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Tumor acidosis, a common phenomenon in solid cancers such as breast cancer, is caused by the abnormal metabolism of cancer cells. The low pH affects cells surrounding the cancer, and tumor acidosis has been shown to inhibit the activity of immune cells. Despite many previous studies, the immune surveillance mechanisms are not fully understood. We found that the expression of PD-L1 was significantly increased under conditions of extracellular acidosis in MDA-MB-231 cells. We also confirmed that the increased expression of PD-L1 mediated by extracellular acidosis was decreased when the pH was raised to the normal range. Gene set enrichment analysis (GSEA) of public breast cancer patient databases showed that PD-L1 expression was also highly correlated with IL-6/JAK/STAT3 signaling. Surprisingly, the expression of both phospho-tyrosine STAT3 and PD-L1 was significantly increased under conditions of extracellular acidosis, and inhibition of STAT3 did not increase the expression of PD-L1 even under acidic conditions in MDA-MB-231 cells. Based on these results, we suggest that the expression of PD-L1 is increased by tumor acidosis via activation of STAT3 in MDA-MB-231 cells., (© 2022. The Author(s).)

Background: Practical guidance is needed regarding the vaccination of coronavirus disease 2019 (COVID-19) convalescent individuals in resource-limited countries. It includes the number of vaccine doses that should be given to unvaccinated patients who experienced COVID-19 early in the pandemic., Methods: We recruited COVID-19 convalescent individuals who received one or two doses of an mRNA vaccine within 6 or around 18 months after a diagnosis of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection. Their samples were assessed for IgG-binding or neutralizing activity and cell-mediated immune responses against SARS-CoV-2 wild-type and variants of concern., Results: A total of 43 COVID-19 convalescent individuals were analyzed in the present study. The results showed that humoral and cellular immune responses against SARS-CoV-2 wild-type and variants of concern, including the Omicron variant, were comparable among patients vaccinated within 6 versus around 18 months. A second dose of vaccine did not significantly increase immune responses., Conclusion: One dose of mRNA vaccine should be considered sufficient to elicit a broad immune response even around 18 months after a COVID-19 diagnosis., (© 2022. The Author(s).)

Background: Despite the fact of ongoing worldwide vaccination programs for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), understanding longevity, breadth, and type of immune response to coronavirus disease-19 (COVID-19) is still important to optimize the vaccination strategy and estimate the risk of reinfection. Therefore, we performed thorough immunological assessments 1 year post-COVID-19 with different severity., Methods: We analyzed peripheral blood mononuclear cells and plasma samples at 1 year post-COVID-19 in patients who experienced asymptomatic, mild, and severe illness to assess titers of various isotypes of antibodies (Abs) against SARS-CoV-2 antigens, phagocytic capability, and memory B- and T-cell responses., Findings: A total of 24 patients (7, 9, and 8 asymptomatic, mild, and severe patients, respectively) and eight healthy volunteers were included in this study. We firstly showed that disease severity is correlated with parameters of immune responses at 1 year post-COVID-19 that play an important role in protecting against reinfection with SARS-CoV-2, namely, the phagocytic capacity of Abs and memory B-cell responses., Interpretation: Various immune responses at 1 year post-COVID-19, particularly the phagocytic capacity and memory B-cell responses, were dependent on the severity of the prior COVID-19. Our data could provide a clue for a tailored vaccination strategy after natural infection according to the severity of COVID-19., 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 Kang, Kim, Hong, Kim, Lee, Chang, Choe, Kim, Kim, Seo, Song, Lee, Shin, Kim, Lee, Park, Kim and Oh.)

