False positive reaction of COVID-19 antibody ELISA screening: internal logic of various methods / 中国输血杂志

【Objective】 To analyze the results of different methods for reactive samples screened by the enzyme linked immunosorbent assay (ELISA) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in blood donors. 【Methods】 From March to April 2020, a total of 8 632 blood samples in Shenzhen were screened for SARS-CoV-2 total antibodies (TAb, including IgG, IgM, IgA) in plasma using ELISA(PC group), the antibody reactivity samples and their follow up plasma samples (FC group), and samples of disease control group(DC group) from January to April 2020 were detected using the following methods: 1) ELISA method for detecting IgG, IgM, and (or without detection) TAb; 2) pseudovirus neutralizing antibody test(pVNT); 3) western blot (WB) of SARS-CoV-2 antibody. The negative control group(NC group) from February to April 2020 performed ELISA and WB testing. 【Results】 Among the 34 total antibody positive samples, 2 were positive for pVNT test, and the total antibody, IgG and WB in the initial screening and tracking testing were positive. Thereafter, it was determined to be confirmed positive. The other 2 cases were positive for pVNT test, while the samples with positive WB results were in the follow-up stage. The TAb, IgG, and pVNT results did not conform to the dynamic evolution of antibodies, and cannot be determined as confirmed positive. 【Conclusion】 The infection status of antibody reactivity samples screened by SARS-CoV-2 ELISA can be judged by the logic of pVNT, WB and the dynamic change of antibody.

HIV prevalence and Western blot analysis of voluntary blood donors in Wuhu area / 中国输血杂志

【Objective】 To investigate the confirmatory status of HIV-1 antibody detection and Western blot (WB) test among voluntary blood donors in Wuhu, and to explore the strategies and methods to further ensure blood quality and safety. 【Methods】 Blood samples were preliminarily screened by ELISA and NAT, and the reactive samples were sent to Wuhu CDC for further WB test of HIV-1 antibody. The confirmation results of HIV-1 antibodies of voluntary blood donors in Wuhu in the past 10 years were retrospectively collected. The characteristics of WB bands of positive samples were analyzed, and the demographic characteristics of HIV-infected voluntary blood donors were sorted out. 【Results】 A total of 354 864 blood samples from voluntary blood donors in Wuhu during January 2011 to May 2021 were investigated, among which 42 were confirmed HIV positive (HIV-1 antibody positive in 41, and solo HIV-RNA reactive in 1), with a total HIV positive rate of 11.8/100 000(42/354 864). Statistical differences were found in gender [males 97.6% (41/42) vs females 2.4% (1/42)], marital status [unmarried 17.3/100 000 vs married 8.0/100 000] and occupation [staff/workers 37.5/100 000 vs students11.4/100 000 vs others 7.7/100 000]. Among the positive samples, the yield rate of WB bands gp160 was 100% (41/41), both gp41 and p24 were 97.6% (40/41),, and p55 was the lowest 46.3% (19/41). P51 and P66 presented the highest yield consistency (Kappa=1.000, P5 000 cps/mL by viral load (VL) testing, indicating HIV window period infection. 【Conclusion】 HIV infection statistically affected male donors more than females in Wuhu area, and most were early infection that revealed by WB band analysis. NAT plays an important role in the detection and confirmation of HIV infection during the window period, and is essential for blood safety.

Prevalencia de cisticercosis en porcinos de la provincia de Tambopata, Perú / Cysticercosis Prevalence in Pigs in the Tambopata Province, Perú

Resumen La cisticercosis es una de las principales enfermedades zoonóticas parasitarias que es causada por el establecimiento de la forma larvaria de Taenia solium. Esta enfermedad se desarrolla principalmente en cerdos que son criados en granjas sin tecnificación, donde el uso de la tecnología y las condiciones sanitarias son mínimas. Este tipo de crianza es muy usual, por lo que representa un riesgo de la salud pública. En ese sentido, se determinó la prevalencia de cisticercosis en porcinos de la provincia de Tambopata, donde fue evaluado un total de 98 porcinos. Se tomaron aproximadamente 5 ml de sangre de la vena cava en animales mayores de 6 meses y hembras que no estuviesen preñadas; posteriormente, se obtuvo el plasma para ser procesado mediante la prueba de enzyme-linked inmunoelectrotransfer bloot assay (EITB) o Western Blot. Se determinó que el 17 % de los cerdos evaluados dio positivo para cisticercosis; con respecto al sexo, se obtuvo una seroprevalencia de 5,21 % ± 0,82 % para machos y 11,45 % ± 1,93 % para hembras. Finalmente, se determinó una seroprevalencia de 10,41 % ± 1,75 % para animales jóvenes de 6 a 11 meses y 6,25 % ± 1,01 % para animales adultos de 12 meses a más. Estos resultados reflejan la importancia de la vigilancia y control de las enfermedades parasitarias en los animales de producción ya que pudo corroborarse que la cisticercosis porcina constituye un serio problema de salud pública.

Abstract Cysticercosis is one of the main zoonotic parasitic diseases caused by the larval settlement of Taenia solium. This disease develops mainly in pigs that are reared in non-technified farms where the use of technology and the sanitary conditions are poor. It is quite usual to rear pigs this way and, therefore, there is a public health risk. In this sense, the cysticercosis prevalence was determined among pigs in the Tambopata Province, including 98 animals in the evaluation. Approximately 5 ml of blood were taken from the vena cava in more than 6-month-old female pigs that were not pregnant. Next, the plasma was taken in order to be processed under an enzyme-linked inmunoelectrotransfer bloot assay (EITB) or western blot. It was found that 17% of pigs were positive to cysticercosis. Regarding the sex, the seroprevalence was 5.21% ± 0.82% in males and 11.45% ± 1.93% in females. Finally, the seroprevalence was determined at 10.41% ± 1.75% in young animals (6 to 11 months old) and 6.25% ± 1.01% in adult animals (12 months old and above). These results show how important it is to monitor and control the parasitic diseases in production animals as this study confirmed that porcine cysticercosis is a serious problem in public health.

