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Systemic sclerosis (SSc) is a complex autoimmune disease with an unclear etiology and no effective treatments. Recent research has suggested involvement of the microbiome in SSc pathogenesis. This study aimed to identify specific microbial species associated with SSc and explore their therapeutic potential. Serum Abs against 384 intestinal microbial species revealed a significant depletion in Abs against Bifidobacterium longum in patients with SSc compared to healthy controls. In a bleomycin-induced SSc mouse model, oral administration of B. longum strain RAPO attenuated skin and lung fibrosis, accompanied by reduced infiltration of inflammatory monocytes/macrophages and downregulation of pro-inflammatory cytokines and chemoattractant Ccl2 genes in lymph nodes and fibrotic tissues. B. longum RAPO treatment restored fecal microbial diversity and augmented short-chain fatty acid (SCFA)-producing bacteria in the gut, leading to increased fecal butyrate levels and upregulated SCFA receptor Gpr41 in the mesenteric lymph node. In vitro , B. longum RAPO and its culture supernatant suppressed the expressions of pro-inflammatory cytokine genes in macrophages and inhibited myofibroblast differentiation in fibroblasts. These findings highlight the probiotic potential of B. longum RAPO in preventing tissue fibrosis by modulating macrophage activity and promoting the growth of SCFA-producing bacteria, underscoring the therapeutic potential of microbial modulation in SSc., Competing Interests: Conflict of Interest: The authors declare no potential conflicts of interest., (Copyright © 2024. The Korean Association of Immunologists.)

Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by skin and internal organ fibrosis and obliterative vasculopathy. Few effective treatments are currently available for fibrosis in SSc, therefore, demand persists for novel therapies. Although use of Ginkgo biloba extract (GBE) has been reported to improve blood circulation and alleviate liver and lung fibrosis, its effect on skin fibrosis in SSc remains unclear. In this study, the effects and underlying mechanisms of GBE on skin fibrosis in bleomycin (BLM)-induced mouse model of SSc was investigated. GBE significantly reduced dermal thickness and protein levels of profibrotic factors in the BLM-induced SSc mouse model. Moreover, GBE inhibited the gene expression of profibrotic factors, such as COL1A1, α-SMA, and connective tissue growth factor (CTGF), in fibroblasts by suppressing transforming growth factor (TGF)-β signaling. Furthermore, GBE inhibited the transdifferentiation of adipocytes into myofibroblasts. Thus, our findings suggest that GBE is a promising therapeutic candidate for the treatment of SSc., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2024 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

A comprehensive understanding of neuronal diversity and connectivity is essential for understanding the anatomical and cellular mechanisms that underlie functional contributions. With the advent of single-cell analysis, growing information regarding molecular profiles leads to the identification of more heterogeneous cell types. Therefore, the need for additional orthogonal recombinase systems is increasingly apparent, as heterogeneous tissues can be further partitioned into increasing numbers of specific cell types defined by multiple features. Critically, new recombinase systems should work together with pre-existing systems without cross-reactivity in vivo. Here, we introduce novel site-specific recombinase systems based on ΦC31 bacteriophage recombinase for labeling multiple cell types simultaneously and a novel viral strategy for versatile and robust intersectional expression of any transgene. Together, our system will help researchers specifically target different cell types with multiple features in the same animal., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Autoimmune diseases are conditions in which the immune system mistakenly targets and damages healthy tissue in the body. In recent decades, the incidence of autoimmune diseases has increased, resulting in a significant disease burden. The current autoimmune therapies focus on targeting inflammation or inducing immunosuppression rather than addressing the underlying cause of the diseases. The activity of metabolic pathways is elevated in autoimmune diseases, and metabolic changes are increasingly recognized as important pathogenic processes underlying these. Therefore, metabolically targeted therapies may represent an important strategy for treating autoimmune diseases. This review provides a comprehensive overview of the evidence surrounding glucose metabolic reprogramming and its potential applications in drug discovery and development for autoimmune diseases, such as type 1 diabetes, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, and systemic sclerosis., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

As our previous study revealed that N -benzyl- N -methyldecan-1-amine (BMDA), a new molecule originated from Allium sativum , exhibits anti-neoplastic activities, we herein explored other functions of the compound and its derivative [decyl-(4-methoxy-benzyl)-methyl-amine; DMMA] including anti-inflammatory and anti-oxidative activities. Pretreatment of THP-1 cells with BMDA or DMMA inhibited tumor necrosis factor (TNF)-α and interleukin (IL)-1β production, and blocked c-jun terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), MAPKAP kinase (MK)2 and NF-κΒ inflammatory signaling during LPS stimulation. Rectal treatment with BMDA or DMMA reduced the severity of colitis in 2,4-dinitrobenzenesulfonic acid (DNBS)-treated rat. Consistently, administration of the compounds decreased myeloperoxidase ( MPO) activity (representing neutrophil infiltration in colonic mucosa), production of inflammatory mediators such as cytokine-induced neutrophil chemoattractant (CINC)-3 and TNF-α, and activation of JNK and p38 MAPK in the colon tissues. In addition, oral administration of these compounds ameliorated collagen-induced rheumatoid arthritis (RA) in mice. The treatment diminished the levels of inflammatory cytokine transcripts, and protected connective tissues through the expression of anti-oxidation proteins such as nuclear factor erythroid-related factor (Nrf)2 and heme oxygenase (HO)1. Additionally, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels did not differ between the BMDA- or DMMA-treated and control animals, indicating that the compounds do not possess liver toxicity. Taken together, these findings propose that BMDA and DMMA could be used as new drugs for curing inflammatory bowel disease (IBD) and RA., Competing Interests: Author I-RC was employed by Hayoung Meditech Inc. The remaining 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 © 2023 Kim, Kang, Budluang, Yawut, Cho, Choi, Kim, Ju, Lee, Sohn, Yim, Lee, Han, Jung, Kang, Park, Jung, Hwang and Chung.)

The WAVE regulatory complex (WRC) is involved in various cellular processes by regulating actin polymerization. The dysregulation of WRC components is associated with cancer development. ABI family member 3 (ABI3)/new molecule including SH3 (NESH) is one of the WRC components and it has been reported that ABI3 phosphorylation can affect WRC function. Although several residues of ABI3 have been reported to be possible phosphorylation sites, it is still unclear which residues are important for the function of ABI3. Furthermore, it is unclear how the phosphorylated form of ABI3 is regulated. Here, we demonstrate that ABI3 is stabilized by its interaction with human leukocyte antigen-F adjacent transcript 10 (FAT10). Using phospho-dead or phospho-mimetic mutants of ABI3, we showed that serine 213 and 216 are important phosphorylation sites of ABI3. In particular, FAT10 has a higher affinity for the phosphorylated form of ABI3 than the non-phosphorylated form, and it stabilizes the phosphorylated form more than the non-phosphorylated form through this differential affinity. The interaction between FAT10 and the phosphorylated form of ABI3 promoted cancer cell migration. Therefore, our results suggest that FAT10 stabilizes the phosphorylated form of ABI3, which may lead to WRC activation, thereby promoting cancer cell migration., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Background: Parkin dysfunction associated with the progression of parkinsonism contributes to a progressive systemic skeletal disease characterized by low bone mineral density. However, the role of parkin in bone remodeling has not yet been elucidated in detail., Result: We observed that decreased parkin in monocytes is linked to osteoclastic bone-resorbing activity. siRNA-mediated knockdown of parkin significantly enhanced the bone-resorbing activity of osteoclasts (OCs) on dentin without any changes in osteoblast differentiation. Moreover, Parkin-deficient mice exhibited an osteoporotic phenotype with a lower bone volume accompanied by increased OC-mediated bone-resorbing capacity displaying increased acetylation of α-tubulin compared to wild-type (WT) mice. Notably, compared to WT mice, the Parkin-deficient mice displayed increased susceptibility to inflammatory arthritis, reflected by a higher arthritis score and a marked bone loss after arthritis induction using K/BxN serum transfer, but not ovariectomy-induced bone loss. Intriguingly, parkin colocalized with microtubules and parkin-depleted-osteoclast precursor cells (Parkin -/- OCPs) displayed augmented ERK-dependent acetylation of α-tubulin due to failure of interaction with histone deacetylase 6 (HDAC6), which was promoted by IL-1β signaling. The ectopic expression of parkin in Parkin -/- OCPs limited the increase in dentin resorption induced by IL-1β, accompanied by the reduced acetylation of α-tubulin and diminished cathepsin K activity., Conclusion: These results indicate that a deficiency in the function of parkin caused by a decrease in parkin expression in OCPs under the inflammatory condition may enhance inflammatory bone erosion by altering microtubule dynamics to maintain OC activity., (© 2023. The Author(s).)

