Preclinical study of LMP1-RNAi-based anti-tumor therapy in EBV-positive nasopharyngeal carcinoma

RNA interference (RNAi) treatment has been proven to be an important therapeutic approach in cancer based on downregulation of target-oncogenes, but its clinical efficacy still needs further investigation. LMP1 is usually presented by Epstein-Barr virus (EBV)-positive tumor cells like EBV-associated nasopharyngeal carcinoma (NPC) and acts as an oncogene in tumorigenesis. However, the mechanism of LMP1 as a proto-oncogene in nasopharyngeal carcinoma is still unclear. Two sequence-specific shRNAs 1 and 2 were designed to target the different nucleotide loci of EBV latent antigen LMP1 gene and a series of in vivo and in vitro experiments were performed to investigate the therapeutic effect of sequence-specific shRNAs targeting LMP1 and its related molecular mechanisms in EBV-positive NPC. LMP1-shRNA2 generated a truncated LMP1 mRNA and protein, whereas LMP1-shRNA1 completely blocked LMP1 mRNA and protein expression. Both LMP1-shRNAs inhibited the proliferation and migration of NPC cells overexpressing LMP1 (NPC-LMP1) as well as the NPC-associated myeloid-derived suppressor cell (MDSC) expansion in vitro. However, LMP1-shRNA2 maintained the immunogenicity of NPC-LMP1 cells, which provoked MHC-class I-dependent T cell recognition. LMP1-shRNAs inhibited tumor growth in nude mice but did not reach statistical significance compared to control groups, while the LDH nanoparticle loaded LMP1-shRNAs and the antigen-specific T cells induced by NPC-LMP1 cells treated with LMP1-shRNA2 significantly reduced tumor growth in vivo. LMP1-RNAi-based anti-tumor therapy could be a new hope for the clinical efficacy of RNAi treatment of tumors like NPC.

Functional investigation of chimeric antigen receptor T cells targeting LMP1 antigen / 中华血液学杂志

Objective: This study aimed to create a type of CAR-T cells that targets LMP1 antigen and study its immunotherapeutic effect on LMP1-positive hematological malignancies. Methods: To generate LMP1 CAR-T cells, a plasmid expressing LMP1 CAR was created using molecular cloning technology, and T cells were infected with LMP1 CAR lentivirus. The effects of LMP1 CAR-T cells on specific cytotoxicity against LMP1-positive tumor cell lines infected with the EB virus had been confirmed. Results: ① LMP1 protein expressing on EB virus-positive lymphoma cells surface was verified. ② The LMP1 CAR-expressing plasmid was created, and LMP1 CAR-T cells were obtained by infecting T cells with a lentivirus packaging system, with an infection efficiency of more than 80% . ③LMP1 CAR-T cells have a 4∶1 effect-to-target ratio in killing LMP1-positive lymphoma cells. The killing effect of LMP1 CAR-T cells on Raji cells was enhanced after 48 h of coculture, but there was no significant killing effect on Ramos, which are LMP1-negative lymphoma cells. ④After coculture with LMP1-positive lymphoma cells at a ratio of 1∶1 for 5 h, the degranulation effect was enhanced. The proportion of CD107a(+) T cells in the LMP1 CAR-T cell treatment group was significantly higher than that in the vector-T cell group [ (13.25±2.94) % vs (1.55±0.05) % , t=3.972, P=0.017]. ⑤After coculture with LMP1-positive lymphoma cells, the proportion of CD69(+) and CD25(+) T cells in the LMP1 CAR-T cell group was significantly higher than that in vector-T cell group [ (7.40±0.41) % vs (3.48±0.47) % , t=6.268, P=0.003; (73.00±4.73) % vs (57.67±2.60) % , t=2.842, P=0.047]. ⑥After coculture with LMP1-positive lymphoma cells, cytokine secretion in the LMP1 CAR-T cell group was higher than that in the vector-T cell group [interferon-gamma: (703±73) ng/L vs (422±87) ng/L, t=2.478, P=0.068; tumor necrosis factor-alpha: (215±35) ng/L vs (125±2) ng/L, t=2.536, P=0.064]. Conclusion: In this study, we found that the LMP1 protein is only found on the surface of the EBV-positive tumor cell. Simultaneously, we created an LMP1 CAR-expressing plasmid and obtained LMP1 CAR-T cells by infecting T cells with a lentivirus packaging system. Furthermore, we demonstrated that LMP1 CAR-T cells could specifically kill LMP1-positive tumor cells in vitro. The degranulation and activation effects of LMP1 CAR-T cells were enhanced after coculture with LMP1-positive tumor cells, indicating a potential clinical application.

