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The purpose of this study is to investigate the clinical manifestations in patients with early primary Sjögren’s syndrome (pSS) based on the severity score found by salivary gland ultrasonography. Thirty-five newly diagnosed patients with early pSS were enrolled and divided into mild (score 0–1) and severe (score 2–3) groups according to the salivary gland ultrasonography grade (SGUS) scores at baseline. Clinical evaluation, ESSPRI and ESSDAI index values, sicca symptoms of the mouth, salivary capacity, and serum autoantibodies and cytokines were investigated. The mean age of pSS patients at diagnosis was 49.9 ± 11.9 years, and the mean duration of sicca symptoms was 0.58 years. ESSPRI (EULAR Sjögren’s syndrome patient report index) and ESSDAI (EULAR Sjögren’s syndrome disease index) scores were 15.97 and 4.77, respectively. Clinical manifestations, including the low production of saliva and autoantibody production, such as antinuclear antibodies, rheumatoid factor, and anti-SSA antibody, were found. A higher prevalence of rheumatoid factor (p = 0.0365) and antinuclear antibody (p = 0.0063) and a higher elevation of total IgG (p = 0.0365) were found in the severe group than in the mild group. In addition, the elevated titer of IL-25 was detected in the severe group than in the mild group. This observation indicated that salivary gland ultrasonography grade (SGUS) scans may help physicians diagnose pSS and the elevated titer of IL-25 in patients may be implicated in the pathogenesis of pSS.

The deficiency of dead cell clearance is a prominent pathogenic factor in systemic lupus erythematosus (SLE). In this study, the overexpression of miR-210-5p resulted in the accumulation of secondary necrotic cells (SNECs) in macrophages through the reduction of protein degradation. The upreguation of miR-210-5p inhibited NADPH oxidase (NOX) activation, reactive oxygen species (ROS) generation, and SNEC clearance. miR-210-5p overexpression suppressed Sp1 and HSCARG expression, and the knockdown of SP1 and HSCARG inhibited NOX expression and superoxide production in macrophages. Furthermore, patients with active SLE expressed a higher level of miR-210-5p and lower expression of SP1 and HSCARG in peripheral blood mononuclear cells. In summary, our findings indicate that the upregulation of miR-210-5p increases the accumulation of SNECs through a decrease in the Sp1-and HSCARG-mediated NOX activity and ROS generation in macrophages. Our results also suggest that targeting miR-210-5p may have therapeutic potential for SLE.

The purpose of this study is to investigate the clinical manifestations in patients with early primary Sjögren’s syndrome (pSS) based on the severity score found by salivary gland ultrasonography. Thirty-five newly diagnosed patients with early pSS were enrolled and divided into mild (score 0–1) and severe (score 2–3) groups according to the salivary gland ultrasonography grade (SGUS) scores at baseline. Clinical evaluation, ESSPRI and ESSDAI index values, sicca symptoms of the mouth, salivary capacity, and serum autoantibodies and cytokines were investigated. The mean age of pSS patients at diagnosis was 49.9 ± 11.9 years, and the mean duration of sicca symptoms was 0.58 years. ESSPRI (EULAR Sjögren’s syndrome patient report index) and ESSDAI (EULAR Sjögren’s syndrome disease index) scores were 15.97 and 4.77, respectively. Clinical manifestations, including the low production of saliva and autoantibody production, such as antinuclear antibodies, rheumatoid factor, and anti-SSA antibody, were found. A higher prevalence of rheumatoid factor (p = 0.0365) and antinuclear antibody (p = 0.0063) and a higher elevation of total IgG (p = 0.0365) were found in the severe group than in the mild group. In addition, the elevated titer of IL-25 was detected in the severe group than in the mild group. This observation indicated that salivary gland ultrasonography grade (SGUS) scans may help physicians diagnose pSS and the elevated titer of IL-25 in patients may be implicated in the pathogenesis of pSS.

A chemiresistive biosensor is described for simple and selective detection of miRNA-21. We developed chemical vapor deposition (CVD) and low-damage plasma treatment (LDPT)-treated bilayer graphene composite of graphene oxide/graphene (GO/GR) for the determination of a reliable biomarker. We have successfully overcome the self-limiting growth mechanism by using CVD method to grow more than one layer of graphene on copper foil. In addition, LDPT can be used to form GO/GR structures for chemiresistive biosensor applications. Due to the direct formation of BLGR (bilayer graphene), the coupling between graphene layers is theoretically superior to that of stacked BLGR, which is also confirmed by the blue shift of the characteristic peak of graphene in Raman spectroscopy. The shift is about double compared with that of stacked BLGR. Based on the results, the limit of detection for the target miRNA-21 was calculated to be 5.20 fM and detection rage is calculated as 100 fM to 10 nM, which is obviously better performance. Compared with previous work, this chemiresistive biosensor has good selectivity, and stability towards detection of miRNA-21. The ability to detect miRNA-21 in different biological fluids was almost identical to that in pH 7.4 phosphate-buffered saline (PBS). Thus, the proposed bilayer GO/GR of modified chemiresistive biosensor may potentially be applied to detect cancer cells in clinical examinations.

