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The muscle LIM protein (MLP) is a member of the cysteine and glycine-rich protein (CSRP) family, composed of CSRP1, CSRP2 and CSRP3/MLP. MLP is involved in a multitude of functional roles, including cytoskeletal organization, transcriptional regulation, and signal transduction. However, the molecular mechanisms underlying its involvement in immune and stress responses remain to be elucidated. This study identified an MnMLP in the freshwater crustacean Macrobrachium nipponense. The isothermal titration calorimetry assay demonstrated that recombinant MnMLP was capable of coordinating with Zn 2+ . Upon challenge by Aeromonas veronii or WSSV, and exposure to CdCl 2 , up-regulation was recorded in the muscle and intestinal tissues, suggesting its involvement in immune and anti-stress responses. MnMLP protein was predominantly expressed in the cytoplasm of the transfected HEK-293T cells, but after treatment with LPS, Cd 2+ or H 2 O 2 , the MnMLP was observed to be transferred into the nucleus. The comet assay demonstrated that the overexpression of MnMLP could mitigate the DNA damage induced by H 2 O 2 in HEK-293T cells, suggesting the potential involvement of MnMLP in the DNA repair process. These findings suggest that DNA repair may represent a possible mechanism by which MnMLP may be involved in the host's defense against pathogens and stress., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Lactate significantly impacts immune cell function in sepsis and septic shock, transcending its traditional view as just a metabolic byproduct. This review summarizes the role of lactate as a biomarker and its influence on immune cell dynamics, emphasizing its critical role in modulating immune responses during sepsis. Mechanistically, key lactate transporters like MCT1, MCT4, and the receptor GPR81 are crucial in mediating these effects. HIF-1α also plays a significant role in lactate-driven immune modulation. Additionally, lactate affects immune cell function through post-translational modifications such as lactylation, acetylation, and phosphorylation, which alter enzyme activities and protein functions. These interactions between lactate and immune cells are central to understanding sepsis-associated immune dysregulation, offering insights that can guide future research and improve therapeutic strategies to enhance patient outcomes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Zhang, Chen, Kueth, Shao, Wang, Ha, Williams, Li, Fan and Yang.)

Dulaglutide, a glucagon-like peptide-1 receptor (GLP-1R) agonist, is widely used to treat diabetes. However, its effects on muscle wasting due to aging are poorly understood. In the current study, we investigated the therapeutic potential and underlying mechanism of dulaglutide in muscle wasting in aged mice. Dulaglutide improved muscle mass and strength in aged mice. Histological analysis revealed that the cross-sectional area of the tibialis anterior (TA) in the dulaglutide-treated group was thicker than that in the vehicle group. Moreover, dulaglutide increased the shift toward middle and large-sized fibers in both young and aged mice compared to the vehicle. Dulaglutide increased myofiber type I and type IIa in young (18.5% and 8.2%) and aged (1.8% and 19.7%) mice, respectively, compared to the vehicle group. Peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α), a master regulator of mitochondrial biogenesis, decreased but increased by dulaglutide in aged mice. The expression of atrophic factors such as myostatin, atrogin-1, and muscle RING-finger protein-1 was decreased in aged mice, whereas that of the myogenic factor, MyoD, was increased in both young and aged mice following dulaglutide treatment. In aged mice, optic atrophy-1 (OPA-1) protein was decreased, whereas Toll-like receptor-9 (TLR-9) and its targeting inflammatory cytokines (interleukin-6 [IL-6] and tumor necrosis factor-α [TNF-α]) were elevated in the TA and quadriceps (QD) muscles. In contrast, dulaglutide administration reversed this expression pattern, thereby significantly attenuating the expression of inflammatory cytokines in aged mice. These data suggest that dulaglutide may exert beneficial effects in the treatment of muscle wasting due to aging.

NLRP3 inflammasome has emerged as a crucial regulator of inflammatory bowel disease (IBD) characterized by a chronic inflammatory disease of the gastrointestinal tract. The expression of MCT4 is significantly increased in intestinal mucosal tissue of IBD, which has been identified to regulate intestinal barrier function. However, the function of MCT4 in cell pyroptosis remained unknown. In this study, we have established a stable cell line with MCT4 overexpression in HT-29 and CaCO2 cells, respectively. Functional analysis revealed that ectopic expression of MCT4 in CaCO2 cells contributed to cell pyroptosis as evidenced by LDH assay, which is largely attributed to Caspase-1-mediated canonical pyroptosis, but not Caspase-4 and Caspase-5, leading to cleave pro-IL-1β and IL-18 into mature form and release mediated by cleaved GSDMD. Mechanically, MCT4 overexpression in HT-29 and CaCO2 cell triggered the phosphorylation of ERK1/2 and NF- κ B p65, while inhibition of MCT4 by MCT inhibitor α -Cyano-4-hydroxycinnamic acid ( α -CHCA) in HT-29 and CaCO2 cells led to a significant downregulation of ERK1/2 and NF- κ B activity. What's more, blockade of ERK1/2-NF- κ B pathway could reverse the promotion effect of MCT4 on IL-1β expression. Importantly, both MCT4 and Caspase-1, GSDMD were significantly increased in patients with IBD, and a positive clinical correlation between MCT4 and Caspase-1 expression was observed (p < 0.001). Taken together, these findings suggested that MCT4 promoted Caspase-1-mediated canonical cell pyroptosis to aggravate intestinal inflammation in intestinal epithelial cells (IECs) through the ERK1/2-NF- κ B pathway., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wang, Zhou, Zou, Chen, Huang, Yang, Pan, Xu, Xu, Chen, Chen, Gong, Zhou, Xu and Zhao.)

High quality, well-validated antibodies are needed to mitigate irreproducibility and clarify conflicting data in science. We describe an epitope-directed monoclonal antibody (mAb) production method that addresses issues of antibody quality, validation and utility. The workflow is illustrated by generating mAbs against multiple in silico-predicted epitopes on human ankyrin repeat domain 1 (hANKRD1) in a single hybridoma production cycle. Antigenic peptides (13-24 residues long) presented as three-copy inserts on the surface exposed loop of a thioredoxin carrier produced high affinity mAbs that are reactive to native and denatured hANKRD1. ELISA assay miniaturization afforded by novel DEXT microplates allowed rapid hybridoma screening with concomitant epitope identification. Antibodies against spatially distant sites on hANKRD1 facilitated validation schemes applicable to two-site ELISA, western blotting and immunocytochemistry. The use of short antigenic peptides of known sequence facilitated direct epitope mapping crucial for antibody characterization. This robust method motivates its ready adoption for other protein targets.

Background: Transgelin-2 is a 22 kDa actin-binding protein that has been proposed to act as an oncogenic factor, capable of contributing to tumorigenesis in a wide range of human malignancies. However, little is known whether this tiny protein also plays an important role in immunity, thereby keeping body from the cancer development and metastasis. Here, we investigated the functions of transgelin-2 in dendritic cell (DC) immunity. Further, we investigated whether the non-viral transduction of cell-permeable transgelin-2 peptide potentially enhance DC-based cancer immunotherapy., Methods: To understand the functions of transgelin-2 in DCs, we utilized bone marrow-derived DCs (BMDCs) purified from transgelin-2 knockout (Tagln2 -/- ) mice. To observe the dynamic cellular mechanism of transgelin-2, we utilized confocal microscopy and flow cytometry. To monitor DC migration and cognate T-DC interaction in vivo, we used intravital two-photon microscopy. For the solid and metastasis tumor models, OVA + B16F10 melanoma were inoculated into the C57BL/6 mice via intravenously (i.v.) and subcutaneously (s.c.), respectively. OTI TCR T cells were used for the adoptive transfer experiments. Cell-permeable, de-ubiquitinated recombinant transgelin-2 was purified from Escherichia coli and applied for DC-based adoptive immunotherapy., Results: We found that transgelin-2 is remarkably expressed in BMDCs during maturation and lipopolysaccharide activation, suggesting that this protein plays a role in DC-based immunity. Although Tagln2 -/- BMDCs exhibited no changes in maturation, they showed significant defects in their abilities to home to draining lymph nodes (LNs) and prime T cells to produce antigen-specific T cell clones, and these changes were associated with a failure to suppress tumor growth and metastasis of OVA + B16F10 melanoma cells in mice. Tagln2 -/- BMDCs had defects in filopodia-like membrane protrusion and podosome formation due to the attenuation of the signals that modulate actin remodeling in vitro and formed short, unstable contacts with cognate CD4 + T cells in vivo. Strikingly, non-viral transduction of cell-permeable, de-ubiquitinated recombinant transgelin-2 potentiated DC functions to suppress tumor growth and metastasis., Conclusion: This work demonstrates that transgelin-2 is an essential protein for both cancer and immunity. Therefore, transgelin-2 can act as a double-edged sword depending on how we apply this protein to cancer therapy. Engineering and clinical application of this protein may unveil a new era in DC-based cancer immunotherapy. Our findings indicate that cell-permeable transgelin-2 have a potential clinical value as a cancer immunotherapy based on DCs.