In vivo and in vitro study of co-expression of LMP1 and Cripto-1 in nasopharyngeal carcinoma / Estudo in vivo e in vitro da coexpressão de LMP1 e Cripto-1 em carcinoma nasofaríngeo

Abstract Introduction: Nasopharyngeal carcinoma, an epithelial-derived malignant tumor which because of its anatomical location and atypical early symptoms, when diagnosed invasion and metastasis often have occurred. This requires a better understanding of the development mechanism, identifying diagnostic markers, and developing new treatment strategies. Objective: To study the relationship of LMP1 and Cripto-1 in nasopharyngeal carcinoma. Methods: The expression of LMP1 and Cripto-1 in specimens obtained from nasopharyngeal carcinoma patients (n = 42) and nasopharyngitis patients (n = 22) were examined. The expression of LMP1 and Cripto-1 in LMP1-negative and LMP1-positive (CNE1-LMP1) cells were also examined. Results: The expression of LMP1 and Cripto-1 was significantly higher in nasopharyngeal carcinoma than in nasopharyngitis (p < 0.05). Their expression in nasopharyngeal carcinoma with metastasis were significantly higher than that without metastasis (p < 0.05), which was correlated with TNM staging (p < 0.05). High Cripto-1 expression and high proliferation rate were seen in CNE1-LMP1 cells. Conclusions: The expression of LMP1 and Cripto-1 in nasopharyngeal carcinoma is positively related. Their co-expression might contribute to the proliferation and metastasis of nasopharyngeal carcinoma.

Resumo Introdução: O carcinoma nasofaríngeo é um tumor maligno derivado do epitélio de localização anatômica recôndita e sintomas iniciais atípicos; quando diagnosticado, frequentemente invasão e metástases já ocorreram. Isso requer uma melhor compreensão do seu mecanismo de desenvolvimento, identificação dos marcadores diagnósticos e desenvolvimento de novas estratégias de tratamento. Objetivo: Estudar a relação de LMP1 e Cripto-1 no carcinoma nasofaríngeo. Método: A expressão de LMP1 e Cripto-1 em espécimes obtidos de pacientes com carcinoma de nasofaringe (n = 42) e pacientes com nasofaringite (n = 22) foi analisada. A expressão de LMP1 e Cripto-1 em células LMP1-negativas e LMP1-positivas (CNE1-LMP1) também foi analisada. Resultados: A expressão de LMP1 e Cripto-1 foi significantemente maior na presença de carcinoma nasofaríngeo do que na nasofaringite (p < 0,05). Sua expressão em carcinomas com metástase foi significantemente maior do que em casos sem metástase (p < 0,05), o que se correlacionou com o estadiamento TNM (p < 0,05). Uma alta expressão de Cripto-1 e alta taxa de proliferação foram observadas nas células CNE1-LMP1. Conclusões: A expressão de LMP1 e Cripto-1 é positivamente relacionada com carcinoma nasofaríngeo. Sua coexpressão pode ser atribuída à proliferação e metástase do tumor.

LMP2-DC Vaccine Elicits Specific EBV-LMP2 Response to Effectively Improve Immunotherapy in Patients with Nasopharyngeal Cancer / 生物医学与环境科学（英文）

Objective@#To evaluate the safety and effectiveness of a vaccine based on latent membrane protein 2 (LMP2) modified dendritic cells (DCs) that boosts specific responses of cytotoxic T lymphocytes (CTLs) to LMP2 before and after intradermal injection in patients with nasopharyngeal carcinoma (NPC).@*Methods@#DCs were derived from peripheral blood monocytes of patients with NPC. We prepared LMP2-DCs infected by recombinant adenovirus vector expressing LMP2 (rAd-LMP2). NPC patients were immunized with 2 × 10 @*Results@#We demonstrated that DCs derived from monocytes displayed typical DC morphologies; the expression of LMP2 in the LMP2-DCs vaccine was confirmed by immunocytochemical assay. Twenty-nine patients with NPC were enrolled in this clinical trial. The LMP2-DCs vaccine was well tolerated in all of the patients. Boosted responses to LMP2 peptide sub-pools were observed in 18 of the 29 patients with NPC. The follow-up data of 29 immunized patients from April, 2010 to April 2015 indicated a five-year survival rate of 94.4% in responders and 45.5% in non-responders.@*Conclusion@#In this pilot study, we demonstrated that the LMP2-DCs vaccine is safe and effective in patients with NPC. Specific CTLs responses to LMP2 play a certain role in controlling and preventing the recurrence and metastasis of NPC, which warrants further clinical testing.

