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1.  Identification of a Group B Streptococcal Fibronectin Binding Protein, SfbA, That Contributes to Invasion of Brain Endothelium and Development of Meningitis 
Infection and Immunity  2014;82(6):2276-2286.
Group B Streptococcus (GBS) is currently the leading cause of neonatal meningitis. This is due to its ability to survive and multiply in the bloodstream and interact with specialized human brain microvascular endothelial cells (hBMEC), which constitute the blood-brain barrier (BBB). The exact mechanism(s) of GBS-BBB penetration is still largely unknown. We and others have shown that GBS interacts with components of the extracellular matrix. In this study, we demonstrate that GBS of representative serotypes binds immobilized and cell surface fibronectin and identify a putative fibronectin binding protein, streptococcal fibronectin binding protein A (SfbA). Allelic replacement of sfbA in the GBS chromosome resulted in a significant decrease in ability to bind fibronection and invade hBMEC compared with the wild-type (WT) parental strain. Expression of SfbA in the noninvasive strain Lactococcus lactis was sufficient to promote fibronectin binding and hBMEC invasion. Furthermore, the addition of an antifibronectin antibody or an RGD peptide that blocks fibronectin binding to integrins significantly reduced invasion of the WT but not the sfbA-deficient mutant strain, demonstrating the importance of an SfbA-fibronectin-integrin interaction for GBS cellular invasion. Using a murine model of GBS meningitis, we also observed that WT GBS penetrated the brain and established meningitis more frequently than did the ΔsfbA mutant strain. Our data suggest that GBS SfbA plays an important role in bacterial interaction with BBB endothelium and the pathogenesis of streptococcal meningitis.
doi:10.1128/IAI.01559-13
PMCID: PMC4019170  PMID: 24643538
2.  Analysis of Two-Component Systems in Group B Streptococcus Shows That RgfAC and the Novel FspSR Modulate Virulence and Bacterial Fitness 
mBio  2014;5(3):e00870-14.
ABSTRACT
Group B Streptococcus (GBS), in the transition from commensal organisms to pathogens, will encounter diverse host environments and, thus, require coordinated control of the transcriptional responses to these changes. This work was aimed at better understanding the role of two-component signal transduction systems (TCS) in GBS pathophysiology through a systematic screening procedure. We first performed a complete inventory and sensory mechanism classification of all putative GBS TCS by genomic analysis. Five TCS were further investigated by the generation of knockout strains, and in vitro transcriptome analysis identified genes regulated by these systems, ranging from 0.1% to 3% of the genome. Interestingly, two sugar phosphotransferase systems appeared to be differentially regulated in the TCS-16 knockout strain (TCS loci were numbered in order of their appearance on the chromosome), suggesting an involvement in monitoring carbon source availability. High-throughput analysis of bacterial growth on different carbon sources showed that TCS-16 was necessary for the growth of GBS on fructose-6-phosphate. Additional transcriptional analysis provided further evidence for a stimulus-response circuit where extracellular fructose-6-phosphate leads to autoinduction of TCS-16, with concomitant dramatic upregulation of the adjacent operon, which encodes a phosphotransferase system. The TCS-16-deficient strain exhibited decreased persistence in a model of vaginal colonization. All mutant strains were also characterized in a murine model of systemic infection, and inactivation of TCS-17 (also known as RgfAC) resulted in hypervirulence. Our data suggest a role for the previously unknown TCS-16, here named FspSR, in bacterial fitness and carbon metabolism during host colonization, and the data also provide experimental evidence for TCS-17/RgfAC involvement in virulence.