Background: Evidence indicates a causal link between cytomegalovirus (CMV) infection and the triggering of systemic lupus erythematosus (SLE). Animal studies have revealed that CMV phosphoprotein 65 (pp65) induces autoantibodies against nuclear materials and causes the autoantibody attack of glomeruli. IgG eluted from the glomeruli of CMVpp65-peptide-immunized mice exhibited cross-reactivity against dsDNA and TATA-box-binding protein associated factor 9 (TAF9). Whether the elevation of anti-TAF9 IgG is associated with anti-CMV reactivity in human lupus remains unclear. Methods: The sera from patients with rheumatic diseases, including ankylosing spondylitis (AS), gout, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and Sjögren syndrome (SS) were examined using ELISA for antibodies of CMV, CMVpp65, and TAF9. Results: In total, 83.8% of the rheumatic patients had acquired CMV infections. The SLE patients had a high prevalence of anti-CMV IgM. The highest seropositivity rates for anti-HCMVpp65 and anti-TAF9 IgG were observed in the SLE patients. Purified anti-CMVpp65 IgG from CMVpp65/TAF9 dual-positive SLE sera reacted to both TAF9 and dsDNA. An increased prevalence of proteinuria and low hemoglobin levels were found in CMV IgG- and CMVpp65 IgG-positive SLE patients. Conclusions: This observation suggests that immunity to CMVpp65 is associated with cross-reactivity with TAF9 and dsDNA and that it is involved in the development of clinical manifestations in SLE.

Background This study investigates the association between exposure to urate-lowering drugs (ULDs) and progression and recovery from chronic kidney disease (CKD). Methods We identified 5860 incident gout patients at Chang Gung Memorial Hospital from 2012 to 2015. Propensity score (PS)-weighted Cox proportional hazards model was used to estimate hazard ratios (HRs) for CKD progression and improvement. A separate analysis was conducted to assess the HR for CKD progression and CKD recovery among those with worsening CKD. Results The incidence of CKD progression among allopurinol, febuxostat and uricosuric agent users were 1.98, 1.88 and 1.64 per 1000 person-days. Compared with allopurinol users, the PS-weighted HR (95% confidence intervals [CIs]) was 1.77 (0.85–1.76) for febuxostat users and 1.37 (0.71–1.37) for uricosuric agent users for CKD progression and 1.43 (1.26–1.62) for febuxostat users and 1.00 (0.88–1.14) for uricosuric agent users for CKD improvement. Compared to allopurinol users, the HRs for CKD progression were 1.14 (0.80–1.66) for febuxostat users and 0.92 (0.67–1.31) for uricosuric agent users. Among 741 patients who had CKD progression, the incidence of CKD recovery was 1.33, 6.21 and 3.53 per 1000 person-days for allopurinol, febuxostat and uricosuric agent users. The HRs (95% CIs) for recovery in febuxostat and uricosuric agent users were 2.17 (1.40–3.47) and 1.80 (1.20–2.83) compared to allopurinol users. Conclusions CKD progression and recovery are common in gout patients using ULDs. Febuxostat and benzbromarone were associated with a similar risk of CKD progression with allopurinol, which has a poorer recovery compared with other ULDs.

