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1.  Phenotypic and functional alterations of pDCs in lupus-prone mice 
Scientific Reports  2016;6:20373.
Plasmacytoid dendritic cells (pDCs) were considered to be the major IFNα source in systemic lupus erythematosus (SLE) but their phenotype and function in different disease status have not been well studied. To study the function and phenotype of pDCs in lupus-prone mice we used 7 strains of lupus-prone mice including NZB/W F1, NZB, NZW, NZM2410, B6.NZMSle1/2/3, MRL/lpr and BXSB/Mp mice and C57BL/6 as control mice. Increased spleen pDC numbers were found in most lupus mice compared to C57BL/6 mice. The IFNα-producing ability of BM pDCs was similar between lupus and C57BL/6 mice, whereas pDCs from the spleens of NZB/W F1 and NZB mice produced more IFNα than pDCs from the spleens of C57BL/6 mice. Furthermore, spleen pDCs from MRL-lpr and NZM2410 mice showed increased responses to Tlr7 and Tlr9, respectively. As the disease progressed, IFN signature were evaluated in both BM and spleen pDC from lupus prone mice and the number of BM pDCs and their ability to produce IFNα gradually decreased in lupus-prone mice. In conclusion, pDC are activated alone with disease development and its phenotype and function differ among lupus-prone strains, and these differences may contribute to the development of lupus in these mice.
doi:10.1038/srep20373
PMCID: PMC4754657  PMID: 26879679
2.  Distribution of ischemic infarction and stenosis of intra- and extracranial arteries in young Chinese patients with ischemic stroke 
Background
The distribution of cerebral ischemic infarction and stenosis in ischemic stroke may vary with age-group, race and gender. This study was conducted to understand the risk factors and characteristics of cerebral infarction and stenosis of vessels in young Chinese patients with ischemic stroke.
Methods
This was a retrospective study, from January 2007 to July 2012, of 123 patients ≤50 years diagnosed with acute ischemic stroke. Patient characteristics were compared according to sex (98 males and 25 females) and age group (51 patients were ≤45 years and 72 patients were 46–50 years). Characteristics of acute ischemic infarction were studied by diffusion weighted imaging. Stenosis of intra- and extracranial arteries was diagnosed by duplex sonography, head magnetic resonance angiography (MRA) or cervical MRA.
Results
Common risk factors were hypertension (72.4 %), dyslipidemia (55.3 %), smoking (54.4 %) and diabetes (33.3 %). Lacunar Infarction was most common in our patients (41.5 %). Partial anterior circulation infarction was predominant in females (52.0 vs 32.7 %; P = 0.073) and posterior circulation infarction in males (19.8 vs 4 %; P = 0.073). Multiple brain infarctions were found in 38 patients (30.9 %). Small artery atherosclerosis was found in 54 patients (43.9 %), with higher prevalence in patients of the 46–50 years age-group. Intracranial stenosis was more common than extracranial stenosis, and middle cerebral artery stenosis was most prevalent (27.3 %). Stenosis in the anterior circulation was more frequent than in the posterior circulation (P < 0.001).
Conclusions
In these young patients, hypertension, smoking, dyslipidemia and diabetes were common risk factors. Intracranial stenosis was most common. The middle cerebral artery was highly vulnerable.
doi:10.1186/s12872-015-0147-5
PMCID: PMC4657340  PMID: 26597918
Young; Stroke; Chinese; Risk factors; Carotid stenosis; Multiple infractions
3.  Berberine may rescue Fusobacterium nucleatum-induced colorectal tumorigenesis by modulating the tumor microenvironment 
Oncotarget  2015;6(31):32013-32026.
Background
Accumulating evidence links colorectal cancer (CRC) with the intestinal microbiota. However, the disturbance of intestinal microbiota and the role of Fusobacterium nucleatum during the colorectal adenoma-carcinoma sequence have not yet been evaluated.
Methods
454 FLX pyrosequencing was used to evaluate the disturbance of intestinal microbiota during the adenoma-carcinoma sequence pathway of CRC. Intestinal microbiota and mucosa tumor-immune cytokines were detected in mice after introducing 1,2-dimethylhydrazine (DMH), F. nucleatum or Berberine (BBR), using pyrosequencing and Bio-Plex Pro™ cytokine assays, respectively. Protein expressions were detected by western blotting.
Results
The levels of opportunistic pathogens, such as Fusobacterium, Streptococcus and Enterococcus spp. gradually increased during the colorectal adenoma-carcinoma sequence in human fecal and mucosal samples. F. nucleatum treatment significantly altered lumen microbial structures, with increased Tenericutes and Verrucomicrobia (opportunistic pathogens) (P < 0.05 = in wild-type C57BL/6 and mice with DMH treatment). BBR intervention reversed the F. nucleatum-mediated increase in opportunistic pathogens, and the secretion of IL-21/22/31, CD40L and the expression of p-STAT3, p-STAT5 and p-ERK1/2 in mice, compared with mice fed with F. nucleatum alone.