Estimation of gingival blood glucose using a sensitive self-monitoring device in periodontitis patients

Background: Diabetes Mellitus is a complex disease with varying degree of systemic and oral complications. The prognosis is quite favorable if a disease is diagnosed in early stages. Since a large number of patients seek dental treatment routinely, screening procedures for early detection of subclinical cases can help in diagnosis of asymptomatic diabetes. Aim: The present study was undertaken to evaluate if gingival crevicular blood can be used for the estimation of blood glucose levels in periodontitis patients. Material and Methods: A prospective study was carried out comprising 150 patients Group A comprised of 75 subjects with gingivitis and group B comprised of 75 subjects with periodontitis. For gingival crevicular blood glucose (GCBG) level estimation, the blood was drawn onto the glucometer strip after gently probing the gingival sulcus and the readings were recorded. At the same time, blood Vijayendra Pandey, Akhilesh Chandra, Deepak Kumar, Anup Kumar Singh, Priyankesh, Alok Kumar Gupta. Estimation of gingival blood glucose using a sensitive self-monitoring device in periodontitis patients. IAIM, 2019; 6(6): 51-56. Page 52 was also collected from the index finger onto the glucometer strip for the capillary finger-prick blood glucose (CFBG) sample. Both the values were compared and statistical analysis of data was performed. Results: The mean GCBGL and CFBGL in group A was 98.43 mg/dl ± 18.62 and 103.48 mg/dl ± 13.90 respectively, while in group B it was 136.37 mg/dl ± 36.95 and 141.62 mg/dl ± 51.84, respectively. There was no statistically significant difference (p> 0.05) between the two values in both the groups. Conclusion: It can be concluded that GCBG levels are positively correlated with CFBG levels. Therefore, clearly indicating that gingival crevicular blood collected during diagnostic periodontal examination may be an excellent source of blood sample for glucometric analysis.

Expression of Immunoproteasome Subunit LMP7 in Breast Cancer and Its Association with Immune-Related Markers / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: In the presence of interferon, proteasome subunits are replaced by their inducible counterparts to form an immunoproteasome (IP) plays a key role in generation of antigenic peptides presented by MHC class I molecules, leading to elicitation of a T cell‒mediated immune response. Although the roles of IP in other cancers, and inflammatory diseases have been extensively studied, its significance in breast cancer is unclear. MATERIALS AND METHODS: We investigated the expression of LMP7, an IP subunit, and its relationship with immune system components in two breast cancer cohorts. RESULTS: In 668 consecutive breast cancer cohort, 40% of tumors showed high level of LMP7 expression, and tumors with high expression of LMP7 had more tumor-infiltrating lymphocytes (TILs) in each subtype of breast cancer. In another cohort of 681 triple-negative breast cancer patients cohort, the expression of LMP7 in tumor cells was significantly correlated with the amount of TILs and the expression of interferon-associated molecules (MxA [p < 0.001] and PKR [p < 0.001]), endoplasmic reticulum stress-associated molecules (PERK [p=0.012], p-eIF2a [p=0.001], and XBP1 [p < 0.001]), and damage-associated molecular patterns (HMGN1 [p < 0.001] and HMGB1 [p < 0.001]). Patients with higher LMP7 expression had better disease-free survival outcomes than those with no or low expression in the positive lymph node metastasis group (p=0.041). CONCLUSION: Close association between the TIL levels and LMP7 expression in breast cancer indicates that better antigen presentation through greater LMP7 expression might be associated with more TILs.

