Secretory Clusterin Inhibits Dopamine Neuron Apoptosis in MPTP Mice by Preserving Autophagy Activity.

Secretory clusterin (sCLU) plays an important role in the research progress of nervous system diseases. However, the physiological function of sCLU in Parkinson's disease (PD) are unclear. The purpose of this study was to examine the effects of sCLU-mediated autophagy on cell survival and apoptosis inhibition in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD. We found that MPTP administration induced prolonged pole-climbing time, shortened traction time and rotarod time, significantly decreased TH protein expression in the SN tissue of mice. In contrast, sCLU -treated mice took less time to climb the pole and had an extended traction time and rotating rod time. Meanwhile, sCLU intervention induced increased expression of the TH protein in the SN of mice. These results indicated that sCLU intervention could reduce the loss of dopamine neurons in the SN area and alleviate dyskinesia in mice. Furthermore, MPTP led to suppressed viability, enhanced apoptosis, an increased Bax/Bcl-2 ratio, and cleaved caspase-3 in the SN of mice, and these effects were abrogated by sCLU intervention. In addition, MPTP increased the levels of P62 protein, decreased Beclin1 protein, decreased the ratio of LC3B-II/LC3B-I, and decreased the numbers of autophagosomes and autophagolysosomes in the SN tissues of mice. These effects were also abrogated by sCLU intervention. Activation of PI3K/AKT/mTOR signaling with MPTP inhibited autophagy in the SN of MPTP mice; however, sCLU treatment activated autophagy in MPTP-induced PD mice by inhibiting PI3K/AKT/mTOR signaling. These data indicated that sCLU treatment had a neuroprotective effect in an MPTP-induced model of PD.

Increased Expression of CD95 in CD4+ Effector Memory T Cells Promotes Th17 Response in Patients with Myasthenia Gravis.

Emerging data have revealed that CD95 can evoke non-apoptotic signals, thereby promoting pro-inflammatory functions that link to the severity of autoimmune disorders. Here, we reported that the expression of CD95 in CD4+ effector memory T (CD4+ TEM) cells was increased in myasthenia gravis (MG) patients. We also found increased expression of CD95 in CD4+ TEM cells from MG patients correlated positively with clinical severity scores (QMGs), serum IL-17 levels and plasma cells (PCs) frequencies. Conventional treatment, such as glucocorticoid, could down-regulate the expression of CD95 in CD4+ TEM cells, QMGs, serum IL-17 levels and PCs frequencies from MG patients. In vitro, low-dose of agonistic anti-CD95 mAb could promote Th17 cell development. This effect was reversed by CD95 siRNA. Moverover, CD95 stimulation induced the phosphorylation of p38 and Erk1/2 and Th17 cell differentiation, and p38 specific inhibitor SB203580 or Erk1/2 specific inhibitor PD98059 could induce opposite changes. However, SB203580 or PD98059 do not abrogate the increase of CCR6+IL-17A+ cells, ROR-γt and IL-17 expression induced by CD95 triggering relatively to each corresponding control. This suggests that p38 and Erk1/2 MAPK pathway plays a role in expression of CCR6+IL-17A+ cells, ROR-γt and IL-17, but not in their increase induced by CD95 triggering. Taken together, this study revealed that increased expression of CD95 in CD4+ TEM cells promotes Th17 response under the microenvironment of MG.

Association between interleukin gene polymorphisms and susceptibility to gastric cancer in the Qinghai population.

OBJECTIVE: To investigate the associations between interleukin (IL) gene polymorphisms and susceptibility to gastric cancer in the Qinghai population, China. METHODS: Patients with gastric cancer and cancer-free controls were enrolled into the study from Qinghai Provincial People's Hospital between September 2016 and September 2018. Single nucleotide polymorphisms (SNPs) were genotyped with the Sequenom MassARRAY® SNP genotype system. The Hardy-Weinberg equilibrium in allele and genotype frequencies, and general characteristics between patients with gastric cancer and cancer-free controls, were evaluated using χ2-test. Potential associations between interleukin gene variants and the risk of gastric cancer were analysed by logistic regression. RESULTS: Among eight candidate SNPs, the allele and genotype frequency distribution of IL-1B rs1143634 polymorphism was significantly different between patients with gastric cancer (n = 190) and cancer-free controls (n = 186). The IL-1B rs1143634 GA genotype and IL-1B rs1143634 GA + AA genotype were associated with a reduced risk of gastric cancer, however, the remaining SNPs were not statistically associated with gastric cancer risk in the Qinghai population. CONCLUSION: The IL-1B rs1143634 polymorphism might be associated with a decreased risk of gastric cancer, and may be a protective factor against gastric cancer.