The RIG-I receptor induces the innate antiviral responses upon sensing RNA viruses. The mechanisms through which RIG-I optimizes the strength of the downstream signaling remain incompletely understood. In this study, we identified that NSUN5 could potentiate the RIG-I innate signaling pathway. Deficiency of NSUN5 enhanced RNA virus proliferation and inhibited the induction of the downstream antiviral genes. Consistently, NSUN5 - deficient mice were more susceptible to RNA virus infection than their wild-type littermates. Mechanistically, NSUN5 bound directly to both viral RNA and RIG-I, synergizing the recognition of dsRNA by RIG-I. Collectively, to our knowledge, this study characterized NSUN5 as a novel RIG-I coreceptor, playing a vital role in restricting RNA virus infection., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Aspartate/asparagine β‑hydroxylase (AspH) is a type II transmembrane protein that catalyzes the post‑translational hydroxylation of definite aspartyl and asparaginyl residues in epidermal growth factor‑like domains of substrates. In the last few decades, accumulating evidence has indicated that AspH expression is upregulated in numerous types of human malignant cancer and is associated with poor survival and prognosis. The AspH protein aggregates on the surface of tumor cells, which contributes to inducing tumor cell migration, infiltration and metastasis. However, small‑molecule inhibitors targeting hydroxylase activity can markedly block these processes, both in vitro and in vivo. Immunization of tumor‑bearing mice with a phage vaccine fused with the AspH protein can substantially delay tumor growth and progression. Additionally, AspH antigen‑specific CD4+ and CD8+ T cells were identified in the spleen of tumor‑bearing mice. Therefore, these agents may be used as novel strategies for cancer treatment. The present review summarizes the current progress on the underlying mechanisms of AspH expression in cancer development.

The present study compares the immunogenic patterns of muscle larvae excretory-secretory proteins (ML E-S) from T. spiralis and T. britovi recognized by Trichinella-infected human sera. Samples were analyzed using two-dimensional electrophoresis (2-DE) coupled with 2D-immunoblot and liquid chromatography-tandem mass spectrometry LC-MS/MS analysis, two ELISA procedures and a confirmatory 1D-immunoblot test. Sera were obtained from nine patients with a history of ingestion of raw or undercooked meat who presented typical clinical manifestations of trichinellosis and from eleven healthy people. Specific anti-Trichinella IgG antibodies were detected in all samples tested with the Home-ELISA kits, but in only four samples for the commercially-available kit. The 1D-immunoblot results indicated that all nine serum samples were positive for T. spiralis ML E-S antigens, expressed as the presence of specific bands. In contrast, eight of the serum samples with T. britovi E-S ML antigens were positive, with one serum sample taken from a patient at 33dpi (days post infection) being negative. To identify immunoreactive proteins that are specifically recognized by host antibodies, both species of ML E-S proteins were subjected to 2D-immunoblotting with human serum taken at 49 dpi. The sera recognized 22 protein spots for T. spiralis and 18 for T. britovi in 2D-immunoblot analysis. Their molecular weights (MW) ranged from 50 to 60 kDa. LC-MS/MS analysis identified both common and specifically-recognized immunoreactive proteins: transmembrane serine protease 9, serine protease, antigen targeted by protective antibodies and Actin-1 partial were shared for both Trichinella species; hypothetical protein T01&#95;7775 and P49 antigen, partial those specific to T. spiralis; deoxyribonuclease-2-alpha and hypothetical protein T03&#95;17187/T12&#95;7360 were specific to T. britovi. Our results demonstrate the value of 2-DE and 2D-immunblot as versatile tools for pinpointing factors contributing to the parasite-host relationship by comparing the secretomes of different Trichinella species., Competing Interests: The authors have declared that no competing interests exist.

Psoriasis is a common chronic inflammatory disease with the prevalence rate of approximately 1-3 %. Currently, it is generally believed that the pathogenesis of psoriasis is a T-cell immune-mediated skin disease mediated by multiple genes and factors, and the interaction between keratinocytes and T cells. TEA domain family member 4 (TEAD4) is a transcription factor which regulates the expression of downstream genes in Hippo pathway and affects several biological processes, such as regulating cell differentiation and embryonic development. However, few studies have reported the role of TEAD4 in psoriasis and its possible regulatory mechanism. In this study, we found the expression level of TEAD4 in the skin of psoriasis was significantly higher than that of normal skin. In patients with the pathological keratinocytes, TEAD4 can transcriptionally regulate the expression of SERPINB3/4 and affect the secretion of chemokines, and the depletion of SERPINB3/4 inhibited the secretion of chemokines. In addition, the supernatant of keratinocytes of patients can significantly increase the migration ability of T cells, and the supernatant of T cells cultured by the supernatant of keratinocytes of patients can significantly enhance the proliferation ability of keratinocytes. Therefore, our results suggested that TEAD4 is a key regulatory factor in progression of psoriasis, and the crosstalk between keratinocytes and T cells mediated by TEAD4 plays a critical role in the psoriasis pathogenesis., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Objective: Direct inhibition of acetylcholine receptor (AChR) function by autoantibodies (Abs) is considered a rare pathogenic mechanism in myasthenia gravis (MG), but is usually studied on AChRs expressed in cell lines, rather than tightly clustered by the intracellular scaffolding protein, rapsyn, as at the intact neuromuscular junction. We hypothesised that clustered AChRs would provide a better target for investigating the functional effects of AChR-Abs., Methods: Acetylcholine-induced currents were measured using whole-cell patch clamping and a fast perfusion system to assess fast (<2 min) functional effects of the serum samples. The sensitivity, specificity and rapidity of the system were first demonstrated by applying maternal AChR-Ab positive plasmas known to inhibit fetal AChR function in TE671 cells. Eleven previously untested AChR-Ab positive MG sera, 10 AChR-Ab negative MG sera and 5 healthy control sera were then applied to unclustered and rapsyn-clustered human adult AChRs in CN21 cells., Results: The maternal AChR-Ab positive plasmas reduced fetal AChR currents, but not adult AChR currents, by >80% within 100 s. Only 2/11 AChR-Ab positive sera inhibited AChR currents in unclustered AChRs, but 6/11 AChR-Ab positive sera compared with none of the 10 AChR-Ab negative sera (p=0.0020) inhibited rapsyn-clustered AChR currents, and current inhibition by the AChR-Ab positive sera was greater when the AChRs were clustered (p=0.0385). None of the sera had detectable effects on desensitisation or recovery from desensitisation., Conclusion: These results show that antibodies can inhibit AChR function rapidly and demonstrate the importance of clustering in exploring pathogenic disease mechanisms of MG Abs., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ.)

