The effect of artesunate on the expression of HO-1 induced by ESAT-6/CFP-10 of mycobacterium tuberculosis / 中国医师杂志

Objective To investigate the effect of artesunate (ASN) on the expression of Heme oxygenase-1 (HO-1) in THP-1 cells induced by the early secretory antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) antigens of Mycobacterium tuberculosis and to investigate its possible mechanism.Methods THP-1 ceils were cultured in vitro.The effects of ESAT-6 and CFP-10 on cell viability were detected by methyl thiazolyl tetrazolium (MTT) assay.THP-1 cells were pre-treated with or without ASN prior to incubation with or without ESAT-6 and CFP-10,the mRNA expression of HO-1 was detected by real time quantitative polymerase chain reaction (RT-qPCR) and Toll-like receptor 2 (TLR2) level was measured by Western blot.Results MTF assay showed that ESAT-6 and CFP-10 were non-toxic to cells in the range of 0-5 μg/ml.Compared with the control group,5 μg/ml ESAT-6 and 5 μg/ml CFP-10 could significantly increased the mRNA expression of HO-1 (P ＜ 0.05).In addition,20 μg/ml ASN could significantly enhance the mRNA expression of HO-1 induced by ESAT-6 and CFP-10,and inhibit the expression of TLR2 induced by ESAT-6.Conclusions ASN in combination with ESAT-6 or CFP-10,may have potential value in treatment of pathogen-associated inflammatory diseases.

Gene cloning and expression of the Tp0453 antigen immuno-dominant epitope fragment of Treponema pallidum and its potential use in serodiagnosis of syphilis / 中华传染病杂志

Objective To clone and express the Tp0453 antigen immuno-dominant epitope fragment of Treponema pallidum (Tp) in Escherichia coli,in an effort to develop serological tests with increased specificity for the diagnosis of syphilis.Methods The gene encoding Tp0453 recombinant outer membrane protein fragment was amplified by polymerase chain reaction (PCR),and inserted into expression vector pQE30 after T-A cloning,then confirmed by restriction map.The constructed recombinant plasmid pQE30-Tp0453 was transformed to E.coli M15 for expression induced by isopropyl β-D-1-thiogalactopyranoside.The expressed product was identified by Western blot,and purified by Ni2+-NTA agarose column chromatography.A double antigen sandwich enzymelinked immunosorbent assays (ELISA) was established by using the recombinant Tp0453 protein to test sera from 48 patients with positive Treponema pallidum particle agglutination test (TPPA),and 40 negative sera as control.Results The PCR amplicon of the target gene was about 490 bp.The recombinant plasmid pQE30-Tp0453 was correctly constructed and successfully expressed in E.coli M15.The expressed product,with a relative molecular of about 21 000,existed in a form of inclusion body,accounting for about 18％ of total somatic protein,and reached a purity of more than 95％ after purification.Western blot showed specific reaction of the expressed protein with Tp positive serum.The ELISA tests with the 88 clinical samples yielded a sensitivity of 97.9％ (47/48),and specificity of 100.0 ％ (40/40).The consistency of results between the ELISA test and the TPPA test was 98.9 ％ (87/88).Conclusion The expressed Tp0453 fragment has showed good immunoreactivity with serum from patients with syphilis,providing the foundation of further development of serological diagnostic kit with increased specificity for the diagnosis of TP infection.

The significance in diagnosing tuberculous meningitis by detecting specific antigen in cerebrospinal fluid / 中华神经科杂志