IL-21 enhances STAT3/Blimp-1 signaling pathway in B cells and contributes to plasma cell differentiation in newly diagnosed patients with myasthenia gravis.

The transcription factor Blimp-1 is necessary for the B cell differentiation toward immunoglobulin-secreting plasma cells. However, the immunopathological mechanisms of Blimp-1 that regulates B cell differentiation remain unclear in MG. The purpose of this study was to perform a quantitative and functional analysis of Blimp-1 in MG. A total of 34 patients with MG (18 ocular MG (OMG) and 16 generalized MG (GMG) and 20 healthy controls (HC) were recruited in this study. CD19+ B cells were isolated by positive selection using CD19 beads. The expression of Blimp-1 and p-STAT3 protein in isolated B cells was assessed by Western blot. Plasma cells were analyzed by flow cytometry. Serum IL-21 levels were detected by ELISA. Our data demonstrated that Blimp-1 in peripheral blood B cell of MG patients was significantly increased compared with HC. The increased expression of Blimp-1 was positively associated with clinical severity score (QMGs), plasma cell frequency, and serum IL-21 levels. Furthermore, glucocorticoid (GC) treatment reduced the expression of Blimp-1 and p-STAT3 in B cells, and this change was accompanied with relieved clinical severity, reduced plasma cell frequency, and decreased serum IL-21 levels. In vitro assay demonstrated that IL-21 stimulation upregulated STAT3 phosphorylation, increased Blimp-1 expression in B cells, and promoted plasma cell differentiation, and these processes could be inhibited by dexamethasone or STAT3 inhibitor stattic. This work indicates for the first time that aberrant expression of Blimp-1 exists on B cells and contributes to the plasma cell differentiation in MG patients. Modulation of IL-21/STAT3/Blimp-1 signaling pathway in B cells may be one of the mechanisms of glucocorticoid in the treatment of MG.

Altered expression of transcription factors IRF4 and IRF8 in peripheral blood B cells is associated with clinical severity and circulating plasma cells frequency in patients with myasthenia gravis.

Previous studies have shown that interferon regulatory factor-4 (IRF4) and IRF8 play critical but distinct roles in the differentiation of B cells into plasma cells (PCs). In the present study, we aimed to measure the expression levels of IRF4 and IRF8 in B cells from patients with myasthenia gravis (MG) and to investigate whether the expression of IRF4 and IRF8 associates with pathogenesis of MG. A total of 35 anti-acetylcholine receptor (AChR) antibody (Ab)-positive patients with MG [20 generalized MG (GMG) and 15 ocular MG (OMG) and 25 healthy donors were recruited in this study. The quantitative myasthenia gravis score (QMGS) was used to evaluate the clinical severity. Real-time PCR and Western blot were used to measure the levels of IRF4 and IRF8 expressed in peripheral blood B cells. Peripheral blood CD138+ PCs were assayed by flow cytometry. Our data demonstrated that the mRNA/protein levels of IRF4 and IRF8 were significantly higher and lower, respectively, in patients with OMG/GMG groups compared with healthy controls. In addition, IRF4 expression was significantly higher and IRF8 expression was significantly lower in GMG group than in OMG group. Pearson's correlation analysis revealed that IRF8 expression was negatively correlated with clinical severity, PCs frequency and anti-AChR Ab levels, while IRF4 expression and IRF4/IRF8 ratio was positively correlated with these parameters in two MG subgroups. Finally, glucocorticoid treatment can relieve the imbalance of IRF4/IRF8 in peripheral blood B cells, and this restoration is accompanied by reduced PCs frequency and clinical symptoms. These evidences suggest that IRF4 and IRF8 are important in the counter-balancing mechanisms controlling differentiation of PCs in MG. The disruption of the balanced IRF4/IFR8 ratio in B cells may play important roles in the pathogenesis of MG and offer a promising therapeutic target for the development of novel immunotherapy for MG patients.

