Interleukin-35 protein inhibits osteoclastogenesis and attenuates collagen-induced arthritis in mice.

Rheumatoid arthritis (RA) is a chronic autoimmune disease. Its pathological features include synovial inflammation, bone erosion, and joint structural damage. Our previous studies have shown that interleukin (IL)-35 is involved in the pathogenesis of bone loss in RA patients. In this study, we are further evaluating the efficacy of IL-35 on collagen-induced arthritis (CIA) in the mouse model. Male DBA/1J mice (n = 10) were initially immunized, 2 µg/mouse IL-35 was injected intraperitoneally every week for 3 weeks after the establishment of the CIA model. Clinical arthritis, histopathological analysis, and three-dimensional micro-computed tomography (3D micro-CT) were determined after the mice were anesthetized on the 42th day. In vitro, RANKL/M-CSF induced mouse preosteoclasts (RAW264.7 cells line) was subjected to antiarthritis mechanism study in the presence of IL-35. The results of clinical arthritis, histopathological analysis, and 3D micro-CT, the expression of RANK/RANKL/OPG axis, inflammatory cytokines, and osteoclastogenesis-related makers demonstrated decreasing severity of synovitis and bone destruction in the ankle joints after IL-35 treatment. Furthermore, IL-35 attenuated inflammatory cytokine production and the expression of osteoclastogenesis-related makers in a mouse preosteoclasts cell line RAW264.7. The osteoclastogenesis-related makers were significantly reduced in IL-35 treated RAW264.7 cells line after blockage with the JAK/STAT1 signaling pathway. These results demonstrated that IL-35 protein could inhibits osteoclastogenesis and attenuates CIA in mice. We concluded that IL-35 can exhibit anti-osteoclastogenesis effects by reducing the expression of inflammatory cytokines and osteoclastogenesis-related makers, thus alleviating bone destruction in the ankle joint and could be a potential therapeutic target for RA.

Executive function impairment is associated with low serum vitamin D levels in children with epilepsy.

PURPOSE: Executive function (EF) impairment and vitamin D deficiency are common clinical features among children with epilepsy (CWE). Recently, vitamin D has become a potential modification factor that affects cognitive status in individuals with neurological disorders. In this study, we investigated the association between EF status and vitamin D levels in patients with CWE. METHODS: In total, 79 CWE patients and 39 healthy controls (HCs) were recruited in this study. Each participant's EF was assessed using the Behavior Rating Inventory of Executive Function-Parent form (Brief-P), and the serum level of 25-OH vitamin D was measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: Compared with those in the HC group, the CWE group had higher T scores of Brief-P scale, including global executive composite (GEC) (51.01(45.12, 60.69) vs. 44.08(39.24, 49.96), p＜0.001), behavioral regulation index (BRI) (51.29(45.67, 59.13) vs. 45.67(40.06, 51.29), p＜0.001), metacognition index (MI) (51.83(46.77, 59.43) vs. 46.13(40.44, 51.83), p＜0.001), and lower serum vitamin D (14.85(10.24,23.2) vs. 22.5(16.91,30), p＜0.001) levels. After adjustment for covariates, multivariate linear regression models suggested that for every 1 ng/ml increase in vitamin D, the GEC, BRI, and MI would decrease by 0.52 (Coeff = -0.48; 95 % CI = -0.69, -0.26; p = 0.000), 0.45 (Coeff = -0.45; 95 % CI = -0.69, -0.20; p = 0.000), and 0.47 (Coeff = -0.45; 95 % CI = -0.67, -0.22; p = 0.000), respectively. CONCLUSION: There may be an association between decreased vitamin D levels and EF impairment in CWE. Future research should consider longitudinal variations in EF related to improving vitamin D deficiency.

Adsorption-Free Self-Priming Direct Digital Dual-crRNA CRISPR/Cas12a-Assisted Chip for Ultrasensitive Detection of Pathogens.

