.0)%, (23.5 +/- 3.7)% (t(d) = 5.79, 3.68, 5.02, all P < 0.05). In Ad-p53-DC immunized mice, anti-CD(8) antibody blocked the cytotoxicity against Ad-p53-P815 (26.7 +/- 2.8)% or D459 (6.1 +/- 1.2)%, but not anti-CD(4) antibody [(59.8 +/- 4.6)%, (18.9 +/- 2.4)%, t(d) = 8.79 or 9.18, all P < 0.05]. Ad-p53-DC immunization provided complete tumor protection in 80% of mice challenged with D459 and in 70% of mice challenged with MethA, while none protected in Ad-DC immunization group (chi(2) = 6.72, 5.86, all P < 0.05). Treated with Ad-p53-DC after D459 inoculation subcutaneously, mice were killed due to the bulky tumor more than 2 weeks later than the mice in the Ad-DC treatment group during 7 week observation (chi(2) = 9.48, P < 0.05). CONCLUSION: DCs transfected with 100 MOI Ad-p53 induced intense CTL responses against P815, D459 and MethA. This CTL response is mediated by CD(8)(+) T cells. Treatment with Ad-p53-DC significantly developed tumor immunology and slowed the growth of established tumors.
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Células Dendríticas/inmunología , Neoplasias Experimentales/terapia , Linfocitos T Citotóxicos/inmunología , Proteína p53 Supresora de Tumor/genética , Adenoviridae/genética , Animales , Células de la Médula Ósea , Línea Celular Tumoral , Citotoxicidad Inmunológica , Vectores Genéticos , Inmunoterapia , Ratones , Ratones Endogámicos C57BL , TransfecciónRESUMEN
OBJECTIVE: To observe the pathological change in the brain of Angiostrongylus cantonensis-infected mouse. METHODS: Forty-eight mice were orally infected each with 40 third stage larvae of A. cantonensis, 3 mice were sacrificed at 7, 10, 13, 16, 19, 22, 25, 28 days postinfection respectively for worm recovery, and another 3 mice were for observing the histopathological change in tissue sections of the brain. RESULTS: Ten days postinfection, worms were found in the brain of the infected mice with a mean worm number of (7.0+/-1.7) per mouse. The highest number of worms was found at 16 days postinfection, with a mean of (23.7+/-4.9) per mouse. Notable symptoms of nervous system were seen on 15 days postinfection. Most mice died around 22 days postinfection. Histological examination revealed mechanical damages. Cavities and inflammation were observed in the brain parenchyma. Worms were seen in the subarachnoid space. Meningitis-like signs started at 13 days and aggravated then. CONCLUSIONS: Infection of A. cantonensis causes pathological change in mouse brain and the process is aggravating with postinfection time.
Asunto(s)
Encéfalo/patología , Infecciones por Strongylida/patología , Angiostrongylus cantonensis , Animales , Masculino , Ratones , Ratones Endogámicos BALB CRESUMEN
BACKGROUND: Restoration of blood flow to the ischemic liver lobes may paradoxically exacerbate tissue injury, which is called hepatic ischemia/reperfusion injury (IRI). Toll-like receptor 4 (TLR4), expressed on several liver cell types, and the nuclear factor-kappa B (NF-kappaB) signaling pathway are crucial to mediating hepatic inflammatory response. Because IRI is essentially a kind of profound acute inflammatory reaction evoked by many kinds of danger signals, we investigated TLR4/NF-kappaB signaling pathway activation in a murine model of partial hepatic IRI. METHODS: Wild-type mice (WT, C3H/HeN) or TLR4 mutant mice (C3H/HeJ) were subjected to 45 minutes of partial hepatic ischemia followed by 1 hour, 3 hours of reperfusion. Sham group accepted the same procedure without the obstruction of blood supply. At the end of reperfusion, the compromise of liver function and the histological change of liver sections were measured as the severity of liver injury. The level of endotoxin in the portal vein was measured by limulus assay. NF-kappaB activation was determined by electrophoretic mobility shift assay (EMSA). The levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) in systemic blood after hepatic IRI were assessed by enzyme-linked immunosorbent assay (ELISA). RESULTS: The compromise of liver function and the morphological injuries in mutant mice were relieved more markedly than those in WT mice after partial hepatic IRI. NF-kappaB activation in WT mice was stronger than that in TLR4 mutant mice, and both were stronger than those in the sham operated mice (P < 0.01). Endotoxin in each group was undetectable. The levels of TNF-alpha and IL-1beta in systemic blood were elevated in both strains, but lower in the sham operated group. These mediators were significantly decreased in TLR4 mutant mice compared with those in WT mice (P < 0.01). CONCLUSIONS: The TLR4/NF-kappaB signaling pathway may mediate hepatic IRI triggered by endogenous danger signals. Inhibition of the TLR4/NF-kappaB pathway may be a potential therapeutic target for attenuating ischemia/reperfusion-induced tissue damage in some clinical settings.
