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Abstract Background This dynamic nomogram model was developed to predict the probability of fetal loss in pregnant patients with systemic lupus erythematosus (SLE) with mild disease severity before conception. Methods An analysis was conducted on 314 pregnancy records of patients with SLE who were hospitalized between January 2015 and January 2022 at Shenzhen People's Hospital, and the Longhua Branch of Shenzhen People's Hospital. Data from the Longhua Branch of the Shenzhen People's Hospital were utilized as an independent external validation cohort. The nomogram, a widely used statistical visualization tool to predict disease onset, progression, prognosis, and survival, was created after feature selection using multivariate logistic regression analysis. To evaluate the model prediction performance, we employed the receiver operating characteristic curve, calibration curve, and decision curve analysis. Results Lupus nephritis, complement 3, immunoglobulin G, serum albumin, C-reactive protein, and hydroxychloroquine were all included in the nomogram model. The model demonstrated good calibration and discriminatory power, with an area under the curve of 0.867 (95% confidence interval: 0.787–0.947). According to decision curve analysis, the nomogram model exhibited clinical importance when the probability of fetal loss in patients with SLE ranged between 10 and 70%. The predictive ability of the model was demonstrated through external validation. Conclusion The predictive nomogram approach may facilitate precise management of pregnant patients with SLE with mild disease severity before conception.

Abstract Introduction Ixekizumab, an interleukin 17A (IL-17A) inhibitor, has demonstrated rapid and sustained improvement in the signs and symptoms in patients with active radiographic axial spondyloarthritis (r-axSpA) in global and Chinese populations. We studied the effect of ixekizumab on patient-reported outcomes (PROs) (including patient global, spinal pain, stiffness, and fatigue) and overall health-related quality of life (HRQoL) of ixekizumab in the phase 3 study in China. Methods In this Chinese phase 3, randomized, double-blind, placebo-controlled study, patients with r-axSpA were randomized (1:1) to receive ixekizumab 80 mg every 4 weeks (IXEQ4W; starting dose 160 mg) or placebo for 16 weeks. At week 16, patients receiving placebo were switched to IXEQ4W, and those receiving IXEQ4W continued, until week 52. Data for patient global, spinal pain, spinal pain at night, stiffness, and fatigue were collected through week 52. Minimally clinical important differences (MCIDs) were determined for spinal pain and spinal pain at night. The subgroup analyses by baseline disease duration since diagnosis and baseline C-reactive protein (CRP) level were conducted post hoc. Results Compared with placebo, patients treated with IXEQ4W reported significantly greater improvement with a rapid onset in changes from baseline of PROs (patient global, spinal pain, spinal pain at night, stiffness, and fatigue) through week 16. Improvements were maintained through week 52. A similar trend of improvement was also observed in MCID response in spinal pain and spinal pain at night. The improvement in overall HRQoL was supported by EQ-5D-5L assessment. Subgroup analyses demonstrated that IXEQ4W provided significantly greater efficacy at week 16 compared with placebo, irrespective of baseline disease duration or baseline CRP level. Conclusion IXEQ4W provided rapid and sustained improvement in clinically relevant PROs and overall HRQoL through 1-year treatment in Chinese patients with r-axSpA. Regardless of the baseline disease duration or baseline CRP level, consistent efficacy was observed. Trial Registration ClinicalTrials.gov identifier NCT04285229.

Background: Accurate assessment of Rheumatoid Arthritis (RA) activity remains a challenge. Multimodal photoacoustic/ultrasound (PA/US) joint imaging emerges as a novel imaging modality capable of depicting microvascularization and oxygenation levels in inflamed joints associated with RA. However, the scarcity of large-scale studies limits the exploration of correlating joint oxygenation status with disease activity. Objective: This study aimed to explore the correlation between multimodal PA/US imaging scores and RA disease activity, assessing its clinical applicability in managing RA. Methods: In this study, we recruited 111 patients diagnosed with RA and conducted examinations of seven small joints on their clinically dominant side using a PA/US imaging system. The PA and power Doppler ultrasound (PDUS) signals were semi-quantitatively assessed using a 0–3 grading system. The cumulative scores for PA and PDUS across these seven joints (PA-sum and PDUS-sum) were calculated. Relative oxygen saturation (So2) values of inflamed joints on the clinically dominant side were measured, and categorized into four distinct PA+So2 patterns. The correlation between PA/US imaging scores and disease activity indices was systematically evaluated. Results: Analysis of 777 small joints in 111 patients revealed that the PA-sum scores exhibited a strong positive correlation with standard clinical scores for RA, including DAS28 [ESR] (ρ = 0.682), DAS28 [CRP] (ρ = 0.683), CDAI (ρ = 0.738), and SDAI (ρ = 0.739), all with p < 0.001. These correlations were superior to those of the PDUS-sum scores (DAS28 [ESR] ρ = 0.559, DAS28 [CRP] ρ = 0.555, CDAI ρ = 0.575, SDAI ρ = 0.581, p < 0.001). Significantly, in patients with higher PA-sum scores, notable differences were observed in the erythrocyte sedimentation rate (ESR) (p < 0.01) and swollen joint count 28 (SJC28) (p < 0.01) between hypoxia and intermediate groups. Notably, RA patients in the hypoxia group exhibited higher clinical scores in certain clinical indices. Conclusion: Multi-modal PA/US imaging introduces potential advancements in RA assessment, especially regarding So2 evaluations in synovial tissues and associated PA scores. However, further studies are warranted, particularly with more substantial sample sizes and in multi-center settings. Summary: This study utilized multi-modal PA/US imaging to analyze Rheumatoid Arthritis (RA) patients' synovial tissues and affected joints. When juxtaposed with traditional PDUS imaging, the PA approach demonstrated enhanced sensitivity, especially concerning detecting small vessels in thickened synovium and inflamed tendon sheaths. Furthermore, correlations between the derived PA scores, PA+So2 patterns, and standard clinical RA scores were observed. These findings suggest that multi-modal PA/US imaging could be a valuable tool in the comprehensive assessment of RA, offering insights not only into disease activity but also into the oxygenation status of synovial tissues. However, as promising as these results are, further investigations, especially in larger and diverse patient populations, are imperative. Key points: ⸸ Multi-modal PA/US Imaging in RA: This novel technique was used to assess the So2 values in synovial tissues and determine PA scores of affected RA joints.⸸ Correlation significantly with Clinical RA Scores: Correlations significantly were noted between PA scores, PA+So2 patterns, and standard clinical RA metrics, hinting at the potential clinical applicability of the technique.