Rapid and sensitive pathogen detection methods are critical for disease diagnosis and treatment. RPA-CRISPR/Cas12 systems have displayed remarkable potential in pathogen detection. A self-priming digital PCR chip is a powerful and attractive tool for nucleic detection. However, the application of the RPA-CRISPR/Cas12 system to the self-priming chip still has great challenges due to the problems of protein adsorption and two-step detection mode of RPA-CRISPR/Cas12. In this study, an adsorption-free self-priming digital chip was developed and a direct digital dual-crRNAs (3D) assay was established based on the chip for ultrasensitive detection of pathogens. This 3D assay combined the advantages of rapid amplification of RPA, specific cleavage of Cas12a, accurate quantification of digital PCR, and point-of-care testing (POCT) of microfluidics, enabling accurate and reliable digital absolute quantification of Salmonella in POCT. Our method can provide a good linear relationship of Salmonella detection in the range from 2.58 × 101 to 2.58 × 104 cells/mL with a limit of detection ∼0.2 cells/mL within 30 min in a digital chip by targeting the invA gene of Salmonella. Moreover, the assay could directly detect Salmonella in milk without nucleic acid extraction. Therefore, the 3D assay has the significant potential to provide accurate and rapid pathogen detection in POCT. This study provides a powerful nucleic detection platform and facilitates the application of CRISPR/Cas-assisted detection and microfluidic chips.

Enzymatic Antimicrobial Susceptibility Testing with Bacteria Identification in 30 min.

Rapid antimicrobial susceptibility testing (AST) with the ability of bacterial identification is urgently needed for evidence-based antibiotic prescription. Herein, we propose an enzymatic AST (enzyAST) that employs ß-d-glucuronidase as a biomarker to identify pathogens and profile phenotypic susceptibilities simultaneously. EnzyAST enables to offer binary AST results within 30 min, much faster than standard methods (>16 h). The general applicability of enzyAST was verified by testing the susceptibility of two Escherichia coli strains to three antibiotics with different action mechanisms. The pilot study also shows that the minimal inhibitory concentrations can be determined by enzyAST with the statistical analysis of enzymatic activity of the bacteria population exposed to varying antibiotic concentrations. With further development of multiple bacteria and sample treatment, enzyAST could be able to evaluate the susceptibility of pathogens in clinical samples directly to facilitate the evidence-based therapy.

Digital Recombinase Polymerase Amplification, Digital Loop-Mediated Isothermal Amplification, and Digital CRISPR-Cas Assisted Assay: Current Status, Challenges, and Perspectives.

Digital nucleic acid detection based on microfluidics technology can quantify the initial amount of nucleic acid in the sample with low equipment requirements and simple operations, which can be widely used in clinical and in vitro diagnosis. Recently, isothermal amplification technologies such as recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), and clustered regularly interspaced short palindromic repeats-CRISPR associated proteins (CRISPR-Cas) assisted technologies have become a hot spot of attention and state-of-the-art digital nucleic acid chips have provided a powerful tool for these technologies. Herein, isothermal amplification technologies including RPA, LAMP, and CRISPR-Cas assisted methods, based on digital nucleic acid microfluidics chips recently, have been reviewed. Moreover, the challenges of digital isothermal amplification and possible strategies to address them are discussed. Finally, future directions of digital isothermal amplification technology, such as microfluidic chip and device manufacturing, multiplex detection, and one-pot detection, are outlined.

Relationship between covid-pandemic anxiety and sleep disorder with menstrual disorders among female medical workers.

BACKGROUND: It has been more than 2 years since the 2019 novel coronavirus disease (COVID-19) pandemic destabilized the world, adversely affecting not only physical health, but also mental health. During this time, frontline medical workers were at a greater health risk, especially female medical workers. Changes or abnormalities in the menstrual cycle-an important indicator of women's health-may jeopardize female reproductive functioning. Considering that emotional health and sleep status may be related to the menstrual cycle, this study aimed to investigate the association between menstrual cycle changes, anxiety, sleep dysfunction, and other factors among female medical workers during the COVID-19 pandemic. METHODS: A cross-sectional survey was conducted by distributing online questionnaires to female medical workers in China from February to May 2022. The study included 160 women aged 18-45 years old. The questionnaires covered data related to the participants' sociodemographic characteristics, medical and reproductive history, and lifestyle. The Rating Scale for Clinical Manifestations of Menopathy (SCMM), Self-Rating Anxiety Scale (SAS), and Sleep Dysfunction Rating Scale (SDRS) were utilized. Data were analyzed using chi-square tests, t-tests, and linear regression analysis. RESULTS: A total of 160 female medical staff were randomly selected in this research, of whom seven scored less than 3 points, 85 scored 3-11 points, and 68 scored more than 11 points on the total score of the SCMM. Compared to pre-pandemic scores, scores of dizziness and tinnitus were significantly higher during the COVID-19 pandemic. Scores corresponding to the following clinical symptoms were also higher during the pandemic: Menopathy, including hypaphrodisia, dim complexion, abnormal urination, languidness, dim menstruation, thin menstruation, dysmenorrhea, and empty or saggy lower abdomen (p < 0.05). However, pre-pandemic scores of vaginal bleeding quantity were significantly higher than those found during the COVID-19 pandemic (p < 0.05). Scores of vaginal bleeding quantity were significantly lower in cabin hospitals than other types of hospitals, and a similar finding was observed for vaginal bleeding duration (all p < 0.05). Moreover, the findings of the univariable and multivariable linear regression analysis revealed a link between consistent exercise, the underlying illness, the SDRS score, the SAS score, and the total score of SCMM (p < 0.05). CONCLUSIONS: In this study, we found that menstruation in female medical workers was affected by the COVID-19 pandemic. Furthermore, regular exercise and good physical condition were protective factors, while anxiety and insomnia were risk factors for menstrual abnormalities.