Background: Periodontitis is a major inflammatory disease of the oral mucosa that is not limited to the oral cavity but also has systemic consequences. Although the importance of chronic periodontitis has been emphasized, the systemic immune response induced by periodontitis and its therapeutic effects remain elusive. Here, we report the transcriptomes of peripheral blood mononuclear cells (PBMCs) from patients with periodontitis., Methods: Using single-cell RNA sequencing, we profiled PBMCs from healthy controls and paired pre- and post-treatment patients with periodontitis. We extracted differentially expressed genes and biological pathways for each cell type and calculated activity scores reflecting cellular characteristics. Intercellular crosstalk was classified into therapy-responsive and -nonresponsive pathways., Results: We analyzed pan-cellular differentially expressed genes caused by periodontitis and found that most cell types showed a significant increase in CRIP1, which was further supported by the increased levels of plasma CRIP1 observed in patients with periodontitis. In addition, activated cell type-specific ligand-receptor interactions, including the BTLA, IFN-γ, and RESISTIN pathways, were prominent in patients with periodontitis. Both the BTLA and IFN-γ pathways returned to similar levels in healthy controls after periodontal therapy, whereas the RESISTIN pathway was still activated even after therapy., Conclusion: These data collectively provide insights into the transcriptome changes and molecular interactions that are responsive to periodontal treatment. We identified periodontitis-specific systemic inflammatory indicators and suggest unresolved signals of non-surgical therapy as future therapeutic targets., (© 2022. The Author(s).)

Competing Interests: The author certifies that there are no funding or commercial associations (consultancies, stock ownership, equity interest, patent/licensing arrangements, etc.) that might pose a conflict of interest in connection with the submitted article related to the author or any immediate family members. All ICMJE Conflict of Interest Forms for authors and Clinical Orthopaedics and Related Research ® editors and board members are on file with the publication and can be viewed on request.

Although recent studies have demonstrated a proinflammatory effect of extracellular histones in sepsis via endothelial cytotoxicity, little is known about their contribution to autoimmune arthritis. Therefore, we investigated the role of extracellular histones in autoimmune arthritis and their cytotoxic effect on synoviocytes and macrophages. We measured histones in the synovial fluid of patients with rheumatoid arthritis (RA) and evaluated arthritis severity in a serum-transfer arthritis (STA) mouse model with intraperitoneal histone injection. Histone-induced cytotoxicity was measured using SYTOX green staining in the synoviocyte cell line MH7A and macrophages differentiated from the monocytic cell line THP-1, and the production of damage-associated molecular patterns (DAMPs) was measured by HMGB1 and ATP. Furthermore, we performed RNA-seq analysis of THP-1 cells stimulated with H2B-α1 peptide or with its citrullinated form. The levels of histones were elevated in RA synovial fluid, and histones aggravated arthritis in the STA model. Histones induced cytotoxicity and DAMP production in synoviocytes and macrophages. Chondroitin sulfate reduced histone-induced cytotoxicity, while lipopolysaccharides aggravated cytotoxicity. Moreover, the cytotoxicity decreased when the arginines in H2B-α1 were replaced with citrullines, which demonstrated its electrostatic nature. In transcriptome analysis, H2B-α1 changed the gene expression pattern of THP-1 cells involving chemokines, interleukin-1, -4, -10, -13, and toll-like receptor (TLR) signaling pathways. Extracellular histones were increased in RA synovial fluid and aggravated synovitis in STA. They induced lytic cell death through electrostatic interaction with synoviocytes and macrophages, leading to the secretion of DAMPs. These findings suggest that histones play a central role in autoimmune arthritis., 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 © 2022 Jung, Lee, Kim, Kim, Jang, Roh, Lee, Robinson, Sohn, Pyun and Song.)

Introduction: The buccal route has been considered an attractive alternative delivery route for injectable formulations. Cell-penetrating peptides (CPPs) are gaining increased attention for their cellular uptake and tissue permeation effects. This study was aimed to evaluate the in vitro and ex vivo permeation-enhancing effect of penetratin-conjugated liposomes for salmon calcitonin (sCT) in TR146 human buccal cells and porcine buccal tissues., Methods: Penetratin was conjugated to phospholipids through a maleimide-thiol reaction. Liposomes were prepared and sCT was encapsulated using a thin-film hydration method. Physical properties such as particle size, zeta potential, encapsulation efficiency, and morphological images via transmission electron microscopy were obtained. Cellular uptake studies were conducted using flow cytometry (FACS) and confocal laser scanning microscopy (CLSM). A cell permeation study was performed using a Transwell ® assay, and permeation through porcine buccal tissue was evaluated. The amount of sCT permeated was quantified using an ELISA kit and was optically observed using CLSM., Results: The particle size of penetratin-conjugated liposomes was approximately 123.0 nm, their zeta potential was +29.6 mV, and their calcitonin encapsulation efficiency was 18.0%. In the cellular uptake study using FACS and CLSM, stronger fluorescence was observed in penetratin-conjugated liposomes compared with the solution containing free sCT and control liposomes. Likewise, the amount of sCT permeated from penetratin-conjugated liposomes was higher than that from the free sCT solution and control liposomes by 5.8-fold across TR146 cells and 91.5-fold across porcine buccal tissues., Conclusion: Penetratin-conjugated liposomes are considered a good drug delivery strategy for sCT via the buccal route., Competing Interests: The authors report no conflicts of interest in this work., (© 2022 Keum et al.)

Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by fibrosis of the skin and internal organs. Despite the recent advances in the pathogenesis and treatment of SSc, effective therapies for fibrosis caused by SSc have not yet been established. In this study, we investigated the potential role of mirodenafil, a potent phosphodiesterase 5 (PDE5) inhibitor, in the treatment of fibrosis in SSc. We used a bleomycin (BLM)-induced SSc mouse model to mimic the typical features of fibrosis in human SSc and examined the dermal thickness to assess the degree of skin fibrosis after staining with hematoxylin and eosin or Masson's trichrome stains. The effect of mirodenafil on the expression of profibrotic genes was also analyzed by treating fibroblasts with transforming growth factor (TGF)-β and mirodenafil. We showed that mirodenafil ameliorated dermal fibrosis and downregulated the protein levels of fibrosis markers including COL1A1 and α-SMA in the BLM-induced SSc mouse model. Further, using mouse embryonic fibroblasts and human lung fibroblasts, we demonstrated that the expression of collagen and profibrotic genes was reduced by treatment with mirodenafil. Finally, we showed that mirodenafil inhibited TGF-β-induced phosphorylation of Smad2/3 in fibroblasts, which suggested that this drug may ameliorate fibrosis by suppressing the TGF-β/Smad signaling pathway. Our findings suggest that mirodenafil possesses a therapeutic potential for treating fibrosis in SSc., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by widespread joint inflammation, which leads to joint damage, disability, and mortality. Among the several types of immune cells, myeloid cells such as macrophages are critical for controlling the pathogenesis of RA. Inositol phosphates are water-soluble signaling molecules, which are synthesized by a series of enzymes including inositol phosphate kinases. Previous studies revealed actions of inositol phosphates and their metabolic enzymes in the modulation of inflammation such as Toll-like receptor-triggered innate immunity. However, the physiological roles of inositol polyphosphate (IP) metabolism in the regulation of RA remain largely uncharacterized. Therefore, our study sought to determine the role of inositol polyphosphate multikinase (IPMK), a key enzyme for IP metabolism and various cellular signaling control mechanisms, in mediating RA. Using myeloid cell-specific IPMK knockout (KO) mice, arthritis was induced via intraperitoneal K/BxN serum injection, after which disease severity was evaluated. Both wild-type and IPMK KO mice developed similar RA phenotypes; however, conditional deletion of IPMK in myeloid cells led to elevated arthritis scores during the resolution phase, suggesting that IPMK deficiency in myeloid cells impairs the resolution of inflammation. Bone marrow-derived IPMK KO macrophages exhibited no apparent defects in immunoglobulin Fc receptor (FcR) activation, osteoclast differentiation, or resolvin signaling. Taken together, our findings suggest that myeloid IPMK is a key determinant of RA resolution., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