BackgroundPrevious studies have revealed that Galectin-9 (Gal-9) acts as an apoptosis modulator in autoimmunity and rheumatic inflammation. In the present study, we investigated the potential role of Gal-9 as a biomarker in patients with rheumatoid arthritis (RA), especially as an indicator of functional limitations and radiographic joint damage.MethodsA total of 146 patients with RA and 52 age- and sex-matched healthy controls were included in this study. Clinical data including disease activity, physical function, and radiographic joint damage were assessed. Functional limitation was defined as the Stanford Health Assessment Questionnaire (HAQ) disability index >1. Subjects with joint erosion >0 or joint space narrowing >0 were considered to have radiographic joint damage. Serum Gal-9 levels were detected by an enzyme-linked immunosorbent assay. Univariate and multivariate logistic regression analysis were used to evaluate the association between Gal-9 and high disease activity and functional limitations, and a prediction model was established to construct predictive nomograms.ResultsSerum levels of Gal-9 were significantly increased in patients with RA compared to those in healthy controls (median 13.1 ng/mL vs. 7.6 ng/mL). Patients with RA who were older (>65 years), had a longer disease duration (>5 years), longer morning stiffness (>60mins), elevated serum erythrocyte sedimentation rate and C-reactive protein, and difficult-to-treat RA had significantly higher Gal-9 levels than those in the corresponding control subgroups (all p 11.6 ng/mL had a significantly higher core clinical disease activity index, HAQ scores, Sharp/van der Heijde modified Sharp scores, as well as a higher percentage of advanced joint damage (all p11.6 ng/mL was an independent risk factor for high disease activity (OR=3.138, 95% CI 1.150–8.567, p=0.026) and presence of functional limitations (OR=2.455, 95% CI 1.017–5.926, p=0.046), respectively.ConclusionGal-9 could be considered as a potential indicator in patients with RA, especially with respect to functional limitations and joint damage.

Abstract Background For the unclear pathogenesis of Sjogren's syndrome (SS), further exploration is necessary. Mesenchymal stem cells (MSCs) and derived exosomes (MSCs‐exo) have exhibited promising results in treating SS. Object This study aimed to investigate the effect and mechanism of human umbilical cord MSCs (UC‐MSCs) on SS. Methods Nonobese Diabetic (NOD) mouse splenic T cells were co‐cultured with UC‐MSCs and UC‐MSCs‐exo, and interferon‐gamma (IFN‐γ), interleukin (IL)‐6, IL‐10, prostaglandin E2 (PGE2), and transforming growth factor‐β1 (TGF‐β1) levels in the supernatant were assessed by quantitative real‐time polymerase chain reaction and enzyme‐linked immunosorbent assay. Co‐cultured T cells were injected into NOD mice via the tail vein. The inflammatory cell infiltration in the intestine and the submandibular gland was characterized by hematoxylin‐eosin staining. Treg/Th17 homeostasis within the spleen was determined by flow cytometry. Gut microbiota was detected by 16S rRNA sequencing, and the relationship between differential microbiota and Treg/Th17 cytokines was analyzed by the Pearson correlation coefficient. Results UC‐MSCs, UC‐MSCs‐exo, and NOD mouse splenic T cells were successfully cultured and identified. After T cells were co‐cultured with UC‐MSCs and UC‐MSCs‐exo, both IFN‐γ and IL‐6 were decreased while IL‐10, PGE2, and TGF‐β1 were increased in transcriptional and translational levels. UC‐MSCs and UC‐MSCs‐exo partially restored salivary secretion function, reduced Ro/SSA antibody and α‐Fodrin immunoglobulin A levels, reduced inflammatory cell infiltration in the intestine and submandibular gland, raised proportion of Treg cells, decreased IFN‐γ, IL‐6, IL‐2, IL‐17, lipopolysaccharide, and tumor necrosis factor‐alpha levels, and raised IL‐10, Foxp3, and TGF‐β1 levels by affecting co‐cultured T cells. The intervention of UC‐MSCs and UC‐MSCs‐exo improved intestinal homeostasis in NOD mice by increasing microbiota diversity and richness. Additionally, differential microbiota was significantly associated with Treg/Th17 cytokine levels. Conclusion Human UC‐MSCs and UC‐MSCs‐exo improved disease characterization of SS in NOD mice through regulation of gut microbiota and Treg/Th17 cellular immunity.

ABSTRACT Global sequencing of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has enabled researchers to monitor the genetic information of new pathogenic variants. However, the integrity and accuracy of most genomic information cannot be guaranteed due to limited sample quality and sequencing technology. This restricts researchers from tracking the latest changes in the virus genome. In this study, we aimed to characterize the diversity and conservation balance of complete SARS-CoV-2 genomes worldwide since the outbreak. To achieve this goal, we collected 5,966,490 genome sequences from various parts of the world, excluding those with incomplete or unknown/degenerate sequences. Our methodology included comparing sequences using BLASTN and BLASTP, analyzing RNA secondary structure, and calculating entropy. Our findings provide insights into the characteristics of SARS-CoV-2 and potential mechanisms of pathogenesis. We found that uracil had the highest proportion of all bases among various coronaviruses and cytosine to uracil mutations had the highest proportion among all point mutations. The consistency in the front part (1–26,599 nt) was significantly higher than that in the back part (26,600–29,903 nt) of the genome. For most genes, the similar consistency characteristics were also observed in their protein families. IMPORTANCE Our results indicate that most severe acute respiratory syndrome coronavirus 2 genomes sampled from patients had a mutation rate ≤1.07 ‰ and genome-tail proteins (including S protein) were the main sources of genetic polymorphism. The analysis of the virus-host interaction network of genome-tail proteins showed that they shared some antiviral signaling pathways, especially the intracellular protein transport pathway.

