Molecular immune mechanisms of HPV-infected HaCaT cells in vitro based on toll-like receptors signaling pathway.

OBJECTIVE: To explore the molecular immune mechanism of HPV-infected HaCaT cells in vitro based on TLRs signaling pathway by analyzing the effects of interfering TLRs on inflammatory and immune factors in the signaling pathway. METHODS: FCM was used to analyze the proportion of Th1, Th2, Th17, and Treg cells in blood samples. HPV-infected HaCaT cells were divided into five groups: A, B, C, D, and E. Group A added TLR3 antagonist, group B added TLR9 antagonist, group C added equivalent saline, group D added IRF3 agonist, and group E added IRF3 inhibitor. Immunohistochemistry was used to analyze the expression of TLR3 and TLR9 in HaCaT cell model; ELISA was used to analyze the expression of inflammatory factors IL-2, TNF-a, and IFN-beta; WB was used to analyze the expression of TRAF3, IKK epsilon, and TBK1; RT-PCR was used to analyze the expression of IRF3 and IRF7 in each cell model. RESULTS: The proportion of blood immune cells in patients with HPV infection was Th1, Th17, Th2, and Treg, with statistical significance (P < .05); the expression of TLR3 and TLR9 in HPV-infected cells was higher than that in negative control group, with statistical significance (P < .05); TLR3 was higher than TLR9, with no significant difference (P > .05); the expression of IL-2, TNF-alpha, IFN-beta in each group, TLR3, and TLR9 was higher than that in negative control group (P < .05). The expression of TRAF3, IKK epsilon, and TBK1 in the control group was higher than that in the TLR3 and TLR9 inhibitor groups, and the expression of IRF3 and IRF7 in the TLR9 inhibitor group was higher than that in the TLR3 inhibitor group (P < .05); the expression of IRF3 and IRF7 in the TLR3i and TLR9i inhibitor groups was lower than that in the TLR3 inhibitor group (P < .05). Compared with the control group, IRF3a group was higher than the control group, IRF3i group was lower than the control group, the difference was statistically significant (P < .05). CONCLUSION: TLR3 and TLR9, the key factors of TLRs, are highly expressed in HaCaT cells infected with HPV. Through TLRs-IKK-e-IRFs-IFN signaling pathway, they can induce high expression of inflammatory factors, IKK-e, IRFs, and IFN, and improve immunity.

Correction: Blood transcriptional profiling reveals IL-1 and integrin signaling pathways associated with clinical response to extracorporeal photopheresis in patients with leukemic cutaneous T-cell lymphoma.

[This corrects the article DOI: 10.18632/oncotarget.26900.].

Blood transcriptional profiling reveals IL-1 and integrin signaling pathways associated with clinical response to extracorporeal photopheresis in patients with leukemic cutaneous T-cell lymphoma.

Extracorporeal photopheresis (ECP) is a frontline therapy for patients with leukemic cutaneous T-cell lymphoma (L-CTCL), but its mechanisms of action are not fully understood. This study was to explore the molecular mechanisms underlying clinical response versus non-response in patients with L-CTCL. We performed blood transcriptional profiling of ten L-CTCL patients at Day 2 and 1 month post- ECP compared to pre-ECP baseline using Agilent Whole Human Genome Microarray technology. Differentially expressed genes (DEGs) between five clinically-responsive patients and five clinically-resistant patients were cross-compared. Higher numbers of genes were modulated in responders than non-responders after ECP at both Day 2 and 1 month, with two thirds of DEGs down-regulated. The down-regulated DEGs at 1 month post-ECP were related to inflammatory, immune and/or stress responses, platelet functions, and chromatin remodeling. Upregulated DEGs were mainly related to functions of the nucleolus. Pathway analysis revealed that integrin and IL-1 signaling pathways were the top pathways affected in responders, which were minimally affected in non-responders. The top upstream transcription regulators affected were IL1B, EGR1, FAS, and TGFB1. Our results suggest that the modulation of cell adhesion and suppression of IL-1ß induced inflammation may underlie the efficacy of ECP in L-CTCL.

Ultraviolet B inhibition of DNMT1 activity via AhR activation dependent SIRT1 suppression in CD4+ T cells from systemic lupus erythematosus patients.

