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1.
Zhongguo Dang Dai Er Ke Za Zhi ; 25(10): 1089-1094, 2023 Oct 15.
Artículo en Chino | MEDLINE | ID: mdl-37905769

RESUMEN

The male patient was referred to the hospital at 44 days old due to dyspnea after birth and inability to wean off oxygen. His brother died three days after birth due to respiratory failure. The main symptoms observed were respiratory failure, dyspnea, and hypoxemia. A chest CT scan revealed characteristic reduced opacity in both lungs with a "crazy-paving" appearance. The bronchoalveolar lavage fluid (BALF) showed periodic acid-Schiff positive proteinaceous deposits. Genetic testing indicated a compound heterozygous mutation in the ABCA3 gene. The diagnosis for the infant was congenital pulmonary alveolar proteinosis (PAP). Congenital PAP is a significant cause of challenging-to-treat respiratory failure in full-term infants. Therefore, congenital PAP should be considered in infants experiencing persistently difficult-to-treat dyspnea shortly after birth. Early utilization of chest CT scans, BALF pathological examination, and genetic testing may aid in early diagnosis.


Asunto(s)
Proteinosis Alveolar Pulmonar , Insuficiencia Respiratoria , Lactante , Recién Nacido , Humanos , Masculino , Lavado Broncoalveolar/efectos adversos , Proteinosis Alveolar Pulmonar/diagnóstico , Proteinosis Alveolar Pulmonar/etiología , Proteinosis Alveolar Pulmonar/patología , Disnea/etiología
2.
J Acquir Immune Defic Syndr ; 91(S1): S27-S34, 2022 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-36094512

RESUMEN

BACKGROUND: With the increasing coverage of antiretroviral therapy, concerns for the emergence and transmission of HIV drug resistance (HIVDR) are arising. HIVDR was divided into 5 levels: sensitive, potentially resistant, low resistant, intermediate resistant, and high resistant. Most of the articles on HIVDR involved low-level, intermediate-level, and high-level drug resistance to antiretroviral drug, and few articles deal with potential drug resistance. Treatment failure associated with the level of low-level, intermediate-level, and high-level resistance to antiretroviral drug has been reported. However, whether virological failure (VF) is related to potential resistance remains unclear. In this study, we aimed to describe the situation of potential resistance to antiretroviral drug and whether it is related to VF. METHODS: We analyzed the demographic, behavioral information, medical history, and drug resistance-associated mutation data from subjects. Drug resistance mutations at baseline and time of failure in patients suffering VF were detected by using the Vela automated next-generation sequencing platform. The χ2 test or Fisher exact test and logistic regression were used to assess the risk factors that contribute to VF in the potential drug-resistant people. RESULTS: The prevalence of overall pretreatment drug resistance was 7.06% (233/3300), and the prevalence of pretreatment potential resistance was 8.79% (290/3300). All these patients with pretreatment potential first-line drugs resistance showed potential resistance to nonnucleoside reverse transcriptase inhibitors (NNRTIs), and some of them had potential drug resistance to NNRTIs and NRTIs or NNRTIs and PIs; among these patients, 94.71% (179/189) had V179 D/E mutations. The VF rate of first-line treatment for potentially resistant people is 17.99%. CD4+ T-cell count ≤200 cells/L at antiretroviral therapy initiation are risk factors for the failure of first-line treatment. CONCLUSIONS: The prevalence of potential drug resistance among individuals with HIV and the VF rate of first-line treatment for potential drug-resistant people were high. To better optimize clinical management, prevention, and control of HIV, attention should be devoted to the potential resistance of nonnucleoside drugs.