Objective: To explore the underlying mechanism of action of Tongxieyaofang decoction in rats with visceral hypersensitivity using proteomics technology., Methods: Twenty-four female Sprague-Dawley rats were randomly divided into three groups: control group, irritable bowel syndrome (IBS) group and Tongxieyaofang treatment group. An IBS model, characterized as visceral hypersensitivity, was established using the odour of mothballs as conditional stimulation and colorectal distension combined with classic physical restraint as non-conditional stimulation. Rats were intragastrically treated with Tongxieyaofang (2 or 4 mL·kg－1·d－1) for 4 weeks. On the 45th day, the rats were dissected and the colonic mucosal proteins were extracted. Differential protein spots were screened by fluorescent two-dimensional differential gel electrophoresis (2D-DIGE), and identified by matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF-MS). Western blotting experiments were performed to verify the changes observed in 2D-DIGE and MALDI-TOF-MS., Results: It was found that the visceral sensitivity of rats in the Tongxieyaofang treatment group (4 mL/kg) was lower than that in the IBS group (P < 0.01). Sixty-one protein spots were differentially expressed between the IBS group and the Tongxieyaofang treatment group. Of these, 23 spots were upregulated in the Tongxieyaofang treatment group, while 38 spots were downregulated. Three specific proteins were successfully identified from the five protein spots with the most obvious changes. The two upregulated proteins were transgelin (TAGLN) and acetaldehyde dehydrogenase 2 (Aldh2) and the downregulated protein was cytokeratin 8 (CK8)., Conclusion: Tongxieyaofang can dose-dependently ameliorate visceral hypersensitivity in rats and the mechanism of action may involve the upregulation of TAGLN and Aldh2 and the downregulation of CK8.

High immunogenicity and systemic toxicity are the main obstacles limiting the clinical use of the therapeutic agents based on Pseudomonas aeruginosa exotoxin A. In this work, we studied the immunogenicity, general toxicity and antitumor effect of the targeted toxin DARPin-LoPE composed of HER2-specific DARPin and a low immunogenic exotoxin A fragment lacking immunodominant human B lymphocyte epitopes. The targeted toxin has been shown to effectively inhibit the growth of HER2-positive human ovarian carcinoma xenografts, while exhibiting low non-specific toxicity and side effects, such as vascular leak syndrome and liver tissue degradation, as well as low immunogenicity, as was shown by specific antibody titer. This represents prospects for its use as an agent for targeted therapy of HER2-positive tumors.

Background Risk-adjusted treatment has led to outstanding improvements of the remission and survival rates of childhood acute lymphoblastic leukemia (ALL). Nevertheless, overtreatment-related toxicity and resistance to therapy have not been fully prevented. In the present study, we evaluated for the first time the clinical impact of the apoptosis-related BCL2L12 gene in prognosis and risk stratification of BFM-treated childhood ALL. Methods Bone marrow specimens were obtained from childhood ALL patients upon disease diagnosis and the end-of-induction (EoI; day 33) of the BFM protocol, as well as from control children. Following total RNA extraction and reverse transcription, BCL2L12 expression levels were determined by qPCR. Patients' cytogenetics, immunophenotyping and minimal residual disease (MRD) evaluation were performed according to the international guidelines. Results BCL2L12 expression was significantly increased in childhood ALL and correlated with higher BCL2/BAX expression ratio and favorable disease markers. More importantly, BCL2L12 expression was associated with disease remission, while the reduced BCL2L12 expression was able to predict patients' poor response to BFM therapy, in terms of M2-M3 response and MRD≥0.1% on day 15. The survival analysis confirmed the significantly higher risk of the BFM-treated patients underexpressing BCL2L12 at disease diagnosis for early relapse and worse survival. Lastly, evaluation of BCL2L12 expression clearly strengthened the prognostic value of the established disease prognostic markers, leading to superior prediction of patients' outcome and improved specificity of BFM risk stratification. Conclusions The expression levels of the apoptosis-related BCL2L12 predict response to treatment and survival outcome of childhood ALL patients receiving BFM chemotherapy.

During inflammation, phagocytes release digestive enzymes from lysosomes to degrade harmful cells such as pathogens and tumor cells. However, the molecular mechanisms regulating this process are poorly understood. In this study, we identified myoferlin as a critical regulator of lysosomal exocytosis by mouse phagocytes. Myoferlin is a type II transmembrane protein with seven C2 domains in the cytoplasmic region. It localizes to lysosomes and mediates their fusion with the plasma membrane upon calcium stimulation. Myoferlin promotes the release of lysosomal contents, including hydrolytic enzymes, which increase cytotoxicity. These data demonstrate myoferlin's critical role in lysosomal exocytosis by phagocytes, providing novel insights into the mechanisms of inflammation-related cellular injuries., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Transgelin-2 is a small 22-kDa actin-binding protein implicated in actin dynamics, which stabilizes actin structures and participates in actin-associated signaling pathways. Much curiosity regarding transgelin-2 has centered around its dysregulation in tumor development and associated diseases. However, recent studies have shed new light on the functions of transgelin-2, the only transgelin family member present in leukocytes, in the context of various immune responses. In this review, we outlined the biochemical properties of transgelin-2 and its physiological functions in T cells, B cells, and macrophages. Transgelin-2 regulates T cell activation by stabilizing the actin cytoskeleton at the immunological synapse. Transgelin-2 in B cells also participates in the stabilization of T cell-B cell conjugates. While transgelin-2 is expressed at trace levels in macrophages, its expression is highly upregulated upon lipopolysaccharide stimulation and plays an essential role in macrophage phagocytosis. Since transgelin-2 increases T cell adhesion to target cells via boosting the "inside-out" costimulatory activation of leukocyte function-associated antigen 1, transgelin-2 could be a suitable candidate to potentiate the antitumor response of cytotoxic T cells by compensating for the lack of costimulation in tumor microenvironment. We discussed the feasibility of using native or engineered transgelin-2 as a synergistic molecule in cell-based immunotherapies, without inducing off-target disturbance in actin dynamics in other cells., (©2018 The Authors. Society for Leukocyte Biology Published by Wiley Periodicals, Inc.)

The skewed T helper (Th) 2 response plays a central role in the pathogenesis of allergic diseases, while its initiating factors remain elusive. Recent studies indicate that Bcl2 like protein-12 (Bcl2L12) is associated with the Th2-biased inflammation. This study is designed to test a hypothesis that Bcl2L12 plays a critical role in the initiation of allergic response. In this study, peripheral CD4 + T cells were isolated from food allergy (FA) patients and healthy subjects; A mouse FA model was developed to test the role of Bcl2L12 in induction of allergic response in the intestine. The results showed that expression of Bcl2L12 by CD4 + T cells was higher in FA patients and FA mice and positively correlated with expression of Th2 cytokines. CD4 + T cells from FA patients showed a Bcl2L12-dependent tendency to differentiate into Th2 cells. Bcl2L12 played a crucial role in induction of allergic response in the intestine. Physical contact between Bcl2L12 and GATA3 facilitated GATA3 to bind Il4 promoter to promote expression of IL-4. Adoptive transfer with Bcl2L12-deficient CD4 + T cells to Rag2¯ / ¯ mice did not reconstitute the efficient CD4 + T cell response as the mice could not be induced FA, while Rag2¯ / ¯ mice received WT CD4 + T cell transfer were induced FA. In conclusion, Bcl2L12 plays a crucial role in the induction of Th2 polarization and allergic response in the intestine. The Bcl2L12 in CD4 + T cells may be a potential target for the treatment of FA., (Copyright © 2018 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

Background: T-helper 2 (Th2) polarization plays a critical role in the pathogenesis of chronic rhinosinusitis (CRS) with accompanying nasal allergy. Recent studies indicate that B cell lymphoma-2-like protein-12 (Bcl2L12) is associated with immune dysregulation. The purpose of this study was to elucidate the role of Bcl2L12 in the pathogenesis of Th2 polarization of CRS patients., Methods: CRS patients with nasal allergy (CRSa) and without nasal allergy (CRSna) were recruited into this study. CD4 + T cells were isolated from the blood samples of human subjects. A variety of immunologic molecular strategies were used to assess Th2 polarization and Bcl2L12 expression., Results: Twenty CRSa patients, 20 CRSna patients, and 20 healthy subjects were recruited into this study. High levels of immunoglobulin E (IgE), interleukin 4 (IL-4), IL-5, IL-13, and Bcl2L12 were detected in nasal extracts of CRSa patients, but not in CRSna patients. The levels of Bcl2L12 were positively correlated with Th2 cytokines. CD4 + T cells from CRSa patients were prone to differentiate into Th2 cells, in which Bcl2L12 was required., Conclusion: Bcl2L12 is positively correlated with Th2 cytokine levels in the nasal mucosa of CRSa patients. Bcl2L12 contributes to the Th2 polarization, which may be a novel therapeutic target in the treatment of CRSa., (© 2018 ARS-AAOA, LLC.)