Effect of 0.01% Atropine on diopter and optic axis in adolescents and children with myopia.

The purpose of this study was to investigate the effect of 0.01% Atropine eye drops on diopter and optic axis in adolescents and children with myopia. A total of 164 children with myopia were randomly divided into two groups, Group A and Group B with 82 patients in each group, according to the digital table method. Group A was treated with 0.01% Atropine eye drops, while Group B was treated with single vision lenses. Before the treatment, there was no significant difference in diopter and axial length between the two groups (P=0.624 and P=0.123). After 12 months of treatment, the diopter and axial length of Group A were lower than those of Group B (P < 0.001 and P = 0.005). There were no obvious adverse reactions during corrective therapy in the two groups. The results show that compared with single vision lenses, 0.01% Atropine is more effective in correcting myopia, and may control the increase of optic axis in adolescents and children with myopia, in a better way, with high safety..

NLRP3 Inflammasome Activation Enhances ADK Expression to Accelerate Epilepsy in Mice.

Epilepsy (SE) is a common and serious neurological disease. NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome participates in the pathogenesis of SE, while its underlying mechanism is still unclear. Here, we attempted to explore the mechanism of action of NLRP3 inflammasome in SE. SE mouse model was constructed by administration of kainic acid (KA). Astrocytes were treated with KA to mimic SE cell model. MCC950 (NLRP3 inhibitor) and Z-YVAD-FMK (Caspase-1 inhibitor) were used to treat astrocytes to inhibit the activity of NLRP3 and Caspase-1. Nissl staining was performed to examine the morphology of neuron. Western blot, enzyme-linked immunosorbent assay and immunofluorescence staining were performed to assess protein expression. SE mouse model exhibited an increase of neuronal loss, and an up-regulation of Cleaved-Caspase-1, IL-1ß and IL-18 in hippocampus. The levels of GFAP+ADK+ cells were significantly increased in SE mice. MCC950 or Z-YVAD-FMK abolished these impacts conferred by KA in SE mice. Moreover, KA treatment enhanced the expression of NLRP3, Cleaved-Caspase-1, IL-1ß and IL-18 in astrocytes, which was rescued by knockdown of NLRP3 or Caspase-1. Additionally, CREB, p-CREB, REST were up-regulated, and SP1 was down-regulated in the KA-treated SE mice and KA-treated astrocytes. Inhibition of NLRP3 or Caspase-1 rescued these proteins expression in KA-treated astrocytes. CREB or REST silencing reduced adenosine kinase (ADK) expression, while SP1 knockdown enhanced ADK expression in KA-treated astrocytes. In conclusion, NLRP3 inflammasome activation enhances ADK expression to accelerate SE in mice through regulating CREB/REST/SP1 signaling pathway. Thus, inhibition of NLRP3 inflammasome may be a treatment for SE.

DCD-chip designed for the digital and ultraprecise quantification of copy number variation.

Copy number variation (CNV), including genomic deletions and duplication, has been associated with many kinds of diseases. It is crucial to precisely quantify the copy number variation of samples among patients, which may guide treatment. Digital PCR (dPCR) enables high-resolution CNV analysis through the ultraprecise absolute quantification of specific nucleic acid sequences. We explored a platform named digital CNV detection chip (DCD-chip), which can simultaneously and absolutely quantify the GPR146 and RPPH1 genes with amounts as low as 1.4 copies per µL. Finally, we verified that DCD-chip was more accurate than qPCR when the samples were diluted to a certain extent, which indicated the powerful quantification capacity of our DCD-chip platform.