Background: Gut microbiota is thought to regulate immune homeostasis. The association between gut microbiota and the development of systemic lupus erythematosus remains unclear. This study aims to investigate the differential alteration of gut microbiota after the induction of lupus in a murine model with viral peptide of human cytomegalovirus (HCMV), which is an established murine model of induced lupus. Results: Specifically, we injected HCMVpp65 peptide in peritoneal space in NZB/W F1 mice to induce lupus. Three arms of animal treatment were prepared: intraperitoneal injection of HCMVpp65 peptide, intraperitoneal injection of adjuvant alone, and none injected. Feces of the study animals were collected before and every two weeks after the lupus induction for 16S rRNA sequencing. At 24 weeks of age, pathological investigation of renal tissue from sacrificed mice was conducted. Statistical analysis for dynamics and alteration of the fecal microbiota and lupus-like-activity-related functional prediction of bacterial communities was performed. HCMVpp65 peptide immunization induced lupus-like activities, with a higher level of anti-dsDNA antibodies, creatinine and proteinuria, and severe glomerular damage, compared to the mice treated with none or adjuvant only. A higher Simpson diversity value was measured in mice with injection of HCMVpp65 peptide than those with injection of adjuvant or none, but there have no difference in the species richness estimates, ACE and Chao1. Statistical Analysis of Metagenomic Profiles (STAMP) showed a higher relative abundance of the family (Saccharimonadaceae, Marinifiaceae, and Desulfovibrionaceae) and genera (Candidatus Saccharimonas, Roseburia, Odoribacter, and Desulfovibrio) that were found in HCMVpp65 peptide treated mice, compared to mice injected with adjuvant or none. The predicted metagenomic taxonomic profile showed statistically significant enrichment of flagellar assembly, bacterial motility, and chemotaxis. Conclusion: A significant correlation between increased related genera abundance (Candidatus Saccharimonas, Roseburia, Odoribacter, and Desulfovibrio) and HCMVpp65 peptide immunization induced lupus-like activities was observed. This study confirmed significant changes in gut microbiota after the onset of lupus in a murine animal model.

Human cytomegalovirus (HCMV) has been linked to the triggering of systemic lupus erythematosus (SLE). We proposed that B cell epitope region of HCMV phosphoprotein 65 (HCMVpp65)422–439 mimics an endogenous antigen and initiates lupus-like autoimmunity. Amino acid homology between HCMVpp65422-439 and TAF9134-144 (TATA-box binding protein associated factor 9, TAF9) was investigated using a similarity search in NCBI protein BLAST program (BLASTP). A murine model was used to confirm their antigenicity and ability to induce lupus-like symptoms. HCMVpp65422-439 induced immune responses with the presence of specific antibodies against HCMVpp65422-439 and TAF9134-144, as well as anti-nuclear and anti-double-stranded (ds)DNA antibodies that are characteristic of SLE. In addition, the majority of HCMVpp65422-439 and TAF9134-144 immunized mice developed proteinuria, and their renal pathology revealed glomerulonephritis with typical abnormalities, such as mesangial hypercellularity and immune complex deposition. Immunoglobulin eluted from the glomeruli of HCMVpp65422-439 immunized mice showed cross-reactivity with TAF9134-144 and dsDNA. Increased anti-TAF9 antibody activity was also observed in the sera from SLE patients compared with healthy people and disease controls. Molecular mimicry between HCMVpp65 peptide and host protein has the potential to drive lupus-like autoimmunity. This proof-of-concept study highlights the mechanisms underlying the link between HCMV infection and the induction of SLE.

Objectives To examine whether gout is an independent risk factor for total joint replacement (TJR) and whether urate-lowering treatment (ULT) reduces this risk. Methods Using the Taiwan National Health Insurance database and the UK Clinical Practice Research Datalink, 74 560 Taiwan patients and 34 505 UK patients with incident gout were identified and age and sex matched to people without gout. Cox proportional hazards models and condition logistic regression were used to examine the risk of TJR in gout patients and the association between cumulative defined daily dose (cDDD) of ULT and TJR. Results The prevalence rates of TJR in the patients at the time of diagnosis of gout and in people without gout were 1.16% vs 0.82% in Taiwan and 2.61% vs 1.76% in the UK. After a gout diagnosis, the incidence of TJR was higher in the patients with gout compared with those without (3.23 vs 1.91 cases/1000 person-years in Taiwan and 6.87 vs 4.61 cases/1000 person-years in the UK), with adjusted HRs of 1.56 (95% CI 1.45, 1.68) in Taiwan and 1.14 (1.05, 1.22) in the UK. Compared with patients with gout with 180 cDDD ULT in Taiwan. In the UK, the respective ORs were 1.09 (0.83, 1.42), 0.93 (0.68, 1.27) and 1.08 (0.94, 1.24). Conclusion This population-based study provides evidence from two nation populations that gout confers significant TJR risk, which was not reduced by current ULT.

