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Inflammatory bowel disease (IBD) is a non-infectious disease characterized by chronic inflammation of the gastrointestinal tract. Currently, management of IBD is still a clinical challenge. The purpose of this study was to investigate the therapeutic potential of surfactin containing

Background Xylaria nigripes (Koltz.) Cooke, also known as Wuling Shen, is a high-value medicinal mushroom. It is a herbal medicine traditionally used for treating insomnia, trauma and depression. However, its toxicity has never been systematically evaluated. This study aimed to evaluate the safety of a standardized aqueous extract (XNE), an ingredient of commercial products, prepared from solid-state cultured X. nigripes in rats. Methods A 90-day subchronic toxicity study was conducted by oral administration of XNE at daily doses of 20, 1000 and 2000 mg/kg body weight to Sprague-Dawley rats of both sexes, and the control group was given distilled water (vehicle). All animals were checked daily for general behavior, body weight changes and signs of toxicity. At the end of the treatment period, hematological analysis, biochemical analysis and histopathological examination of organs were conducted. Results At tested concentrations, oral XNE administration caused no treatment-induced adverse effects on general health, body weight gain, relative organ weights, and hematological and biochemical parameters. Histopathological results also showed no significant structural changes in organs even in high-dose XNE-treated animals. Conclusion This study suggests that treatment with XNE for 90 days does not produce significant toxicity, even up to 100 fold (2000 mg/kg body weight/day) of the recommended daily intakes. Therefore, the use of XNE as herbal medicines is considered to be relatively safe.

IL-23 promotes autoimmune disease, including Th17 CD4 T cell development and autoantibody production. In this study, we show that a deficiency of the p19 component of IL-23 in the autoimmune BXD2 (BXD2-p19−/−) mouse leads to a shift of the follicular T helper cell program from follicular T helper (Tfh)–IL-17 to Tfh–IFN-γ. Although the germinal center (GC) size and the number of GC B cells remained the same, BXD2-p19−/− mice exhibited a lower class-switch recombination (CSR) in the GC B cells, leading to lower serum levels of IgG2b. Single-cell transcriptomics analysis of GC B cells revealed that whereas Ifngr1, Il21r, and Il4r genes exhibited a synchronized expression pattern with Cxcr5 and plasma cell program genes, Il17ra exhibited a synchronized expression pattern with Cxcr4 and GC program genes. Downregulation of Ighg2b in BXD2-p19−/− GC B cells was associated with decreased expression of CSR-related novel base excision repair genes that were otherwise predominantly expressed by Il17ra+ GC B cells in BXD2 mice. Together, these results suggest that although IL-23 is dispensable for GC formation, it is essential to promote a population of Tfh–IL-17 cells. IL-23 acts indirectly on Il17ra+ GC B cells to facilitate CSR-related base excision repair genes during the dark zone phase of GC B cell development.

Objective: To determine if circulating autoantibodies (autoAbs) and T-helper cell cytokines correlate with a different class of lupus nephritis (LN), including class III/IV, compared to class V and other manifestations of systemic lupus erythematosus (SLE). Methods: All patients (N=62) met the SLE classification criteria of the American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) or Systemic Lupus International Collaborating Clinics (SLICC) classification criteria. Demographic, clinical data, and serologic manifestations included anti-DNA, anti-Smith (Anti-Sm), C3, and C4 were included. Plasma levels of interferon gamma (IFNɣ), interleukin 17 (IL-17), interleukin 10 (IL-10) isotype-specific (IgG) anti-DNA, anti-Ro/SSA (SSA), and anti-Sm were measured using enzyme-linked immunosorbent assay (ELISA).Results: The most common manifestations by history were arthritis in 61% (N=38), photosensitivity rash in 53% (N=33), LN in 44% (N=27), and oral/nasal ulcer in 31% (N=19) SLE patients. AutoAbs (anti-DNA, anti-SSA, and anti-Sm), IFNɣ, and IL-17, but not IL-10, were significantly elevated in SLE patients compared to healthy controls. There were elevated plasma levels of anti-DNA (p = 0.0101) and anti-Sm (p = 0.0499) in patients with a history of LN compared to patients without LN. In contrast, plasma levels of anti-Sm were decreased in patients who had a history of acute mucocutaneous manifestations, including photosensitivity rash and/or malar rash (p = 0.0152). Among the three cytokines that were analyzed, IL-10 was significantly elevated in patients with a history of LN compared to patients without LN (p = 0.0216). IL-17 was positively correlated with anti-SSA (p = 0.0130) and was significantly higher in patients with discoid rash (p = 0.0238) and history of class V LN (p = 0.0055). IFNɣ was positively correlated with anti-DNA (p = 0.0355) and anti-SSA (p = 0.0402). Conclusion: This cross-sectional study supports the role of different T-helper cell cytokines that may be associated with the development of different autoAbs in influencing the diversity of SLE clinical manifestations. The results suggests that elevated IFNɣ and IL-17 are more generalized features in SLE patients. In contrast, higher levels of IL-10 were observed in patients with a history of LN. This provide insights into the pathogenic mechanisms of LN that can help guide future diagnosis and therapies.

