Targeting HIF-1α alleviates the inflammatory responses and rebuilds the CD4+ T cell subsets balance in the experimental autoimmune myasthenia gravis inflammation model via regulating cellular and humoral immunity.

BACKGROUND: Cells and tissues in an inflammatory state are usually hypoxic. The hypoxic environment can affect the differentiation of immune cells and produce Hypoxia-inducible Factor-1α (HIF-1α). Inflammation is also a major contributor to the development and deterioration of Myasthenia Gravis (MG). There are limited studies on the immunopathological mechanism and targeted therapy associated with MG exacerbated with inflammation. This research aimed to explore whether BAY 87-2243 (HIF-1α inhibitor) ameliorates the symptoms of the Experimental Autoimmune Myasthenia Gravis (EAMG) inflammation model and study its regulatory mechanism on cellular immunity and humoral immunity. METHODS: We first establish the EAMG inflammation model using Lipopolysaccharide (LPS), BAY 87-2243 was applied to the EAMG inflammation model and its therapeutic effects were evaluated in vivo and in vitro experiments. RESULTS: The proportion of Treg cells was increased whereas Th1, Th17, and Th1/17 cells were decreased in BAY 87-2243-treated EAMG inflammation model. BAY 87-2243 ameliorated the acetylcholine receptors (AChRs) loss and the complement deposited at the neuromuscular junction of the EAMG inflammation model, declined the levels of IFN-γ, IL-17, and IL-6 in serum, and further attenuated responses in the germinal center and reduced the antibody levels by inhibiting the IL-6-dependent STAT3 axis. CONCLUSION: BAY 87-2243 restored the balance of CD4+T cell subsets and reduced the production of the pro-inflammatory cytokines, thus acting as both an immune imbalance regulator and anti-inflammatory. The current study suggests that HIF-1α might be a potential target for the treatment of MG exacerbated with inflammation.

JAK2 inhibitor ameliorates the progression of experimental autoimmune myasthenia gravis and balances Th17/Treg cells via regulating the JAK2/STAT3-AKT/mTOR signaling pathway.

BACKGROUND: An imbalance in Th17/regulatory T (Treg) cells is the major pathogenic mechanism underlying myasthenia gravis (MG). JAK2 inhibitors selectively inhibit JAK2 and reduce inflammatory responses. However, there have been no studies examining the therapeutic effects of JAK2 inhibitors in the context of MG. METHODS: Here, an experimental autoimmune MG (EAMG) rat model was established to explore the therapeutic effect of JAK2 inhibitors on EAMG rats immunized with the AChR α-subunit (97-116 peptide). A JAK2 inhibitor was administered to EAMG rats both in vivo and in vitro. The following experimental methods were used to evaluate the effects of JAK2 inhibitors. The behavioral scores and body weights of the rats were assessed on alternate days. Serum anti-AChR (97-116) IgG and cytokine levels were detected using ELISA. CD4+ T cell subsets and related transcription factors in mononuclear cells were detected using flow cytometry and qPCR, respectively. The expression levels of protein molecules in the signaling pathway were detected by western blotting, and the neuromuscular junctions were observed using immunofluorescence. RESULTS: The results revealed that JAK2 inhibitors could regulate Th17/Treg balance in vivo and in vitro. JAK2 inhibitors reduced the immune response in EAMG rats (including reducing pro-inflammatory cytokines and postsynaptic membrane complement deposition), improved clinical symptoms, and increased AChR aggregation in the postsynaptic membrane. Meanwhile, this study demonstrated that JAK2 inhibitor treatment suppressed the phosphorylation of JAK2/STAT3 and AKT/mTOR pathways and decreased the expression level of the IL-23 receptor. CONCLUSIONS: This study reveals that there is crosstalk between the JAK2/STAT3 and AKT/mTOR pathways in EAMG rats. JAK2 inhibitors can ameliorate EAMG by regulating Th17/Treg balance by inhibiting both signaling pathways. Our study provides new potential therapeutic targets for MG immunotherapy.