Objective To evaluate the detection of culture filtrate protein 10 (CFP10) and 6000 early secretory antigenic target (ESAT-6) in cerebrospinal fluid to be used in diagnosing tuberculous meningitis. Methods Dot enzyme linked immunosorbent assay ( Dot ELISA) method that was improved by applying concentrated cerebrospinal fluid was used to detect CFP10 and ESAT-6 in cerebrospinal fluid to analyze small protein antigen secreted by M. tuberculosis. Cerebrospinal fluid of 111 subjects were collected,in which 58 specimens were clinically diagnosed as tuberculous meningitis and 53 as non-tuberculous.CFP10 and ESAT-6 were detected in cerebrospinal fluid using Dot ELISA method and the results were analyzed. Results The sensitivities of detecting CFP10 and ESAT-6 antigen were 93.1% and 91.4% respectively, and the specificities were 92. 5% and 94. 3% respectively. The sensitivities and specificities are generally higher compared with the other methods of detecting M. tuberculosis or materials of M. tuberculosis by acid-fast staining or mycobacterium tuberculosis culture and polymerase chain reaction.Conclusions Using Dot ELISA method to detect CFP10 and ESAT-6 in cerebrospinal fluid to diagnose tuberculous meningitis has a high sensitivity and specificity. Our study provided the evidence of detecting the specific antigen of M. tuberculosis to be used in diagnosing tuberculosis.

Preparation of recombinant PPE65 protein of Mycobacterium tuberculosis and its applications in serodiagnosis of patients with pulmonary tuberculosis / 中华检验医学杂志

Objective To evaluate the potential value of IgG antibodies against recombinant PPE65 protein (rPPE65) of Mycobacterium tuberculosis in serodiagnosis of tuberculosis.Methods The gene encoding PPE65 protein of M.tuberculosis was cloned into the PET-28a vector and then expressed in Escherichia coli.The rPPE65 was purified with Ni-NTA affinity and ion exchange chromatography.After dialysis renaturation, the concentration of rPPE65 was determined using Lowry assay.ELISA was used to detect the levels of specific IgG against rPPE65 and recombinant PstS1 protein (rPstS1) in sera from 144 patients with pulmonary tuberculosis (PTB patients), 144 health controls, and 56 patients with non-tuberculosis pulmonary diseases.ROC curves were used to determine cut-off values with the results of IgG antibodies against rPPE65 and rPstS1 for 144 PTB patients and 97 controls with negative PPD skin test.The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of rPPE65 and the combination of rPPE65 and rPstS1 were counted.Results The PPE65 protein of M.tuberculosis was successfully expressed in E.coli. The purity and concentration of rPPE65 were 95% and 0.5 mg/ml, respectively.ROC analysis showed that the cut-off of ELISA using rPPE65 was 0.64.The sensitivity, specificity, PPV, NPV, and accuracy of rPPE65 were 34.7%(50/144), 93.5%(187/200), 79.4%(50/63), 66.5%(187/287), and 68.9%(237/344), respectively.The sensitivity, specificity, PPV, NPV, and accuracy of the combination of rPPE65 and rPstS1 were 59.0%, 91.0%, 82.5%, 75.5%, 77.6%, respectively.Conclusions The rPPE65 of M.tuberculosis appears to be a candidate antigen for serodiagnosis of tuberculosis.Detection of IgG antibodies against the combination of rPPE65 and rPstS1 can increase the sensitivity of serological test for tuberculosis.

Estudo retrospectivo das infecções pós-operatórias em cirurgia de coluna: correlação com o número de limpezas cirúrgicas realizadas / Estudio retrospectivo de infecciones postoperatorias en cirugía de espina dorsal: correlación con el número de desbridamientos quirúrgicos realizados / Retrospective study of post-operative infections in spine surgery: correlation with the number of surgical debridement performed

OBJETIVO: Avaliar as características das infecções pós-operatórias e determinar a resolução das mesmas em relação ao número de limpezas e de agentes infectantes. MÉTODO: Foram avaliados todos os prontuários dos pacientes que evoluíram com infecção pós-operatória durante 30 meses para análise e correlação de diversas variáveis. Nesses 30 meses, 40 pacientes evoluíram com infecção pós-operatória de um total de 410 cirurgias. Foram excluídos os casos de infecção primária da coluna (osteomielite ou espondilodiscite) totalizando três casos. Variáveis relacionadas ao paciente, ao procedimento e à evolução foram avaliadas e correlacionadas com as variáveis chaves: número de limpezas cirúrgicas e de agentes infectantes isolados nas culturas. RESULTADOS: A taxa de infecção pós-operatória foi de 9,83 por cento. Foram relacionadas as diversas variáveis estudadas com o número de limpezas cirúrgicas realizadas e não foi possível estabelecer uma relação. No entanto verificou-se que os pacientes com maior número de procedimentos cirúrgicos apresentavam maior taxa de dor pós-operatória. CONCLUSÃO: Pacientes submetidos a um maior número de procedimentos apresentaram mais dor na evolução pós-operatória. Não houve correlação estatisticamente significativa entre o número de limpezas ou de agentes com as demais variáveis. Um maior número de pacientes no estudo pode ser necessário para identificar outras relações.

