TMED2 is Regulated by miR-5583-5p to Elicit an Oncogene Effect in Tongue Squamous Cell Carcinoma / 中国生物化学与分子生物学报

Tongue squamous cell carcinoma (TSCC) is the most common type of oral squamous cell carcinoma (OSCC) with high morbidity and mortality. Many studies have shown that microRNA (miRNA) are small non-coding RNA that regulate the post-transcriptional processing of target genes, resulting in the degradation and translation inhibition of target mRNA. However, how the transmembrane p24 trafficking protein 2 (TMED2) is regulated by miR-5583-5p on migration, invasion, proliferation and epithelial-mesenchymal transition (EMT) of TSCC Cal-27 cells is unclear. In this study, a database was used to analyze the expression of TMED2 in HNSCC (P <0. 001) in head and neck cancer (HNC). Western blot showed that the expression of TMED2 protein was up-regulated in 6 cases of TSCC tissues and cell lines such as SCC-9, SCC-25 and CAL-27. After the Cal-27 cells transfected with TMED2 interference plasmid (SiTMED2) the expression of E-cadherin was up-regulated, and N-cadherin and Vimentin was down-regulated. Migration and invasion experiments showed that the number of cells transfused into the basement membrane of the cells was lower than that of the control group (P<0. 05). The results of EdU showed that the proliferation of Cal-27 cells transfected with SiTMED2 was decreased (P<0. 05). The results of dual luciferase experiment showed that TMED2 had a binding target to miR-5583-5p, and the expression of miR-5583-5p in Cal-27 cell was lower than that in Hoec cells. The expression of miR-5583-5p was increased and TMED2 protein was decreased after the Cal-27 cells were transfected with miR-5583-5p plasmid (P < 0. 05). In conclusion, TMED2 is regulated by miR-5583-5p and promoted the migration, invasion, proliferation and EMT of tongue squamous cell carcinoma cell Cal-27.

Eukaryotic expression and serodiagnostic potential of HIV-1 p24 antigen / 中华微生物学和免疫学杂志

Objective To express HIV-1 capsid p24 antigen in an eukaryotic expression system and to evaluate its antigenicity and potential in the early diagnosis of HIV. Methods The full-length gene of HIV-1 p24 and the signal peptide DRVI gene were amplified by PCR. The signal peptide DRVI preceding the p24 gene was introduced using fusion PCR, and cloned into vector pDRVI1. 0. Two recombinant plas-mids pDRVI-p24 and pDRVI-p24s were constructed and transfected into 293F cells. Expression and secre-tion of p24 protein were detected by SDS-PAGE, Ni-NTA column chromatography and molecular sieve were used to purify p24s protein. The purified protein was identified by Western blot and indirect ELISA using hu-man/mouse HIV-1-positive serum samples. Results The eukaryotic expression system for HIV-1 p24 anti-gen was successfully established with high efficiency. The target protein of interest with the signal peptide DRVI was obviously detected in the supernatants of cell culture. The recombinant protein had good specifici-ty and sensitivity based on the results of serological tests using serum samples of five HIV-1-positive and five HIV-negative mice , 30 HIV-1-positive patients and 50 HIV-1-negative healthy individuals . Conclusions The eukaryotic expression system for HIV-1 p24 antigen was successfully established. The purified HIV-1 p24s antigen had good antigenicity. An indirect ELISA assay with good specificity and sensitivity for the de-tection of HIV-1 was preliminarily constructed and showed great potential for application.

The expression characteristics and co-localization of HIV capsid protein p24 and TRIM22 in U-251 glioma cells / 西安交通大学学报(医学版)

Objective: To observe the expression characteristics and co-localization of exogenous TRIM22 and HIV capsid protein p24 in glioma cells. Methods: The vectors of pEGFP-N3-TRIM22 or pDsRed1-p24 were transfected into U-251 glioma cells respectively to examine the expression of TRIM22-EGFP or p24-DsRed1 by confocal microscopy. Moreover, we used a confocal z-stacking program to achieve series of optical sections and to rebuild 3-D images by ImageJ 1.50i software to detect the expression characteristics of p24-DsRed1 in U-251 cells. In the end, the vectors of pEGFP-N3-TRIM22 and pDsRed1-p24 were co-transfected into U-251 cells to detect the co-localization between TRIM22 and p24 by confocal microscopy. Results: Confocal microscopy results showed that TRIM22-EGFP or p24-Dsred1 was localized to the cell body as well as to protuberance in U-251 cells, and 3D structural reconstruction showed that p24-Dsred1 could be transferred to foot processes of U-251 cells. Simultaneously, confocal microscopy results also showed that TRIM22 and p24 could be co-localized and their combination could be released by budding in U-251 cells co-transfected with pEGFP-N3-TRIM22 and pDsRed1-p24. Conclusion: TRIM22 co-localized to HIV capsid protein p24 and their combination can be released by budding in glioma cells.

