Gene cloning, protein expression and examination of biological activity of chicken CD40L / 生物工程学报

To obtain chicken CD40L protein, the cDNA was prepared from chicken splenic cells and used as a template to clone and amplify CD40L by PCR. The target gene was cloned into pFastBac vector to construct a pFastBac-chCD40L donor plasmid. Recombinant plasmid was transformed into DH10Bac and recombinant Bacmid-chCD40L was obtained. The Bacmid-chCD40L plasmid was transfected into sf9 insect cells to obtain His-chCD40L protein. In addition, the target gene was cloned into pQM01 vector to construct a pQM01-chCD40L plasmid, recombinant plasmid was transfected into HEK 293T cells to obtain Strep-chCD40L protein. The chCD40L protein was purified by affinity chromatography, and the concentration of purified chCD40L protein was determined to be 0.01 mg/mL. Primary cells were isolated from the bursal tissue of 3-week old SPF chickens, and the chCD40L protein was added to the culture medium to stimulate cells. The chCD40L could bind to CD40 on B cells as examined by Western blotting, indirect immunofluorescence assay and flow cytometry, suggesting that chCD40L protein is biologically active. We successfully obtained chicken CD40L protein of biological activity, which laid the foundation in the in vitro culture of primary B lymphocytes for the isolation and diagnosis of virulent IBDV.

The Expression of CD40/CD40L in MM Patients and Its Significance to the Prognosis of MM Patients / 中国实验血液学杂志

OBJECTIVE@#To explore the expression of CD40/CD40L in multiple myeloma(MM) patients and its influence on prognosis.@*METHODS@#Thirty patients with MM treated in Cangzhou People's Hospital from May 2016 to June 2017 were selected and divided into MM group, then 30 healthy people with a physical examination in our hospital at the same time were selected as the normal group. The serum CD40/CD40L levels of the patients in the two groups was detected by flow cytometry, and its correlation with the lymphocyte population, pathological grade and prognostic significance of MM patients was anaysis.@*RESULTS@#The expression of CD40 in serum of the patients in MM group was significantly higher than those in normal group (P0.05). The levels of CD40 and CD40L in the patients before and after chemotherapy showed no difference(P>0.05). The levels of Ts and NK cells in the patients of MM group were lower than those in normal group (P0.05). The CD40 level was correlated with the serum total B lymphocyte level of the patients in MM group (r=0.877, P=0.005). There was a correlation with CD40L and Th cells in the serum of MM patients (r=-0.783, P=0.035). The expression of serum CD40 in the patients at phase III-IV was higher than those of the patients at phase I-II, the levels of serum CD40L in MM patients at different periods showed no significant difference(P>0.05). The survival rate of MM patients with high CD40 expression was lower than that of MM patients with low CD40 expression (χ@*CONCLUSION@#The increasing of CD40 level in MM patients is related to the pathological grade of the patients. Chemotherapy can reduce the level of CD40. The increasing of CD40 is an important factor for the poor prognosis of MM patients. CD40L level is not meaningful for MM treatment and prognosis.

Genetic analysis of a child with co-commitment progressive multifocal leukoencephalopathy and X-linked hyper IgM syndrome / 中华医学遗传学杂志

OBJECTIVE@#To detect variant of the CD40L gene and infection of Jamestown Canyon virus (JCV) in a 7-year-and-9-month-old boy with co-commitment progressive multifocal leukoencephalopathy (PML) and X-linked hyper IgM syndrome (XHIGM).@*METHODS@#Peripheral blood samples of the child and his parents were collected for the extraction of genomic DNA. The 5 exons and exon/intronic boundaries of the CD40L gene were subjected to PCR amplification and sequencing. Suspected variants were analyzed by using bioinformatic software. The JCV gene was amplified from genomic DNA by nested PCR and sequenced.@*RESULTS@#The child was found to harbor a hemizygous c.506 A>C (p.Y169S) missense variant in exon 5 of the CD40L gene. The variant may affect the TNFH domain of the CD40L protein and result in structural instability and loss of hydrophobic interaction between CD40L and CD40. As predicted by PolyPhen2 and SIFT software, the variant was probably damaging (score = 1.00) and deleterious (score= -8.868). His mother was found to be a heterozygous carrier, while the same variant was not found in his father. Gel electrophoresis of the nested PCR product revealed presence of target JCV band, which was confirmed to be 99% identical with the JCV gene by sequencing.@*CONCLUSION@#The patient was diagnosed with co-commitment XHIGM and PML based on the testing of the CD40L gene and JCV infection.

Avaliação do perfil de expressão das moléculas OX40, 4-1BB e CD40/CD40L no mieloma múltiplo / Evaluation of the expression profile of the molecules OX40, 4-1BB and CD40/CD40L in multiple myeloma