Conclusions
F. nucleatum colonization in the intestine may prompt colorectal tumorigenesis. BBR could rescue F. nucleatum-induced colorectal tumorigenesis by modulating the tumor microenvironment and blocking the activation of tumorigenesis-related pathways.
PMCID: PMC4741656  PMID: 26397137
colorectal tumorigenesis; intestinal microbiota; fusobacterium nucleatum; berberine; tumor-immune cytokine
4.  MiR-125a-5p Decreases the Sensitivity of Treg cells Toward IL-6-Mediated Conversion by Inhibiting IL-6R and STAT3 Expression 
Scientific Reports  2015;5:14615.
The transcription factor FOXP3 is essential for the differentiation and function of regulatory T cells (Treg). It is established that the transcription factor GATA-3 is induced in Treg cells under inflammatory conditions. GATA-3 stabilizes FOXP3 levels to avoid the differentiation of Treg cells into inflammatory-like T cells. The IL-6 signal pathway influences the sensitivity of Treg cells towards instability. The mechanism of GATA-3 in regulating FOXP3 and its relation to the IL-6 pathway remains unclear. Here we report how miR-125a-5p plays an important role in regulating the conversion of Treg cells by IL-6. miR-125a-5p expression is low in Treg cells under steady state conditions and can be induced by GATA-3 to inhibit the expression of IL-6R and STAT3. This finding reveals a GATA3/miR-125a-5p/IL-6R and STAT3/FOXP3 regulatory pathway, which determines how Treg cells respond to inflammatory IL-6-rich conditions.
doi:10.1038/srep14615
PMCID: PMC4589732  PMID: 26424054
5.  Identification of MTMR3 as a novel susceptibility gene for lupus nephritis in northern Han Chinese by shared-gene analysis with IgA nephropathy 
Objectives
Recent genome-wide association studies have identified several novel susceptibility genes for systemic lupus erythematosus (SLE) and IgA nephropathy (IgAN). Since both lupus nephritis (LN) and IgAN are autoimmune kidney diseases, they may share common disease mechanisms that overlap with genetic susceptibility. To test this hypothesis, we sought to identify genetic variants associated with IgAN in LN.
Methods
In the first stage, 500 LN patients, 240 SLE patients without LN, and 500 healthy controls were enrolled. Fifteen reported SNPs with top association signals with IgAN were selected for further testing in LN. Three independent cohorts from Beijing, Shanghai and Hong Kong were included as replicates. We also analyzed the functional significance of identified non-coding variants on regulatory motifs and gene expression.
Results
Except for associations with HLA gene polymorphisms, genetic variants of MTMR3 in 22q12 showed associations with LN (rs9983A with P = 2.07×10−3; OR 1.61; 95% CI 1.19–2.19) compared to healthy controls in the first stage. In replications, associations were replicated and reinforced with northern (LN vs. non-LN patients, P = 0.01) but not southern Han Chinese, although significant genetic heterogeneity was observed. In silico analyses predicted conservative and regulatory features of rs9983. In expression analysis, we observed lower MTMR3 transcription levels in blood samples with rs9983A and renal biopsies from LN and IgAN.
Conclusions
Our results suggested that the MTMR3 gene was shared between IgAN and LN in the northern Chinese, further highlighting the role of autophagy in SLE. However, widespread replication of these experiments, fine mapping, and functional assays are required to establish this connection.
doi:10.1002/art.38749
PMCID: PMC4180767  PMID: 24943867
IgA nephropathy; lupus nephritis; MTMR3; SLE
6.  miR-744 enhances type I interferon signaling pathway by targeting PTP1B in primary human renal mesangial cells 
Scientific Reports  2015;5:12987.
Renal mesangial cells (RMCs) constitute a population of cells in glomerular mesangium. Inflammatory cytokines produced by RMCs play a vital role in renal inflammation. miRNAs are key regulators of inflammatory cytokine expression. The abnormal expression of renal miRNAs and the consequent changes in inflammatory signal transduction are closely associated with renal inflammation. However, our knowledge of the functions of renal miRNAs is still limited. In this study, we investigated the role of miR-744 in type I interferon (IFN) signaling pathway in primary human RMCs. We show that overexpression of miR-744 enhances IFN-induced CCL2, CCL5, CXCL10, and IL6 expression specifically in RMCs. We found that the activation of TYK2, STAT1 and STAT3 was significantly enhanced by miR-744. miR-744 also enhanced the activation of non-classical signal components, such as ERK and p38. We then identified PTP1B, a ubiquitously expressed phosphatase, as the target of miR-744 that is responsible for enhancing type I IFN response. Finally, miR-744 expression was induced by type I IFN in RMCs. Collectively, our data indicate that by targeting PTP1B, miR-744 plays a feed-forward role in regulating type I IFN signaling pathway. These findings give us new insights into the functions of renal miRNAs in regulating important signaling pathways.
doi:10.1038/srep12987
PMCID: PMC4531339  PMID: 26259828
7.  The microbiome of uncontacted Amerindians 
Science advances  2015;1(3):e1500183.