Clinical evaluation of efficacy of leflunomide combined with low-dose prednisone for treatment of myasthenia gravis.

This study evaluated the clinical efficacy of leflunomide combined with low-dose prednisone (0.25 mg/kg/day) for treatment of myasthenia gravis (MG). We enrolled 32 MG patients treated with leflunomide combined with low-dose prednisone. In the control group, 14 patients were treated with low-dose prednisone. Improvement in MG composite (MGC) score of ≥ 3 points from enrollment to 12-week follow-up indicated that the treatment was effective. In the leflunomide combined low-dose prednisone group, the median of MGC score at the time of enrollment was 8.5 points. After 12 weeks, the MGC score dropped to four points. There was statistically significant difference in MGC score before and after treatment (p < 0.001). In the low-dose prednisone group also followed up for 12 weeks, the median of MGC score of the patients decreased from 7 to 4 points, and the change was not statistically significant (p = 0.05). In the leflunomide combined low-dose prednisone group, the improvement of clinical symptoms occurred mainly in the first 4 weeks and the last 4 weeks. Relatively, the decline of the score was mostly seen during the first 8 weeks in the low-dose prednisone group. In leflunomide combined with low-dose prednisone group, the effective rate of generalized MG(gMG) was significantly higher than ocular MG(oMG) (χ2 test, p = 0.036). However, there is no significant difference in the effective rate between AChR-Ab-positive and -negative groups (Fisher's Exact Test, p = 0.625). No serious side effects were observed in any of the subjects. Leflunomide combined with low-dose prednisone rapidly improved the clinical symptoms of patients with MG. It may be a promising treatment for gMG.

Is myasthenia gravis a contraindication for botulinum toxin?

Botulinum toxin (BTX) is a neurotoxin that has been used to treat various disorders and has also become a popular choice for cosmetic indications, yet traditionally, myasthenia gravis (MG) is considered a contraindication for BTX. To determine whether BTX should be avoided in MG patients, clinical data from our MG and dystonia specialist clinic were analyzed retrospectively. In addition, a systematic literature review was conducted to identify all published cases associated with the co-existence of MG and BTX treatments. Here, we described one patient from our clinic, who received BTX injections before being given MG diagnosis. After the literature review, 8 cases with subclinical MG previously treated with BTX for dystonia or cosmetic reasons ("BTX injections before MG diagnosis") were identified. Markedly, 8 out of 8 (100%) patients developed obvious muscle weakness. In contrast, 10 patients presenting MG as comorbidity had received BTX for dystonia or overactive bladder ("BTX injection after MG diagnosis"), and 8 out of 10 (80%) experienced improved symptoms through appropriate dose modifications and adequate treatment for MG before receiving BTX injections. These findings support that, under proper management of co-existing MG, BTX could be used safely and successfully in patients presenting MG comorbidities in the future.

Targeted and Untargeted Metabolomics Profiling of Wheat Reveals Amino Acids Increase Resistance to Fusarium Head Blight.

Fusarium head blight (FHB), a notorious plant disease caused by Fusarium graminearum (F. graminearum), is severely harmful to wheat production, resulting in a decline in grain quality and yield. In order to develop novel control strategies, metabolomics has been increasingly used to characterize more comprehensive profiles of the mechanisms of underlying plant-pathogen interactions. In this research, untargeted and targeted metabolomics were used to analyze the metabolite differences between two wheat varieties, the resistant genotype Sumai 3 and the susceptible genotype Shannong 20, after F. graminearum inoculation. The untargeted metabolomics results showed that differential amino acid metabolic pathways existed in Sumai 3 and Shannong 20 after F. graminearum infection. Additionally, some of the amino acid contents changed greatly in different cultivars when infected with F. graminearum. Exogenous application of amino acids and F. graminearum inoculation assay showed that proline (Pro) and alanine (Ala) increased wheat resistance to FHB, while cysteine (Cys) aggravated the susceptibility. This study provides an initial insight into the metabolite differences of two wheat cultivars under the stress of F. graminearum. Moreover, the method of optimization metabolite extraction presents an effective and feasible strategy to explore the understanding of the mechanisms involved in the FHB resistance.