Prueba de anticuerpos anti-VIH / Anti-HIV antibody test

Prueba de anticuerpos anti-VIHAnti-HIV antibody testCódigo SCPC (Sociedad Colombiana de Patología Clínica): 66600. Código CUPS (Codificación Única de Procedimientos en Salud): 906249. Sección: Endocrinología. Nivel de complejidad: media. Metodología: CMIA (Ensayo quimioluminiscente de micropartículas) Sinónimos: anticuerpos anti-HIV1/HIV2/Agp24.DefiniciónEl ensayo para la detección cualitativa y simultánea del antígeno p24 del virus de la inmunodeficiencia humana (VIH) y los anticuerpos frente al VIH tipo 1 y 2 (VIH-1 y VIH-2) es un ensayo quimioluminiscente de micropartículas (CMIA) realizado en suero o plasma humano como ayuda diagnóstica de las infecciones por el VIH-1/VIH2-2. Espectro clínico de aplicación El virus de la inmunodeficiencia humana (VIH) es el agente causal del síndrome de la Inmunodeficiencia humana adquirida (SIDA), un retrovirus del género Lentivirus que ataca el sistema inmune, debilitándolo, volviendo gradualmente a la persona infectada inmunodeficiente, lo que conduce a una mayor sensibilidad a infecciones y enfermedades.La pandemia del VIH/SIDA genera numerosas pérdidas humanas, deterioro de la calidad de vida de millones de personas, consecuencias económicas negativas debido al incremento de recursos necesarios para la atención en salud y la incapacidad y muerte de miles de personas en edad productiva, lo que en conjunto la sitúan como uno de los mayores desafíos para la salud humana.

Study on HIV diagnosis of early detection of infection of three HIV antigen/antibody detection method / 中国免疫学杂志

Objective:Comparison of commercially available three HIV antibody detection kit for HIV infection,found as early HIV infection provides a reference method.Methods:By using two fourth generation HIV Kit ( Murex HIV Ag/Ab Kit:British Abbott company production numbers for A;Holland Organon company Vironostika HIV Uni-Form II Ag/Ab Kit:No.B;) a third generation HIV Kit ( British Abbott company produced Murex HIV-1.2.O Kit:No.C) and P24 antigen detection kit for 3 863 blood samples and BBI positive blood winding detection,sensitivity analysis,the specificity for the two fourth generation HIV kit for detecting,at the same time analysis of three kinds of antibody detection kit for detection of HIV infection window period of time whether the advance.Results:A kit,B were all positive blood samples from 54 patients with HIV infection,the sensitivity of A detection kit=100%,the specificity =99.61%, the rate of missed diagnosis=0, misdiagnosis rate=0.39%;the sensitivity of B detection kit=100%, the specificity =99.37%,the rate of missed diagnosis=0,misdiagnosis rate=0.63%;reagent A and B detection results are compared,the results did not show significant difference (P>0.05) ;A kit,B kit respectively compared with C reagent detection window period ahead of 5.5 and 3.7 d,but compared with P24 antigen kit detection window period was a lag of 4.25 to 6.05 d.Conclusion:In this study,the ability of two fourth generation HIV kit for detection of HIV infection in the strong sensitivity,reached 100%,but can be HIV infection window period in advance,to ensure the safe use of blood.

Anatomopathological characterization of placentas from HIV+ patients associated with p24 expression / Caracterização anatomopatológica da placenta de pacientes HIV+ associada à expressão do p24

INTRODUCTION: The study of placentas from pregnant human immunodeficiency virus (HIV) positive women has become the subject of numerous studies in the literature. Morphological, viral, immune and inflammatory placental aspects have been analyzed in order to grasp the vertical transmission of the virus. OBJECTIVE: To identify the most frequent findings in the placentas by associating them with a viral antigen and correlating them with the infection of newborns. MATERIAL AND METHODS: Thirty-five placentas from HIV- positive pregnant women were pathologically and immunohistochemically analyzed with the use of p24 antibody in the period from 1992 to1997 in accordance with the routine laboratory testing from the Anatomopathological Department - Hospital Universitário Antônio Pedro - Universidade Federal Fluminense (APD/HUAP/UFF). RESULTS: The microscopic alterations detected in all cases, including those with vertical transmission, were arteriopathy in the fetal blood circulation, chorioamnionitis, perivillous fibrin deposition, syncytial knotting, villous edema and villous immaturity. No specific macroscopic or histopathological changes were found in these placentas. The neonatal infection was observed in five cases. Vertical transmission was identified in two out of five placentas that had low weight for the respective stage of pregnancy. Immunohistochemical analysis revealed 14 positive cases, two of which showed vertical transmission. The viral protein was not identified in 10 out of 14 placentas from patients who had been medicated with zidovudine (AZT). CONCLUSION: Our study has contributed to the anatomopathological investigation into placentas from HIV-positive patients, although p24 expression per se did not allow a definite and early diagnosis of the vertical transmission.