Introdução: O mieloma múltiplo é uma desordem clonal das células plasmocitárias e, responde por 10-15% das neoplasias hematológicas. Apresenta diversas alterações no sistema imune, caracterizadas por déficits na produção de anticorpos; alterações do perfil imunológico das células T; aumento da expressão do PD-L1; modificações no microambiente medular favorecendo o recrutamento de populações imunossupressoras como as Treg e disfunção nas células dendríticas. Manter um sistema imune ativo é fundamental para o controle da doença, pacientes com manutenção de células T efetoras apresentam maiores taxas de remissão e sobrevida. Receptores coestimuladores como o OX40, CD40/CD40L e 4-1BB, participam na ativação, proliferação e amplificação da resposta imune. Objetivo: Avaliar os níveis de linfócitos B e T e das moléculas coestimuladoras OX40, CD40, CD40L e 4-1BB no sangue e medula óssea dos pacientes com mieloma múltiplo. Métodos: Trata-se de estudo exploratório, realizado entre 2016 e 2019 no Hospital de Câncer de Pernambuco (HCP) e Laboratório de Pesquisa Translacional do Instituto de Medicina Integral Prof. Fernando Figueira (IMIP). Foram incluídas 40 pacientes, até 79 anos de idade, com diagnóstico de Mieloma Múltiplo. Coletas de sangue periférico e medula óssea foram realizadas ao diagnóstico. As mensurações dos níveis de expressão de proteínas de membrana CD20, CD3, OX40, CD40/CD40L foram detectadas pela técnica de Cytometric Bead Array por citometria de fluxo. A dosagem dos níveis solúveis de s4-1BB, sOX40 e sCD40L foi realizada por enzyme linked immunonoSorbent assay (ELISA). Foi realizada análise de curva Receiver Operating Characteristic (ROC) para determinar o melhor valor de acurácia de cada marcador estudado assim como, a ocorrência de óbito. A análise estatística foi realizada no programa GraphPadPrism v8.0. O nível de significância estatística foi de p<0,05. Resultados: Em sangue periférico, comparando-se pacientes e controles, verificou-se níveis menores de CD20 (p<0,0001) e CD20low (p<0,0001), CD40+ em leucócitos totais (p=0,0005), CD40+ em linfócitos (0,0006) e CD40/CD3+ (p<0,0001) no grupo de pacientes. Mas, em contrapartida, os pacientes apresentaram níveis mais elevados de OX40+ (p=0,0012), CD40L+ em leucócitos totais (p=0,002) e OX40+/CD3+ (p<0,0001). Os níveis séricos de s4-1BB (p=0,03) e sOX40 (p=0,01) estavam reduzidos no grupo de MM quando comparado aos controles. Na análise segundo o ISS, somente os níveis de expressão de CD40L+ em leucócitos (p=0,01) e de CD40+ em linfócitos (p=0,0045), mostraram níveis superiores nos pacientes com ISS1-2 em relação ao ISS-3. As medidas de expressão de OX40+ e CD40L+ em leucócitos totais eram inferiores nos casos com evolução para óbito (p<0,0006 e p=0,002, respectivamente). Os pacientes que apresentavam níveis de expressão de OX40 em leucócitos totais ≥2,93% tiveram maior sobrevida em relação àqueles com valores <2,93% (p=0,03), bem como aqueles com CD40L em leucócitos totais com valores ≥3,09% (p=0,001). Na análise da medula óssea, segundo o ISS, somente os níveis de expressão de OX40/CD3+, mostraram níveis superiores nos pacientes com ISS1-2 em relação ao ISS-3 (p<0,0017). Não foram observadas diferenças significativas entre os valores de expressão dos diversos marcadores em medula óssea, com relação ao desfecho óbito. Na análise de correlação de Spearman, os valores de CD20 em sangue e medula óssea, apresentam uma correlação moderada entre si (r=0,64 e p<0,0001). Conclusão: Os resultados deste estudo permitem concluir que existem alterações de mecanismos celulares envolvidos na regulação e ativação da resposta imune no MM quando comparados aos controles. A manutenção de níveis mais elevados de moléculas coestimuladoras (OX40 ≥2,93% e CD40L≥ 3,09%), foi preditiva de melhor sobrevida no MM

Introduction: Multiple myeloma (MM) is a malignant plasma cell (PC) disorder, accounting for approximately 10-15% of all hematological cancers. Multiple myeloma presents several immune system alterations, characterized by deficits in antibody production, disruption of the T-cell immune profile, increased expression of cell death ligand 1 (PD-L1), changes in the bone marrow microenvironment favoring the recruitment of immunosuppressive populations such as Tregs and dysfunction in dendritic cells. It is important to preserve the integrity of the active immune system for the control of disease progression and patients with maintenance of T-cell cytotoxic activities improve rates of remission and overall survival. Co-stimulating receptors such as OX40, CD40/CD40L and 4-1BB, cooperate in the activation, proliferation, and amplification of the immune response. Objective: To evaluate T and B lymphocyte levels as well as co-stimulating molecules OX40, CD40, CD40L and 4-1BB in the blood and bone marrow of multiple myeloma patients. Methods: This is a cross-sectional and exploratory study, conducted between 2016 and 2019 at Pernambuco Cancer Hospital (HCP) and Translational Research Laboratory of Instituto de Medicina Integral Prof. Fernando Figueira (IMIP). Forty patients, up to 79 years of age, diagnosed with Multiple Myeloma were included. Peripheral blood and bone marrow samples were collected at diagnosis. Serum concentrations of CD20, CD3, OX40, CD40/CD40L were detected through the Cytometric Bead Array technique by flow cytometry, and the soluble forms of s4-1BB, sOX40 e sCD40L by enzyme linked immunosorbent assays. Receiver Operating Characteristic (ROC) curve analysis was performed to determine not only the best accuracy value of each studied marker but also, mortality. Statistical analysis was performed in the GraphPadPrism v8.0 program and the level of statistical significance was p <0.05. Results: In peripheral blood, comparing patients and controls, there were lower levels of CD20 (p<0.0001) and CD20low (p<0.0001), CD40+ in total leukocytes (p=0.0005), CD40+ in lymphocytes (0.0006) and CD40/CD3+ (p<0.0001) in the patient group. However, on the other hand, patients had higher levels of OX40+ (p=0.0012), CD40L+ in total leukocytes (p=0.002) and OX40+/CD3+ (p<0.0001). Serum levels of s4-1BB (p=0.03) and sOX40 (p=0.01) were reduced in the MM group compared to controls. According to the ISS, CD40L+ in leukocytes (p=0.01) and CD40+ in lymphocytes (p=0.0045) showed higher levels in patients with ISS1-2 compared to ISS-3. Regarding the outcome death, levels of OX40+ and CD40L+ in total leukocytes were lower (p<0.0006 and p=0.002, respectively). In survival analyses, patients who had OX40+ levels in total leukocytes ≥2.93% had higher survival compared to those with levels <2.93% (p=0.03), as well as those with CD40L+ in total leukocytes with values ≥3.09% (p=0.001). In the bone marrow only the OX40/CD3+ levels were higher in patients with ISS1-2 compared to ISS-3 (p<0.0017). No significant differences were observed between values of other bone marrow markers in relation to the outcome death. In Spearman's correlation analysis, CD20 levels in blood and bone marrow present moderate correlation between them (r=0.64 and p<0.0001). Conclusion: This study shows differences in cellular mechanisms involved in the regulation and activation of immune response in MM patients in comparison to healthy controls. The maintenance of higher levels of co-stimulating molecules (OX40 ≥2.93% and CD40L≥ 3.09%) is associated with better survival in multiple myeloma

