Expression and Aggregation of Recombinant Human Consensus Interferon-α Mutant by Pichia pastoris

A recombinant human consensus interferon-α mutant (cIFN) was expressed in Pichia pastoris. The maximum dry cell weight, cIFN concentration and antiviral activity were 160 g l⁻¹, 1.24 g l⁻¹ and 4.1 × 10⁷ IU ml⁻¹, respec tively. The cIFN secreted into the medium was in the form of aggregates dominantly by non-covalent interaction and partially by disulphide bond. When the fermentation supernatant was disaggregated with 6 M guanidine hydrochloride, the antiviral activity of cIFN achieved 2.2 × 10⁸ IU ml⁻¹.     

Gene–Gene and Gene-Sex Epistatic Interactions of MiR146a,IRF5, IKZF1, ETS1 and IL21 in Systemic Lupus Erythematosus

Several confirmed genetic susceptibility loci involved in the interferon signaling and Th17/B cell response for SLE in Chinese Han populations have been described. Available data also indicate that sex-specific genetic differences contribute to SLE susceptibility. The aim of this study was to test for gene–gene/gene-sex epistasis (interactions) in these known lupus susceptibility loci. Six single-nucleotide polymorphisms (SNPs) in MiR146a, IRF5, IKZF1, ETS1 and IL21 were genotyped by Sequenom MassArray system. A total of 1,825 subjects (858 SLE patients and 967 controls) were included in the final analysis. Epistasis was tested by additive model, multiplicative model and multifactor dimensionality reduction (MDR) method. Additive interaction analysis revealed interactions between IRF5 and IKZF1 (OR 2.26, 95% CI 1.48–3.44 [P = 1.21×10⁴]). A similar tendency was also observed between IL21 and ETS1 by parametric methods. In addition, multiple high dimensional gene-gene or gene-sex interactions (three-and four-way) were identified by MDR analysis. Our study identified novel gene–gene/gene-sex interactions in lupus. Furthermore, these findings highlight sex, interferon pathway, and Th17/B cells as important contributors to the pathogenesis of SLE.     

The Effect of Mycophenolate Mofetil on Disease Development in the gld.apoE ?/? Mouse Model of Accelerated Atherosclerosis and Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that is characterized by autoantibody production and inflammatory disease involving multiple organs. Premature atherosclerosis is a common complication of SLE and results in substantial morbidity and mortality from cardiovascular disease (CVD). The reasons for the premature atherosclerosis in SLE are incompletely understood, although chronic inflammation is thought to play an important role. There is currently no known preventative treatment of premature atherosclerosis in SLE. Mycophenolate mofetil (MMF) is an immunosuppressive agent that is commonly used for treatment of patients with SLE. In order to study the impact of this drug on murine lupus disease including premature atherosclerosis development, we treated gld.apoE^−/− mice, a model of SLE and accelerated atherosclerosis, with MMF. We maintained seven-week old gld.apoE^−/− mice on a high cholesterol Western diet with or without MMF. After 12 weeks on diet, mice receiving MMF showed decreased atherosclerotic lesion area compared to the control group. MMF treatment also improved the lupus phenotype, indicated by a significant decrease circulating autoantibody levels and ameliorating lupus nephritis associated with this model. This data suggests that the effects of MMF on the immune system may not only be beneficial for lupus, but also for inflammation driving lupus-associated atherosclerosis.     

Intractable Headaches,Ischemic Stroke,and Seizures Are Linked to the Presence of Anti-β2GPI Antibodies in Patients with Systemic Lupus Erythematosus

Background

Neuropsychiatric systemic lupus erythematosus (NPSLE) is a common and potentially fatal manifestation of SLE. Antiphospholipid antibodies (aPL) such as lupus anticoagulant (LA), anticardiolipin (aCL) and antibodies to β2glycoprotein I (anti-β2GPI), the most important aPL antigen, are thought to play a role in some forms of NPSLE. As of yet, their specific roles in NPSLE manifestations remain to be elucidated.

Methodology/Principal Findings

57 SLE patients (53 women) were assessed for LA, aCL and anti-β₂GPI twice, to determine persistent positivity. All patients were examined by neurology and psychiatry specialists. 69 healthy subjects were assessed as controls. NPSLE was diagnosed in 74% of patients. Headaches were the most prevalent manifestation of NPSLE (39%), followed by cerebrovascular disease (CVD) (23%), depressive disorders (19.0%), and seizures (14%). NPSLE and non-NPSLE patients showed comparable SLE activity and corticosteroid use. In 65% of patients neuropsychiatric manifestations preceded SLE diagnosis. aPL profiles of NPSLE patients and non-NPSLE patients were similar. Headaches and ischemic stroke were independently associated with anti-β₂GPI-IgM (OR=5.6; p<0.05), and seizures were linked to anti-β₂GPI-IgG (OR=11.3; p=0.01).