Presencia del marcador lmp-1 del virus epstein-barr en linfomas de caninos / Presence of lmp-1 protein of epstein-barr virus in lymphomas of canine origin

RESUMEN El virus Epstein-Barr (EBV) es un virus de alta prevalencia en humanos que se asocia con tumores de la línea linfoide B. En caninos se dispone de pocos reportes sobre la presencia del EBV y su rol en esta especie. El objetivo del presente estudio fue determinar la presencia de la proteína latente de membrana del EBV (LMP-1) en tejidos obtenidos de 20 linfomas de caninos cuyo diagnóstico se había realizado durante un periodo de 10 años, entre 2004 y 2014. Los linfomas se reclasificaron mediante las nuevas clasificaciones histopatológicas para linfomas y se sometieron a inmunohistoquímica (IHQ) con los anticuerpos anti-CD79a, anti-CD3, anticuerpos específicos para linfocitos B y T, además de un anti-LMP-1 como marcador de la presencia del EBV. Se encontró que el linfoma más común fue el linfoma nodal de zona T con un 75% de los casos. Al realizar la inmunomarcación se encontraron 18 casos positivos a CD3, 2 casos positivos a CD79a y 6 casos positivos a LMP-1, lo que representa el 30% de positividad del EBV en linfomas. El análisis Ji cuadrado demostró significancia estadística entre la presencia del virus y la presencia del linfoma lo que sugiere, no solamente que el virus está circulando en la población canina, sino que además puede tener relación con la ocurrencia de esta neoplasia. Con relación a las variables demográficas, sólo en la raza Golden Retriever se demostró relación con la presencia del linfoma, pero no con la presencia del virus.

ABSTRACT Epstein Barr virus (EBV) is a human high prevalent virus associated with lymphoid B cells tumors development. In canines, few reports have been published regarding the presence of the virus in dogs but its role in this species remains unclear. The aim of this study was to determine the presence of LMP-1 protein of EBV in 20 canine lymphomas tissues which were previously diagnosed in a period of time between 2004 -2014. Lymphomas were reclassified in accordance with the new histopathological classifications for lymphomas and were stained by IHQ with anti-CD79a, anti-CD3 and anti-LMP-1; in addition, specific antibodies for B lymphocytes, T lymphocytes and EBV biomarker, respectively. It was found that the most common lymphoma was T-zone lymphoma in 75% of the cases of the study. The distribution of the cases regarding the immunostaining was: 18 positive cases with anti-CD3, 2 positive cases with anti-CD79a and 6 positive cases with anti- LMP-1. Positive cases of LMP-1 as a biomarker of the presence of EBV corresponded to the 30% of the cases of the study. Chi-square test showed statistical significance between the presence of the virus and the presence of lymphomas, which suggests not only that the virus is circulating in the canine population but also that could have implications in the development of the disease. Regarding demographic parameters, only the Golden Retriever breed showed a relationship with the presence of lymphoma, but not with the presence of the virus.

Subversion of immunoproteasome subunit expression in dengue virus serotype 2-infected HepG2 cells

Abstract: INTRODUCTION: Infection with all serotypes of dengue virus (DV) results in augmented antigen presentation by MHC class I molecules. However, the upregulation of immunoproteasome subunits only results from infection with two serotypes. This study aims to elucidate changes in the expression of immunoproteasome subunits resulting from infection with DV, particularly DV serotype 2 (DV2). METHODS: HepG2 cells were grown in various culture milieu. Total cellular RNA and proteins were extracted and quantified. RESULTS: Results demonstrated sequestration of immunoproteasome subunits LMP2 and LMP7 in DV2-infected cells. CONCLUSIONS: This study provides insights into the mechanisms underlying immune evasion by DV.

Effects of vIL-10 on MHC-I antigen processing“the operon”in nasopharyngeal carcinoma cell lines / 西安交通大学学报(医学版)

ABSTRACT:Objective To explore the effects of virus interleukin‐10 (vIL‐10 ) on different expressions of MHC‐I antigen processing “the operon” .Methods We collected nasopharyngeal carcinoma cells (CNE‐1 and CNE‐2) treated by vIL‐10 at different time points ,and detected the changes of MHC‐I antigen processing “the operons” (TAP‐1 ,TAP‐2 ,LMP‐2 ,LMP‐7 and HLA‐I) by RT‐PCR and Western blot .Results ① mRNA level :There was no difference in the expression of TAP‐1 in CNE‐1 and CNE‐2 cells at various time points .The expressions of TAP‐2 and LMP‐2 in CNE‐1 and CNE‐2 did not change at 1 ,4 ,6 ,12 h ,but downregulated and even disappeared at 24 h .The expression of LMP‐7 in CNE‐1 decreased 4 h after vIL‐10 was added ,and that in CNE‐2 decreased at 6 h .The expression of HLA‐I in CNE‐1 and CNE‐2 showed significant decrease at 24 h .② Protein expression :The expression of TAP‐1 in CNE‐1 and CNE‐2 showed significant decrease at 24 h .The expression of TAP‐2 in CNE‐1 and CNE‐2 was gradually downregulated at different time points .The expressions of LMP‐2 and LMP‐7 in CNE‐2 were gradually downregulated at different periods ,while that in CNE‐1 was only decreased at 12 h .The expression of HLA‐I in CNE‐1 and CNE‐2 was gradually downregulated ,but there was no significant difference at each period in CNE‐1 ,while the expression of HLA‐I in CNE‐2 at 24 h was significantly downregulated .Conclusion vIL‐10 can inhibit the expression of MHC‐I antigen processing “the operon” in NPC in the time‐dependent manner .