BACKGROUND: Previous studies have reported that ultraviolet B (UVB) inhibits DNA methyltransferase1 (DNMT1) activity in CD4+ T cells from systemic lupus erythematosus (SLE) patients. Silent mating type information regulation 2 homolog 1 (SIRT1) is a type of Class III histone deacetylases (HDACs), and has been reported to play roles in the pathogenesis of different autoimmune diseases and can modulate DNMT1 activity. Moreover, aryl hydrocarbon receptor (AhR) has been reported to link UVB with SLE. However, the exact mechanisms by which DNMT1 activity is inhibited by UVB in lupus CD4+ T cells remain largely unknown. OBJECTIVE: To elucidate the exact mechanisms by which DNMT1 activity is inhibited by UVB in lupus CD4+ T cells. METHODS: Twenty-two newly diagnosed active SLE patients and 30 healthy controls were enrolled in the study. CD4+ T cells were isolated, cultured and treated. DNMT1 activity assay, quantitative real-time PCR (qRT-PCR), Western blotting, RNA interference using small interfering RNA and Chromatin Immunoprecipitation (ChIP) assay were employed. RESULTS: DNMT1 activity was inhibited in si-SIRT1-transfected CD4+ T cells, and increased by the established SIRT1 activator, SRT1720. Moreover, the mRNA and protein expression of SIRT1 were suppressed by UVB exposure in lupus CD4+ T cells. UVB-inhibited DNMT1 activity was reversed by SRT1720 in si-control-transfected lupus CD4+ T cells, but not in si-SIRT1-transfected lupus CD4 + T cells. Furthermore, AhR activation by VAF347 reduced the mRNA and protein expression of SIRT1. ChIP using an antibody against AhR in normal CD4+ T cells revealed a 16-fold stronger signal at the site about 1.6kb upstream from the translation start site of the SIRT1 promoter. Finally, UVB could activate AhR and inhibit the mRNA and protein expression of SIRT1. AhR knockdown abrogated the inhibition of UVB-mediated SIRT1 mRNA and protein expression and DNMT1 activity in lupus CD4+ T cells. CONCLUSION: UVB suppressed SIRT1 expression via activating AhR, and subsequently inhibited DNMT1 activity in CD4+ T cells from SLE patients.

NF-κB inhibition rescues Toll-like receptor 9 expression in human papillomavirus type 11 infected HaCaT cells.

OBJECTIVES: Toll-like receptors (TLRs) are related to human papillomavirus (HPV) infections including condyloma acuminatum (CA). This study was designed to investigate the mechanism of TLR9 and nuclear factor κB (NF-κB) in CA. METHODS: Expression of TLR9 protein in CA patients was detected and compared with those in CA relapse-free (CaRF) patients and normal control. HaCaT cells were transfected with HPV11 genome and NF-κB p65 siRNA or IκB kinase inhibitor BMS345541. Expression of NF-κB and TLR9 were detected using both PCR and Western blot methods. RESULTS: TLR9 was downregulated in CA specimens as compared to CaRF and normal controls. HPV11 transfection into HaCaT (HPV11.HaCaT) cells reduced TLR9 expression and activated NF-κB p65 expression. However, administration of NF-κB p65 siRNA or IκB kinase inhibitor BMS345541 significantly inhibited NF-κB p65 expression and rescued the expression of TLR9. CONCLUSION: Inhibition of NF-κB activation could rescue TLR9 expression in HPV11.HaCaT cells. TLR9/NF-κB mechanism may provide new target for clinical treatment of CA.

Effect of Hath1 on the proliferation and apoptosis of cutaneous squamous cell carcinoma in vitro.

Increasing evidence has demonstrated that the tumor suppressor gene Hath1 is implicated in the development and progression of tumors and is verified to be downregulated in several types of tumor. However, the roles and precise molecular mechanisms of Hath1 in cutaneous squamous cell carcinoma (SCC) remain to be elucidated. In the present study, two approaches were used to investigate the tumor­suppressing effect of Hath1 in cutaneous SCC. Firstly, the effect of inhibiting Hath1 expression with short hairpin RNA (shRNA) on tumor growth and apoptosis was investigated. KUMA5 cells were stably transfected with a plasmid expressing Hath1 shRNA (pGenesil­1­Hath1). Secondly, the anti­tumor effect of Hath1 was investigated in KUMA5 cells following transfection with pcDNA3.1­Hath1. The mRNA and protein expression of Hath1 was detected by reverse transcription quantitative polymerase chain reaction and western blot analysis, respectively. Cell proliferation in vitro was assessed using an MTT assay. Flow cytometry was used to detect cell apoptosis. The results demonstrated that compared with the control groups, the expression of Hath1 was significantly reduced in the KUMA5/pGenesil­1­Hath1 cells and markedly increased in the KUMA5/pcDNA3.1­Hath1 cells. Cell proliferation was markedly increased in the KUMA5/pGenesil­1­Hath1 cells in a time­dependent manner; however, it was markedly inhibited in the KUMA5/pcDNA3.1­Hath1 cells. Flow cytometry revealed that apoptosis decreased in KUMA5/pGenesil­1­Hath1 cells and increased in KUMA5/pcDNA3.1­Hath1 cells. Downregulation of Hath1 expression promoted the proliferation and reduced the apoptosis of KUMA5 cells. By contrast, overexpression of Hath1 inhibited proliferation and induced the apoptosis of KUMA5 cells. These findings provide possible new strategies and therapeutic targets for the treatment and diagnosis of cutaneous SCC.