INTRODUÇÃO: A importância do estudo da placenta de gestantes com o vírus da imunodeficiência humana (HIV) soropositivas tornou-se alvo de inúmeros trabalhos na literatura. Aspectos morfológicos, virais, imunes e inflamatórios intrínsecos ao tecido placentário foram analisados para o entendimento da transmissão vertical do vírus. OBJETIVO: Identificar as lesões mais frequentes nas placentas, associando-as ao antígeno viral e correlacionando-as com a infecção dos recém-nascidos. MATERIAL E MÉTODOS: Trinta e cinco placentas de gestantes HIV soropositivas foram analisadas por estudo anatomopatológico e imuno-histoquímico, utilizando o anticorpo p24, no período de 1992 a 1997, segundo a rotina do laboratório do Serviço Anatomia Patológica/Hospital Universitário Antônio Pedro/Universidade Federal Fluminense (SAP/HUAP/UFF). RESULTADOS: As alterações microscópicas registradas em todos os casos, inclusive nos de transmissão vertical, foram arteriopatia no circuito vascular fetal, corionamnionite, depósito fibrinoide perivilositário, excesso de nós sinciciais, edema do estroma viloso e dismaturidade vilosa. Nenhuma alteração microscópica ou macroscópica específica do HIV foi encontrada nas placentas. A infecção neonatal pôde ser constatada em cinco casos. A transmissão vertical foi identificada em duas placentas entre cinco que tinham baixo peso para a idade gestacional. Análise da imuno-histoquímica do p24 mostrou 14 casos positivos, dois dos quais apresentaram transmissão vertical. A proteína viral não foi identificada em 10 das 14 placentas cujas pacientes foram medicadas com zidovudina (AZT). CONCLUSÃO: Nosso estudo contribuiu para o estudo anatomopatológico da placenta de pacientes soropositivas para o HIV, porém a expressão do p24 por si só não permitiu um diagnóstico definitivo e precoce da transmissão vertical.

Evaluation of two indigenous rapid and two ELISA assays for the diagnosis of HIV infection India.

Purpose: Human immunodeficiency virus (HIV) diagnostic tests are being used extensively in India. However, the evaluation data on these assays are very limited. The present study evaluates indigenous HIV test kits manufactured in India. Materials and Methods: A total of 200 characterised specimens were assayed with Comb AIDS - RS Advantage HIV 1+2 Immunodot Test, Enzaids HIV 1+2 ELISA test, Enzaids Duet HIV Antigen+antibody ELISA test and Signal HIV Flow Through HIV 1+2 test kits. Performance characteristics of these assays were calculated. Results: Sensitivity, specificity, positive predictive value, negative predictive value and efficiency of all the assays were 100% except for Signal HIV Flow Through HIV 1+2 test kit. The specificity, positive predictive value and efficiency of the Signal HIV Flow Through HIV 1+2 test kit were 98.9%, 98.9% and 99.4%, respectively. The Enzaids Duet HIV kit was found to be extremely sensitive in detecting p24 Ag with the sensitivity of 1.5 pg/mL. Conclusions: To conclude, selection of better diagnostic assay is very much important to resolve discrepancies in HIV diagnosis. All these assays under evaluation in this report have got excellent performance characteristics and much suitable to use in serial testing algorithms in use for resources limited settings.

Detection of HIV-1 p24 based on gold nanoparticle probe and PCR / 中华检验医学杂志

ObjectiveTo establish a novel assay for HIV-1 p24 ultrasensitive detection based on Gold Nanoparticle Probe (GNP) and PCR.MethodsSandwich ELISA method was established by a pair of anti-p24 monoclonal antibodies (mAbs),1G12 and 1D4,and was used to detect recombinant HIV-1 p24 antigen.The bio-barcode DNA was 47 bp,selected from genome of Arabidopsis,and formed double-stranded DNA by hybridization with the capture DNA (complementary with bio-barcode DNA) modified with sulfhydryl.Then double-stranded DNA were conjugated on the surface of 1D4-modified gold nanoparticles by sulfhydryl,and the Gold Nanoparticle Probe was produced.1G12 was precoated in the micropaltes,and in the presence of target recombinant HIV-1 p24 protein,a sandwich immuno-complex would form by adding GNP.Then the bio-barcode DNA in the immuno-complex were released by heating as detection signal,and consequently characterized by the polymerase chain reaction (PCR) with synthesized special primers and analyzed by 4％ agar gel electrophoresis,so HIV-1 p24 antigen could be evaluated.The sensitivity comparison between the new assay and ELISA can be done.ResultsSandwich ELISA was used to quantify HIV-1 p24 antigen by monoclonal antibodies 1G12 and 1D4,and the limit of detection (LOD) was 1000 pg/ml.The new GNP assay was established by the same pair of antibodies,combined with PCR and agar gel electrophoresis,and was used to indirectly detect HIV-1 p24 antigen.The band intensity of PCR products paralleled with the quantity of HIV-1 p24 antigen,and the limit of detection (LOD) could reach down to 1 pg/ml.ConclusionThe new assay based on GNP and PCR was efficient in the detection of HIV-1 p24,which is at least 3 orders of magnitude more sensitive than traditional ELISA.