Asunto(s)
Síndrome de Inmunodeficiencia Adquirida , Fármacos Anti-VIH , Infecciones por VIH , VIH-1 , Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/uso terapéutico , Antirretrovirales/uso terapéutico , Farmacorresistencia Viral/genética , Infecciones por VIH/epidemiología , VIH-1/genética , Humanos , Inhibidores de la Transcriptasa Inversa/farmacología , Inhibidores de la Transcriptasa Inversa/uso terapéutico
3.
J Inflamm Res ; 15: 4751-4761, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36017172

RESUMEN

Objective: To explore the long-term effects of SARS-Cov-2 infection on the pulmonary function in the severe convalescent COVID-19 patients for 6 to 9 months follow-up in Beijing, China. Methods: A total of 64 cases of COVID-19 patients were recruited for the study and discharged from the Beijing Ditan Hospital, Capital Medical University, for 6 to 9 months. COVID-19 patients were divided into non-severe (mild and moderate) and severe groups. The follow-up investigated the lung function tests, the novel coronavirus antibody (IgM and IgG), chest CT and blood tests. Results: About 25.00% (16/64) patients had pulmonary ventilation dysfunction and 35.9% (23/64) had diffusion dysfunction. In the severe group, 56.50% (13/23) individuals showed decreased diffusion function. The diffusion dysfunction of the severe group was significantly decreased than the non-severe group (P = 0.01). Among 56 cases, the positive rate of IgG titers was 73.2% (41/56). The result of chest CT showed 55.36% (31/56) cases in nodules, 44.64% (25/56) in strip-like changes, 37.5% (21/56) in-ground glass shadow, and 5.36% (3/56) in grid shadow, which was significantly different between the severe group and the non-severe group. Patients tended to have ground glass changes in the severe group while nodules in the non-severe group. Conclusion: For the 6 to 9 months in convalescent COVID-19 patients, 56.50% (13/23) of severe patients had pulmonary diffusion dysfunction. Convalescent COVID-19 patients should have their pulmonary function regularly tested, especially those with severe illness.

4.
Food Chem ; 212: 403-10, 2016 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-27374549

RESUMEN

Cavitation-accelerated aqueous enzymatic extraction (CAEE) of seed oil from Cucurbita pepo was performed. An enzyme cocktail comprised of cellulose, pectinase and proteinase can work synergistically in releasing the oil. The CAEE extraction conditions were optimized by a Plackett-Burman design followed by a central composite methodology. A maximal extraction yield of 58.06% was achieved under optimal conditions of vacuum degree -0.07, enzyme amount 1.05% and extraction time 69min. As compared to soxhlet extraction (SE)-derived oil, CAEE-derived oil exhibited similar physical properties and better oxidation stability. In addition, chemical composition analyzing showed that the content of linoleic acid obtained by CAEE (47.67%) was higher than that of SE (44.51%). Moreover, the IC50 of oil obtained by CAEE and SE, as measured by α-amylase inhibition assay, were 40.68µg/mL and 45.46µg/mL. All results suggest that CAEE represents an excellent alternative protocol for production of oil from oil-bearing materials.


Asunto(s)
Cucurbita , Cromatografía de Gases y Espectrometría de Masas/métodos , Hipoglucemiantes/análisis , Aceites de Plantas/análisis , Semillas/química , Agua/análisis , Hipoglucemiantes/farmacología , Ácido Linoleico/análisis , Extracción Líquido-Líquido/métodos , Oxidación-Reducción , Aceites de Plantas/farmacología , alfa-Amilasas/análisis , alfa-Amilasas/antagonistas & inhibidores
5.
Zhongguo Zhong Xi Yi Jie He Za Zhi ; 34(9): 1096-102, 2014 Sep.
Artículo en Chino | MEDLINE | ID: mdl-25335334