IMPORTANCE
Two-component systems have been evolved by bacteria to detect environmental changes, and they play key roles in pathogenicity. A comprehensive analysis of TCS in GBS has not been performed previously. In this work, we classify 21 TCS and present evidence for the involvement of two specific TCS in GBS virulence and colonization in vivo. Although pinpointing specific TCS stimuli is notoriously difficult, we used a combination of techniques to identify two systems with different effects on GBS pathogenesis. For one of the systems, we propose that fructose-6-phosphate, a metabolite in glycolysis, is sufficient to induce a regulatory response involving a sugar transport system. Our catalogue and classification of TCS may guide further studies into the role of TCS in GBS pathogenicity and biology.
doi:10.1128/mBio.00870-14
PMCID: PMC4030450  PMID: 24846378
3.  Genetic variant in IL33 is associated with susceptibility to rheumatoid arthritis 
Arthritis Research & Therapy  2014;16(2):R105.
Introduction
Interleukin (IL)-33 is a proinflammatory cytokine contributing to the pathogenesis of rheumatoid arthritis (RA). The gene encoding IL-33 may serve as a genetic factor and be associated with the risk of RA. To investigate the potential association between IL33 and RA, we performed a case–control study based on Chinese Han population.
Methods
A three-stage case–control study was performed. Two tag single-nucleotide polymorphisms (SNPs) (rs7044343 and rs10975514), mapping to the IL33 gene, were first genotyped in the discovery population. We further genotyped rs7044343 and rs10975514 in the validation and replication population. The associations between the two tag SNPs and phenotypic subgroups of RA and levels of serum IL-33 were assessed with a logistic regression model.
Results
In the discovery population, the CC genotype of rs7044343 was associated with RA patients (odds ratio (OR) = 0.777, 95% confidence interval (CI), 0.611 to 0.988; P = 0.040). After anti-citrullinated peptide antibody (ACPA) stratification, the CC genotype of rs7044343 was also shown to be a protective genotype in RA without ACPA (OR = 0.610; 95% CI, 0.379 to 0.982; P = 0.042). In the validation population and replication population, the association between rs7044343 and RA, especially ACPA-negative RA, was still significant. A meta-analysis of discovery, validation, and replication panels confirmed the association between CC genotype of rs7044343 and RA (Pcombined = 0.0004; ORcombined = 0.77; 95% CI, 0.67 to 0.89). No evidence was found for heterogeneity between three sample sets (Phet = 0.99; I2 = 0%). Similar results were also obtained in ACPA-negative RA (Pcombined = 0.0002; ORcombined = 0.57; 95% CI, 0.43 to 0.77). No association was detected between rs10975514 polymorphism and RA susceptibility in the discovery and validation population. The serum levels of IL-33 were significantly lower in the patients with the rs7044343 CC genotype.
Conclusion
The CC genotype of rs7044343 in IL33 is associated with RA patients and downregulates IL-33 expression in RA.
doi:10.1186/ar4554
PMCID: PMC4075243  PMID: 24779919
4.  Antilymphocyte Antibodies in Systemic Lupus Erythematosus: Association with Disease Activity and Lymphopenia 
Journal of Immunology Research  2014;2014:672126.
Purpose. We analyzed the prevalence, clinical correlation, and the functional significance of ALA in patients with systemic lupus erythematosus (SLE). Methods. ALA IgG was detected by indirect immunofluorescence in the serum of 130 SLE patients, 75 patients with various rheumatic diseases, and 45 healthy controls (HC). Results. The sensitivity and specificity of ALA IgG in SLE were 42.3% and 96.7%, respectively. ALA was observed in 55.6% (50/90) of patients with lymphopenia, which was significantly higher than in patients with normal lymphocytes (5/40, 12.5%; P < 0.001). Patients with active SLE showed higher ALA positivity (60.9%) than those with inactive disease (24.2%; χ2 = 17.925; P < 0.001). ALA correlated significantly with hypocomplementemia, anti-dsDNA antibodies, and higher SLEDAI scores. The incidences of ALA in SLE patients who were seronegative for anti-dsDNA, anti-Sm, or both antibodies were 32.9% (26/79), 41.0% (43/105), and 32.4% (22/68), respectively. The ALA-positive group also had higher incidences of neuropsychiatric SLE (NPSLE) and lupus nephritis (LN). In multivariate analyses, ALA was independently associated with lymphopenia, higher SLEDAI scores, and increased risk for LN. ALA titers significantly decreased as clinical disease was ameliorated following treatment. Conclusions. ALA occurred more frequently in patients with active SLE and was independently associated with lymphopenia, disease activity, and LN.
doi:10.1155/2014/672126
PMCID: PMC4016860  PMID: 24860837
6.  Conserved and variable structural elements in the 5′ untranslated region of two hypoviruses from the filamentous fungus Cryphonectria parasitica 
Virus research  2011;161(2):10.1016/j.virusres.2011.07.023.