Evaluation of the immunogenicity of a rBCG expressing GM-CSF and LMP2A / 中华微生物学和免疫学杂志

Objective To identify the expression of a fusion gene GCA formed from GM-CSF gene and LMP2A gene of Epstein-Barr virus (EBV) in a recombinant BCG (rBCG) and to study its immunoge-nicity.Methods The rBCG was constructed to express the fusion gene GCA and the expressed products were detected by Western blot assay .ELISA was performed to measure specific antibody titers in serum sam-ples from mice immunized with rBCG .Lactate dehydrogenase assay was used to analyze the cellular immuni-ty of mice.A mouse model of EBV-positive gastric carcinoma was established to evaluate the therapeutic effects of rBCG.Results The target proteins of GM-CSF and LMP2A were successfully expressed in rBCG . The specific antibodies were detected in rBCG immunized mice as indicated by ELISA .The maximum anti-body titer reached 1 ∶27 900 [(326.5±7.8) pg/ml] as injection with rBCG 5×108/mouse.The rBCG in-duced cytotoxicity of cytotoxic lymphocytes (CTLs) to EBV-positive gastric carcinoma cells (GT39) (with a killing rate of 89.6%±6.8%) was significantly higher than that of control group (P<0.05) The sizes of tumor in PBS control group [(1964.0±548.7) mm3] and BCG group [(1268.65±72.4) mm3] were big-ger than those in rBCG group [(168.64±78.80) mm3].Conclusion The rBCG expressing GM-CSF and LMP2A fusion gene was successfully constructed .The rBCG could induce humoral and cellular immune re-sponses in mice and inhibit the growth of tumor .

Advance on subtypes/variant of Epstein-Barr virus and relationship between subtypes/variants andEpstein-Barr virus related diseases / 国际儿科学杂志

Epstein-Barr virus( EBV )is a ubiquitous human gamma-1 herpesvirus,which is associated with human malignancies,such as nasopharyngeal carcinoma,Burkitt's lymphoma and Hodgkin lymphoma.Based on the polymorphism of amino acids in the polymorphic region of EBV genome,it can be classified into different subtypes/variants.By now,whether the subtypes/variants of EBV preferentially is associated with particular malignancies or represent geographical polymorphism remains controversial.This review summarized the literature on sequence variation in EBV genes,focusing on LMP-1,EBNA-1,and BZLF-1 and their distribution by geography and disease.

Investigação da LMP1 do EBV e a coinfeçcão do HPV em lesões genitais de pacientes infectados ou não pelo HIV / Investigation of the LMP1 EBV and co-infection by HPV in genital lesions of patients infected or not by HIV