Conclusions

In SLE patients, neuropsychiatric manifestations occur frequently and early, often before the disease is diagnosed. Autoantibodies to β2GPI are linked to non-specific headaches, ischemic stroke and seizures, and show a better predictive value than aCL and LA. These findings may help to improve the diagnosis of NPSLE and should prompt further studies to characterize the role of anti-β2GPI in the pathogenesis of this condition.     

Tongshu Capsule Down-Regulates the Expression of Estrogen Receptor α and Suppresses Human Breast Cancer Cell Proliferation

The Tongshu Capsule (TSC) is a prevalent form of traditional Chinese medicine widely used for its purported effects in treating mammary gland hyperplasia and inflammation. Though successful in several clinical studies, there is no clear evidence as to why TSC has a positive treatment effect, and little known about underlying mechanism that may account for it. In this study, we examined the effects of TSC and found that it has a comparatively strong growth inhibition on ERα positive breast cancer cells. TSC seems to cause G1 cell cycle arrest instead of apoptosis. Interestingly, TSC also down-regulated the expression of ERα and Cyclin D1. Consistently, TSC suppressed E2 mediated ERα downstream gene expression and cell proliferation in ERα positive breast cancer cell lines MCF7 and T47D. Depletion of ERα partially abolished the effects of TSC on the decrease of Cyclin D1 and cell viability. Our findings suggest that TSC may have therapeutic effects on ERα positive breast cancers and moreover that TSC may suppress breast epithelial cell proliferation by inhibiting the estrogen pathway.     

A Case Report of Widespread Majocchi’s Granuloma in a Patient with Systemic Lupus Erythematosus

Majocchi’s granuloma is an intracutaneous or subcutaneous granulomatous inflammation caused by invasion of dermatophytic fungus, especially Trichophyton rubrum. This type of lesion is misdiagnosed frequently without proper auxiliary examination. Here, we report a case of widespread Majocchi’s granuloma caused by T. rubrum in a 35-year-old woman with systemic lupus erythematosus for 9 years. The patient was initially misdiagnosed as SLE-associated skin lesions, which delayed her treatment and resulted in severe multiple disseminated lesions. After confirmed as Majocchi’s granuloma, the patient was cured after 11-month treatment with terbinafine.     

Interferon-β Suppresses Murine Th1 Cell Function in the Absence of Antigen-Presenting Cells

Interferon (IFN)-β is a front-line therapy for the treatment of the relapsing-remitting form of multiple sclerosis. However, its immunosuppressive mechanism of function remains incompletely understood. While it has been proposed that IFN-β suppresses the function of inflammatory myelin antigen-reactive T cells by promoting the release of immunomodulatory cytokines such as IL-27 from antigen-presenting cells (APCs), its direct effects on inflammatory CD4⁺ Th1 cells are less clear. Here, we establish that IFN-β inhibits mouse IFN-γ⁺ Th1 cell function in the absence of APCs. CD4⁺ T cells express the type I interferon receptor, and IFN-β can suppress Th1 cell proliferation under APC-free stimulation conditions. IFN-β-treated myelin antigen-specific Th1 cells are impaired in their ability to induce severe experimental autoimmune encephalomyelitis (EAE) upon transfer to lymphocyte-deficient Rag1^-/- mice. Polarized Th1 cells downregulate IFN-γ and IL-2, and upregulate the negative regulatory receptor Tim-3, when treated with IFN-β in the absence of APCs. Further, IFN-β treatment of Th1 cells upregulates phosphorylation of Stat1, and downregulates phosphorylation of Stat4. Our data indicate that IFN-γ-producing Th1 cells are directly responsive to IFN-β and point to a novel mechanism of IFN-β-mediated T cell suppression that is independent of APC-derived signals.     

