Xia Ke

Impaired Balance of Th17/Treg in Patients with Nasal Polyposis

Nasal polyposis (NP) is a chronic inflammatory disease of the nasal cavity and sinuses that is regulated by T lymphocyte subsets. Imbalance of Th17/Treg has been considered critical in the development of inflammation and atopic reactions. To assess whether the balance of Th17/Treg is disrupted in patients with NP, we evaluated the distribution of Th17 and Treg cells among peripheral blood mononuclear cells (PBMCs) in atopic patients with NP, non‐atopic patients with NP and controls. We then determined mRNA levels of RORc and Foxp3 and protein levels of IL‐17, TGF‐β and IL‐10 in polyp tissue among the three groups. Finally, we investigated the correlation between Th17‐, Treg‐ and Th1‐, Th2‐related cytokines (INF‐γ, IL‐4, IL‐5). The results demonstrated that both atopic and non‐atopic patients with NP revealed significantly increased Th17 proportion and decreased Treg proportion in PBMCs, as well as significantly increased RORc and IL‐17 levels and decreased Foxp3 and TGF‐β levels in polyp tissue. Furthermore, these differences were significant between atopic and non‐atopic groups. The frequency of Treg in PBMCs was found to be negatively correlated with Th1 and Th2 cytokines in polyps. These results indicated that an impaired balance of Th17/Treg existed in patients with NP and was more severe in atopic patients, suggesting that the imbalance of Treg/Th17 may play an important role in the development of NP and that atopy may aggravate NP by promoting the imbalance of Th17/Treg.

Association between Interleukin-27 gene polymorphisms and susceptibility to allergic rhinitis

OBJECTIVES Allergic rhinitis (AR) is an inflammatory disorder of the upper airway. Interleukin-27 (IL-27), a novel IL-12 family member, has recently been reported to play a role in some immune-related disorders. This study was performed to evaluate the potential association of IL-27 polymorphisms with AR in a Chinese Han population. DESIGN AND METHODS A case-control study was performed in 445 Chinese AR patients and 691 healthy controls. Three SNPs in the IL-27p28 gene, including rs153109, rs17855750 and rs181206, were detected using a polymerase chain reaction-restriction fragment length polymorphism assay (PCR-RFLP). RESULTS A significantly increased prevalence of the rs153109 TT genotype and the T allele was found in AR patients, while a decreased prevalence of the CT and CC genotypes and the C allele was found. For rs153109, the TT genotype and the T allele were significantly associated with the risk of AR, but the CT and CC genotypes and the C allele decreased the risk of AR; for rs17855750, the TT genotype and T allele were risk factors for AR, and the GT genotype and G allele provided protection. TTT and TTC haplotypes in the IL-27p28 gene were positively correlated with AR, while CGT, CTC and CTT haplotypes were associated with a significantly decreased risk of AR. CONCLUSION This study indicates that IL-27p28 polymorphisms rs153109 and rs17855750 are likely involved in AR susceptibility, making them potentially useful genetic biomarkers for AR susceptibility in the Chinese Han population.

MicroRNA expression profile of mature dendritic cell in chronic rhinosinusitis

ObjectiveChronic rhinosinusitis (CRS), which includes CRS without nasal polyposis (CRSsNP) and with nasal polyposis (CRSwNP), shows imbalance of helper T cells (Th) and regulatory T cells (Treg). The balance of Th and Treg cells is orchestrated by dendritic cells (DCs). Recent studies show functions of DCs can be regulated by microRNAs (miRNAs or miRs). This study is aimed to investigate miRNAs expression profiles of peripheral blood DCs in CRS.MethodsPeripheral blood samples of 30 patients with CRS and 7 patients with nasal septum deviation alone were collected. CD14+ monocytes were isolated from these samples and differentiated into dendritic cells (DCs). Small RNAs were extracted from mature DCs and reversely transcribed into cDNA by Mir-XTM miRNA First-Strand synthesis method. MiRNA microarrays were used for miRNA expression analysis. Microarray results were validated by real-time PCR performed on five top list target genes.ResultsMiRNA microarrays showed that DCs from different types of patients have different sets of differential expressed miRNAs when comparing with Controls; they also share 31 commonly changed miRNAs among all three groups of CRS patients. Of these 31 miRNAs, 5 miRNAs were up-regulated and 25 miRNAs were down-regulated in all three types of CRS, while MiR-1290 was down-regulated in CRSsNP but up-regulated in both atopic CRSwNP and non-atopic CRSwNP.ConclusionsBy comparing miRNA gene expression patterns in 3 types of CRS patients, we have been able to identify candidate miRNAs that might mediate the core pathogenesis of CRS through regulating dendritic cells. These miRNAs could serve as potential therapeutic targets for CRS.