Most studies of the human microbiome have focused on westernized people with life-style practices that decrease microbial survival and transmission, or on traditional societies that are currently in transition to westernization. We characterize the fecal, oral, and skin bacterial microbiome and resistome of members of an isolated Yanomami Amerindian village with no documented previous contact with Western people. These Yanomami harbor a microbiome with the highest diversity of bacteria and genetic functions ever reported in a human group. Despite their isolation, presumably for >11,000 years since their ancestors arrived in South America, and no known exposure to antibiotics, they harbor bacteria that carry functional antibiotic resistance (AR) genes, including those that confer resistance to synthetic antibiotics and are syntenic with mobilization elements. These results suggest that westernization significantly affects human microbiome diversity and that functional AR genes appear to be a feature of the human microbiome even in the absence of exposure to commercial antibiotics. AR genes are likely poised for mobilization and enrichment upon exposure to pharmacological levels of antibiotics. Our findings emphasize the need for extensive characterization of the function of the microbiome and resistome in remote nonwesternized populations before globalization of modern practices affects potentially beneficial bacteria harbored in the human body.
doi:10.1126/sciadv.1500183
PMCID: PMC4517851  PMID: 26229982
8.  NF-κB-induced microRNA-31 promotes epidermal hyperplasia by repressing protein phosphatase 6 in psoriasis 
Nature Communications  2015;6:7652.
NF-κB is constitutively activated in psoriatic epidermis. However, how activated NF-κB promotes keratinocyte hyperproliferation in psoriasis is largely unknown. Here we report that the NF-κB activation triggered by inflammatory cytokines induces the transcription of microRNA (miRNA) miR-31, one of the most dynamic miRNAs identified in the skin of psoriatic patients and mouse models. The genetic deficiency of miR-31 in keratinocytes inhibits their hyperproliferation, decreases acanthosis and reduces the disease severity in psoriasis mouse models. Furthermore, protein phosphatase 6 (ppp6c), a negative regulator that restricts the G1 to S phase progression, is diminished in human psoriatic epidermis and is directly targeted by miR-31. The inhibition of ppp6c is functionally important for miR-31-mediated biological effects. Moreover, NF-κB activation inhibits ppp6c expression directly through the induction of miR-31, and enhances keratinocyte proliferation. Thus, our data identify NF-κB-induced miR-31 and its target, ppp6c, as critical factors for the hyperproliferation of epidermis in psoriasis.
Psoriasis is accompanied by NF-κB activation and hyperplasia. Here the authors show that NF-κB transcriptionally activates miR-31, which downregulates a negative cell cycle regulator protein phosphatase 6, and that this is critical for NF-κB to drive keratinocyte hyperproliferation.
doi:10.1038/ncomms8652
PMCID: PMC4506511  PMID: 26138368
9.  Single molecule-level detection and long read-based phasing of epigenetic variations in bacterial methylomes 
Nature Communications  2015;6:7438.
Beyond its role in host defense, bacterial DNA methylation also plays important roles in the regulation of gene expression, virulence and antibiotic resistance. Bacterial cells in a clonal population can generate epigenetic heterogeneity to increase population-level phenotypic plasticity. Single molecule, real-time (SMRT) sequencing enables the detection of N6-methyladenine and N4-methylcytosine, two major types of DNA modifications comprising the bacterial methylome. However, existing SMRT sequencing-based methods for studying bacterial methylomes rely on a population-level consensus that lacks the single-cell resolution required to observe epigenetic heterogeneity. Here, we present SMALR (single-molecule modification analysis of long reads), a novel framework for single molecule-level detection and phasing of DNA methylation. Using seven bacterial strains, we show that SMALR yields significantly improved resolution and reveals distinct types of epigenetic heterogeneity. SMALR is a powerful new tool that enables de novo detection of epigenetic heterogeneity and empowers investigation of its functions in bacterial populations.