We investigated decaying post-seismic deformation observed on the Korean Peninsula associated with the 2011 Mw 9.0 Tohoku-Oki earthquake using Global Navigation Satellite System (GNSS). The GNSS velocity vectors were estimated in five periods from 2005 to 2019. A co-seismic offset of the Korean Peninsula caused by the 2011 earthquake was inversely proportional to epicentral distances. According to the temporal variations of two components (magnitude and direction) of the GNSS velocity vector with the epicentral distance, the difference between the eastern and western regions for the two components becomes smaller over time. For approximately nine years after the 2011 event, the direction for the crustal movement in South Korea showed a recovery pattern returning to the pre-earthquake motion. In addition, the recovery patterns of the crustal movement were observed differently with the regional geologic structure (e.g., the crustal thickness) and each period. Our estimates of the decay in post-seismic deformation of the Korean Peninsula suggest that post-seismic relaxation will be complete within 5-20 years after the 2011 earthquake. The results suggest that the crustal movement on the Korean Peninsula is gradually recovering to its pre-earthquake motion.

We examined whether it is possible to directly detect citrullinated antigens in the serum of rheumatoid arthritis (RA) patients using a monoclonal antibody (mAb) designed to be specific for citrullinated peptides. In order to confirm the potential of the mAb as a direct arthritis-inducing substance through experimental model of RA, a monoclonal antibody (mAb) 12G1 was generated using by immunization of mice with a challenging cyclic citrullinated peptide. Immunohistochemical analysis of RA-affected synovial tissue showed that our mAb 12G1 could indeed detect citrullinated proteins in target tissues. Subsequently, serum levels of citrullinated type II collagen and filaggrin were measured in healthy volunteers, patients with RA, ankylosing spondylitis (AS), and systemic lupus erythematosus (SLE) using a 12G1-based sandwich ELISA. This showed that citrullinated filaggrin showed 78.9% sensitivity and 85.9% specificity for RA diagnosis with a cutoff optical density (OD) value of 1.013, comparable with the results from a second-generation anti-citrullinated protein antibody (ACPA) test. Circulating citrullinated collagen and filaggrin were detected even in sera of RA patients who were negative for both rheumatoid factor (RF) and ACPA. ELISA results also showed that RF and ACPA titers showed significantly positive correlation with both citrullinated collagen and filaggrin OD values in sera of RA patients. 12G1 challenging aggravated the severity of murine arthritis. In summary, mAb 12G1 can directly detect citrullinated proteins in RA target tissue and in sera of RA patients and 12G1 showed direct arthritogenic potential in vivo . This, 12G1 might be useful for diagnosis of RA including seronegative RA and may help to elucidate the pathophysiological role of citrullination in RA., 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 Won, Kim, Jung, Rim, Sohn, Robinson, Moon and Ju.)

We investigated whether the bone-synthetic activities of vertebral bodies or vertebral corners quantified using 18 F-fluoride positron emission tomography (PET) was associated with bone mineral density (BMD) at the corresponding lumbar vertebrae in ankylosing spondylitis (AS) at each vertebra level. We analyzed 48 lumbar vertebrae in 12 AS patients who underwent 18 F-fluoride PET and dual energy X-ray absorptiometry (DXA). The mean standardized uptake values (SUVmean) of the vertebral body and corners from L1 to L4 were measured using the spatially separated region of interest (ROI). The L1-L4 BMDs were calculated based on the DXA ("conventional BMD"). The BMD of the internal vertebral bodies was measured by manually drawing ROIs to represent the trabecular BMD ("alternative BMD"). After adjusting the within-patient correlation, the 18 F-fluoride SUVmean of the vertebral corners but not that of vertebral bodies was significantly related with the conventional BMD of the vertebra. Otherwise, the 18 F-fluoride uptake of both the vertebral and vertebral bodies was significantly related with the alternative BMD. The bone-synthetic activities of the vertebral corners may be more closely related with BMD than those of the vertebral bodies, suggesting that the effects of regional bone metabolism at the vertebral corners and bodies on BMD differ in AS.

We estimate precipitable water vapor (PWV) from data collected by the low-cost Global Navigation Satellite System (GNSS) receiver at a vessel. The dual-frequency GNSS receiver that the vessel ISABU is equipped with that is operated by the Korea Institute of Ocean Science and Technology. The ISABU served in the Pacific Ocean for scientific research during a period from August 30 to September 21, 2018. It also performs radiosonde observations to obtain a vertical profile of troposphere on the vessel's path. The GNSS-derived PWV is compared to radiosonde observations and the Atmospheric Infrared Sounder (AIRS) on NASA's Aqua satellite output. A bias and root-mean-square (RMS) error between shipborne GNSS-PWV and radiosonde-PWV were -1.48 and 5.22 mm, respectively. When compared to the ground GNSS-PWV, shipborne GNSS-PWV has a relatively large RMS error in comparison with radiosonde-PWV. However, the GNSS observations on the vessel are still in good agreement with radiosonde observations. On the other hand, the GNSS-PWV is not well linearly correlated with AIRS-PWV. The RMS error between the two observations was approximately 8.97 mm. In addition, we showed that the vessel on the sea surface has significantly larger carrier phase multipath error compared to the ground-based GNSS observations. This also can result in reducing the accuracy of shipborne GNSS-PWV. However, we suggest that the shipborne GNSS has sufficient potential to derive PWV with the kinematic precise point positioning (PPP) solution on the vessel.

Although the positive correlation between serum uric acid (UA) levels and bone mineral density (BMD) has been reported in the general population, there are little data regarding the effect of serum UA levels on bone loss in patients with rheumatoid arthritis (RA).We investigated whether increased serum UA levels were associated with a reduced risk of osteoporosis in postmenopausal women with RA.In this retrospective cross-sectional study, 447 postmenopausal female patients with RA and 200 age-matched, postmenopausal healthy controls underwent BMD examination by dual energy x-ray absorptiometry and serum UA levels measurement. Osteoporosis was diagnosed when the T-score was <-2.5.The median UA level in postmenopausal RA patients was found to be significantly lower than that in the healthy women (4 vs 4.1 mg/dL, P = .012) and the frequency of osteoporosis incidence in the lumbar spine, hip, and either site in RA patients was 25.5%, 15.9%, and 32.5%, respectively; the values were significantly higher than those of the controls. After adjusting for confounding factors, a significantly lower risk for osteoporosis of the hip in RA patients was observed within the highest quartile (odds ratio [OR] = 0.37, 95% confidence interval [CI] = 0.16-0.72, P = .021) and the second highest quartile (OR = 0.44, 95% CI = 0.2-0.95, P = .038) of serum UA levels as compared with the lowest quartile, but this association was not found to be consistent with respect to the lumbar spine. Serum UA levels also showed an independently positive correlation with femoral neck BMD (β = 0.0104, P = .01) and total hip BMD (β = 0.0102, P = .017), but not with lumbar BMD.Our data suggest that UA may exert a protective effect on bone loss in RA, especially in the hip.