To determine the extent to which the gut microbiome influences systemic autoimmunity in a mouse model of lupus.We generated germ-free (GF) lupus-prone BXD2 mice, which under normal conditions develop spontaneous germinal centers (GCs) and high titers of serum autoantibodies. GF status was confirmed by gut bacterial culture. The autoimmune phenotypes of 6- and 12-month-old gnotobiotic GF BXD2 mice and specific pathogen-free (SPF) BXD2 mice were compared. Serum levels of autoantibodies were measured by enzyme-linked immunosorbent assay. Histologic sections of the mouse kidney and joints were evaluated. Flow cytometry was used to analyze GCs and age-associated B cells. CD4+ T cells were analyzed for PD-1+ICOS+ activated T cells, T follicular regulatory (Tfr) cells (Foxp3+CD25+ PD-1+CXCR5+), and PD-1+ICOS+ T cells expressing interleukin-17A (IL-17A) or interferon-γ (IFNγ) after stimulation with phorbol myristate acetate (PMA)/ionomycin.In 6-month-old mice, GF status did not affect splenomegaly, GC B cells, age-associated B cells, or serum autoantibody levels, except for IgG antihistone. GF BXD2 mice exhibited a significantly higher percentage of Tfr cells compared to their SPF counterparts (P0.05). At 12 months of age, however, GF BXD2 mice had significantly diminished IgG autoantibody levels and a lower percentage of GC B cells and age-associated B cells (P0.05). Following stimulation with PMA/ionomycin, PD-1+ICOS+ CD4+ T cells expressed significantly lower IL-17A, but not IFNγ, levels in GF BXD2 mice compared to SPF BXD2 mice (P0.01). SPF BXD2 mice and GF BXD2 mice developed equivalent renal and joint disease with no significant differences in severity.Our results suggest a model in which genetics plays a dominant role in determining the initial development of autoimmunity. In contrast, gut microbiomes may regulate the persistence of certain aspects of systemic autoimmunity.

Objective: To compare the differences between the work competencies self-reported by nurse aides’ and those perceived by nurses. Method: A cross-sectional survey was employed. The settings were units implemented a skill mix model institution in Taiwan. The instruments consisted of the participants’ demographic data and a nurse aide work competence scale. Results: The results indicated that the nurse aides had room for improvement in terms of “problem solving” and “activity design”. The nurse aides and nurses differed significantly in terms of the nurse aides’ competence in “activity design”, as the nurse aides reported themselves to be more competent in “activity design” than reported by the nurses. Conclusion: Nurse aides should be incorporated into cross-disciplinary teams. Activity design should be handled by other healthcare providers such as physical therapists or senior social workers.

Background In systemic lupus erythematosus (SLE), detection of interferon-β (IFNβ) in B cells was found to be most prominent in patients with high anti-Smith (Sm) and renal disease, but a mechanistic connection was not clear. The objective of the present study is to determine the association of IFNβ in peripheral blood naïve B cells with the histopathological features of lupus nephritis (LN). Methods The percentage of IFNβ+ cells in IgD+CD27− naïve CD19+ B cells (B cell IFNβ) among peripheral blood mononuclear cells (PBMCs) from 80 SLE patients were analyzed using flow cytometry. Serological and clinical data were collected. The correlations of B cell IFNβ with LN classification and with histopathological findings (light, electron, and immunofluorescence [IF] microscopic analyses for deposition of IgM, IgG, IgA, C1q, and C3) were determined in 23 available biopsy specimens. Results B cell IFNβ is positively associated with anti-Sm (p = 0.001), anti-DNA (p = 0.013), and LN (p < 0.001) but was negatively associated with oral/nasal ulcer (p = 0.003) and photosensitivity (p = 0.045). B cell IFNβ positively correlated with immune complex (IC) deposit in the glomerular basement membrane (GBM) (p = 0.002) but not in the mesangial (p = 0.107) or tubular region (p = 0.313). Patients with high B cell IFNβ had statistically increased development of the proliferative LN (Classes III, IV and/or V), compared to patients with low B cell IFNβ (p p = 0.023), chronic glomerular lesions indicated by segmental sclerosis (p = 0.033), and a membranous pattern of renal damage indicated by spike/holes (p = 0.015). Conclusion B cell IFNβ correlates with history of severe LN, glomerular basement membrane (GBM) IC deposition, and anatomical features of both active and chronic glomerular lesions.

Background: Benign immunoglobulin G4 (IgG4)-related orbital disease (IgG4-ROD)&mdash, characterized as tumors mimicking malignant orbital lymphoma (OL)&mdash, responds well to steroids, instead of chemotherapy, radiotherapy and/or surgery of OL. The objective of this study was to report the differences in computed tomography (CT) features and- serum IgG4 levels of IgG4-ROD and OL. Methods: This study retrieved records for patients with OL and IgG4-ROD from a pathology database during an eight-year-and-five-month period. We assessed the differences between 16 OL patients with 27 lesions and nine IgG4-ROD patients with 20 lesions according to prebiopsy CT features of lesions and prebiopsy serum IgG4 levels and immunoglobulin G (IgG) levels This study also established the receiver-operating curves (ROC) of precontrast and postcontrast CT Hounsfield unit scales (CTHU), serum IgG4 levels, serum IgG levels and their ratios. Results: Significantly related to IgG4-ROD (all p &lt, 0.05) were the presence of lesions with regular borders, presence of multiple lesions&mdash, involving both lacrimal glands on CT scans&mdash, higher median values of postcontrast CTHU, postcontrast CTHU/precontrast CTHU ratios, serum IgG4 levels and serum IgG4/IgG level ratios. Compared to postcontrast CTHU, serum IgG4 levels had a larger area under the ROC curve (0.847 [95% confidence interval (CI): 0.674&ndash, 1.000, p = 0.005] vs. 0.766 [95% CI: 0.615&ndash, 0.917, p = 0.002]), higher sensitivity (0.889 [95% CI: 0.518&ndash, 0.997] vs. 0.75 [95% CI: 0.509&ndash, 0.913]), higher specificity (0.813 [95% CI: 0.544&ndash, 0.960] vs. 0.778 [95% CI: 0.578&ndash, 0.914]) and a higher cutoff value (&ge, 132.5 mg/dL [milligrams per deciliter] vs. &ge, 89.5). Conclusions: IgG4-ROD showed distinct CT features and elevated serum IgG4 (&ge, 132.5 mg/dL), which could help distinguish IgG4-ROD from OL.