Background: Atopic dermatitis (AD) is a common inflammatory skin disease. A subset of patients with AD are susceptible to disseminated herpes simplex virus (HSV) infection, a complication termed eczema herpeticum (ADEH+). The immune mechanisms causing ADEH+ remain elusive. Using RNA sequencing, we recently found that ankyrin repeat domain 1 (ANKRD1) was significantly induced in human PBMCs upon HSV-1 stimulation, and its induction in patients with ADEH+ was significantly reduced compared with that seen in AD patients without a history of eczema herpeticum (ADEH-)., Objective: We sought to validate ANKRD1 gene expression in nonatopic (NA) subjects, patients with ADEH-, and patients with ADEH+ and to delineate the biological function of ANKRD1 and the signaling pathway or pathways involved., Methods: Purification of human PBMCs, monocytes, B cells, dendritic cells, T cells, and natural killer cells; RNA extraction and quantitative RT-PCR; small interfering RNA technique; co-immunoprecipitation; and Western blot assays were used., Results: ANKRD1 expression was significantly reduced in PBMCs from patients with ADEH+ after HSV-1 stimulation compared with PBMCs from patients with ADEH-. We found that the induction of ANKRD1 by HSV-1 and multiple pattern recognition receptor agonists are mediated by inflammatory cytokines. Silencing ANKRD1 gene expression in antigen-presenting cells led to increased viral load and reduced IFNB1 and IL29 production. Using co-immunoprecipitation methods, we demonstrated that ANKRD1 formed protein complexes with interferon regulatory factor (IRF) 3 and IRF7, which are important transcription factors regulating signaling transduction of pattern recognition receptors. Overexpression of ANKRD1 enhanced the IRF3-mediated signaling pathways., Conclusion: ANKRD1 is involved in IRF3-mediated antiviral innate immune signaling pathways. Its reduced expression in patients with ADEH+ might contribute to the pathogenesis of ADEH+., (Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Shigella deploys a unique mechanism to manipulate macrophage pyroptosis by delivering the IpaH7.8 E3 ubiquitin ligase via its type III secretion system. IpaH7.8 ubiquitinates glomulin (GLMN) and elicits its degradation, thereby inducing inflammasome activation and pyroptotic cell death of macrophages. Here, we show that GLMN specifically binds cellular inhibitor of apoptosis proteins 1 and 2 (cIAP1 and cIAP2), members of the inhibitor of apoptosis (IAP) family of RING-E3 ligases, which results in reduced E3 ligase activity, and consequently inflammasome-mediated death of macrophages. Importantly, reducing the levels of GLMN in macrophages via IpaH7.8, or siRNA-mediated knockdown, enhances inflammasome activation in response to infection by Shigella, Salmonella, or Pseudomonas, stimulation with NLRP3 inflammasome activators (including SiO 2 , alum, or MSU), or stimulation of the AIM2 inflammasome by poly dA:dT GLMN binds specifically to the RING domain of both cIAPs, which inhibits their self-ubiquitination activity. These findings suggest that GLMN is a negative regulator of cIAP-mediated inflammasome activation, and highlight a unique Shigella stratagem to kill macrophages, promoting severe inflammation., (© 2017 The Authors.)

The pathogenesis of the immune regulation dysfunction is unclear. Bcl2-like protein 12 (Bcl2L12) has immune suppression function. This study tests a hypothesis that tumor necrosis factor (TNF) increases Bcl2L12 to suppress the expression of interleukin (IL) 10 in peripheral B cells of patients with inflammatory bowel disease (IBD). In this study, peripheral blood samples were collected from IBD patients and healthy controls. B cells were isolated from the blood samples. The expression of IL-10 and Bcl2L12 in B cells was analyzed by quantitative reverse transcription polymerase chain reaction and Western blotting. We observed that the expression of Bcl2L12 in the peripheral B cells was higher in IBD patients than that in healthy controls. The IL-10 levels in B cells were negatively correlated with the expression of Bcl2L12. Exposure of B cells to TNF in the culture enhanced the expression of Bcl2L12. The Bcl2L12 mediated the effects of TNF on suppression of IL-10 in B cells. In conclusion, Bcl2L12 mediates the effects of TNF to suppress the expression of IL-10 in B cells. The data suggest that Bcl2L12 may be a therapeutic target for the treatment of IBD., (Copyright © 2017 John Wiley & Sons, Ltd.)

Skeletal muscle insulin resistance is considered to be the primary defect involved in type 2 diabetes mellitus (T2DM). Despite transcriptome studies in limited T2DM human subjects suggesting an association of T2DM with impaired oxidative phosphorylation in muscle, its molecular pathogenesis remains largely unknown. To identify dysregulated genes and gene networks that are associated with T2DM in human skeletal muscle, we examined expression patterns of 56,318 transcribed genes on 92 T2DM cases and 184 gender-, age- and race-matched non-diabetic controls from the Genotype-Tissue Expression (GTEx) database. RNA-Sequencing data suggest that diabetic skeletal muscle is characterized by decreased expression of genes that are related to insulin resistance (IRS2, MTOR, SLC2A4, and PPARA), carbohydrate, energy, and amino acid metabolism pathways (NDUFS1, NDUFA10, NDUFB4, NDUFB5, NDUFA5, NDUFB10, SDHB, SDHC, ATP5H, ATP5A, and ATP5J). Up-regulated genes in T2DM are mainly enriched in apoptosis pathways (TP53, GADD45A, TNFRSF10B, TP53AIP1, and PMAIP1), and notably include immune-related pathways suggestive of a response to various infectious diseases (C2, CFB, C4A, C4B, C1S, C1R, C3, HLA-DRA, HLA-DMA, HLA-DOA, and HLA-DPB1). These results confirm the essential regulation of impaired insulin signaling and oxidative phosphorylation in the muscle of T2DM patients, and provide novel molecular insights into the pathophysiological mechanisms of T2DM., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: RCAN1 (regulator of calcineurin 1) has been shown to be involved in various physiological and pathological processes. However, the biological implications of RCAN1 during gastrointestinal tract infection remain unclear. In this study, we tried to determine the role of RCAN1 in acute Salmonella infectious colitis., Methods: Wild type and RCAN1-deficient mice or macrophages were used to characterize the impacts of RCAN1 on intestinal inflammation, inflammatory cytokines production, animal survival, and pathogen clearance following Salmonella challenge., Results: Histologic and quantitative assessments showed increased inflammation and elevated proinflammatory cytokines production in RCAN1-deficient mice. The aberrant inflammatory response was recapitulated in primary bone marrow-derived macrophages. In addition, we reveal a novel regulatory role for RCAN1 in the proinflammatory JNK signaling both in vitro and in vivo. Further analysis showed that the increased inflammation in RCAN1-deficient mice contributed to pathogen clearance and host survival., Conclusions: The present study demonstrates that RCAN1 deficiency protects against Salmonella intestinal infection by enhancing proinflammatory JNK signaling., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSCs) expand in the spleen during cancer and promote progression through suppression of cytotoxic T cells. An anti-inflammatory reflex arc involving the vagus nerve and memory T cells is necessary for resolution of acute inflammation. Failure of this neural circuit could promote procarcinogenic inflammation and altered tumour immunity. Here we show that splenic TFF2, a secreted anti-inflammatory peptide, is released by vagally modulated memory T cells to suppress the expansion of MDSCs through CXCR4. Splenic denervation interrupts the anti-inflammatory neural arc, resulting in the expansion of MDSCs and colorectal cancer. Deletion of Tff2 recapitulates splenic denervation to promote carcinogenesis. Colorectal carcinogenesis could be suppressed through transgenic overexpression of TFF2, adenoviral transfer of TFF2 or transplantation of TFF2-expressing bone marrow. TFF2 is important to the anti-inflammatory reflex arc and plays an essential role in arresting MDSC proliferation. TFF2 offers a potential approach to prevent and to treat cancer.