Effects of Soluble CD40 Ligand on Non-Hodgkin Lymphoma Cells and Its Relative Mechanism / 中国实验血液学杂志

OBJECTIVE@#To explore the effect of soluble CD40 ligand (sCD40L) on the proliferation, apoptosis, and cell cycle of human non-Hodgkin lymphoma (NHL) cells, and analyze its possible mechanism.@*METHODS@#NHL CA46 cell and Raji cell were treated with different concentrations of sCD40L for 48 h, CCK-8 was used to detect the effect of sCD40L on cell proliferation in vitro, flow cytometry on apoptosis and cycle of NHL cells, and Western blot on the expression of PTEN, BCL-2, and BAX in NHL cells.@*RESULTS@#Compared with the control group, 4 and 8 μg/ml sCD40L could significantly inhibit the proliferation of lymphoma Raji cell and CA46 cell (P<0.05). The test results of flow cytometry showed that 4 μg/ml sCD40L could significantly promote the apoptosis of CA46 and Raji cells, and significantly inhibit the S phase proportions (P<0.05). Western blot results showed that sCD40L could promote the expression of PTEN and BAX, while inhibit the expression of BCL-2 (P<0.05).@*CONCLUSION@#sCD40L can promote the apoptosis and inhibit the proliferation of NHL cells through the PTEN signaling pathway.

Enhancing immune effects of a DNA vaccine against kidney cancer using CD40L as an adjuvant

The use of specific combinations of antigens and adjuvant represents a promising approach for increasing the immunogenicity of DNA vaccines. In the present study, we evaluated the immunity and antitumor effects of DNA vaccines with G250 as the target antigen in a mouse model of renal cell carcinoma. We constructed two recombinant plasmids, pVAX1-G250 and pVAX1-CD40L. The recombinant plasmids were injected into mice by intramuscular injection and electrical pulse stimulation. ELISA and ELISPOT experiments were performed to evaluate the corresponding humoral and cellular immune responses following immunization. To further investigate the antitumor potential of the DNA vaccines, we established a tumor-bearing mouse model expressing G250 target antigen. Our results showed that immunization with the combination of the two plasmids exerted the strongest anti-tumor effects. Therefore, our findings demonstrated the effectiveness of CD40L as an adjuvant for DNA vaccines and highlighted the promising use of these vaccines for the treatment of tumors.

Análise da expressão de citocinas e quimiocinas, dos agregados de plaquetas-leucócitos e dos mediadores solúveis sCD40L e sCD62P no melanoma cutâneo / Analysis of the expression of cytokines and chemokines, platelet-leukocyte aggregates and soluble mediators sCD40L and sCD62P in cutaneous melanoma

Introdução: O melanoma cutâneo é uma neoplasia maligna dos melanócitos da pele com incidência variável no mundo e o prognóstico é desfavorável nos estágios mais avançados. O desenvolvimento do melanoma está associado ao sistema imunológico e a maior evolução no seu tratamento se deu a partir de medicamentos baseados no estímulo da resposta imune contra o tumor. Objetivo: Avaliar a expressão de citocinas e quimiocinas, dos agregados de plaquetas-leucócitos e de mediadores solúveis sCD40L e sCD62P no sangue de pacientes com melanoma cutâneo. Métodos: Foi realizado um estudo transversal em pacientes com melanoma cutâneo admitidos para tratamento cirúrgico no Hospital de Câncer de Pernambuco (HCP), entre os anos de 2015 e 2018. Foram incluídos 51 pacientes (média de 57,6 anos) com diagnóstico de melanoma cutâneo, e como grupo controle, 30 indivíduos saudáveis (média de 56,7 anos). As coletas de sangue foram realizadas antes da ressecção do melanoma. A determinação dos níveis de citocinas IL6, IL10, TNF, IL1 e IL12p70 e das quimiocinas CXCL10 (IP10), CCL2 (MCP-1), CXCL9 (MIG), CCL5 (RANTES) e CXCL8 (IL8), sCD40L e sCD62P, e agregados plaquetas-leucócitos foi realizada por citometria de fluxo. Foram realizadas análises entre os grupos de pacientes e controles, e pelos parâmetros como tipo histológico, estadio, espessura de Breslow e presença de metástases linfonodais. O teste de Mann-Whitney foi utilizado para comparação entre dois grupos, e de Kruskal-Wallis para as análises entre três ou mais grupos. Foi considerado significativo p<0,05. Resultados: Não houve detecção de IL1 e IL12 no sangue dos pacientes e controles. Verificou-se níveis elevados das citocinas IL10 e diminuídos de TNF nos pacientes comparados ao grupo controle, (p<0,0001). A IL6 esteve aumentada nos pacientes com estadio II em relação ao III (p=0,017) e em pacientes com linfonodos negativos (p<0,0001). Foram encontrados níveis reduzidos da quimiocina CCL5 (p=0,009) nos pacientes quando comparados ao grupo controle. O percentual de agregação plaquetária em linfócitos, monócitos e neutrófilos também foi elevado nos pacientes comparado ao grupo controle (p<0,0001, p=0,009 e p<0,0001 respectivamente). Foram encontrados níveis elevados de agregados plaquetas-monócitos nos pacientes com linfonodos positivos (p=0,008). Os níveis solúveis sCD40L e sCD62P foram elevados nos pacientes comparados aos controles (p=0,03 e p=0,006, respectivamente). Conclusão: Os dados obtidos das análises realizadas mostram que os pacientes com melanoma cutâneo apresentam um perfil imunossupressor com a participação de plaquetas e monócitos/macrófagos que favorecem a progressão tumoral