RESUMEN

OBJECTIVE: To observe the effect of Huanglian Jiedu Decoction (HLJDD) in in vivo regulating differentiation of monocytes in an apolipoprotein E knockout (ApoE(-/-)) mouse model, and to observe the effect of HLJDD-containing serum in in vitro regulating differentiation of macrophages and foam cells. METHODS: Fifteen apoE(-/-) mice were randomly divided into the common diet group, the hyperlipidemia group, and the hyperlipidemia +HLJDD treatment group, 5 in each group. Mice in the common diet group were fed with a chow diet. Mice in the hyperlipidemia group were fed with high cholesterol wild diet (WD). Those in the hyperlipidemia +HLJDD treatment group were fed with high cholesterol WD supplemented with HLJDD. All mice were fed for 4 weeks. Five C57BL/6 wild types were recruited as the wild common diet control group. HLJDD was administered to mice in the hyperlipidemia + HLJDD treatment group by gastrogavage at the daily dose of 5 g/kg. Equal volume of purified water was given by gastrogavage to mice in the rest 3 groups. Four weeks later, subtypes of monocytes in the peripheral blood were detected by FACS. HLJDD administered to another 30 SD rats by gastrogavage at the daily dose of 5 g/kg, once for every 12 h for 5 times in total, thereby preparing 5% HLJDD containing serum to intervene the differentiation of in vitro primary bone marrow-derived macrophage (BMDM) and foam cells. The M2 subtype surface receptor CD206 of macrophages and foam cells were detected by FACS. The expression of Nos2 and Arg1 genes were assayed by Real-time PCR. RESULTS: The ratio of inflammatory subset of monocytes (Ly6C(high)) increased in the peripheral blood after ApoE(-/-) mice were fed with high fat diet for 4 weeks. HLJDD significantly decreased the ratio of inflammatory subset of monocytes (P < 0.05). Compared with the vehicle serum, 5% HLJDD containing serum significantly increased differentiation of CD206 + M2 BMDM (P = 0.034). Results of real-time quantitative PCR showed that the expression level of Arg1 mRNA could be up-regulated by HLJDD containing serum (P < 0.05), and that of Nos2 mRNA down-regulated (P = 0.017). ox-LDL induced the differentiation of M2 subtype foam cells from BMDM, and HLJDD containing serum could further elevate the ratio of CD206 + M2 foam cells and increase the Arg1 mRNA expression level (both P < 0.01). HLJDD containing serum could inhibit the inversion of M2 subtype of foam cells to M1 subtype induced by Th1 factors, significantly elevate the Arg1 mRNA expression level, and decrease the Nos2 mRNA expression level (all P < 0.01). CONCLUSIONS: HLJDD could lower hyperlipidemia induced inflammatory monocyte subtype ratios in the peripheral blood of ApoE(-/-) mice. HLJDD containing serum promoted in vitro differentiation of M2 macrophages and foam cells. HLJDD attenuated and inhibited the occurrence and development of atherosclerosis induced by hyperlipidemia possibly through regulating the functional differentiation of monocytes, macrophages, and foam cells.


Asunto(s)
Diferenciación Celular/efectos de los fármacos , Medicamentos Herbarios Chinos/farmacología , Células Espumosas/citología , Macrófagos/citología , Monocitos/citología , Animales , Apolipoproteínas E/genética , Femenino , Células Espumosas/efectos de los fármacos , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/efectos de los fármacos
6.
Yao Xue Xue Bao ; 49(3): 322-8, 2014 Mar.
Artículo en Chino | MEDLINE | ID: mdl-24961102

RESUMEN

In order to obtain the lead compound for treatment of rheumatoid arthritis (RA), in this study, therapeutic efficacy of three bispecific antibodies (BsAB-1, BsAB-2 and BsAB-3) against both hIL-1beta and hIL-17 were compared on CIA model mice. First, by ELISA method we compared the binding capacity of the three bispecific antibodies to the two antigens. The results showed that all three antibodies could simultaneously bind both antigens, among these antibodies, BsAB-1 was superior over BsAB-2 and BsAB-3. CIA model was established with chicken type II collagen (CII) and developed RA-like symptoms such as ankle swelling, skin tight, hind foot skin hyperemia. The CIA mice were treated with three antibodies once every two days for total of 29 days. Compared with the CIA model mice, the RA-like symptoms of the antibody treated-mice significantly relieved, while the BsAB-1 treated-mice were almost recovered. CII antibody level in the serum and cytokines (IL-2, IL-1beta, IL-17A and TNF-alpha) expression in the spleen were examined. Compared with the CIA model mice, all three antibodies could significantly reduce CII antibody and cytokine expression levels. BsAB-1 antibody was more potent than BsAB-2 and BsAB-3. In summary, BsAB-1 is superior over BsAB-2 and BsAB-3 in amelioration of RA symptoms and regulation of CII antibody production and pro-inflammatory cytokine expression, therefore, BsAB-1 can be chosen as a lead compound for further development of drug candidate for treatment of RA.