BackgroundSjögren’s syndrome (SS) is a systemic autoimmune disease that affects about 0.04-0.1% of the general population. SS diagnosis depends on symptoms, clinical signs, autoimmune serology, and even invasive histopathological examination. This study explored biomarkers for SS diagnosis.MethodsWe downloaded three datasets of SS patients’ and healthy pepole’s whole blood (GSE51092, GSE66795, and GSE140161) from the Gene Expression Omnibus (GEO) database. We used machine learning algorithm to mine possible diagnostic biomarkers for SS patients. Additionally, we assessed the biomarkers’ diagnostic value using the receiver operating characteristic (ROC) curve. Moreover, we confirmed the expression of the biomarkers through the reverse transcription quantitative polymerase chain reaction (RT-qPCR) using our own Chinese cohort. Eventually, the proportions of 22 immune cells in SS patients were calculated by CIBERSORT, and connections between the expression of the biomarkers and immune cell ratios were studied.ResultsWe obtained 43 DEGs that were mainly involved in immune-related pathways. Next, 11 candidate biomarkers were selected and validated by the validation cohort data set. Besides, the area under curves (AUC) of XAF1, STAT1, IFI27, HES4, TTC21A, and OTOF in the discovery and validation datasets were 0.903 and 0.877, respectively. Subsequently, eight genes, including HES4, IFI27, LY6E, OTOF, STAT1, TTC21A, XAF1, and ZCCHC2, were selected as prospective biomarkers and verified by RT-qPCR. Finally, we revealed the most relevant immune cells with the expression of HES4, IFI27, LY6E, OTOF, TTC21A, XAF1, and ZCCHC2.ConclusionIn this paper, we identified seven key biomarkers that have potential value for diagnosing Chinese SS patients.

Systemic lupus erythematous (SLE) is a complex chronic autoimmune disease with difficult early treatment and accurate diagnosis. Circulating exosomes containing proteins, lipids and nucleic acids can be ideal diagnostic biomarkers and disease management strategies for SLE. Our aim was to examine the unique expression profiles of circulating exosomal miRNAs and proteins in patients with SLE patients.Using RNA-sequencing and proteomic approaches, we compared the expression patterns of exosomal miRNAs and proteins in the plasma of SLE patients and healthy subjects, and discussed the underlying signaling network of circulating exosomes. We also summarize common molecules (miRNAs and proteins) and pathways shared by our plasma exosomes, as well as previously reported data (PBMC, T cells, B cells and plasma).We identified groups of differentially expressed exosomal miRNAs and proteins in the plasma of SLE patients and healthy controls. We obtained consensus molecules (39 miRNAs, 14 proteins) and 21 signaling pathways that are common in our current study and previous reports.Common molecules (miRNAs and proteins) and pathways shared by our plasma exosomes data and other circulating components data reported previously indicate their potential application in the clinical treatment and diagnosis of SLE disease.

IntroductionSystemic lupus erythematosus (SLE) is a chronic autoimmune disease for which there is no cure. Effective diagnosis and precise assessment of disease exacerbation remains a major challenge.MethodsWe performed peripheral blood mononuclear cell (PBMC) proteomics of a discovery cohort, including patients with active SLE and inactive SLE, patients with rheumatoid arthritis (RA), and healthy controls (HC). Then, we performed a machine learning pipeline to identify biomarker combinations. The biomarker combinations were further validated using enzyme-linked immunosorbent assays (ELISAs) in another cohort. Single-cell RNA sequencing (scRNA-seq) data from active SLE, inactive SLE, and HC PBMC samples further elucidated the potential immune cellular sources of each of these PBMC biomarkers.ResultsScreening of the PBMC proteome identified 1023, 168, and 124 proteins that were significantly different between SLE vs. HC, SLE vs. RA, and active SLE vs. inactive SLE, respectively. The machine learning pipeline identified two biomarker combinations that accurately distinguished patients with SLE from controls and discriminated between active and inactive SLE. The validated results of ELISAs for two biomarker combinations were in line with the discovery cohort results. Among them, the six-protein combination (IFIT3, MX1, TOMM40, STAT1, STAT2, and OAS3) exhibited good performance for SLE disease diagnosis, with AUC of 0.723 and 0.815 for distinguishing SLE from HC and RA, respectively. Nine-protein combination (PHACTR2, GOT2, L-selectin, CMC4, MAP2K1, CMPK2, ECPAS, SRA1, and STAT2) showed a robust performance in assessing disease exacerbation (AUC=0.990). Further, the potential immune cellular sources of nine PBMC biomarkers, which had the consistent changes with the proteomics data, were elucidated by PBMC scRNAseq.DiscussionUnbiased proteomic quantification and experimental validation of PBMC samples from two cohorts of patients with SLE were identified as biomarker combinations for diagnosis and activity monitoring. Furthermore, the immune cell subtype origin of the biomarkers in the transcript expression level was determined using PBMC scRNAseq. These findings present valuable PBMC biomarkers associated with SLE and may reveal potential therapeutic targets.