Increased expression of P2X7 receptor in peripheral blood mononuclear cells correlates with clinical severity and serum levels of Th17-related cytokines in patients with myasthenia gravis.

OBJECTIVES: P2X7R is a well-documented activator of innate and adaptive immune responses. We aimed to measure the expression levels of P2X7R in peripheral blood mononuclear cells (PBMCs) from patients with myasthenia gravis (MG) and to investigate whether the expression of P2X7R is associated with pathogenesis of MG. PATIENTS AND METHODS: A total of 32 patients with MG (12 generalized MG (GMG) and 20 Ocular MG (OMG) and 22 healthy donors were recruited in this study. The quantitative MG score was used to evaluate the clinical severity. Real-time PCR and western blot were used to measure the levels of P2X7R expressed in PBMCs. Serum Th17-related cytokines (IL-1ß, IL-6, IL-17 and IL-21) were tested by ELISA. PBMCs from MG patients were purified and challenged by LPS with or without a selective P2X7R inhibitor (BBG). RESULTS: Our results showed that the expression of P2X7R mRNA and protein in PBMCs was increased in MG patients compared with healthy controls, with higher expression in generalized patients (GMG) than in ocular patients (OMG). In addition, P2X7R expression presents a significantly positive correlation with clinical severity and serum levels of IL-1ß, IL-6, IL-17 and IL-21 in MG. In cultured MG PBMC, LPS challenge led to up-regulated P2X7R expression accompanied with increased production of IL-1ß, IL-6, IL-17 and IL-21. Importantly, P2X7R blockade with BBG significantly attenuates the LPS-induced production of cytokines. CONCLUSION: P2X7R expression was up-regulated in MG and LPS-P2X7R axis may be involved in the pathogenesis of MG by promoting Th17 immune response.

CD19+ Tim-1+ B cells are decreased and negatively correlated with disease severity in Myasthenia Gravis patients.

T cell immunoglobulin mucin domain-1(Tim-1) was recently identified to be critical and essential for optimal regulatory B cells function in maintaining immune tolerance. We aimed to measure the expression levels of Tim-1 on B cells from patients with Myasthenia Gravis (MG) and to investigate whether the expression of Tim-1 is associated with pathogenesis of MG. A total of 34 patients with MG (18 generalized MG (GMG) and 16 ocular MG (OMG) and 24 healthy donors were recruited in this study. The quantitative myasthenia gravis score (QMGS) was used to evaluate the clinical severity. Real-time PCR and flow cytometry were used to measure the levels of Tim-1 expressed on peripheral B cells. Peripheral CD138+ plasma cells were assayed by flow cytometry. Serum Th17-related cytokines (IL-6, IL-1ß and IL-17) and anti-AChR antibody (Ab) titers were tested by enzyme-linked immunosorbent assay (ELISA). Our data demonstrated that the mRNA and protein expression levels of B cell Tim-1 in both the GMG and OMG groups were significantly lower than those in healthy controls, with lower expression in GMG than in OMG. Tim-1 expression on B cells from OMG/GMG was negatively correlated with clinical severity, plasma cells frequency, serum Th17-related cytokines and anti-AChR Ab levels. Our results indicated that aberrant expression of Tim-1 exists on B cells and may contribute to the Th17 polarization and antibody-secreting plasma cells differentiation in MG patients.