Asunto(s)
Anticuerpos Biespecíficos/uso terapéutico , Artritis Experimental/terapia , Artritis Reumatoide/terapia , Animales , Anticuerpos/metabolismo , Anticuerpos Biespecíficos/inmunología , Reacciones Antígeno-Anticuerpo , Artritis Experimental/inducido químicamente , Artritis Experimental/metabolismo , Artritis Reumatoide/inducido químicamente , Artritis Reumatoide/metabolismo , Colágeno Tipo II/inmunología , Interleucina-17/metabolismo , Interleucina-1beta/metabolismo , Interleucina-2/metabolismo , Masculino , Ratones , Bazo/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo
7.
Zhongguo Zhong Xi Yi Jie He Za Zhi ; 33(11): 1520-5, 2013 Nov.
Artículo en Chino | MEDLINE | ID: mdl-24483114

RESUMEN

OBJECTIVE: To observe the effect of Huanglian Jiedu IJecoction (HJU) on systemic and vascular immune responses of high fat diet fed apoE deficient (apoE(-/-)) mice. METHODS: Eight wild type C57BL6 mice were recruited as the wild type common food group. Totally 24 apoE(-/-) mice were randomly divided into the ApoE'common food group, the ApoE(-/-) hyperlipidemia group, and the ApoE(-/-) hyperlipidemia plus HJD group, 8 in each group. In the present study, the common food mice and high fat fed mice were fed with a chow diet or a high cholesterol diet for 4 weeks. HJD was given to mice in the ApoE(-/-) hyperlipidemia plus HJD group at the daily dose of 5 g/kg by gastrogavage, while equal volume of pure water was given to mice in the rest groups by gastrogavage. Four weeks later, the plasma levels of blood lipids, the ratio of peripheral blood mononuclear cells, and expressions of Toll-like receptor 4 (TLR-4) and CD36 on the monocytes were detected. The pathological changes and expressions of cytokines in local aorta were detected. The plasma cytokine levels in response to lipopolysaccharide (LPS) were analyzed. Results (1) Compared with the wild type common food group, TO, TG, and LDL-O significantly increased in the ApoE(-/-) common food group (P < 0. 05, P < 0.01). Compared with the ApoE(-/-) common food group, TC and LDL-C significantly increased in the hyperlipidemia group (P < 0. 05). There was no statistical difference in each index between the ApoE(-/-) hyperlipidemia group and the ApoE(-/-) hyperlipidemia plus HJD group (P > 0.05). (2) Compared with the wild type common food group, no obvious change of the ratio of peripheral blood mononuclear cells happened, the TLR4 expression level significantly increased in the ApoE'common food group (P < 0. 05). Compared with the ApoE common food group, the ratio of peripheral blood mononuclear cells and the TLR4 expression level significantly increased in the ApoE' hyperlipidemia group (P < 0.05). Compared with the ApoE(-/-) hyperlipidemia group, the ratio of peripheral blood mononuclear cells and the TLR4 expression level significantly decreased. Besides, the CD36 expression level also significantly decreased (P<0.05). (3) After stimulated by LPS for 3 h, compared with the wild type common food group, plasma TNF-ct and IL-b expressions significantly increased in the ApoE(-/-) common food group (P < 0.05). Compared with the ApoE(-/-) common food group, plasma expressions of IL-12, TNF-alpha, MCP-1, and IL-10 increased, but with no statistical difference in the ApoE(-/-) hyperlipidemia group (P > 0.05). After 4-week intervention of HJD, compared with the ApoE(-/-) hyperlipidemia group, the MCP-1 expression was significantly down-regulated, while the IL-10 expression significantly increased, showing statistical difference (P < 0.05). Compared with the wild type common food group, mRNA expression levels of IFN-gamma, MCP-1 , TNF-alpha, IL-10, and IL-1beta significantly increased (P < 0. 05, P < 0.01). Compared with the ApoE(-/-) common food group, not only mRNA expression levels of IFN-gamma, MCP-1, TNF-alpha, and IL-1beta, further significantly increased, but also IL-12, IL-10, and TGF-beta significantly increased (P < 0. 05, P < 0. 01). After 4-week intervention of HJD, compared with the ApoE(-/-) hyperlipidemia group, mRNA expression levels of MCP-1, TNF-alpha, IL-1beta, and IL-12 significantly decreased in the ApoE(-/-) hyperlipidemia plus HJD group (P < 0.05, P < 0.01). CONCLUSIONS: High fat diet induced systemic reaction and inflammatory reactions of local vessels. The local inflammatory response of vessels exceeded systemic inflammatory response. Intervention of HJD could attenuate inflammatory response, especially in local arteries. Meanwhile, it enhanced systemic anti-inflammatory reactions.