Phenotypic Characterization of Autoreactive B Cells—Checkpoints of B Cell Tolerance in Patients with Systemic Lupus Erythematosus

DNA-reactive B cells play a central role in systemic lupus erythematosus (SLE); DNA antibodies precede clinical disease and in established disease correlate with renal inflammation and contribute to dendritic cell activation and high levels of type 1 interferon. A number of central and peripheral B cell tolerance mechanisms designed to control the survival, differentiation and activation of autoreactive B cells are thought to be disturbed in patients with SLE. The characterization of DNA-reactive B cells has, however, been limited by their low frequency in peripheral blood. Using a tetrameric configuration of a peptide mimetope of DNA bound by pathogenic anti-DNA antibodies, we can identify B cells producing potentially pathogenic DNA-reactive antibodies. We, therefore, characterized the maturation and differentiation states of peptide, (ds) double stranded DNA cross-reactive B cells in the peripheral blood of lupus patients and correlated these with clinical disease activity. Flow cytometric analysis demonstrated a significantly higher frequency of tetramer-binding B cells in SLE patients compared to healthy controls. We demonstrated the existence of a novel tolerance checkpoint at the transition of antigen-naïve to antigen-experienced. We further demonstrate that patients with moderately active disease have more autoreactive B cells in both the antigen-naïve and antigen-experienced compartments consistent with greater impairment in B cell tolerance in both early and late checkpoints in these patients than in patients with quiescent disease. This methodology enables us to gain insight into the development and fate of DNA-reactive B cells in individual patients with SLE and paves the way ultimately to permit better and more customized therapies.     

Can Micro RNA-24 Affect the Cardiovascular Morbidity in Systemic Lupus Erythematosus by Targeting YKL-40?

Background:Systemic lupus erythematosus (SLE) is an autoimmune disease with inflammatory nature. One of the leading causes of death in SLE patients is cardiovascular (CVS) morbidity. MiRNA-24 is highly expressed in vascular endothelial cells (VECs). This dysregulated expression pattern is associated with dysfunction or even damage of VECs and leads to the occurrence of cardiovascular diseases. YKL- 40 is an inflammatory glycoprotein involved in the pathogenesis of endothelial dysfunction and thereby atherosclerosis. In this work, we aimed at illustrating the possible role of miR-24 and its target YKL-40 in the pathogenesis of the CVS morbidity associated with SLE.Methods:This work was conducted on 40 SLE patients and 40 healthy controls. Quantitative real-time PCR (qPCR) was done to estimate the expression level of miRNA-24 in serum. In addition, we measured the serum level of YKL-40 using ELISA.Results:miR-24-fold change was found to be down-regulated, whereas serum YKL- 40 was up-regulated among SLE patients with observed significant and negative correlation between the two parameters.Conclusion:Our study provided an insight about the role of miR-24 and its target serum YKL-40 protein in the development of SLE-related inflammation and atherosclerosis.Key Words: miRNA-24, SDC-1, SLE, VCAM, YKL-40     

Expression of Recombinant Human Interferon-γ with Antiviral Activity in the Bi-Cistronic Baculovirus-Insect/Larval System

A bi-cistronic baculovirus-insect/larval system containing a polyhedron promoter, an internal ribosome entry site (IRES), and an egfp gene was developed as a cost-effective platform for the production of recombinant human interferon gamma (rhIFN-γ). There was no significant difference between the amounts of rhIFN-γ produced in the baculovirus-infected Spodoptera frugiferda 21 cells grown in serum-free medium and the serum-supplemented medium, while the Trichoplusia ni (T. ni) and Spodoptera exigua (S. exigua) larvae afforded rhIFN-γ amounting to 1.08±0.04 and 9.74±0.35 μg/mg protein respectively. The presence of non-glycosylated and glycosylated rhIFN-γ was confirmed by immunoblot and lectin blot. The immunological activity of purified rhIFN-γ, with 96% purity by Nickel (II)-nitrilotriacetic acid (Ni-NTA) affinity chromatography, was similar to that commercially available. Moreover, the rhIFN-γ protein from T. ni had more potent antiviral activity. These findings suggest that this IRES-based expression system is a simple and inexpensive alternative for large-scale protein production in anti-viral research.     

Pregnancy-Related Systemic Lupus Erythematosus: Clinical Features,Outcome and Risk Factors of Disease Flares — A Case Control Study

Objective

To investigate the clinical features, outcome, and risk factors of disease flares in patients with pregnancy-related lupus (PRL).

Methods

Medical charts of 155 consecutive PRL inpatients were systematically reviewed, including demographic data, clinical features, laboratory findings, treatment, complications, and outcome.