Endoplasmic reticulum-specific fluorescent probe for the two-photon imaging of endogenous superoxide anion (O2•-) in live cells and zebrafishes.

ER stress has close relation with various metabolic diseases including obesity and insulin resistance, and could result in the abnormal production of ROS including O2-. Real-time and in situ detection of endogenous O2- in ER is vitally important for revealing the physiological roles of O2- during ER stress. Herein, we present an ER-specific two-photon probe (ER-Rs) for the detection of endogenous O2- in living cells and zebrafishes. The probe ER-Rs employed triflate as the response site for O2-, and used p-methylsulfonamide as the ER-specific moiety. In response to O2-, the triflate group of the probe ER-Rs was transformed to hydroxyl and the turn-on fluorescence was produced. The probe ER-Rs displayed highly sensitive and selective response to O2-, and could be employed as an ER-specific two-photon probe for the visualization of endogenous O2- in live cells, tissues and zebrafishes.

Revealing the Viscosity Changes in Lipid Droplets during Ferroptosis by the Real-Time and In Situ Near-Infrared Imaging.

Ferroptosis is characterized by the massive lipid peroxidation, and recently has been demonstrated to be closely associated with lipid droplets (LDs). However, the changes of LDs viscosity during ferroptosis are still unrevealed. Herein, we present the changes of the LDs viscosity during ferroptosis by a novel viscosity-sensitive near-infrared (NIR) fluorescent probe (BDHT). Probe BDHT (2-(benzo[d]thiazol-2-yl)-7-(4-(dimethylamino)phenyl)hepta-2,4,6-trienenitrile, C22H19N3S) showed highly sensitive and selective response to viscosity, mainly distributed in cellular LDs. By means of the real-time and in situ NIR imaging, we discovered that the LDs viscosity showed an obvious increase in HeLa cells during the erastin-induced ferroptosis process, while it displayed nearly no change when the cells were simultaneously treated with ferrostatin-1, which is a common inhibitor of ferroptosis. It is also confirmed that the LDs viscosity increased in several types of the cancer cells of erastin-induced and RSL3-induced ferroptosis. We expect that this new NIR probe could provide an effective approach to rapidly monitor ferroptosis, and these findings could greatly promote the in-depth understanding of the biological effects of LDs during ferroptosis.

Circulating Th1/17 cells serve as a biomarker of disease severity and a target for early intervention in AChR-MG patients.

Interleukin-17-expressing CD4+ T helper 17 (Th17) cells are considered to be critical regulators of thymic inflammation in AChR-MG patients. However, Th17 cells are functionally heterogeneous and circulating Th17 subsets are incompletely understood in AChR-MG patients. Here, we studied characteristics of Th17 subsets in peripheral blood from treatment-naïve AChR-MG patients, patients treated with immunosuppressants, as well as healthy controls. We found increased frequencies of circulating Th1-like Th17 (Th1/17) (IFN-γ + IL-17 + CD4 + CD3+) cells, which declined earlier than conventional Th17 (IFN-γ - IL-17 + CD4 + CD3+) cells in patients who respond well to immunosuppression treatment. Additionally, circulating Th1/17 cell frequencies were found to correlate positively with disease severity. Further, compared to conventional Th17 cells, Th1/17 cells showed an elevated expression of IFNG, TBX21, IL23R, CSF2, and a reduced expression of AHR and IL10. Taken together, our results suggest circulating Th1/17 cells may serve as a biomarker of disease severity and provide a strong rationale for early intervention in AChR-MG patients.

Glutamate synthase 1 is involved in iron-deficiency response and long-distance transportation in Arabidopsis.