Asunto(s)
Medicamentos Herbarios Chinos/farmacología , Hiperlipidemias/inmunología , Síndrome de Respuesta Inflamatoria Sistémica/inmunología , Animales , Aorta/patología , Apolipoproteínas E/genética , Antígenos CD36/metabolismo , Quimiocina CCL2/metabolismo , Grasas de la Dieta/efectos adversos , Femenino , Hiperlipidemias/sangre , Hiperlipidemias/etiología , Inflamación , Interleucina-10/sangre , Interleucina-12/sangre , Interleucina-1beta/sangre , Leucocitos Mononucleares/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Síndrome de Respuesta Inflamatoria Sistémica/sangre , Síndrome de Respuesta Inflamatoria Sistémica/etiología , Receptor Toll-Like 4/metabolismo , Factor de Crecimiento Transformador beta/sangre , Factor de Necrosis Tumoral alfa/sangre
8.
Zhongguo Zhong Xi Yi Jie He Za Zhi ; 31(1): 23-7, 2011 Jan.
Artículo en Chino | MEDLINE | ID: mdl-21434338

RESUMEN

OBJECTIVE: To investigate the distribution of monocyte subpopulations and the changes of Toll-like receptor 4 (TLR4), CD163 and CD36 expressions in the peripheral blood of patients with hyperlipidemia of phlegm-stagnancy obstruction syndrome pattern (HLE-PSO), and to evaluate the intervening effects of Qutan Huayu Jiedu (QHJ) herbs on these parameters. METHODS: Monocytes in the peripheral blood were sorted using flow cytometry into 3 subpopulations: the CD14high CD16- (Mo1), the CD14high CD16+ (Mo2a) and the CD14low CD16+ (Mo2b) subpopulation. The percentages of various monocyte subpopulations in 83 patients and 42 matched healthy controls were determined and the levels of their surface receptors TLR4, CD163 and CD36 expressions were assayed with flow cytometer. Furthermore, patients allocated in the tested group (10 patients) and the control group (11 patients) were treated with QHJ Herbs and Qutan Huayu (QH, removing Phlegm and dissolving stagnancy but without detoxication) herbs respectively. The changes of monocyte subpopulations percentage and TLR4, CD163 and CD36 expressions were determined 4 weeks after they received treatment. RESULTS: Percentage of Mo2a subpopulation was significantly higher in HLE-PSO patients than the normal range (5.35 +/- 2.57 vs. 3.09 +/- 2.38, P < 0.01), but the deviation of the other two subpopulations in percentage was insignificant. The TLR4 expression on Mo1 monocyte in patients was lower than normal (50.73 +/- 24.45 vs. 69.92 +/- 21.06, P < 0.01), while CD163 and CD36 expressions of all three subpopulations in patients were similar to those in healthy persons respectively. After 4 weeks of treatment, a significant lowered Mo2a proportions were observed in the tested group (3.73 +/- 1.05 vs. 5.50 +/- 2.06, P = 0.043); but not in the control group (4.20 +/- 1.81 vs. 5.65 +/- 1.89, P = 0.097), while the levels of TLR4, CD163 and CD36 were not significantly altered after treatment in both groups. CONCLUSIONS: The elevated proportions of Mo2a subpopulation and the lowered TLR4 expression in Mol subpopulation are the characteristic changes in HLE-PSO patients, which might be related with the hyperlipidemia caused immune injury in patients. QHJ herbs could effectively improve the disproportion of Mo2a subpopulation.