Reactividad de sueros de ratones inmunizados con vesículas de membrana externa derivadas de Salmonella entérica / Reactivity of sera from mice immunized with outer membrane vesicles derived from Salmonella enterica

La fiebre tifoidea causada por Salmonella Paratyphi A (fiebre paratifoidea) es indistinguible de la producida por Salmonella Typhi y el grado de incidencia ha aumentado en los últimos años, especialmente en el sudeste asiático. Por otro lado, la diarrea y otras complicaciones entéricas causadas por Salmonella Enteritidis y Salmonella Typhimurium continúan siendo un problema de salud grave, especialmente en países subdesarrollados. Las vacunas continúan siendo la forma más efectiva de prevenir estas enfermedades. Existen vacunas basadas en el polisacárido capsular de Salmonella Typhi que protegen contra la fiebre tifoidea; sin embargo, no hay vacunas efectivas licenciadas para uso en humanos que prevengan las enfermedades producidas por los serotipos de Salmonella no tifoideas. El desarrollo de una formulación con capacidad para proteger contra estas enfermedades sigue siendo un desafío para la comunidad científica. En este trabajo se evaluó, mediante Western blot, la reactividad de los sueros de ratones inmunizados por vía subcutánea con formulaciones basadas en vesículas de membrana externa derivadas de Salmonella Paratyphi A, Salmonella Enteritidis y Salmonella Typhimurium, contra los respectivos lisados celulares, para identificar la formulación que induce la mejor respuesta inmunológica cruzada. Los resultados obtenidos indicaron una alta reactividad de todos los sueros a los lisados, sin una diferencia aparente entre ellos. Sin lugar a dudas, se deberán realizar pruebas de inmunogenicidad seguidas de pruebas de retos cruzados para identificar un candidato vacunal. Estos resultados sugieren que las vesículas de membrana externa empleadas en este estudio están compuestas por antígenos posiblemente conservados en los tres serotipos de Salmonella y que pueden inducir una respuesta inmune de amplio espectro y protección cruzada(AU)

Typhoid fever caused by Salmonella Paratyphi A (paratyphoid fever) is indistinguishable from that caused by Salmonella Typhi and the degree of incidence has increased in recent years, especially in Southeast Asia. On the other hand, diarrhea and other enteric complications caused by Salmonella Enteritidis and Salmonella Typhimurium continue to be a serious health problem, especially in underdeveloped countries. Vaccines continue to be the most effective way to prevent these diseases. There are vaccines based on Salmonella Typhi capsular polysaccharide, which protects against typhoid fever; however, there are no effective vaccines licensed for use in humans to prevent disease caused by nontyphoidal Salmonella serotypes. Developing a formulation capable of protecting against these diseases remains a challenge for the scientific community. In this work, the reactivity of the sera of mice immunized subcutaneously with formulations based on Outer Membrane Vesicles (OMV) derived from Salmonella Paratyphi A, Salmonella Enteritidis and Salmonella Typhimurium, was evaluated by Western blot, against the respective cell lysates to identify the formulation that induces the best cross immune response. The results obtained indicated a high reactivity of all the sera to the lysates; without an apparent difference between them. Undoubtedly, immunogenicity tests followed by cross-challenge tests should be performed to identify a vaccine candidate. These results suggest that the OMV used in this study are composed of possibly conserved antigens in the three Salmonella serotypes and that they can induce a broad-spectrum immune response and cross protection(AU)

Prevalencia de anticuerpos anti-hepatitis E de tipo IgG evaluados a través de tres ensayos comerciales / Prevalence of anti-Hepatitis E IgG analyzed in sera through three commercial kits

Resumen Introducción: El virus de la Hepatitis E (HVE) es de ácido ribonucleico desnudo, los genotipos 3 y 4 pueden presentarse como una zoonosis transmitida por agua o alimentos contaminados. En la zona del eje cafetero-Colombia, no se ha descrito la presencia de anticuerpos para este virus en la comunidad. Objetivo: Determinar la prevalencia de anticuerpos anti-HVE de tipo Inmuniglobulinas G (IgG) en muestras de suero de un laboratorio clínico del Eje Cafetero. Materiales y métodos: En un periodo de dos meses se analizaron 90 sueros de pacientes atendidos en un laboratorio clínico de la ciudad de Armenia, se utilizaron tres técnicas diferentes para la caracterización de los anticuerpos y se compararon sus resultados. Resultados: De los 90 sueros evaluados, la técnica de ELISA de anticuerpos totales ELISA IgG anti HVE Recom Well marca Mikrogen identificó 2 sueros positivos (2,2%), la Prueba ELISA IgG HVE versión ULTRA® marca Diapro evidenció una muestra equivoca (1,1%). La prueba western blot Recom line HVE marca Mikrogen detectó 4 muestras positivas (4,4%). Conclusiones: Se encontró una prevalencia de anticuerpos HVE IgG que oscila entre 0 y 4,4% dependiendo de la prueba comercial utilizada, evidenciando circulación del virus y un posible ciclo infecciosos en la región.

Abstract Introduction: Hepatitis E virus (HEV) is a nonenveloped, RNA virus. HEV genotypes 3 and 4 are considered zoonosis transmitted by contaminated water and/or food. The presence of antibodies against this virus have not been described in communities inhabiting the "Coffee Axis" region of Colombia. Objective: To determine the prevalence of anti-Hepatitis E IgG in serum samples analyzed in a clinical laboratory from the Colombian Coffee Axis. Materials and methods: 90 serum samples from patients treated at a clinical laboratory in the city of Armenia (Quindio) were analyzed and compared through three different methods that characterize antibodies. Results: The Mikrogen recomWell ELISA kit (IgG anti-HEV) identified two positive sera (2.2%). The Diapro HEV IgG ELISA (version ULTRA®) test registered a false positive sample (1.1%). The Mikrogen recom Line HVE western blot assay detected 4 positive samples (4.4%). Conclusions: Depending on the commercial kit used, the prevalence of anti-HEV IgG antibodies fluctuated between 0% to 4.4%, which demonstrates that the virus is circulating and that a possible infectious cycle in this region exists.