The study investigated changes in myokines, heat shock proteins, and growth factors in highly ranked, young, male tennis players in response to physical workload during the competitive season and their potential correlations with match scores. Blood collections were carried out at the beginning, the midpoint, and the end of the tournament season. Data analysis revealed a significant increase in interleukin 6 and its inverse correlation with the number of lost games (r = -0.45; 90% CI -0.06 to 0.77). Neither the irisin nor BDNF level changed notably, yet delta changes of irisin across the season significantly correlated with the number of games won. The concentration of HSP27 recorded a small increase (31.2%; 90% CI 10.7 to 55.5, most likely). A negative correlation was noted between IGF-1 and HSP27 concentration at baseline (-0.70 very high; 90% CI -0.89 to -0.31, very likely). At the end of the season IGF-1 correlated positively with the number of games won (r = 0.37 moderate, 90% CI -0.16 to 0.73, likely) but negatively with the number of games lost (r = -0.39, 90% CI -0.14 to -0.74, likely). In conclusion our data indicated that Il-6, irisin, and growth factor IGF-1 may modify overall performance during a long lasting season, expressed in the amount of games won or lost.

Mutations of the gene encoding four-and-a-half LIM domain 1 (FHL1) are the causative factor of several X-linked hereditary myopathies that are collectively termed FHL1-related myopathies. These disorders are characterized by severe muscle dysfunction and damage. Here, we have shown that patients with idiopathic inflammatory myopathies (IIMs) develop autoimmunity to FHL1, which is a muscle-specific protein. Anti-FHL1 autoantibodies were detected in 25% of IIM patients, while patients with other autoimmune diseases or muscular dystrophies were largely anti-FHL1 negative. Anti-FHL1 reactivity was predictive for muscle atrophy, dysphagia, pronounced muscle fiber damage, and vasculitis. FHL1 showed an altered expression pattern, with focal accumulation in the muscle fibers of autoantibody-positive patients compared with a homogeneous expression in anti-FHL1-negative patients and healthy controls. We determined that FHL1 is a target of the cytotoxic protease granzyme B, indicating that the generation of FHL1 fragments may initiate FHL1 autoimmunity. Moreover, immunization of myositis-prone mice with FHL1 aggravated muscle weakness and increased mortality, suggesting a direct link between anti-FHL1 responses and muscle damage. Together, our findings provide evidence that FHL1 may be involved in the pathogenesis not only of genetic FHL1-related myopathies but also of autoimmune IIM. Importantly, these results indicate that anti-FHL1 autoantibodies in peripheral blood have promising potential as a biomarker to identify a subset of severe IIM.

The inner mitochondrial membrane (IMM) invaginates to form cristae and the maintenance of cristae depends on the mitochondrial contact site (MICOS) complex. Mitofilin and CHCHD6, which physically interact, are two components of the MICOS. In this study, we performed immunoprecipitation experiments with Mitofilin and CHCHD6 antibodies and identified a complex containing Mitofilin, Sam50, and CHCHD 3 and 6. Using transcription activator-like effector nucleases (TALENs), we generated knockdown/knockout clones of Mitofilin and CHCHD6. Transmission electron microscopy (TEM) revealed that vesicle-like cristae morphology appeared in cell lines lacking Mitofilin, and mitochondria exhibited lower cristae density in CHCHD6-knockout cells. Immunoblot analysis showed that knockdown of Mitofilin, but not knockout of CHCHD6, affected their binding partners that control cristae morphology. We also demonstrated that Mitofilin and CHCHD6 directly interacted with Sam50. Additionally, we observed that Mitofilin-knockdown cells showed decreased mitochondrial membrane potential (ΔΨm) and intracellular ATP content, which were minimally affected in CHCHD6-knockout cells. Taken together, we conclude that the integrity of MICOS and its efficient interaction with Sam50 are indispensable for cristae organization, which is relevant to mitochondrial function.

LIM-only protein FHL2 is associated with several immune and inflammatory diseases such as arthritis, influenza A virus infection, and lung inflammation. However, the role of FHL2 in macrophage differentiation and in the development of granuloma formation is unknown. Here, we show that expression of FHL2 is induced in mouse bone marrow derived macrophages (BMMs) following stimulation with M2 cytokines such as IL-4 and IL-10. FHL2-knockout (FHL2-KO) BMMs exhibit a proinflammatory M1 phenotype after LPS treatment and display a reduced anti-inflammatory M2 phenotype following IL-4 treatment. Furthermore, thioglycollate-induced migration of macrophages and B cells is enhanced in FHL2-KO mice. To evaluate the importance of FHL2 in the development of pulmonary granuloma formation, FHL2-KO mice were challenged with Schistosoma mansoni eggs. FHL2-KO mice show an enhanced number of granulomas and display decreased expression of Th2 markers and an exacerbated Th1 type of inflammation, characterized by enhanced expression of neutrophil markers and Th1 cytokines. Furthermore, the expression of barrier proteins is reduced in FHL2-KO lung compared to WT. Collectively, these data identify a previously unrecognized role for FHL2 in the pathogenesis of pulmonary granulomatous inflammation, partly through its effect on macrophage polarization, modulation of the Th1/Th2 balance and regulation of permeability in lung., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

"Iodine allergy" does not exist. The concept of "iodine allergy" should be abandoned since it may result in inappropriate measures such as drug, food or environmental eviction. Immediate or non-immediate allergic hypersensitivity to iodinated contrast media is not infrequent. The corresponding allergens have not been identified. Iodine is not involved. Immediate or non-immediate allergic hypersensitivity to povidone iodine is rare. The corresponding allergen is povidone in case of immediate hypersensitivity while nonoxynol might be involved during non-immediate hypersensitivity. Seafood allergens belong to a group of muscle proteins. Immediate drug hypersensitivity or food hypersensitivity is assessed by immediate-reading skin tests while non-immediate drug hypersensitivity is investigated by delayed-reading skin testing. Combined histamine and tryptase measurement is invaluable during the diagnostic approach of immediate hypersensitivity. Other biological tests are being evaluated. Allergic hypersensitivity to iodinated contrast agents does not contraindicate the use of other iodinated drugs., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

Response gene to complement 32 (RGC-32) is a cell cycle regulator involved in the proliferation, differentiation and migration of cells and has also been implicated in angiogenesis. Here we show that RGC-32 expression in macrophages is induced by IL-4 and reduced by LPS, indicating a link between RGC-32 expression and M2 polarization. We demonstrated that the increased expression of RGC-32 is characteristic of alternatively activated macrophages, in which this protein suppresses the production of pro-inflammatory cytokine IL-6 and promotes the production of the anti-inflammatory mediator TGF-β. Consistent with in vitro data, tumor-associated macrophages (TAMs) express high levels of RGC-32, and this expression is induced by tumor-derived ascitic fluid in an M-CSF- and/or IL-4-dependent manner. Collectively, these results establish RGC-32 as a marker for M2 macrophage polarization and indicate that this protein is a potential target for cancer immunotherapy, targeting tumor-associated macrophages.