Results

PRL cases were divided into active (a-PRL) (n = 82, 53.0%) and stable lupus (s-PRL) (n = 73, 47.0%). Compared with nonpregnant active female systemic lupus erythematosus (SLE) patients, a-PRL including new-onset lupus (n-PRL) and flare lupus (f-PRL) (n = 41 respectively), had a higher incidence of renal and hematological involvement but less mucocutaneous and musculoskeletal involvement (p<0.05). The incidence of preeclampsia/eclampsia, fetal loss, and preterm birth were significantly higher in a-PRL than in s-PRL (p<0.05). Despite receiving a more vigorous glucocorticoid treatment, a-PRL mothers had a poorer prognosis (p<0.001). Five (6.1%) of them died and 13 (15.9%) developed severe irreversible organ failure, whereas none of these events was observed in the s-PRL group. Multivariate logistic analysis indicated that a history of lupus flares and serological activity (hypocomplementemia and/or anti-dsDNA positivity) at the time of conception were associated with lupus flares in PRL mothers.

Conclusions

SLE patients with a flare history and serological activity at the time of conception were at an increased risk of disease flares during pregnancy and puerperium. a-PRL patients were more prone to renal and hematological involvement, pregnancy complications, and a poorer prognosis despite more vigorous glucocorticoid treatment.     

Association of Increased Frequencies of HLA-DPB1*05∶01 with the Presence of Anti-Ro/SS-A and Anti-La/SS-B Antibodies in Japanese Rheumatoid Arthritis and Systemic Lupus Erythematosus Patients

Introduction

Autoantibodies to ribonucleoprotein are associated with a variety of autoimmune diseases, including rheumatoid arthritis (RA). Many studies on associations between human leukocyte antigen (HLA) alleles and RA have been reported, but few have been validated in RA subpopulations with anti-La/SS-B or anti-Ro/SS-A antibodies. Here, we investigated associations of HLA class II alleles with the presence of anti-Ro/SS-A or anti-La/SS-B antibodies in RA.

Methods

An association study was conducted for HLA-DRB1, DQB1, and DPB1 in Japanese RA and systemic lupus erythematosus (SLE) patients that were positive or negative for anti-Ro/SS-A and/or anti-La/SS-B antibodies.

Results

An increased prevalence of certain class II alleles was associated with the presence of anti-Ro/SS-A antibodies as follows: DRB1*08∶03 (Pc = 3.79×10⁻⁵, odds ratio [OR] 3.06, 95% confidence interval [CI] 1.98–4.73), DQB1*06∶01 (Pc = 0.0106, OR 1.70, 95%CI 1.26–2.31), and DPB1*05∶01 (Pc = 0.0040, OR 1.55, 95%CI 1.23–1.96). On the other hand, DRB1*15∶01 (Pc = 0.0470, OR 3.14, 95%CI 1.63–6.05), DQB1*06∶02 (Pc = 0.0252, OR 3.14, 95%CI 1.63–6.05), and DPB1*05∶01 (Pc = 0.0069, OR 2.27, 95% CI 1.44–3.57) were associated with anti-La/SS-B antibodies. The DPB1*05∶01 allele was associated with anti-Ro/SS-A (Pc = 0.0408, OR 1.69, 95% CI 1.19–2.41) and anti-La/SS-B antibodies (Pc = 2.48×10⁻⁵, OR 3.31, 95%CI 2.02–5.43) in SLE patients.

Conclusion

HLA-DPB1*05∶01 was the only allele associated with the presence of both anti-Ro/SS-A and anti-La/SS-B antibodies in Japanese RA and SLE patients.     

Characterization and Evaluation of Key Sites in the Peptide Inhibitor of TAB1/p38α Interaction

A combination of computation techniques and peptide mutants have been used to determine the binding site and amino acid residues on the inhibitor peptide that are critical for binding to Mitogen-activated protein kinase 14 (p38α). In our previous research work, the functional peptide, named as PT5, target to p38α, was obtained based on the theoretical complex structure of p38α and [transforming growth factor-β (TGF-β)-activated protein kinase 1 (TAK1)-binding protein 1] (TAB1). Based on the computer-guided ab initio modeling method, the inhibitor peptide PT5 and its mutants were modeled. Furthermore, the 3-D complex structures of PT5 or its mutants and p38α were constructed using molecular docking and dynamics simulation methods. The key residues in the peptide PT5 involved in binding interaction to p38α were predicted. According to the 3-D theoretical complex structure PT5/ p38α, the interaction binding mode between PT5 and p38α was analyzed using distance geometry technology. Mutants of the peptide PT5 was used to evaluate the bio-function when the critical residues were mutated. The mutant experimental results identified the key residues in PT5, i.e. Thr¹¹ and Asp¹² and determined the core sequence of PT5 binding to p38α. Based on the results, optimized peptides compounds could be developed for treating myocardial ischemia/reperfusion (I/R) injury in clinical.     