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Hígado/irrigación sanguínea , FN-kappa B/fisiología , Daño por Reperfusión/etiología , Transducción de Señal/fisiología , Receptor Toll-Like 4/fisiología , Alanina Transaminasa/sangre , Animales , Interleucina-1beta/biosíntesis , Ratones , Ratones Endogámicos C3H , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
AIM: To investigate the biological characteristics and effect of TNF-alpha binding peptide (TBP) and TNFR blocking peptide (TRBP) in vitro. METHODS: The binding specificity of TBP or TRBP was tested by competition experiment using GFP-TNF fusion protein and detected by FCM and fluorescent microscope. The interaction between TBP and TRBP was determined by non-denatured PAGE and the inhibitory effect of TBP and TRBP on TNF-alpha cytotoxicity against U937 was carried out by MTT colorimetry. The effects of TBP and TRBP on the functions of human monocytes activated by TNF-alpha and IFN-gamma in vitro were detected by nitrotetrazolium blue chloride (NBT) reduction assay for evaluating respiratory burst and by RT-PCR for evaluating IL-1beta and IL-8 mRNA transcription. RESULTS: It was showed that TBP and TRBP was able to block the GFP-TNF binding to the TNFR on the surface of cells and no binding interaction took place between TBP and TRBP. Both TBP and TRBP were able to inhibit the TNF-alpha cytotoxicity against U937 and this inhibitory effect was dose-dependent and the combination of TBP and TRBP (pep.38+X4) was able to inhibit the TNF-alpha cytotoxicity more efficiently than the individual peptide at all tested concentrations. The combination of TBP and TRBP was able to obviously inhibit both respiratory burst of monocytes induced by TNF-alpha and IFN-gamma and transcription level of IL-1beta and IL-8 mRNA induced by TNF-alpha. CONCLUSION: TBP or TRBP can bind with their ligands specifically and don't interact with each other. They can also block the biological activity of the TNF-alpha in vitro, and the combination of TBP and TRBP is able to inhibit the biological activity of the TNF-alpha more efficiently. This research will provide an experimental basis for the clinical treatment of the inflammatory disease.
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Monocitos/efectos de los fármacos , Péptidos/metabolismo , Péptidos/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Línea Celular Tumoral , Células Cultivadas , Colorimetría , Citometría de Flujo , Humanos , Interferón gamma/farmacología , Interleucina-1beta/genética , Interleucina-8/genética , Microscopía Fluorescente , Monocitos/metabolismo , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
OBJECTIVE: To analyze the difference of GRA7 gene of Toxoplasma gondii different isolated strains and express GRA7 in Escherichia coli. METHODS: The GRA7 gene was amplified from genomes of T. gondii isolates by PCR and was cloned into pGEX-4T-1. The recombinant plasmid was transformed into JM109 and sent to be sequenced. The sequence was analyzed with CLUSTALW (an internet tool). The recombinant plasmid was induced by IPTG to express the fusion protein,which was identified by SDS-PAGE and Western blot with positive sera. The protein was purified and used as a diagnostic antigen for ELISA to test serum samples. RESULTS: There was no difference among the sequences of T. gondii GRA7 gene from different isolates. The recombinant plasmid pGEX-4T-1/GRA7 induced by IPTG was expressed in E. coli. It was a GST fusion protein and could react with human and rabbit positive sera analyzed by Western blot. CONCLUSION: The GRA7 gene of T. gondii isolates is highly conservative. The GRA7 is expressed as a recombinant protein in Escherichia coli, which shows an immunoreactivity.