Background:Systemic lupus erythematosus (SLE) is a multifactorial disease. Gut microbiota is an important environmental factor for SLE1. The perturbation of gut microbiota is often observed at onset or during the disease course. The fragment of HCMV phosphoprotein 65422-439(HCMVpp65422-439) containing B cell epitopes has been reported to elicit humoral immunity and accelerate the autoimmune response in murine lupus2,3. However, little is there to know about the interplay between viral trigger for SLE and the change of gut microbiota during lupus progression.Objectives:By using a murine lupus model with NZB/W F1, we investigated the differential alteration in gut microbiota associated with the progression of lupus disease in HCMVpp65422-439immunized mice and control mice.Methods:Ten weeks-old NZB/W F1 mice were given or not given an intraperitoneal injection of 100-μg HCMVpp65422-439peptide biweekly for four times. Fecal samples, urine and blood of mice were collected once every two weeks followed by 16S rRNA genes sequencing and ELISA tests. The pathological investigation of renal tissue from sacrificed mice was conducted at 24 weeks of mice age. Statistical analysis for dynamics and alteration of the gut microbiota as well as functional prediction of bacterial communities related to the progression of lupus-like activity was performed.Results:HCMVpp65422-439immunization results in the onset of lupus-like activities in NZB/W F1 mice with a higher titer of anti-dsDNA antibody, creatinine and proteinuria, and severe glomerular damage (figure 1). Also, higher diversity and increased family abundance of several bacterial species were observed in HCMVpp65422-439immunized mice (Table 1 and Figure 2a). The predicted metagenomic taxonomic profile in NZB/W F1 mice showed statistically significant enrichment of flagellar assembly, bacterial motility, and chemotaxis (Figure 2b). Spearman’s correlation analysis revealed that a significant association between the increased relative family abundance forSaccharimonadaceae, Marinifilaceae, Desulfovibrionaceae,andRikenellaceaeand HCMVpp65422-439induced lupus-like activity in NZB/W F1 mice (Figure 2c).Table 1.Significant test of microbial community structure between two groupsMRPPAdonisAnosimGroupsE-ΔPR2PRPPre-disease vs. Control0.430.10.770.110.112Pre-disease vs. HCMVpp65422-4390.520.0070.550.0090.970.01Control vs. HCMVpp65422-4390.450.0130.350.0140.580.019Conclusion:Our results demonstrated that HCMVpp65422-439immunization induced the change in gut microbiota composition and suggested the association of gut microbiota alteration with lupus-like activity in NZB/W F1 mice.References:[1]Azzouz D, Omarbekova A, Heguy A, Schwudke D, Gisch N, Rovin BH, et al. Lupus nephritis is linked to disease-activity associated expansions and immunity to a gut commensal. Ann Rheum Dis. 2019;78(7):947-56.[2]Sebastiani GD, Iuliano A, Canofari C, and Bracci M. Cytomegalovirus infection in Systemic Lupus Erythematosus: report of four cases challenging the management of the disease, and literature review. Lupus. 2019;28(3):432-7.[3]HoHsieh A, Wang CM, Wu YJ, Chen A, Chang MI, and Chen JY. B cell epitope of human cytomegalovirus phosphoprotein 65 (HCMV pp65) induced anti-dsDNA antibody in BALB/c mice. Arthritis Res Ther. 2017;19(1):65Disclosure of Interests: :None declared

Human cytomegalovirus (HCMV) infection has been implicated in the development of autoimmunity, including systemic lupus erythematosus (SLE). Previously we reported that HCMV phosphoprotein 65 (pp65) could induce early onset of autoantibody and glomerulonephritis on lupus-prone NZB/W mice. This study further examined whether the B cell epitope(s) in pp65 is able to drive the development of autoantibody. Sera from SLE patients or HCMVpp65-immunized mice were analyzed for anti-nuclear antibody by immunoblotting, enzyme-linked immunosorbent assay (ELISA), immunofluorescent stain and Crithidia luciliae stain. The deposition of immunoglobulin to the kidney was also examined by immunofluorescent stain. The interactions between pp65 sub-fragment to cellular proteins were revealed by yeast two-hybrid analyses. Our results showed that most SLE patients possessed antibodies to the C-terminal half of the HCMVpp65 antigen. Of these positive sera, 73% were also positive to the pp65336-439 sub-fragment. The immunization of pp65336-439 induced formation of multiple anti-nuclear antibodies, including anti-chromatin, anti-centriole, anti-mitotic spindle type I/II (MSA I/II) and a significant elevation of anti-double-stranded DNA (anti-dsDNA) antibodies on BALB/c mice. Yeast two-hybrid analyses revealed the binding of pp65336-439 sub-fragment to cellular proteins. Immunoglobulin deposition on glomeruli was also detected on pp65336-439-immunized mice. Our data suggested that HCMVpp65336-439 sub-fragment may induce cross-reactive antibodies to several nuclear antigens, which could contribute to the development of autoimmunity in genetic-suspected individuals.