A retrospective analysis of 2 728 HIV antibody screening positive samples / 公共卫生与预防医学

Objective To analyze the positive results of HIV antibody screening in the laboratory of AIDS confirmation center of Hubei Provincial Center for Disease Control and Prevention from 2014 to 2020, and to provide a basis for improving detection strategies. Methods A total of 2 728 primary screening positive specimens received by the laboratory of Hubei confirmation center from 2014 to 2020 were retested with two reagents. Specimens with at least one reactive result were confirmed with western blot (WB). The samples with uncertain or negative WB results were further confirmed by nucleic acid quantitative detection. The test results were analyzed retrospectively. Results A total of 2 297 specimens with positive retest results were confirmed by WB, with a positive rate of 93.47%. The highest proportion of patients was from medical institutions. The positive rate detected by 4 diagnostic kits was apparently higher in S/CO＞10 cases than that in S/CO≤10, and the difference was statistically significant (P 5 000cps / ml, and 12 cases were TND. 13 of the 30 WB negative samples had nucleic acid test results＞5 000CPs/mL . Conclusions The coverage of HIV screening laboratories in hospitals at all levels should be further increased to find more HIV infected persons. The anti-HIV ELISA S/CO ratio is correlated with the positive results confirmed by western blot. Therefore, ELISA S/CO ratio can be used to predict anti-HIV antibody positivity. For samples with uncertain or negative WB detection, supplemental nucleic acid test should be carried out timely for early diagnosis.

Epidemiological investigation and follow-up study of indeterminate HIV antibody test results in Jiading, Shanghai / 上海预防医学

Objective To investigate the rate and the population distribution of subjects with indeterminate result of HIV antibody test and to understand the relationships between the western blot(WB)banding patterns and HIV infection through follow-up reexamination. Methods Samples with indeterminate results of HIV antibody test were collected by Jiading Center for Disease Control and Prevention from 2013 to 2017. They were used for analysis of the source, the distribution of Western blotting band pattern and the follow-up results. Results Among 698 samples required to be re-tested for confirmation of HIV infection, 151(21.63%)showed indeterminate WB test results. There were 18 types of WB band in 151 HIV antibody-indeterminate samples. The most common band types, accounting for 79.47%, were p24, gp160, and gp160p24. One hundred(among 151)subjects were followed up and the success follow-up rate was 50.00%. Among them, 28(56.00%)samples were still with indeterminate results of HIV antibody, 11(22.00% turned to be negative and 11(22.00%)turned to positive. The follow-up confirmatory tests showed that 67.86% of the samples with p24 band were still with indeterminate results and 40.00% of the samples with gp160 band became HIV antibody-negative. The samples with one of the three band patterns of gp160gp120p24, gp160p24p17 and gp160gp120p66p51 all became HIV antibody-positive. Conclusion The detection rate of indeterminate HIV antibody results varies in different populations. Positive conversion rates with different WB band patterns are different. Follow-up of the populations with specific WB band patterns should be strengthened to detect HIV infection cases as early as possible.

HIV antibody screening and confirmative tests of voluntary blood donors in a hospital in Beijing: A retrospective analysis / 中国输血杂志

【Objective】 To retrospectively analyze the epidemiological characteristics and regularity of HIV among voluntary blood donors in our hospital, so as to provide help for the formulation of effective coping strategies for voluntary blood donation, reduce the incidence of blood transmitted diseases, and improve blood safety. 【Methods】 HIV infection and population characteristics of voluntary blood donors in our hospital from January 2010 to December 2019 were statistically analyzed. 【Results】 A total of 330 000 blood donations occurred during 2010 to 2019, and 1 024 HIV-infected blood donors were screened out, with a positive rate of 0.31%. The detection rate was the highest in 2016, with 158 cases infected(158/35 889, 0.44%), followed by 151 in 2015(151/37 586, 0.40%), and 42 in 2010(42/20 824, 0.20%). The difference was statistically significant (χ2=88.754, P<0.001). Among the 1 024 HIV-infected patients, 876 were males and 148 females, with a gender ratio close to 6∶1. The majority were aged between 18~35 years old, accounting for 86.13%. 【Conclusion】 The HIV infection rate among voluntary blood donors had been increasing year by year in recent years. Major blood centers should strengthen the health information before blood donation, carry out HIV screening strictly, select blood donors appropriately, establish a stable blood donation team, so as to reduce the discarding rate of blood.

Characterization of the sensitization profile of bee venom allergic patients ­ Association with the severity of reaction? / Caracterização do perfil de sensibilização molecular de doentes alérgicos à picada de abelha - Associação com a gravidade da reação?

Introduction: Bee venom (BV) allergy, a common cause of anaphylaxis in adults, is often associated with severe reactions. The use of component-resolved diagnostics (CRD) increases diagnostic accuracy. Objectives: To characterize the sensitization profile of BV allergic patients and a possible correlation with the severity of reaction. Materials and methods: We selected patients with a clinical history of BV allergy, positive skin tests, and specific IgE (sIgE) for BV. The allergenic profile was analyzed by both CRD and Western blot using a well-defined and properly characterized BV extract. Results: Forty-four patients were included, 30 (68.2%) were men. Mean age was 48.9 (SD 17.9) years. Eleven (25%) had large local reactions (LLRs) and 33 (75%) had systemic sting reactions (SSRs). One patient with negative sIgE for BV had positive sIgE for Api m 1, Api m 5, and Api m 10. The sensitization frequency for BV, Api m 1, Api m 2, Api m 3, Api m 5, and Api m 10 was 97.7%, 75%, 47.7%, 20.5%, 40.9%, and 61.4%, respectively. Five patients (11.4%) were sensitized to all BV components. CRD association showed that 5 patients (11.4%) were sensitized only to Api m 1, 8 (18.2%) to Api m 1/Api m 3/Api m 10, and 16 (36.6%) to Api m 1/ Api m 10. Twenty-eight patients (84.8%) with SSRs were sensitized to Api m 1, and concomitant sensitization to Api m 1/Api m 10 was detected in 20 (60.6%). There was a significant difference in Api m 1 between patients with LLRs and SSRs (p = 0.0104). Similar profiles were identified by Western blot analysis, with relevance for the detection of Api m 6 in 28 (64%) and Api m 4 in 16 (36%) patients. Conclusion: The analysis of the sensitization profile using CRD and the association of several of these components can increase diagnostic accuracy in BV allergy. Our data showed that concomitant sensitization to Api m 1 and Api m 10, detected by both CRD and electrophoretic profile, may be associated with SSRs. We emphasize the identification of sensitization to Api m 6 in > 50% of patients, which may be considered a major allergen, and to Api m 4, which may be related to reactions during BV immunotherapy.