Background: The pathophysiology of osteoarthritis (OA) involves wear and tear, and a state of low-grade inflammation. Tissue repair responses include transforming growth factor beta (TGFβ)-induced myofibroblast production of extracellular matrix. Fibronectins are an essential part of the extracellular matrix, and injection of fibronectin fragments into rabbit joints is a previously established animal model of OA. Fibronectin containing the ED-A domain is currently being used as drug delivery target in the development of anti-inflammatory drugs (e.g. Dekavil)., Methods: In this study, samples of synovial membrane were obtained from patients with knee OA undergoing joint replacement surgery. Immunostaining for ED-A fibronectin and the myofibroblast marker alpha smooth muscle actin (αSMA) was performed on fibroblast-like synovial cells (FLS) and synovial membranes. RAW 264.7 macrophages were incubated with recombinant ED-A fibronectin., Results: The staining of ED-A fibronectin in OA FLS was increased by TGFβ but not by TNFα, lipopolysaccharide, or IL-6 ( n  = 3). ED-A fibronectin co-stained with the myofibroblast marker αSMA in both the OA FLS ( n  = 3) and in the OA synovial membranes ( n  = 8). ED-A fibronectin staining was associated with both number of lining layer cells (rho = 0.85 and p  = 0.011) and sublining cells (rho = 0.88 and p  = 0.007) in the OA synovium ( n  = 8), and co-distributed with TNFα ( n  = 5). Recombinant ED-A fibronectin increased the production of TNFα by RAW 264.7 macrophages ( n  = 3)., Conclusions: The disease process in OA shares features with the chronic wound healing response. Our findings support utilizing ED-A fibronectin for drug delivery or therapeutic targeting to reduce pro-inflammatory responses in OA., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2019.)

Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney cancer. Novel biomarkers of ccRCC may provide crucial information on tumor features and prognosis. The present study aimed to determine whether the expression of γ-aminobutyric acid (GABA) A receptor subunit θ ( GABRQ ) could serve as a novel prognostic marker of ccRCC. GABA is the main inhibitory neurotransmitter in the brain that activates the receptor GABA A , which is comprised of three subunit isoforms: GABRA3, GABRB3 and GABRQ. A recent study reported that GABRQ is involved in the initiation and progression of hepatocellular carcinoma; however, the role of GABRQ in ccRCC remains unknown. In the present study, clinical and transcriptomic data were obtained from cohorts of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). Differential GABRQ expression levels among early (TI and II), late (TIII and IV), nonmetastatic (M0) and metastatic (M1, primary tumor) stages of ccRCC samples were then identified. Furthermore, the use of GABRQ as a prognostic gene was analyzed using Uno's C-index based on the time-dependent area under the curve (AUC), the AUC of the receiver operating characteristic curve at 5 years, the Kaplan-Meier survival curve and multivariate analysis. The survival curve analysis revealed that low GABRQ mRNA expression was significantly associated with a poor prognosis of ccRCC (P<0.001 and P=0.0012 for TCGA and ICGC data, respectively). In addition, analyses of the C-index and AUC values further supported this discriminatory power. Furthermore, the prognostic value of GABRQ mRNA expression was confirmed by multivariate Cox regression analysis. Taken together, these results suggested that GABRQ mRNA expression may be considered as a novel prognostic biomarker of ccRCC., (Copyright: © Lee et al.)

Three-dimensional (3D) printing has been recently employed in the design and formulation of various dosage forms with the aim of on-demand manufacturing and personalized medicine. In this study, we formulated a floating sustained release system using fused deposition modeling (FDM). Filaments were prepared using hypromellose acetate succinate (HPMCAS), polyethylene glycol (PEG 400) and pregabalin as the active ingredient. Cylindrical tablets with infill percentages of 25%, 50% and 75% were designed and printed with the FDM printer. An optimized formulation (F6) was designed with a closed bottom layer and a partially opened top layer. Filaments and tablets were characterized by means of fourier-transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), X-ray powder diffraction (XRPD), and thermogravimetric analysis (TGA). The results show that the processing condition did not have a significant effect on the stability of the drug and the crystallinity of the drug remained even after printing. A dissolution study revealed that drug release is faster in an open system with low infill percentage compared to closed systems and open systems with a high infill ratio. The optimized formulation (F6) with partially opened top layer showed zero-order drug release. The results show that FDM printing is suitable for the formulation of floating dosage form with the desired drug release profile., Competing Interests: The authors declare no conflict of interest.

Osteoarthritis (OA) is the leading cause of joint failure, yet the underlying mechanisms remain elusive, and no approved therapies that slow progression exist. Dysregulated integrin function was previously implicated in OA pathogenesis. However, the roles of integrin αVβ3 and the integrin-associated receptor CD47 in OA remain largely unknown. Here, transcriptomic and proteomic analyses of human and murine osteoarthritic tissues revealed dysregulated expression of αVβ3, CD47, and their ligands. Using genetically deficient mice and pharmacologic inhibitors, we showed that αVβ3, CD47, and the downstream signaling molecules Fyn and FAK are crucial to OA pathogenesis. MicroPET/CT imaging of a mouse model showed elevated ligand-binding capacities of integrin αVβ3 and CD47 in osteoarthritic joints. Further, our in vitro studies demonstrated that chondrocyte breakdown products, derived from articular cartilage of individuals with OA, induced αVβ3/CD47-dependent expression of inflammatory and degradative mediators, and revealed the downstream signaling network. Our findings identify a central role for dysregulated αVβ3 and CD47 signaling in OA pathogenesis and suggest that activation of αVβ3 and CD47 signaling in many articular cell types contributes to inflammation and joint destruction in OA. Thus, the data presented here provide a rationale for targeting αVβ3, CD47, and their signaling pathways as a disease-modifying therapy.

Sirtuin 1 (SIRT1) is a histone deacetylase implicated in stem cell homeostasis. Conditional Sirt1 deletion in the hematopoietic stem and progenitor system promotes hematopoietic stem and progenitor cell (HSPC) expansion under stress conditions. In addition, SIRT1 activators modulate the capacity and HSPC numbers in the bone marrow (BM). To investigate the role of SIRT1 in the BM niche, a conditional Sirt1 deletion in the BM niche was generated in a mouse model for the present study. Multicolor flow cytometric analyses were performed to determine HSC cell populations. Using 5-fluorouracil-induced proliferative stress, a survival curve was produced. In the present study, Sirt1 deletion in the BM niche demonstrated that the production of mature blood cells, lineage distribution within hematopoietic organs and frequencies of HSPC populations were comparable to those of controls. Additionally, Sirt1 deletion in the BM niche did not perturb HSC maturation under stress induced by transplantation. Therefore, these observations suggest that SIRT1 serves a dispensable role in HSC maturation in the BM niche.

3D printing is a method of rapid prototyping and manufacturing in which materials are deposited onto one another in layers to produce a three-dimensional object. Although 3D printing was developed in the 1980s and the technology has found widespread industrial applications for production from automotive parts to machine tools, its application in pharmaceutical area is still limited. However, the potential of 3D printing in the pharmaceutical industry is now being recognized. The ability of 3D printing to produce medications to exact specifications tailored to the needs of individual patients has indicated the possibility of developing personalized medicines. The technology allows dosage forms to be precisely printed in various shapes, sizes and textures that are difficult to produce using traditional techniques. However, there are various challenges associated with the proper application of 3D printing in the pharmaceutical sector which should be overcome to exploit the scope of this technology. In this review, an overview is provided on the various 3D printing technologies used in fabrication of complex dosage forms along with their feasibility and limitations., (© 2019 Shenyang Pharmaceutical University. Published by Elsevier B.V.)