Immune checkpoint inhibitors (ICIs) have significantly improved the survival of patients with advanced tumors. However, immune-related adverse events (irAEs) caused by ICIs, especially high-grade irAEs, are of growing concern. High-grade multisystem irAEs due to toripalimab, a programmed cell death-1 (PD-1) inhibitor, have been rarely reported. Two patients with malignant metastatic tumors were treated with anti-PD-1 immunotherapy. However, both patients developed high-grade multisystem irAEs based on myocarditis, with chest discomfort and malaise as the main clinical manifestation. Both patients had an elevation of cardiac enzymes, abnormal electrocardiography and left ventricular wall motion. Patient 2 was also diagnosed with organizing pneumonia. Immunotherapy was suspended. High-dose intravenous methylprednisolone was immediately initiated. The patients’ symptoms were significantly relieved in a short period of time. Immunosuppressants were discontinued at the 6th month follow-up in patient 1 without relapse. However, patient 2 was lost to follow up due to financial reasons. To the best of our knowledge, this is the first report regarding ICI-associated myocarditis-pneumonia due to toripalimab, indicating the significance of early recognition and management of high-grade multisystem irAEs in clinical practice.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with multi-organ inflammation and defect, which is linked to many molecule mediators. Oxylipins as a class of lipid mediator have not been broadly investigated in SLE. Here, we applied targeted mass spectrometry analysis to screen the alteration of oxylipins in serum of 98 SLE patients and 106 healthy controls. The correlation of oxylipins to lupus nephritis (LN) and SLE disease activity, and the biomarkers for SLE classification, were analyzed. Among 128 oxylipins analyzed, 92 were absolutely quantified and 26 were significantly changed. They were mainly generated from the metabolism of several polyunsaturated fatty acids, including arachidonic acid (AA), linoleic acid (LA), docosahexanoic acid (DHA), eicosapentanoic acid (EPA) and dihomo-γ-linolenic acid (DGLA). Several oxylipins, especially those produced from AA, showed different abundance between patients with and without lupus nephritis (LN). The DGLA metabolic activity and DGLA generated PGE1, were significantly associated with SLE disease activity. Random forest-based machine learning identified a 5-oxylipin combination as potential biomarker for SLE classification with high accuracy. Seven individual oxylipin biomarkers were also identified with good performance in distinguishing SLE patients from healthy controls (individual AUC > 0.7). Interestingly, the biomarkers for differentiating SLE patients from healthy controls are distinct from the oxylipins differentially expressed in LN patients vs. non-LN patients. This study provides possibilities for the understanding of SLE characteristics and the development of new tools for SLE classification.

Abstract Background Systemic lupus erythematosus (SLE) is an autoimmune disease with a complicated pathogenesis, and its aetiology has not been clearly unveiled. The lack of effective diagnosis and treatment methods makes it necessary to explore the molecular mechanism of SLE. We aimed to identify some critical signalling pathways and key competing endogenous RNAs (ceRNAs) underlying the molecular mechanism of SLE and to map out the systematic signalling networks by integrating the data on different kinds of RNAs. Methods Peripheral blood mononuclear cells (PBMCs) were collected from both SLE patients and healthy subjects, RNA was extracted from the PBMCs, and RNA libraries including ribosomal RNA-depleted strand-specific libraries and small RNA libraries were built for deep RNA sequencing (RNA-seq). RNA-seq yielded differential expression profiles of lncRNAs/circRNAs/miRNAs/mRNAs related to SLE. The DAVID database (v. 6.8) was employed for Gene Ontology (GO) and KEGG pathway analysis. ceRNA networks (circRNA/lncRNA-miRNA-mRNA) were constructed and visualized using Cytoscape software (v. 3.5.0). The TargetScan and miRanda databases were used to predict target relationships in ceRNA networks. qRT-PCR was used to verify our data. Results Differential expression of ceRNAs related to SLE was detected in SLE patients’ PBMCs: 644 mRNAs (384 upregulated, 260 downregulated), 326 miRNAs (223 upregulated, 103 downregulated), 221 lncRNAs (79 upregulated, 142 downregulated), and 31 circRNAs (21 upregulated, 10 downregulated). We drew ceRNA signalling networks made up of the differentially expressed mRNAs/miRNAs/lncRNAs/circRNAs mentioned above, and the hub genes included IRF5, IFNAR2, TLR7, IRAK4, STAT1, STAT2, C2, and Tyk2. These hub genes were involved in ceRNA signalling pathways, such as the IL-17 signalling pathway and type I interferon signalling pathway. Conclusions We explored the differential expression profiles of various kinds of ceRNAs and integrated signalling networks constructed by ceRNAs. Our findings offer new insights into the pathogenesis of SLE and hint at therapeutic strategies.

Traditional Chinese herbal medicine aiming at nourishing yin formed a distinctive school of thought in history to achieve anti-aging and longevity. In the formula Gancao nourishing yin (GCNY) decoction, all of the ingredients show antioxidant properties. However, in real clinical practice, extractions of herbs are rarely applied alone but are prescribed as the integrated formula. To investigate whether GCNY possesses anti-oxidation potential, we applied GCNY to treat rats to acquire medicated serum, which was then added on H2O2 (200 μM)-modeled human microglial cell line HMC-3 in comparison with its control serum. The results revealed that GCNY-medicated serum decreased reactive oxygen species (ROS) levels. Inflammatory cytokines such as pNF-κB p65 (ser536) and IL-6 were also decreased. Nrf2 and its pathway-related molecules, such as HO1, ABCC2, GLCM, ME1, NQO1, and TKT, were activated by H2O2 modeling while declined by treating with GCNY-medicated serum, which indicated attenuated oxidative stress of GCNY. Furthermore, mRNA-seq analysis showed 58 differential expressed genes (DEGs), which were enriched in pathways including antigen processing and presentation, longevity regulation, oxidative phosphorylation, and Parkinson’s disease progression. DEGs that were downregulated by H2O2 modeling but upregulated by GCNY treatment include CENPF, MKI67, PRR11, and TOP2A. Those targets were reported to be associated with the cell cycle and cell proliferation and belong to the category of growth factor genes. In conclusion, this study verified anti-oxidation effects of GCNY and indicated its promising application for cognitive degeneration and aging-related disorders.

Detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) needs human samples, which inevitably contain trace human DNA and RNA. Sequence similarity may cause invalid detection results; however, there is still a lack of gene similarity analysis of SARS-CoV-2 and humans. All publicly reported complete genome assemblies in the Entrez genome database were collected for multiple sequence alignment, similarity and phylogenetic analysis. The complete genomes showed high similarity (>99.88% sequence identity). Phylogenetic analysis divided these viruses into three major clades with significant geographic group effects. Viruses from the United States showed considerable variability. Sequence similarity analysis revealed that SARS-CoV-2 has 612 similar sequences with the human genome and 100 similar sequences with the human transcriptome. The sequence characteristics and genome distribution of these similar sequences were confirmed. The sequence similarity and evolutionary mutations provide indispensable references for dynamic updates of SARS-CoV-2 detection primers and methods.

Abstract Therapeutic proteins have exhibited promising clinical applications in the diagnosis and treatment of some diseases. Prior to the detection of analytes using enzyme-linked immunosorbent assay, biological samples of therapeutic proteins are conventionally frozen at temperatures ranging from − 20 to − 80 °C to increase the stability of analytes. However, therapeutic proteins destabilization and aggregation may occur during the frozen storage or the freeze-thawing step. In this work, an effective method was proposed to freeze-dry therapeutic protein samples to allow subsequent storage or transport of samples without freezing them. This new method was validated with quality control samples of adalimumab and etanercept, and it was also used in the bioanalysis of adalimumab and etanercept in pharmacokinetic (PK) studies. Adalimumab and etanercept were stable for 14 days at 4 °C after being prepared and stored using the new method, with detection that was accurate and repeatable. Studies of adalimumab and etanercept in animals and humans showed that the PK parameters of the analytes stored with the new method were consistent with those of analytes stored using the conventional method. This effective method will be attractive for facilitating the storage and transport of plasma samples containing therapeutic proteins.

HAPLN1 maintains aggregation and the binding activity of extracellular matrix (ECM) molecules (such as hyaluronic acid and proteoglycan) to stabilize the macromolecular structure of the ECM. An increase in HAPLN1 expression is observed in a few types of musculoskeletal diseases including rheumatoid arthritis (RA); however, its functions are obscure. This study examined the role of HAPLN1 in determining the viability, proliferation, mobility, and pro-inflammatory phenotype of RA- fibroblast-like synoviocytes (RA-FLSs) by using small interfering RNA (siHAPLN1), over-expression vector (HAPLN1OE), and a recombinant HAPLN1 (rHAPLN1) protein. HAPLN1 was found to promote proliferation but inhibit RA-FLS migration. Metformin, an AMPK activator, was previously found by us to be able to inhibit FLS activation but promote HAPLN1 secretion. In this study, we confirmed the up-regulation of HAPLN1 in RA patients, and found the positive relationship between HAPLN1 expression and the AMPK level. Treatment with either si-HAPLN1 or HAPLN1OE down-regulated the expression of AMPK-ɑ gene, although up-regulation of the level of p-AMPK-ɑ was observed in RA-FLSs. si-HAPLN1 down-regulated the expression of proinflammatory factors like TNF-ɑ, MMPs, and IL-6, while HAPLN1OE up-regulated their levels. qPCR assay indicated that the levels of TGF-β, ACAN, fibronectin, collagen II, and Ki-67 were down-regulated upon si-HAPLN1 treatment, while HAPLN1OE treatment led to up-regulation of ACAN and Ki-67 and down-regulation of cyclin-D1. Proteomics of si-HAPLN1, rHAPLN1, and mRNA-Seq analysis of rHAPLN1 confirmed the functions of HAPLN1 in the activation of inflammation, proliferation, cell adhesion, and strengthening of ECM functions. Our results for the first time demonstrate the function of HAPLN1 in promoting the proliferation and pro-inflammatory phenotype of RA-FLSs, thereby contributing to RA pathogenesis. Future in-depth studies are required for better understanding the role of HAPLN1 in RA.

BackgroundSystemic lupus erythematosus (SLE) is an autoimmune illness caused by a malfunctioning immunomodulatory system. China has the second highest prevalence of SLE in the world, from 0.03% to 0.07%. SLE is diagnosed using a combination of immunological markers, clinical symptoms, and even invasive biopsy. As a result, genetic diagnostic biomarkers for SLE diagnosis are desperately needed.MethodFrom the Gene Expression Omnibus (GEO) database, we downloaded three array data sets of SLE patients’ and healthy people’s peripheral blood mononuclear cells (PBMC) (GSE65391, GSE121239 and GSE61635) as the discovery metadata (nSLE = 1315, nnormal = 122), and pooled four data sets (GSE4588, GSE50772, GSE99967, and GSE24706) as the validate data set (nSLE = 146, nnormal = 76). We screened the differentially expressed genes (DEGs) between the SLE and control samples, and employed the least absolute shrinkage and selection operator (LASSO) regression, and support vector machine recursive feature elimination (SVM-RFE) analyze to discover possible diagnostic biomarkers. The candidate markers’ diagnostic efficacy was assessed using the receiver operating characteristic (ROC) curve. The reverse transcription quantitative polymerase chain reaction (RT-qPCR) was utilized to confirm the expression of the putative biomarkers using our own Chinese cohort (nSLE = 13, nnormal = 10). Finally, the proportion of 22 immune cells in SLE patients was determined using the CIBERSORT algorithm, and the correlations between the biomarkers’ expression and immune cell ratios were also investigated.ResultsWe obtained a total of 284 DEGs and uncovered that they were largely involved in several immune relevant pathways, such as type І interferon signaling pathway, defense response to virus, and inflammatory response. Following that, six candidate diagnostic biomarkers for SLE were selected, namely ABCB1, EIF2AK2, HERC6, ID3, IFI27, and PLSCR1, whose expression levels were validated by the discovery and validation cohort data sets. As a signature, the area under curve (AUC) values of these six genes reached to 0.96 and 0.913, respectively, in the discovery and validation data sets. After that, we checked to see if the expression of ABCB1, IFI27, and PLSCR1 in our own Chinese cohort matched that of the discovery and validation sets. Subsequently, we revealed the potentially disturbed immune cell types in SLE patients using the CIBERSORT analysis, and uncovered the most relevant immune cells with the expression of ABCB1, IFI27, and PLSCR1.ConclusionOur study identified ABCB1, IFI27, and PLSCR1 as potential diagnostic genes for Chinese SLE patients, and uncovered their most relevant immune cells. The findings in this paper provide possible biomarkers for diagnosing Chinese SLE patients.