In systemic lupus erythematosus (SLE), type I IFNs promote induction of type I IFN–stimulated genes (ISG) and can drive B cells to produce autoantibodies. Little is known about the expression of distinct type I IFNs in lupus, particularly high-affinity IFN-β. Single-cell analyses of transitional B cells isolated from SLE patients revealed distinct B cell subpopulations, including type I IFN producers, IFN responders, and mixed IFN producer/responder clusters. Anti-Ig plus TLR3 stimulation of SLE B cells induced release of bioactive type I IFNs that could stimulate HEK-Blue cells. Increased levels of IFN-β were detected in circulating B cells from SLE patients compared with controls and were significantly higher in African American patients with renal disease and in patients with autoantibodies. Together, the results identify type I IFN–producing and –responding subpopulations within the SLE B cell compartment and suggest that some patients may benefit from specific targeting of IFN-β.

Myeloid-derived suppressor cells (MDSCs) are known suppressors of antitumor immunity, affecting amino acid metabolism and T cell function in the tumor microenvironment. However, it is unknown whether MDSCs regulate B cell responses during tumor progression. Using a syngeneic mouse model of lung cancer, we show reduction in percentages and absolute numbers of B cell subsets including pro–, pre–, and mature B cells in the bone marrow (BM) of tumor-bearing mice. The kinetics of this impaired B cell response correlated with the progressive infiltration of MDSCs. We identified that IL-7 and downstream STAT5 signaling that play a critical role in B cell development and differentiation were also impaired during tumor progression. Global impairment of B cell function was indicated by reduced serum IgG levels. Importantly, we show that anti–Gr-1 Ab-mediated depletion of MDSCs not only rescued serum IgG and IL-7 levels but also reduced TGF-β1, a known regulator of stromal IL-7, suggesting MDSC-mediated regulation of B cell responses. Furthermore, blockade of IL-7 resulted in reduced phosphorylation of downstream STAT5 and B cell differentiation in tumor-bearing mice and administration of TGF-β–blocking Ab rescued these IL-7–dependent B cell responses. Adoptive transfer of BM-derived MDSCs from tumor-bearing mice into congenic recipients resulted in significant reductions of B cell subsets in the BM and in circulation. MDSCs also suppressed B cell proliferation in vitro in an arginase-dependent manner that required cell-to-cell contact. Our results indicate that tumor-infiltrating MDSCs may suppress humoral immune responses and promote tumor escape from immune surveillance.

Fucosylation is a biological process broadly observed in vertebrates, invertebrates, plants, bacteria, and fungi. Fucose moieties on cell-surface glycans are increasingly recognized as critical to many cell-cell interaction and signaling processes. One of the characteristic roles of fucose is its regulation of selectin-dependent leukocyte adhesion that has been well studied over the last two decades. Recent studies of fucose in immune cell development and function regulation have significantly expanded the contemporary understanding of fucosylation. From cellular adhesion to immune regulation, herein we discuss the use of gene knockout studies, competitive inhibitors of fucose-containing glycan, and metabolic inhibitors of fucose biosynthesis to probe fucosylated glycan biosynthesis and signaling and its functional consequences. Promising clinical and preclinical applications in sickle cell disease, rheumatoid arthritis, tumor inhibition, metastasis prevention, antibody-dependent cell-mediated cytotoxicity, chemoresistance reversal, and in improving chemotherapy-related side effects and recovery are reviewed.

The transitional stage of B cell development is a formative stage in the spleen where autoreactive specificities are censored as B cells gain immune competence, but the intrinsic and extrinsic factors regulating survival of transitional stage 1 (T1) B cells are unknown. We report that B cell expression of IFN-β is required for optimal survival and TLR7 responses of transitional B cells in the spleen and was overexpressed in T1 B cells from BXD2 lupus-prone mice. Single-cell gene expression analysis of B6 Ifnb+/+ versus B6 Ifnb–⁄– T1 B cells revealed heterogeneous expression of Ifnb in wild-type B cells and distinct gene expression patterns associated with endogenous IFN-β. Single-cell analysis of BXD2 T1 B cells revealed that Ifnb is expressed in early T1 B cell development with subsequent upregulation of Tlr7 and Ifna1. Together, these data suggest that T1 B cell expression of IFN-β plays a key role in regulating responsiveness to external factors.

The current concepts for development of autoreactive B cells in SLE (systemic lupus erythematosus) focus on extrinsic stimuli and factors that provoke B cells into tolerance loss. Traditionally, major tolerance loss pathways are thought to be regulated by factors outside the B cell including autoantigen engagement of the B-cell receptor (BCR) with simultaneous type I interferon (IFN) produced by dendritic cells, especially plasmacytoid dendritic cells (pDCs). Later, in autoreactive follicles, B-cells encounter T-follicular helper cells (Tfh) that produce interleukin (IL)-21, IL-4 and pathogenic cytokines, IL-17 and IFN gamma (IFNɣ). This review discusses these mechanisms and also highlights recent advances pointing to the peripheral transitional B-cell stage as a major juncture where transient autocrine IFNβ expression by developing B-cells imprints a heightened susceptibility to external factors favoring differentiation into autoantibody-producing plasmablasts. Recent studies highlight transitional B-cell heterogeneity as a determinant of intrinsic resistance or susceptibility to tolerance loss through the shaping of B-cell responsiveness to cytokines and other environment factors.