IL-32 exists as seven mRNA transcripts that can translate into distinct individual IL-32 variants with specific protein domains. These translated protein domains of IL-32 variants code for specific functions that allow for interaction with different molecules intracellularly or extracellularly. The longest variant is IL-32γ possessing 234 amino acid residues with all 11 protein domains, while the shortest variant is IL-32α possessing 131 amino acid residues with three of the protein domains. The first domain exists in 6 variants except IL-32δ variant, which has a distinct translation initiation codon due to mRNA splicing. The last eleventh domain is common domain for all seven IL-32 variants. Numerous studies in different fields, such as inflammation, autoimmunity, pathogen infection, and cancer biology, have claimed the specific biological activity of individual IL-32 variant despite the absence of sufficient data. There are 4 additional IL-32 variants without proper transcripts. In this review, the structural characteristics of seven IL-32 transcripts are described based on the specific protein domains., Competing Interests: Conflict of Interest: The authors declare no potential conflicts of interest.

Although pathological observations provide approximate prognoses, it is difficult to achieve prognosis in patients with existing prognostic factors. Therefore, it is very important to find appropriate biomarkers to achieve accurate cancer prognosis. Renal cell carcinoma (RCC) has several subtypes, the discrimination of which is crucial for proper treatment. Here, we present a novel biomarker, VNN3, which is used to prognose clear cell renal cell carcinoma (ccRCC), the most common and aggressive subtype of kidney cancer. Patient information analyzed in our study was extracted from The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) cohorts. VNN3 expression was considerably higher in stages III and IV than in stages I and II. Moreover, Kaplan-Meier curves associated high VNN3 expression with poor prognoses (TCGA, p  < .0001; ICGC, p  = .00076), confirming that ccRCC prognosis can be predicted via VNN3 expression patterns. Consistent with all patient results, the prognosis of patients with higher VNN3 expression was worse in both low stage (I and II) and high stage (III and IV) (TCGA, p < 0.0001 in stage I and II; ICGC, p = 0.028 in stage I and II; TCGA, p = 0.005 in stage III and IV). Area under the curve and receiver operating characteristic curves supported our results that highlighted VNN3 expression as a suitable ccRCC biomarker. Multivariate analysis also verified the prognostic performance of VNN3 expression (TCGA, p  < .001; ICGC, p  = .017). Altogether, we suggest that VNN3 is applicable as a new biomarker to establish prognosis in patients with ccRCC.

IL-17 produced by Th17 cells has been implicated in the pathogenesis of rheumatoid arthritis (RA). It is important to prevent the differentiation of Th17 cells in RA. Homodimeric soluble γc (sγc) impairs IL-2 signaling and enhances Th17 differentiation. Thus, we aimed to block the functions of sγc by inhibiting the formation of homodimeric sγc. The homodimeric form of sγc was strikingly disturbed by sγc-binding DNA aptamer. Moreover, the aptamer effectively inhibited Th17 cell differentiation and restored IL-2 and IL-15 signaling impaired by sγc with evidences of increased survival of T cells. sγc was highly expressed in SF of RA patients and increased in established CIA mice. The therapeutic effect of PEG-aptamer was tested in CIA model and its treatment alleviated arthritis pathogenesis with impaired differentiation of pathogenic Th17, NKT1, and NKT17 cells in inflamed joint. Homodimeric sγc has pathogenic roles to exacerbate RA progression with differentiation of local Th17, NKT1, and NKT17 cells. Therefore, sγc is suggested as target of a therapeutic strategy for RA.

Protein kinase casein kinase 2 (CK2) is important in the regulation of cell proliferation and death, even under pathological conditions. Previously, we reported that CK2 regulates the expression of heme oxygenase‑1 (HO‑1) in stress‑induced chondrocytes. In the present study, it was shown that CK2 is involved in the dedifferentiation and cellular senescence of chondrocytes. Treatment of primary articular chondrocytes with CK2 inhibitors, 4,5,6,7‑terabromo‑2‑azabenzimidazole (TBB) or 5,6‑dichlorobenzimidazole 1‑β‑D‑ribofuranoside (DRB), induced an increase in senescence‑associated β‑galactosidase (SA‑β‑gal) staining. In addition, TBB reduced the expression of type II collagen and stimulated the accumulation of β‑catenin, phenotypic markers of chondrocyte differentiation and dedifferentiation, respectively. It was also observed that the abrogation of CK2 activity by CK2 small interfering RNA induced phenotypes of chondrocyte senescence. The association between HO‑1 and cellular senescence was also examined in CK2 inhibitor‑treated chondrocytes. Pretreatment with 3‑morpholinosydnonimine hydrochloride, an inducer of the HO‑1 expression, or overexpression of the HO‑1 gene significantly delayed chondrocyte senescence. These results show that CK2 is associated with chondrocyte differentiation and cellular senescence and that this is due to regulation of the expression of HO‑1. Furthermore, the findings suggest that CK2 is crucial as an anti‑aging factor during chondrocyte senescence.

Inflammatory bowel disease (IBD) is chronic inflammation of the gastrointestinal tract caused by high levels of pro-inflammatory cytokines and epithelial barrier dysfunction. Alnus japonica Steud. (Betulaceae) has been used in traditional Asian medicine. However, the potential of A. japonica for the treatment of intestinal inflammation has not been investigated. This study investigated the effects of ethanol extract from A. japonica bark (AJE) on colonic mucosa injury in mice with dextran sodium sulfate (DSS)-induced colitis. Treatment with AJE ameliorated pathological damage and the histopathologic features of DSS-induced colitis. The administration of AJE also inhibits DSS-induced pro-inflammatory cytokines expression, including interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and cyclooxygenase (COX)-2. Notably, AJE administration attenuated the reduction of tight junction proteins, zonula occludens (ZO)-1 and occludin, in DSS-induced colitis. In addition, AJE increased heme oxygenase (HO)-1 expression and prevented DSS-induced apoptosis in colonic epithelial cells. Furthermore, in vitro studies demonstrated that AJE inhibits TNF-α-induced IL-8, IL-1β, and COX-2 expression in human intestinal epithelial HT-29 cells and tert-butyl hydroperoxide-induced reduction of ZO-1 and occludin expression in human intestinal epithelial Caco-2 cells. AJE-induced HO-1 protein expression was also found in both HT-29 and Caco-2 cells. Taken together, our findings demonstrated that AJE inhibits intestinal inflammation and protects against intestinal barrier disruption in mice with DSS-induced colitis in vivo and human intestinal epithelial cells in vitro. These results suggest that AJE might have beneficial effects for the treatment of IBD., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

In oriental medicine, centipede Scolopendra subspinipes mutilans has long been used as a remedy for rheumatoid arthritis (RA), a well-known chronic autoimmune disorder. However, the molecular identities of its bioactive components have not yet been extensively investigated. We sought to identify bioactive molecules that control RA with a centipede. A novel antimicrobial peptide (AMP) (scolopendrasin IX) was identified from Scolopendra subspinipes mutilans. Scolopendrasin IX markedly activated mouse neutrophils, by enhancing cytosolic calcium increase, chemotactic cellular migration, and generation of superoxide anion in neutrophils. As a target receptor for scolopendrasin IX, formyl peptide receptor (FPR)2 mediates neutrophil activation induced by the AMP. Furthermore, scolopendrasin IX administration strongly blocked the clinical phenotype of RA in an autoantibody-injected model. Mechanistically, the novel AMP inhibited inflammatory cytokine synthesis from the joints and neutrophil recruitment into the joint area. Collectively, we suggest that scolopendrasin IX is a novel potential therapeutic agent for the control of RA via FPR2.