OBJECTIVE: To evaluate the characteristics of post-operative infections and determine their resolution in relation to the number of surgical debridement and infectious agents. METHOD: We collected all records of patients who developed post-operative infection for 30 months and several variables were analyzed and correlated. In those 30 months, 40 patients developed post-operative infection of a total of 410 surgeries. We excluded cases of primary infection of the spine (osteomyelitis or spondylodiscitis) totaling 3 cases. Variables related to the patient, procedure and outcome were evaluated and correlated with the key variables: number of surgical debridement and infectious agents isolated from cultures. RESULTS: The rate of infection after surgery was 9.83 percent. Several variables were related to the number of surgical debridement performed and it was not possible to establish any relationship. However, it was found that patients with higher number of surgical procedures had a higher rate of post-operative pain. CONCLUSION: Patients receiving a greater number of procedures had more post-operative pain . There was no statistically significant correlation between the number of debridement or infectious agents or with other variables. A study with a larger number of patients may be needed to identify other relationships.

OBJETIVO: Evaluar las características de infecciones postoperatorias y determinar la solución de ellas con relación al número de limpiezas y desbridamientos quirúrgicos, y agentes infecciosos. MÉTODO: Recolectamos, para un período de 30 meses, todos los registros de pacientes que tuvieron infección postoperatoria y varias variables fueron analizadas y correlacionadas. En esos 30 meses, 40 pacientes, de un total de 410 cirugías, tuvieron infección postoperatoria. Excluimos casos de infección primaria de la espina dorsal (osteomielitis o espondilodiscitis) totalizando 3 casos. Variables relativas al paciente, realización de tratamientos y resultados fueron evaluadas y correlacionadas con las variables clave: número de desbridamientos quirúrgicos y cultivos aislados de agentes infecciosos. RESULTADOS: La tasa de infección, después de la cirugía, fue 9,83 por ciento. Algunas variables fueron relacionadas con el número de desbridamientos quirúrgicos realizados y no fue posible establecer alguna relación. No obstante, se descubrió que pacientes con un mayor número de tratamientos quirúrgicos realizados tuvieron una tasa mayor de dolor postoperatorio. CONCLUSIÓN: Pacientes que recibieron un mayor número de tratamientos realizados tuvieron más dolor en el período postoperatorio. No hubo correlación, estadísticamente significativa, entre el número de desbridamientos o agentes infecciosos, o con otras variables. Puede ser necesario tener un mayor número de pacientes en el estudio, a fin de identificar otras relaciones.

Comparison of three different novel Chlamydophila pneumonia recombinant antigens for serodiagnosis / 中华检验医学杂志

Objective To evaluate three different Chlamydophila pneumoniae recombinant antigens for use in Chlamydophila pneumoniae serodiagnosis. Methods The recombinant plasmids pGEX6p-2/ Cpn0146,Cpn0147 and Cpn0308 were constructed and expressed as GST fusion proteins. The immunogenicity and the immunocompetence of these recombinant protein were analyzed by Western-blot and indirect ELISA. A total of 183 sera samples of patients with respiratory tract infection and 32 sera samples of patients with Chlamydia trachomatis infection were detected with indirect ELISA coated microwell plates with the purified recombinant proteins comparing with SeroCP-TM IgG ELISA kits. The positive recognition rate, sensitivity and specificity of each method were analyzed. Results GST-Cpn0146, Cpn0147 and Cpn0308 were obtained after expression and purification. The titers of the specific IgG antibodies against Cpn0146, Cpn0147 and Cpn0308 were higher than 1:6 400, 1:128 00 and 1:128 00, respectively. When the indirect ELISA was developed to detect the IgG antibody against Chlamydophila pneumoniae in 183 samples, the concordance rate between the indirect ELISA test and SeroCP-TM IgG ELISA kits were 92. 3% (Cpn0146) , 94.5% (Cpn0147) and 96.7% (Cpn0308), respectively. The recombinant Cpn0146, Cpn0147 and Cpn0308 were recognized by 71 (38.8% positive recognition rate), 75 (40.9%), and 82 (44.8%) samples, respectively. The recombinant antigen-based detection assays displayed > 97% of detection specificity and>87%of sensitivity.Condusion GST-Cpn0308 shows a better sensitivity and specificity,which suggests it could be used for developing serodiagnosis kits of Chlamydophila pneumoniae infection.