INTRODUÇÃO: Vários estudos têm demonstrado associação do vírus Epstein-Barr (EBV) com neoplasias malignas, inclusive genitais, em que o papilomavírus humano (HPV) é o principal vírus associado às neoplasias epiteliais benignas e malignas. OBJETIVO: Investigar a presença do EBV e do HPV em lesões genitais de ambos os sexos, em pacientes soropositivos (grupo A) ou não (grupo B) para o vírus da imunodeficiência humana (HIV). MATERIAL E MÉTODO: Selecionados 126 pacientes e 135 lesões anogenitais, sendo 67 pacientes (53 por cento) e 75 lesões (56 por cento) no grupo A e 59 pacientes (47 por cento) e 60 lesões (44 por cento) no grupo B, para análise imuno-histoquímica (IHQ) por meio dos anticorpos monoclonais antiproteína latente de membrana 1 (LMP1) e HPV (DAKO®). RESULTADOS: A análise mostrou que o número total de lesões com imunopositividade para o HPV e para a LMP1 foi maior no grupo A (32 e 35, respectivamente) quando comparado ao B (16 e seis, respectivamente). A análise estatística (nível de significância de 5 por cento) mostrou que as proporções para o HPV não são estatisticamente significativas (z = 1,93; valor p = 0,053). Entretanto, para a LMP1, a diferença (47 por cento no grupo A e 10 por cento no B) é significativa (z = 4,60; valor p = 4,2×10-6). Do mesmo modo, a associação HPV-LMP1 (21 por cento no grupo A e 7 por cento no B) também mostrou diferença estatisticamente significativa (z = 2,38; valor p = 0,017). CONCLUSÃO: Esses resultados indicam a possibilidade de sinergismo da infecção pelo EBV e a coinfecção EBV-HPV em lesões epiteliais genitais, particularmente em pacientes soropositivos para o HIV. Entretanto, investigações com metodologia de maior especificidade e sensibilidade são necessárias para a verificação da real participação do EBV na patogênese de lesões epiteliais genitais.

INTRODUCTION: Several studies have demonstrated the association between Epstein-Barr virus (EBV) and malignant neoplasias, including genital lesions, in which the human papillomavirus (HPV) is the main virus associated with both benign and malignant epithelial neoplasias. OBJECTIVE: Investigate the presence of EBV and HPV in genital lesions in HIV-infected patients (group A) or HIV non-infected patients (group B) from both genders. MATERIAL AND METHOD: We selected 126 patients and 135 anogenital lesions, comprising 67 patients (53 percent) and 75 lesions (56 percent) from group A and 59 patients (47 percent) and 60 lesions (44 percent) from group B, to histopathological and immunohistochemical analyses through latent membrane protein 1 (LMP1) monoclonal antibodies and HPV (DAKO®). RESULTS: The analysis showed that the total number of lesions with immunopositivity for HPV and for LMP1 was higher in group A (32 and 35 respectively) in comparison with B (16 and six respectively). Statistical analysis (significance level of 5 percent) showed that the proportions for HPV are not statistically significant (z = 1.93; value p = 0.053). However, the difference (47 percent in group A and 10 percent in B) is significant for LMP1 (z = 4.60; value p = 4.2×10-6). Similarly, the association of HPV and LMP1 (21 percent in group A and 7 percent in B) also showed a significant statistical difference (z = 2.38; value p = 0.017). CONCLUSION: The results demonstrated the possibility of synergism between EBV infection and EBV-HPV co-infection in genital epithelial lesions, mainly among HIV-infected patients. However, further investigations with a more specific and sensitive methodology are required in order to assess the real influence of EBV on the pathogenesis of genital epithelial lesions.

Extraction model of low methoxyl pectin from apple pomace effects of acid concentration and time on the process and the product

In Brazil, one of the top apple producing countries in the world, apple processing is an increasing activity, with pomace as the main by-product. To extract pectin from pomace, factors affecting process and product should be studied for optimization. A model to produce LMP directly from dried apple pomace was established observing the effects of HNO3 concentration and the time of reaction at 97ºC, analyzed from a statistical and practical point of view. The model for gravimetric yield (R² =0.9834) predicts the highest value of 20.07 g/100 g (126 mM; 14.07 min) of a pectin with a degree of esterification of 48.49 percent. The model for degree of esterification of extracted pectin (R²= 0.9797) predicts the lowest value of 43.73 percent (200 mM; 10.07 min) with a yield of 16.77g/100 g. The results using the central coordinates (100 mM; 10 min) for gravimetric yield were 19.01 g/100 g and for the degree of esterification, 50.79 percent.

No Brasil, um dos países com alta produção de maçã, o seu processamento é uma atividade em crescimento tendo o bagaço como principal sub-produto. Para extrair pectina do bagaço os fatores que afetam o processo e o produto devem ser otimizados. Um modelo para extrair LMP foi estabelecido observando os efeitos de concentrações de HNO3 e do tempo de reação a 97ºC, analisados estatística e praticamente. O modelo para o rendimento (R² =0,9834) prediz o mais elevado valor de 20,07 g/100 g (126 mM; 14,07 min) de pectina com grau de esterificação de 48,49 por cento. O modelo para o grau de esterificação de pectina extraída (R²= 0,9797) prediz o mais baixo valor de 43,73 por cento (200 mM; 10,07 min) com um rendimento de 16,77g/100 g. Os resultados usando as coordenadas do ponto central (100 mM; 10 min) para o rendimento foram 19,01 g/100 g e para o grau de esterificação, 50,79 por cento.