Introdução: A alergia ao veneno de abelha (VA) é uma causa frequente de anafilaxia em adultos e está muitas vezes associada a reações graves. O diagnóstico por componentes moleculares (CRD) contribui para uma melhor caracterização desta alergia. Objetivos: Caracterização do perfil de sensibilização molecular de doentes alérgicos ao veneno de abelha e possível correlação com a gravidade da reação. Material e métodos: Selecionaram-se doentes com história de alergia a VA, testes cutâneos e IgE específica (sIgE) positivos para VA. Avaliou-se o perfil alergênico por CRD e por Western Blot, utilizando extrato de VA bem caracterizado. Resultados: 44 doentes, 30 (68,2%) sexo masculino. Média de idades 48,9 ± 17,9 anos, 11 (25%) com reacções locais exuberantes e 33 (75%) com reações sistêmicas à picada (SSR). Um doente tinha sIgE negativa para VA, mas Api m 1, Api m 5 e Api m 10 positivas. A frequência de sensibilização para VA, Api m 1, Api m 2, Api m 3, Api m 5 e Api m 10 foi 97,7%; 75%; 47,7%; 20,5%; 40,9% e 61,4%, respectivamente. Cinco (11,4%) doentes estavam sensibilizados a todos os componentes. Por associação de CRD, detectaram-se 5 (11,4%) doentes sensibilizados apenas a Api m 1, 8 (18,2%) a Api m 1/Api m 3/Api m 10, e 16 (36,6%) a Api m 1/Api m 10. Vinte e oito (84,8%) doentes com SSR tinham Api m 1 positiva e 20 (60,6%) tinham Api m 1/Api m 10 simultaneamente positivas. Observou-se uma diferença estatisticamente significativa para a Api m 1 entre doentes com reações locais exuberantes e sistêmicas (p = 0,0104). Os perfis detectados por Western Blot foram semelhantes, de referir, à detecção de Api m 6 em 28 (64%) e Api m 4 em 16 (36%) dos doentes. Conclusão: A análise do perfil de sensibilização através de CRD e a sua associação aumentam a precisão do diagnóstico de alergia a VA. Sensibilização simultânea a Api m 1 e Api m 10 identificados tanto por CRD como por perfil eletroforético, pode estar associada à ocorrência de SSR. Destaca-se a sensibilização a Api m 6 em > 50% dos doentes, podendo ser considerado um alergênio major, e a Api m 4, possivelmente associado a reações durante a imunoterapia com VA.

Seroprevalence of Lymes disease in the Nagarahole and Bandipur forest areas of South India

Purpose: The aim of this study was to determine the seroprevalence of Lymes disease in a population at risk in south India. Methods: Prospective ongoing study and included screening of forest workers and staff of Nagarahole and Bandipur forest ranges in South India for Lymes disease. Screening included a detailed questionnaire for Lymes disease, complete ocular and systemic examination by an ophthalmologist and infectious disease specialist and blood collection. ELISA for IgM and IgG antibodies for Borrelia burgdorferi were performed on the collected sera samples. Western blot confirmation was done on the seropositive samples. Ticks were also collected from these forest areas for future studies to detect if they harbor B. burgdorferi. Results: Seroprevalence of 19.9% was noted by ELISA. Western blot confirmation was seen in 15.6% of the seropositive samples. There was significant correlation between seropositivity and exposure to tick bites (P = 0.023). Conclusion: There is a high seroprevalence of infection with B. burgdorferi in the forest areas of Nagarahole and Bandipur ranges in south India.

Serological evaluation of Neospora caninum in pregnant women treated at referral center for prenatal screening in Mato Grosso do Sul, Brazil / Avaliação sorológica de Neospora caninum em gestantes atendidas em centro de referência para triagem pré-natal em Mato Grosso do Sul, Brasil

Abstract Neospora caninum is an obligate intracellular protozoan with canids (Canis domesticus, Canis lupus dingo, Canis latrans, Canis lupus) as its definitive hosts. The objective of this study was to detect anti-N. caninum antibodies in pregnant women seen at referral center for prenatal screening in the state of state Mato Grosso do Sul, Brazil. A total of 188 serum samples from pregnant women provided by the Instituto de Pesquisa, Ensino e Diagnósticos da APAE de Campo Grande (IPED/ APAE) were subjected to IFA test and western blot analysis. The samples were divided into three groups: 23/99 samples from the seropositive group for toxoplasmosis were positive for anti-N. caninum IgG antibodies, and 9/99 positive for IgM; in the HIV group, 7/33 were positive for IgG; and in the HIV+toxoplasmosis group, 13/56 were positive for IgG and two positive for IgM. The seropositivity for IgG was assessed by western blot by testing 43 IFA test positive samples using rNcSRS2 (Nc-p43) as antigen. The serological results of the present study suggest that exposure of these pregnant women to the parasite N. caninum and presence of IgM antibodies are indicative of recent infection. Further studies are needed to establish the possibility of active infection.

