A20 promoted the phagocytosis of lymphoma cells by dendritic cells from activated B-cell-like non-Hodgkin lymphoma.

Non-Hodgkin lymphoma (NHL) is a lymphoproliferative disorder derived from either B or T lymphocytes. Among NHL, activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL) and T cell non-Hodgkin lymphomas (T-NHL) are poor prognosis and aggressive subtypes. Macrophages are professional phagocytic cells and dendritic cells (DCs) are professional antigen-presenting cells in immune system. Doxorubicin (Dox) and Etoposide (ET) are the most effective anti-cancer drugs. A20 and CYLD are negative regulators of NF-κB-dependent functions in many cell types. Little is known about the roles of A20 and CYLD in regulating functions of DCs and macrophages from NHL. The present study, therefore, explored whether A20/CYLD expression contributes to functions of DCs and macrophages from NHL. To this end, blood samples of seventy-nine patients with ABC DLBCL and T-NHL were examined. Gene expression profile was determined by quantitative RT-PCR and immunophenotype, cell apoptosis and phagocytosis by flow cytometry. As a result, immunophenotypic analysis showed that the numbers of CD13+CD117-, CD56+CD40+ and CD23+CD40+ expressing cells were significantly elevated in ABC DLBCL cases compared to healthy individuals and T-NHL patients. Interestingly, upon treatment of Dox and ET, the phagocytosis of lymphoma cells was significantly reduced by CD11c+CD123- DCs and the percentage of CD56+ mature DCs was significantly enhanced in ABC DLBCL patients only in the presence of A20 siRNA, but not CYLD siRNA. In conclusion, ABC DLBCL patients with low A20 expression were defective in elimination of lymphoma cells by DCs and linked to killer DC expansion in circulation.

Effects of Vinblastine and Vincristine on the function of chronic myeloid leukemic cells through expression of A20 and CYLD.

Chronic myelogenous leukemia (CML) is characterised by the translocation of regions of the BCR and ABL genes, leading to the fusion gene BCR-ABL forming the Philadelphia (Ph) chromosome. Vinblastine (Vinb) and Vincristine (Vinc) are Vinca alkaloids and frequently used in combination chemotherapy in leukemias and lymphomas. Deubiquitinating enzyme (DUB) genes such as A20, Otubain 1 and CYLD are known as inhibitors of functional activation of immune cells mediated through the NF-κB/STAT pathway. Little is known about the regulatory role of Vinb/Vinc on the function of CML cells and the contribution of the DUBs to those effects. In the end, the gene expression profile was determined by quantitative RT-PCR, physiological properties of CML cells by flow cytometry and cytokine production by ELISA. As a result, inactivated expression of the DUBs A20, CYLD, Otubain 1 and Cezanne and enhanced activation of CD11b+ and CD4T cells were observed in CML patients. Importantly, Vinc enhanced the expression of A20 and CYLD and inhibited the proliferation and survival of CML (K562) cells. The effects were abolished in the presence of A20 siRNA, while cell proliferation only depended on the presence of CYLD. In conclusion, the up-regulation of A20 by Vinc could involve inhibitory effects on the proliferation and survival of K562 cells. The events might contribute to the anticancer effect of Vinc on A20-sensitive CML cells.

Regulation of dendritic cell function by A20 through high glucose-induced Akt2 signaling.

A20 is a negative regulator of nuclear factor (NF)-κB-dependent inflammatory reaction in response to different stimuli by immune cells including dendritic cells (DCs), the most potent antigen-presenting cells involved in both the innate and adaptive immune response. Dendritic cells use glucose as carbon source to synthesize fatty acid and generate energy. Glucose enhances cell apoptosis mediated through PI3K/Akt, ERK1/2, and Bax/Bcl-2 pathways. The protein kinase Akt2/PKBß is expressed in DCs and a regulator of Ca2+ influx, Na+/H+ exchanger activity, and migration of DCs. This study explored whether regulation of high glucose-induced DC function through Akt2 signaling is influenced by overexpression of A20. To this end, A20 protein expression was determined by western blotting and immunoprecipitation, secretion of inflammatory cytokines by ELISA, and expression of apoptotic markers by flow cytometry. As a result, treatment of mice with 10% high glucose enriched water increased secretion of insulin/IGF1 and reduced A20 protein level, the effects were blunted in Akt2-/- mice. Incubation of DCs with high glucose significantly decreased A20 protein expression in both control and Akt1-silenced DCs, but not in Akt2-/- DCs. Importantly, treatment of DCs with high glucose increased ceramide synthesis, caspase 8 activity, and annexin V binding in control DCs, the effects were abolished in Akt2-/- DCs or by A20 overexpression. In conclusion, regulation of A20 sensitive DC function by high glucose is mediated through insulin/IGF-1/Akt2 signaling.