Iron is an essential microelement for plant growth. After uptake from the soil, iron is chelated by ligands and translocated from roots to shoots for subsequent utilization. However, the number of ligands involved in iron chelation is unclear. In this study, we identified and demonstrated that GLU1, which encodes a ferredoxin-dependent glutamate synthase, was involved in iron homeostasis. First, the expression of GLU1 was strongly induced by iron deficiency condition. Second, lesion of GLU1 results in reduced transcription of many iron-deficiency-responsive genes in roots and shoots. The mutant plants revealed a decreased iron concentration in the shoots, and displayed severe leaf chlorosis under the condition of Fe limitation, compared to wild-type. Third, the product of GLU1, glutamate, could chelate iron in vivo and promote iron transportation. Last, we also found that supplementation of glutamate in the medium can alleviate cadmium toxicity in plants. Overall, our results provide evidence that GLU1 is involved in iron homeostasis through affecting glutamate synthesis under iron deficiency conditions in Arabidopsis.

Quantitative features and clinical significance of two subpopulations of AChR-specific CD4+ T cells in patients with myasthenia gravis.

Acetylcholine receptor (AChR)-specific CD4+ T cells play a driving role in myasthenia gravis (MG) by regulating the production of autoantibodies. However, the quantitative features of AChR-specific T cells and their clinical significance in MG are unclear. In this study, we adopted standard and cultured enzyme-linked immunosorbent spot (ELISPOT) assays to quantify subpopulations of AChR-specific CD4+ T cells in MG patients, and evaluate their correlation with clinical characteristics. The results showed that Th1- and Th17-AChR-specific CD4+ T cells were detectable by standard and cultured ELISPOT assay respectively, with higher levels observed in MG patients comparing with healthy controls. The number of Th17-AChR-specific CD4+ T cells was positively correlated with anti-AChR antibody titer and quantitative MG score and may have latent capacity to reflect responses to immunosuppressants. These results highlight the differences in quantitative features of AChR-specific CD4+ T cells and imply Th17-AChR-specific CD4+ T cells can serve as a biomarker in MG.

AChR myasthenia gravis switching to MuSK or double antibody positive myasthenia gravis in two children and literature review.

Muscle-specific tyrosine kinase antibody (MuSK-Ab) and acetylcholine receptor antibody (AChR-Ab) coexistence in myasthenia gravis (MG) is very rare. In this report, two children with AChR-Ab switching to double antibody positive MG (DP-MG) or MuSK-Ab positive MG (MuSK-MG) are described. Six similar cases were found in the literature via online database search. Therefore, this study describes eight patients in total, six female and two male. The average age of onset was 7.25 ± 5.95 years. Four AChR-MG patients switched to DP-MG with no known precipitating factor and four switched after thymectomy (two to MuSK-MG and two to DP-MG). After the serological switch, the patients transitioned to the phenotype of MuSK-MG and responded poorly to cholinesterase inhibitors and well to corticosteroids and plasma exchange.

Prolonging and enhancing the protective efficacy of the EtMIC3-C-MAR against eimeria tenella through delivered by attenuated salmonella typhimurium.

The microneme adhesive repeats (MAR) of Eimeria tenella microneme protein 3 (EtMIC3) are associated with binding to and invasion of host cells. Adhesion and invasion-related proteins or domains are often strongly immunogenic, immune responses mounted against these factors that play a key role in blocking invasion. In the present study, an oral live vaccine consisting of attenuated Salmonella typhimurium X4550 carrying two MAR domains fragment (St-X4550-MAR) was constructed and its protective efficacies were evaluated. The results showed that St-X4550-MAR was more immunogenic and conferred a higher degree of protection than recombinant MAR polypeptide as reflected by increased body weight, decreased oocyst shedding and lesion scores, increased serum IgG and cecal sIgA antibody production, and increasing levels of interferon-γ and interleukin-10. Thus, MAR domains are highly immunogenic and St-X4550-MAR had moderate activity against E. tenella infection by stimulating humoral, mucosal and cellular immunity. Chickens immunized with our constructed live vaccine provided considerable protections as early as at 10 d post-immunization (ACI: 155.17), and maintained higher protection levels at 20 d post-immunization (ACI: 173.66), and at 30 d post-immunization (ACI: 162.4). While the protective efficacy of chickens immunized with the recombinant MAR peptides showed a decreased trend as the post immunization time prolonging. Thus, using live-attenuated S. typhimurium X4550 as a vaccine expression and delivery system can significantly improve the protective efficacy and duration of protective immunity of MAR of EtMIC3.