Cutaneous melanoma is a malignancy originated from the skin melanocytes, with a variable incidence worldwide and a poor prognosis in advanced stages. Melanoma growth is closely associated with the immune system and the most important treatment advances resulted from stimulation of immune response against the tumor. Objective: To evaluate the expression of cytokines and chemokines, platelet-leukocyte aggregates and soluble mediators sCD40L and sCD62P in the blood of patients with cutaneous melanoma. Methods: A cross-sectional study was performed in cutaneous melanoma patients admitted for surgical treatment at the Hospital de Câncer de Pernambuco (HCP) between 2015 and 2018. Fifty-one patients (mean age 57.6 years) with a diagnosis of melanoma were included, and 30 healthy individuals (mean age 56.7 years) were chosen as the control group. The blood samples were taken before resection of the melanoma. The determination of cytokines IL6, IL10, TNFα, IL1ß and IL12p70 and chemokines CXCL10 (IP10), CCL2 (MCP-1), CXCL9 (MIG), CCL5 (RANTES) and CXCL8 (IL8), sCD40L and sCD62P, and platelet-leukocyte aggregate was performed by flow cytometry. Analysis were performed between patient and control groups, and by parameters such as histological type, stage, Breslow thickness, and presence of lymph node metastases. Mann-Whitney test was used for comparison between the two groups, and Kruskal-Wallis test for analysis between three or more groups. It was considered significant p <0.05. Results: There was no detection of IL1ß and IL12 in the blood of patients and controls. Elevated levels of IL10 and decreased TNFα cytokines were found in patients compared to the control group (p <0.0001). IL6 was increased in patients with stage II compared to III (p=0.017) and in patients with negative lymph nodes (p <0.0001). Reduced CCL5 chemokine levels (p=0.009) were found in patients compared to the control group. The percentage of platelet aggregation in lymphocytes, monocytes and neutrophils was also high in patients when compared to the control group (p <0.05). High levels of platelet-monocyte aggregates were found in patients with positive lymph nodes (p=0.008). Soluble sCD40L and sCD62P levels were elevated in patients compared to controls (p=0.03 and p=0.006, respectively). Conclusion: The data obtained from the analysis performed show that patients with cutaneous melanoma have an immunosuppressive profile with platelet participation and monocytes/macrophages that favor tumor progression

Análise da expressão de microRNAs, receptores OX40 e CD40L, e do agregado de plaquetas-leucócitos no câncer gástrico / Analysis of the expression of microRNAs, OX40 and CD40L receptors, and platelets-leukocytes aggregation in gastric cancer

O prognóstico dos portadores de câncer gástrico tem melhorado pouco nas últimas décadas e o melhor entendimento das vias moleculares e as interações imunes no microambiente tumoral podem revelar novas possibilidades de tratamento. O ambiente tumoral é composto por células do sistema imune, que refletem a tentativa desse sistema em promover uma resposta antitumoral. Complexas interações entre células e mediadores imunes no microambiente tecidual regulam o crescimento de tumores, progressão, metástase e angiogênese. O entendimento das alterações da imunidade na população com câncer gástrico (CG) permitirá a intervenção terapêutica para melhorar a resposta à cirurgia e à quimioterapia. Objetivo: comparar a expressão de miRNA em tecidos de pacientes com câncer gástrico e controles saudáveis para encontrar miRNAs desregulados no câncer gástrico e usar ferramentas de bioinformática para determinar a possível influência desses miRNAs no sistema imunológico. Avaliar a formação de agregados plaquetas-leucócitos circulantes, bem como os níveis de ativação plaquetária (CD62p+, CD40L) desses conjugados a leucócitos em pacientes com CG. Quantificar a expressão de moléculas costimulatórias da resposta imune (OX40) em linfócitos T de pacientes com CG. Métodos: é um estudo do tipo corte transversal, exploratório e translacional realizado no Hospital de Câncer de Pernambuco, Laboratório de Pesquisa Translacional do Instituto de Medicina Integral Prof. Fernando Figueira e Centro Internacional de Pesquisa (CIPE) do AC Camargo entre 2015 a 2018. Foram avaliados 83 pacientes com câncer gástrico e 69 controles. Foram determinados os níveis de expressão de microRNAs no tecido tumoral gástrico em comparação aos da mucosa gástrica normal por técnica de reação em cadeia da polimerase (qPCR - TaqMan). Foram realizadas as análises dos niveis linfócitos T e B, OX40, CD40L e de agregado de plaquetas no sangue periférico por citometria de fluxo. Resultados: As análises revelaram um miRNA mais expresso (miR-196a-5p) e dois significativamente menos expressos (miR-374a-5p e miR-375) em comparação ao grupo controle. Pacientes com estádio IV (metástatico) apresentaram uma diminuição significativa na expressão do miR-374-5p quando comparados aos pacientes não metastáticos (p=0.03). Com uso de plataformas de bioinformática, foram observadas várias vias que sofrem influência dos microRNAs desregulados e que interagem com genes envolvidos com a resposta imune celular, moléculas de adesão celular e migração celular. Foram encontrados níveis elevados de expressão de OX40 em linfócitos T, monócitos e neutrófilos de pacientes com neoplasia gástrica (p<0.0001), entretanto, os níveis de OX40 foram reduzidos nos grupos com neoplasia estádio III/IV quando comparados ao estádio I/II. Observamos níveis elevados de agregados de plaquetas-linfócitos T e plaquetas-linfócitos B no sangue de pacientes GC com estágio IV quando comparados com os estágios I, II e III, e grupo controle (p <0,05). Níveis reduzidos de agregados plaquetas-linfócitos totais com expressão de CD40L foram observados no estádio IV da doença (p<0,05). Níveis elevados de plaquetas ativadas e agregados de plaquetas-monócitos ativados (CD62p+) foram observados em pacientes GC quando comparados ao grupo controle (p<0,05). Conclusão: Os resultados deste estudo permitem concluir que existem alterações de mecanismos moleculares (miRNA) e celulares envolvidos na regulação e ativação da resposta imune, sendo associadas à progressão e metástase no GC