Transdermal drug administration presents several advantages and it is therefore favorable as an alternative drug delivery route. However, transdermal delivery of biopharmaceutical drugs is made difficult by the skin barrier. Microneedle application and iontophoresis are strategies which can be used to overcome this barrier. Therefore, recombinant human growth hormone (rhGH) was used as a model macromolecular drug and was transdermally delivered using microneedle application and iontophoresis. Methylene blue staining, stereomicroscopy and scanning electron microscope (SEM) imaging were used to characterize the microchannels produced. To optimize the iontophoresis protocol, the effects of molecular charge and current density on transdermal delivery were evaluated in an in vitro permeation study using excised rat skin tissues. Using the optimized iontophoresis protocol, the combination effects of iontophoretic delivery via microchannels were evaluated in three different experimental designs. The flux obtained with anodal iontophoresis in citrate buffer was approximately 10-fold higher that that with cathodal iontophoresis in phosphate buffered saline (PBS). Flux also increased with current density in anodal iontophoresis. The combination of iontophoresis and microneedle application produced higher flux than single application. These results suggest that anodal iontophoresis with higher current density enhances the permeation of macromolecules through microchannels created by microneedles. In conclusion, the combination of iontophoresis and microneedles is a potential strategy for the enhancement of transdermal delivery of macromolecular drugs.

Damage-associated molecular patterns (DAMPs) are endogenous danger molecules that are released from damaged or dying cells and activate the innate immune system by interacting with pattern recognition receptors (PRRs). Although DAMPs contribute to the host's defense, they promote pathological inflammatory responses. Recent studies have suggested that various DAMPs, such as high-mobility group box 1 (HMGB1), S100 proteins, and heat shock proteins (HSPs), are increased and considered to have a pathogenic role in inflammatory diseases. Here, we review current research on the role of DAMPs in inflammatory diseases, including rheumatoid arthritis, systemic lupus erythematosus, osteoarthritis, atherosclerosis, Alzheimer's disease, Parkinson's disease, and cancer. We also discuss the possibility of DAMPs as biomarkers and therapeutic targets for these diseases., Competing Interests: Conflict of Interest: The authors declare no potential conflicts of interest.

Virus-like particles (VLPs) derived from human papillomavirus (HPV) L1 capsid proteins were used for HPV quadrivalent recombinant vaccine. The HPV quadrivalent vaccine is administrated in a 3-dose regimen of initial injection followed by subsequent doses at 2 and 6 months to prevent cervical cancer, vulvar, and vaginal cancers. The type 6, 11, 16, or 18 of HPV infection is associated with precancerous lesions and genital warts in adolescents and young women. The HPV vaccine is composed of viral L1 capsid proteins are produced in eukaryotic expression systems and purified in the form of VLPs. Four different the L1 protein of 3 different subtypes of HPV: HPV11, HPV16, and HPV18 were expressed in Escherichia coli divided into 2 fragments as N- and C-terminal of each protein in order to examine the efficacy of HPV vaccine. Vaccinated sera failed to recognize N-terminal L1 HPV type 16 and type 18 by western blot while they detected N-terminal L1 protein of HPV type 11. Moreover, the recombinant C-terminal L1 proteins of type 16 was non-specifically recognized by the secondary antibody conjugated with horseradish peroxidase. This expression and purification system may provide simple method to obtain robust recombinant L1 protein of HPV subtypes to improve biochemical analysis of antigens with immunized sera., Competing Interests: Conflict of Interest: The authors declare no potential conflicts of interest.

Most previous research investigating osteoporosis in rheumatoid arthritis (RA) has focused on female patients and there is a lack of data regarding clinical characteristics of osteoporosis in male patients with RA.The aim of this study was to compare the frequency of osteoporosis between male patients with RA and healthy patients, and to identify the risk factors for osteoporosis and low bone mineral density (BMD) in male patients with RA.We conducted a retrospective, cross-sectional study including 76 South Korean male patients with RA aged over 50 years and 76 age-matched male healthy individuals. BMD was measured at the lumbar spine (L1-4) and left hip (femoral neck and total hip) using dual energy X-ray absorptiometry. Osteoporosis was defined as a T-score of ≤ -2.5 according to the World Health Organization (WHO) classification.The frequency of osteoporosis at either the spine or the hip among male patients with RA was significantly higher than that among controls (22.4% vs 10.5%, P = .049) and RA patients had a significantly lower total hip BMD than healthy individuals (0.92 ± 0.14 vs 0.96 ± 0.1 g/cm, P = .027). For male RA patients, the mean 28-joint Disease Activity Scores using erythrocyte sedimentation rate (DAS28-ESR) and body mass index (BMI) were 3.28 and 22 kg/m, respectively. In multivariable logistic regression models, BMI ≤ 22 kg/m (odds ratio = 3.43, P = .043) and DAS28-ESR > 3.2 (odds ratio = 3.85, P = .032) were independent risk factors for osteoporosis at either site in male patients with RA.Our data demonstrate that male patients with RA had a 2.1 times higher risk for osteoporosis compared with healthy individuals. This suggests that appropriate management of osteoporosis in patients with RA is crucial not only for postmenopausal women but also for men aged over 50 years, especially those with low BMI and higher disease activity.

To define whether individual human leukocyte antigen (HLA) class I allotypes are used preferentially in human cytomegalovirus (CMV)-specific cytotoxic T lymphocyte responses, CD8 + T cell responses restricted by up to six HLA class I allotypes in an individual were measured in parallel using K562-based artificial antigen-presenting cells expressing both CMV pp65 antigen and one of 32 HLA class I allotypes (7 HLA-A, 14 HLA-B, and 11 HLA-C) present in 50 healthy Korean donors. The CD8 + T cell responses to pp65 in the HLA-C allotypes were lower than responses to those in HLA-A and -B allotypes and there was no difference between the HLA-A and HLA-B loci. HLA-A*02:01, -B*07:02, and -C*08:01 showed the highest magnitude and frequency of immune responses to pp65 at each HLA class I locus. However, HLA-A*02:07, -B*59:01, -B*58:01, -B*15:11, -C*03:02, and -C*02:02 did not show any immune responses. Although each individual has up to six different HLA allotypes, 46% of the donors showed one allotype, 24% showed two allotypes, and 2% showed three allotypes that responded to pp65. Interestingly, the frequencies of HLA-A alleles were significantly correlated with the positivity of specific allotypes. Our results demonstrate that specific HLA class I allotypes are preferentially used in the CD8 + T cell immune response to pp65 and that a hierarchy among HLA class I allotypes is present in an individual.

Previously, we found that most patients with acute myeloid leukemia (AML) expressed at least one of the leukemic associated antigens (LAAs) WT1, survivin and TERT, and different combinations of the three LAAs predicted negative clinical outcomes. Multi-tumor antigen-specific T cells were generated to overcome antigenic variation and may be sufficient to maximize antitumoral effects. To generate triple antigen-specific (Tri)-T cells that recognize three LAAs, dendritic cells (DCs) were transfected with three tumor antigen-encoding RNAs. These DCs were used to stimulate both CD8 and CD4 T cells and to overcome the limitation of known human leukocyte antigen-restricted epitopes. The sum of the antigen-specific T cell frequencies was higher in the Tri-T cells than in the T cells that recognized a single antigen. Furthermore, the Tri-T cells were more effective against leukemic blasts that expressed all three LAAs compared with blasts that expressed one or two LAAs, suggesting a proportional correlation between IFN-γ secretion and LAA expression. Engrafted leukemic blasts in the bone marrow of mice significantly decreased in the presence of Tri-T cells. This technique represents an effective immunotherapeutic strategy in AML.