Effect of Morphine on the replication of HIV-1 in MT2 and macrophage / 中华检验医学杂志

Objective To determine whether Morphine has the ability to enhance HIV-1 replication in MT2 and Macrophage in vitro and assess the influence of Naloxone on Morphine2s effect.Methods MT2 cells were randomly assigned into 4 groups: (1) Morphine treatment for MT2 group, (2) Morphine+Naloxone co-treatment for MT2 group, (3) Naloxone treatment for MT2 group and (4) MT2 Control;Macrophages were also randomly assigned into 4 groups: (5) Morphine treatment for Macrophage group, (6) Morphine+Naloxone co-treatment for Macrophage group, (7) Naloxone treatment for Macrophage group and (8) Macrophage Control. Group (2), (3), (6) and (7) were pre-treated with 10-8 mol/L Naloxone for 0.5 h, and then group (1) and (2) were treated with 10-12, 10-10 and 10-8 mol/L Morphine for 24 h;group (5) and (6) were disposed of 10-10 mol/L Morphine for 24 h.All 8 groups were added in HIV-1 viral strain with 50% tissue culture infective dose(TCID50).P24 antigen in MT2 cells culture supernatant at day 3, 4, 5 and 6, and in Macrophages culture supernatant at day 4, 6, 8, 10 and 12 after infection were determined with ELISA.Student2s t-test and ANOVA were used to compare the differential expression in different groups, and repeated measures ANOVA was used to compare the increasing or decreasing expression of p24 antigen in morphine treatment groups than that in the control group at different time points.Results On the 3rd day of infection with HIV-1 in MT2 cells, the expression of p24 antigen in 10-12, 10-10 and 10-8mol/L dose of group (1) were (4.44?.30), (5.59?.25) and (4.60?.24) ng/ml respectively, compared to control[(1.93?.05) ng/ml, t= 14.15, 24.74 and 19.14, all P<0.01].On the 4th day, 10-12, 10-10 and 10-8mol/L dose of group (1) resulted in a significant increase of p24 antigen expression [(24.30?.66), (31.73?.17) and (26.02?.37) ng/ml]in culture supernatants compared to control[(8.03?.09) ng/ml, t=10.59, 34.92 and 81.2, all P<0.01].On the 5th day, the expression of p24 antigen in 10-12, 10-10 and 10-8 mol/L dose of group (1) were (56.30?.26), (81.77?.49) and (63.66?.57) ng/ml respectively, compared to control [(15.30?.91) ng/ml, t= 45.83, 43.51 and 30.07, all P<0.01].On the 6th day, the expression of p24 antigen in 10-12, 10-10 and 10-8 mol/L dose of group (1) were (150.70?.97), (243.09?.93) and (173.72?.73) ng/ml respectively, compared to control [(41.01?.84) ng/ml, t= 21.09, 39.02 and 29.55, all P<0.01].The enhanced multiple of p24 antigen expression in three doses of morphine treatment group compared to control increased with HIV-1 infected MT2 cells time, trend analysis of repeated measurements showed statistically significant time effect (F=842.18, P<0.01). On the 4th day of infection with HIV-1 in Macrophage cells, the expression of p24 antigen in 10-12, 10-10 and 10-8 mol/L dose of group (5) were (0.68?.15), (0.87?.41) and (0.75?.09) ng/ml respectively, compared to control [(0.60?.01) ng/ml, t= 7.27, 11.06 and 3.02, all P<0.05]. On the 6th day, 10-12, 10-10 and 10-8 mol/L dose of group (5) resulted in a significant increase of p24 antigen expression[(1.64?.57) , (2.07?.12 ) and (1.75?.17) ng/ml]in culture supernatants compared to control [(1.16?.07) ng/ml, t=8.93, 11.3 and 5.45, all P<0.01].On the 8th day, the expression of p24 antigen in 10-12, 10-10 and 10-8 mol/L dose of group (5) were (6.31?.17), (8.81?.34) and (7.19?.11) ng/ml respectively, compared to control [(3.84?.45) ng/ml, t=8.83, 15.11 and 12.42, all P<0.01]. On the 10th day, the expression of p24 antigen in 10-12, 10-10 and 10-8 mol/L dose of Morphine treated group were (32.30?7.55), (50.74?7.55) and (39.74?.56) ng/ml respectively, compared to control [(17.55?.86) ng/ml, t= 13.65, 17.84 and 36.69, all P<0.01].The enhanced multiple of p24 antigen expression in three doses of group (5) compared to control increased with HIV-1 infected Macrophage cells time, trend analysis of repeated measurements showed statistically significant time effect (F=135.58, P<0.01).Conclusions Morphine has the ability to enhance HIV-1 replication in MT2 cell and Macrophage. This Morphine-mediated increase of p24 antigen expression can be blocked by Naloxone.