The prognosis of gastric cancer patients has not been improved in the last decades and the the understanding of the molecular immune pathways and how immune interactions happens on tumoral microenvironment, open new possibilities of treatment. The tumor environment is infiltrated by cells of the immune system, which reflect an antitumor response. Complex interactions between microenvironmental cells and mediators regulate tumor growth, progression, metastasis and angiogenesis. The knowledge of the immunity in the population with gastric cancer will allow therapeutic intervention to improve the response to surgery and the chemotherapy. Objective: our primary goal was to compare the miRNA expression in tissues from primary gastric cancer patients and healthy controls to find miRNAs dysregulated in gastric cancer and used bioinformatics tools to determine potential roles of these miRNAs in the immune system. We conducted a secondary analysis to evaluate the formation of circulating platelet-leucocyte conjugates as well as the CD40L levels conjugate to leucocytes in GC and finally, samples were analysed for levels of costimulatory molecules related to the immune response (OX40) in GC. Methods: A cross-sectional, translational and exploratory study carried out at the Hospital de Cancer de Pernambuco, Translational Research Laboratory of the Instituto de Medicina Integral Prof. Fernando Figueira and AC Camargo International Research Center (CIPE) from 2015 to 2018, involving 83 patients with gastric cancer and 69 controls. Expression levels of microRNAs in gastric tumor tissue and normal gastric mucosa were determined by polymerase chain reaction technique (qPCR - TaqMan). Analyzes of T and B lymphocytes, OX40, CD40L and platelet aggregate levels were performed in peripheral blood by flow cytometry. Results: The results revealed a more express miRNA (miR-196a-5p) and two significantly less expressed (miR-374a-5p and miR-375) compared to the control group. Patients with stage IV (metastasis) showed a significant decrease in miR-374-5p expression when compared to non-metastatic patients (p = 0.03). Bioinformatics analysis suggested that the pathways regulated by these differentially expressed miRNAs were related to the immune response, cell adhesion, and cell migration. High levels of OX40 expression were found in T lymphocytes, monocytes and neutrophils of patients with gastric neoplasia (p <0.0001); however, OX40 levels were reduced in groups with stage III / IV neoplasia when compared to stage I / II. We observed higher levels of platelet-T lymphocyte aggregate (P-T lymp) and platelet-B lymphocyte aggregate (P-B lymp) in the peripheral blood (PB) of GC patients with stage IV when compare with stages I-II-III, and control group (p<0,05). Reduced levels of CD40L+ Platelet-total lymphocyte (P-lymp) were observed at stage IV of the disease (p<0.05). High levels of CD62p+ platelets and CD62p+ platelets-monocyte aggregate were observed GC patients when compare to control group (p<0.05). Conclusion: The results of this study allow us to conclude that there are alterations of molecular mechanisms (miRNA) and cellular mechanisms involved in the regulation and activation of the immune response and associated to the progression and metastasis in GC

A Disintegrin and Metalloproteinase 8 as a Potential Blood Biomarker for Early Diagnosis of Gastric Cancer

PURPOSE: We aimed to evaluate the clinical significance of a disintegrin and metalloproteinase 8 (ADAM 8) as a potential blood biomarker for gastric cancer (GC). MATERIALS AND METHODS: Blood ADAM 8 was measured by ELISA. Cytokines/chemokines [interleukin-23 (IL-23), stromal cell-derived factor 1α/CXC chemokine ligand 12 (SDF-1α/CXCL12), interleukin-8 (IL-8), and soluble CD40 ligand (sCD40L)] were measured by chemiluminescent immunoassay. They were compared among five groups; normal/gastritis, high-risk, early GC (EGC), advanced GC (AGC) without distant metastasis, and AGC with distant metastasis by one-way analysis of variance in both training (n=80) and validation dataset (n=241). Clinicopathological features of GC and GC-associated cytokines were evaluated for their correlations with blood ADAM 8. To evaluate the diagnostic accuracy to predict GC, receiver operating characteristic (ROC) curve and logistic regression were used. RESULTS: Blood ADAM 8 significantly increased along GC carcinogenesis in both training (ANOVA, p<0.001) and validation dataset (p<0.001). It was significantly higher in EGC compared to high-risk (post-hoc Bonferroni, p=0.041) and normal (p<0.001). It was also higher in AGC compared with high-risk (p<0.001) and normal (p<0.001) groups. However, no significant difference was found between cancer groups. Blood ADAM 8 was correlated with N-stage (Spearman's correlation, γs=0.320, p=0.011), but not with T-stage or M-stage. Pearson's correlations showed blood ADAM 8 was closely correlated with pre-inflammatory cytokines, IL-23 (p=0.036) and SDF-1α/CXCL12 (p=0.037); however, it was not correlated with pro-angiogenic cytokine IL-8 (p=0.313), and sCD40L (p=0.702). ROC curve and logistic regression demonstrated that blood ADAM 8 showed higher diagnostic accuracy (sensitivity, 73.7%; specificity, 86.2%) than CEA (sensitivity, 23.1%; specificity, 91.4%). Combination of ADAM 8 and CEA further increased the diagnostic accuracy to predict GC (sensitivity, 81.8%; specificity, 84.0%). CONCLUSION: Blood ADAM 8 is a promising biomarker for early detection of GC.