Background: The reverse Warburg effect describes the phenomenon that epithelial cancer cells take advantage of the metabolic machinery from nearby cancer-associated fibroblast, inducing them to produce lactate and ketones to fuel the high metabolic demands of the epithelial tumour tissues. This is in breast cancer observed as a lack of stromal caveolin-1 (CAV-1) and an increased expression of monocarboxylate transporter 4 (MCT-4) in the tumour stroma, with a concomitant increase in the expression of monocarboxylate transporter 1 (MCT-1) in the epithelial, tumour compartment. The lack of CAV-1 and increased expression of MCT-4 have been shown to have prognostic importance, primarily in patients with breast cancer. However, this phenomenon has only scarcely been described in oral squamous cell carcinoma (OSCC). Given the prognostic importance of myofibroblasts in OSCC, we also examined a potential relationship between the expression of MCT-4 and the presence of myofibroblasts., Methods: Paraffin-embedded tissues from 30 patients with OSCC were immunostained with antibodies towards MCT-1, MCT-4, Cav-1, GLUT-1, α-SMA, TOMM20 and KI-67, and evaluated for their specific epithelial and stromal expression., Results and Conclusions: In patients with OSCC, we find an increased expression of MCT-1 and MCT-4 in both the epithelial and stromal compartment, with almost no overlap in their spatial expression. We found a large spatial overlap between α-SMA and MCT-1 in the stroma compartment, but no relationship between MCT-4 and myofibroblasts. Interestingly, we did not observe any relationship between the absence of CAV-1 and the presence of MCT-4 as has been shown in breast carcinomas., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Serological testing for anti-neural autoantibodies is important in patients presenting with idiopathic cerebellar ataxia, since these autoantibodies may indicate cancer, determine treatment and predict prognosis. While some of them target nuclear antigens present in all or most CNS neurons (e.g. anti-Hu, anti-Ri), others more specifically target antigens present in the cytoplasm or plasma membrane of Purkinje cells (PC). In this series of articles, we provide a detailed review of the clinical and paraclinical features, oncological, therapeutic and prognostic implications, pathogenetic relevance, and differential laboratory diagnosis of the 12 most common PC autoantibodies (often referred to as 'Medusa-head antibodies' due to their characteristic somatodendritic binding pattern when tested by immunohistochemistry). To assist immunologists and neurologists in diagnosing these disorders, typical high-resolution immunohistochemical images of all 12 reactivities are presented, diagnostic pitfalls discussed and all currently available assays reviewed. Of note, most of these antibodies target antigens involved in the mGluR1/calcium pathway essential for PC function and survival. Many of the antigens also play a role in spinocerebellar ataxia. Part 1 focuses on anti-metabotropic glutamate receptor 1-, anti-Homer protein homolog 3-, anti-Sj/inositol 1,4,5-trisphosphate receptor- and anti-carbonic anhydrase-related protein VIII-associated autoimmune cerebellar ataxia (ACA); part 2 covers anti-protein kinase C gamma-, anti-glutamate receptor delta-2-, anti-Ca/RhoGTPase-activating protein 26- and anti-voltage-gated calcium channel-associated ACA; and part 3 reviews the current knowledge on anti-Tr/delta notch-like epidermal growth factor-related receptor-, anti-Nb/AP3B2-, anti-Yo/cerebellar degeneration-related protein 2- and Purkinje cell antibody 2-associated ACA, discusses differential diagnostic aspects and provides a summary and outlook.

Alpha-syntrophin (SNTA) is an adaptor protein that regulates several signaling pathways. To analyze expression of SNTA immunoblot assays must be performed. Here, the specificity of four commercially available SNTA antibodies has been evaluated in immunoblot experiments using liver tissues of wild-type and SNTA-deficient mice. While one of the antibodies reacts with SNTA, two antibodies specifically recognize beta 2 syntrophin (SNTB2). The antigen detected by the fourth antibody has not been identified but is different from SNTA and SNTB2. Therefore, only one of the four tested antibodies is appropriate to analyze SNTA protein levels by immunoblot., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Cooperation between calcineurin (CN)-NFATc and RAF-MEK-ERK signaling pathways is essential in thymocyte positive selection. It is known that the Regulators of Calcineurin (RCAN) proteins can act either facilitating or suppressing CN-dependent signaling events. Here, we show that RCAN genes are expressed in lymphoid tissues, and address the role of RCAN proteins in T cell development. Overexpression of human RCAN3 and RCAN1 can modulate T cell development by increasing positive selection-related surface markers, as well as the "Erk(hi) competence state" in double positive thymocytes, a characteristic molecular signature of positive selection, without affecting CN activity. We also found that RCAN1/3 interact with RAF kinases and CN in a non-exclusive manner. Our data suggests that the balance of RCAN interactions with CN and/or RAF kinases may influence T cell positive selection., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background and Objectives: IgA plays a key role in IgA nephropathy (IgAN) by forming immune complexes and depositing in the glomeruli, leading to an inflammatory response. However, the antigenic targets and functional characterization of IgA have been incompletely defined in this disease., Design, Setting, Participants, & Measurements: This study was performed in sera from patients who were studied as part of a prospective, observational study of IgAN. These patients (n=22) all had biopsy-proven IgAN within 3 years of study initiation, complete clinical data, annual urinary inulin clearance for GFRs, and at least 5 years of follow-up. Progression was defined as loss of >5 ml/min per 1.73 m(2) per year of inulin clearance measured over at least 5 years. A protein microarray was used for detection of IgAN-specific IgA autoantibodies in blood across approximately 9000 human antigens to specifically identify the most immunogenic protein targets that drive IgA antibodies in IgAN (n=22), healthy controls (n=10), and non-IgAN glomerular diseases (n=17). Results were validated by ELISA assays in sera and by immunohistochemistry in IgAN kidney biopsies. IgA-specific antibodies were correlated with clinical and histologic variables to assess their effect on disease progression and prognosis., Results: Fifty-four proteins mounted highly significant IgA antibody responses in patients with IgAN with a false discovery rate (q value) of ≤10%; 325 antibodies (P≤0.05) were increased overall. Antitissue transglutaminase IgA was significantly elevated in IgAN (P<0.001, q value of 0%). IgA antibodies to DDX4 (r=-0.55, P=0.01) and ZADH2 (r=-0.48, P=0.02) were significantly correlated with the decline of renal function. Specific IgA autoantibodies are elevated in IgAN compared with normal participants and those with other glomerular diseases., Conclusions: In this preliminary study, IgA autoantibodies target novel proteins, highly expressed in the kidney glomerulus and tubules. These IgA autoantibodies may play important roles in the pathogenesis of IgAN., (Copyright © 2015 by the American Society of Nephrology.)

Myasthenia gravis (MG) is an autoimmune disorder caused by autoantibodies targeting proteins expressed at the neuromuscular junction (NMJ). In most cases the targets are acetylcholine receptor (AChR), muscle-specific tyrosine kinase (MuSK), or occasionally low-density lipoprotein receptor-related protein 4 (LRP4), but there is still a group of patients, often called seronegative MG (SNMG), with unknown antibody targets. One potential target is collagen Q (COLQ), which is restricted to the NMJ and is crucial for anchoring the NMJ-specific form of acetylcholinesterase (AChE). 415 serum samples with a clinical diagnosis of MG and 43 control samples were screened for the presence of COLQ autoantibodies using a cell-based assay (CBA) with HEK293 cells overexpressing COLQ at the cell surface. COLQ antibodies were detected in 12/415 MG sera and in one/43 control samples. Five of the COLQ-Ab+individuals were also positive for AChR-Abs and 2 for MuSK-Abs. Although the COLQ antibodies were only present at low frequency, and did not differ significantly from the small control cohort, further studies could address whether they modify the clinical presentation or the benefits of anti-cholinesterase therapy., (Copyright © 2014 Elsevier B.V. All rights reserved.)

There has been fast growing evidence showing that glycolysis plays a critical role in the activation of immune cells. Enhanced glycolysis leads to increased formation of intracellular lactate that is exported to the extracellular environment by monocarboxylate transporter 4 (MCT4). Although the biological activities of extracellular lactate have been well studied, it is less understood how the lactate export is regulated or whether lactate export affects glycolysis during inflammatory activation. In this study, we found that MCT4 is up-regulated by TLR2 and TLR4, but not TLR3 agonists in a variety of macrophages. The increased expression of MCT4 was mediated by MYD88 in a NF-κB-dependent manner. Furthermore, we found that MCT4 is required for macrophage activation upon TLR2 and TLR4 stimulations, as evidenced by attenuated expression of proinflammatory mediators in macrophages with MCT4 knockdown. Mechanistically, we found that MCT4 knockdown leads to enhanced intracellular accumulation of lactate and decreased glycolysis in LPS-treated macrophages. We found that LPS-induced expression of key glycolytic enzymes hexokinase 2 and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 is diminished in macrophages with MCT4 knockdown. Our data suggest that MCT4 up-regulation represents a positive feedback mechanism in macrophages to maintain a high glycolytic rate that is essential to a fully activated inflammatory response., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: There is a paucity of information on structural organization of muscular bundles in the interatrial septum (IAS). The aim was to investigate histologic and ultrastructural organization of muscular bundles in human IAS, including fossa ovalis (FO) and flap valve., Methods: Macroscopic and light microscopy evaluations of IAS were performed from postmortem studies of 40 patients. Twenty three IAS specimens underwent serial transverse sectioning, and 17--longitudinal sectioning. The transverse sections from 10 patients were immunolabeled for HCN4, Caveolin3 and Connexin43. IAS specimens from 6 other patients underwent electron microscopy., Results: In all IAS specimens sections the FO, its rims and the flap valve had muscle fibers consisting of working cardiac myocytes. Besides the typical cardiomyocytes there were unusual cells: tortuous and horseshoe-shaped intertangled myocytes, small and large rounded myocytes with pale cytoplasm. The cells were aggregated in a definite structure in 38 (95%) cases, which was surrounded by fibro-fatty tissue. The height of the structure on transverse sections positively correlated with age (P = 0.03) and AF history (P = 0.045). Immunohistochemistry showed positive staining of the cells for HCN4 and Caveolin3. Electron microscopy identified cells with characteristics similar to electrical conduction cells., Conclusions: Specialized conduction cells in human IAS have been identified, specifically in the FO and its flap valve. The cells are aggregated in a structure, which is surrounded by fibrous and fatty tissue. Further investigations are warranted to explore electrophysiological characteristics of this structure.