Resultados preliminares del perfeccionamiento del DAVIH-AgP24 con el empleo del sistema de amplificación biotina-tiramina/estreptavidina-peroxidasa / Preliminary results of DAVIH-AgP24 with the biotin-tiramyne/streptavidine-peroxidase amplification system

Se modificó el ELISA DAVIH-Ag P24 con la introducción de la disociación por calor de las muestras de plasma y el empleo de un sistema de amplificación biotina-tiramina/estreptavidina-peroxidasa, para incrementar su sensibilidad. Se determinó la repetibilidad interensayo en el DAVIH-Ag P24 amplificado. Se evaluaron 32 muestras de plasma de individuos infectados por el VIH-1 en 3 categorías clínicas (caso SIDA, asintomßticos y con infecciones oportunistas menores). En la determinación de la repetibilidad interensayo se obtuvo un coeficiente de variación entre 4 y 10,3 por ciento. Con el DAVIH-Ag P24 amplificado se incrementó el nivel de detección de P 24 hasta 0,5 pg/mL. El DAVIH-Ag P24 amplificado alcanzó 66 por ciento de sensibilidad, mientras que el DAVIH-Ag P24 obtuvo 31 por ciento. Este estudio preliminar permitió demostrar que la incorporación de las nuevas modificaciones al sistema DAVIH-Ag P24 amplificado logró aumentar los niveles de detección de P24 y ganar en sensibilidad.

ELISA DAVIH-Ag p24 was modified by introducing heat dissociation of plasma samples and a tyramine/streptavidine-peroxidase amplification system, with the objective of increasing sensitivity. Between-assay repeatability was determined in amplified DAVIH-Ag p24. Thirty two plasma samples from HIV-1-infected individuals classified in three clinical categories (AIDS case, asymptomatic and minor opportunistic infections) were evaluated. The variation coefficient ranged 4-10.3 percent in between-assay repeatability. With the amplified DAVIH-p24 Ag, the p24 antigen detection level increased to 0.5 pg/mL. Amplified DAVIH-p24 Ag reached 66 percent sensitivity whereas standard DAVIH-p24 Ag sensitivity rate was 31 percent. This preliminary study proved that the introduction of new modifications in amplified DAVIH-p24 Ag managed to increase the p24 antigen detection levels and to gain sensitivity.

Probabilidad de infección reciente por VIH en pacientes con presencia de antígeno p24 y prueba confirmatoria negativa / Probability of recent HIV infection in patients with presence of p24 antigen and negative confirmation test

Introducción: La inclusión en las pruebas de detección del VIH de la capacidad de determinar el antígeno p24 ha potenciado su capacidad diagnóstica para infecciones recientes. Objetivos: Evaluar el antígeno p24 como predictor de infección reciente por VIH en pacientes con prueba confirmatoria negativa. Método: Estudio descriptivo de seroconversión en 245 muestras de personas desde los 14 años de edad con resultado confirmatorio negativo para el VIH, en muestras de las serotecas de los laboratorios de Salud Pública Distrital y el Centralizado de VIH en la ciudad de Bogotá, Colombia. Se encontraron en 12 de ellas y se estudió la seroconversión en 6. Resultados: Se confirmó seroconversión en el 60% de pacientes positivos para prueba presuntiva de p24 y en el 75% de los positivos para confirmatoria del mismo antígeno. Conclusiones: Estos resultados sugieren la necesidad de realizar pruebas diagnósticas adicionales a todos los casos con resultado reactivo en prueba presuntiva y negativo para confirmatoria, en los que la reactividad de la primera pueda estar determinada por la presencia del antígeno p24 en a muestra, a fin de establecer una posible infección reciente por este virus.

Background: The inclusion of the capacity to generate p24 antigen in presumptive tests to detect the Human Immunodeficiency Virus (HIV) have enhanced its diagnosing potential in recent infection cases. The absence of this condition in confirmatory tests creates a risk of false negatives. Aims: To assess p24 antigen as a predictor of recent HIV infection in patients with negative confirmation test. Methods and design: Descriptive study of seroconversion of patients with confirmed negative test for HIV. Scenario: A study based on samples taken from the erum banks of the District Public Health Lab and Centralized HIV Lab in Bogotá, D.C., Colombia. Participants: p24 antigen was sought in 245 samples of people aged 14 or older, either reactive for presumptive tests or negative for confirmatory tests. The antigen was found in 12 of them and seroconversion took place in 6 of them. Interventions: 253 blood samples, either reactive for presumptive tests or negative for confirmatory tests were obtained out of 393,247 samples taken between January 2006 and November 2007. Finally, seroconversion was studied in patients with reactive test for p24 antigen. Outcome measurement: Seroconversion took place in patients with reactive tests for p24 antigen test for presumptive test and negative confirmatory test. Results: Seroconversion was confirmed in 60% of the patients tested positive for presumptive p24 antigen and in 75% of the patients tested positive for confirmatory test of the same antigen. Conclusions: These results suggest the need to carry out additional diagnostic tests to all cases with reactive results in presumptive testing and negative confirmatory testing in which the reactivity of the presumptive testing can be determined by the presence of p24 antigen in the sample, in order to establish a possible recent infection by this virus.