Decreased microRNA miR-181c expression in peripheral blood mononuclear cells correlates with elevated serum levels of IL-7 and IL-17 in patients with myasthenia gravis.

miR-181c is a newly identified negative regulator of immune cell activation. In this study, we aimed to investigate the expression and functional role of miR-181c in myasthenia gravis (MG). miR-181c showed significant downregulation in peripheral blood mononuclear cells (PBMCs) from MG patients compared with healthy controls, with lower expression in generalized patients than in ocular ones. MG patients also had increased serum IL-7 and IL-17 levels. Additionally, serum IL-7 level presents a positive correlation with the serum IL-17 level. miR-181c levels were negatively correlated with serum levels of IL-7 and IL-17 in either generalized patients or ocular patients. A luciferase reporter assay revealed that miR-181c could directly bind to the 3'-UTR of interleukin-7. Forced expression of miR-181c led to decreased IL-7 and IL-17 release in cultured PBMCs, while depletion of miR-181c increased the secretion of these two proinflammatory cytokines. The results from our study suggested for the first time that miR-181c was able to negatively regulate the production of proinflammatory cytokines IL-7 and IL-17 in MG patients, and it is a novel potential therapeutic target for MG.

TIPE2 Play a Negative Role in TLR4-Mediated Autoimmune T Helper 17 Cell Responses in Patients with Myasthenia Gravis.

Th17-related cytokines have been suggested to play a crucial role in myasthenia gravis (MG) pathogenesis.The tumor necrosis factor (TNF)-α-induced protein 8-like-2 (TNFAIP8L2 or TIPE2), is a newly identified member of the tumor necrosis TNFAIP8 family which is an essential negative regulator of both innate and adaptive immunity. In the present study, the expression of TIPE2 mRNA and protein in peripheral blood mononuclear cells (PBMC) from healthy and MG subjects were detected by Real-time PCR and Western blotting.The serum IL-6, IL-17 and IL-21 levels were tested by ELISA. Furthermore, PBMC from MG patients were purified and stimulated with LPS (TLR4 agonist) with or without transfection of TIPE2 expressing adenovirus, then the expression of TIPE2 and Th17-specific transcriptional factor RORγt and the IL-6, IL-17 and IL-21 levels of supernatant were analized. Our data demonstrated that the expression of TIPE2 mRNA and protein was reduced in MG compared with normal controls, with lower expression in generalized patients than in ocular ones. Furthermore, TIPE2 mRNA presents a significantly negative correlation with the serum levels of IL-6, IL-17 and IL-21 in either generalized patients or ocular patients. In cultured MG PBMC, TLR4 activation led to the down-regulation of TIPE2, while the expression of RORγt and production of IL-6, IL-17 and IL-21 were significantly increased. However, when TIPE2 was overexpressed, these TLR4 activation-induced effects were significantly abrogated. Overall, our results indicated for the first time that TIPE2 may participate in the development of MG through negatively regulation of TLR4-mediated autoimmune T helper 17 cell responses.

Artesunate Attenuates Lipopolysaccharide-Stimulated Proinflammatory Responses by Suppressing TLR4, MyD88 Expression, and NF-κB Activation in Microglial Cells.

Microglia are considered as a major target in the prevention of neuroinflammation by modulating the production of pro-inflammatory mediators. Artesunate, a water-soluble artemisinin derivative, exerts an anti-inflammatory effect. In the present study, we showed artesunate dose-dependently suppressed the lipopolysaccharide (LPS)-induced production of nitric oxide (NO), inducible nitric oxide synthase (iNOS), and interleukin-1beta (IL-1ß) in BV2 microglial cells. In addition, artesunate inhibited LPS-induced expression of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and activation of nuclear factor kappa B (NF-κB) by blockade of inhibitor of NF-κB (IκB) degradation. This data indicate that artesunate attenuates the generation of proinflammatory mediators on LPS-stimulated BV-2 microglial cells. And this effect may be associated with the suppression of TLR4/MyD88/NF-κB signaling pathways. Therefore, artesunate may be a potential anti-neuroinflammatory agent.

RNA interference targeting Bcl-6 ameliorates experimental autoimmune myasthenia gravis in mice.