Background Dysregulated responses to type I interferons (IFNs) is a hallmark of autoreactive B-cell development in SLE. This study investigated the source of IFN, the major type I IFN responsive B cells, and the disparities associated with B-cell IFN production and type I IFN responses. Methods IFN expression in B, CD4 T and plasmacytoid dendritic cells (pDCs) in PBMCs were analyzed by flow cytometry. Single cell gene expression analysis was carried out using the Fluidigm/BioMark system for targeted expression of low abundance genes, and the 10x Chromium platform for unbiased transcriptome and BCR V(D)J analysis of approximately 2,000 B cells per subject. Autoantigen epitope targets were analyzed using a 4287 high-throughput PEPperPrint Autoimmune Epitope Microarray and a conventional ELISA analysis. Results IFN was analyzed in B cells, CD4 T cells and pDCs in PBMCs of SLE patients and healthy controls (HCs). Endogenous IFN was significantly increased in transitional (Tr), mature naive, and memory B cells of SLE patients compared to HCs. Endogenous IFN in B cells was equivalent to that in pDCs. B-cell endogenous IFN was highly correlated with renal disease, anti-dsDNA, anti-Sm and anti-SSA. Strikingly, the highest correlation of IFN with clinical manifestations was observed in African-American (AA) patients with IgG autoAbs against snRNP323-339, U1snRNP-C97-113. At the single cell transcriptome levels, Tr B cells could be divided into type I IFN expressing (IFN+) or type I IFN stimulated gene (ISG+) subpopulations. TLR7 and TLR3 were mainly expressed by IFN +cells whereas TLR9 was mainly expressed by ISG +B cells. Unbiased single cells analysis of B cells indicated highly expressed ISG gene set in IGHM+, IGHD+, and IGHG +B cells in AA patients with autoantibodies and renal disease. Further, ISG highly expressing SLE B cells exhibited unique heavy- and light-chain repertoires including expression of the autoreactive IGHV4-34 gene, targeted with the 9 G4 anti-idiotype antibody that recognizes DNA- and RBP-autoreactive B cells. Conclusions (i) B cells are an important source of type I IFNs in modulating TLR and BCR responses in SLE; (ii) there are well-orchestrated distinct programs in type I IFN expression and response genes in subsets of B cells, (iii) distinct pathways of autoreactive B cell survival and activation are effected by combined signaling through BCR, TLR, and IFNAR with resultant distinct BCR heavy- and light-chain repertoire. Funding Source(s): R01-AI-071110, R01 AI134023, LRA Distinguished Innovator Award and Novel Research Award, VA Merit Review Award I01B × 004049, Immunology T32 Training Grant 2T32AI007051-39, the LFA Finzi Summer Fellowship.

Background: Self-reflection (also known as reflection) is an internal process that is difficult to perceive or assess. An instrument that is able to measure self-reflection may serve as a resource for educators to assess the learning process of students and to tailor education approaches to student needs. Purpose: The aim of this study was to translate the Self- Reflection and Insight Scale (SRIS) into Chinese and evaluate its psychometric properties for use with Taiwanese nursing students. Methods: For this cross-sectional study, nursing students were recruited from two nursing schools in southern Taiwan in two phases: Phase 1, which included 361 fourth-year students, and Phase 2, which included 703 fifth-year students. Data were collected in December 2012 and May 2013 using the Chinese version of the SRIS (SRIS-C), Taiwan Critical Thinking Disposition Inventory, and the Perceived Identity as a Nurse Questionnaire, which was developed by the author. In Phase 1, exploratory factor analysis was used to explore the factor structure of the SRIS-C in the fourth-year student participants. In Phase 2, confirmatory factor analysis was used to determine the fitness of the model for the fifth-year student participants. Results: Eight items were deleted from the original SRIS to create the SRIS-C. Thus, the Chinese-version measure had 12 items and two factors (self-reflection and insight) that fit the data well. The Cronbach's alpha coefficients for the total scale and its two subscales were .79, .87, and .83, respectively. The 3-week test-retest reliability was .74. SRIS-C scores correlated significantly with scores on the Taiwan Critical Thinking Disposition Inventory and the Perceived Identity as a Nurse Questionnaire, indicating good convergent validity for the SRIS-C. Conclusions: The current study showed that the SRIS-C has sound psychometric properties. This instrument provides nurse educators with information that may be used to evaluate the self-reflection and insight of students and to develop interventions to effectively improve these skills in Chinese-language-based nursing education.

Tamoxifen is the most widely used drug to treat women with estrogen receptor α (ERα)-positive breast cancer. Endoxifen is recognized as the active metabolite of tamoxifen in humans. We studied endoxifen effects on ERα-positive MCF-7 breast cancer cells. Estradiol increased the proliferation of MCF-7 cells by two- to threefold and endoxifen suppressed its effects. Endoxifen suppressed c-myc, c-fos and Tff1 oncogene expression, as revealed by RT-PCR. Estradiol increased the activity of ornithine decarboxylase (ODC) and adenosyl methioninedecarboxylase (AdoMetDC), whereas endoxifen suppressed these enzyme activities. Endoxifen increased activities of spermine oxidase (SMO) and acetyl polyamine oxidase (APAO) significantly, and reduced the levels of putrescine and spermidine. These data suggest a possible mechanism for the antiestrogenic effects of tamoxifen/endoxifen, involving the stimulation of polyamine oxidase enzymes. Therefore, SMO and APAO stimulation might be useful biomarkers for the efficacy of endoxifen treatment of breast cancer.

Although multiple and overlapping mechanisms are ultimately responsible for the immunopathology observed in patients with systemic lupus erythematosus, autoreactive Abs secreted by autoreactive plasma cells (PCs) are considered to play a critical role in disease progression and immunopathology. Given that PCs derive from the germinal centers (GC), long-term dysregulated GC reactions are often associated with the development of spontaneous autoantibody responses and immunopathology in systemic lupus erythematosus patients. In this review, we summarize the emerging evidence concerning the roles of T follicular helper cells in regulating pathogenic GC and autoreactive PC responses in lupus.