A near-infrared and two-photon dual-mode fluorescent probe for the colorimetric monitoring of SO2in vitro and in vivo.

SO2 has been recently identified as an essential gas messenger followed by NO, CO and H2S. However, abnormal concentrations of SO2 in our bodies can cause many diseases. Thus, the real-time monitoring of SO2 to well define the generation, physiological and pathological functions of SO2 is urgently needed. In this work, we developed a novel SO2 fluorescent probe on the basis of the conjugation of semi-cyanine and coumarin derivate dyes with superior features, such as near-infrared (NIR) and two-photon dual-mode monitoring, a large Stokes shift (175 nm), ultrafast response towards SO2 (within 10 s), high selectivity and photostability. Furthermore, this probe could sense SO2 by dual colorimetric and fluorescence means. In biological imaging, the probe was able to trace exogenous and endogenous SO2 in living cells, mitochondria, E. coli, zebrafish and mice under an NIR and two-photon dual-mode. These results demonstrated that the probe has strong potential for studying the physiological and pathological functions of SO2in vitro and in vivo.

Ratiometric Imaging of Cysteine Level Changes in Endoplasmic Reticulum during H2O2-Induced Redox Imbalance.

Cysteine (Cys) is an important mediator to regulate the redox state of endoplasmic reticulum (ER), and its level is closely related with many ER stress induced serious diseases. Herein, we present an ER-specific fluorescent probe for the ratiometric imaging of cellular Cys for the first time. The probe exhibited desirable selectivity and sensitivity to Cys. Biological imaging experiments demonstrated that the probe possessed an ER-targeting property, showed ratiometric response to Cys in ER, and could be applied for the ratiometric imaging of Cys level changes during H2O2-induced redox imbalance in living cells.

Predictive Value of Hepatorenal Status in Contrast-Induced Nephropathy Among Patients Receiving Coronary Angiography and/or Intervention: A Systematic Review and Meta-Analysis.

BACKGROUND: Low serum albumin, high blood urea nitrogen (BUN), and uric acid are regarded closely related to the incidence of contrast-induced nephropathy (CIN), whereas it remains unclear whether they can function as predictors of CIN onset. The objective of this systematic review and meta-analysis was to explore the association between abovementioned indicators and CIN incidence rate in patients receiving coronary angiography (CAG) and/or intervention. METHODS: Clinical studies were retrieved from the electronic databases of PubMed, EMBASE, Google Scholar, Clinical Trials, and ScienceDirect from their inception to July 13, 2018. Meta-analysis was performed on pooled eligible studies to determine whether these hepatorenal indicators were associated with CIN. RESULTS: A total of 18 studies involving 16 171 patients were included in the meta-analysis. Pooled analysis results revealed that patients with hypoalbuminemia (odds ratio [OR] = 3.09, 95% confidence interval [CI] = 1.44-6.64, P = .004) and hyperuricemia (OR = 1.32, 95% CI = 1.15-1.50, P < .0001) both exhibited significantly higher CIN rates, regardless of the study design, renal function, and whether urgent clinical situation or not. However, there was no significant association between serum BUN and CIN risk. CONCLUSION: Hypoalbuminemia and hyperuricemia are independently associated with the occurrence of CIN among the patients undergoing CAG and/or intervention.