Abstract Background The incidence and mortality of lung cancer are the highest among all cancers. Patients with systemic sclerosis show a four-fold greater risk of lung cancer than the general population. However, the underlying mechanism remains poorly understood. Methods The expression profiles of 355 peripheral blood samples were integratedly analyzed, including 70 cases of lung cancer, 61 cases of systemic sclerosis, and 224 healthy controls. After data normalization and cleaning, differentially expressed genes (DEGs) between disease and control were obtained and deeply analyzed by bioinformatics methods. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed online by DAVID and KOBAS. The protein–protein interaction (PPI) networks were constructed from the STRING database. Results From a total of 14,191 human genes, 299 and 1644 genes were identified as DEGs in systemic sclerosis and lung cancer, respectively. Among them, 64 DEGs were overlapping, including 36 co-upregulated, 10 co-downregulated, and 18 counter-regulated DEGs. Functional and enrichment analysis showed that the two diseases had common changes in immune-related genes. The expression of innate immune response and response to virus-related genes increased significantly, while the expression of negative regulation of cell cycle-related genes decreased notably. In contrast, the expression of mitophagy regulation, chromatin binding and fatty acid metabolism-related genes showed distinct trends. Conclusions Stable differences and similarities between systemic sclerosis and lung cancer were revealed. In peripheral blood, enhanced innate immunity and weakened negative regulation of cell cycle may be the common mechanisms of the two diseases, which may be associated with the high risk of lung cancer in systemic sclerosis patients. On the other hand, the counter-regulated DEGs can be used as novelbiomarkers of pulmonary diseases. In addition, fat metabolism-related DEGs were consideredto be associated with clinical blood lipid data.

Abstract Background The diagnosis of systemic lupus erythematosus (SLE) is complicated. This study explores the expression of circular RNAs (circRNAs), which are closed non-coding RNAs in which the 5′ and 3′ ends are covalently linked and which work by sponging microRNAs. CircRNAs were extracted from peripheral blood mononuclear cells (PBMCs) of SLE patients to identify novel circRNA species that might be used for SLE diagnosis. Methods Microarray was applied to screening circRNAs changes in PBMCs obtained from SLE patients (n = 10) and healthy participants (n = 10), paired for age and sex. We then verified the selected circRNAs in PBMCs using quantitative reverse transcription-polymerase chain reaction amplification (qRT-PCR) in another cohort, including ten paired SLE patients and healthy participants. The correlation between the differential circRNAs and clinical pathology of SLE were analyzed. Results 182 up-regulated and 563 significantly down-regulated circRNAs in PBMCs of patients with SLE were identified. Besides, the qRT-PCR results were consistent with the microarray results. The correlation analysis revealed that has_circRNA_100236, has_circRNA_102489, and has_circRNA_101413 were correlated with positive anti-dsDNA, thrombocytopenia, and positive IgG, respectively. Lastly, their miRNAs targets and the binding sites were predicted. Conclusion We identified some dysregulated circRNAs in PBMCs from SLE patients, and these circRNAs may be developed as the novel biomarkers for the diagnosis of SLE.

BackgroundAnkylosing spondylitis (AS) is a chronic inflammatory disease with serious consequences and a high rate of morbidity and mortality, In our previous work, we reveal the key features of proteins in new-onset ankylosing spondylitis patients.Material and MethodsAnkylosing spondylitis (AS) is a chronic inflammatory condition that affects the spine, and inflammation plays an essential role in AS pathogenesis. The inflammatory process in AS, however, is still poorly understood due to its intricacy. Systematic proteomic and phosphorylation analyses of peripheral blood mononuclear cells (PBMCs) were used to investigate potential pathways involved in AS pathogenesis.ResultsLiquid chromatography-tandem mass spectrometry (LC–MS/MS) analysis was performed and discovered 782 differentially expressed proteins (DEPs) and 122 differentially phosphorylated proteins (DPPs) between 9 new-onset AS patients and 9 healthy controls. The DEPs were further verified using parallel reaction monitoring (PRM) analysis. PRM analysis verified that 3 proteins (HSP90AB1, HSP90AA1 and HSPA8) in the antigen processing and presentation pathway, 6 proteins (including ITPR1, MYLK and STIM1) in the platelet activation pathway and 10 proteins (including MYL12A, MYL9 and ROCK2) in the leukocyte transendothelial migration pathway were highly expressed in the PBMCs of AS patients.ConclusionThe key proteins involved in antigen processing and presentation, platelet activation and leukocyte transendothelial migration revealed abnormal immune regulation in patients with new-onset AS. These proteins might be used as candidate markers for AS diagnosis and new therapeutic targets, as well as elucidating the pathophysiology of AS.

Delanzomib is a novel proteasome inhibitor initially developed for treating multiple myeloma. It was found to inhibit the expression of tumor necrosis factor alpha (TNF-α). This study aimed to investigate the ameliorating effect of delanzomib on collagen-induced arthritis (CIA) and to explore the pharmacodynamics and pharmacokinetics (PK) interactions between delanzomib and adalimumab. Rats with CIA were randomly assigned to receive the treatment with delanzomib, adalimumab, delanzomib combined with adalimumab, or placebo. Visual inspection and biochemical examinations including TNF-α, interleukin 6, and C-reactive protein were performed to assess arthritis severity during the treatment. The adalimumab concentration in rats was determined to evaluate the PK interaction between delanzomib and adalimumab. Also, the levels of neonatal Fc receptor (FcRn) and FcRn mRNA were measured to explore the role of FcRn in the PK interaction between delanzomib and adalimumab. As a result, delanzomib combined with adalimumab exhibited stronger anti-arthritis activity than a single drug because both drugs synergistically reduced TNF-α level in vivo. Delanzomib also decreased adalimumab elimination in rats by increasing the level of FcRn. The slower elimination of adalimumab in rats further prolonged the anti-TNF-α effect of adalimumab. Moreover, FcRn level was increased by delanzomib via suppressing FcRn degradation rather than promoting FcRn production. In conclusion, delanzomib combined with adalimumab may be a potential therapeutic approach for treating rheumatoid arthritis. The initial finding that the PK interaction occurred between delanzomib and adalimumab may have clinical relevance for patients who simultaneously take proteasome inhibitors and anti-TNF-α therapeutic proteins.