GPR151 in nociceptors modulates neuropathic pain via regulating P2X3 function and microglial activation.

Neuropathic pain is a major health problem that affects up to 7-10% of the population worldwide. Currently, neuropathic pain is difficult to treat because of its elusive mechanisms. Here we report that orphan G protein-coupled receptor 151 (GPR151) in nociceptive sensory neurons controls neuropathic pain induced by nerve injury. GPR151 was mainly expressed in non-peptidergic C-fibre dorsal root ganglion neurons and highly upregulated after nerve injury. Importantly, conditional knockout of Gpr151 in adult nociceptive sensory neurons significantly alleviated chronic constriction injury-induced neuropathic pain-like behaviour but did not affect basal nociception. Moreover, GPR151 in DRG neurons was required for chronic constriction injury-induced neuronal hyperexcitability and upregulation of colony-stimulating factor 1 (CSF1), which is necessary for microglial activation in the spinal cord after nerve injury. Mechanistically, GPR151 coupled with P2X3 ion channels and promoted their functional activities in neuropathic pain-like hypersensitivity. Knockout of Gpr151 suppressed P2X3-mediated calcium elevation and spontaneous pain behaviour in chronic constriction injury mice. Conversely, overexpression of Gpr151 significantly enhanced P2X3-mediated calcium elevation and dorsal root ganglion neuronal excitability. Furthermore, knockdown of P2X3 in dorsal root ganglia reversed chronic constriction injury-induced CSF1 upregulation, spinal microglial activation and neuropathic pain-like behaviour. Finally, the coexpression of GPR151 and P2X3 was confirmed in small-diameter human dorsal root ganglion neurons, indicating the clinical relevance of our findings. Together, our results indicate that GPR151 in nociceptive dorsal root ganglion neurons plays a key role in the pathogenesis of neuropathic pain and could be a potential target for treating neuropathic pain.

A multiplex and fast detection platform for microRNAs based on a self-priming microfluidic chip and duplex-specific nuclease.

MicroRNA expression levels highly correlate with the occurrence, diagnosis and prognosis of disease. However, challenges remain in establishing a multiplex and fast detection method. Here, we developed a multiplex and fast detection platform for microRNAs based on a self-priming microfluidic chip and duplex-specific nuclease. It can detect three types of miRNAs, including miR-100, miR-155, and Let-7a, simultaneously at 50 °C within 1 h. The probes are pre-introduced into the chip using the self-priming method and cross-contamination can be avoided by using a screw valve. The reagent consumption and cost have been largely reduced in this work compared to the traditional detection method. This chip also exhibits good quantitative performance and specificity. As a proof of concept, we detect three types of microRNAs from different cancer cell lines, which demonstrates its potential in real sample analysis. In summary, this microfluidic chip shows great advantages in multiplex, fast and simple detection of microRNAs, and possesses great potential in the early diagnosis of miRNA-related diseases, especially for point-of-care application.

The diagonal branches and outcomes in patients with anterior ST- elevation myocardial infarction.

BACKGROUND: The management of diagonal branch (D) occlusion is still controversary. The association between the flow loss of D and the prognosis remains unclear. We aim to detect the impact of D flow on cardiac function and clinical outcomes in patients with anterior ST-segment elevation myocardial infarction (STEMI). METHODS: Patients with anterior STEMI undergoing primary percutaneous coronary intervention (PCI) at our clinic between October 2015 and October 2018were reviewed. Anterior STEMI due to left anterior descending artery (LAD) occlusion with or without loss of the main D flow (TIMI grade 0-1 or 2-3) was enrolled in the analysis. The short- and long-term incidence of major adverse cardiac events (MACEs, a composite of all-cause death, target vessel revascularization and reinfarction) and left ventricular ejection fraction (LVEF) were analyzed. RESULTS: A total of 392 patients (mean age of 63.9 years) with anterior STEMI treated with primary PCI was enrolled in the study. They were divided into two groups, loss (TIMI grade 0-1, n = 69) and no loss (TIMI grade2-3, n = 323) of D flow, before primary PCI. Compared with the group without loss of D flow, the group with loss of D flow showed a lower LVEF post PCI (41.0% vs. 48.8%, p = 0.003). Meanwhile, loss of D flow resulted in the higher in-hospital, one-month, and 18-month incidence of MACEs, especially in all-cause mortality (all p < 0.05). Landmark analysis further indicated that the significant differences in 18-month outcomes between the two groups mainly resulted from the differences during the hospitalization. In addition, multivariate Cox proportional hazards analysis found that D flow loss before primary PCI was independent factor predicting short- and long-term outcomes in patients with anterior STEMI. CONCLUSION: Loss of the main D flow in anterior STEMI patients was independently associated with the higher in-hospital incidences of MACEs and all-cause death as well as the lower LVEF.