Background Increased type I interferon (IFN) has been shown to affect survival and activation of B cells in SLE. This study investigated novel mechanisms of endogenous production and autocrine activity of IFNβ in SLE B cells at the single-cell level. Methods IFNβ in B cells from SLE patients was analyzed using t-SNE platform based high dimensional flow cytometry. Intracellular IFNβ expression was visualized and analyzed by super-resolution confocal imaging and ImageStream analysis. Single cell gene expression analysis was carried out using the Fluidigm/BioMark system for targeted expression of low abundance genes, and the 10x Chromium platform for unbiased transcriptome analysis of up to 4,000 B cells per subject. Functional production of type I IFNs by B cells was analyzed using a human type I IFNs SEAP reporter HEK293 cell line. Results High dimension flow cytometry analysis identified intracellular IFNβ expression in pDCs, B cells, and CD4 T cells. B-cell endogenous IFNβ was required for optimal in vitro BCR and TLR7-induced activation and survival of B cells. Using a Fluidigm targeted-gene approach, B cells could be divided into type I IFN expressing (IFN+) or type I IFN stimulated gene (ISG+) subpopulations, suggesting B cells not only respond to type I IFNs but also express type I IFNs including IFNB and different IFNA genes. TLR7 and TLR3 were mainly expressed by IFN +cells whereas TLR9 was mainly expressed by ISG +B cells. The production of functional IFNβ and IFNα protein by single B cells from SLE subjects and was verified using a novel alkaline phosphatase live staining of HEK-blue reporter cells. There was enhanced IFNAR signaling by reporter cells in direct contact with SLE B cells which was blocked by anti-IFNβ and anti-IFNα. Interesting, anti-Ig crosslinking was required for optimal B-cell endogenous type I IFNs to stimulate responder cells. Unbiased single cells transcriptome analysis of SLE B cells using the 5’ 10X Chromium platform and Loupe V(D)J Browser indicated that gene clusters in type I IFN expressing or responding SLE B cells exhibited unique heavy- and light-chain gene expression repertoires. Conclusion (i) B cells are an important source of type I IFNs in modulating TLR and BCR responses in SLE; (ii) well-orchestrated and distinct programs in type I expression and responses genes in subsets of B cells, and (iii) distinct pathways of B cell survival and activation based on combined signaling through BCR, TLR, and IFNAR with a distinct BCR heavy- and light-chain repertoire. Acknowledgements This work was supported by grants from R01-AI-071110, R01 AI134023, Lupus Research Alliance Distinguished Innovator Award, I01B × 004049, and 1I01B × 000600 to J.D.M, 2T32AI007051–39 Immunology T32 Training Grant and the LFA Finzi Summer Fellowship to J.A.H, and the LRA Novel Research Award to H.-C.H.

Background Dysregulated responses to type I interferons (IFNs) is a hallmark of autoreactive B cell development in SLE patients. High sera levels of type I IFN protein were recently shown to occur in the absence of increased circulating pDCs and in the absence of increased pDC IFNs, suggesting the likelihood of other important sources of type I IFN that may act on B cells. The present study determined the cellular source of IFNβ and its association with disease activities in SLE. Methods Peripheral blood mononuclear cells (PBMCs) were obtained from 31 SLE patients meeting ACR 1997 revised criteria for SLE and 9 healthy controls. Intracellular IFNβ was determined using flow cytometry with FITC-anti-IFNβ mAb. Comprehensive clinical data was recorded for each SLE subject and the clinical data and laboratory analysis of B-cell intracellular IFNβ expression were collected in a double-blind fashion. Results IFNβ was detected in various cell types including CD4 T cells, B cells and pDCs in PBMCs of SLE patients. Endogenous IFNβ in B cells was significantly higher than endogenous IFNβ in CD4 T cells and were equivalent to that seen in pDCs. Within B cells, there was a significant increase in endogenous IFNβ in all B cell subpopulations of SLE patients compared to healthy controls. The most significant increase was found in the CD27+IgD– memory subpopulation. B-cell endogenous IFNβ was not a result of B-cell uptake of exogenous IFNβ as coculture of SLE B cells with HEK293 reporter cells resulted in induction of interferon stimulatory genes as determined by the secreted alkaline phosphatase assay. This was further blocked by an anti-IFNβ neutralization antibody. Interestingly B-cell endogenous IFNβ was highly correlated with clinical disease including renal disease and autoantibodies including anti-dsDNA, anti-Sm and anti-SSA. Strikingly, the highest correlation of IFNβ with clinical manifestations was observed in African-American patients. B-cell IFNβ expression was significantly correlated with CD19loCD38hiCD27+ plasma cell formation. Conclusion Intracellular IFNβ production by B cells is a novel and important B-cell intrinsic factor that may be essential for B-cell development into autoantibody producing B cells. The present work suggests a need for future human lupus studies into type I IFN dysregulation that pioneer beyond the view of pDC produced IFNα. These results also provided a mechanistic basis for development of more effective therapies to target the high-affinity IFNβ or the enhanceosome components that promote its induction in a subgroup of lupus patients. Acknowledgements This work was supported by grants from R01-AI-071110, R01 AI134023, Lupus Research Alliance Distinguished Innovator Award, I01B × 004049, and 1I01B × 000600 to J.D.M, 2T32AI007051–39 Immunology T32 Training Grant and the LFA Finzi Summer Fellowship to J.A.H, and the LRA Novel Research Award to H.-C.H.