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Antígenos de Protozoos/biosíntesis , Proteínas Protozoarias/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Toxoplasma/genética , Animales , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Secuencia de Bases , Clonación Molecular , Escherichia coli/genética , Glutatión Transferasa/genética , Datos de Secuencia Molecular , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Proteínas Recombinantes de Fusión/inmunología , Toxoplasma/inmunologíaRESUMEN
BACKGROUND: A monoclonal antibody would be an effective tool for the detection of circulating antigens in the serum of patients with schistosomiasis, but the traditional way of producing monoclonal antibodies is not cost-effective. The objective of this study was to find a new method for the large-scale production of monoclonal antibodies against Schistosoma japonicum (Sj). METHODS: A phage display antibody library for Sj was constructed. To obtain a single-chain variable fragment antibody (scFv) against Sj, the library was screened with metabolic antigens from adult Sj worms (Sj-MAg) using enzyme-linked immunosorbent assay. The soluble scFvs selected were used to detect Sj antigens in the serum of acute and chronic schistosomiasis patients. RESULTS: Six positive clones with good reactivity to Sj-MAg were obtained from the phage display antibody library of about 1.07 x 10(6) individual clones. Only two of these six clones bound specifically to Sj-MAg and were chosen for further analysis. Specific soluble anti-Sj-MAg scFvs were produced by inducing the 2 clones with isopropyl-D-thiogalactopyranoside. The characteristics of the scFvs were then determined. The results of Western blot showed that these scFvs could bind to Sj-MAg specifically and had a molecular weight of about 31 kD. When testing serum from schistosomiasis patients with one of the two specific scFvs, its sensitivity was found to be 60% and 37% in acute and chronic patients, respectively, with a specificity of 90%. When the two specific scFvs were combined, their sensitivity was found to be 75% and 57% in acute and chronic patients, respectively, with a specificity of 85%. CONCLUSIONS: The results indicate that the scFvs are potentially useful for the diagnosis of schistosomiasis. The library construction also provides a useful tool for the further screening of other antibodies for both diagnostic and immunotherapeutic applications and for epitope analysis and vaccine design.
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Anticuerpos Antihelmínticos/inmunología , Anticuerpos Monoclonales/inmunología , Antígenos Helmínticos/sangre , Biblioteca de Péptidos , Esquistosomiasis Japónica/diagnóstico , Animales , Secuencia de Bases , Fragmentos de Inmunoglobulinas/inmunología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Conejos , Sensibilidad y Especificidad , Pruebas SerológicasRESUMEN
AIM: To compare the changes of free calcium concentration in target cells killed by transmembrane tumor necrosis factor-alpha(TM-TNF-alpha) and secretory TNF-alpha(S-TNF-alpha). METHODS: The cytotoxicity of two types of TNF-alpha was tested by bioassay. The intracellular Ca(2+) concentration was determined by Frua-2. RESULTS: TM-TNF-alpha had cytotoxic effect on all 6 kinds of target cells, whereas S-TNF-alpha could kill only two of them. The cytotoxic activity of both types of TNF-alpha was accompanied by a dramatically increase of intracellular free Ca(2+) concentration. The intracellular Ca(2+) concentration in the target cells treated with S-TNF-alpha was obviously reduced by pretreating target cells with 10 micromol/L calcium chelator EGTA for 30 minutes (P<0.01) and the cytotoxicity of S-TNF-alpha was significantly inhibited (P<0.01), while the pretreatment with EGTA had no effect on the intracellular Ca(2+) concentration in the target cells treated with TM-TNF-alpha and the cytotoxicity of TM-TNF-alpha. CONCLUSION: These results suggest that both types of TNF-alpha increase Ca(2+) concentration in target cells by promoting the redistribution of intracellular free calcium and only S-TNF-alpha seems to be able to accelerate the influx of extracellular calcium into the target cells.
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Apoptosis/efectos de los fármacos , Calcio/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Ácido Egtácico/farmacología , Femenino , Células HL-60 , Hepatoblastoma/patología , Humanos , Neoplasias Hepáticas/patología , Linfoma de Células B/patología , Factor de Necrosis Tumoral alfa/clasificaciónRESUMEN
OBJECTIVE: To express the proteinase cathepsin L1 gene of Schistosoma japonicum (SjCL1) in Escherichia coli JM109 cells. METHODS: The SjCL1 gene was amplified from the recombinant plasmid pcDNA3-SjCL1 by PCR. The gene was cloned into a prokaryotic expression vector pGEX4T-1 to construct a recombinant plasmid pGEX-SjCL1. The E. coli JM109 cells were transformed with the recombinant plasmid pGEX-SjCL1 and the transformants were induced by IPTG to express the recombinant protein, the target protein was then identified by SDS-PAGE and Western blotting. RESULTS: A 1 kb length PCR product was obtained and a recombinant plasmid pGEX-SjCL1 was constructed. The expression product was detected by SDS-PAGE and Western blotting and an expression band about 62000 was found. CONCLUSION: The SjCL1 gene is effectively expressed in the E. coli JM109 cells.