Virulence-attenuating viruses (hypoviruses) of the filamentous fungus Cryphonectria parasitica, the causative agent of chestnut blight, have become a premier model for understanding the molecular biology of mycoviruses. However, a major gap exists in current understanding of structure and function of the untranslated regions (UTRs) of the hypovirus RNA genome, despite considerable evidence that secondary and tertiary UTR structure plays a crucial role in the control of translation and genome replication in other systems. In this study we have used structure prediction software coupled with RNase digestion studies to develop validated structural models for the 5′ UTRs of the two best-characterized members of the Hypoviridae, CHV1-EP713 and CHV1-Euro7. These two hypovirus strains exhibit significant variation in virulence attenuation despite sharing >90 % sequence identity. Our models reveal highly structured regions in the 5′ UTR of both strains, with numerous stem-loops suggestive of internal ribosome entry sites. However, considerable differences in the size and complexity of structural elements exist between the two strains. These data will guide future, mutagenesis-based studies of the structural requirements for hypovirus genome replication and translation.
doi:10.1016/j.virusres.2011.07.023
PMCID: PMC3837689  PMID: 21884737
mycovirus; secondary structure; Cryphonectria parasitica; hypovirus; Mfold; RNA virus
7.  Increased IL-33 in Synovial Fluid and Paired Serum Is Associated with Disease Activity and Autoantibodies in Rheumatoid Arthritis 
Objectives. IL-33, a newly found cytokine which is involved in joint inflammation, could be blocked by a decoy receptor—sST2. The expression and correlation of IL-33 and sST2 in rheumatoid arthritis (RA) are of great interest. Methods. Synovial fluid (SF) was obtained from 120 RA and 30 osteoarthritis (OA) patients, and paired sera were collected from 54 of these RA patients. The levels of IL-33 and sST2 were measured by ELISA. Results. SF IL-33 was significantly higher in RA than in OA, which was correlated with disease activity score 28, erythrocyte sedimentation rate, rheumatoid factor (RF)-IgM, RF-IgG, glucose phosphate isomerase (GPI), and immunoglobulin. Serum IL-33 was correlated positively with SF IL-33 in RA. Furthermore, it was correlated with RF-IgM and GPI. sST2 was partly detectable in RA (13 out of 54, 24.1%), while not in OA. Serum sST2 in RA had no significant correlation with serum IL-33 or SF IL-33. However, SFs from both RA and OA patients did not express sST2. Conclusions. This study supported that IL-33 played an important role in the local pathogenesis of RA. Considering the tight correlation between IL-33 and clinical features, it may become a new target of local treatment.
doi:10.1155/2013/985301
PMCID: PMC3782822  PMID: 24106520
8.  Correction: Hypoxia-Inducible Factor-1α and Interleukin 33 Form a Regulatory Circuit to Perpetuate the Inflammation in Rheumatoid Arthritis 
PLoS ONE  2013;8(8):10.1371/annotation/f61a5d49-25e7-47ce-8509-6478df526886.
doi:10.1371/annotation/f61a5d49-25e7-47ce-8509-6478df526886
PMCID: PMC3758480
9.  Hypoxia-Inducible Factor-1α and Interleukin 33 Form a Regulatory Circuit to Perpetuate the Inflammation in Rheumatoid Arthritis 
PLoS ONE  2013;8(8):e72650.