Objective Follicular regulatory T (Tfr) cells act as the regulatory counterpart of follicular helper T (Tfh) cells to suppress germinal center (GC) B cell differentiation. We recently showed that interleukin-21 (IL-21) promoted Tfh cell differentiation in autoimmune BXD2 mice that develop spontaneous GCs. This study was undertaken to determine the modulatory effects of IL-21 on Tfr cells and the Tfr cell to Tfh cell balance in BXD2 mice. Methods The percentage and phenotype of Tfr cells were determined in BXD2 and BXD2-IL21−/− mice. The effects of IL-21 on Tfr cells and the Tfr cell:Tfh cell ratio were evaluated. Sorted Tfr cells from BXD2-IL21−/− mice were cocultured with Tfh cells and B cells, or transferred into BXD2 mice to determine their function. Results The percentages and numbers of GC B cells and Tfh cells were significantly reduced, but the percentage of Tfr cells was 2-fold higher in BXD2-IL21−/− mice than in wild-type BXD2 mice. Administration of AdIL-21 to BXD2-IL21−/− mice decreased the percentages and numbers of Tfr cells and the Tfr cell:Tfh cell ratio but increased the number of GC B cells in the spleen. Recombinant murine IL-21 suppressed FoxP3 and significantly reduced Tgfb1, Il2, and Gitr but enhanced Il21, Il6, Pd1, Cxcr5, and Icos expression in Tfr cells. IL-21 also counteracted Tfr cell–mediated inhibition of antibody secretion in the Tfh cell–B cell coculture system. Transfer of Tfr cells into young BXD2 mice reduced GC size and decreased the numbers of autoantibody-producing B cells. Conclusion Our findings indicate that high levels of IL-21 selectively enhance Tfh cell differentiation but inhibit Tfr cell commitment and the suppressive function of Tfr cells on Tfh cells and B cells, suggesting that IL-21 skews the balance from Tfr cells to Tfh cells to promote autoreactive GC reactions in BXD2 mice.

Objective Fucosylation catalyzed by fucosyltransferases (FUTs) is an important post-translational modification involved in a variety of biological processes. The purpose of the current study is to determine the roles of fucosylation in rheumatoid arthritis (RA) and the efficacy of reestablishing the immune homeostasis by using 2-Deoxy-D-galactose (2-D-gal), a fucosylation inhibitor.

Germinal centers (GCs) provide a microenvironment that promotes and regulates the interactions of B cells with follicular Th (TFH) cells. In this study, we show that there are significantly higher frequencies of CXCR5+ICOS+ TFH cells in autoimmune BXD2 mice, and these cells express both IL-21R and IL-17RA. Although IL-17 and IL-21 are both important for the formation of spontaneous GCs and development of pathogenic autoantibodies, IL-21, but not IL-17, is required for the proper development of TFH cells in BXD2 mice. The total numbers of TFH cells and their ability to induce B cell responses in vitro were not affected by a deficiency of IL-17RA in BXD2-Il17ra−/− mice, the majority of CXCR5+ TFH cells from BXD2-Il17ra−/− mice were, however, not localized in the GC light zone (LZ). Interruption of IL-17 signaling, either acutely by AdIL-17R:Fc or chronically by Il17ra−/−, disrupted TFH–B interactions and abrogated the generation of autoantibody-forming B cells in BXD2 mice. IL-17 upregulated the expression of regulator of G-protein signaling 16 (RGS16) to promote the ability of TFH to form conjugates with B cells, which was abolished in TFH cells from BXD2-Rgs16−/− mice. The results suggests that IL-17 is an extrinsic stop signal that it acts on postdifferentiated IL-17RA+ TFH to enable its interaction with responder B cells in the LZ niche. These data suggest a novel concept that TFH differentiation and its stabilization in the LZ are two separate checkpoints and that IL-21 and IL-17 act at each checkpoint to enable pathogenic GC development.

Marginal zone macrophages (MZMs) act as a barrier to entry of circulating apoptotic debris into the follicles of secondary lymphoid organs. In autoimmune BXD2 mice, there is a progressive reduction in the function and numbers of MZMs. Absence of MZMs results in retention of apoptotic cell (AC) debris within the marginal zone (MZ) and increased loading of AC Ags on MZ B cells and MZ-precursor (MZ-P) B cells. The MZ-P B cells are capable of translocating the AC Ags to the follicular zone and stimulating T cells. Both MZMs and MZ-P B cells from BXD2 mice express low levels of tolerogenic signals and high levels of inflammatory signals. Thus, the current study suggests a multifaceted mechanism in which MZMs maintain tolerance to apoptotic autoantigens and suppress their translocation to follicles. Lack of clearance of apoptotic debris by MZMs drives follicular Ag–transportation by MZ-P B cells to stimulate an autoimmune response.

Given the protective roles of 25-hydroxyvitamin D (25(OH)D or vitamin D) in musculoskeletal health and the potential beneficial effects of vitamin D supplementation in reducing the risk of various chronic diseases, intensive repletion of vitamin D has been widely advocated. Of note, CD8 T cells have the highest levels of the vitamin D receptor compared with other major immune cells. The effects of vitamin D on CD8 T cells during aging, however, remain unclear. This study determined the relationship between vitamin D levels and CD8 T cell status in 34 healthy female subjects (all >60 years old). The CD8 T-cell phenotype was defined by the surface expression of CD28 and CD95. The low-25(OH)D serum groups (≤30 ng/ml) had higher percentages of CD28+CD95–CD8+ (na?ve) T cells and lower percentages of CD28+CD95+CD8+ (effector) T cells. By contrast, subjects with high levels of 25(OH)D had very low percentages of na?ve CD8 T cells but very high percentages of effector CD8 T cells. There was a significant inverse correlation between 25(OH)D levels and the frequency of na?ve CD8 T cells. The results show that higher levels of vitamin D are correlated with decreased frequencies of na?ve CD8 T cells during early aging, suggesting that higher levels of 25(OH)D accelerate CD8 T cell senescence. These results warrant further evaluation of the effects of vitamin D supplementation in immune aging.