Changes in the Anatomic and Microscopic Structure and the Expression of HIF-1α and VEGF of the Yak Heart with Aging and Hypoxia

The study aimed to identify the changes of anatomic and microscopic structure and the expression and localization of hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) in the myocardium and coronary artery of the yak heart adapted to chronic hypoxia with aging. Thirty-two yaks (1 day, 6 months, 1 year, 2 years, and 5 year old) were included, and immunoelectronmicroscopy, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) were used. Right ventricular hypertrophy was not present in yaks with aging. There was no intima thickening phenomenon in the coronary artery. The ultrastructure of myofibrils, mitochondria, and collagen fibers and the diameter and quantity of collagen changed significantly with aging. The enzymatic activity of complexes I, II, and V increased with age. Immunogold labeling showed the localization of HIF-1α protein in the cytoplasm and nuclei of endothelial cells and cytoplasm of cardiac muscle cells, and VEGF protein in the nuclei and perinuclei areas of smooth muscle cells of coronary artery, and in the cytoplasm and nuclei of endothelial cells. ELISA results showed that HIF-1α secretion significantly increased in the myocardium and coronary artery from an age of 1 day to 2 years of yaks and decreased in old yaks. However, VEGF protein always increased with aging. The findings of this study suggest that 6 months is a key age of yak before which there are some adaptive changes to deal with low-oxygen environment, and there is a maturation of the yak heart from the age of 6 months to 2 years.     

The Effect of TGF-β1 on Expression of Chemokine and Its Receptor in Human Decidual Stromal Cells

为探讨转化生长因子β1(TGF-β1)在蜕膜基质细胞中发挥免疫调节作用的机制,本研究以人妊娠初期的蜕膜基质细胞为研究对象,经0 ng/ml、1 ng/ml、5 ng/ml和10 ng/ml的TGF-β1处理后,运用RT-PCR方法检测趋化因子mRNA的表达,Western-blot检测趋化因子蛋白质的表达.结果表明:在mRNA水平和蛋白水平,高浓度的TGF-β1能够显著的下调蜕膜基质细胞中趋化因子配体CX3CL1、CXCL12和CXCL16的表达,有意义的上调趋化因子受体CXCR4和CXCR6的表达.研究结果提示,TGF-β1对趋化因子配体/受体有显著的调节作用,并通过趋化因子参与母胎界面的免疫调节.     

Elemental Analysis of Whole and Protein Separated Blood Serum of Patients with Systemic Lupus Erythematosus and Sjögren’s Syndrome

Data on the effects of selenium supplementation on clinical signs and metabolic profiles in women at risk for intrauterine growth restriction (IUGR) are scarce. This study was designed to assess the effects of selenium supplementation on clinical signs and metabolic status in pregnant women at risk for IUGR. This randomized double-blind placebo-controlled clinical trial was performed among 60 women at risk for IUGR according to abnormal uterine artery Doppler waveform. Participants were randomly assigned to intake either 100 μg selenium supplements as tablet (n = 30) or placebo (n = 30) for 10 weeks between 17 and 27 weeks of gestation. After 10 weeks of selenium administration, a higher percentage of women in the selenium group had pulsatility index (PI) of <1.45) (P = 0.002) than of those in the placebo group. In addition, changes in plasma levels of total antioxidant capacity (TAC) (P < 0.001), glutathione (GSH) (P = 0.008), and high-sensitivity C-reactive protein (hs-CRP) (P = 0.004) in the selenium group were significant compared with the placebo group. Additionally, selenium supplementation significantly decreased serum insulin (P = 0.02), homeostasis model of assessment-estimated insulin resistance (HOMA-IR) (P = 0.02), and homeostatic model assessment for B-cell function (HOMA-B) (P = 0.02) and significantly increased quantitative insulin sensitivity check index (QUICKI) (P = 0.04) and HDL-C levels (P = 0.02) compared with the placebo. We did not find any significant effect of selenium administration on malondialdehyde (MDA), nitric oxide (NO), fasting plasma glucose (FPG), and other lipid profiles. Overall, selenium supplementation in pregnant women at risk for IUGR resulted in improved PI, TAC, GSH, hs-CRP, and markers of insulin metabolism and HDL-C levels, but it did not affect MDA, NO, FPG, and other lipid profiles.Clinical trial registration number http://www.irct.ir: IRCT201601045623N64.