Immuno-PCR Assay on Detection of HIV-1 p24 Antigen:A Primary Evaluation / 中华医院感染学杂志

OBJECTIVE To evaluate primarily the detection limit,specificity and reproducibility of immuno-PCR assay on HIV-1 p24 antigen.METHODS We p24 antigen were detected by established immuno-PCR system,and then the detection limit,specificity and reproducibility were discussed.We quantitatively analyzed the nonspecific amplified bands with fluorescence intensity(FI),and a preliminary determination of the lower specific amplification limit was made.RESULTS We taken(x-+3s)FI of nonspecific amplification as the lower limit of specific amplification signal.The detection limit of immuno-PCR assay was 0.1 ng/L.CONCLUSIONS The detection limit,specificity and reproducibility can meet the needs of HIV-1 p24 antigen detection.

Epidemiological investigation on natural infection of different canine breeds with Borna disease virus in Ili, China / 中华流行病学杂志

Objective To investigate the epidemioiogical pattern of Borna disease virus (BDV) among different canine breeds in Ili, China, and to analyze its potential phylogeny. Methods BDV p24 RNA fragments were detected from peripheral blood mononuclear cells (PBMCs) of canine by modified nested RT-PCR (nRT-PCR). Possible false positives were excluded by determination of both BDV p40 RNA fragments and PMD19 plasmid standards. Analysis were performed on genetic sequence, homologous comparison, amino acid sequence and phylogeny after p24 positive products were validated. Results BDV p24 RNA fragments were found only in Kazakh Tobet (a shepherd dog) in 8 breeds of 150 cases and their overall positive rate was 11.0% (10/91). Compared with the strain of He/80 from horse and that of S6 from sheep in Germany, the homologous similarities of Kazakh Tobet was 99.2% and 95.7%, and that of amino acid as 100% and 89.3%, respectively. The kinship of Kazakh Tobet was close to He/80 and next to S6. Conclusion There was potential natural BDV infection in Kazakh Tobet in Ili, and its endemic strain was concerned with He/ 80 infecting Ili horse and S6 of German Merino sheep introduced into the region from Germany.

Repair of Rabbit Femoral Defects with a Novel BMP2-derived Oligopeptide P24 / 华中科技大学学报（医学）（英德文版）

In this study, the bioactivity of a novel BMP2-derived oligopeptide P24 was investigated by using the model of rabbit femoral defect after loaded in the biodegradable poly (lactic acid / glycolic acid / asparagic acid-co-polyethylene glycol) (PLGA-[ASP-PEG]). A 1.5-cm unilateral segmental bone defect was created in the left femoral diaphysis in each of the 30 new zealand white rabbits.The defects of 18 legs filled with BMP2-derived peptide P24 combined with PLGA-[ASP-PEG]scaffold serves as the experimental group, and the defects in the rest 12 rabbits filled with(PLGA-[ASP-PEG]) without P24 as control group. The bone-repairing capability in the target region of the two group was grossly, radiologically, histopathologically and biomechanically evaluated 4, 8and 12 weeks after the operation. Our results showed that in each group, primary healing of incision was achieved in the two groups. Radiographically, in experimental group, defects were filled with induced callus within 8 weeks, and a cortical bone-like structure was observed in some animals at the12th week. According to the standardized stage of bone defect repair, 9 (64.28%) achieved grade-4healing. In contrast, little bone formation was seen in the defects even 12 weeks after the operation,and 5 (62.50%) had grade 0 healing in this group. Histologically, tissue engineering material was mostly absorbed and cartilage was found around implants in the experimental group at the 4th week;8 weeks after operation, the engineering material was completely absorbed, and formation of woven bone was observed and typical trabecular bone structure could be seen. In control group, 8 weeks after operation, the defect was filled with fibrous tissues, and no bone-like structure was observed. Statistical analysis showed very significant difference in biomechanical indicators between the two groups (P＜0.05). It is concluded that new oligopeptide P24 can induce excellent bone regeneration and promote bone repair.