EBV-LMP1-targeted DNAzyme restrains nasopharyngeal carcinoma growth in a mouse C666-1 xenograft model

Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) has been demonstrated to be linked to the pathogenesis of nasopharyngeal carcinoma (NPC), one of the most common cancers in southeast Asia as well as in the areas of southern China. RNA-cleaving DNAzymes are catalytic nucleic acids that bind and cleave a target RNA and have been increasingly used to inhibit gene expression. In this study, we aimed to explore the effects of down-regulation of LMP1 by DNAzymes on the NPC growth using a mouse xenograft model derived from human NPC C666-1 cells that constitutively express the LMP1. A specific LMP1-targeted DNAzyme, named DZ509, was synthesized, showing high activities in the inhibition of LMP1 expression, while the control DNAzyme (mutDZ509) had little effect on LMP1 expression. Knockdown of the LMP1 expression by DZ509 reduced cell proliferation and increased apoptosis compared to the cells transfected with mutDZ509. Intratumoral injections of DZ509 into C666-1 xenografts caused a significant suppression of tumor growth compared to mutDZ509-treated xenografts. Examination of the LMP1 expression in the xenografts demonstrated a marked inhibition of LMP1 by DZ509. An antisense oligonucleotide targeting the same site of the LMP1 transcripts was employed in parallel and produced a comparable inhibition on cell proliferation in vitro and tumor growth in vivo. These data demonstrate that LMP1-targeted DNAzyme is efficient in controlling tumor growth in vivo, and thus may have therapeutic implications in the treatment of NPC.

FCRL3 Gene Polymorphisms Confer Autoimmunity Risk for Allergic Rhinitis in a Chinese Han Population.

Background Heredity and environmental exposures may contribute to a predisposition to allergic rhinitis (AR). Autoimmunity may also involve into this pathologic process. FCRL3 (Fc receptor-like 3 gene), a novel immunoregulatory gene, has recently been reported to play a role in autoimmune diseases. Objective This study was performed to evaluate the potential association of FCRL3 polymorphisms with AR in a Chinese Han population. Methods Five single-nucleotide polymorphisms of FCRL3, rs945635, rs3761959, rs7522061, rs10489678 and rs7528684 were genotyped in 540 AR patients and 600 healthy controls using a PCR-restriction fragment length polymorphism assay. Allele, genotype and haplotype frequencies were compared between patients and controls using the χ2 test. The online software platform SHEsis was used to analyze their haplotypes. Results This study identified three strong risk SNPs rs7528684, rs10489678, rs7522061 and one weak risk SNP rs945635 of FCRL3 in Chinese Han AR patients. For rs7528684, a significantly increased prevalence of the AA genotype and A allele in AR patients was recorded. The frequency of the GG genotype and G allele of rs10489678 was markedly higher in AR patients than those in controls. For rs7522061, a higher frequency of the TT genotype, and a lower frequency of the CT genotype were found in AR patients. Concerning rs945635, a lower frequency of the CC genotype, and a higher frequency of G allele were observed in AR patients. According to the analysis of the three strong positive SNPs, the haplotype of AGT increased significantly in AR cases (AR = 38.8%, Controls = 24.3%, P = 8.29×10-14, OR [95% CI] 1.978 [1.652~2.368]). Conclusions This study found a significant association between the SNPs in FCRL3 gene and AR in Chinese Han patients. The results suggest these gene polymorphisms might be the autoimmunity risk for AR.