We developed and validated a pseudo-range correction (PRC) modeling system that can prevent degradation of positioning accuracy even in situations where one cannot obtain PRC messages for Differential Global Navigation Satellite System (DGNSS). A PRC modeling scheme was devised based on the repeat time of GNSS satellites and previously-collected PRC data. The difference between the modeled and real PRC values observed at the reference station showed a bias error of about ±1.0 m and a root mean square error (RMSE) less than 1.5 m. When we applied the predicted PRC to Differential Global Positioning System (DGPS) and Differential BeiDou (DBDS) positioning, horizontal RMSE values were at a level of 1.0 m, while vertical RMSE was in the range of 1.8-3.0 m. We found that modelled PRCs can provide positioning results similar to those based on real PRCs and can provide significant improvement over standalone positioning without PRCs.

Objectives: The aim was to investigate the effects of nicotine on neutrophil extracellular traps (NETs) formation in current and non-smokers and on a murine model of RA., Methods: We compared spontaneous and phorbol 12-myristate 13-acetate-induced NETosis between current and non-smokers by DNA release binding. Nicotine-induced NETosis from non-smokers was assessed by DNA release binding, NET-specific (myeloperoxidase (MPO)-DNA complex) ELISA and real-time fluorescence microscopy. We also used immunofluorescent staining to detect nicotinic acetylcholine receptors (nAChRs) on neutrophils and performed a functional analysis to assess the role of nAChRs in nicotine-induced NETosis. Finally, we investigated the effects of systemic nicotine exposure on arthritis severity and NETosis in the CIA mouse model., Results: Neutrophils derived from current smokers displayed elevated levels of spontaneous and phorbol 12-myristate 13-acetate-induced NETosis. Nicotine induced dose-dependent NETosis in ex vivo neutrophils from healthy non-smokers, and co-incubation with ACPA-immune complexes or TNF-α facilitated a synergistic effect on NETosis. Real-time fluorescence microscopy revealed robust formation of NET-like structures in nicotine-exposed neutrophils. Immunofluorescent staining demonstrated the presence of the α7 subunit of the nAChR on neutrophils. Stimulation of neutrophils with an α7-specific nAChR agonist induced NETosis, whereas pretreatment with an nAChR antagonist attenuated nicotine-induced NETosis. Nicotine administration to mice with CIA exacerbated inflammatory arthritis, with higher plasma levels of NET-associated MPO-DNA complex., Conclusion: We demonstrate that nicotine is a potent inducer of NETosis, which may play an important role in accelerating arthritis in the CIA model. This study generates awareness of and the mechanisms by which nicotine-containing products, including e-cigarettes, may have deleterious effects on patients with RA., (Published by Oxford University Press 2016. This work is written by US Government employees and is in the public domain in the United States.)

Introduction/purpose: Quadriceps strength is a useful clinical predictor of self-reported function after anterior cruciate ligament reconstruction (ACLR). However, it remains unknown if quadriceps strength normalized to body mass (QBM) or quadriceps strength limb symmetry index (QLSI) is the best predictor of self-reported function in individuals with ACLR. We sought to determine whether QBM and QLSI are able to predict individuals with ACLR who self-report high function (≥90% on the international knee documentation committee (IKDC) index)., Methods: Ninety-six individuals with a history of a primary unilateral ACLR were recruited for a multisite cross-sectional descriptive laboratory experiment. Bilateral isometric quadriceps strength was collected at 90° of knee flexion to calculate QBM and QLSI (ratio of the ACLR limb to the contralateral limb). Area under the curve (AUC) values were calculated using receiver operating characteristic curve analyses to determine the capacity of QBM and QLSI to predict individuals with high self-reported function on the IKDC index., Results: QBM displayed high accuracy (AUC = 0.76; 95% confidence interval, 0.66-0.86) for identifying participants with an IKDC index ≥90%. A QBM cutoff score of 3.10 N·m·kg was found to maximize sensitivity (0.61) and specificity (0.84), and displayed 8.15 (3.09-21.55) times higher odds of reporting high function. QLSI displayed a moderate accuracy (AUC = 0.62, 0.50-0.73) for identifying participants with an IKDC index ≥90%. A QLSI cutoff score of 96.5% maximized sensitivity (0.55) and specificity (0.70), and represented 2.78 (1.16-6.64) times higher odds reporting high function., Conclusion: QBM is a stronger predictor of high self-reported function compared with QLSI in individuals with ACLR. Rehabilitation guidelines may benefit from incorporating the use of QBM measurements for the purpose of predicting participants that may maintain high self-reported function.

Human γδ T cells play important roles in the regulation of infection and cancer. To understand the roles of costimulatory signals in activation and expansion ex vivo, Vγ9Vδ2 T cells were grown with artificial APCs that express CD83, 4-1BB ligand, and/or CD32, which allowed a loading of αCD3 and αCD28 antibodies. The costimulatory signals through CD80, 4-1BB, and CD83 ligand in low levels of IL-2 triggered an explosive ex vivo proliferation of Vγ9Vδ2 T cells capable of secreting high levels of IL-2, IFN-γ, and TNF-α. Moreover, the triple-costimulatory signals cause augmented cell viabilities for long-term growth of Vγ9Vδ2 T cells, resulting in phenotypic changes to CD27(-)CD45RA(+) effector memory-like cells. Notably, we observed that CD83 ligand signaling is crucial to promote ex vivo expansion, survival, and cytolytic effector functions of Vγ9Vδ2 T cells. In contrast, 4-1BB signaling is moderately important in up-regulating surface molecules on Vγ9Vδ2 T cells. Consequently, γδ T cells stimulated in the presence of triple-costimulatory signals have diverse cytolytic effector molecules, including perforin, granzyme A, granzyme B, and Fas ligand, eliciting potent cytolytic activities against tumor cells. Overall, our results provide insights into the roles of costimulatory signals in manufacturing long-lived and fully functional Vγ9Vδ2 T cells that could be useful against cancers., (© Society for Leukocyte Biology.)

Purpose: After anterior cruciate ligament (ACL) injury and reconstruction, abnormal biomechanics during daily tasks may have prominent and detrimental long-term consequences on knee joint health. The purpose of this study was to longitudinally evaluate hip and knee joint biomechanics during stair ascent and descent in patients with acute ACL injury and at return to activity after ACL reconstruction., Methods: Twenty individuals with unilateral ACL injury (age, 20.9 ± 4.4 yr; height, 172.4 ± 7.5 cm; mass, 76.2 ± 12.2 kg) that were scheduled to undergo surgical reconstruction were compared with 20 healthy matched controls (age, 21.7 ± 3.7 yr; height, 173.7 ± 9.9 cm; mass, 76.1 ± 19.7 kg). Lower extremity biomechanics were recorded using three-dimensional motion analysis during stair ascent and descent at two testing sessions (before surgery and at approximately 6 months after surgery or when they were allowed to return to unrestricted physical activity). Time between sessions for healthy participants was matched on the basis of the ACL group. Peak sagittal and frontal plane knee and hip joint angles and moments, joint angles at initial contact, and joint excursions across stance phase were evaluated., Results: The ACL-injured limb of patients experienced smaller knee extension moments than the uninjured limb and healthy controls during stair ascent and descent (P < 0.05) before and 6 months after ACL reconstruction. During stair ascent, ACL patients experienced more extended knee joint positions and less sagittal plane knee joint excursions, coupled with greater frontal plane hip joint excursions (P < 0.05)., Conclusions: Patients with ACL injury experience reductions in knee flexion angle and knee extension moments during stair walking. These alterations were observed both before and after reconstruction, suggesting that early gait retraining interventions may be beneficial in these patients.