Objective: Although the majority of available evidence suggests that vertebroplasty and kyphoplasty can relieve pain associated with vertebral compression fractures (VCFs) and improve function, some studies have suggested results are similar to those of placebo. The purpose of this meta-analysis was to compare the outcomes of vertebroplasty and kyphoplasty with conservative treatment in patients with osteoporotic VCFs. Methods: Medline, Cochrane, and Embase databases were searched until January 31, 2015 using the keywords: vertebroplasty, kyphoplasty, compression fracture, osteoporotic, and osteoporosis. Inclusion criteria were randomized controlled trials (RCTs) in which patients with osteoporosis, and VCFs were treated with vertebroplasty/kyphoplasty or conservative management. Outcome measures were pain, function, and quality of life. Standardized differences in means were calculated as a measure of effect size. Main results: Ten RCTs were included. The total number of patients in the treatment and control groups was 626 and 628, respectively, the mean patient age ranged from 64 to 80 years, and the majority was female. Vertebroplasty/kyphoplasty was associated with greater pain relief (pooled standardized difference in means = 0.82, 95% confidence interval [CI]: 0.374–1.266, P

Macrophages play a central role in the pathogenesis of rheumatoid arthritis (RA). There is an imbalance of inflammatory and antiinflammatory macrophages in RA synovium. Although the polarization and heterogeneity of macrophages in RA have not been fully uncovered, the identity of macrophages in RA can potentially be defined by their products, including the co-stimulatory molecules, scavenger receptors, different cytokines/chemokines and receptors, and transcription factors. In the last decade, efforts to understand the polarization, apoptosis regulation, and novel signaling pathways in macrophages, as well as how distinct activated macrophages influence disease progression, have led to strategies that target macrophages with varied specificity and selectivity. Major targets that are related to macrophage development and apoptosis include TNF-α, IL-1, IL-6, GM-CSF, M-CSF, death receptor 5 (DR5), Fas, and others, as listed in Table 1. Combined data from clinical, preclinical, and animal studies of inhibitors of these targets have provided valuable insights into their roles in the disease progression and, subsequently, have led to the evolving therapeutic paradigms in RA. In this review, we propose that reestablishment of macrophage equilibrium by inhibiting the development of, and/or eliminating, the proinflammatory macrophages will be an effective therapeutic approach for RA and other autoimmune diseases.

Objective To determine whether functional suppression of the catalytic domain of activation-induced cytidine deaminase (AID) can suppress the hyperreactive germinal center (GC) responses in BXD2 mice. Methods We generated transgenic BXD2 mice expressing a dominant-negative (DN) form of Aicda at the somatic hypermutation site (BXD2-Aicda-DN–transgenic mice). Real-time quantitative reverse transcriptase–polymerase chain reaction was used to determine the expression of Aicda and DNA damage/repair genes. Enzyme-linked immunosorbent assay was used to measure serum levels of autoantibodies and immune complexes (ICs). Development of GCs and antibody-containing ICs as well as numbers of proliferative and apoptotic cells were determined using flow cytometry and/or immunohistochemical analyses. Development of arthritis and kidney disease was evaluated histologically in 6–8-month-old mice. Results Suppression of the somatic hypermutation function of AID resulted in a significant decrease in autoantibody production without affecting the expression of DNA damage–related genes in GC B cells of BXD2-Aicda-DN–transgenic mice. There was decreased proliferation, increased apoptosis, increased expression of caspase 9 messenger RNA in GC B cells, and lower numbers of GCs in the spleens of BXD2-Aicda-DN–transgenic mice. Decreased GC response was associated with lower levels of IgG-containing ICs. Anti-IgM– and anti-CD40 plus anti-Ig–induced B cell proliferative responses were decreased in BXD2-Aicda-DN–transgenic mice. Conclusion Inhibition of the AID somatic hypermutation function in BXD2 mice suppressed development of spontaneous GCs, generation of autoantibody-producing B cells, and autoimmunity in BXD2 mice. Suppression of AID catalytic function to limit selection-based survival of GC B cells could become a novel therapy for the treatment of autoimmune disease.

We previously identified that autoreactive B cells from BXD2 mice can be targeted by IL-17, leading to upregulation of the expression of regulators of G-protein signaling (Rgs) genes that facilitated the development of spontaneous germinal centers. Little is known about the signaling pathway used by IL-17 to upregulate RGS. In the current study, we found that IL-17 rapidly activates the canonical NF-κB signaling pathway and that BXD2 B cells exhibit higher basal and activated phosphorylated p65 levels than B6 or BXD2-Il17ra−/− B cells. Inhibition of p65 phosphorylation downregulated RGS16 expression and abrogated the IL-17‑induced chemotactic arrest of B cells in response to CXCL12. Knockdown of TNFR-associated factor 6 or NF-κB activator 1 in 70Z/3 pre-B cells led to decreased Rgs16 expression, indicating that both of these two genes are involved in IL-17‑mediated activation of NF-κB signaling in B cells. These findings identify the signaling pathway regulated by IL-17 to contribute to the development of spontaneous germinal centers in autoimmune BXD2 mice.

The pathogenic connection of type I IFN and its role in regulating the migration response of Ag delivery by B cells into lymphoid follicles in an autoimmune condition has not been well-identified. Here, we show that there was a significantly larger population of marginal zone precursor (MZ-P) B cells, defined as being IgMhiCD1dhiCD21hiCD23hi in the spleens of autoimmune BXD2 mice compared with B6 mice. MZ-P B cells were highly proliferative compared with marginal zone (MZ) and follicular (FO) B cells. The intrafollicular accumulation of MZ-P B cells in proximity to germinal centers (GCs) in BXD2 mice facilitated rapid Ag delivery to the GC area, whereas Ag-carrying MZ B cells, residing predominantly in the periphery, had a lower ability to carry Ag into the GCs. IFN-α, generated by plasmacytoid dendritic cells, induced the expression of CD69 and suppressed the sphingosine-1-phosphate-induced chemotactic response, promoting FO-oriented Ag transport by MZ-P B cells. Knockout of type I IFN receptor in BXD2 (BXD2-Ifnαr−/−) mice substantially diffused the intrafollicular MZ-P B cell conglomeration and shifted their location to the FO-MZ border near the marginal sinus, making Ag delivery to the FO interior less efficient. The development of spontaneous GCs was decreased in BXD2-Ifnαr−/− mice. Together, our results suggest that the MZ-P B cells are major Ag-delivery B cells and that the FO entry of these B cells is highly regulated by type I IFN–producing plasmacytoid dendritic cells in the marginal sinus in the spleens of autoimmune BXD2 mice.