Replacement of HIV p24 Ag test by a multiplex RT-PCR method for primary screening of blood donors

Nucleic Acid Testing (NAT) as a tool for primary screening of blood donors became a reality in the end of the 1990 decade. We report here the development of an "in-house" RT-PCR method that allows the simultaneous (multiplex) detection of HCV and HIV-RNA in addition to an artificial RNA employed as an external control. This method detects all HIV group M subtypes, plus group N and O, with a detection threshold of 500 IU/mL. After validation, the method replaced p24 Ag testing, in use for blood donation screening since 1996 at our services. From July 2001 to February 2006, 102,469 donations were tested and 41 (0.04 percent) were found HIV-RNA reactive. One NAT-only reactive donation (antibody non-reactive) was observed, with subsequent seroconversion of the implied donor, giving a yield of 1:102,469. This rate is in contrast to the international experience that reports a detection of approximately 1:600,000 - 1:3,100,000 of isolated HIV-RNA donations.

O uso de testes de ácidos nucleicos (NAT) na rotina de triagem de doadores de sangue tornou-se uma realidade ao final da década de 1990. Descreve-se aqui uma metodologia de RT-PCR multiplex "in-house" que permite a detecção simultânea dos RNAs dos vírus HIV e HCV além de uma molécula artificial de RNA usada como controle externo. O método detecta todos os subtipos de HIV do grupo M e também do grupo N e O, com uma sensibilidade de 500 UI/mL. Após validação, este teste substituiu o do antígeno p24, até então na rotina de triagem em nosso laboratório, desde 1996. De julho de 2001 a fevereiro de 2006 foram testadas 102.469 doações e 41 (0.04 por cento) foram NAT reativas. Uma doação NAT isoladamente reativa (anticorpo não-reativa) foi detectada com soroconversão subseqüente do doador, portanto, o rendimento do NAT nesta população até o presente momento é de 1:102.469. Este número contrasta com a experiência obtida internacionalmente, onde taxas de 1:600.000 - 1:3.100.000 foram descritas.

Experimental Research on Ectopic Osteogenesis of BMP2-derived Peptide P24 Combined with PLGA Copolymers / 华中科技大学学报（医学）（英德文版）

To experimentally evaluate the ectopic osteogenetic capacity of synthesized BMP2-derived peptide P24 combined with poly lactic-co-glycolic acid (PLGA), Wistar rats were divided into two groups: group A, in which BMP2-derived peptide P24/PLGA complex was implanted,and group B which received simple PLGA implant. The complex was respectively implanted into the back muscles of rats. Samples were taken the 1 st, 4 th, 8 th, and the 12 th week after the implantation.Their bone formation was detected by X-ray examination, and tissue response was histologically observed. Western blotting was used for the detection of the expression of collagen Ⅰ (Col- Ⅰ ) and osteopontin (OPN). There was acute inflammation in the tissue around both types of implants at early stage. The cartilage was found around implant areas 4 weeks after the implantation of BMP2-derived peptide p24/PLGA complex, 8 weeks after the implantation, osteoblasts were found, and 12 weeks after the implantation, typical trabecular bone structure was observed. In group B, after 12 weeks, no osteoblasts were found. It is concluded that PLGA is an ideal scaffold material for bone tissue engineering. BMP2-derived peptide can start endochondral ossification and is more effective in inducing ectopic osteogenesis.

Detection of HIV-1 p24 by Immuno-PCR Assay Using Gold-magnetic Particles as Carriers / 中华医院感染学杂志

OBJECTIVE To establishan immuno-PCR assay with the carriers of gold-magnetic particles for detection of HIV-1 p24. METHODS The feasibility of using gold-magnetic particles as the carriers was verified. The gold-magnetic particles were coated with mouse anti-p24 monoclonal antibody as the capture antibody. The reporter DNA was initially generated by PCR amplification using a biotinylated primer, and was bound through streptavidin to biotinylated polyclonal antibody as the detection antibody. HIV-1 p24 sandwiched by two antibodies was detected by amplifying the reporter DNA using PCR. RESULTS The efficiency of gold-magnetic particles coated with mouse anti-p24 monoclonal antibody could reach up to 95%. Furthermore, the amount of antibodies immobilization was consistent among different batches of gold-magnetic particles and there was nearly without nonspecific adsorption. The detection limit of immuno-PCR assay was 0.1 ng/L, an approximately 1.5?104-fold higher compared with an enzyme-linked immunosorbent assay. The linear range of p24 concentration was 0.1-100 ng/L. CONCLUSIONS Gold-magnetic particle is one of the ideal immuno-PCR reaction carriers. The immuno-PCR for detection of HIV-1 p24 reported in this article is indicated to be a promising detection method.