Tumor Size Improves the Accuracy of the Prognostic Prediction of Lymph Node-Negative Gastric Cancer.

BACKGROUND: Tumor size has been regarded as the "T" stage of many solid tumors because of its effect on prognosis. However, the prognostic impact of tumor size in gastric cancer (GC) is still controversial. MATERIALS AND METHODS: A total of 436 patients with curatively resected GC and those without lymph node metastasis in our center were retrospectively enrolled. The appropriate cutoff points for tumor size were determined. Potential prognostic factors were analyzed. In addition, a pathological tumor-size (pTS) classification system was proposed to evaluate the superiority of its prognostic prediction of node-negative GC patients compared with that of the pT staging system. RESULTS: The ideal cutoff points for tumor size were 4 and 8 cm. In the multivariate analysis, tumor size was identified as an independent prognostic factor for node-negative GC patients after surgery, as was pT stage. The pTS classification was found to be more appropriate for predicting the overall survival of node-negative GC patients after curative surgery than pT stage, and the -2 log-likelihood of the pTS classification (1680.782) was smaller than the value of pT (1695.239). CONCLUSIONS: As an independent prognostic factor, tumor size should be incorporated into the pT staging system to enhance the accuracy of the prognostic prediction of node-negative GC patients.

IL-35 prevent bone loss through promotion of bone formation and angiogenesis in rheumatoid arthritis.

OBJECTIVES: Angiogenesis in bone and osteogenesis appear to be closely linked, suggesting the existence of molecular crosstalk between pro-angiogenic molecules and osteoblasts. The pro-angiogenic molecules vascular endothelial growth factor (VEGF) with its receptors Flt-1, Flk-1 and fibroblast growth factor (FGF)-2 play a crucial role in born formation, an early and critical event in the pathogenesis of rheumatoid arthritis (RA). Interleukin (IL)-35 is demonstrated to be an anti-inflammatory cytokine in RA. However, the mechanisms involved are not fully understood. This study aims to investigate whether IL-35 has an impact on angiogenesis in osteoblasts and its related signalling pathway in RA. METHODS: The effects of IL-35 on osteoblasts proliferation, apoptosis and pro-angiogenic molecules mRNA and protein were examined using osteoblast-like MC3T3E1 cells in vitro. The effects of IL-35 on proliferation and apoptosis were examined using cell counting kit-8 (CCK-8) assay and flow cytometry, respectively. Pro-angiogenic molecules expression were assessed by real time PCR and ELISA, respectively. The signalling pathway between IL-35, bone formation, angiogenesis and signalling pathway was also investigated. RESULTS: IL-35 promoted osteoblasts proliferation and inhibited apoptosis in a dose-dependent manner in vitro. IL-35 increased basal and TNF-α induced pro-angiogenic molecules expression by osteoblasts. Blocking the Th17/IL-17 signalling pathway with plumbagin inhibited the pro-angiogenic effects of IL-35 in osteoblasts. CONCLUSIONS: These results suggested that IL-35 promotes bone formation and angiogenesis by fostering osteoblasts proliferation, inhibiting apoptosis and upregulating pro-angiogenic molecules through Th17/IL-17 related-signalling pathway. Our findings extend the current understanding of mechanisms modulating bone formation and angiogenesis in RA.

Correlation between Serum IL-35 Levels and Bone Loss in Postmenopausal Women with Rheumatoid Arthritis.

