Two Novel Variants of the CAPN3 Gene in Chinese Patients with Limb-Girdle Muscular Dystrophy Recessive 1.

INTRODUCTION: Recessive mutations in the CAPN3 gene can lead to limb-girdle muscular dystrophy recessive 1 (LGMD R1). Targeted next-generation sequencing facilitates the discovery of new mutations linked with disease, owing to its ability to selectively enrich specific genomic regions. METHODS: We performed targeted next-generation sequencing of all exons of the CAPN3 gene in 4 patients with sporadic limb-girdle muscular dystrophy (LGMD) and further analyzed the effects of the novel identified variant using various software tools. RESULTS: We found 5 variants in CAPN3 gene in 4 patients, c.82_83insC (insertion mutation) and c.1115+2T>C (splicing mutation) are reported for the first time in CAPN3 (NM_000070.2). The bioinformatics analysis indicated that these two novel variants affected CAPN3 transcription as well as translation. DISCUSSION: Our findings reveal previously unreported splicing mutation and insertion mutation in CAPN3 gene, further expanding the pathogenic gene profile of LGMD.

Effects of silent brain infarction on the hemorrhagic transformation and prognosis in patients with acute ischemic stroke after intravenous thrombolysis.

Background: Silent brain infarction (SBI) is a special type of stroke with no definitive time of onset, which can be found on pre-thrombolysis imaging examination in some patients with acute ischemic stroke (AIS). However, the significance of SBI on intracranial hemorrhage transformation (HT) and clinical outcomes after intravenous thrombolysis therapy (IVT) is uncertain. We aimed to explore the effects of SBI on intracranial HT and the 3-month clinical outcome in patients with AIS after IVT. Methods: We consecutive collected patients who were diagnosed with ischemic stroke and received IVT from August 2016 to August 2022, and conducted a retrospective analysis in this study. The clinical and laboratory data were obtained from hospitalization data. Patients were divided into SBI and Non-SBI groups based on clinical and neuroimaging data. We use Cohen's Kappa to assess the interrater reliability between the two evaluators, and multivariate logistic regression analysis was used to further assess the association between SBI, HT and clinical outcomes at 3 months after IVT. Results: Of the 541 patients, 231 (46.1%) had SBI, 49 (9.1%) had HT, 438 (81%) had favorable outcome, 361 (66.7%) had excellent outcome. There was no significant difference in the incidence of HT (8.2 vs. 9.7%, p = 0.560) and favorable outcome (78.4% vs. 82.9%, p = 0.183) between patients with SBI and Non-SBI. However, patients with SBI had a lower incidence of excellent outcome than the patients with Non-SBI (60.2% vs. 71.6%%, p = 0.005). After adjustment for major covariates, multivariate logistic regression analysis disclosed that SBI was independently associated with the increased risk of worse outcome (OR = 1.922, 95%CI: 1.229-3.006, p = 0.004). Conclusion: We found that SBI was no effect for HT after thrombolysis in ischemic stroke patients, and no effect on favorable functional outcome at 3 months. Nevertheless, SBI remained an independent risk factor for non-excellent functional outcomes at 3 months.

Inducible costimulator ligand (ICOSL) on CD19+ B cells is involved in immunopathological damage of rheumatoid arthritis (RA).

Inducible costimulator (ICOS) and its ligand (ICOSL) are critical to regulate the immune response in autoimmune diseases. The participation of B lymphocytes exhibits pathogenic potential in the disease process of rheumatoid arthritis (RA). However, the precise role of ICOSL in RA remains unclear. In this study, we aimed to explore the regulatory effects of CD19+ICOSL+ B cells in the pathogenesis of RA. We demonstrated the increased expression of ICOS and ICOSL in patients with RA and collagen-induced arthritis (CIA) mice. The population of CD19+ICOSL+ B-cell subset was significantly correlated with clinicopathological characteristics of RA patients and CIA mice. Adoptive transfer of CD19+ICOSL+ B cells aggravated arthritic progression in CIA mice. Moreover, microarray analysis revealed that CD19+ICOSL+ cells could exert pivotal effect in pathological process of RA. Further blocking of ICOSL significantly inhibited proinflammatory responses and ameliorated arthritic progression. Therefore, CD19+ICOSL+ B-cell subset could be defined as a specific pathogenic cell subpopulation involved in immunopathological damage of RA. Blockade of ICOSL is promising to be a potential new approach for RA therapy.