Mucosal Immunity Related to FOXP3⁺ Regulatory T Cells, Th17 Cells and Cytokines in Pediatric Inflammatory Bowel Disease

BACKGROUND: We aimed to investigate mucosal immunity related to forkhead box P3 (FOXP3+) regulatory T (Treg) cells, T helper 17 (Th17) cells and cytokines in pediatric inflammatory bowel disease (IBD). METHODS: Mucosal tissues from terminal ileum and colon and serum samples were collected from twelve children with IBD and seven control children. Immunohistochemical staining was done using anti-human FOXP3 and anti-RORγt antibodies. Serum levels of cytokines were analyzed using a multiplex assay covering interleukin (IL)-1β, IL-4, IL-6, IL-10, IL-17A/F, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, interferon (IFN)-γ, soluble CD40L, and tumor necrosis factor-α. RESULTS: FOXP3+ Treg cells in the lamina propria (LP) of terminal ileum of patients with Crohn's disease were significantly (P < 0.05) higher than those in the healthy controls. RORγt+ T cells of terminal ileum tended to be higher in Crohn's disease than those in the control. In the multiplex assay, serum concentrations (pg/mL) of IL-4 (9.6 ± 1.5 vs. 12.7 ± 3.0), IL-21 (14.9 ± 1.5 vs. 26.4 ± 9.1), IL-33 (14.3 ± 0.9 vs. 19.1 ± 5.3), and IFN-γ (15.2 ± 5.9 vs. 50.2 ± 42.4) were significantly lower in Crohn's disease than those in the control group. However, serum concentration of IL-6 (119.1 ± 79.6 vs. 52.9 ± 39.1) was higher in Crohn's disease than that in the control. Serum concentrations of IL-17A (64.2 ± 17.2 vs. 28.3 ± 10.0) and IL-22 (37.5 ± 8.8 vs. 27.2 ± 3.7) were significantly higher in ulcerative colitis than those in Crohn's disease. CONCLUSION: Mucosal immunity analysis showed increased FOXP3+ T reg cells in the LP with Crohn's disease while Th17 cell polarizing and signature cytokines were decreased in the serum samples of Crohn's disease but increased in ulcerative colitis.

Cytidine-phosphate-guanosine oligodeoxynucleotides in combination with CD40 ligand decrease periodontal inflammation and alveolar bone loss in a TLR9-independent manner

Abstract Local administration of toll-like receptor 9 (TLR9), agonist cytidine-phosphate-guanosine oligodeoxynucleotide (CpG ODNs), and CD40 ligand (CD40L) can decrease ligature-induced periodontal inflammation and bone loss in wild type (WT) mouse. Objective: This study aimed to explore whether such effect is dependent on TLR9 signaling. Material and Methods: Purified spleen B cells isolated from WT C57BL/6J mice and TLR9 knockout (KO) mice were cultured for 48 hours under the following conditions: CD40L, CpG+CD40L, CpG at low, medium and high doses. We determined B cell numbers using a hemocytometer at 24 h and 48 h. Percentages of CD1dhiCD5+ B cells were detected by flow cytometry. Interleukin-10 (IL-10) mRNA expression and protein secretion were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and by ELISA, respectively. The silk ligature was tied around the maxillary second molars for 14 days, during which the CpG+CD40L mixture or PBS was injected into palatal gingiva on days 3, 6, and 9. Results: For both WT and TLR9 KO mice, CpG significantly induced B cell proliferation, increased IL-10 mRNA expression and protein secretion of IL-10 but reduced CD1dhiCD5+ B cells population; local injection of CpG+CD40L mixture significantly decreased alveolar bone loss and the number of TRAP-positive cells adjacent to the alveolar bone surface, and significantly increased the gingival mRNA expression of IL-10 and decreased RANKL and IFN-γ mRNA expression. Conclusions: These results indicated that CpG plus CD40L decreased periodontal inflammation and alveolar bone loss in a TLR9-independent manner in ligature-induced experimental periodontitis.

Benzodiazepine-Associated Carcinogenesis: Focus on Lorazepam-Associated Cancer Biomarker Changes in Overweight Individuals

OBJECTIVE: Cellular, animal, and human epidemiological studies suggested that benzodiazepines increase the risk of cancer and cancer mortality. Obesity is also clearly linked to carcinogenesis. However, no human studies have examined benzodiazepine-associated carcinogenesis as assessed by changes in cancer biomarkers. METHODS: A total of 19 patients were recruited, and received a 6-week treatment of 0.5 mg lorazepam. The measured cancer biomarkers were angiopoietin-2 (ANG-2), soluble CD40 ligand, epidermal growth factor, endoglin, soluble Fas ligand (sFASL), heparin-binding EGF-like growth factor (HB-EGF), insulin-like growth factor binding protein, interleukin (IL)-6, IL-8, IL-18, plasminogen activator inhibitor (PLGF), placental growth factor, transforming growth factor (TGF)-α, tumor necrosis factor (TNF)-α, urokinase-type plasminogen (uPA), vascular endothelial growth factor (VEGF)-A, VEGF-C, and VEGF-D. RESULTS: Six cancer biomarkers were significantly increased in all patients as a whole. The subgroup analysis revealed a distinct pattern of change. Overweight patients showed a significant increase in 11 cancer biomarkers, including ANG-2, sFASL, HB-EGF, IL-8, PLGF, TGF-α, TNF-α, uPA, VEGF-A, VEGF-C, and VEGF-D. However, normal-weight patients did not show any changes in cancer biomarkers. CONCLUSION: Adiposity may have primed the carcinogenic potential, leading to lorazepam-associated carcinogenesis in overweight patients. Epidemiological studies addressing this issue should consider the potential modulator contributing to benzodiazepine-associated carcinogenesis.