Objective: Anti-citrullinated protein antibodies (ACPAs) are characteristic of rheumatoid arthritis (RA). However, their presence years before the onset of clinical RA is perplexing. Although multiple putative citrullinated antigens have been identified, no studies have demonstrated the specific capacity of these antigens to initiate inflammatory arthritis. This study was undertaken to recapitulate the transition from preclinical to clinical RA and to demonstrate the capacity of local citrullination to facilitate this transition., Methods: We performed proteomic analysis of activated human neutrophils to identify citrullinated proteins, including those targeted as part of the RA immune response. Using enzyme-linked immunosorbent assay, we compared RA and osteoarthritis synovial fluid for levels of citrullinated histone H2B and its immune complex. Using macrophage activation assays, we assessed the effect of histone citrullination on immunostimulatory capacity and evaluated the stimulatory capacity of native and citrullinated H2B immune complexes. Finally, we assessed the potential for anti-citrullinated H2B antibodies to mediate arthritis in vivo., Results: We identified robust targeting of neutrophil-derived citrullinated histones by the ACPA immune response. More than 90% of the RA patients had anti-citrullinated H2B antibodies. Histone citrullination increased innate immunostimulatory capacity, and immune complexes containing citrullinated histones activated macrophage cytokine production and propagated neutrophil activation. Finally, we demonstrated that immunization with H2B was arthritogenic, but only in the setting of underlying articular inflammation., Conclusion: Our findings indicate that citrullinated histones, specifically citrullinated H2B, are an antigenic target of the ACPA immune response. Furthermore, local generation of citrullinated antigen during low-grade articular inflammation provides a mechanistic model for the conversion from preclinical autoimmunity to inflammatory arthritis., (Published 2015. This article is a U.S. Government work and is in the public domain in the USA.)

Heme oxygenase-1 (HO-1) is upregulated in colorectal carcinoma (CRC) cells. However, the role of HO-1 in the metastatic potential of CRC remains to be elucidated. In this study, we investigated the potential of HO-1 to control the antitumor immunity of CRC. Intercellular adhesion molecule-1 (ICAM-1) plays an important role in the immune surveillance system. Hemin-induced HO-1 expression suppressed the expression of ICAM-1 in human CRC cells. HO-1 regulated ICAM-1 expression via tristetraprolin, an mRNA-binding protein, at the posttranscriptional level in CRC cells. The upregulated HO-1 expression in CRC cells markedly decreased the adhesion of peripheral blood mononuclear lymphocytes (PBMLs) to CRC cells and PBML-mediated cytotoxicity against CRC cells. Production of CXCL10, an effector T cell-recruiting chemokine, was significantly reduced by the increased HO-1 expression. The expression of the CXCL10 receptor, CXCR3, decreased significantly in PBMLs that adhered to CRC cells. HO-1 expression correlated negatively, although nonsignificantly, with ICAM-1 and CXCL10 expression in xenograft tumors. Taken together, our data suggest that HO-1 expression is functionally linked to the mediation of tumor progression and metastasis of CRC cells by inhibiting antitumor immunity.

Context: Neuromuscular dysfunction is common after anterior cruciate ligament reconstruction (ACL-R). However, little is known about quadriceps spinal-reflex and descending corticomotor excitability after ACL-R. Understanding the effects of ACL-R on spinal-reflex and corticomotor excitability will help elucidate the origins of neuromuscular dysfunction., Objective: To determine whether spinal-reflex excitability and corticomotor excitability differed between the injured and uninjured limbs of patients with unilateral ACL-R and between these limbs and the matched limbs of healthy participants., Design: Case-control study., Setting: Laboratory., Patients or Other Participants: A total of 28 patients with unilateral ACL-R (9 men, 19 women; age = 21.28 ± 3.79 years, height = 170.95 ± 10.04 cm, mass = 73.18 ± 18.02 kg, time after surgery = 48.10 ± 36.17 months) and 29 participants serving as healthy controls (9 men, 20 women; age = 21.55 ± 2.70 years, height = 170.59 ± 8.93 cm, mass = 71.89 ± 12.70 kg) volunteered., Main Outcome Measure(s): Active motor thresholds (AMTs) were collected from the vastus medialis (VM) using transcranial magnetic stimulation. We evaluated VM spinal reflexes using the Hoffmann reflex normalized to maximal muscle responses (H : M ratio). Voluntary quadriceps activation was measured with the superimposed-burst technique and calculated using the central activation ratio (CAR). We also evaluated whether ACL-R patients with high or low voluntary activation had different outcomes., Results: The AMT was higher in the injured than in the uninjured limb in the ACL-R group (t27 = 3.32, P = .003) and in the matched limb of the control group (t55 = 2.05, P = .04). The H : M ratio was bilaterally higher in the ACL-R than the control group (F1,55 = 5.17, P = .03). The quadriceps CAR was bilaterally lower in the ACL-R compared with the control group (F1,55 = 10.5, P = .002). The ACL-R group with low voluntary activation (CAR < 0.95) had higher AMT than the control group (P = .02), whereas the ACL-R group with high voluntary activation (CAR ≥ 0.95) demonstrated higher H : M ratios than the control group (P = .05)., Conclusions: The higher VM AMT in the injured limbs of ACL-R patients suggested that corticomotor deficits were present after surgery. Higher bilateral H : M ratios in ACL-R patients may be a strategy to reflexively increase excitability to maintain voluntary activation.

An EBV-specific cellular immune response is associated with the control of EBV-associated malignancies and lymphoproliferative diseases, some of which have been successfully treated by adoptive T cell therapy. Therefore, many methods have been used to measure EBV-specific cellular immune responses. Previous studies have mainly used autologous EBV-transformed B-lymphoblastoid cell lines (B-LCLs), recombinant viral vectors transfected or peptide pulsed dendritic cells (DCs) as stimulators of CD8(+) and CD4(+) T lymphocytes. In the present study, we used an interferon-γ (IFN-γ) enzyme-linked immunospot (ELISPOT) assay by using isolated CD8(+) and CD4(+) T cells stimulated with mRNA-transfected DCs. The frequency of latent membrane protein 1 (LMP1)-specific IFN-γ producing CD4(+) T cells was significantly higher than that of LMP2a. The frequency of IFN-γ producing CD4(+) T cells was significantly correlated with that of CD8(+) T cells in LMP1-specific immune responses (r = 0.7187, Pc < 0.0001). To determine whether there were changes in LMP1- or LMP2a-specific immune responses, subsequent peripheral blood mononuclear cells (PBMCs) samples were analyzed. Significant changes were observed in 5 of the 10 donors examined, and CD4(+) T cell responses showed more significant changes than CD8(+) T cell responses. CD8(+) and CD4(+) T cells from EBV-seropositive donors secreted only the Th1 cytokines IFN-γ, TNF-α, and IL-2, while Th2 (IL-4) and Th17 (IL-17a) cytokines were not detected. CD4(+) T cells secreted significantly higher cytokine levels than did CD8(+) T cells. Analysis of EBV-specific T cell responses using autologous DCs transfected with mRNA might provide a comprehensive tool for monitoring EBV infection and new insights into the pathogenesis of EBV-associated diseases.

Leptin, a hormone mainly produced from adipose tissue, has been shown to induce proliferation of cancer cells. However, the molecular mechanisms underlying leptin-induced tumor progression have not been clearly elucidated. In the present study, we investigated the role of autophagy in leptin-induced cancer cell proliferation using human hepatoma (HepG2) and breast cancer cells (MCF-7), and tumor growth in a xenograft model. Herein, we showed that leptin treatment caused autophagy induction as assessed by increase in expression of autophagy-related genes, including beclin-1, Atg5 and LC3 II, further induction of autophagosome formation and autophagic flux. Interestingly, inhibition of autophagic process by treatment with inhibitors and LC3B gene silencing blocked leptin-induced increase in cell number and suppression of apoptosis, indicating a crucial role of autophagy in leptin-induced tumor progression. Moreover, gene silencing of p53 or FoxO3A prevented leptin-induced LC3 II protein expression, suggesting an involvement of p53/FoxO3A axis in leptin-induced autophagy activation. Leptin administration also accelerated tumor growth in BALB/c nude mice, which was found to be autophagy dependent. Taken together, our results demonstrate that leptin-induced tumor growth is mediated by autophagy induction and autophagic process would be a promising target to regulate development of cancer caused by leptin production.