Resumo Neospora caninum é um protozoário intracelular obrigatório que possui os canídeos (Canis domesticus, Canis lupus dingo, Canis latrans, Canis lupus) como seus hospedeiros definitivos. O objetivo deste trabalho foi detectar anticorpos anti-N. caninum em gestantes, atendidas em centro de referência para triagem pré-natal, em Mato Grosso do Sul, Brasil. Um total de 188 amostras de soro de gestantes, cedidas pelo Instituto de Pesquisa, Ensino e Diagnósticos da APAE de Campo Grande (IPED/ APAE), foram submetidas ao teste de RIFI e western blot para a detecção de anticorpos anti-N. caninum. As amostras foram divididas em três grupos: 23/99 amostras do grupo soropositivo para toxoplasmose demonstraram positividade para anticorpos IgG anti-N. caninum e 9/99 positivos para IgM. No grupo HIV 7/33 apresentaram positividade para IgG. No grupo HIV+Toxoplasmose 13/56 apresentaram positividade para IgG e duas para IgM. A soropositividade para IgG foi avaliada por western blot, testando 43 amostras positivas para RIFI, usando-se rNcSRS2 (Nc-p43) como antígeno. Os resultados sorológicos do presente estudo sugerem exposição dessas gestantes ao parasita N. caninum e a positividade para anticorpos IgM são indicativos de infecção recente. Mais estudos na área são necessários para estabelecer a possibilidade de infecção ativa.

Effect and Mechanism of Midostaurin Derivatives 5'''-methoxyfradcarbazole A on Mouse Leukemia CB3 Cells / 中国药学杂志

OBJECTIVE: To investigate the mechanism of midostaurin derivative 5'''-methoxyfradcarbazole A of action in inhibiting mouse leukemia cells (CB3) growth. METHODS: MTT assay was employed to evaluate the effect of compound 5'''-methoxyfradcarbazole A on the proliferation of CB3 cells, and generate the growth inhibition curves. Flow cytometry and Annexin V-FITC /PI double staining were used to determine the changes of the cell cycle, cell differentiation and apoptosis. Western blot analysis was applied to test the effects of 5'''-methoxyfradcarbazole A on cyclin and apoptosis-related proteins. RESULTS: The compound 5'''-methoxyfradcarbazole A could significantly inhibit the growth of CB3 cells, and the half maximal inhibitory concentration (IC50) of the compound was (0.587±0.135)μmol•L-1. 5'''-Methoxyfradcarbazole A was able to induce early apoptosis and late apoptosis of CB3 cells in a time- and dose-dependent manner. At the same time, it also affected the cell cycle of CB3 and significantly increased the proportion of G2 phase. The expression of CD41, a platelet differentiation marker, and Ter119, an erythrocyte differentiation marker, were also increased in CB3 cells treated with 5'''-methoxyfradcarbazole A. Besides, the expressions of apoptosis-related proteins Bim, PARP1 and cyclin-related protein P21 were significantly increased, and the phosphorylated ERK protein was decreased. CONCLUSION: Midostaurin derivative 5'''-methoxyfradcarbazole A could increase the apoptosis of CB3 cells by promoting the expression of apoptotic protein Bim, and cause G2 phase arrest by increasing the expression of cyclin protein P21. Also, the changes of the expression levels of PARP1 and phosphorylated ERK protein also partly explain the inhibition of 5'''-methoxyfradcarbazole A on CB3 cells growth.

Lycium barbarum polysaccharide intervenes with CD151 and matrix metalloproteinase 3 in the articular cartilage in a rabbit model of knee osteoarthritis / 中国组织工程研究

BACKGROUND: Studies have shown that Lycium barbarum polysaccharide (LBP) has the functions of anti-aging, nerve protection, anti-fatigue, blood sugar control, anti-oxidation, and anti-tumor. It may have some protective effects against osteoarthritis of the knee, but have been rarely reported. CD151 and matrix metalloproteinase 3 (MMP-3) are two common cytokines for assessing knee osteoarthritis. OBJECTIVE: To observe the effect of LBP on the expression of CD151 and MMP-3 in rabbit osteoarthritis. METHODS: Sixty-four healthy 6-month-old white rabbits were randomly divided into four groups: blank group, model group, LBP group and normal saline group. Animal models of knee osteoarthritis were made using Hulth method in the rabbits except those in the blank group. The rats in the LBP and normal saline groups were fed with normal dose of LBP and normal saline for 4 weeks, and then the articular cartilage tissues were taken from the affected side at 12 weeks after modeling. The morphological changes of the articular cartilage were observed by hematoxylin-eosin staining. The expression levels and spatial distribution of CD151 and MMP-3 in articular cartilage was observed by immunohistochemical staining and western blot. Ethic approval was given by the People’s Hospital of Ningxia Hui Autonomous Region (approval No. 2014-30817). RESULTS AND CONCLUSION: immunohistochemistry staining and western blot results showed that the absorbance values and protein expression of MMP-3 and CD-151 were significantly lower in the LBP group than the normal saline and model groups (P < 0.05). Therefore, the expression of CD151 and MMP-3 in the articular cartilage of osteoarthritis was increased, and LBP could inhibit the expression of CD151 and MMP-3 in osteoarthritis, so as to slow down the occurrence of osteoarthritis.

Effect of Yinfupian and Yangfupian on Brown Adipose Tissue of Yang Deficiency Mice / 中国实验方剂学杂志