Characteristics of the antigenic genes of four clinical Bordetella pertussis strains / 中华检验医学杂志

Objectives To analyze the characteristics of antigenic genes of clinical Bordetella pertussis strains recently isolated by analyzing the sequence of pertussis toxin S1 subunit(ptxS1) , pertactin (Prn) , fimbriae 2 (Fim2) and fimbriae 3 (Fim3 ) genes of four clinical isolates. Methods The 4 clinical isolates were collected in 2002 in Shijiazhuang of Hebei province. Four strains were isolated from pertussis patient's nasopharyngeal aspirate. ptxS1, Prn, Fim2 and Fim3 genes of these strains were amplified and sequenced. The sequences of those genes were compared with those of the isolates in GenBank and the isoaltes used in the production of pertussis vaccine in China. Results The results of the gene sequencing showed the four clinical isolates belonged to ptxS1 A type, which were different from those in vaccine strains. In addition, three Prn and three Fim'3 variants were observed in the four clinical isolates. Sequence analysis showed that the nucleotide sequence and deduced amino acid sequence of those strains had more than 99% identity with those in vaccine strains. The phylogenetic trees of those genes also showed these strains had a higher level of similarity with other Bordetella pertussis strains. Conclusion The four clinical isolates are different from vaccine strains in four antigenic genes, which laid a foundation for further studies on pertussis epidemiology,quality control and development of pertussis vaccine in China.

Could gastric histology be a useful marker for making decision on Helicobacter pylori eradication therapy in patients with dyspepsia? / É a histologia gástrica um marcador útil na decisão de erradicar o Helicobacter pylori nos pacientes com dispepsia?

CONTEXT: It still remains an open debate whether Helicobacter pylori eradication is beneficial or not for the improvement of symptoms in functional dyspepsia. Differences in geographic distribution, the worldwide H. pylori genetic variability and the fact that the outcome of infection is strongly related to the virulence of the infecting strain are factors that might be driving ongoing controversies. OBJECTIVE: To study the correlation between gastric histology and H. pylori serology status in patients with dyspepsia. METHODS: This is a cross-sectional study where 40 consecutive dyspeptic patients (28 women and 12 men, mean age 48.5 years) with endoscopically normal stomachs were selected from the endoscopy unit at a university hospital in Recife, PE, Northeast of Brazil, between March 1998 and July 1999. Patients underwent gastric mucosal biopsy and serological tests (anti-Hp and anti-CagA antibodies). Gastric biopsies were examined using H-E and Giemsa stains and gastritis was classified and graded (mild, moderate or severe) according to "the updated Sydney System - Houston, 1994". RESULTS: Among 40 patients with dyspepsia the gastric histology revealed that about » had moderate (25 percent) or severe (2.5 percent) gastritis. This subgroup of patients also had a greater positive frequency of anti-Hp (100 percent vs 41 percent; P = 0.0005) and anti-CagA (91 percent vs 58 percent; P = 0.09) antibodies when compared with those with normal histology (27.5 percent) or mild gastritis (45 percent). CONCLUSION: Since upper gastrointestinal endoscopy is part of the functional dyspepsia investigation and serology for anti-CagA antibody is not available in daily clinical practice, by biopsying gastric mucosa we would only be able to selectively apply H. pylori eradication therapy for those with histology that best correlate with virulent infecting strains (moderate or severe gastritis) - around » of our study patients with dyspepsia.