Allergen induced Th17 response in the peripheral blood mononuclear cells (PBMCs) of patients with nasal polyposis

BACKGROUND Nasal polyposis (NP) is a chronic inflammatory disease of the nasal cavity and sinuses. Th17 cells have been considered to play roles in allergic airway diseases and various chronic inflammatory disorders. AIM OF THE STUDY This study aimed to investigate the population and function of peripheral Th17 cells in response to house dust mite extracts (HDM) allergen in NP patients, and evaluate the possible correlation between Th17 cells and atopy, to explore the role of atopy in the pathogenesis of NP. METHODS Peripheral blood mononuclear cells (PBMCs) obtained from atopic NP patients, non-atopic NP patients, and controls were stimulated by phytohemagglutinin (PHA) or HDM plus PHA. The resulting frequency of Th17 cells was detected by flow cytometry and the expression of RORc was measured by real-time PCR. Then the concentrations of IL-17A, INF-γ, IL-4 and IL-5 in the supernatants were assayed by specific ELISAs. RESULTS The population and function of Th17 cells in allergen stimulated PBMCs were significantly higher in atopic NP patients. In addition, in atopic group, HDM+PHA stimulation induced significant increase of Th17 population and IL-17A production versus those in PHA stimulated ones. However, the frequency of Th17 cells was not correlated with Th1, Th2 cytokine productions. CONCLUSION Th17 immunity is involved in the systemic immune responses to allergen in atopic NP and atopy may aggravate NP by stimulating the increase of Th17 population and IL-17A production. The mechanism of Th17 cells response to allergen may be regulated differently from the regulation of Th1 and Th2 immunity in NP.

Genetic risk of TNFSF4 and FAM167A-BLK polymorphisms in children with asthma and allergic rhinitis in a Han Chinese population

Abstract Background: Asthma and allergic rhinitis (AR) frequently occur as comorbid diseases of the upper airways. Single-nucleotide polymorphisms (SNPs) in the TNFSF4 and FAM167A-BLK genes have recently been shown to be associated with various immune-related disorders. Objective: Our aim was to determine whether TNFSF4 or FAM167A-BLK polymorphisms confer genetic susceptibility to asthma and AR in a Han Chinese population. Methods: We performed a case–control study of 290 asthmatic children and 252 healthy controls. Nine SNPs in the TNFSF4 region (rs1234313, rs1234314, rs1234315, rsl 2039904, rs844648 and rsl 0912580) and the FAM167A-BLK region (rs2254546, rs13277113 and rs1600249) were detected using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. Results: This study revealed that three SNPs in TNFSF4 (rsl 234313, rsl 234314 and rsl 234315) and two SNPs in FAM167A-BLK (rs2254546 and rsl 600249) were significantly correlated with asthma and AR, while SNP rsl600249 was associated with asthma without allergic rhinitis as a risk factor. Further, we demonstrated synergistic effects between the TNFSF4 and FAM167A-BLK SNPs. Conclusion: This study supports that the SNPs in TNFSF4 and FAM167A-BLK may be involved in asthma and AR gene risk in the Han Chinese cohort.

High frequency of T helper type 9 cells in Chinese patients with allergic rhinitis.