Association of UGT1A1 gene variants, expression levels, and enzyme concentrations with 2,3,7,8-TCDD exposure in individuals exposed to Agent Orange/Dioxin.

Among the congener of dioxin, 2,3,7,8-TCDD is the most toxic, having a serious long-term impact on the environment and human health. UDP-glucuronosyltransferase 1A1 (UGT1A1) plays a crucial role in the detoxification and excretion of endogenous and exogenous lipophilic compounds, primarily in the liver and gastrointestinal tract. This study aimed to investigate the association of UGT1A1 gene polymorphisms, expression levels, and enzyme concentration with Agent Orange/Dioxin exposure. The study included 100 individuals exposed to Agent Orange/Dioxin nearby Da Nang and Bien Hoa airports in Vietnam and 100 healthy controls. UGT1A1 SNP rs10929303, rs1042640 and rs8330 were determined by Sanger sequencing, mRNA expression was quantified by RT-qPCR and plasma UGT1A1 concentrations were measured by ELISA. The results showed that UGT1A1 polymorphisms at SNPs rs10929303, rs1042640 and rs8330 were associated with Agent Orange/Dioxin exposure (OR = 0.55, P = 0.018; OR = 0.55, P = 0.018 and OR = 0.57, P = 0.026, respectively). UGT1A1 mRNA expression levels and enzyme concentration were significantly elevated in individuals exposed to Agent Orange/Dioxin compared to controls (P < 0.0001). Benchmark dose (BMD) analyses showed that chronic exposure to 2,3,7,8-TCDD contamination affects the UGT1A1 mRNA and protein levels. Furthermore, UGT1A1 polymorphisms affected gene expression and enzyme concentrations in individuals exposed to Agent Orange/Dioxin. In conclusion, UGT1A1 gene polymorphisms, UGT1A gene expression levels and UGT1A1 enzyme concentrations were associated with Agent Orange/Dioxin exposure. The metabolism of 2,3,7,8-TCDD may influence UGT1A gene expression and enzyme concentrations.

Association of PKLR gene copy number, expression levels and enzyme activity with 2,3,7,8-TCDD exposure in individuals exposed to Agent Orange/Dioxin in Vietnam.

2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) is the most toxic congener of dioxin and has serious long-term effects on the environment and human health. Pyruvate Kinase L/R (PKLR) gene expression levels and gene variants are associated with pyruvate kinase enzyme deficiency, which has been identified as the cause of several diseases linked to dioxin exposure. In this study, we estimated PKLR gene copy number and gene expression levels using real-time quantitative PCR (RT-qPCR) assays, genotyped PKLR SNP rs3020781 by Sanger sequencing, and quantified plasma pyruvate kinase enzyme activity in 100 individuals exposed to Agent Orange/Dioxin near Bien Hoa and Da Nang airfields in Vietnam and 100 healthy controls. The means of PKLR copy numbers and PKLR gene expression levels were significantly higher, while pyruvate kinase enzyme activity was significantly decreased in Agent Orange/Dioxin-exposed individuals compared to healthy controls (P < 0.0001). Positive correlations of PKLR gene copy number and gene expression with 2,3,7,8-TCDD concentrations were observed (r = 0.2, P = 0.045 and r = 0.54, P < 0.0001, respectively). In contrast, pyruvate kinase enzyme activity was inversely correlated with 2,3,7,8-TCDD concentrations (r = -0.52, P < 0.0001). PKLR gene copy number and gene expression levels were also inversely correlated with pyruvate kinase enzyme activity. Additionally, PKLR SNP rs3020781 was found to be associated with 2,3,7,8-TCDD concentrations and PKLR gene expression. In conclusion, PKLR copy number, gene expression levels, and pyruvate kinase enzyme activity are associated with 2,3,7,8-TCDD exposure in individuals living in Agent Orange/Dioxin-contaminated areas.