Primary biliary cirrhosis associated with myasthenia gravis after postpartum: a case report.

BACKGROUND: Autoimmune diseases refers to a class of diseases involving abnormal immune response of human body and tissue damage caused by the dysregulation of autoimmune balance or destruction of immune tolerance. Recent research has revealed that the occurrence of autoimmune diseases is influenced by genetic, hormonal, immunological, and environmental factors. As sex hormone levels change obviously during pregnancy and postpartum, the morbidity and recurrence rate of autoimmune diseases increase during this period. CASE PRESENTATION: A 31-year-old Asian woman was admitted to our hospital for myasthenia gravis and treated with methylprednisolone and pyridostigmine bromide 3 months postpartum. Physical examination and laboratory inspection after admission suggested that the patient had primary biliary cirrhosis. Subsequently, azathioprine was added to the treatment, and the symptoms of both diseases were successfully controlled. CONCLUSIONS: This case exhibits a rare condition of myasthenia gravis combined with primary biliary cirrhosis postpartum. Given the fluctuation of the immune status during the postpartum period, combined autoimmune diseases need to be taken into account when patients develop clinical symptoms of an autoimmune disease. Therefore, detailed physical and laboratory examination can help to prevent the missed diagnosis of these diseases.

hsa-miR-7 Is a Potential Biomarker for Idiopathic Inflammatory Myopathies with Interstitial Lung Disease in Humans.

OBJECTIVES: microRNAs (miRNAs) have been identified as biomarkers for various diseases. However, the significance of circulating miRNAs for the diagnosis of idiopathic inflammatory myopathies (IIM) is still unknown. In this study, we aim to investigate the significance of miRNAs as potential biomarkers for predicting the patients with IIM and interstitial lung disease (ILD) METHODS: Total RNA was isolated from plasma of 43 IIM patients and 43 healthy people. The expression of miRNAs was analyzed by miRNA microarray and validated by qRT-PCR RESULTS: Microarray shows more differentially expressed circulating miRNAs found in IIM patients compared with healthy controls (P<0.05). qRT-PCR confirmed miR-7 and miR-21 showes significantly changed levels in plasma samples between IIM patients and healthy controls (P<0.05). However, only miR-7 was the sole miRNA lower expression in each IIM patient (P<0.05), which is also lower expression in IIM/ILD patients than that without ILD (P<0.05). The area under curve (AUC) for distinguishing IIM/ILD patients from IIM without ILD is 0.8978, and the 95% confidence interval is 0.7961 to 0.9995. The receiver operating characteristic (ROC) curve analysis showes miR-7 cutoff value is 0.0063. Further, AUC analysis showes both miR-7 and miR-21 have diagnostic value as biomarkers for IIM patients form health controls; however, miR-7 is more sensitive CONCLUSION: miR-7 is a potential biomarker as a diagnostic indicator for IIM patients and can be used to distinguish IIM/ILD patients from IIM without ILD.

OX40 signaling is involved in the autoactivation of CD4+CD28- T cells and contributes to the pathogenesis of autoimmune arthritis.

BACKGROUND: CD4+CD28- T cells exhibit autoreactive potential in autoimmune disorders, including rheumatoid arthritis (RA). It is not well known which costimulator functions as an alternative second signal in the activation of this subset after CD28 expression is downregulated. Tumor necrosis factor receptor superfamily member OX40 is a key costimulator in the activation of T cells. The aim of this study was to investigate the costimulatory effects of OX40 on CD4+CD28- T cells in autoimmune arthritis. METHODS: Clinical samples were collected from patients with RA and control subjects. Collagen-induced arthritis (CIA) was induced with collagen type II (CII) in DBA/1 mice. The CD4+CD28-OX40+ T-cell subset and its cytokine production were detected by flow cytometry. After T-cell purification, adoptive transfer was performed in CIA mice. The regulatory role of OX40 was determined by blocking experiments in vitro and in vivo. RESULTS: OX40 and OX40L were abnormally expressed in patients with RA and CIA mice. Further analysis showed that CD4+CD28-OX40+ T cells accumulated in patients with RA and in animal models. These cells produced higher levels of proinflammatory cytokines and were closely correlated with the clinicopathological features of the affected individuals. Adoptive transfer of CII-specific CD4+CD28-OX40+ T cells remarkably aggravated arthritic development and joint pathology in CIA mice. Moreover, OX40 blockade significantly reduced the proinflammatory responses and ameliorated arthritis development. CONCLUSIONS: OX40 acts as an alternative costimulator of CD4+CD28- T cells and plays a pathogenic role in autoimmune arthritic development, suggesting that it is a potential target for immunomodulatory therapy of RA.