Hyperplasia of synovial fibroblasts, infiltration with inflammatory cytokines, and tissue hypoxia are the major characteristics of rheumatoid arthritis (RA). Interleukin 33 (IL-33) is a newly identified inflammatory cytokine exacerbating the disease severity of RA. Hypoxia-inducible factor-1α (HIF-1α) showed increased expression in RA synovium and could regulate a number of inflammatory cytokine productions. Nevertheless, its correlation with IL-33 remains largely unknown. Here, we showed that elevated levels of IL-33 were demonstrated in RA patient synovial fluids, with upregulated expression of HIF-1α and IL-33 in the synovial fibroblasts. Knocking down HIF-1α compromised IL-33 expression in rheumatoid arthritis synovial fibroblasts (RASF), while enforcing HIF-1α expression in RASF substantially upregulated IL-33 levels. HIF-1α promoted the activation of the signalling pathways controlling IL-33 production, particularly the p38 and ERK pathways. Moreover, we showed for the first time that IL-33 in turn could induce more HIF-1α expression in RASF, thus forming a HIF-1α/IL-33 regulatory circuit that would perpetuate the inflammatory process in RA. Targeting this pathological pathway and HIF-1α may provide new therapeutic strategies for overcoming the persistent and chronic inflammatory disease.
doi:10.1371/journal.pone.0072650
PMCID: PMC3744448  PMID: 23967327
10.  Human and Methodological Sources of Variability in the Measurement of Urinary 8-Oxo-7,8-dihydro-2′-deoxyguanosine 
Antioxidants & Redox Signaling  2013;18(18):2377-2391.
Abstract
Aims: Urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) is a widely used biomarker of oxidative stress. However, variability between chromatographic and ELISA methods hampers interpretation of data, and this variability may increase should urine composition differ between individuals, leading to assay interference. Furthermore, optimal urine sampling conditions are not well defined. We performed inter-laboratory comparisons of 8-oxodG measurement between mass spectrometric-, electrochemical- and ELISA-based methods, using common within-technique calibrants to analyze 8-oxodG-spiked phosphate-buffered saline and urine samples. We also investigated human subject- and sample collection-related variables, as potential sources of variability. Results: Chromatographic assays showed high agreement across urines from different subjects, whereas ELISAs showed far more inter-laboratory variation and generally overestimated levels, compared to the chromatographic assays. Excretion rates in timed ‘spot’ samples showed strong correlations with 24 h excretion (the ‘gold’ standard) of urinary 8-oxodG (rp 0.67–0.90), although the associations were weaker for 8-oxodG adjusted for creatinine or specific gravity (SG). The within-individual excretion of 8-oxodG varied only moderately between days (CV 17% for 24 h excretion and 20% for first void, creatinine-corrected samples). Innovation: This is the first comprehensive study of both human and methodological factors influencing 8-oxodG measurement, providing key information for future studies with this important biomarker. Conclusion: ELISA variability is greater than chromatographic assay variability, and cannot determine absolute levels of 8-oxodG. Use of standardized calibrants greatly improves intra-technique agreement and, for the chromatographic assays, importantly allows integration of results for pooled analyses. If 24 h samples are not feasible, creatinine- or SG-adjusted first morning samples are recommended. Antioxid. Redox Signal. 18, 2377–2391.
doi:10.1089/ars.2012.4714
PMCID: PMC3671631  PMID: 23198723
11.  Binding of Glycoprotein Srr1 of Streptococcus agalactiae to Fibrinogen Promotes Attachment to Brain Endothelium and the Development of Meningitis 
PLoS Pathogens  2012;8(10):e1002947.