Introduction: In rheumatoid arthritis (RA), several genetic risk factors and smoking are strongly associated with the presence of anticitrullinated protein antibodies (ACPA), while much less is known about risk factors for ACPA-negative RA. Antibodies against carbamylated proteins (anti-CarP) have been described in both ACPA-positive and ACPA-negative RA patients. In this study, we have analysed the relationships among anti-CarP antibodies, ACPA, genetic risk factors (HLA-DRB1 alleles and PTPN22) and smoking in RA., Methods: Presence of antibodies to carbamylated fetal calf serum (CarP-FCS) and fibrinogen (CarP-Fib) was determined by inhouse ELISAs among RA cases in the Leiden Early Arthritis Clinic (n=846) and in the Swedish Epidemiological Investigation of Rheumatoid Arthritis (n=1985) cohorts. ORs for associations with different HLA-DRB1 alleles, PTPN22 genotypes and smoking were calculated separately for each cohort as well as in meta-analysis in RA subsets defined by the presence/absence of anti-CarP and anticyclic citrullinated peptide (anti-CCP) antibodies., Results: In both cohorts, anti-CarP antibody positivity was mainly detected in the anti-CCP-positive population (49%-73%), but also in the anti-CCP-negative population (8%-14%). No associations between anti-CarP antibodies and HLA-DRB1 shared epitope alleles could be identified, while there were data to support an association between anti-CarP-FCS and HLA-DRB1*03. Further analyses did not reveal any specific associations of anti-CarP antibodies with other HLA-DRB1 alleles, PTPN22 genotypes or smoking., Conclusions: Anti-CarP antibodies were present in both ACPA-positive and ACPA-negative RA. There were no significant associations among anti-CarP antibodies and HLA-DRB1 alleles, PTPN22 or smoking. These data suggest that different biological mechanisms may underlie anti-CarP versus anti-CCP antibody formation., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Objective: In idiopathic inflammatory myopathies (IIM) infiltration of immune cells into muscle and upregulation of MHC-I expression implies increased antigen presentation and involvement of the proteasome system. To decipher the role of immunoproteasomes in myositis, we investigated individual cell types and muscle tissues and focused on possible immune triggers., Methods: Expression of constitutive (PSMB5, -6, -7) and corresponding immunoproteasomal subunits (PSMB8, -9, -10) was analyzed by real-time RT-PCR in muscle biopsies and sorted peripheral blood cells of patients with IIM, non-inflammatory myopathies (NIM) and healthy donors (HD). Protein analysis in muscle biopsies was performed by western blot. Affymetrix HG-U133 platform derived transcriptome data from biopsies of different muscle diseases and from immune cell types as well as monocyte stimulation experiments were used for validation, coregulation and coexpression analyses., Results: Real-time RT-PCR revealed significantly increased expression of immunoproteasomal subunits (PSMB8/-9/-10) in DC, monocytes and CD8+ T-cells in IIM. In muscle biopsies, the immunosubunits were elevated in IIM compared to NIM and exceeded levels of matched blood samples. Proteins of PSMB8 and -9 were found only in IIM but not NIM muscle biopsies. Reanalysis of 78 myositis and 20 healthy muscle transcriptomes confirmed these results and revealed involvement of the antigen processing and presentation pathway. Comparison with reference profiles of sorted immune cells and healthy muscle confirmed upregulation of PSMB8 and -9 in myositis biopsies beyond infiltration related changes. This upregulation correlated highest with STAT1, IRF1 and IFNγ expression. Elevation of T-cell specific transcripts in active IIM muscles was accompanied by increased expression of DC and monocyte marker genes and thus reflects the cell type specific involvement observed in peripheral blood., Conclusions: Immunoproteasomes seem to indicate IIM activity and suggest that dominant involvement of antigen processing and presentation may qualify these diseases exemplarily for the evolving therapeutic concepts of immunoproteasome specific inhibition.

We have recently identified disorganized muscle protein-1 (DIM-1) in one of the proinflammatory fractions of the human filaria Brugia malayi adult worm. The present study was undertaken to characterize B. malayi DIM-1 (DIM-1bm) and explore its vaccine potential. In this study we cloned and expressed the DIM-1bm gene, investigated its sequence homology with other nematodes, constructed in silico structural model, purified the recombinant DIM-1bm (rDIM-1bm) protein, and studied the effect of immunization with rDIM-1bm on the establishment of B. malayi infection in Mastomys coucha. DIM-1bm showed similarity with DIM-1 of Caenorhabditis elegans, Ascaris suum and Loa loa. Structural modeling revealed three immunoglobulin domains in DIM-1bm indicating that it is a member of immunoglobulin superfamily (IgSF) and 'blastn' results showed that DIM-1bm coding sequence (CDS) have almost no homology with human and mouse nucleotide sequences. Immunization with rDIM-1bm partially protected M. coucha against establishment of infection as inferred by a low recovery of microfilariae (37-64%) and parasite burden (∼50%). The enhanced activity of macrophages, and IFN-γ and NO responses, and elevated levels of specific IgG, IgG1, IgG2a and IgG2b correlated with parasitological findings. This is the first report on cloning, expression, structural modeling and purification of rDIM-1bm and its ability to partially prevent establishment of B. malayi infection. DIM-1bm's almost complete lack of homology with the human counterpart makes it an attractive protein for exploring its vaccine potential., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Adaptive responses of skeletal muscle regulate the nuclear shuttling of the sarcomeric protein Ankrd2 that can transduce different stimuli into specific adaptations by interacting with both structural and regulatory proteins. In a genome-wide expression study on Ankrd2-knockout or -overexpressing primary proliferating or differentiating myoblasts, we found an inverse correlation between Ankrd2 levels and the expression of proinflammatory genes and identified Ankrd2 as a potent repressor of inflammatory responses through direct interaction with the NF-κB repressor subunit p50. In particular, we identified Gsk3β as a novel direct target of the p50/Ankrd2 repressosome dimer and found that the recruitment of p50 by Ankrd2 is dependent on Akt2-mediated phosphorylation of Ankrd2 upon oxidative stress during myogenic differentiation. Surprisingly, the absence of Ankrd2 in slow muscle negatively affected the expression of cytokines and key calcineurin-dependent genes associated with the slow-twitch muscle program. Thus, our findings support a model in which alterations in Ankrd2 protein and phosphorylation levels modulate the balance between physiological and pathological inflammatory responses in muscle.