Follicular helper T (Tfh) cells are dedicated to providing help to B cells and are strongly associated with antibody-mediated autoimmune disease. B cell lymphoma 6 (Bcl-6) is a key transcription factor of Tfh cells, and IL-21 is known to be a critical cytokine produced by Tfh cells. We silenced Bcl-6 gene expression using RNA interference (RNAi) delivered by a lentiviral vector, to evaluate the therapeutic role of Bcl-6 short hairpin RNAs (shRNAs) in experimental autoimmune myasthenia gravis (EAMG). Our data demonstrate that CD4(+)CXCR5(+)PD-1(+) Tfh cells, Bcl-6 and IL-21 were significantly increased in EAMG mice, compared with controls. In addition, we found that frequencies of Tfh cells were positively correlated with the levels of serum anti-AChR Ab. In-vivo transduction of lenti-siRNA-Bcl6 ameliorates the severity of ongoing EAMG with decreased Tfh cells, Bcl-6 and IL-21 expression, and leads to decreased anti-AChR antibody levels. Furthermore, we found that siRNA knockdown of Bcl-6 expression increases the expression of Th1(IFN-γ, T-bet) and Th2 markers (IL-4 and GATA3), but failed to alter the expression of Th17-related markers (RORγt, IL-17) and Treg markers (FoxP3). Our study suggests that Tfh cells contribute to the antibody production and could be one of the most important T cell subsets responsible for development and progression of EAMG or MG. Bcl-6 provides a promising therapeutic target for immunotherapy not only for MG, but also for other antibody-mediated autoimmune diseases.

Identification of novel MicroRNA signatures linked to experimental autoimmune myasthenia gravis pathogenesis: down-regulated miR-145 promotes pathogenetic Th17 cell response.

Emerging evidence demonstrates that miRNAs, a new family of key mRNA regulatory molecules, have crucial roles in controlling and modulating immunity. Their contribution to myasthenia gravis (MG), a T cell-dependent, antibody-mediated nervous system autoimmune disease, has not been thoroughly investigated. In the present study, using a highly sensitive microarray-based approach, we identified 11 miRNAs with differential expression between Peripheral Blood Mononuclear Cells (PBMC) from experimental autoimmune MG (EAMG) rats and control rats. miR-145 is one of the most significantly down-regulated miRNAs in PBMC from EAMG rats. Down-regulation of miR-145 expression was confirmed in PBMC and CD4+CD25- T cells (T effector cells) from both EAMG rats and MG patients by real-time PCR. Bioinformatics target prediction identified two crucial immune-related molecules-CD28 and NFATc1, as putative targets of miR-145. Furthermore, miR-145 inhibited CD28 and NFATc1 expression by directly targeting their 3'-UTRs, which was abolished by mutation of the miR-145 and CD28/NFATc1 binding sites. In vitro up-regulation of miR-145 in CD4+ T cells can significantly reduce CD28 protein levels accompanied by decreased proliferative response. In a dendritic cell (DC)-T cell coculture system, overexpression of miR-145 in AChR-specific CD4+ T cells suppresses NFATc1 expression and T Helper 17 cells level. Finally, we observed that administration of lentiviral-miR-145 decreased the severity of ongoing, established EAMG with decreased IL-17 production, and also decreased serum anti-AChR IgG levels. Our studies provide an important new insight into the pathogenesis of EAMG and MG, which may open a new perspective for the development of effective gene therapy for EAMG/MG.

Role of calbindin-D28K in estrogen treatment for Parkinson's disease.

Studies have shown that estrogen has neuroprotective effects on the nigrostriatal system. The present study established a Parkinson's disease model in C57BL/6 mice by intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrapyridine. The mice were subjected to 17ß estradiol injection into the lateral ventricle. Immunofluorescence double staining showed that estrogen increased tyrosine hydroxylase and calbindin-D28K expression and co-expression in dopaminergic neurons of midbrain substantia nigra pars compacta of model mice. Behavior experiments showed that estrogen improved swimming and hanging behaviors in this mouse model of Parkinson's disease.

Dopamine content in the striatum and expression changes of Bad and Bcl-2 in elderly rats with abnormal behavior.

