Lanfang Cao

MYCN Transgenic Zebrafish Model with the Characterization of Acute Myeloid Leukemia and Altered Hematopoiesis

Background Amplification of MYCN (N-Myc) oncogene has been reported as a frequent event and a poor prognostic marker in human acute myeloid leukemia (AML). The molecular mechanisms and transcriptional networks by which MYCN exerts its influence in AML are largely unknown. Methodology/Principal Findings We introduced murine MYCN gene into embryonic zebrafish through a heat-shock promoter and established the stable germline Tg(MYCN:HSE:EGFP) zebrafish. N-Myc downstream regulated gene 1 (NDRG1), negatively controlled by MYCN in human and functionally involved in neutrophil maturation, was significantly under-expressed in this model. Using peripheral blood smear detection, histological section and flow cytometric analysis of single cell suspension from kidney and spleen, we found that MYCN overexpression promoted cell proliferation, enhanced the repopulating activity of myeloid cells and the accumulation of immature hematopoietic blast cells. MYCN enhanced primitive hematopoiesis by upregulating scl and lmo2 expression and promoted myelopoiesis by inhibiting gata1 expression and inducing pu.1, mpo expression. Microarray analysis identified that cell cycle, glycolysis/gluconeogenesis, MAPK/Ras, and p53-mediated apoptosis pathways were upregulated. In addition, mismatch repair, transforming and growth factor β (TGFβ) were downregulated in MYCN-overexpressing blood cells (p<0.01). All of these signaling pathways are critical in the proliferation and malignant transformation of blood cells. Conclusion/Significance The above results induced by overexpression of MYCN closely resemble the main aspects of human AML, suggesting that MYCN plays a role in the etiology of AML. MYCN reprograms hematopoietic cell fate by regulating NDRG1 and several lineage-specific hematopoietic transcription factors. Therefore, this MYCN transgenic zebrafish model facilitates dissection of MYCN-mediated signaling in vivo, and enables high-throughput scale screens to identify the potential therapeutic targets.

The Efficacy and Safety of Subcutaneous Immunotherapy in Mite-Sensitized Subjects With Asthma: A Meta-Analysis

BACKGROUND: Subcutaneous immunotherapy (SCIT) is widely used in the management of allergic diseases such as allergic asthma. We aimed to conduct a systematic review and meta-analysis to evaluate the efficacy and safety of SCIT in mite-sensitized subjects with asthma. METHODS: Literature from January 1990 to February 2013 on the efficacy and safety of SCIT for mite-sensitized asthma patients was searched in electronic databases, including the Cochrane Library, MEDLINE, Embase, PubMed, China Knowledge Resource Integrated Database, Wanfang, and Vendor Information Pages. Data were extracted from randomized controlled trials (RCTs) according to the selection criteria by 2 investigators independently. The quality of included trials was evaluated according to the Jadad scale standard. RESULTS: A total of 796 subjects from 19 different RCTs were included in this analysis. SCIT significantly reduced the asthma symptom scores (standardized mean difference of −0.94, 95% CI −1.58 to −0.29, P = .004) and the asthma medication scores (standardized mean difference of −1.06, 95% CI −1.70 to −0.42, P = .001) compared with the control group. However, there were no significant differences between subjects receiving SCIT and the control group in lung function (peak expiratory flow, percent-of-predicted FEV1, percent-of-predicted FVC) and specific antibody (allergen-specific immunoglobulin E) levels of blood serum (P > .05). In the studies containing data on safety, the incidences of systemic and local adverse reactions were 9.1% (8/88) and 17.2% (23/134), respectively, in subjects treated with SCIT, and no severe adverse events were reported. CONCLUSIONS: Our results suggest that SCIT is helpful in alleviating symptoms and reducing medication used in mite-sensitive asthma subjects, but with no improvement in lung function. The safety of SCIT is acceptable.

Bacterial lysate increases the percentage of natural killer T cells in peripheral blood and alleviates asthma in children.