The serine-rich repeat glycoprotein Srr1 of Streptococcus agalactiae (GBS) is thought to be an important adhesin for the pathogenesis of meningitis. Although expression of Srr1 is associated with increased binding to human brain microvascular endothelial cells (hBMEC), the molecular basis for this interaction is not well defined. We now demonstrate that Srr1 contributes to GBS attachment to hBMEC via the direct interaction of its binding region (BR) with human fibrinogen. When assessed by Far Western blotting, Srr1 was the only protein in GBS extracts that bound fibrinogen. Studies using recombinant Srr1-BR and purified fibrinogen in vitro confirmed a direct protein-protein interaction. Srr1-BR binding was localized to amino acids 283–410 of the fibrinogen Aα chain. Structural predictions indicated that the conformation of Srr1-BR is likely to resemble that of SdrG and other related staphylococcal proteins that bind to fibrinogen through a “dock, lock, and latch” mechanism (DLL). Deletion of the predicted latch domain of Srr1-BR abolished the interaction of the BR with fibrinogen. In addition, a mutant GBS strain lacking the latch domain exhibited reduced binding to hBMEC, and was significantly attenuated in an in vivo model of meningitis. These results indicate that Srr1 can bind fibrinogen directly likely through a DLL mechanism, which has not been described for other streptococcal adhesins. This interaction was important for the pathogenesis of GBS central nervous system invasion and subsequent disease progression.
Author Summary
Streptococcus agalactiae (Group B streptococcus, GBS) is a leading cause of meningitis in newborns and infants. This life-threatening infection of the brain and surrounding tissues continues to result in a high incidence of morbidity and mortality, despite antibiotic therapy. A key factor in disease production is the ability of this organism to invade the central nervous system, via the bloodstream. We now report that a GBS surface protein called Srr1 binds fibrinogen, a major protein in human blood. This interaction enhances the attachment of GBS to brain vascular endothelial cells, and contributes to the development of meningitis. A mutation in Srr1 that specifically disrupted binding to fibrinogen significantly reduced GBS attachment to brain endothelium, and markedly reduced virulence in an in vivo model of GBS disease. These studies have identified a new mechanism by which Srr1 contributes to GBS invasion of the central nervous system and may provide a basis for novel therapies targeting Srr1 binding.
doi:10.1371/journal.ppat.1002947
PMCID: PMC3464228  PMID: 23055927
12.  Therapeutic potential of human umbilical cord mesenchymal stem cells in the treatment of rheumatoid arthritis 
Arthritis Research & Therapy  2010;12(6):R210.
Introduction
Rheumatoid arthritis (RA) is a T-cell-mediated systemic autoimmune disease, characterized by synovium inflammation and articular destruction. Bone marrow mesenchymal stem cells (MSCs) could be effective in the treatment of several autoimmune diseases. However, there has been thus far no report on umbilical cord (UC)-MSCs in the treatment of RA. Here, potential immunosuppressive effects of human UC-MSCs in RA were evaluated.
Methods
The effects of UC-MSCs on the responses of fibroblast-like synoviocytes (FLSs) and T cells in RA patients were explored. The possible molecular mechanism mediating this immunosuppressive effect of UC-MSCs was explored by addition of inhibitors to indoleamine 2,3-dioxygenase (IDO), Nitric oxide (NO), prostaglandin E2 (PGE2), transforming growth factor β1 (TGF-β1) and interleukin 10 (IL-10). The therapeutic effects of systemic infusion of human UC-MSCs on collagen-induced arthritis (CIA) in a mouse model were explored.
Results
In vitro, UC-MSCs were capable of inhibiting proliferation of FLSs from RA patients, via IL-10, IDO and TGF-β1. Furthermore, the invasive behavior and IL-6 secretion of FLSs were also significantly suppressed. On the other hand, UC-MSCs induced hyporesponsiveness of T cells mediated by PGE2, TGF-β1 and NO and UC-MSCs could promote the expansion of CD4+ Foxp3+ regulatory T cells from RA patients. More importantly, systemic infusion of human UC-MSCs reduced the severity of CIA in a mouse model. Consistently, there were reduced levels of proinflammatory cytokines and chemokines (TNF-α, IL-6 and monocyte chemoattractant protein-1) and increased levels of the anti-inflammatory/regulatory cytokine (IL-10) in sera of UC-MSCs treated mice. Moreover, such treatment shifted Th1/Th2 type responses and induced Tregs in CIA.