Development of a Novel Functional Monoclonal Antibody to Human CD275: Characterization and Biological Activity.

CD275 (B7-H2, ICOSL), a co-stimulatory molecule of the B7 superfamily, plays a critical role in immune response. In this report, a novel mouse anti-human CD275 monoclonal antibody (MAb) was prepared using hybridoma technology, and immunological characteristics of the MAb were determined. The results showed that the MAb (clone 13D11) was IgG2(κ) and bound specifically to human CD275. By mutual competition, we found that the antibody recognized different epitopes of CD275 antigen compared with commercial antibodies and could block ICOS-CD275 interaction. Crosslinking of CD275 with MAb 13D11 markedly blocked ICOS positive signal and inhibited T cell proliferation and cytokine production. In addition, the 13D11 MAb was suitable for indirect ELISA detection. Thus, the MAb against human CD275 with high specificity and different activity would be useful for further study of this molecule.

Soluble PD-1 aggravates progression of collagen-induced arthritis through Th1 and Th17 pathways.

INTRODUCTION: The programmed cell death 1 (PD-1) protein is a critical regulator of T-cell activation and is also an important therapeutic target for autoimmune diseases. Little is known about the regulation and functional properties of the soluble PD-1 (sPD-1) variant. The aim of this study was to examine the role of sPD-1 in the regulation of human and murine rheumatoid arthritis (RA). METHODS: Expression of cytokines and sPD-1 in sera, synovial fluid, and peripheral blood (PB) mononuclear cells of patients with RA were analyzed by enzyme-linked immunosorbent assay and quantitative polymerase chain reaction. PD-1 function was assessed in PB T cells after stimulation of the cells with anti-CD3 and PD-L1-Fc to crosslink PD-1. Recombinant PD-1-Fc was injected intraperitoneally into DBA/1 mice with collagen-induced arthritis (CIA) to analyze the function of sPD-1 in vivo. RESULTS: High concentrations of sPD-1 were found in sera and synovial fluid of patients with RA. The levels of serum sPD-1 were significantly correlated with titers of rheumatoid factor (RF) (r = 0.306, p = 0.005) and 28-joint Disease Activity Score (r = 0.545, p < 0.001). Further characterization of sPD-1 revealed that it functionally blocked the inhibitory effect of membrane-bound PD-1 on T-cell activation. Interferon γ, tumor necrosis factor α, and interleukin 17A were identified as inducers of sPD-1 in vitro. Moreover, PD-1-Fc enhanced proinflammatory cytokine expression, generation of Th1 cells and Th17 cells, and joint pathology in a CIA model. CONCLUSIONS: sPD-1 regulates peripheral T-cell responses in both human and murine RA. Thus, sPD-1 may represent an additional biomarker or target in immunomodulatory therapy for RA.

Correlation between B7-H3 expression and rheumatoid arthritis: A new polymorphism haplotype is associated with increased disease risk.

CD276 (B7-H3) is a costimulatory molecule that plays a potent role in T cell responses, however, the role of B7-H3 in autoimmune diseases has not been elucidated. We analyzed B7-H3 expression in rheumatoid arthritis (RA) for the first time and found B7-H3 was significantly up-regulated on monocytes in RA patients, while the levels of soluble B7-H3 in serum were lower than in controls (P < 0.0001). These differences correlated with clinical and laboratory disease parameters and informatory factor TNF-α. Through in vitro experiments, we demonstrated that B7-H3 promoted TNF-α secretion. In addition, a new polymorphism variant, B7-H3-T-A-C-T, was identified and shown to be associated with the incidence of RA and the decreased release of sB7-H3. These results suggest that B7-H3 may be a promising biomarker associated with the pathogenesis of RA. Notably, the new B7-H3-T-A-C-T polymorphism variant is associated with RA risk and might be associated with the release of soluble B7-H3.