Aims: To assess the efficacy of conventional treatment combined with bacterial lysate [OM-85 Broncho-Vaxom (BV)] in the prevention of asthma in children as well as its influence on the number of natural killer T (NKT) cells and their cytokine production. Materials and Methods: Sixty children diagnosed with asthma were divided into either a BV-treated group (with oral OM-85 BV) or a conventional inhaled corticosteroid (ICS) group. The numbers of NKT cells and CD4+ NKT cells were measured in the peripheral blood by flow cytometry. The levels of IFN-γ, IL-4, and IL-10 after the blood cells had been cultured with an NKT cell agonist were detected by ELISA. Results: After therapy, asthma attacks were significantly decreased compared with before therapy in both groups. However, after therapy, respiratory tract infections were reduced compared with before therapy in the BV-treated group only. Additionally, the frequency of asthma attacks and use of antibiotics in the BV-treated group were lower than in the ICS group. With BV treatment, the numbers of peripheral blood NKT cells and CD4+ NKT cells were higher after therapy than before therapy. After therapy, the ratio of IFN-γ/IL-4 and IL-10 levels were increased in the BV-treated group, whereas IL-4 was reduced in the BV-treated group compared with the ICS group. Conclusion: BV combined with conventional asthma treatment can prevent recurrent respiratory tract infections and suppress the severity of asthma attacks, possibly by altering the rates and cytokines of NKT cells.

Using modified whole-mount in situ hybridization to study mpo expression in zebrafish

In this study, we cloned the myeloperoxidase (mpo) gene of zebrafish and prepared a digoxigenin-labeled mpo RNA probe to investigate mpo gene expression in zebrafish during embryonic development by whole-mount in situ hybridization (WISH). The earliest mpo expression was detected in cells of the intermediate cell mass (ICM) at 18 h post-fertilization (hpf). It was detected 1 to 2 h later in cells in the rostral blood island (RBI) and strong signals were observed in the anterior ICM. Then, it spread over the yolk sac. By 72 hpf these mpo-expressing cells were in the circulation and distributed throughout the embryo. We identified that the level of mpo expression detected by WISH at an early stage was consistent with the data of cytological analyses of adult fish. The use of this method enabled us to track the gene changes that took place before morphological phenotypes were detected, as well to as investigate the hematopoietic cell fate in mutational or transgenic models in vivo. In this study, we modified several steps of WISH. The improved hybridization results demonstrated high specificity, distinct coloration and low background figures.

RANTES gene polymorphisms and risk of pediatric asthma: A meta-analysis

Numerous studies have evaluated the association between regulated upon activation, normal T cells expressed and secreted (RANTES) gene polymorphisms (−403G/A and −28C/G) and risk of pediatric asthma. However, the results have been inconsistent. A meta-analysis of the association between RANTES gene polymorphisms and pediatric asthma risk was performed in the current study. A search for published literature was conducted in the Google Scholar, PubMed and the CNKI databases (January 2000 to April 2012) and seven studies were retrieved. The associations between RANTES gene polymorphisms and pediatric asthma risk were estimated by pooled odds ratio (OR) and 95% confidence interval (CI) using a fixed- or random-effects model. Meta-analysis results revealed no significant association between the −403G/A polymorphism and risk of pediatric asthma. In the subgroup analysis by ethnicity, no association was identified between the −403G/A polymorphism and pediatric asthma risk in Caucasian and Asian populations. In the −28C/G group, the meta-analysis indicated a significant association between the −28C/G polymorphism and pediatric asthma susceptibility among the total population (recessive model: OR, 1.34; 95% CI, 1.04–1.72). However, when the subgroup analysis was performed by ethnicity, no significant associations were identified in Asians and Europeans. This result suggests that the −28C/G polymorphism may not be associated with pediatric asthma risk, while the observed increase in the risk of pediatric asthma may be due to racial differences. Additional large-scale studies are required to provide conclusive evidence on the effects of RANTES gene polymorphisms on the risk of pediatric asthma.

