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author:("Lu, bufei")
1.  Autoantibody‐Positive Healthy Individuals Display Unique Immune Profiles That May Regulate Autoimmunity 
Antinuclear antibodies (ANAs) are detected in ∼18% of females, yet autoimmune disease develops in only 5–8%. Immunologic differences between ANA‐positive healthy individuals and patients with systemic lupus erythematosus (SLE) may elucidate the regulatory mechanisms by which ANA‐positive individuals avoid transition to clinical autoimmune disease.
Healthy individuals (n = 790) were screened for autoantibodies specific for 11 antigens associated with lupus, systemic sclerosis, and Sjögren's syndrome. From this screening, 31 European American ANA‐positive healthy individuals were selected and demographically matched to ANA‐negative controls and SLE patients. Serum cytokine profiles, leukocyte subset frequency, and reactivity were analyzed by multiplex assays, immunophenotyping, and phosphospecific flow cytometry.
Of 790 individuals screened, 57 (7%) were ANA‐positive. The majority of proinflammatory cytokines, including interferon‐γ (IFNγ), tumor necrosis factor, interleukin‐17 (IL‐17), and granulocyte colony‐stimulating factor, exhibited a stepwise increase in serum levels from ANA‐negative controls to ANA‐positive healthy individuals to SLE patients (P < 0.0001). IFNα, IFNβ, IL‐12p40, and stem cell factor/c‐Kit ligand were increased in SLE patients only (P < 0.05). B lymphocyte stimulator (BlyS) was elevated in SLE patients but decreased in ANA‐positive individuals (P < 0.001). Further, IL‐1 receptor antagonist (IL‐1Ra) was down‐regulated in SLE patients only (P < 0.0001). ANA‐positive individuals had increased frequencies of monocytes, memory B cells, and plasmablasts and increased levels of pSTAT‐1 and pSTAT‐3 following IFNα stimulation compared with ANA‐negative controls (P < 0.05).
ANA‐positive healthy individuals exhibit dysregulation in multiple immune pathways yet differ from SLE patients by the absence of elevated IFNs, BLyS, IL‐12p40, and stem cell factor/c‐Kit ligand. Further, severely decreased levels of IL‐1Ra in SLE patients compared with ANA‐positive individuals may contribute to disease development. These results highlight the importance of IFN‐related pathways and regulatory elements in SLE pathogenesis.
PMCID: PMC5042816  PMID: 27059145
2.  Vitamin D Deficiency in a Multiethnic Healthy Control Cohort and Altered Immune Response in Vitamin D Deficient European-American Healthy Controls 
PLoS ONE  2014;9(4):e94500.
In recent years, vitamin D has been shown to possess a wide range of immunomodulatory effects. Although there is extensive amount of research on vitamin D, we lack a comprehensive understanding of the prevalence of vitamin D deficiency or the mechanism by which vitamin D regulates the human immune system. This study examined the prevalence and correlates of vitamin D deficiency and the relationship between vitamin D and the immune system in healthy individuals.
Healthy individuals (n = 774) comprised of European-Americans (EA, n = 470), African–Americans (AA, n = 125), and Native Americans (NA, n = 179) were screened for 25-hydroxyvitamin D [25(OH)D] levels by ELISA. To identify the most noticeable effects of vitamin D on the immune system, 20 EA individuals with severely deficient (<11.3 ng/mL) and sufficient (>24.8 ng/mL) vitamin D levels were matched and selected for further analysis. Serum cytokine level measurement, immune cell phenotyping, and phosphoflow cytometry were performed.
Vitamin D sufficiency was observed in 37.5% of the study cohort. By multivariate analysis, AA, NA, and females with a high body mass index (BMI, >30) demonstrate higher rates of vitamin D deficiency (p<0.05). Individuals with vitamin D deficiency had significantly higher levels of serum GM-CSF (p = 0.04), decreased circulating activated CD4+ (p = 0.04) and CD8+ T (p = 0.04) cell frequencies than individuals with sufficient vitamin D levels.
A large portion of healthy individuals have vitamin D deficiency. These individuals have altered T and B cell responses, indicating that the absence of sufficient vitamin D levels could result in undesirable cellular and molecular alterations ultimately contributing to immune dysregulation.
PMCID: PMC3984168  PMID: 24727903
3.  Comparison of autoantibody specificities between traditional and bead-based assays in a large, diverse collection of SLE patients and family members 
Arthritis and rheumatism  2012;64(11):3677-3686.
The replacement of standard immunofluorescence anti-nuclear antibody (ANA) methods with bead-based assays is a new clinical option. A large, multi-racial cohort of SLE patients, blood relatives and unaffected control individuals was evaluated for familial aggregation and subset clustering of autoantibodies by high-throughput serum screening technology and traditional methods.