A Promoter Region Polymorphism in PDCD-1 Gene Is Associated with Risk of Rheumatoid Arthritis in the Han Chinese Population of Southeastern China.

Objective. Programmed cell death 1 (PD-1) induces negative signals to T cells during interaction with its ligands and is therefore a candidate gene in the development of autoimmune diseases such as rheumatoid arthritis (RA). Herein, we investigate the association of PDCD-1 polymorphisms with the risk of RA among Chinese patients and healthy controls. Methods. Using the PCR-direct sequencing analysis, 4 PDCD-1 SNPs (rs36084323, rs11568821, rs2227982, and rs2227981) were genotyped in 320 RA patients and 309 matched healthy controls. Expression of PD-1 was determined in peripheral blood lymphocytes by flow cytometry and quantitative real-time reverse transcriptase polymerase chain reaction. Results. We observed that the GG genotype of rs36084323 was associated with a increased risk for developing RA (OR 1.70, 95% 1.11-2.61, P = 0.049). Patients carrying G/G genotype displayed an increased mRNA level of PD-1 (P = 0.04) compared with A/A genotype and healthy controls. Meanwhile, patients homozygous for rs36084323 had induced basal PD-1 expression on activated CD4+ T cells. Conclusion. The PDCD-1 polymorphism rs36084323 was significantly associated with RA risk in Han Chinese population. This SNP, which effectively influenced the expression of PD-1, may be a biomarker of early diagnosis of RA and a suitable indicator of utilizing PD-1 inhibitor for treatment of RA.

[Expressions and clinical significance of OX40 and OX40L in peripheral blood of patients with primary Sjogren's syndrome].

OBJECTIVE: To investigate the expressions of costimulatory molecules OX40 and OX40L on peripheral blood mononuclear cells (PBMC) and their relationship with clinical characteristics of patients with primary Sjogren's syndrome (pSS). METHODS: Peripheral blood samples were collected from 51 pSS patients and 36 healthy subjects (HC). The expressions of OX40 and OX40L on PBMC were detected by immunofluorescence and flow cytometry. In addition, we observed the changes in the levels of OX40 and OX40L after treatment in 11 patients with primary pSS and searched for the relationship between their expression levels and patients' clinical manifestations. RESULTS: The expression of OX40 on CD4(+);T cells in pSS patients was significantly higher than that in the HC group (8.65%±3.51% vs 5.68%±1.68%, P<0.01). However, there was no significant difference in OX40 expression on CD8(+);T cells between patient group and HC group. In comparison with HC group, the expression of OX40L on CD14(+); monocytes (6.76%±3.60% vs 3.15%±1.89%, P<0.01) and CD19(+);B cells (4.69%±2.40% vs 2.76%±1.33%, P<0.01) significantly increased in pSS patients. Moreover, OX40 expression on CD4(+);T cells and OX40L expression on monocytes and B cells rose significantly in active pSS patients compared with those in inactive patients. The expression levels of OX40 and OX40L were higher in pSS patients with multiple system damage than in patients with simple exocrine gland injury. In addition, immunosuppressive therapy significantly reduced the expressions of OX40 and OX40L. CONCLUSION: The expressions of OX40 and OX40L on peripheral lymphocytes was upregulated in pSS patients. The high levels of OX40 and OX40L expression were significantly correlated with clinical outcome and therapeutic response, suggesting that OX40/OX40L pathway may play a critical role in pSS pathogenesis.