Conclusions
In conclusion, human UC-MSCs suppressed the various inflammatory effects of FLSs and T cells of RA in vitro, and attenuated the development of CIA in vivo, strongly suggesting that UC-MSCs might be a therapeutic strategy in RA. In addition, the immunosuppressive activitiy of UC-MSCs could be prolonged by the participation of Tregs.
doi:10.1186/ar3187
PMCID: PMC3046518  PMID: 21080925
13.  Common Virulence Factors and Genetic Relationships between O18:K1:H7 Escherichia coli Isolates of Human and Avian Origin 
Journal of Clinical Microbiology  2006;44(10):3484-3492.
Extraintestinal pathogenic (ExPEC) Escherichia coli strains of serotype O18:K1:H7 are mainly responsible for neonatal meningitis and sepsis in humans and belong to a limited number of closely related clones. The same serotype is also frequently isolated from the extraintestinal lesions of colibacillosis in poultry, but it is not well known to what extent human and avian strains of this particular serotype are related. Twenty-two ExPEC isolates of human origin and 33 isolates of avian origin were compared on the basis of their virulence determinants, lethality for chicks, pulsed-field gel electrophoresis (PFGE) patterns, and classification in the main phylogenetic groups. Both avian and human isolates were lethal for chicks and harbored similar virulence genotypes. A major virulence pattern, identified in 75% of the isolates, was characterized by the presence of F1 variant fimbriae; S fimbriae; IbeA; the aerobactin system; and genomic fragments A9, A12, D1, D7, D10, and D11 and by the absence of P fimbriae, F1C fimbriae, Afa adhesin, and CNF1. All but one of the avian and human isolates also belonged to major phylogenetic group B2. However, various subclonal populations could be distinguished by PFGE in relation to animal species and geographical origin. These results demonstrate that very closely related clones can be recovered from extraintestinal infections in humans and chickens and suggest that avian pathogenic E. coli isolates of serotype O18:K1:H7 are potential human pathogens.
doi:10.1128/JCM.00548-06
PMCID: PMC1594794  PMID: 17021071
14.  Immunohistochemical assessment of angiogenesis in hepatocellular carcinoma and surrounding cirrhotic liver tissues 
AIM: To investigate whether vascular endothelial growth factor (VEGF) was over-expressed in hepatocellular carcinoma (HCC) or in surrounding cirrhotic liver tissues.
METHODS: Immunohistochemistry was performed to investigate the expression of VEGF proteins in HCC tissues from 105 consecutive patients undergoing curative resection for HCC. The immunostaining results and related clinicopathologic materials were analyzed with statistical methods. Kaplan–Meier method was used to calculate survival curves, and Log-rank test was performed to compare differences in survival rates of the patients with positive HCC staining and negative VEGF.
RESULTS: VEGF-positive expression was found in 72 of 105 HCC patients (68.6%). Capsular infiltration (P = 0.005), vascular invasion (P = 0.035) and intrahepatic metastasis (P = 0.008) were observed more frequently in patients with VEGF-positive expression than in those with VEGF-negative expression. Kaplan–Meier curves showed that VEGF-positive expression was associated with a shorter overall survival (P = 0.014). VEGF-positive expression was found in 47 of tissues 68 HCC (69.1%), and VEGF-positive expression was found in 54 of 68 surrounding cirrhotic liver tissues (79.4%). VEGF-positive expression was significantly higher in surrounding cirrhotic liver tissues than in HCC (P = 0.017).
CONCLUSION: VEGF may play an important role in the angiogenesis and prognosis of HCC, as well as in the angiogenesis of liver cirrhosis.
doi:10.3748/wjg.v11.i7.960
PMCID: PMC4250785  PMID: 15742396
Angiogenesis; Vascular endothelial growth factor; Hepatocellular carcinoma; Surrounding cirrhotic liver tissues

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