No Evidence for Linkage of Chromosome 6p24-22, The Locus of Dysbindin Gene, to Schizophrenia in Korean Families / 신경정신의학

OBJECTIVES: Chromosome 6p24-22 has been identified as a disease locus with a high probability for schizophrenia based on several genomewide linkage scans with Caucasian families. The recent association studies suggest that the dysbindin gene located at chromosome 6p22.3 may be a candidate gene of schizophrenia. The purpose of this study was to investigate the linkage of chromosome 6p24.3-22.3 locus to schizophrenia in Korean families. METHODS: We recruited one hundred fifty-seven family members from forty-six multiplex schizophrenia families. One hundred three of them were affected individuals. four microsatellite markers with 4.8 cM intervals on 6p24.3-22.3 were genotyped. Nonparametric linkage analysis was performed by evaluating the levels of allele sharing between the affected relative pairs. RESULTS: In the single point analysis, no markers on chromosome 6p24.3-22.3 locus showed statistical evidence for linkage. Significant evidence for linkage was not found in the multi-point analysis. CONCLUSION: These results do not support the previous evidence from Caucasian families for a locus predisposing to schizophrenia at 6p24.3-22.3, the locus of dysbindin gene. We conclude that if there is a susceptibility locus for schizophrenia in this region then its effect size is so small as to render our study insufficiently powerful to detect it and schizophrenia susceptibility loci in Korean families likey have different ethnicity-specific effects from Caucasian families.

Modified boosted – p24 antigen can predict HIV-1 RNA viral load (Ampiclor HIV-1 Monitor).

Objective : To evaluate the prediction of HIV-1 RNA viral load (Ampiclor) in log_copies per ml by each level of modified boosted-p24 antigen in log_ fg per ml. , Methods : 283 plasma samples were collected and blindly determined the HIV-1 RNA Amplicor Monitor, Roche as standard test with modified boosted p24 antigen assay. Likelihood ratio positive analyses of multiple levels of p24 were calculated as well as the post-test probability in predicting the amount of virus (log_copies/ ml). Results: Subject were between 18 to 73 years old with the range of virus 1.75 to 5.92 log-copies per ml. By the calculation of likelihood ratio positive and positive predictive value, it was demonstrated that modified boosted p24 antigen (Ag in log-fg per ml) might predict the viral load (VL in log-copies per ml) as follow. Ag 2.0-3.0 log-fg per ml corresponding to 2.0 or lower log-copies per ml VL Ag 3.0-3.5 log-fg per ml corresponding to 2.5 or lower log-copies per ml VL Ag 3.5-4.0 log-fg per ml corresponding to 3.5 or lower log-copies per ml VL Ag 4.0-over log-fg per ml corresponding to 4.5 or lower log-copies per ml VL Conclusions: In countries with limited financial resources, the modified p24 antigen may be clinically applied in antiretroviral management programmes, instead of the HIV-1 RNA Amplicor Monitor, Roche.

Antibody recognition of synthetic peptides mimicking immunodominant regions of HIV-1 p24 and p17 proteins / Reconocimiento por anticuerpos de péptidos sintéticos que imitan regiones inmunodominantes de las proteínas p24 y p17 de VIH-1

The gag gene of HIV-1 encodes a single open reading frame of 55 kDa that contains three subdomains: the matrix domain (p17), the capsid domain (p24) and the nucleocapsid domain (p15). The p24 and p17 proteins have a predominant a-helical structure and perform important functions throughout thevirallife-cycle. The determination of gag-specific antibodies is important because declining titers of these antibodies herald clinical deterioration.In this work we present the results obtained on immunoreactiviy of synthetic peptides that mimic immunogenic a-helical regions of p24 and p17. The influence on the immunoreactivity of structural modifications in native sequences, including the addition of non immunogenic side chains: AAAC- and -CAAA on both side of minimal epitopes was evaluated in indirect and competitive enzymeimmunoassays. The conformational characteristcs to the peptides were analysed by circular dichroism and these results were correlated with that obtained in the immunoassays. It was shown that the reactivity of peptides mimicking short a-helical regions of p24 and p17 is improved by adding short non immunogenic chains on both N- and C- terminus. These modifications enhanced the immobilization of the peptides onto the solid support and allowed more accesibility to the minimal epitopes byspecific antibodies, in solution.

El gen gag del VIH-1 codifica una región de 55kDA que contiene tres subdominios: matriz (p17), cápside (p24) y nucleocápside (p15). Las proteínas p24 y p17 tienen una estructura predominante helicoidal y cumplen un rol importante en el ciclo de vida del virus. En este trabajo presentamos los resultados de inmunorreactividad de péptidos sintéticos que imitan regiones helicoidales de p24 y p17. Utilizando enzimoinmunoensayos se evaluó la influencia de modificaciones en las secuencias nativas sobre la capacidad de reconocimiento de anticuerpos específicos en solución y en fase sólida, incluyendo el agregado de cadenas no inmunogénicas en ambos extremos de los epitopes mínimos. La conformación de los péptidos se determinó por dicroísmo circular y los resultados se correlacionaron con los de inmunorreactividad. Se observó que la capacidad de reconocimiento de anticuerpos por péptidos pequeños que imitan estructuras helicoidales de p24 y p17 mejoró con el agregado de cadenas no inmunogénicas en ambos extremos de los epitopes. Estas modificaciones mejoran la inmovilización sobre las superficies sólidas y permiten una mayor accesibilidad de los anticuerpos a los epitopes mínimos en solución.