ObjectiveGenetic studies on ankylosing spondylitis (AS) have identified more than 100 pathogenic genes. Building a bridge between these genes and biologically targeted therapies is the current research hotspot.MethodsWe integrated single-cell assaying transposase-accessible chromatin sequencing (scATAC-seq) and single-cell RNA sequencing (scRNA-seq) to explore the key genes and related mechanisms associated with AS pathogenesis.ResultsWe identified 18 cell types in peripheral mononuclear cells from patients with AS and normal controls and summarized the cell-type-specific abnormal genes by scRNA-seq. Interestingly, we found that the pathogenic gene NFKB involved in AS progression originated from CD8+ T cells. Moreover, we observed an abnormal tumor TNF pathway mediated by abnormal expression of TNF, NFKB, FOS, JUN, and JUNB, and scATAC-seq results confirmed the abnormal accessible binding sites of transcriptional factors FOS, JUN, and JUNB. The final magnetic bead sorting and quantitative real-time PCR(RT-qPCR) confirmed that NFKB, FOS, JUN, and JUNB in CD8+ T cells differed in the AS group.ConclusionsOur results revealed a possible mechanism by which NFKB abnormally regulates FOS, JUN, and JUNB and drives AS progression, providing a novel perspective from a single cell point of view in AS.

ObjectiveSystemic lupus erythematosus (SLE) is a complex autoimmune disease, and various immune cells are involved in the initiation, progression, and regulation of SLE. Our goal was to reveal the chromatin accessibility landscape of peripheral blood mononuclear cells (PBMCs) in SLE patients at single-cell resolution and identify the transcription factors (TFs) that may drive abnormal immune responses.MethodsThe assay for transposase accessible chromatin in single-cell sequencing (scATAC-seq) method was applied to map the landscape of active regulatory DNA in immune cells from SLE patients at single-cell resolution, followed by clustering, peak annotation and motif analysis of PBMCs in SLE.ResultsPeripheral blood mononuclear cells were robustly clustered based on their types without using antibodies. We identified twenty patterns of TF activation that drive abnormal immune responses in SLE patients. Then, we observed ten genes that were highly associated with SLE pathogenesis by altering T cell activity. Finally, we found 12 key TFs regulating the above six genes (CD83, ELF4, ITPKB, RAB27A, RUNX3, and ZMIZ1) that may be related to SLE disease pathogenesis and were significantly enriched in SLE patients (p 2). With qPCR experiments on CD83, ELF4, RUNX3, and ZMIZ1 in B cells, we observed a significant difference in the expression of genes (ELF4, RUNX3, and ZMIZ1), which were regulated by seven TFs (EWSR1-FLI1, MAF, MAFA, NFIB, NR2C2 (var. 2), TBX4, and TBX5). Meanwhile, the seven TFs showed highly accessible binding sites in SLE patients.ConclusionsThese results confirm the importance of using single-cell sequencing to uncover the real features of immune cells in SLE patients, reveal key TFs in SLE-PBMCs, and provide foundational insights relevant for epigenetic therapy.

Systemic lupus erythematosus (SLE) is a heterogeneous, autoimmune disease that can affect multiple organs and systems such as skin, joints, kidneys, hematologic system or central nervous system. Women of childbearing age are the predominate population affected by SLE. In this study, we generated an iPS cell line from a 30-year-old female who was pregnant with a gestational age of 27 weeks and diagnosed with severe preeclampsia, SLE and psoriasis. This patient-specific iPSC line will be useful to create the specific disease model of systemic lupus erythematosus to elucidate the pathological mechanisms and develop drug screening.

ObjectivePrimary Sjögren’s syndrome (pSS) is a systemic autoimmune disease, and its pathogenetic mechanism is far from being understood. In this study, we aimed to explore the cellular and molecular mechanisms that lead to pathogenesis of this disease.MethodsWe applied single-cell RNA sequencing (scRNA-seq) to 57,288 peripheral blood mononuclear cells (PBMCs) from five patients with pSS and five healthy controls. The immune cell subsets and susceptibility genes involved in the pathogenesis of pSS were analyzed. Flow cytometry was preformed to verify the result of scRNA-seq.ResultsWe identified two subpopulations significantly expand in pSS patients. The one highly expressing cytotoxicity genes is named as CD4+ CTLs cytotoxic T lymphocyte, and another highly expressing T cell receptor (TCR) variable gene is named as CD4+ TRAV13-2+ T cell. Flow cytometry results showed the percentages of CD4+ CTLs, which were profiled with CD4+ and GZMB+ staining; the total T cells of 10 patients with pSS were significantly higher than those of 10 healthy controls (P= 0.008). The expression level of IL-1β in macrophages, TCL1A in B cells, as well as interferon (IFN) response genes in most cell subsets was upregulated in the patients with pSS. Susceptibility genes including HLA-DRB5, CTLA4, and AQP3 were highly expressed in patients with pSS.ConclusionsOur data revealed disease-specific immune cell subsets and provided some potential new targets of pSS. Specific expansion of CD4+ CTLs may be involved in the pathogenesis of pSS, which might give valuable insights for therapeutic interventions of pSS.