Bacterial DNA methylation is involved in many processes, from host defense to antibiotic resistance, however current methods for examining methylated genomes lack single-cell resolution. Here Beaulaurier et al. present Single Molecule Modification Analysis of Long Reads, a new tool for de novo detection of epigenetic heterogeneity.
doi:10.1038/ncomms8438
PMCID: PMC4490391  PMID: 26074426
10.  Association of large intergenic noncoding RNA expression with disease activity and organ damage in systemic lupus erythematosus 
Introduction
Despite growing evidence that large intergenic noncoding RNAs (lincRNAs) can regulate gene expression and widely take part in normal physiological and disease conditions, our knowledge of systemic lupus erythematosus (SLE)-related lincRNAs remains limited. The aim of this study was to detect the levels of four lincRNAs (ENST00000500949: linc0949, ENST00000500597: linc0597, ENST00000501992: linc1992, and ENST00000523995: linc3995) involved in innate immunity in the peripheral blood mononuclear cells (PBMCs) of patients with SLE and correlate these lincRNA levels with disease activity, organ damage, clinical features and medical therapies.
Methods
PBMCs were obtained from 102 patients with SLE, 54 patients with rheumatoid arthritis (RA) and 76 healthy donors. lincRNA expression levels were measured by real-time quantitative polymerase chain reaction. Disease activity was assessed using the Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) scores, and organ damage was evaluated with the Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index.
Results
linc0949 and linc0597 were significantly decreased in patients with SLE compared with patients with RA and healthy control subjects. linc0949 was correlated with SLEDAI-2K score (r = −0.329, P = 0.0007), as well as with complement component C3 level (r = 0.348, P = 0.0003). The level of linc0949 was also reduced in patients with SLE who had the presence of cumulative organ damage. In addition, decreasing expression of linc0949 was associated with lupus nephritis. linc0949 expression significantly increased after treatment, whereas neither disease activity nor organ damage correlated with linc0597 expression.
Conclusions
Our results provide novel empirical evidence that linc0949 could be a potential biomarker for diagnosis, disease activity and therapeutic response in SLE.
Electronic supplementary material
The online version of this article (doi:10.1186/s13075-015-0632-3) contains supplementary material, which is available to authorized users.
doi:10.1186/s13075-015-0632-3
PMCID: PMC4440330  PMID: 25994030
11.  Equine Viperin Restricts Equine Infectious Anemia Virus Replication by Inhibiting the Production and/or Release of Viral Gag, Env, and Receptor via Distortion of the Endoplasmic Reticulum 
Journal of Virology  2014;88(21):12296-12310.
ABSTRACT
Viperin is an endoplasmic reticulum (ER)-associated multifunctional protein that regulates virus replication and possesses broad antiviral activity. In many cases, viperin interferes with the trafficking and budding of viral structural proteins by distorting the membrane transportation system. The lentivirus equine infectious anemia virus (EIAV) has been studied extensively. In this study, we examined the restrictive effect of equine viperin (eViperin) on EIAV replication and investigated the possible molecular basis of this restriction to obtain insights into the effect of this cellular factor on retroviruses. We demonstrated that EIAV infection of primary equine monocyte-derived macrophages (eMDMs) upregulated the expression of eViperin. The overexpression of eViperin significantly inhibited the replication of EIAV in eMDMs, and knockdown of eViperin transcription enhanced the replication of EIAV in eMDMs by approximately 45.8%. Further experiments indicated that eViperin restricts EIAV at multiple steps of viral replication. The overexpression of eViperin inhibited EIAV Gag release. Both the α-helix domain and radical S-adenosylmethionine (SAM) domain were required for this activity. However, the essential motifs in SAM were different from those reported for the inhibition of HIV-1 Gag by human viperin. Furthermore, eViperin disrupted the synthesis of both EIAV Env and receptor, which consequently inhibited viral production and entry, respectively, and this disruption was dependent on the eViperin α-helix domain. Using immunofluorescence assays and electron microscopy, we demonstrated that the α-helix domain is responsible for the distortion of the endoplasmic reticulum (ER). Finally, EIAV did not exhibit counteracting eViperin at the protein level.
IMPORTANCE In previous studies, viperin was indicated as restricting virus replications primarily by the inhibition of virus budding. Here, we show that viperin may have multiple antiviral mechanisms, including the reduction of EIAV Gag budding and Env expression, and these activities are dependent on different viperin domains. We especially demonstrate that the overexpression of viperin inhibits EIAV entry by decreasing the level of virus receptor. Therefore, viperin restriction of viruses is determined largely by the dependence of virus on the cellular membrane transportation system.
doi:10.1128/JVI.01379-14
PMCID: PMC4248950  PMID: 25122784
12.  miRNAs in the Pathogenesis of Systemic Lupus Erythematosus 
MicroRNAs (miRNAs) were first discovered as regulatory RNAs that controlled the timing of the larval development of Caenorhabditis elegans. Since then, nearly 30,000 mature miRNA products have been found in many species, including plants, warms, flies and mammals. Currently, miRNAs are well established as endogenous small (~22 nt) noncoding RNAs, which have functions in regulating mRNA stability and translation. Owing to intensive investigations during the last decade, miRNAs were found to play essential roles in regulating many physiological and pathological processes. Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by elevated autoantibodies against nuclear antigens and excessive inflammatory responses affecting multiple organs. Although efforts were taken and theories were produced to elucidate the pathogenesis of SLE, we still lack sufficient knowledge about the disease for developing effective therapies for lupus patients. Recent advances indicate that miRNAs are involved in the development of SLE, which gives us new insights into the pathogenesis of SLE and might lead to the finding of new therapeutic targets. Here, we will review recent discoveries about how miRNAs are involved in the pathogenesis of SLE and how it can promote the development of new therapy.