KDM4B and KDM4A promote endometrial cancer progression by regulating androgen receptor, c-myc, and p27kip1.

Epidemiological evidence suggests that elevated androgen levels and genetic variation related to the androgen receptor (AR) increase the risk of endometrial cancer (EC). However, the role of AR in EC is poorly understood. We report that two members of the histone demethylase KDM4 family act as major regulators of AR transcriptional activityin EC. In the MFE-296 cell line, KDM4B and AR upregulate c-myc expression, while in AN3CA cells KDM4A and AR downregulate p27kip1. Additionally, KDM4B expression is positively correlated with AR expression in EC cell lines with high baseline AR expression, while KDM4A and AR expression are positively correlated in low-AR cell lines. In clinical specimens, both KDM4B and KDM4A expression are significantly higher in EC tissues than that in normal endometrium. Finally, patients with alterations in AR, KDM4B, KDM4A, and c-myc have poor overall and disease-free survival rates. Together, these findings demonstrate that KDM4B and KDM4A promote EC progression by regulating AR activity.

Saikosaponin-d affects the differentiation, maturation and function of monocyte-derived dendritic cells.

Saikosaponin-d (Ssd) is a triterpenoid saponin derived from Bupleurum falcatum L., which has been shown to exhibit a variety of pharmacological properties, including anti-inflammatory, antibacterial and antiviral properties. The aim of the present study was to investigate the effect of Ssd on the differentiation, maturation and function of human monocyte-derived dendritic cells (DCs) isolated from condylomata acuminata patients. The results of the present study demonstrated that Ssd reduced the differentiation of DCs, as evidenced by decreased expression levels of cluster of differentiation (CD)1a, CD80 and CD86 molecules and increased CD14 expression. Expression levels of the mannose receptor and CD32 were also significantly elevated, which was associated with enhanced fluorescein isothiocyanate-dextran endocytic activity. Furthermore, Ssd treatment promoted DC maturation by increasing the expression levels of CD40, CD83, CD80 and CD86. In addition, the function of mature DCs, including the secretion of IL-12 and the stimulation of lymphocyte proliferation, was significantly increased following Ssd administration. In conclusion, the present study indicated that Ssd exhibited immunomodulatory effects and may be a novel potent chemopreventive drug candidate for the treatment of condylomata acuminata.

5-aminolevulinic acid-based photodynamic therapy for the treatment of condylomata acuminata in Chinese patients: a meta-analysis.

OBJECTIVE: This meta-analysis was designed to assess the efficacy of topical 5-aminolaevulinic acid (ALA) photodynamic therapy (PDT) in Chinese patients with condylomata acuminata (CA). METHODS: Electronic literature databases (Medline, Embase, Cochrane Library, China National Knowledge Infrastructure, and Wanfang database) were searched for relevant randomized controlled trials (RCTs) published prior to October 2012. Only RCTs that compared ALA-PDT to non-ALA-PDT for patients with genital condylomata were selected. The outcomes included the recurrence rate and adverse events. The risk ratios (RRs) and 95% confidence intervals (CIs) were calculated as the ALA-PDT vs. without ALA-PDT. RESULTS: Twenty RCTs composed of 1903 patients (ALA-PDT, n = 1106; non-ALA-PDT, n = 797) were included in the meta-analysis. ALA-PDT decreased the recurrence rate within 12 week after treatment (vs. without ALA-PDT, RR: 0.28, 95% CI: 0.22-0.35) and 24 week after treatment (vs. without ALA-PDT, RR: 0.24, 95% CI: 0.17-0.34) in a fixed-effect model. The common adverse events related ALA-PDT included a mild burning and/or stinging sensation, erythema, mild edema, erosion, and hyperpigmentation. CONCLUSION: Local application of ALA-PDT reduced recurrence rate vs. without ALA-PDT. The use of ALA-PDT should be considered as a feasible therapy for the treatment of CA.

Detection of telomerase activity in patients with mycosis fungoides / 中国医学科学杂志(英文版)

<p><b>OBJECTIVES</b>To detect telomerase activity in patients with mycosis fungoides (MF) and to study the role of telomerase in the tumorigenesis of MF.</p><p><b>METHODS</b>The technique of PCR-ELISA was employed to detect telomerase activity in 35 patients with various stages of MF.</p><p><b>RESULTS</b>92.3% tumor stage of MF, 78.6% plaque stage of MF and 75.0% patch stage of MF had positive telomerase activity. The control samples had no telomerase activity. Telomerase activity in tumor stage of MF was significantly higher than that in plaque stage, while the latter was higher than that in patch stage. Telomerase activity was correlated with the stage of MF.</p><p><b>CONCLUSION</b>High level of telomerase activity frequently occurred in patients with MF, suggesting that telomerase might play an important role in the tumorigenesis of MF and is a useful marker for the diagnosis of MF possibly.</p>