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Catepsinas/biosíntesis , Proteínas Recombinantes/biosíntesis , Schistosoma japonicum/enzimología , Animales , Catepsinas/genética , Clonación Molecular , Escherichia coli/genética , Expresión Génica , Plásmidos/genética , Schistosoma japonicum/genéticaRESUMEN
AIM: To explore the relationship of p53 and the cytotoxicity mediated by TM-TNF-alpha and S-TNF-alpha. METHODS: P53 mutation in tumor cell was detected by PCR-SSCP. Wide type p53 expression plasmid was transfected to tumor cells with mutant p53 gene, while the mutant type p53 plasmid to tumor cell with wide type p53 gene.Then,the effects of transfection on the cytotoxicity of two types of TNF were detected. RESULTS: Mutation in the p53 gene were found in most of tumor cells (Raji, HL-60, K562) which were resistant to S-TNF-alpha. The tumor cells transfected with wild type p53 plasmid were more sensitive to S-TNF-alpha while the tumor cells transfected with mutant type p53 plasmid were less sensitive to S-TNF-alpha. But the cytotoxicity of TM-TNF-alpha was not affected by the transfection. CONCLUSION: The cytotoxicity mediated by TM-TNF-alpha is not dependent on wide type p53, which may account for the broader tumorcidal spectrum of TM-TNF-alpha than that of S-TNF-alpha.
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Línea Celular Tumoral/efectos de los fármacos , Genes p53 , Mutación , Factor de Necrosis Tumoral alfa/farmacología , Proteína p53 Supresora de Tumor/genética , Neoplasias de la Mama/patología , Exones , Células HL-60/efectos de los fármacos , Humanos , Plásmidos , Transfección , Factor de Necrosis Tumoral alfa/clasificaciónRESUMEN
AIM: To identify membrane-associated molecules involved in the tumoricidal cytotoxicity of TM-TNF-alpha and to explore the molecular mechanism underlying the tumoricidal cytotoxicity of TM-TNF-alpha and differences of biological effects between TM-TNF-alpha and sTNF. METHODS: Antibody blocking test and RT-PCR were used to evaluate the role of ICAM-1 and VCAM-1 in the cytotoxicity of TM-TNF-alpha mediated by TNFR I or TNFR II. RESULTS: After the ICAM-1 expressed on the membrane of MCF-7 cells(expressing TNFRI) was blocked, the cytotoxicity of TM-TNF-alpha to this cell lines decreased significantly, while blocking VCAM-1 had no effect. Blocking ICAM-1 or VCAM-1 expressed on the membrane of HL-60 cells(expressing TNFRII)had no significant effects on the cytotoxicity of TM-TNF-alpha to this cell lines. CONCLUSION: ICAM-1 participates in the cytotoxicity of TM-TNF mediated by TNFRI.
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Antígenos CD/fisiología , Molécula 1 de Adhesión Intercelular/fisiología , Receptores del Factor de Necrosis Tumoral/fisiología , Factor de Necrosis Tumoral alfa/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Humanos , Ratones , Receptores Tipo I de Factores de Necrosis Tumoral , Molécula 1 de Adhesión Celular Vascular/fisiologíaRESUMEN
AIM: To study the effect of rosiglitazone (RSG) on insulin secretion in isolated pancreatic islets under chronic exposure to free fatty acid (FFA) and to investigate the potential signaling mechanism of RSG action. METHODS: Rat pancreatic islets were cultured with or without FFA (2 mmol/L, oleate:palmitate, 2:1) in the presence or absence of RSG (0.05-10 micromol/L). The insulin release was measured by radioimmuoassay, the expression level of insulin receptor substrate-2 (IRS-2) protein and the association of IRS-2 with p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) were determined by immunoprecipitation and Western blot. RESULTS: The islets exposed to high FFA concentration showed an increased basal and a decreased glucose-induced insulin release as compared with control islets (P<0.01). IRS-2 protein level was decreased by 65 % (P<0.01) and the association of IRS-2 with p85 subunit of PI 3-kinase and was decreased by 73 % (P<0.01). When islets were cultured with FFA in the presence of RSG 5 micromol/L, both basal and glucose-induced insulin secretion were reversed to a pattern of control islets (P<0.01, P<0.05). The addition of RSG in the cultured medium increased significantly the expression of IRS-2 protein by 2.6 fold (P<0.01) and the association of IRS-2 with p85 by 2.7-fold (P<0.01) as compared with islets incubated with FFA alone. The effects of RSG on insulin secretion were blocked by a PI 3-kinase inhibitor, wortmannin. CONCLUSION: The effects of RSG on insulin secretion could be mediated through an IRS-2-associated PI 3-kinase signaling pathway.