doi:10.3390/ijms16059557
PMCID: PMC4463604  PMID: 25927578
microRNA; systemic lupus erythematosus; innate immunity; adaptive immunity; lupus nephritis; biomarker; therapy
13.  The microbiome of uncontacted Amerindians 
Science Advances  2015;1(3):e1500183.
Fecal, oral, and skin biomes of isolated Amerindians show higher human bacterial diversity including antibiotic resistance genes.
Most studies of the human microbiome have focused on westernized people with life-style practices that decrease microbial survival and transmission, or on traditional societies that are currently in transition to westernization. We characterize the fecal, oral, and skin bacterial microbiome and resistome of members of an isolated Yanomami Amerindian village with no documented previous contact with Western people. These Yanomami harbor a microbiome with the highest diversity of bacteria and genetic functions ever reported in a human group. Despite their isolation, presumably for >11,000 years since their ancestors arrived in South America, and no known exposure to antibiotics, they harbor bacteria that carry functional antibiotic resistance (AR) genes, including those that confer resistance to synthetic antibiotics and are syntenic with mobilization elements. These results suggest that westernization significantly affects human microbiome diversity and that functional AR genes appear to be a feature of the human microbiome even in the absence of exposure to commercial antibiotics. AR genes are likely poised for mobilization and enrichment upon exposure to pharmacological levels of antibiotics. Our findings emphasize the need for extensive characterization of the function of the microbiome and resistome in remote nonwesternized populations before globalization of modern practices affects potentially beneficial bacteria harbored in the human body.
doi:10.1126/sciadv.1500183
PMCID: PMC4517851  PMID: 26229982
Antibiotic; Amerindian; Microbiome; Resistome; Westernization
14.  Effect of dyslipidemia on intima-media thickness of intra- and extracranial atherosclerosis by regulating the expression of hsp70 in rabbits 
The aim of this study was to explore the effect of dyslipidemia on intima-media thickness (IMT) of Intra- and extracranial atherosclerosis by regulating the expression of heat shock protein 70 (HSP70) in rabbits. Twenty-seven male white rabbits were randomly divided into normal control group A, high fat group B and high fat + endothelial injury operation group C (each group was 9), we measured lipids and obtained tissues from different cerebral arteries including Bilateral common carotid artery (CCA), Internal carotid artery (ICA), middle cerebral artery (MCA) and vertebral artery (VA). Pathological analysis were done, western blot analysis was used to detect the expression of HSP70 in CCA and MCA. The Serum lipid levels were overall significantly increased at 12th week in Group B and Group C compared to normal control (P < 0.05); at 12th week, the IMT of CCA and MCA in group B and C were showed significant increment compared with Group A; the correlation between HDL/CHOL/LDL and IMT of different cerebral arteries are as follows: MCA > ICA > CCA > VA; between TG and IMT of different cerebral arteries: VA > ICA > MCA > CCA; the expression of HSP70 from MCA were increased compared with CCA in group B and group C (P < 0.05). Significant positive correlations were observed between hyperlipidemia and different cerebral arteries. Hyperlipidemia has more impact on IMT of intracranial cerebral arteries. The expression of HSP70 from intracranial cerebral arteries is significantly increased. The mechanisms underlied was speculated that might be involved in inhibiting the inflammatory via HSP70.
PMCID: PMC4484004  PMID: 26131122
High fat; HSP70; dyslipidemia; cerebral atherosclerosis; intima
15.  Peste des Petits Ruminants Virus in Heilongjiang Province, China, 2014 
Emerging Infectious Diseases  2015;21(4):677-680.
During March 25–May 5, 2014, we investigated 11 outbreaks of peste des petits ruminants in Heilongjiang Province, China. We found that the most likely source of the outbreaks was animals from livestock markets in Shandong. Peste des petits ruminants viruses belonging to lineages II and IV were detected in sick animals.
doi:10.3201/eid2104.141627
PMCID: PMC4378501  PMID: 25811935
peste des petits ruminants; PPR; peste des petits ruminants virus; PPRV; viruses; sheep; goats; ruminants; Heilongjiang Province; China
16.  Lupus risk variants in the PXK locus alter B-cell receptor internalization 
Frontiers in Genetics  2015;5:450.