Foxp3 regulates ratio of Treg and NKT cells in a mouse model of asthma.

Chronic inflammatory disorder of the airways causes asthma. Regulatory T cells (Treg cells) and Natural killer T cells (NKT cells) both play critical roles in the pathogenesis of asthma. Activation of Treg cells requires Foxp3, whereas whether Foxp3 may regulate the ratio of Treg and NKT cells to affect asthma is uncertain. In an ovalbumin (OVA)-induced mouse model of asthma, we either increased Treg cells by lentivirus-mediated forced expression of exogenous Foxp3, or increased NKT cells by stimulation with its activator α-GalCer. We found that the CD4+CD25+ Treg cells increased by forced Foxp3 expression, and decreased by α-GalCer, while the CD3+CD161+ NKT cells decreased by forced Foxp3 expression, and increased by α-GalCer. Moreover, forced Foxp3 expression, but not α-GalCer, significantly alleviated the hallmarks of asthma. Furthermore, forced Foxp3 increased levels of IL_10 and TGFβ1, and α-GalCer increased levels of IL_4 and INFγ in the OVA-treated lung. Taken together, our study suggests that Foxp3 may activate Treg cells and suppress NKT cells in asthma. Treg and NKT cells may antagonize the effects of each other in asthma.

Fever as an Initial Manifestation of Enthesitis-Related Arthritis Subtype of Juvenile Idiopathic Arthritis: Retrospective Study

Objective We wished to determine the prevalence of fever as one of the first symptoms of the enthesitis-related arthritis (ERA) subtype of juvenile idiopathic arthritis. Also, we wished to ascertain if ERA patients with fever at disease onset differed from those without fever. Methods Consecutive cases of ERA were diagnosed and followed in a retrospective observational study from 1998 to 2013. Information about clinical/laboratory data, medications, magnetic resonance imaging (MRI), and disease activity during the study period was also recorded. Results A total of 146 consecutive ERA patients were assessed. Among them, 52 patients (35.6%) had fever as one of the first symptoms at disease onset. Compared with ERA patients without fever at disease onset, patients with fever had significantly more painful joints (3.5 vs. 2.8), more swollen joints (1.1 vs. 0.8), and more enthesitis (1.0 vs. 0.4) (p<0.05 for all comparisons). Patients with fever had significantly higher mean values of erythrocyte sedimentation rate, C-reactive protein, platelet count, and child health assessment questionnaire (CHAQ) scores (40.8 vs. 26.4 mm/h; 20.7 vs. 9.7 mg/dL; 353.2×109/L vs. 275.6×109/L; 1.0 vs. 0.8, respectively; all p<0.05). During two-year follow-up, CHAQ score, number of flares, as well as the number of patients treated with oral non-steroidal anti-inflammatory drugs, corticosteroids and combination therapy with disease-modifying anti-rheumatic drugs, were significantly higher in ERA patients with fever. Conclusions Fever was a frequent manifestation of ERA. ERA patients with fever had more active disease at disease onset and poorer outcomes than ERA patients without fever.

Changes of Treg and Th17 cells as well as cytokines in children with acute bronchitis

The present study aimed to investigate changes of T-regulatory (Treg) and T-helper (Th)17 cells as well as cytokines in peripheral blood of children with acute bronchitis, and to explore the roles of these cells in the pathogenesis of acute bronchitis. A total of 126 children who had presented at Renji Hospital (Shanghai, China) with acute bronchitis were selected as the observation group and 30 healthy children were selected as the control group. Th17/Tregs in the peripheral blood of the children of the observation group and the control group was detected by flow cytometry. The levels of cytokines interleukin (IL)-17, IL-22, IL-10 and transforming growth factor (TGF)-β in peripheral blood serum were detected by ELISA. Compared with those in the control group, Treg cells, the Treg/Th17 ratio as well as serum IL-10 and TGF-β levels were significantly decreased in the observation group (P<0.05), while Th17 cells as well as serum levels of IL-17 and IL-22 were significantly increased (P<0.05). In conclusion, Treg/Th17 and the expression of associated cytokines lost their balance in children with acute bronchitis, suggesting that Treg and Th17 cells as well as their cytokines may be involved in the pathogenesis of acute bronchitis. It may be of certain guiding significance to detect Treg/Th17 and levels of serum cytokines in peripheral blood for clinical treatment.