Asunto(s)
Medicamentos Herbarios Chinos/uso terapéutico , Hiperlipidemias/tratamiento farmacológico , Monocitos/citología , Fitoterapia , Anciano , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Antígenos CD36/metabolismo , Estudios de Casos y Controles , Medicamentos Herbarios Chinos/farmacología , Femenino , Citometría de Flujo , Humanos , Hiperlipidemias/diagnóstico , Hiperlipidemias/metabolismo , Masculino , Medicina Tradicional China , Monocitos/efectos de los fármacos , Receptores de Superficie Celular/metabolismo , Receptor Toll-Like 4/metabolismo
9.
J Bone Miner Res ; 23(7): 1084-96, 2008 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-18302500

RESUMEN

High-mobility group box 1 (HMGB1), a nonhistone nuclear protein, is released by macrophages into the extracellular milieu consequent to cellular activation. Extracellular HMGB1 has properties of a pro-inflammatory cytokine through its interaction with receptor for advanced glycation endproducts (RAGE) and/or toll-like receptors (TLR2 and TLR4). Although HMGB1 is highly expressed in macrophages and differentiating osteoclasts, its role in osteoclastogenesis remains largely unknown. In this report, we present evidence for a function of HMGB1 in this event. HMGB1 is released from macrophages in response to RANKL stimulation and is required for RANKL-induced osteoclastogenesis in vitro and in vivo. In addition, HMGB1, like other osteoclastogenic cytokines (e.g., TNFalpha), enhances RANKL-induced osteoclastogenesis in vivo and in vitro at subthreshold concentrations of RANKL, which alone would be insufficient. The role of HMGB1 in osteoclastogenesis is mediated, in large part, by its interaction with RAGE, an immunoglobin domain containing family receptor that plays an important role in osteoclast terminal differentiation and activation. HMGB1-RAGE signaling seems to be important in regulating actin cytoskeleton reorganization, thereby participating in RANKL-induced and integrin-dependent osteoclastogenesis. Taken together, these observations show a novel function of HMGB1 in osteoclastogenesis and provide a new link between inflammatory mechanisms and bone resorption.


Asunto(s)
División Celular/fisiología , Proteína HMGB1/fisiología , Osteoclastos/citología , Ligando RANK/fisiología , Receptores Inmunológicos/fisiología , Animales , Secuencia de Bases , Western Blotting , Cartilla de ADN , Técnica del Anticuerpo Fluorescente , Ratones , Ratones Noqueados , Receptor para Productos Finales de Glicación Avanzada
10.
Neurosci Bull ; 22(1): 29-33, 2006 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-17684536

RESUMEN

Objective To clone the cDNA of rat alpha-Syn gene, investigate its prokaryotic expression and produce purified recombinant rat alpha-Syn protein. Methods Rat alpha-Syn cDNA was amplified from the rat brain total RNA by RT-PCR and was cloned into pGEX-4T-1, a prokaryotie expressing vector. The recombinant plasmid containing rat alpha-Syn gene was transformed into E. Coli BL21 to express a fusion protein with rat alpha-Syn protein tagged by glutathione-S-transferase (GST). The fusion protein was then cleaved by thrombin during passing through the GST-agarose 4B column to release the recombinant rat alpha-Syn protein. The recombinant rat alpha-Syn protein was further purified using Superdex S200 gel filtration.Results DNA sequencing confirmed that the cloned cDNA contained 420 base pairs encoding 140 amino acids, which was identical to the reported amino acid sequence of rat alpha-Syn. After transformation, the recombinant plasmid pGEX-raSyn expressed a soluble protein that was inducible by IPTG. The purified recombinant protein was shown to be single band on SDS-PAGE, with a molecular size of around 18000, which was identical to the reported molecular size of rat alpha-Syn.Western blot analysis demonstrated that the recombinant protein was recognized by specific antibody against alpha-Syn. Conclusion The rat alpha-Syn gene was successfully expressed in prokaryotic expression system and highly purified rat alpha-Syn recombinant protein was produced.