Background: Biomarkers allowing the characterization of malignancy and therapy response of oral squamous cell carcinomas (OSCC) or other types of carcinomas are still outstanding. The biochemical suicide molecule endonuclease DNaseX (DNaseI-like 1) has been used to identify the Apo10 protein epitope that marks tumor cells with abnormal apoptosis and proliferation. The transketolase-like protein 1 (TKTL1) represents the enzymatic basis for an anaerobic glucose metabolism even in the presence of oxygen (aerobic glycolysis/Warburg effect), which is concomitant with a more malignant phenotype due to invasive growth/metastasis and resistance to radical and apoptosis inducing therapies., Methods: Expression of Apo10 and TKTL1 was analysed retrospectively in OSCC specimen (n = 161) by immunohistochemistry. Both markers represent independent markers for poor survival. Furthermore Apo10 and TKTL1 have been used prospectively for epitope detection in monocytes (EDIM)-blood test in patients with OSCC (n = 50), breast cancer (n = 48), prostate cancer (n = 115), and blood donors/controls (n = 74)., Results: Positive Apo10 and TKTL1 expression were associated with recurrence of the tumor. Multivariate analysis demonstrated Apo10 and TKTL1 expression as an independent prognostic factor for reduced tumor-specific survival. Apo10+/TKTL1+ subgroup showed the worst disease-free survival rate in OSCC.EDIM-Apo10 and EDIM-TKTL1 blood tests allowed a sensitive and specific detection of patients with OSCC, breast cancer and prostate cancer before surgery and in after care. A combined score of Apo10+/TKTL1+ led to a sensitivity of 95.8% and a specificity of 97.3% for the detection of carcinomas independent of the tumor entity., Conclusions: The combined detection of two independent fundamental biophysical processes by the two biomarkers Apo10 and TKTL1 allows a sensitive and specific detection of neoplasia in a noninvasive and cost-effective way. Further prospective trials are warranted to validate this new concept for the diagnosis of neoplasia and tumor recurrence.

Whey protein and leucine ingestion following exercise increases muscle protein synthesis and could influence neutrophil function during recovery from prolonged intense exercise. We examined the effects of whey protein and leucine ingestion post-exercise on neutrophil function and immunomodulators during a period of intense cycling. In a randomized double-blind crossover, 12 male cyclists ingested protein/leucine/carbohydrate/fat (LEUPRO 20/7.5/89/22 g h(-1), respectively) or isocaloric carbohydrate/fat control (CON 119/22 g h(-1)) beverages for 1-3 h post-exercise during 6 days of high-intensity training. Blood was taken pre- and post-exercise on days 1, 2, 4 and 6 for phorbol myristate acetate (PMA)-stimulated neutrophil superoxide (O2 (-)) production, immune cell counts, amino acid and lipid metabolism via metabolomics, hormones (cortisol, testosterone) and cytokines (interleukin-6, interleukin-10). During recovery on day 1, LEUPRO ingestion increased mean concentrations of plasma amino acids (glycine, arginine, glutamine, leucine) and myristic acid metabolites (acylcarnitines C14, myristoylcarnitine; and C14:1-OH, hydroxymyristoleylcarnitine) with neutrophil priming capacity, and reduced neutrophil O2 production (15-17 mmol O2 (-) cell(-1) ± 90 % confidence limits 20 mmol O2 (-) cell(-1)). On day 2, LEUPRO increased pre-exercise plasma volume (6.6 ± 3.8 %) but haematological effects were trivial. LEUPRO supplementation did not substantially alter neutrophil elastase, testosterone, or cytokine concentrations. By day 6, however, LEUPRO reduced pre-exercise cortisol 21 % (±15 %) and acylcarnitine C16 (palmitoylcarnitine) during exercise, and increased post-exercise neutrophil O2 (-) (33 ± 20 mmol O2 (-) cell(-1)), relative to control. Altered plasma amino acid and acylcarnitine concentrations with protein-leucine feeding might partly explain the acute post-exercise reduction in neutrophil function and increased exercise-stimulated neutrophil oxidative burst on day 6, which could impact neutrophil-dependent processes during recovery from intense training.

Four-and-a-half LIM-only protein 2 (FHL2) is an important mediator in many signaling pathways. In this study, we analyzed the functions of FHL2 in nuclear factor κB (NF-κB) signaling in the liver. We show that FHL2 enhanced tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) activity in transcriptional activation of NF-κB targets by stabilizing the protein. TRAF6 is a binding partner of FHL2 and an important component of the Toll-like receptor-NF-κB pathway. Knockdown of FHL2 in 293-hTLR4/MD2-CD14 cells impaired lipopolysaccharide (LPS)-induced NF-κB activity, which regulates expression of inflammatory cytokines. Indeed, FHL2(-/-) macrophages showed significantly reduced production of TNF and interleukin 6 (IL-6) following LPS stimulation. TNF and IL-6 are the key cytokines that prime liver regeneration after hepatic injury. Following partial hepatectomy, FHL2(-/-) mice exhibited diminished induction of TNF and IL-6 and delayed hepatocyte regeneration. In the liver, NF-κB signaling orchestrates inflammatory cross talk between hepatocytes and hepatic immune cells that promote chemical hepatocarcinogenesis. We found that deficiency of FHL2 reduced susceptibility to diethylnitrosamine-induced hepatocarcinogenesis, correlating with the activator function of FHL2 in NF-κB signaling. Our findings demonstrate FHL2 as a positive regulator of NF-κB activity in liver regeneration and carcinogenesis and highlight the importance of FHL2 in both hepatocytes and hepatic immune cells.

Patients with myasthenia gravis(MG) are divided into three groups: (1) acetylcholine receptor antibody positive MG: 80%, (2) muscle-specific receptor tyrosine kinase (MuSK) antibody positive MG: 5-10%, and (3) double seronegative MG. In 2011, autoantibodies (Abs) against low-density lipoprotein receptor-related protein 4(Lrp4) were identified in Japanese MG patients and thereafter have been reported in Germany and USA. In other Lrp4 Ab papers, Lrp4 Ab positive sera inhibited agrin-induced aggregation of AChRs in cultured myotubes, suggesting a pathogenic role regarding the dysfunction of the neuromuscular endplate. Anti-MuSK autoantibodies were revealed to block binding of collagen Q (ColQ) to MuSK. Anti-Kv1.4 antibodies targeting alpha-subunits(Kv1.4) of the voltage-gated potassium channel occurs frequently among MG patients with thymoma. Further understandings of neuromuscular junction structure and functions through newly discovered autoantibodies may provide more specific clinical information and treatments in MG.

Myasthenia gravis is an autoimmune disease. An autoantibody directed toward acetylcholine receptor (AChR) causes the destruction of the postsynaptic membrane and a reduction of the number of AChRs at neuromuscular junctions. A very puzzling, but interesting characteristic of myasthenia gravis is that many of the patients have an abnormality in their thymus. Many have a hyperplastic thymus with germinal centers, while others have a thymic tumor. How is the abnormality of the thymus related to myasthenia gravis? This review will summarize the existing evidence and try to find the missing link between the thymus and myasthenia gravis. The review will also comment on two distinct populations of myasthenia gravis patients without thymoma. The autoimmunity found in elderly patients is nonspecific and initiated via a different mechanism from the initiation of myasthenia gravis in younger patients.

Obscurins comprise a family of giant (~870- to 600-kDa) and small (~250- to 55-kDa) proteins that play important roles in myofibrillogenesis, cytoskeletal organization, and cell adhesion and are implicated in hypertrophic cardiomyopathy and tumorigenesis. Giant obscurins are composed of tandem structural and signaling motifs, including 2 serine/threonine kinase domains, SK1 and SK2, present at the COOH terminus of giant obscurin-B. Using biochemical and cellular approaches, we show for the first time that both SK1 and SK2 possess enzymatic activities and undergo autophosphorylation. SK2 can phosphorylate the cytoplasmic domain of N-cadherin, a major component of adherens junctions, and SK1 can interact with the extracellular domain of the β1-subunit of the Na(+)/K(+)-ATPase, which also resides in adherens junctions. Immunostaining of nonpermeabilized myofibers and cardiocytes revealed that some obscurin kinase isoforms localize extracellularly. Quantification of the exofacial expression of obscurin kinase proteins indicated that they occupy ~16 and ~5% of the sarcolemmal surface in myofibers and cardiocytes, respectively. Treatment of heart lysates with peptide-N-glycosidase F revealed that while giant obscurin-B localizes intracellularly, possessing dual kinase activity, a small obscurin kinase isoform that contains SK1 localizes extracellularly, where it undergoes N-glycosylation. Collectively, our studies demonstrate that the obscurin kinase domains are enzymatically active and may be involved in the regulation of cell adhesion.