Identification of potential peripheral blood diagnostic biomarkers for patients with juvenile idiopathic arthritis by bioinformatics analysis

Juvenile idiopathic arthritis (JIA) is common childhood rheumatic disease harming children health. However, there is still lack of effective biomarkers for diagnosis JIA at early onset. We aim to construct a classification model to predict JIA disease. The peripheral blood gene expression profile data of JIA were downloaded from GEO database. We compared and analyzed differentially expressed genes (DEGs) between different JIA samples through Pearson’s correlation coefficient method and unsupervised clustering analysis. Diagnostic model were constructed based on the deviation pathway through bioinformatics method. Eighteen specific correlated DEGs were obtained, but the correlations altered in different disease states. Although most JIA and control samples were clustered by unsupervised clustering analysis, respectively, a few JIA samples could not be clustered well. Four co-expression networks were next constructed with gene connections dynamically altered under variable conditions. Eight signaling pathways were significantly enriched including B/T cell receptor, ErbB and MAPK signaling pathways. The deviation scores of pathways were calculated. Applying these eight signaling pathways as feature to construct a classification model could predict JIA disease with high accuracies. Our data provide some light into pathogenic mechanism of JIA, the specific gene sets and the related signaling pathways may be potential biomarkers for diagnosis or therapeutic targets of JIA.

Atopy in children with juvenile systemic lupus erythematosus is associated with severe disease

The influence of co-existing atopy on the prognosis of juvenile systemic lupus erythematosus (JSLE) was assessed in this study. Patients diagnosed with JSLE between October 2005 and April 2016 were enrolled in a prospective study and followed up for 2 years. Management of patients was evaluated using the systemic lupus erythematosus disease activity index 2000 (SLEDAI-2K) score and laboratory variables. Eighty JSLE patients were enrolled at diagnosis and divided into those with (n = 35) and without (n = 45) atopy. When compared with the non-atopic group, atopic patients showed higher SLEDAI-2K score at disease onset (16.09 vs. 11.18), higher erythrocyte sedimentation rate (52.89 vs. 38.27 mm/h), higher percentage of total B-cells (25.85 vs. 19.51%), lower percentage (7.26 vs. 9.03%) and cytotoxicity (9.92 vs. 11.32%) of natural killer cells, and lower complement C3 (0.51 vs. 0.69 g/L) (all p<0.05). At 1, 3, 6, 12, 18, and 24 months, JSLE patients with atopy reached higher SLEDAI-2K score and lower ΔSLEDAI-2K improvement rate (at 1 month, 8.34 vs. 4.71 and 43.63 vs. 57.95%, respectively; at 3 months, 8.57 vs. 2.62 and 48.39 vs. 75.10%, respectively; at 6 months, 6.91 vs. 2.38 and 53.59 vs. 77.26%, respectively; at 12 months, 4.71 vs. 1.80 and 69.54 vs. 84.10%, respectively; at 18 months, 4.66 vs. 2.02 and 68.14 vs. 82.93%, respectively; at 24 months, 8.57 vs. 2.62 and 70.00 vs. 81.88%, respectively; all p<0.05). During the 24 months of follow-up, the total number of disease flares was higher in JSLE patients with co-existing atopy (3.77 vs. 1.51, p<0.05), and the atopic group needed much more time to reach the stable condition of the disease (6.88 vs. 4.65 months, p<0.05). JSLE patients combined with co-existing atopy had more severe disease at diagnosis and poorer outcomes than JSLE patients without atopy.