JAK2 rs10974944 is associated with both V617F-positive and negative myeloproliferative neoplasms in a Vietnamese population: A potential genetic marker.

The JAK2 gene encodes for a non-receptor tyrosine kinase that plays a key role in the JAK/STAT signaling transfer pathway. Genetic polymorphisms of this gene have been indicated to be associated with myeloproliferative neoplasm-associated thrombosis in recent studies. This research aimed to evaluate the association between the variant rs10974944 and different types of Myeloproliferative neoplasms disorders in the Vietnamese population. DNA samples were obtained from 172 essential thrombocythemia patients, 14 primary myelofibrosis patients, 76 polycythemia vera patients, and 192 healthy controls. The JAK2 rs10974944 and V617F genotypes were identified by the polymerase chain reaction-restriction fragment length polymorphism genotyping and Sanger sequencing methods. Results showed that there was a strong association between rs10974944 and Myeloproliferative neoplasms phenotype (p < .0001) and the most significant association was observed in the recessive model of the mutant allele (G). The G allele carriers had a 1.74, 2.86, and 3.03 higher risk of getting essential thrombocythemia, primary myelofibrosis, and polycythemia vera, respectively. Interestingly, this effect of rs10974944 seemed to be independent of the JAK2 V617F genotype. The distribution of rs10974944 genotypes were significantly different between V617F-positive and negative groups (p = .008). Moreover, the GG genotype of rs10974944 was observed to be associated with the risk of getting Myeloproliferative neoplasms both in JAK2 V617F-positive group, and for the first time in JAK2 V617F-negative patients. A systematic meta-analysis in different populations strengthened the evidence regarding the correlation between rs10974944 and myeloproliferative neoplasm disorders. To sum up, our results suggested that rs10974944 can be used as a predisposition screening marker for predicting Myeloproliferative neoplasms susceptibility.

Prevalence of CYLD mutations in Vietnamese patients with polycythemia vera.

BACKGROUND: Polycythemia vera (PV) is characterized by increased proliferation and accumulation of erythroid and mature myeloid cells and megakaryocyte in the bone marrow and peripheral blood. The JAK2V617F mutation is present in most PV patients. Deubiquitinase (DUB) genes, including TNFAIP3 (A20), CYLD and Cezanne, function as negative regulators of inflammatory reaction through nuclear factor kappa-light-chain-enhancer of activated B cells (NF-eB) signaling. OBJECTIVES: To determine single nucleotide polymorphisms (SNPs) profiling and gene expression of the DUB genes as well as the immunophenotype of PV cells. MATERIAL AND METHODS: Seventy-seven patients with PV and 55 healthy individuals with well-characterized clinical profiles were enrolled. Gene expression profile was determined using quantitative real-time polymerase chain reaction (qRT-PCR), the immunophenotype with flow cytometry, secretion of cytokines using enzyme-linked immunosorbent assay (ELISA), and gene polymorphisms using direct DNA sequencing. RESULTS: Inactivation of A20, CYLD and Cezanne, and increases in interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-á) levels, as well as the enhanced number of CD25+CD4 T, Th1 and regulatory T cells were observed in PV patients. The genetic analysis of the CYLD gene identified 11 SNPs, in which a novel W736G nsSNP in exon 15 and a SNP c.2483+6 T>G in intron 15 were observed in PV cases with the frequencies of 18.2% and 5.2%, respectively. The W736G non-synonymous SNP (nsSNP) was found to be most likely to exert deleterious effect and the intronic SNP c.2483+6 T>G was identified as aberrant splicing. Sequencing of Cezanne gene identified 7 SNPs in intron 10 and PV carriers of the SNPs had at least 2 SNPs in this gene. Importantly, PV carriers of the W736G nsSNP had multiple SNPs in CYLD, but not in A20 or Cezanne gene. CONCLUSIONS: Two identified SNPs, including the W736G nsSNP and the SNP c.2483+6 T>G, in CYLD gene might be associated with a risk of PV disease, in which the deleterious effect of the W736G nsSNP in CYLD gene could contribute to the pathogenesis of PV.