Toll-like receptor agonists Porphyromonas gingivalis LPS and CpG differentially regulate IL-10 competency and frequencies of mouse B10 cells

Abstract IL-10 expressing regulatory B cells (B10) play a key role in immune system balance by limiting excessive inflammatory responses. Effects of toll-like receptor signaling and co-stimulatory molecules on B10 activity during innate and adaptive immune responses are not fully understood. Objective This study is to determine the effects of P. gingivalis LPS and CpG on B10 cell expansion and IL-10 competency in vitro. Material and Methods Spleen B cells were isolated from C57BL/6J mice with or without formalin-fixed P. gingivalis immunization. B cells were cultured for 48 hours under the following conditions: CD40L, CD40L+LPS, CD40L+CpG, and CD40L+LPS+CpG in the presence or absence of fixed P. gingivalis. Percentages of CD1dhiCD5+ B cells were measured by flow cytometry. IL-10 mRNA expression and secreted IL-10 were measured by real-time quantitative PCR and by ELISA respectively. Results P. gingivalis LPS plus CD40L significantly increased CD1dhiCD5+ B cell percentages and secreted IL-10 levels in both immunized and non-immunized mice B cells in the presence or absence of P. gingivalis, compared with control group. Secreted IL-10 levels were significantly increased in CD40L+LPS treated group compared with CD40L treatment group in the absence of P. gingivalis. CpG plus CD40L significantly decreased CD1dhiCD5+ B cell percentages, but greatly elevated secreted IL-10 levels in immunized and non-immunized mice B cells in the absence of P. gingivalis, compared with CD40L treatment group. Conclusions P. gingivalis LPS and CpG differentially enhance IL-10 secretion and expansion of mouse B10 cells during innate and adaptive immune responses.

Correlation between Intravoxel Incoherent Motion Magnetic Resonance Imaging Derived Metrics and Serum Soluble CD40 Ligand Level in an Embolic Canine Stroke Model

OBJECTIVE: To determine the relationship between intravoxel incoherent motion (IVIM) imaging derived quantitative metrics and serum soluble CD40 ligand (sCD40L) level in an embolic canine stroke model. MATERIALS AND METHODS: A middle cerebral artery occlusion model was established in 24 beagle dogs. Experimental dogs were divided into low- and high-sCD40L group according to serum sCD40L level at 4.5 hours after establishing the model. IVIM imaging was scanned at 4.5 hours after model establishment using 10 b values ranging from 0 to 900 s/mm². Quantitative metrics diffusion coefficient (D), pseudodiffusion coefficient (D*), and perfusion fraction (f) of ischemic lesions were calculated. Quantitative metrics of ischemic lesions were normalized by contralateral hemisphere using the following formula: normalized D = D(stroke) / D(contralateral). Differences in IVIM metrics between the low- and high-sCD40L groups were compared using t test. Pearson's correlation analyses were performed to determine the relationship between IVIM metrics and serum sCD40L level. RESULTS: The high-sCD40L group showed significantly lower f and normalized f values than the low-sCD40L group (f, p 0.05). Both f and normalized f values were negatively correlated with serum sCD40L level (f, r = −0.789, p 0.05). CONCLUSION: The f value derived from IVIM imaging was negatively correlated with serum sCD40L level. f value might serve as a potential imaging biomarker to assess the formation of microvascular thrombosis in hyperacute period of ischemic stroke.

Evaluation of serum levels and significance of soluble CD40 ligand in screening patients with hepatitis C virus-related hepatocellular carcinoma

The study's objective was to evaluate the clinical significance of sCD40L in HCV- associated hepatocellular carcinoma [HCV-HCC] patients. Sera concentration of circulating sCD40L and IL-10 were assayed using ELISA in 30 HCV positive patients with HCC, 30 HCV-positive patients with liver cirrhosis and 30 age-matched healthy volunteers with negative anti-HCV-Ab as a control group. Serum sCD40Lshowed statistically-significant high levels in HCV-HCC patients compared to HCV-cirrhotic patients and normal controls [P < 0.001]. Serum sCD40L had higher diagnostic value in HCC patients compared with serum AFP. High sensitivity and specificity of sCD40L was observed compared to AFP [90%, 86.7% and 83% and 80% respectively]. Significant positive correlation was detected between serum sCD40L and IL-10[r = 0.85 P < 0.001], AFP [r = 0.62 P < 0.05] and tumour staging [r = 0.5 P < 0.05]. The study concluded that sCD40L is a valuable diagnostic tool in early diagnosis and screening for HCV and HCC as well as routine follow up of HCV cirrhosis patients. Assessment of serum IL-10 levels in HCV patients may provide a possible predictive marker for disease progression

Recurrent fever, hepatosplenomegaly and eosinophilia in a boy / 中国当代儿科杂志

A 2-year-old boy was admitted into the hospital because of cough and fever. Lymph node tuberculosis was noted when he was 2 months old and he was subsequently hospitalized several times because of cough and fever. After hospitalization the laboratory examination showed an increased eosinophia level in blood. The immune function tests shows decreased levels of IgG, IgA, and IgM. The patient had no response to anti-tuberculosis, anti-bacterial, and anti-fungal treatment, resulting in recurrent fever and progressive enlargement of the liver and spleen. Jam-like stools were noted 35 days after admission. B ultrasonography showed suspected intussusception. Laparotomy, reduction of intussusception and ileocecum angioplasty, biopsies of intestinal wall nodules and lymphoglandulae mesentericae, and hepatic biopsy were then performed under general anesthesia. The patient eventually died because of postoperative severe liver damage, disseminated intravascular coagulation and electrolyte disorder. Both the blood culture and hepatic biopsy tests showed Penicillium marneffei infecton. Immunodeficiency gene test was performed on the patient, his bother and their parents. T→G base substitution mutation (IVS1-3 T→G) in the CD40L gene was found in the patient. X-linked hyper-IgM syndrome was thus diagnosed in the patient. His mother was a carrier of the mutated CD40L gene, but his father was normal in the gene test. Hemizygous mutation in the CD40L gene was found in both the patient and his bother.