BACKGROUND T helper type 9 cells (Th9) are the most recently discovered subset of Th cells, and are involved in the pathology of several autoimmune and allergic diseases. The significance of Th9 cells in allergic rhinitis (AR) in Chinese patients is unclear. OBJECTIVE The aim of this study was to investigate the possible role of Th9 cells in AR in Chinese patients. METHODS Th9 cells and related factors were assessed by measuring levels of interleukin-9 (IL-9), PU.1, interferon-regulatory factor 4 (IRF4), and numbers of Th9 cells. A Th9-polarized milieu was evaluated by determining the levels of IL-4 and transforming growth factor-β1 (TGF-β1). Disease severity was assessed by rhino-conjunctivitis quality of life questionnaires (RQLQ), visual analog scale scores (VAS), and peripheral eosinophils (EOS) count. RESULTS Levels of IL-4 and TGF-β1 were elevated in AR groups versus healthy controls (P < 0.05). Levels of IL-9, PU.1, IRF4, and the numbers of Th9 cells were also significantly higher in the AR groups (P < 0.05). Furthermore, positive correlations were identified between IL-9 levels and EOS expression, RQLQ, and VAS scores (P < 0.01). CONCLUSIONS Th9 cells and their relative factors were elevated in AR patients. Levels of Th9 polarization-related factors were much higher in AR patients, and the severity of disease was associated with a more severe Th9 response. These results suggest that AR patients present a favorable environment for Th9 differentiation, and that Th9 cells may play a crucial role in the pathology of AR in Chinese patients.

Fluorescence Visualization Screening for EBV-LMP1-Targeted DNAzymes

Objectives To develop a novel screening method for DNAzymes targeting the LMP1 carboxy region. Study Design To design a method to screen special DNAzymes toward the Epstein-Barr virus (EBV)–associated carcinoma before clinic use. Setting Key Laboratory of the Ministry of Education–Molecular Biology of Infectious Diseases in Chongqing Medical University. Subjects and Methods Four novel 10-23 DNAzymes (DZ509, DZ1037, DZ893, and DZ827) targeting the EBV-LMP1 gene were designed and evaluated by detecting enhanced green fluorescence protein (EGFP) expression of LMP1 mRNA and the protein in the nasopharyngeal carcinoma (NPC) cell line CNE2 transfected with the pEGFP-C1-LMP1c vector. The screened specific DNAzymes were then transfected into NPC cell lines C666-1 while a mutant oligonucleotide mutDZ509 and an antisense oligonucleotide ASODN509 were designed as positive and negative controls. Cell proliferation, cell apoptosis, LMP1 mRNA, and the protein were assessed using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, Annexin V-fluorescence isothiocyanate (FITC), reverse transcription polymerase chain reaction (RT-PCR), and Western blots. Results The inhibition rates of fluorescence expression of the DNAzymes DZ509, DZ1037, DZ893, and DZ827 were 91.25%, 65.84%, 49.02%, and 44.56%, respectively. The results were in accordance with the inhibition effects of mRNA and protein expression. The screened DZ509 could effectively knock down endogenous LMP1 expression in C666-1 cells, inhibit cell proliferation, and induce cell apoptosis compared with mutDZ509 and ASODN509. Conclusion LMP1 could present a potential target for DNAzymes toward the EBV-associated carcinoma, and the EGFP expression vector could be a visible method for screening special DNAzymes before clinic use.

Association between JAK1 gene polymorphisms and susceptibility to allergic rhinitis.

OBJECTIVES Allergic rhinitis (AR) is an inflammatory disorder of the upper airway. Janus kinase 1 (JAK1), a member of JAK family, has recently been found to participate in the immune response and the development of allergic airway disease. This study was performed to evaluate the potential association of JAK1 polymorphisms with AR in a Chinese Han population. METHODS A case-control study was performed in 450 Chinese AR patients and 615 healthy controls. Three SNPs in the JAK1 gene, rs3790532, rs310241 and rs2780815, were analyzed using a polymerase chain reaction-restriction fragment length polymorphism assay (PCR-RFLP). RESULTS An association was detected between SNP rs310241 in the JAK1 gene and AR in a Chinese Han population. However, no significant association was observed between the polymorphisms rs3790532 and rs2780815 and AR. For rs310241, the CC genotype and the C allele significantly increased the risk of AR. Furthermore, we found that the ACG haplotype in JAK1 gene was positively correlated with AR, while the GTG haplotype was associated with a significantly decreased risk of AR. CONCLUSION This study indicates that the JAK1 rs310241 C-related genotype and allele are involved in AR susceptibility, making them potentially useful genetic biomarkers for AR susceptibility in the Chinese Han population.