CONTEXTO: O benefício da terapia de erradicação do H. pylori como parte do tratamento da dispepsia funcional ainda é uma questão em aberto. Diferenças na distribuição geográfica, a ampla variabilidade genética e o fato de que a expressão clínica da infecção está fortemente relacionada com a virulência das cepas infectantes, são fatores que provavelmente guiam as controvérsias. OBJETIVO: Estudar a correlação entre histologia gástrica e sorologia para H. pylori em doentes com dispepsia. MÉTODOS: Estudo descritivo-transversal com 40 pacientes consecutivos com sintomas dispépticos (28 mulheres e 12 homens, média de idade de 48,5 anos) e achado endoscópico de estômago normal, selecionados a partir da sala de endoscopia (Hospital das Clínicas da Universidade Federal de Pernambuco, Recife, PE.) entre março de 1998 e julho de 1999. Todos foram submetidos a biopsias gástricas e testes sorológicos (anti-Hp e anti-CagA). As biopsias foram analisadas pelos métodos de H-E e Giemsa e os achados de gastrite classificados de acordo com o Sistema Sydney atualizado. RESULTADOS: A histologia dos 40 pacientes revelou que cerca de » apresentava gastrite moderada (25 por cento) ou severa (2,5 por cento). Esse grupo também apresentava maior frequência de positividade anti-Hp (100 por cento vs 41 por cento; P = 0,0005) e anti-CagA (91 por cento vs 58 por cento; P = 0,09) quando comparado com os casos com histologia normal (27,5 por cento) ou gastrite leve (45 por cento). CONCLUSÃO: Considerando que a endoscopia digestiva alta é parte da rotina de investigação da dispepsia funcional e que a sorologia anti-CagA não está disponível na prática clínica diária, através da histologia pode-se selecionar e aplicar terapia de erradicação do H. pylori apenas para os casos que muito provavelmente estão associados a cepas patogênicas de H. pylori (doentes com gastrite moderada ou severa) - cerca de » da presente amostra.

The stability of Vietnam-producted poliomyelitis vaccine from the year 2000 to 2002

Whole studied poliomyelitis vaccine bulk lots had had sensitivity in high temperature and low concentration of NaHCO3. They all met WHO standards

Comparative study on two DNA vaccines of tuberculosis Ag85A antigen / 中华传染病杂志

Objective To explore new method for enhancing the efficacy of tuberculosis DNA vaccine. Methods Two recombinant plasmids were constructed, one named as pBK GM/85A encoding mouse granulocyte macrophage colony stimulating factor(GM CSF) fused to Mycobacterium tuberculosis Ag85A antigen, the other named as pBK 85A encoding Mycobacterium tuberculosis Ag85A antigen alone. Subsequently, the two plasmids were transferred into cultured COS7 cells by using cationic liposomes. The expression products were identified by Western blotting. Then, in a murine model, we compared the immunogenicity and protective immunity of the two recombinant plasmids following genetic immunization. Results All pBK GM/85A injected mice elicited higher antibody titres than that for pBK 85A injected mice. Lymphocytes obtained from the spleen of pBK GM/85A immunized mice exhibited higher lymphocyte proliferative response、IFN ? production and CTL activity than that for pBK 85A immunized mice. The protective efficacy was also higher for pBK GM/85A immunized mice than that for pBK 85A immunized mice. However, The protective efficacy for pBK GM/85A immunized mice was lower than that for BCG immunized mice. Conclusion These results showed that DNA vaccines with GM CSF/antigen fusion constructs could greatly improve the immunogenicity of DNA vaccine against Mycobacterium tuberculosis.

Detection of M. leprae-specific Antigen with M-Dot-ELISA in Sera from Household Contacts of Leprosy Patients / 中华皮肤科杂志

We report the results of detecting M. lepraespecific antigen--phenolic glycolipid I (PGL--I) with modified M-Dot-ELISA in sera from 75 cases of household contacts (HC)of leprosy patients, which were all seropositive by Gelatin Particle Agglutination Test (MLPA)and ELISA. The results indicate that: (1 ) 16/75 (21. 3 % ) are antigen positive. The rates of positivity in HC of MB patients are much higher than those in HC of PB patients,the difference (P