Genome wide association studies have identified variants in PXK that confer risk for humoral autoimmune diseases, including systemic lupus erythematosus (SLE or lupus), rheumatoid arthritis and more recently systemic sclerosis. While PXK is involved in trafficking of epidermal growth factor Receptor (EGFR) in COS-7 cells, mechanisms linking PXK to lupus pathophysiology have remained undefined. In an effort to uncover the mechanism at this locus that increases lupus-risk, we undertook a fine-mapping analysis in a large multi-ancestral study of lupus patients and controls. We define a large (257kb) common haplotype marking a single causal variant that confers lupus risk detected only in European ancestral populations and spans the promoter through the 3′ UTR of PXK. The strongest association was found at rs6445972 with P < 4.62 × 10−10, OR 0.81 (0.75–0.86). Using stepwise logistic regression analysis, we demonstrate that one signal drives the genetic association in the region. Bayesian analysis confirms our results, identifying a 95% credible set consisting of 172 variants spanning 202 kb. Functionally, we found that PXK operates on the B-cell antigen receptor (BCR); we confirmed that PXK influenced the rate of BCR internalization. Furthermore, we demonstrate that individuals carrying the risk haplotype exhibited a decreased rate of BCR internalization, a process known to impact B cell survival and cell fate. Taken together, these data define a new candidate mechanism for the genetic association of variants around PXK with lupus risk and highlight the regulation of intracellular trafficking as a genetically regulated pathway mediating human autoimmunity.
doi:10.3389/fgene.2014.00450
PMCID: PMC4288052  PMID: 25620976
lupus; PXK; fine-mapping; B cells; BCR
17.  Predominant Role of Plasmacytoid Dendritic Cells in Stimulating Systemic Autoimmunity 
Plasmacytoid dendritic cells (pDCs), which are prominent type I interferon (IFN-I)-producing immune cells, have been extensively implicated in systemic lupus erythematosus (SLE). However, whether they participate critically in lupus pathogenesis remains unknown. Recent studies using various genetic and cell type-specific ablation strategies have demonstrated that pDCs play a pivotal role in the development of autoantibodies and the progression of lupus under diverse experimental conditions. The findings of several investigations highlight a notion that pDCs operate critically at the early stage of lupus development. In particular, pDCs have a profound effect on B-cell activation and humoral autoimmunity in vivo. This deeper understanding of the vital role of pDCs in lupus pathogenesis supports the therapeutic targeting of the pDC-IFN-I pathway in SLE.
doi:10.3389/fimmu.2015.00526
PMCID: PMC4601279  PMID: 26528288
plasmacytoid dendritic cells; systemic lupus erythematosus; type I interferon; amyloid; autoantibody; autoimmunity; animal models; B cells
18.  A Recombinant Avian Leukosis Virus Subgroup J for Directly Monitoring Viral Infection and the Selection of Neutralizing Antibodies 
PLoS ONE  2014;9(12):e115422.
Avian leukosis virus subgroup J (ALV-J) has induced serious clinical outbreaks and has become a serious infectious disease of chickens in China. We describe here the creation of a recombinant ALV-J tagged with the enhanced green fluorescent protein (named rHPRS-103EGFP). We successfully utilize the rHPRS-103EGFP to visualize viral infection and for development of a simplified serum-neutralization test.
doi:10.1371/journal.pone.0115422
PMCID: PMC4270768  PMID: 25522008
19.  Inhibition of p53 deSUMOylation Exacerbates Puromycin Aminonucleoside-Induced Apoptosis in Podocytes 
Apoptosis is a major cause of reduced podocyte numbers, which leads to proteinuria and/or glomerulosclerosis. Emerging evidence has indicated that deSUMOylation, a dynamic post-translational modification that reverses SUMOylation, is involved in the apoptosis of Burkitt’s lymphoma cells and cardiomyocytes; however, the impact of deSUMOylation on podocyte apoptosis remains unexplored. The p53 protein plays a major role in the pathogenesis of podocyte apoptosis, and p53 can be SUMOylated. Therefore, in the present study, we evaluated the effect of p53 deSUMOylation, which is regulated by sentrin/SUMO-specific protease 1 (SENP1), on podocyte apoptosis. Our results showed that SENP1 deficiency significantly increases puromycin aminonucleoside (PAN)-induced podocyte apoptosis. Moreover, SENP1 knockdown results in the accumulation of SUMOylated p53 protein and the increased expression of the p53 target pro-apoptotic genes, BAX, Noxa and PUMA, in podocytes during PAN stimulation. Thus, SENP1 may be essential for preventing podocyte apoptosis, at least partly through regulating the functions of p53 protein via deSUMOylation. The regulation of deSUMOylation may provide a novel strategy for the treatment of glomerular disorders that involve podocyte apoptosis.
doi:10.3390/ijms151121314
PMCID: PMC4264227  PMID: 25411797
apoptosis; deSUMOylation; p53; podocyte; sentrin/SUMO-specific protease 1
21.  Generation and characterization of a new mammalian cell line continuously expressing virus-like particles of Japanese encephalitis virus for a subunit vaccine candidate 
BMC Biotechnology  2014;14:62.