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Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/metabolismo , Tiazoles/farmacología , Tiazolidinedionas , Animales , Células Cultivadas , Ácidos Grasos , Hipoglucemiantes/farmacología , Proteínas Sustrato del Receptor de Insulina , Secreción de Insulina , Péptidos y Proteínas de Señalización Intracelular , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Rosiglitazona , Transducción de Señal/efectos de los fármacosRESUMEN
AIM: To compare the effect of the two types of TNF-alpha, transmembrane TNF-alpha (TM-TNF-alpha) and secreted TNF-alpha (sTNF-alpha) on the biological functions of monocytic cell line U937 so as to explore the role of TM-TNF-alpha in inflammation. METHODS: Effect of the two types of TNF-alpha on the functions of U937 cells, such as phagocytosis, accumulation of cytokines' mRNA, the level of cytoplasmic IkappaB-alpha and expression of ICAM-1, was compared by phagocytosis, RT-PCR, Western blot and FACS. RESULTS: sTNF-alpha was found to be able to promote markedly phagocytosis by monocytic U937 cells, enhance the mRNA accumulation of cytokines, such as TNF-alpha, IL-1beta and IL-8 mRNA, induce the degradation of IkappaB-alpha and the expression of adhesion molecule ICAM-1; while TM-TNF-alpha was shown to have no effect on the functions above of U937 cells. Furthermore, it seemed TM-TNF-alpha did not have a synergic action with sTNF-alpha when U937 cells was treated with both types of TNF-alpha. CONCLUSION: The data above shows that sTNF-alpha can activate U937 cells, while TM-TNF-alpha has no influence. It suggests that the two types of TNF-alpha may play the different role in inflammation.
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Monocitos , Factor de Necrosis Tumoral alfa , Humanos , Molécula 1 de Adhesión Intercelular , Interleucina-8/metabolismo , Monocitos/metabolismo , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/genética , Células U937RESUMEN
AIM: To study the effects of TNF receptor blocking peptide on adjuvant arthritis in rats. METHODS: The model of rat adjuvant arthritis was induced by injection of complete Freund's adjuvant. The TNF receptor blocking peptide was injected locally in the ankle. The ankle swelling, the pathologic changes in the ankle joint and the expression of IL-1 beta mRNA and TNF-alpha mRNA by peritoneal macrophages (RT-PCR) were observed. RESULTS: The model of rat adjuvant arthritis induced by injection of complete Freund's adjuvant was similar to human rheumatoid arthritis. The treatment with TNF receptor blocking peptide for 10 days resulted in complete inhibition of joint swelling, a decrease in infiltration of inflammatory cell into joint tissue, an obvious alleviation of inflammatory pathological damages and an apparent decline of TNF-alpha mRNA and IL-1 beta mRNA of peritoneal macrophages of rats. CONCLUSION: The TNF receptor blocking peptide can protect the joint from inflammatory damage induced by adjuvant arthritis by suppression of TNF-alpha and IL-1 production, thereby alleviating the pathological injury of joint and controlling effectively the clinic course of arthritis.
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Articulación del Tobillo/patología , Artritis Experimental/inmunología , Macrófagos Peritoneales/metabolismo , Receptores del Factor de Necrosis Tumoral/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/biosíntesis , Animales , Artritis Experimental/patología , Interleucina-1/biosíntesis , Interleucina-1/genética , Masculino , Péptidos/farmacología , ARN Mensajero/genética , Ratas , Ratas Wistar , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
OBJECTIVE: To provide a theoretical basis for the study of vaccines against Schistosoma japonicum, the receptor for human LDL in the tegument of adult Schistosoma japonicum was investigated. METHODS: Proteins existed in adult Schistosoma japonicum membrane were extracted by Triton X-100 and purified through reverse-phase high performance liquid chromatography (HPLC), and the main protein peaks were then collected separately. 125I-LDL of human plasma as the ligand, through the radioautography and radioligand binding assay, the protein which can bind human serum 125I-LDL specifically was identified. The molecular weight and IEF were detected by SDS-PAGE. RESULTS: According to the radioautography and radioligand binding assay, a protein with retention time of 10.5 min was proved to be able to bind human serum 125I-LDL specifically. SDS-PAGE revealed that the molecular weight of the purified protein is 60-65 kDa, and its IEF is 6.7. CONCLUSION: LDL binding protein may exist on the surface of both male and female adult Schistosoma japonicum with the function of obtaining cholesterol from host circulating system.