OBJECTIVE: IL-35 was reported as a crucial anti-inflammatory cytokine and could efficiently regulate bone metabolism in murine collagen-induced arthritis model. However, the relationship between IL-35 and bone health in human rheumatoid arthritis (RA) has not been clarified. In this study, the aim was to explore the correlations between IL-35 and bone loss in postmenopausal women with RA. METHODS: The study included 76 postmenopausal women with RA and 53 healthy postmenopausal women as healthy controls (HCs). Serum IL-35 levels were detected by enzyme-linked immunosorbent assay. Bone mineral density (BMD) at lumbar spine 1-4 and at total hip was measured using dual-energy X-ray absorptiometry. Alkaline phosphatase (ALP), ß-isomerised carboxy-terminal cross-linking telopeptide of type I collagen (ß-CTX), and 25-(OH) VitD3 were measured by turbidimetric inhibition immunoassay. RESULTS: Serum IL-35 levels were increased compared with HCs, and it positively correlated with BMD and 25-(OH) VitD3 and negatively correlated with ß-CTX in postmenopausal women with RA. Furthermore, serum IL-35 levels in the increased ALP group were higher than those in the normal ALP group. CONCLUSIONS: IL-35, an important anti-inflammatory cytokine, may participate in the pathogenesis of bone loss in postmenopausal women with RA.

Changes in Serum Cytokines May Predict Therapeutic Efficacy of Tofacitinib in Rheumatoid Arthritis.

OBJECTIVE: Tofacitinib is a novel therapy for rheumatoid arthritis (RA). The aim of this study was to measure various serum cytokines levels and to explore potential markers predictive of therapeutic efficacy of tofacitinib for RA patients. METHODS: Thirty-two patients with RA were given tofacitinib (5 mg bid). Serum cytokines levels of Th1 (IFN-γ), Th2 (IL-6), Th17 (IL-17), Tregs (IL-35), and TNF-α were detected by enzyme-linked immunosorbent assays. RESULTS: Disease activity was significantly decreased as early as week 4 after tofacitinib treatment. Serum IL-35 levels were significantly increased and serum levels of TNF-α, IL-17, IL-6, and IFN-γ were significantly reduced in response to tofacitinib since week 4. CONCLUSIONS: After treatment with tofacitinib, RA patients may benefit from monitoring of disease activity as early as week 4. IL-35 also might be a predictive indicator of the disease activity and drug efficacy. Meanwhile, tofacitinib might be CS-sparing in RA.

Elevated frequency of IL-37- and IL-18Rα-positive T cells in the peripheral blood of rheumatoid arthritis patients.

OBJECTIVE: To detect the expression of IL-37 and its receptors IL-18Rα and IL-1R8 in CD4+ T cells and total lymphocytes in rheumatoid arthritis (RA) patients and the relationship between autoantibodies and disease activity. To investigate the mechanism of IL-37 and its receptors involved in the pathogenesis of RA. To evaluate the effects of different concentrations of rhIL-37 on peripheral blood mononuclear cells (PBMCs) in RA patients with TNF-α, and IL-6. METHODS: The expression of IL-37 and its receptor IL-18Rα and IL-1R8 in peripheral blood CD4+ T cells and total lymphocytes in RA patients and healthy controls were measured by flow cytometry. The levels of TNF-α and IL-6 in the supernatant were measured by ELISA after rhIL-37 stimulation with PBMCs. RESULTS: The expression of IL-37 and IL-18Rα in the total lymphocytes, especially in CD4+ T cells in RA patients, was significantly higher than in the healthy control group. There was a positive correlation between the frequency of IL-37- or IL-18Rα-positive CD4+ T cells and ESR, CRP, and DAS28 values. Additionally, rhIL-37 significantly down-regulated TNF-α and IL-6 production in RA patients' PBMCs. CONCLUSIONS: IL-37 plays an important role in the regulation of inflammation in RA. IL-37 and its receptors may play an immunoregulatory role in the activation of lymphocytes, especially CD4+ T cells, in RA patients. IL-37 may represent a therapeutic target for rheumatoid arthritis.

Interleukin-35 inhibits angiogenesis through STAT1 signalling in rheumatoid synoviocytes.