Serum samples (1,540 SLE patients, 1,127 unaffected relatives, and 906 healthy, population-based controls) were analyzed for SLE autoantibodies using a bead-based assay, immunofluorescence, and immunodiffusion. Autoantibody prevalence, disease sensitivity, clustering, and association with standard immunodiffusion results were evaluated.
ANA frequency in SLE patient sera were 89%, 73%, and 67% by BioPlex 2200 and 94%, 84%, and 86% by immunofluorescence in African-American, Hispanic, and European-American patients respectively. 60kD Ro, La, Sm, nRNP A, and ribosomal P prevalence were compared across assays, with sensitivities ranging from 0.92 to 0.83 and specificities ranging from 0.90 to 0.79. Cluster autoantibody analysis showed association of three subsets: 1) 60kD Ro, 52kD Ro and La, 2) spliceosomal proteins, and 3) dsDNA, chromatin, and ribosomal P. Familial aggregation of Sm/RNP, ribosomal P, and 60kD Ro in SLE patient sibling pairs was observed (p ≤ 0.004). Simplex pedigree patients had a greater prevalence for dsDNA (p=0.0003) and chromatin (p=0.005) autoantibodies than multiplex patients.
ANA frequencies detected by a bead-based assay are lower in European-American SLE patients compared to immunofluorescence. These assays have strong positive predictive values across racial groups, provide useful information for clinical care, and provide unique insights to familial aggregation and autoantibody clustering.
PMCID: PMC3490432  PMID: 23112091
systemic lupus erythematosus; autoantibodies; ancestry
4.  Genetic and Physical Interaction of the B-Cell SLE-Associated Genes BANK1 and BLK 
Annals of the Rheumatic Diseases  2011;71(1):136-142.
Altered signaling in B-cells is a predominant feature of systemic lupus erythematosus (SLE). The genes BANK1 and BLK were recently described as associated with SLE. BANK1 codes for a B-cell-specific cytoplasmic protein involved in B-cell receptor signaling and BLK codes for an Src tyrosine kinase with important roles in B-cell development. To characterize the role of BANK1 and BLK in SLE, we performed a genetic interaction analysis hypothesizing that genetic interactions could reveal functional pathways relevant to disease pathogenesis.
We Used the method GPAT16 to analyze the gene-gene interactions of BANK1 and BLK. Confocal microscopy was used to investigate co-localization, and immunoprecipitation was used to verify the physical interaction of BANK1 and BLK.
Epistatic interactions between BANK1 and BLK polymorphisms associated with SLE were observed in a discovery set of 279 patients and 515 controls from Northern Europe. A meta-analysis with 4399 European individuals confirmed the genetic interactions between BANK1 and BLK.
As BANK1 was identified as a binding partner of the Src tyrosine kinase LYN, we tested the possibility that BANK1 and BLK could also show a protein-protein interaction. We demonstrated co-immunoprecipitation and co-localization of BLK and BANK1. In a Daudi cell line and primary naïve B-cells the endogenous binding was enhanced upon B-cell receptor stimulation using anti-IgM antibodies.
Here, we show a genetic interaction between BANK1 and BLK, and demonstrate that these molecules interact physically. Our results have important consequences for the understanding of SLE and other autoimmune diseases and identify a potential new signaling pathway.
PMCID: PMC3268679  PMID: 21978998
systemic lupus erythematosus; genetics; polymorphism; B-cells; autoantibodies
5.  Multiple Autoantibodies Display Association with Lymphopenia, Proteinuria, and Cellular Casts in a Large, Ethnically Diverse SLE Patient Cohort 
Autoimmune Diseases  2012;2012:819634.
Purpose. This study evaluates high-throughput autoantibody screening and determines associated systemic lupus erythematosus (SLE) clinical features in a large lupus cohort. Methods. Clinical and demographic information, along with serum samples, were obtained from each SLE study participant after appropriate informed consent. Serum samples were screened for 10 distinct SLE autoantibody specificities and examined for association with SLE ACR criteria and subcriteria using conditional logistic regression analysis. Results. In European-American SLE patients, autoantibodies against 52 kD Ro and RNP 68 are independently enriched in patients with lymphopenia, anti-La, and anti-ribosomal P are increased in patients with malar rash, and anti-dsDNA and anti-Sm are enriched in patients with proteinuria. In African-American SLE patients, cellular casts associate with autoantibodies against dsDNA, Sm, and Sm/nRNP. Conclusion. Using a high-throughput, bead-based method of autoantibody detection, anti-dsDNA is significantly enriched in patienets with SLE ACR renal criteria as has been previously described. However, lymphopenia is associated with several distinct autoantibody specificities. These findings offer meaningful information to allow clinicians and clinical investigators to understand which autoantibodies correlate with select SLE clinical manifestations across common racial groups using this novel methodology which is expanding in clinical use.