11.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 21(6): 676-8, 2005 Nov.
Artículo en Chino | MEDLINE | ID: mdl-16256023

RESUMEN

AIM: To study the effect of HLA-G1 molecule expressed by an endothelial cell line (ECV304) on the cytotoxic activity of allogeneic NK cells. METHODS: ECV304 cells were transfected with recombinant plasmid pcDNA3-HLA-G1 by the liposome transfection, and the expressed HLA-G1 on the cell surface was detected by indirect immunofluorescent assay and flow cytometry. The cytotoxic activity of allogeneic NK cells against ECV304 cells was analyzed by the MTT method. RESULTS: HLA-G1 was expressed on the surface of the transfected ECV304 cells. The specific lysis of NK cells against plasmid pcDNA3 transfected ECV304 was (50.6+/-18.1)%, while the specific lysis against pcDNA3-HLA-G1 transfected ECV304 was (29.7+/-11.4)%, which was significantly lower than the former (P<0.001). CONCLUSION: HLA-G1 expressed by the ECV304 cells can inhibit cytotoxicity of allogeneic NK cells.


Asunto(s)
Antígenos HLA/inmunología , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/fisiología , Línea Celular , Células Cultivadas , Pruebas Inmunológicas de Citotoxicidad , Citometría de Flujo , Técnica del Anticuerpo Fluorescente Indirecta , Vectores Genéticos/genética , Antígenos HLA/genética , Antígenos HLA-G , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Liposomas , Transfección
12.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 21(3): 265-8, 2005 May.
Artículo en Chino | MEDLINE | ID: mdl-15862135

RESUMEN

AIM: To form soluble HLA-G1-peptide complex by refolding in vitro, and to study its immune function. METHODS: The heavy chain and beta(2m) of sHLA-G1 were expressed as insoluble aggregates in E. coli, and then the two subunits were refolded to form HLA-G1-peptide complex by dilution method in the presence of specific peptide. The refolded product was purified through Sephadex G-75 gel filtration. The purified product was identified by Western blot with mAb W6/32. The function of soluble HLA-G1 was explored from following three aspects, namely, the influences on cytotoxicity of NK cells, on proliferation of T cells in mixed lymphocyte culture and apoptosis of activated T cells. RESULTS: The refolded complex was recognized by mAb W6/32. It effectively inhibited cytotoxicity of NK cells and proliferation of T cells, and induced apoptosis of activated T cells. CONCLUSION: The refolding of soluble HLA-G1-peptide complex has been successfully realized in vitro. The complex can inhibit the functions of NK cells and T cells.


Asunto(s)
Antígenos HLA/inmunología , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Péptidos/química , Péptidos/metabolismo , Renaturación de Proteína , Animales , Apoptosis/inmunología , Western Blotting , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Proliferación Celular , Antígenos HLA/química , Antígenos HLA-G , Antígenos de Histocompatibilidad Clase I/química , Humanos , Células Asesinas Naturales/inmunología , Pliegue de Proteína/efectos de los fármacos , Renaturación de Proteína/efectos de los fármacos , Urea/farmacología
13.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 21(2): 167-70, 2005 Mar.
Artículo en Chino | MEDLINE | ID: mdl-15766400

RESUMEN

AIM: To improve the refolding efficiency of soluble HLA-A2-peptide complex in vitro. METHODS: The heavy chain (HC) of MHC class I was extracted from bacteria under denaturing and non-reducing conditions. Anion-exchange and (NH4)2SO4 precipitation were applied to purify the HC. Then the purified HC, beta2m and an antigenic peptide (N-YMDGTMSQV-COOH of Try(369-377)) were refolded to form an HLA-A2-peptide complex by dilution method in the buffer of pH 6.6. The refolded products were detected by Western blot and ELISA with W6/32 and anti-human beta2m antibody. RESULTS: The refolded products consisted of HLA-A2-peptide complex, beta2m, and a little amount of HC polymer. The refolding efficiency was 2.5 fold higher than that of the conventional method. CONCLUSION: This study confirmed that the refolding efficiency of the method reported in this paper is higher as compared with the conventional method, which is of importance to the preparation of HLA-peptide tetramers and artificial antigen presenting cells.