OBJECTIVES: We studied the anti-angiogenic effect of interleukin-35 (IL-35) by investigating its effects on signal transmission through the Janus kinase signal transducer and activator of transcription (JAK-STAT) pathway in fibroblast-like synoviocytes (FLS). METHODS: Using the collagen-induced arthritis (CIA) model of rheumatoid arthritis (RA), we derived and cultured FLS, stimulated FLS with IL-35 at different concentrations and examined the expression levels of mRNA and protein of both vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), endostatin, TNF-α, and IL-6 using reverse transcription polymerase chain reaction (RT-PCR) and immunoblotting. We used Western blotting to study the effects of IL-35 on the function of the JAK-STAT pathway in FLS. RESULTS: IL-35 treatment inhibited the expression of VEGF, FGF-2, TNF-α and IL-6, and increased the expression of endostatin in FLS. Western blotting showed that IL-35 treatment of CIA-derived FLS resulted in signalling through STAT1, but not through STAT3 or STAT5. CONCLUSIONS: IL-35 signalling through STAT1 and inhibition of the expression of mediators of angiogenesis and inflammation in FLS provide a likely mechanism for anti-angiogenic effects seen in experimental models of RA. Our data suggest that IL-35 and its signalling pathway represent a therapeutic target for the treatment of RA and other angiogenesis-related diseases.

Peripheral immune tolerance alleviates the intracranial lipopolysaccharide injection-induced neuroinflammation and protects the dopaminergic neurons from neuroinflammation-related neurotoxicity.

BACKGROUND: Neuroinflammation plays a critical role in the onset and development of neurodegeneration disorders such as Parkinson's disease. The immune activities of the central nervous system are profoundly affected by peripheral immune activities. Immune tolerance refers to the unresponsiveness of the immune system to continuous or repeated stimulation to avoid excessive inflammation and unnecessary by-stander injury in the face of continuous antigen threat. It has been proved that the immune tolerance could suppress the development of various peripheral inflammation-related diseases. However, the role of immune tolerance in neuroinflammation and neurodegenerative diseases was not clear. METHODS: Rats were injected with repeated low-dose lipopolysaccharide (LPS, 0.3 mg/kg) intraperitoneally for 4 days to induce peripheral immune tolerance. Neuroinflammation was produced using intracranial LPS (15 µg) injection. Inflammation cytokines were measured using enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR). Microglial activation were measured using immunostaining of Iba-1 and ED-1. Dopaminergic neuronal damage was evaluated using immunochemistry staining and stereological counting of TH-positive neurons. Behavioral impairment was evaluated using amphetamine-induced rotational behavioral assessment. RESULTS: Compared with the non-immune tolerated animals, pre-treatment of peripheral immune tolerance significantly decreased the production of inflammatory cytokines, suppressed the microglial activation, and increased the number of dopaminergic neuronal survival in the substantia nigra. CONCLUSIONS: Our results indicated that peripheral immune tolerance attenuated neuroinflammation and inhibited neuroinflammation-induced dopaminergic neuronal death.

Peripheral Blood Monocyte Tolerance Alleviates Intraperitoneal Lipopolysaccharides-Induced Neuroinflammation in Rats Via Upregulating the CD200R Expression.

Neuroinflammation is an important pathogenesis of Parkinson's disease (PD). The peripheral immune system could produce profound effects on central immunities. The peripheral blood monocyte (PBM) immune tolerance is the refractoriness of immune system to avoid overactive peripheral inflammation. The PBM are also actively involved in central immune activities. There is evidence implying the probable failure of immune tolerance and impairment of CD200/CD200R signaling in PD patients. Here we aimed to explore the effects of PBM tolerance in peripheral LPS-induced neuroinflammation as well as the specific roles of CD200/CD200R pathway in PBM tolerance. We found that repeated intraperitoneal administration of 0.3 mg/kg LPS was able to induce the PBM tolerance. PBM tolerance reduced peripheral LPS-induced elevation of serum TNF-α, IL-1ß expression and TLR4 expression in PBM. PBM tolerance and PBM depletion alleviated peripheral LPS-induced neuroinflammation demonstrated by reduced proinflammatory cytokines in brain and blocked microglia activation. The CD200R expression in PBM was upregulated in PBM tolerance group after intraperitoneal administration of high-dose LPS in vivo and the blockade of CD200/CD200R interaction induced the failure of PBM tolerance in vitro. These results suggested the PBM tolerance could attenuate the peripheral LPS-induced neuroinflammation via upregulating the CD200R expression and the CD200/CD200R signaling played a key role in PBM tolerance. Effective regulation of the PBM in periphery may be a potential way to limit neuroinflammation while the CD200R on PBM could be used as a potential therapeutic target to alleviate neuroinflammation.