Objective:To observe the expression of brown adipose tissue (BAT), cells, proteins and corresponding genes in Yang deficiency model mice induced by Rhei Radix et Rhizoma suspension, and to explore the thermogenesis of processed products of Aconiti Lateralis Radix Praeparata with Jianchang faction characteristics. Method:Twenty mice, half male and half female, were randomly selected as the normal female and male groups. And the other 80 mice were administrated with Rhei Radix et Rhizoma suspension (the content of 0.25 g·mL-1) to establish Yang deficiency model, after the model was established, they were randomly divided into the model female and male groups, female and male groups of Shengfupian, female and male groups of Yinfupian, female and male groups of Yangfupian, 10 mice in each group. Mice were intragastric administrated with corresponding medical solution for two weeks (1.54 g·kg-1·d-1) according to groups. Normal group and model group were given equal volume distilled water. After administration, BAT of scapular region of mice was collected and the changes of BAT cells were observed by hematoxylin-eosin (HE) staining. The expression of uncoupling protein 1 (UCP1) and its mRNA were detected by Western blot and real-time fluorescence quantitative polymerase chain reaction (Real-time PCR). Result:Compared with the normal group of the same sex, the proportion of BAT in the model group decreased significantly (P<0.01). Compared with the model group of the same sex, the proportion of BAT in female mice from Shengfupian and Yinfupian groups increased significantly (P<0.01), while there was no significant difference between each administration group and model group in the male mice. Compared with normal mice of the same sex, there were many scattered vacuoles in BAT cells of the model group, and fewer cells could be observed due to larger vacuoles. Compared with the model group of the same sex, BAT cells in mice from the Shengfupian group showed fewer vacuoles, smaller cells and tight arrangement, the density of BAT cells in mice from the Yangfupian group also increased significantly, while the vacuoles in BAT cells of mice from the Yinfupian group decreased relatively and the cells did not increase significantly. Compared with the same sex mice, the expression level of UCP1 in the model group and the normal group was statistically significant (P<0.05, P<0.01). In the female mice, the expression level of UCP1 in Yangfupian group was significantly higher than that in the model group (P<0.05), each administration group of male mice was significantly different from that of the model group of the same sex (P<0.05), of which Yangfupian was the most significant. The relative expression of UCP1 mRNA in the model group was significantly lower than that in the normal group of the same sex (P<0.05, P<0.01). In the female mice, compared with the model group, the relative expression levels of UCP1 mRNA in Yangfupian group, Shengfupian group and Yinfupian group increased significantly (P<0.05, P<0.01), compared with Yangfupian group, the relative expression levels of UCP1 mRNA in Shengfupian and Yinfupian were also significantly different (P<0.05). In the male mice, compared with the model group, the relative expression of UCP1 mRNA in Yangfupian group was significantly increased (P<0.01), but there was no significant difference in Shengfupian group and Yinfupian group, in addition, compared with Yangfupian group, the relative expression of UCP1 mRNA in Shengfupian group and Yinfupian group had significant difference (P<0.05). Conclusion:Shengfupian, Yinfupian and Yangfupian all have obvious improvement on Yang deficiency syndrome induced by Rhei Radix et Rhizoma suspension. The mechanism may be to promote the expression of UCP1 protein and its mRNA and enhance the activity of BAT. And the effect of Yangfupian is the best.

Inhibitory Effect of Essential Oil from Fructus Alpinia zerumbet on Endothelial-to-mesenchymal Transition Induced by High Glucose via Nrf2/Notch1 Signaling Pathway / 中国实验方剂学杂志

Objective:To investigate the inhibitory effect and the possible mechanism of essential oil from fructus Alpinia zerumbet （EOFAZ） on endothelial-to-mesenchymal transition （EndMT） induced by high glucose （HG）. Method:Human umbilical vein endothelial cells （HUVECs） was cultured in vitro to analyze the pharmacodynamic effects of EOFAZ on EndMT and oxidative stress damage induced by HG. The experiment was set the blank group， HG group （35 mmol·L-1）， EOFAZ low dose group （1 μg·L-1） and EOFAZ high dose group （4 μg·L-1）. After EOFAZ intervention for 2 h， HG was added to incubate for 72 h in order to establish EndMT cell model. Western blot was used to detect the protein expression of vimentin and platelet endothelial cell adhesion molecule （CD31）. Angiogenesis experiment was used to detect the ability of cell migration ability in order to analyze the effect of EOFAZ on EndMT. The changes of reactive oxygen species （ROS） levels were detected by 2'，7'-dichlorodihydrofluorescein diacetate （DCFH-DA） fluorescence probe and the contents of malondialdehyde （MDA）， superoxide dismutase （SOD） and catalase （CAT） in cells were detected by the kit method to analyze the effect of EOFAZ on oxidative stress. Western blot was used to detect the protein expression levels of nuclear transcription factor E2 related factor 2 （Nrf2） and Notch1. The overexpression of Nrf2 was achieved by adenovirus （AD） transfection and the mechanism of EOFAZ inhibiting EndMT was further analyzed. The experiment was set the blank group， HG group （35 mmol·L-1）， AD-Nrf2 group， EOFAZ group （4 μg·L-1）， AD-Nrf2+EOFAZ group （4 μg·L-1）. The cells were infected with recombinant adenovirus overexpression plasmid of Nrf2 gene for 6 h， then replaced with normal medium for 24 h. After EOFAZ intervention for 2 h， HG was added to co-incubate for 72 h to induce EndMT. Western blot was used to detect the protein expressions of Nrf2， CD31， vimentin， Notch1 and Snail. Result:Compared with the HG group， after treatment with EOFAZ， the protein expression of CD31 was significantly up-regulated （P<0.05）， the protein expression of vimentin was significantly down-regulated （P<0.01）， the ability of cell migration was decreased （P<0.01）， and the contents of ROS and MDA were decreased （P<0.05， P<0.01）， the levels of CAT and SOD were increased （P<0.01）. In addition， EOFAZ could significantly up-regulate the protein expression of antioxidant signal Nrf2 （P<0.01） and down-regulate the protein expression of Notch1 （P<0.01）. High expression of Nrf2 was achieved by stable AD transfection into HUVECs. The results of Western blot showed that， compared with the HG group， the protein expression levels of Nrf2 and CD31 in each treatment group were significantly increased （P<0.01）， while the protein expression levels of vimentin， Notch1 and Snail were down-regulated （P<0.01）. At the same time， compared with the AD-Nrf2 group， the AD-Nrf2+EOFAZ group could further up-regulate the protein expressions of Nrf2 and CD31 （P<0.05， P<0.01）， while decrease the protein expression levels of vimentin， Notch1 and Snail （P<0.01）. Conclusion:EOFAZ ameliorates oxidative stress injury of vascular endothelial cells induced by HG and inhibits EndMT， which is related to Nrf2/Notch1 signaling pathway.