Asunto(s)
Disulfuros/química , Antígeno HLA-A2/química , Antígeno HLA-A2/metabolismo , Péptidos/química , Péptidos/metabolismo , Renaturación de Proteína/efectos de los fármacos , Secuencia de Aminoácidos , Sulfato de Amonio/farmacología , Animales , Células Presentadoras de Antígenos/citología , Electroforesis en Gel de Poliacrilamida , Humanos , Concentración de Iones de Hidrógeno , Cuerpos de Inclusión/metabolismo , Pliegue de Proteína , Solubilidad
14.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 20(3): 265-8, 2004 May.
Artículo en Chino | MEDLINE | ID: mdl-15193213

RESUMEN

AIM: To refold and biotinylate HLA-A2-peptide complex in-vitro. METHODS: The BirA substrate peptide (BSP) containing H chain of HLA-A2 and beta(2m) were expressed highly as insoluble aggregates in E.coli, and then the two subunits were refolded to form an HLA-A2-peptide complex by dilution method in the presence of an antigenic peptide (NH(2)-CLGGLLTMV-COOH of EB virus latent membrane protein 2A LMP2A). Then the BirA enzyme was used to biotinylate the refolded complex. The refolded and biotinylated products were detected by ELISA and Western blot with mAb W6/32 and rabbit anti-human beta(2m) antibody and streptavidin. RESULTS: The refolded complex was composed of H chain aggregate, HLA-A2-peptide complex and beta(2m). Both HLA-A2-peptide complex and the H chain aggregate could be biotinylated. CONCLUSION: The refolding and biotinylation of HLA-A2-peptide complex were successfully performed and the products were confirmed by our practical immunological method. This study laid the foundation for the preparation of HLA-peptide tetramer and artificial antigen presenting cells.


Asunto(s)
Biotinilación , Escherichia coli/metabolismo , Antígeno HLA-A2/metabolismo , Pliegue de Proteína , Microglobulina beta-2/metabolismo , Ligasas de Carbono-Nitrógeno/metabolismo , Proteínas de Escherichia coli/metabolismo , Antígeno HLA-A2/genética , Proteínas Represoras/metabolismo , Solubilidad , Factores de Transcripción/metabolismo , Microglobulina beta-2/genética
15.
Zhonghua Yi Xue Za Zhi ; 83(7): 584-7, 2003 Apr 10.
Artículo en Chino | MEDLINE | ID: mdl-12887750

RESUMEN

OBJECTIVE: To investigate the immune tolerance inducing effects of soluble human leucocyte antigen G1 (sHLA-G1) on natural killer (NK) cells and T cells. METHODS: A recombinant plasmid expressing sHLA-G1 was constructed and transfected into human lymphoblastoid cells LCL721.221. sHLA-G1 in the supernatant was purified by immuno-affinity chromatography and then added into the culture of NK cells obtained from the peripheral blood mononuclear cells of 3 unrelated individuals. Target cells, K562 cells, were added too. The killing rate of NK was calculated. Peripheral blood lymphocytes (PBLs) were obtained and stimulated by Ebstein-Barr-virus-transformed B lymphoblastoid cell line (EBV-LCL). The proliferation of the T cells in the mixed lymphocyte culture was examined by enzyme linked immunosorbent assay. The antigen-specific T cells in the peripheral blood was activated. sHLA-G1 was added into the culture. Then the T cells were suspended in the solution of fluorescence isothiocyanate (FITC)-annexin-V. Flow cytometry was used to detect the fluorescent intensity of FITC so as to examine the apoptosis of T cells. RESULTS: sHLAG-1 inhibited the cytotoxicity of NK cells dose-dependently. sHLAG-1 inhibited the proliferation of activated T cells, and induced the apoptosis of T cells dose-dependently, with a dose-saturation character and without antigen-specificity. CONCLUSION: sHLAG-1 is a kind of immune tolerance inducing molecule.


Asunto(s)
Antígenos HLA/fisiología , Antígenos de Histocompatibilidad Clase I/fisiología , Tolerancia Inmunológica , Células Asesinas Naturales/inmunología , Linfocitos T/inmunología , Apoptosis , Citotoxicidad Inmunológica , Antígenos HLA-G , Humanos , Prueba de Cultivo Mixto de Linfocitos
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