Background
Japanese encephalitis virus (JEV) is the most important cause of epidemic encephalitis in most Asian regions. There is no specific treatment available for Japanese encephalitis, and vaccination is the only effective way to prevent JEV infection in humans and domestic animals. The purpose of this study is to establish a new mammalian cell line stably and efficiently expressing virus-like particle of JEV for potential use of JEV subunit vaccine.
Results
We generated a new cell clone (BJ-ME cells) that stably produces a secreted form of Japanese encephalitis virus (JEV) virus-like particle (VLP). The BJ-ME cells were engineered by transfecting BHK-21 cells with a code-optimized cDNA encoding JEV prM and E protein expression plasmid. Cell line BJ-ME can stably produces a secreted form of Japanese encephalitis virus virus-like particle (JEV-VLP) which contains the JEV envelope glycoprotein (E) and membrane protein (M). The amount of JEV-VLP antigen released into the culture fluid of BJ-ME cells was as high as 15–20 μg/ml. JEV-VLP production was stable after multiple cell passages and 100% cell expression was maintained without detectable cell fusion or apoptosis. Cell culture fluid containing the JEV-VLP antigen could be harvested five to seven times continuously at intervals of 4–6 days while maintaining the culture. Mice immunized with the JEV-VLP antigen with or without adjuvant developed high titers of neutralizing antibodies and 100% protection against lethal JEV challenge.
Conclusion
These results suggest that the recombinant JEV-VLP antigen produced by the BJ-ME cell line is an effective, safe and affordable subunit Japanese encephalitis vaccine candidate, especially for domestic animals such as pig and horse.
doi:10.1186/1472-6750-14-62
PMCID: PMC4094896  PMID: 25011456
Japanese encephalitis virus; Mammalian cell line; Virus-like particle; Subunit vaccine
22.  Gene–Gene Interaction of BLK, TNFSF4, TRAF1, TNFAIP3, and REL in Systemic Lupus Erythematosus 
Arthritis and rheumatism  2012;64(1):222-231.
Objective
Although the number of convincingly established genetic associations with systemic lupus erythematosus (SLE) has increased sharply over the last few years, refinement of these associations is required, and their potential roles in gene–gene interactions need to be further investigated. Recent genome-wide association studies (GWAS) in SLE have produced renewed interest in B cell/T cell responses and the NF-κB signaling pathway. The aim of this study was to search for possible gene–gene interactions based on identified single-nucleotide polymorphisms (SNPs), in using an approach based on the role of signaling pathways.
Methods
The SNPs in BLK, TNFSF4, TRAF1, TNFAIP3, and REL were replicated in order to evaluate genetic associations with SLE. TaqMan genotyping was conducted in 804 Chinese patients with SLE and 722 matched control subjects. A multiple logistic regression model was used to estimate the multiplicative interaction effect of the SNPs, and additive interactions were analyzed by 2 × 2 factorial designs. Data from a previously published GWAS conducted by the International Consortium on the Genetics of Systemic Lupus Erythematosus were derived for comparison and validation.
Results
Single-marker analysis validated the association of BLK rs2736340 (P = 4.25 × 10–6) as well as TNFSF4 rs2205960 (P = 2.82 × 10–5) and TNFAIP3 rs5029939 (P = 1.92 × 10–3) with SLE susceptibility in Chinese. Multiplicative interaction analysis indicated that BLK had an interactive effect with TNFSF4 in Chinese patients with SLE (P = 6.57 × 10–4). Additive interaction analysis revealed interactions between TRAF1 and TNFAIP3 in both Chinese (P = 2.18 × 10–3) and Caucasians (P = 2.86 × 10–4). In addition, multiple tendencies toward interactions were observed, and an additive effect was observed as the number of risk genotypes increased.
Conclusion
The results of this study provide evidence of the possible gene–gene interactions of BLK, TNFSF4, TRAF1, TNFAIP3, and REL in SLE, which may represent a synergic effect of T cells and B cells through the NF-κB pathway in determining immunologic aberration.