Effect of Soluble CD40L on Leukemia K562 Cells and Its Mechanism / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of sCD40L on biological behavior of leukemia cell line K562 and the possible mechanism.</p><p><b>METHODS</b>The different concentration of sCD40L was used to treat K562 cells, and the optimum concentration of sCD40L was screened by detecting the proliferation inhibition rate of K562 cells. The optimum concentration of sCD40L was used to treat K562 cells, the cell apoptosis rate and expression level of P53 and BCL-2 were detected by flow cytometry and the expression levels of Caspase 8 and Caspase 3 were detected by ELISA.</p><p><b>RESULTS</b>The optimum concentration of sCD40L was 4 µg/ml. After treated with sCD40L, the cell apoptosis rate, the expression of apoptosis-related factor P53 and the expression of Caspase 8 and Caspase 3 were significantly up-regulated in K562 cells,but the expression of BCL-2 was significantly down-regulated.</p><p><b>CONCLUSION</b>4 µg/ml sCD40L can inhibit the cell proliferation and promote the apoptosis of K562 cells, its mechanism may be related with mitochondrial and P53 pathway.</p>

Qingre quyu granule stabilizes plaques through inhibiting the expression of tenascin-C in patients with severe carotid stenosis / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the therapeutic effects of Qingre Quyu Granule (QQG) on the patients with severe carotid stenosis, and to explore the mechanism of it.</p><p><b>METHODS</b>Ninety-six patients with severe carotid stenosis were enrolled in the study and were classified into a QQG group (n=48) and a control group (n=48) randomly using consecutively numbered envelopes. The patients in the QQG group were given QQG and Western medicine, those in the control group were given Western medicine merely, the course of treatment was 16 weeks. All patients went through endarterectomy after treatment. Plaques were subjected to the analysis of CD3, CD68, soluble intercellular adhesion molecule 1 (ICAM-1), matrix metalloprotease-9 (MMP-9), CD40L, tenascin-C, and collagen content lipid content by immunohistochemistry or polarized light analysis.</p><p><b>RESULTS</b>By the end of experiment, the expressions of CD3, CD68, ICAM-1, MMP9, CD40L and tenascin-C on the plaques were statistically significant lower in the QQG group compared with the control group(P<0.01). The lipid content of the plaque was also significantly lower in the QQG group compared with the control group (P<0.01). The interstitial collagen in the tissue sections of the plaques was also significantly higher in the QQG group in comparison with the control group (P<0.01).</p><p><b>CONCLUSION</b>QQG could stabilize carotid artery plaques through inhibiting pro-inflammation factors and restraining the tenascin-C and MMP9 pathway.</p>

Phenotypic Tfh development promoted by CXCR5-controlled re-localization and IL-6 from radiation-resistant cells

How follicular T-helper (Tfh) cells develop is incompletely understood. We find that, upon antigen exposure in vivo, both naïve and antigen-experienced T cells sequentially upregulate CXCR5 and Bcl6 within the first 24 h, relocate to the T-B border, and give rise to phenotypic Bcl6(+)CXCR5(+) Tfh cells before the first cell division. CXCR5 upregulation is more dependent on ICOS costimulation than that of Bcl6, and early Bcl6 induction requires T-cell expression of CXCR5 and, presumably, relocation toward the follicle. This early and rapid upregulation of CXCR5 and Bcl6 depends on IL-6 produced by radiation-resistant cells. These results suggest that a Bcl6(hi)CXCR5(hi) phenotype does not automatically define a Tfh lineage but might reflect a state of antigen exposure and non-commitment to terminal effector fates and that niches in the T-B border and/or the follicle are important for optimal Bcl6 induction and maintenance.

Increase in Anti-Gal IgM Level is Associated With Early Graft Failure in Intraportal Porcine Islet Xenotransplantation

BACKGROUND: Anti-Gal is a major antibody induced in non-human primates (NHPs) after xenotransplantation. To understand the mechanism of graft rejection, we investigated the association between anti-Gal responses and graft failure in NHP recipients of porcine islet transplantation (PITx). METHODS: Intraportal PITx was performed in 35 diabetic NHPs, and graft function was monitored. Early graft failure (EGF) was defined as loss of graft function within a month after PITx. Seven, 19, nine NHPs received immunosuppression (IS) without CD40 pathway blockade (Group I), with anti-CD154 (Group II), and with anti-CD40 (Group III), respectively. The anti-Gal levels on day 0 and day 7 of PITx were measured by ELISA. RESULTS: The frequency of EGF was significantly lower in Group II (26.3%) than in Group I (100%, P=0.0012) and Group III (77.8%, P=0.0166). While levels of anti-Gal IgG in Group I and anti-Gal IgM in Group III increased on day 7 compared with day 0 (P=0.0156 and 0.0273), there was no increase in either on day 7 in Group II. The ratio of anti-Gal IgM or IgG level on day 7 to that on day 0 (Ratio7/0) was significantly higher in recipients with EGF than without EGF (P=0.0009 and 0.0027). ROC curve analysis of anti-Gal IgM Ratio7/0 revealed an area under the curve of 0.789 (P=0.0003). CONCLUSIONS: IS with anti-CD154 suppressed anti-Gal responses and prevented EGF in PITx. Anti-Gal IgM Ratio7/0, being associated with EGF, is a predictive marker for EGF.