Background Chemokines are critical mediators of T-cell homing into inflamed skin. The complex nature of this multicellular response makes it difficult to analyse mechanisms mediating the early responses in vivo.

Development of adenovirus (Ad) vectors in the clinical context has highlighted that vector efficacy may be limited by the host humoral response due to pre-existing titers of neutralizing antibodies against the vector itself in humans. Further, multiple dosing of Ad vectors based on serotype 5 would be limited. Current immune evasion strategies being investigated by other laboratories are only applicable to non-replicating vectors. Therefore we have proposed ge- netic shielding as an alternate that would be applicable to both non-replicating and conditionally replicating Ad vectors. Genetic shielding would encapsulate fusion of a self-protein to Ad minor capsid protein, pIX, as a means to cloak immu- nogenic capsid epitopes and prevent neutralization of Ad vectors through Ad specific antibodies. In the development of a suitably shielded Ad vector we choose several self-proteins that we attempted to fuse to pIX. We have also used an indirect method to conjugate albumin to the capsid through an albumin binding domain fused to pIX. Despite attaining novel pIX modified Ad vectors we found that none of the pIX attached molecules in this study prevented neutralizing an- tibodies from halting gene transfer.

Beclin 1 is an essential mediator of autophagy and a regulator of cell growth and cell death. We examined the effect of Beclin 1 overexpression on the action of estradiol (E2) and two antiestrogens, raloxifene and 4-hydroxytamoxifen, in estrogen receptor α (ERα)-positive MCF-7 breast cancer cells. [3H]-thymidine incorporation studies showed that Beclin 1–overexpressing cells (MCF-7.beclin) had a lower proliferative response to E2 compared with cells transfected with vector control (MCF-7.control). There was only a 35% increase in [3H]-thymidine incorporation, after 24 hours of E2 treatment of MCF-7.beclin cells compared with untreated cells, whereas this increase was 2-fold for MCF-7.control cells. E2-induced changes in the expression of early-response genes were examined by real-time quantitiative PCR. There were significant differences in the pattern of expression of E2-induced genes c-myc, c-fos, Erg-1, and Nur77 between MCF-7.beclin and MCF-7.control cells two hours after treatment. Although E2-induced growth of MCF-7.control cells was completely inhibited by 500 nmol/L raloxifene or 500 nmol/L 4-hydroxytamoxifen, these concentrations of antiestrogens had no significant effect on the growth of MCF-7.beclin cells. Confocal microscopic and coimmunoprecipitation studies showed evidence for colocalization and association of Beclin 1 and ERα. In addition, E2 caused a decrease in Akt phosphorylation in MCF-7.beclin cells, compared with a 3-fold increase in MCF-7 cells, five minutes after treatment. These results indicate that Beclin 1 can down-regulate estrogenic signaling and growth response, and contribute to the development of antiestrogen resistance. This observation might be useful to define and overcome antiestrogen resistance of breast cancer. [Cancer Res 2008;68(19):7855–63]

A decrease in the expression of Th1 cytokines has been associated with age-related decrease in cytotoxic T-lymphocyte (CTL) function. We utilized an E1-deleted adenovirus (Ad) vector to deliver the murine interleukin-12 (IL-12) gene in order to enhance the antivirus CTL response. Wild-type (WT) Ad was administered 3 days after AdIL-12 treatment, when IL-12 production was at its peak and the anti-Ad antibody response had not yet begun to develop. Before receiving AdIL-12 treatment, aged (18 month old) mice exhibited a 58% decrease in the number of virus-specific CTLs, and a 30% decrease in in vivo CTL activity as compared to young (2 month old) mice. After AdIL-12 treatment, aged mice displayed a greater increase in IL-12 expression and endogenous production of interferon-gamma than observed in young mice. When infected with WT Ad, these AdIL-12-treated aged mice exhibited an increased in vivo CTL response and an in vitro proliferative response that was similar to those in young mice. The frequencies of occurrence of D(b)-E1Bp(+)CD8(+) T cells in the spleen, liver, and lung in aged mice were higher than the corresponding values in young mice. These results indicate that IL-12 treatment significantly promotes the virus-specific CTL response in aged mice and, more importantly, specifically targets the virally infected organs, such as the liver and lung, promoting enhanced CTL activity against the virus.

Interleukin 17 (IL-17) is a cytokine associated with inflammation, autoimmunity and defense against some bacteria. Here we show that IL-17 can promote autoimmune disease through a mechanism distinct from its proinflammatory effects. As compared with wild-type mice, autoimmune BXD2 mice express more IL-17 and show spontaneous development of germinal centers (GCs) before they increase production of pathogenic autoantibodies. We show that blocking IL-17 signaling disrupts CD4+ T cell and B cell interactions required for the formation of GCs and that mice lacking the IL-17 receptor have reduced GC B cell development and humoral responses. Production of IL-17 correlates with upregulated expression of the genes Rgs13 and Rgs16, which encode regulators of G-protein signaling, and results in suppression of the B cell chemotactic response to the chemokine CXCL12. These findings suggest a mechanism by which IL-17 drives autoimmune responses by promoting the formation of spontaneous GCs.