Construction and identification of recombinant retroviral vector and stable cell line expressing latent membrane protein 2A of Epstein-Barr virus / 细胞与分子免疫学杂志

AIM: To clone EBV-LMP2A gene, construct and identify the recombinant retroviral vector and stable cell strains expressing EBV LMP2A. METHODS: The full-length EBV LMP2A gene was generated by RT-PCR amplification from B95.8 cells which contain complement nucleotide sequence of EBV LMP2A gene. The gene was ligated to T-vector and sequenced to construct retroviral vector consisting with LMP2A. To produce retroviral virus, packing cells, 293T cells were co-transfected with recombinant retroviral expression vector pGEZ-LMP2A and two auxiliary viral vectors pHIT456 and pHIT60 by lipofectAMINE2000. Viral titration was performed according to the instructions of the manufacturer. To establish L929 cell line stable expressing LMP2A, L929 cells were infected with recombinant retrovirus three times and selected by Zeocine. The Zeocine-resistant clones (L929/LMP2A) were screened for LMP2A expression by RT-PCR and Western blot. RESULTS: The recombinant retrovirus vector carrying LMP2A gene was constructed successfully. Transfection yield a titer of 5×10~8 infectious particles/L. The infected L929 cells were selected by Zeocine. Results of RT-PCR and Western blot indicated that L929 transgenetic cells could stably express EBV-LMP2A. CONCLUSION: The L929 cell line stably expressing LMP2A provides suitability for extraction of the LMP2A protein and preparations of the vaccine for the therapy of EBV-associated diseases.

Construction of co-expression plasmid pBudCE4.1-LMP-1-LMP-3 and its expression in vitro / 重庆医科大学学报

Objective:To construct the mammalian CO-expression plasmid pBudCE4.1-LMP-1-LMP-3,and to detect the expression of the plasmid in vitro.Methods:Fragments of LMP-1 gene and Fragments of LMP-3 gene were gained from the Company,and were constructed respectively into the plasmid vector Puc57,The inserted target genes in plasmid were verified by nucleotide sequencing and enzymes.fragments of LMP-1 gene were constructed firstly into the plasmid vector pBudCE4.1,fragments of LMP-3 gene were constructed into the plasmid vector pBudCE4.1-LMP-1 after iden tification with nucleotide sequencing and enzymes.MSCs cell line was transfected with this Co-expression plasmid using lipofectin reagent.according to the transfect situation,the MSCs were divided into 5 groups,the non-transfected group(Group A),the group transfected by empty vector(Group B),the group transfected by LMP-1(Group C),the group transfected by LMP-3(Group D)and the group transfected both LMP-1 and LMP-3(Group E).the expression of LMP-1 and LMP-3 were detected by RT-PCR and Western blot technique.Results:The plasmids Puc57-LMP-1、Puc57-LMP-3 and pBudCE4.1-LMP-1-LMP-3 were obtained successfully and verified by nucleotide sequencing and enzymes.After transfection with this mammalian Co-expression plasmid,the LMP-1 and LMP-3 molecules were expressed in MSCs cells.The results of RT-PCR and Western Blot were measured with the grey value.To the expression of mRNA and protein of LMP-1,the diferences between groups A、B and groups C、D、E were significant(P0.05);To the expression of mRNA and protein of LMP-3,the diferences between groups A、B and groups C、D、E were significant(P0.05).Conclusion:the constructed mammalian Co-expression plasmid pBudCE4.1-LMP-1-LMP-3 can express LMP-1 and LMP-3 molecules in vitro at the same time.

Expression of LMP-1、EBNA-2 in the Follicular Epithelium Neoplasm of the Thyroid / 医学研究杂志

0.05);LMP-1 and EBNA-2 in papillary thyroid carcinoma had no correlation.Conclusion Some thyroid papillary carcinomas may be related to EB virus infection,the relationship has yet to be further studied.