Effect of myricetinon enterovirus 71 infection in vitro / 中国药理学通报

Aim To investigate the antiviral activity and mechanism of myricetin against enterovirus 71 (E V 7 1) infection. Methods The cytopathic effect (CPE) and plaque assay were used to observe the antiviral effect of myricetin against EV71 in Vero cell. The cells were treated with myricetin at different concentrations combined with crystal violet staining to detectthe cytotoxicity of myricetin. The effect of myricetin on VP1 protein expression was detected by Western blot. The effect of myricetin on VP1 gene expressionwas evaluated byRT-PCR. Results Myricetin pretreatment at 2. 5-20 fimol L-1' significantly inhibitedcell death induced by EV71 infection in a dose-dependent manner with the IC50 value of 5. 6 jxmol • L-1. Compared to virus control group, myricetin could significantly reduce the viral titer at the concentration of 2. 5 ~ 20 u,mol • L-1. The results of Western blot and RT-PCR showed that myricetin could markedlyreduce the gene and protein expression levels of viral capsid protein VP1. Conclusion Myricetin has significant antiEV71 activity in vitro.

A new tricyclic abietane diterpenoid from Salvia chloroleuca and evaluation of cytotoxic and apoptotic activities

Abstract In this study, sahandone (1) and a new diterpenoid named sahandol II (2) were isolated from the roots of Salvia chloroleuca Rech. f. & Aellen, Lamiaceae. The absolute configurations of compounds 1 and 2 were assigned by comparison of experimental electronic circular dichroism spectra and comparing with published data. Cytotoxic and apoptotic evaluation of the isolated compounds and the methanol crude extract and its subfractions including petroleum ether, dichloromethane, ethyl acetate, n-butanol and aqueous fraction on two human prostate cancer cell lines and a breast cancer cell lines, showed that non-polar and semi-polar subfractions had the potent cytotoxic effect on PC3 cells with the IC50 values of 24.19, 33.59, and 47.15 µg/ml, respectively. Sub-G1 peak in flow cytometry histogram of cells treated with petroleum ether, dichloromethane and ethyl acetate subfractions showed the induction of apoptosis. Change in the Bax/Bcl-2 ratio and cleavage of poly ADP-ribose polymerase were observed.

Análise da relação entre a expressão de marcadores de células tronco tumorais (ALDH-1 e SOX-2) e as características clinico patológicas de neoplasias de glândulas salivares / Analysis of the relationship between the expression of tumor stem cell markers (ALDH-1 and SOX-2) and the clinicopathological characteristics of salivary gland neoplasms

Os mecanismos moleculares e celulares que estão associados a patogênese, baixa resposta ao tratamento, recidiva e óbito em tumores de glândula salivar não são totalmente conhecidos. Nesse sentido, é importante ressaltar que as células-tronco (CT) dentro de um tumor (CTT) estão relacionadas com a tumorigenicidade e progressão em neoplasias humanas. Diante do exposto, o objetivo desse trabalho foi avaliar a expressão de marcadores relacionados às CT (ALDH-1 e SOX-2) em neoplasias de glândula salivar e verificar se suas expressões apresentam associação com dados clinicopatológicos e desfecho dos pacientes. Foram selecionados 103 casos de neoplasias malignas (25 CME; 15 CCA; 13 CAC; 10 ACP; 13 ACSOE; 8 CEME; 7 CAEXAP; 5 CDS; 4 ACCB; 3 CCC) e 51 casos de neoplasias benignas (25 AP; 9 MIO; 7 TW; 5 ACA; 5 ACB). Os dados obtidos foram analisados no software Statistical Package for Social Science, GraphPad Prism e STATA. O nível de significância de 5% foi adotado para os testes estatísticos (p<0.05). Os pacientes do estudo foram principalmente do sexo masculino, com média de idade de 52 anos; a parótida foi o sítio anatômico mais afetado. A maioria das neoplasias malignas, foi classificada como T1-T2, N0 e M0. A expressão das proteínas foi avaliada através de imuno-histoquímica e confirmadas por meio de Western-blot, verificando-se que os resultados foram semelhantes entre as técnicas e que estavam correlacionados estatisticamente, tanto para SOX-2 (p<0.001) quanto para ALDH-1 (p=0.039). Em relação a expressão da SOX-2, a maioria dos tumores benignos foi negativa (n=39; 76.5%), sendo constatada expressão apenas nos tumores sem diferenciação mioepitelial (ACA e TW) (p<0.0001). Em contraparte a maioria dos tumores malignos estudados foi positiva para SOX-2 (n=54; 52.4%) sendo esse resultado estatisticamente significativo (p=0.002). Também foi evidenciada que essa expressão ocorreu em casos sem diferenciação mioepitelial (p=0.006) principalmente em CME e CCC. Não foram evidenciadas associações estatisticamente significativas entre a expressão de SOX-2 e parâmetros clínicos. A proteína ALDH-1 esteve frequentemente expressa no parênquima de neoplasias malignas (n=88; 85.6%) e benignas (100%). De maneira geral, a expressão da ALDH-1 no parênquima não se associou com parâmetros clínicos das neoplasias malignas, entretanto, os casos de CME com alta expressão no parênquima estavam associados com tumores de estadiamento clínico avançado (p=0.047). Foi constatada expressão da ALDH-1 em células do estroma tumoral, principalmente de neoplasias malignas (n=67; 65.0%), estando associada com metástase em linfonodos (p=0.032), estadiamento clínico avançado (p=0.008), recorrência tumoral (p=0.006) e óbito (p=0.013). A sobrevida global e livre de doença em 5 e 10 anos foi menor em pacientes diagnosticados com CAC, estadiamento clínico avançado, que apresentaram recorrência e com expressão estromal de ALDH-1. Destaca-se que na análise multivariada, o estadiamento clínico avançado e expressão estromal da ALDH-1 representaram fatores prognósticos independentes na sobrevida livre de doença. Com base nos resultados apresentados pode-se concluir que o perfil que caracteriza as CTT apresenta variações nos diferentes tumores de glândula salivar. A expressão diferencial da SOX-2 e ALDH-1 nessas neoplasias sugere que existem subtipos diferentes de CTT que podem ser ativadas por vias moleculares distintas. Conclui-se também que a presença de marcação estromal para ALDH-1 caracteriza células com perfil de CT mesenquimais que podem estar diretamente relacionada com o comportamento biológico e progressão de tumores malignos em glândula salivar (AU).