Rapamycin protects the mitochondria against oxidative stress and apoptosis in a rat model of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disease, in which oxidative stress and mitochondrial dysfunction are responsible for neuronal apoptosis. Rapamycin plays a crucial role in reducing oxidative stress and protecting the mitochondria. However, its protective role in PD has not yet been fully elucidated. In this study, we report that pre-treatment with rapamycin provides behavioral improvements, protects against the loss of dopaminergic neurons, and alleviates mitochondrial ultrastructural injuries in a rat model of PD. Peroxide levels were lower and antioxidant activities were higher in PD rats pre-treated with rapamycin compared to the PD rats pre-treated with the vehicle. Furthermore, pre-treatment with rapamycin significantly elevated the expression of anti-apoptotic markers and reduced the levels of pro-apoptotic markers compared to pre-treatment with the vehicle. In conclusion, our results demonstrated that rapamycin reduced oxidative stress and alleviated mitochondrial injuries in the 6-hydroxydopamine (6-OHDA)-induced rat model of PD, which may subsequently contribute to its anti-apoptotic effects. The ability of rapamycin to exhibit neuroprotection in a rat model of PD may be related to its antioxidant capabilities.

Enhancement of membrane B7-H3 costimulatory molecule but reduction of its soluble form in multiple sclerosis.

PURPOSE: Multiple sclerosis (MS) is an autoimmune disease of the central nervous system mediated by T cells. B7-H3 plays a diverse role in regulating T cell responses. However, its expression and clinical significance in MS are not well known. This study analyzed the expression of membrane B7-H3 (mB7-H3) and levels of soluble B7-H3 (sB7-H3) in MS patients to determine its clinical significance. METHODS: Peripheral blood (PB) or cerebrospinal fluid (CSF) samples from healthy controls, other noninflammatory neurological disorders, viral encephalitis, and MS patients were collected. Expression of mB7-H3 on immune cells was detected by flow cytometry. Levels of sB7-H3 in serum or CSF samples were measured by ELISA. RESULTS: mB7-H3 expression was up-regulated in CSF from MS patients compared to PB (p<0.001). However, serum or CSF levels of sB7-H3 in MS patients were significantly lower than those in controls (p<0.05). Relapsing-MS patients had higher CSF mB7-H3 expression than the remitting subgroup. Relapsing-MS patients had decreased serum and CSF sB7-H3 levels compared with the remitting subgroup. Neurological deficits showed negative correlations with serum or CSF sB7-H3 levels, but a positive correlation with CSF mB7-H3 expression. Methylprednisolone therapy significantly elevated sB7-H3 levels and reduced mB7-H3 expression compared with pre-therapy levels. sB7-H3 levels did not correlate with mB7-H3 expression. CONCLUSIONS: We demonstrated enhanced mB7-H3 expression and reduced sB7-H3 levels in MS patients which correlated with the clinical characteristics of MS patients. These results suggest that B7-H3 may be a promising biomarker and associated with the pathogenesis of MS.

[Expression of B7-H3 costimulatory molecule in peripheral blood of myasthenia gravis patients].

AIM: To reveal the clinical significance of B7-H3 costimulatory molecule in myasthenia gravis (MG) patients by analyzing membranous B7-H3 (mB7-H3) and soluble B7-H3 (sB7-H3) expressions. METHODS: We collected peripheral blood samples of 35 MG patients and 44 health controls (HC) and detected the expression of mB7-H3 on peripheral blood mononuclear cells (PBMCs) using flow cytometry. ELISA was performed to analyze the levels of sB7-H3 in plasma samples from MG patients and HC. RESULTS: There was no significant difference in the expressions of mB7-H3 on T lymphocytes, monocytes or B cells between MG patients and HC. However, the level of sB7-H3 from MG patients was (2.166±0.958) ng/mL, significantly lower than that from HC (3.379±0.768) ng/mL. The level of sB7-H3 in general MG (GMG) patients (1.664±0.699) ng/mL was lower than that in ocular MG (OMG) patients (2.396±0.985) ng/mL. In MG patients complicated with abnormal thymus, the level of sB7-H3 was (1.593±0.441) ng/mL, also lower than that in MG patients with normal thymus (2.364±1.014) ng/mL. In addition, a significant negative correlation was found between the levels of sB7-H3 and QMGS in MG patients (r=-0.4189, P=0.012), but sB7-H3 was not associated with mB7-H3 in MG patients. CONCLUSION: In MG patients, down-regulation of sB7-H3 is finely correlated to the severity of the disease. Its different expression levels in various types of MG patients indicate that this costimulatory molecule may be involved in the immunopathogenesis of MG.