Abstract Objectives This study aims to determine a role of interleukin‐17A (IL‐17) in salivary gland (SG) dysfunction and therapeutic effects of targeting IL‐17 in SG for treating autoimmune sialadenitis in primary Sjögren’s syndrome (pSS). Methods Salivary IL‐17 levels and IL‐17‐secreting cells in labial glands of pSS patients were examined. Kinetic changes of IL‐17‐producing cells in SG from mice with experimental Sjögren’s syndrome (ESS) were analysed. To determine a role of IL‐17 in salivary secretion, IL‐17‐deficient mice and constructed chimeric mice with IL‐17 receptor C (IL‐17RC) deficiency in non‐hematopoietic and hematopoietic cells were examined for saliva flow rates during ESS development. Both human and murine primary SG epithelial cells were treated with IL‐17 for measuring cholinergic activation‐induced calcium movement. Moreover, SG functions were assessed in ESS mice with salivary retrograde cannulation of IL‐17 neutralisation antibodies. Results Increased salivary IL‐17 levels were negatively correlated with saliva flow rates in pSS patients. Both IL‐17‐deficient mice and chimeric mice with non‐hematopoietic cell‐restricted IL‐17RC deficiency exhibited no obvious salivary reduction while chimeric mice with hematopoietic cell‐restricted IL‐17RC deficiency showed significantly decreased saliva secretion during ESS development. In SG epithelial cells, IL‐17 inhibited acetylcholine‐induced calcium movement and downregulated the expression of transient receptor potential canonical 1 via promoting Nfkbiz mRNA stabilisation. Moreover, local IL‐17 neutralisation in SG markedly attenuated hyposalivation and ameliorated tissue inflammation in ESS mice. Conclusion These findings identify a novel function of IL‐17 in driving salivary dysfunction during pSS development and may provide a new therapeutic strategy for targeting SG dysfunction in pSS patients.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that affects thousands of people worldwide. Recently, alterations in metabolism and gut microbiome have emerged as key regulators of SLE pathogenesis. However, it is not clear about the coordination of gut commensal bacteria and SLE metabolism. Here, by integrating 16S sequencing and metabolomics data, we characterized the gut microbiome and fecal and serum metabolome alterations in patients with SLE. Microbial diversity sequencing revealed gut microflora dysbiosis in SLE patients with significantly increased beta diversity. The metabolomics profiling identified 43 and 55 significantly changed metabolites in serum and feces samples in SLE patients. Notably, lipids accounted for about 65% altered metabolites in serum, highlighted the disruption of lipid metabolism. Integrated correlation analysis provided a link between the gut microbiome and lipid metabolism in patients with SLE, particularly according to regulate the conversion of primary bile acids to secondary bile acids. Overall, our results illustrate the perturbation of the gut microbiome and metabolome in SLE patients which may facilitate the development of new SLE interventions.

Abstract Background Interleukin (IL)-37 has been known to play an immunosuppressive role in various inflammatory disorders, but whether it participates in the regulation of pathogenesis of adult-onset Still’s disease (AOSD) has not been investigated. In this study, we examined serum IL-37 levels and their clinical association with AOSD, and we explored the anti-inflammatory effects of IL-37 on peripheral blood mononuclear cells (PBMCs) from patients with AOSD. Methods Blood samples were collected from 62 patients with AOSD and 50 healthy control subjects (HC). The serum IL-37 levels were determined using an enzyme-linked immunosorbent assay (ELISA). The correlations of serum IL-37 levels with disease activity, laboratory values, and inflammatory cytokines in AOSD were analyzed by Spearman’s correlation test. The correlations between serum IL-37 levels and clinical manifestations were analyzed by Mann-Whitney U test. PBMCs from ten patients with AOSD were stimulated with recombinant human IL-37 protein, and expression levels of tumor necrosis factor (TNF)-α, IL-6, IL-10, IL-1β, and IL-18 were determined by qRT-PCR and ELISA. Results A significantly higher IL-37 protein level was observed in patients with AOSD than in HC. Serum IL-37 levels correlated with systemic score, laboratory values, IL-1β, IL-18, and IL-10 in patients with AOSD. The expression levels of IL-37 were closely related to the patients with AOSD who also had fever, skin rash, lymphadenopathy, splenomegaly, myalgia, and arthralgia. Moreover, the production of proinflammatory cytokines such as IL-6, IL-1β, TNF-α, and IL-18 in PBMCs from patients with AOSD was obviously attenuated after recombinant human IL-37 stimulation. Conclusions Increased expression of IL-37 and its positive correlation with disease activity suggest its involvement in AOSD pathogenesis. More importantly, IL-37 inhibits the expression of proinflammatory cytokines in PBMCs from patients with AOSD, indicating the potential anti-inflammatory role of IL-37 in AOSD. Thus, IL-37 may be a novel disease activity biomarker and research target in AOSD.

Objectives: Assays for transposase-accessible chromatin with single-cell sequencing (scATAC-seq) contribute to the progress in epigenetic studies. The purpose of our project was to discover the transcription factors (TFs) that were involved in the pathogenesis of rheumatoid arthritis (RA) at a single-cell resolution using epigenetic technology. Methods: Peripheral blood mononuclear cells of seven RA patients and seven natural controls were extracted nuclei suspensions for library construction. Subsequently, scATAC-seq was performed to generate a high-resolution map of active regulatory DNA for bioinformatics analysis. Results: We obtained 22 accessible chromatin patterns. Then, 10 key TFs were involved in RA pathogenesis by regulating the activity of mitogenactivated protein kinase. Consequently, two genes (PTPRC and SPAG9) regulated by 10 key TFs were found, which may be associated with RA disease pathogenesis, and these TFs were obviously enriched in RA patients (P < .05, fold change value > 1.2). With further quantitative polymerase chain reaction validation on PTPRC and SPAG9 in monocytes, we found differential expression of these two genes, which were regulated by eight TFs [ZNF384, HNF1B, DMRTA2, MEF2A, NFE2L1, CREB3L4 (var. 2), FOSL2::JUNB (var. 2), and MEF2B], showing highly accessible binding sites in RA patients. Conclusions: These findings demonstrate the value of using scATAC-seq to reveal transcriptional regulatory variation in RA-derived peripheral blood mononuclear cells, providing insights into therapy from an epigenetic perspective. [ABSTRACT FROM AUTHOR]