doi:10.1002/art.33318
PMCID: PMC3994469  PMID: 21905002
23.  Ginsenosides Rb1 and Rg1 Stimulate Melanogenesis in Human Epidermal Melanocytes via PKA/CREB/MITF Signaling 
Reduced or defective melanin skin pigmentation may cause many hypopigmentation disorders and increase the risk of damage to the skin triggered by UV irradiation. Ginsenosides Rb1 and Rg1 have many molecular targets including the cAMP-response element-binding protein (CREB), which is involved in melanogenesis. This study aimed to investigate the effects of ginsenosides Rb1 and Rg1 on melanogenesis in human melanocytes and their related mechanisms. The effects of Rb1 and Rg1 on cell viability, tyrosinase activity, cellular melanin content and protein levels of tyrosinase, microphthalmia-associated transcription factor (MITF), and activation of CREB in melanocytes were assessed. Results showed that Rb1 or Rg1 significantly increased cellular melanin content and tyrosinase activity in a dose-dependent manner. By contrast, the cell viability of melanocytes remained unchanged. After exposure to Rb1 or Rg1, the protein levels of tyrosinase, MITF, and phosphorylated CREB were significantly increased. Furthermore, pretreatment with the selective PKA inhibitor H-89 significantly blocked the Rb1- or Rg1-induced increase of melanin content. These findings indicated that Rb1 and Rg1 increased melanogenesis and tyrosinase activity in human melanocytes, which was associated with activation of PKA/CREB/MITF signaling. The effects and mechanisms of Rb1 or Rg1 on skin pigmentation deserve further study.
doi:10.1155/2014/892073
PMCID: PMC3988736  PMID: 24799945
24.  Targeting Orthotopic Glioma in Mice with Genetically Engineered Salmonella typhimurium 
Objective
With the growing interests of bacteria as a targeting vector for cancer treatment, diverse genetically engineered Salmonella has been reported to be capable of targeting primary or metastatic tumor regions after intravenous injection into mouse tumor models. The purpose of this study was to investigate the capability of the genetically engineered Salmonella typhimurium (S. typhimurium) to access the glioma xenograft, which was monitored in mouse brain tumor models using optical bioluminescence imaging technique.
Methods
U87 malignant glioma cells (U87-MG) stably transfected with firefly luciferase (Fluc) were implanted into BALB/cAnN nude mice by stereotactic injection into the striatum. After tumor formation, attenuated S. typhimurium expressing bacterial luciferase (Lux) was injected into the tail vein. Bioluminescence signals from transfected cells or bacteria were monitored using a cooled charge-coupled device camera to identify the tumor location or to trace the bacterial migration. Immunofluorescence staining was also performed in frozen sections of mouse glioma xenograft.
Results
The injected S. typhimurium exclusively localized in the glioma xenograft region of U87-MG-bearing mouse. Immunofluorescence staining also demonstrated the accumulation of S. typhimurium in the brain tumors.
Conclusion
The present study demonstrated that S. typhimurium can target glioma xenograft, and may provide a potentially therapeutic probe for glioma.
doi:10.3340/jkns.2014.55.3.131
PMCID: PMC4024811  PMID: 24851147
Cancer targeting; Mouse glioma model; Optical bioluminescence imaging; Salmonella typhimurium; U87-MG
25.  SNPs in VKORC1 are Risk Factors for Systemic Lupus Erythematosus in Asians 
Arthritis and rheumatism  2013;65(1):211-215.
OBJECTIVE
The increased risk of thrombosis in systemic lupus erythematosus (SLE) may be partially explained by interrelated genetic pathways for thrombosis and SLE. In a case-control analysis, we investigated whether 33 established and novel single nucleotide polymorphisms (SNP) in 20 genes involved in hemostasis pathways that have been associated with deep venous thrombosis in the general population were risk factors for SLE development among Asians.
METHODS
Patients in the discovery cohort were enrolled in one of two North American SLE cohorts. Patients in the replication cohort were enrolled in one of four Asian or two North American cohorts. SLE cases met American College of Rheumatology classification criteria. We first genotyped 263 Asian SLE and 357 healthy Asian control individuals for 33 SNPs using Luminex multiplex technology in the discovery phase, and then used Taqman and Immunochip assays to examine 5 SNPs in up to an additional 1496 cases and 993 controls in the Replication phase. SLE patients were compared to healthy controls for association with minor alleles in allelic models. Principal components analysis was used to control for intra-Asian ancestry in an analysis of the replication cohort.
RESULTS
Two genetic variants in the gene VKORC1, rs9934438 and rs9923231, were highly significant in both the discovery and replication cohorts: OR(disc) = 2.45 (p=2×10−9), OR(rep) = 1.53 (p=5×10−6) and OR(disc) = 2.40 (p=6×10−9), OR(rep) = 1.53 (p=5×10−6), respectively. These associations were significant in the replication cohort after adjustment for intra-Asian ancestry: rs9934438 OR(adj) = 1.34 (p=0.0029) and rs9923231 OR(adj) = 1.34 (p=0.0032).
CONCLUSION
Genetic variants in VKORC1, involved in vitamin K reduction and associated with DVT, are associated with SLE development in Asians. These results suggest intersecting genetic pathways for the development of SLE and thrombosis.
doi:10.1002/art.37751
PMCID: PMC3670944  PMID: 23124848
systemic lupus erythematosus; single nucleotide polymorphisms; genetic risk factors

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