To determine the dopamine (DA) content in the striatum and the expression changes of the apoptosis-associated proteins Bad and Bcl-2 in the substantia nigra compacta (SNc) in elderly rats with abnormal behavior. Fifty three Sprague-Dawley rats were divided into three groups: adult, age-motorplus (normal behavior) and aged-motorminus (abnormal behavior) using the hanger test. The DA content in the striatum and the expression of tyrosine hydroxylase (TH), Bad and Bcl-2 in the SNc were measured by HPLC/MS (high performance liquid chromatogram-mass spectra) and Western Blot. (1) The results from the hanger test demonstrated that the scores and latency of aged-motorminus group were lower than the age-motorplus group. (2) Results from HPLC-MS showed that, compared with the age-motorplus and adult group, the content of DA in elderly rat striata decreased significantly, with a statistically significant difference. (3) The Western Blot demonstrated that, compared with the adults, the expression of TH in elderly rats significantly decreased, but the difference was not significant between the aged-motorminus group and the age-motorplus group. Compared with the age-motorplus and the adult group, the expression of Bad increased but Bcl-2 decreased in the aged-motorminus group. The decrease in TH content in the SNc correlated with the aging of rats. The decrease in DA content in the striatum may correlate with the abnormal behavior in elderly rats, which could be ascribed to the variations in Bad and Bcl-2.

Influence of aging on the dopaminergic neurons in the substantia nigra pars compacta of rats.

To study the relationship between aging and the vulnerability of substantia nigra pars compacta (SNc) tyrosine hydroxylase immunoreactive positive (TH+) dopaminergic (DA) neurons. We determined the number of TH(+) DA neurons in aged rats (24 mon) compared to adult rats (5 mon) using immunohistochemistry and cell counting. Furthermore, the expression of TH mRNA and protein levels in SN was studied by semi-quantitative RT-PCR and Western blotting. A 13.6% loss of neurons was detected in rostral segment of SNc, and the expression of TH mRNA levels was also reduced (P < 0.05), however, no difference was detected in TH protein levels (P > 0.05). These data suggest that expression of TH protein may increase in the existing SNc DA neurons, which may compensate for the partial loss of TH+ DA neurons.

Influence of aging on the calbindin-D-28k immunoreactive positive dopaminergic neurons in the substantia nigra pars compacta of rats.

We studied the relationship between aging and the vulnerability of substantia nigra pars compacta (SNc) calbindin-D-28k immunoreactive positive (CB+) dopaminergic (DA) neurons. Immunohistochemistry and cell counting were used to determine the number of CB+ DA neuron in aged rats (24 mon) compared to adult rats (5 mon). Furthermore, the expression of CB mRNA and protein levels in SN was studied by semi-quantitative RT-PCR and Western blotting. An 11% loss of CB+ DA neurons was detected in both the rostral (8.9%) and caudal (1.7%) segments but not in the intermedial segment of SNc in aged rats compared to adult rats (P<0.05). No difference was detected in CB mRNA and protein levels between aged and adult rats (P>0.05). These data suggest that expression levels of CB mRNA and protein may increase in the existing SNc DA neurons, which may compensate for the partial age dependent loss of CB+ DA neurons in the SNc.

Involvement of NCAM in the effects of GDNF on the neurite outgrowth in the dopamine neurons.

Glial cell line-derived neurotrophic factor (GDNF) exerts its biological effects via a multi-component receptor system including the ligand binding receptor--GDNF family receptor-alpha1 (GFRalpha1) and the signaling receptor--RET tyrosine kinase. Recently, the neural cell adhesion molecule (NCAM) has been identified as an alternative signaling receptor for GDNF. The purpose of this study was to investigate whether NCAM could mediate the protective effect of GDNF on injured dopamine (DA) neurons and to determine which cytoplasmic signal molecule associated with NCAM was activated while GDNF performing this effect. The results showed that the phosphorylation of NCAM-associated Fyn was upregulated with GDNF treatment, and this upregulation was inhibited by pre-treatment with the NCAM function-blocking antibody. Moreover, pre-treatment with the antibody could abolish the effect of GDNF on promoting the neurite outgrowth of these DA neurons, except for the effect of GDNF on promoting the expression of tyrosine hydroxylase (TH) in these DA neurons. These results suggest that NCAM is involved in the promotive effect of GDNF on the neurite outgrowth in lesioned DA neurons, but not involved in the promotive effect of GDNF on TH expression in these neurons.