The molecular and cellular mechanisms that are associated with pathogenesis, poor treatment response, recurrence, and death in salivary gland tumors are not fully known. In this matter, stem cells (SC) within a tumor (TSC) are related to tumorigenicity and progress in human neoplasms. As such, the aim of this study was to evaluate the expression of SC-related markers (ALDH-1 and SOX-2) in salivary gland neoplasms and their possible association with clinicopathological data and patient outcomes. We selected 103 cases of malignant neoplasms (25 mucoepidermoid carcinoma; 15 acinic cell carcinoma; 13 adenoid cystic carcinoma; 10 polymorphous adenocarcinoma; 13 adenocarcinoma NOS; 8 epithelial-myoepithelial carcinoma; 7 carcinoma ex pleomorphic adenoma; 5 salivary duct carcinoma; 4 basal cell adenocarcinoma; 3 clear cell carcinoma) and 51 cases of benign neoplasms (25 pleomorphic adenoma; 9 myoepithelioma; 7 Warthin tumor; 5 canalicular adenoma; 5 basal cell adenoma). Data were analyzed in Statistical Package for Social Science, GraphPad Prism and STATA softwares. A significance level of 5% was adopted for the statistical tests (p<0.05). Most patients were male, with a mean age of 52 years, and the parotid was the most common anatomical site. Most malignant neoplasms were classified as T1-T2, N0 and M0. Protein expression assessed by immunohistochemistry and confirmed by western blot showed similar results that were statistically correlated for both SOX-2 (p<0.001) and ALDH-1 (p=0.039). Regarding the expression of SOX-2, most benign tumors were negative (n=39; 76.5%), and expression was only observed in tumors without myoepithelial differentiation (p<0.0001). In the other hand, most of the malignant tumors were positive for SOX-2 (n=54; 52.4%), being statistically significant (p=0.002). The expression occurred in cases without myoepithelial differentiation (p=0.006) mainly in mucoepidermoid carcinoma and clear cell carcinoma. No association was found between SOX-2 expression and clinical parameters. ALDH-1 was frequently expressed in the parenchyma of malignant (n=88; 85.6%) and benign (100%) neoplasms. Overall, the presence of ALDH-1 in the parenchyma was not associated with clinical data of malignant neoplasms; nevertheless, the cases of mucoepidermoid carcinoma with high expression in the parenchyma were associated with advanced clinical stage (p=0.047). The expression of ALDH-1 in tumor stroma cells occurred mainly in malignant neoplasms (n=67; 65.0%), being associated with lymph node metastasis (p=0.032), advanced clinical stage (p=0.008), recurrence (p=0.006) and death (p=0.013). Overall survival and Diseasefree survival at 5 and 10 years were lower in patients diagnosed with adenoid cystic carcinoma, advanced clinical stage, recurrence and stromal expression of ALDH-1. Multivariate analysis showed advanced clinical stage and stromal expression of ALDH1 were independent prognostic factors for disease-free survival. Based on the results, the profile of TSC presents variations in different salivary gland tumors. The differential expression of SOX-2 and ALDH-1 in these neoplasms suggests that there are different subtypes of TSC that can be activated by distinct molecular pathways. Also, the presence of ALDH-1 stromal expression characterizes cells with mesenchymal CT profile that may be directly related to the biological behavior and progress of malignant tumors in the salivary gland (AU).

The expression and clinical significance of migration inhibitory factor in human pulmonary tissues with chronic obstructive pulmonary disease / 中华急诊医学杂志

Objective@#To investigate the expression of macrophage migration inhibitory factor (MIF) in pulmonary tissues from patients with chronic obstructive pulmonary disease (COPD) and the relationship with its clinical features.@*Methods@#One hundred and eighty patients who underwent pulmonary bullectomy lobectomy due to pneumatocele from January 2015 to September 2018 in Longgang Central Hospital were enrolled and classified into patients without COPD (control group)and patients with COPD (COPD group), with 90 patients each group. According to the lung function parameters, 90 patients with COPD were divided into the mild COPD group, the moderate COPD group, and the severe COPD group. The levels of mRNA and protein of MIF were measured with RT-PCR, ELISA and Western blot. One-way ANOVA, Pearson correlation analysis and SNK-q test were used to analyze the results with SPSS 18.0, and P<0.05 was considered statistically significant.@*Results@#The level of MIF in pulmonary tissues from the control group was obviously lower than those in the COPD group (P<0.05). The level of MIF in pulmonary tissues in the severe COPD group was obviously higher than those in pulmonary tissues in the mild COPD, moderate COPD and control groups (P<0.05). MIF was positively correlated with the lung function parameters (P<0.05).@*Conclusion@#The high expression of MIF in pulmonary tissues is closely related to the severity of COPD.

Expression and clinical significance of FGF8b in ovarian cancer tissues / 中华内分泌外科杂志

Objective To investigate the expression of FGF8b in ovarian cancer tissue and its relationship with clinical characteristics of patients.Methods Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the FGF8b mRNA level in 28 cases of human ovarian cancer tissues and the adjacent normal tissues.Western blot method was used to detect the FGF8b protein.The relationship between the clinical characteristics of the patients was analyzed.Results The expression of FGF8b mRNA in ovarian cancer tissue (0.23±0.08) was higher than that of paraplastic tissue (0.71±0.11)(P＜0.05),and FGF8b protein expression in cancerous tissue (0.27±0.03) was higher than that in cancerous tissue (0.44±0.03)(P＜0.05).In ovarian cancer tissues,FGF8b positive rate (76.9 ％) in patients with stage Ⅲ to Ⅳ was higher than in patients with stage Ⅰ to Ⅱ (33.3％) (P＜0.05) The positive rate of lymph node metastasis (60.0％) was higher than that of non-lymphatic metastasis (27.8％)(P＜0.05).Conclusion FGF8b is significantly highly expressed in ovarian cancer tissues,which is correlated with the clinical characteristics of the ovarian cancer patients.