PMCID: PMC3439936  PMID: 22988489
6.  Effects of IRF5 Lupus Risk Haplotype on Pathways Predicted to Influence B Cell Functions 
Both genetic and environmental interactions affect systemic lupus erythematosus (SLE) development and pathogenesis. One known genetic factor associated with lupus is a haplotype of the interferon regulatory factor 5 (IRF5) gene. Analysis of global gene expression microarray data using gene set enrichment analysis identified multiple interferon- and inflammation-related gene sets significantly overrepresented in cells with the risk haplotype. Pathway analysis using expressed genes from the significant gene sets impacted by the IRF5 risk haplotype confirmed significant correlation with the interferon pathway, Toll-like receptor pathway, and the B-cell receptor pathway. SLE patients with the IRF5 risk haplotype have a heightened interferon signature, even in an unstimulated state (P = 0.011), while patients with the IRF5 protective haplotype have a B cell interferon signature similar to that of controls. These results identify multiple genes in functionally significant pathways which are affected by IRF5 genotype. They also establish the IRF5 risk haplotype as a key determinant of not only the interferon response, but also other B-cell pathways involved in SLE.
PMCID: PMC3304673  PMID: 22500098
7.  Genetic Associations of LYN with Systemic Lupus Erythematosus 
Genes and immunity  2009;10(5):397-403.
We targeted LYN, a src-tyosine kinase involved in B cell activation, in case-control association studies using populations of European American, African American and Korean subjects. Our combined European-derived population, consisting of 2463 independent cases and 3131 unrelated controls, demonstrates significant association with rs6983130 in a female-only analysis with 2254 cases and 2228 controls (p=1.1 × 10−4, OR=0.81 (95% CI: 0.73 – 0.90)). This SNP is located in the 5′ UTR within the first intron near the transcription initiation site of LYN. Additional SNPs upstream of the first exon also show weak and sporadic association in subsets of the total European American population. Multivariate logistic regression analysis implicates rs6983130 as a protective factor for SLE susceptibility when anti-dsDNA, anti-chromatin, anti-52 kDa Ro or anti-Sm autoantibody status were used as covariates. Subset analysis of the European American female cases by ACR classification criteria reveals a reduction in the risk of hematologic disorder with rs6983130 compared to cases without hematologic disorders (p=1.5 × 10−3, OR=0.75 (95% C.I.=0.62-0.89)). None of the 90 SNPs tested demonstrate significant association with SLE in the African American or Korean populations. These results support an association of LYN with European-derived individuals with SLE, especially within autoantibody or clinical subsets.
PMCID: PMC2750001  PMID: 19369946
systemic lupus erythematosus; association; LYN; SNP
8.  Replication of the BANK1 genetic association with systemic lupus erythematosus in a European-Derived Population 
Genes and immunity  2009;10(5):531-538.
Systemic lupus erythematosus (SLE) is an autoimmune disease with highly variable clinical presentation. Patients suffer from immunological abnormalities that target T cell, B cell and accessory cell functions. B cells are hyperactive in SLE patients. An adaptor protein expressed in B cells called BANK1 (B-cell scaffold protein with ankyrin repeats) was reported in a previous study to be associated with SLE in a European population. The objective of this study is to assess the BANK1 genotype-phenotype association in an independent replication sample. We genotyped 38 single nucleotide polymorphisms (SNPs) in BANK1 on 1892 European-derived SLE patients and 2652 European-derived controls. The strongest associations with SLE and BANK1 were at rs17266594 (corrected p-value=1.97 × 10−5, OR=1.22, 95% C.I.(1.12–1.34)) and rs10516487 (corrected p-value=2.59 × 10−5, OR=1.22, 95% C.I.(1.11–1.34)). Our findings suggest that the association is explained by these two SNPs, confirming previous reports that these polymorphisms contribute to the risk of developing lupus. Analysis of patient subsets enriched for hematological, immunological and renal ACR criteria or the levels of autoantibodies, such as anti-RNP A and anti-SmRNP, uncovers additional BANK1 associations. Our results suggest that BANK1 polymorphisms alter immune system development and function to increase the risk for developing lupus.
PMCID: PMC2736873  PMID: 19339986
systemic lupus erythematosus; replication; association; European; BANK1

Results 1-8 (8)