The influence of siRNA targeting LMPI gene on expression of AP-1 and its related factors / 中国癌症杂志

Background and purpose:LMP1 was one of the protein encoded by EBV latent gene,which was found to be able to transform cell lines and alter the phenotype of cells due to its oncogenic potential.In human epithelial cells,LMP1 alters many functional properties that are involved in tumor progression and invasions.In this study we investigated the influence of LMP1 silence on AP-1 signal transduction pathway and its downstream factors involved with cell transformation,proliferation and apoptosis.Methods:The chemically synthetic siRNA targeting LMP1 was transfected into EBV positive gastric carcinoma epithelial cell line by lipofectamine 2000 at 50 nmol/L final concentration.The protein expression of c-Jun,JunB and CDK4 was tested by,Western blotting.The mRNA of c-Jun,surviving,CDK4 and MMP9 mRNA were tested by RT-PCR.The expression of survivin were tested by immunohistochemistry.Results:Compared with the cell control,CDK4 mRNA was up regulated(P

Expression of LMP-1,NET-1,and VEGF in non-keratin nasopharyngeal carcinoma and its clinical significance / 中国肿瘤生物治疗杂志

Objective:To investigate the expression of latent membrane protein-1(LMP-1),NET-1,and vascular endothelial growth factor(VEGF)in non-keratin nasopharyngeal carcinoma(NK-NPC)and its clinical significance.Methods :Sixty biopsy specimens from pathologically-confirmed NK-NPC patients,who were treated in the Affilitated Hospital of Nantong University from May 1999 to May 2003,were included in the present study.Using immunohistochemical techniques (Envison two-step),we examined the expression of LMP-1,NET-1 and VEGF protein in the specimens.The relationship between their expression and elnicopathological parameters and the prognosis was anayzed.Ten specimens of chronic nasopharyngitis served as control.Results:(1)The positive rates of LMP-1,NET-1 and VEGF in NK-NPC were significantly higher than those in the chronic nasopharyngitis(P0.05).(3)NET-1 and VEGF expression in NK-NPC specimens were positively correlated with lymph node metastasis and distant metastasis(P

Expression of STAT3 and its relation with LMP1 expression in human nasopharyngeal carcinoma / 西安交通大学学报(医学版)

Objective To study the expression and activation of signal transducer and activator of transcription 3(STAT3) in human nasopharyngeal carcinoma (NPC) tissues and its relation with the expression of latent membrane protein 1(LMP1) encoded by Epstein-Barr virus.Methods The expressions of LMP1,STAT3 and phosphated STAT3(p-STAT3) in 45 cases of NPC and 27 cases of chronic nasopharyngitis were studied by immunohistochemical method.Correlation between protein expressions was analyzed.Results The positive rates of LMP1,STAT3 and p-STAT3 in NPC were significantly higher than those in chronic nasopharyngitis(P0.05).Conclusion There are overexpression and abnormal activation of STAT3 protein in NPC tissues.LMP1 may play a role in the activation of STAT3.

The Synergistic Effect of LMP1 and IRF7 on the Expression of TAP1 in DG 75 Cell Line

In this study, the author explored the role of interferon regulatory factor 7 (IRF7) and latent membrane protein 1 (LMP1) on the regulation of antigen presenting molecules in B-lymphoblastoid cell lines. First, the author observed the endogenous expression of IRF7 and LMP1 in paired EBV-positive B-lymphoblastoid cell lines, Sav I and Sav III, which represent EBV type I and type III latency, respectively. The Sav I cell, which does not express LMP1, showed very low levels of endogenous IRF7 in the cytoplasm. However, Sav III cells, which express large amounts of LMP1, contained high levels of endogenous IRF7 in both the cytoplasm and the nucleus. The expression of surface MHC class I antigen was 7.8-fold higher in Sav III compared with Sav I cells when measured by fluorescence activated cell sorter (FACS) analysis. To understand whether IRF7 is involved in regulation of MHC I and TAP1, LMP1 or IRF7 were expressed by cotransfection in DG75 cells. Levels of TAP1 protein were up-regulated by LMP1 and IRF7 alone, and by LMP1 co expression with IRF7, the expression level was highest after co-transfection of LMP1 with IRF7. TAP1 promoter activity was also up-regulated to 2.4, 2.0, 3.2-fold by LMP1, by IRF7, and by LMP1 plus IRF7, respectively. Surface expression of MHC class I antigen was up-regulated by LMP1 alone and LMP1 plus IRF7, but not by IRF7 alone. These results suggest that IRF7 induces the expression of TAP1, but not MHC class I antigen and that LMP1 and IRF7 have additive effects on the expression of TAP1 protein.