Diabetes mellitus (DM) characterized by hyperglycemia is a chronic metabolic disorder that leads to many symptoms and vascular complications. Despite the close association between DM and cancer progression, the response and role of endothelial cells (ECs) under diabetic conditions in tumor metastasis remain to be elucidated. In this study, we sought to determine whether and how ECs under diabetic conditions contribute to tumor metastasis. We have taken advantage of syngeneic mouse tumor models of Lewis lung carcinoma (LLC) and melanoma (B16F10) cells and a streptozotocin (STZ)-induced hyperglycemia model. We demonstrated that hyperglycemia increased the metastasis of LLC and B16F10 cells in an experimental metastasis model with an intravenous injection of the tumor cells. We also found that hyperglycemia promoted lung metastasis of tumor cells by increasing the adhesiveness of ECs to facilitate the adhesion of tumor cells to ECs rather than affecting the metastatic behavior of tumor cells themselves. From the analysis of gene expression in primary lung ECs from STZ-treated mice, we identified that vWF promoted the adhesion of tumor cells to ECs and the transendothelial migration of tumor cells. Mechanistically, hyperglycemia-induced oxidative stress in ECs, and increased oxidative stress enhanced vWF expression in ECs through an increase in the transcription factor GATA1. These results provide evidence for the role of vWF in ECs in promoting hyperglycemia-induced tumor metastasis and potential therapeutic targets for the regulation of vWF expression in ECs and hyperglycemia-induced tumor metastasis., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Mutation is the primary determinant of genetic diversity in influenza viruses. The rate of mutation, measured in an absolute time-scale, is likely to be dependent on the rate of errors in copying RNA sequences per replication and the number of replications per unit time. Conditions for viral replication are probably different among host taxa, potentially generating the host specificity of the viral mutation rate, and possibly between highly and low pathogenic (HP and LP) viruses. This study investigated whether mutation rates per year in avian influenza A viruses depend on host taxa and pathogenicity. We inferred mutation rates from the rates of synonymous substitutions, which are assumed to be neutral and thus equal to mutation rates, at four segments that code internal viral proteins (PB2, PB1, PA, NP). On the phylogeny of all avian viral sequences for each segment, multiple distinct subtrees (clades) were identified that represent viral subpopulations, which are likely to have evolved within particular host taxa. Using simple regression analysis, we found that mutation rates were significantly higher in viruses infecting chickens than domestic ducks and in those infecting wild shorebirds than wild ducks. Host dependency of the substitution rate was also confirmed by Bayesian phylogenetic analysis. However, we did not find evidence that the mutation rate is higher in HP than in LP viruses. We discuss these results considering viral replication rate as the major determinant of mutation rate per unit time., (© The Author(s) 2022. Published by Oxford University Press.)

The resistance of highly aggressive glioblastoma multiforme (GBM) to chemotherapy is a major clinical problem resulting in a poor prognosis. GBM contains a rare population of self-renewing cancer stem cells (CSCs) that proliferate, spurring the growth of new tumors, and evade chemotherapy. In cancer, major vault protein (MVP) is thought to contribute to drug resistance. However, the role of MVP as CSCs marker remains unknown and whether MVP could sensitize GBM cells to Temozolomide (TMZ) also is unclear. We found that sensitivity to TMZ was suppressed by significantly increasing the MVP expression in GBM cells with TMZ resistance. Also, MVP was associated with the expression of other multidrug-resistant proteins in tumorsphere of TMZ-resistant GBM cell, and was highly co-expressed with CSC markers in tumorsphere culture. On the other hands, knockdown of MVP resulted in reduced sphere formation and invasive capacity. Moreover, high expression of MVP was associated with tumor malignancy and survival rate in glioblastoma patients. Our study describes that MVP is a potentially novel maker for glioblastoma stem cells and may be useful as a target for preventing TMZ resistance in GBM patients., (Copyright © 2021. Published by Elsevier Inc.)

Tendinopathy, the most common disorder affecting tendons, is characterized by chronic disorganization of the tendon matrix, which leads to tendon tear and rupture. The goal was to identify a rational molecular target whose blockade can serve as a potential therapeutic intervention for tendinopathy. We identified C1q/TNF-related protein-3 (CTRP3) as a markedly up-regulated cytokine in human and rodent tendinopathy. Overexpression of CTRP3 enhanced the progression of tendinopathy by accumulating cartilaginous proteoglycans and degenerating collagenous fibers in the mouse tendon, whereas CTRP3 knockdown suppressed the tendinopathy pathogenesis. Functional blockade of CTRP3 using a neutralizing antibody ameliorated overuse-induced tendinopathy of the Achilles and rotator cuff tendons. Mechanistically, CTRP3 elicited a transcriptomic pattern that stimulates abnormal differentiation of tendon stem/progenitor cells and ectopic chondrification as an effect linked to activation of Akt signaling. Collectively, we reveal an essential role for CTRP3 in tendinopathy and propose a potential therapeutic strategy for the treatment of tendinopathy.

Background: Understanding the memory T-cell response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial for assessing the longevity of protective immunity after SARS-CoV-2 infection or coronavirus disease 2019 (COVID-19) vaccination. However, the longitudinal memory T-cell response up to 8 months post-symptom onset (PSO) according to the severity of illness is unknown., Methods: We analyzed peripheral blood mononuclear cells (PBMCs) from healthy volunteers or patients with COVID-19 who experienced asymptomatic, mild, or severe illness at 2, 5, and 8 months PSO. SARS-CoV-2 spike, nucleocapsid, and membrane protein-stimulated PBMCs were subjected to flow cytometry analysis., Results: A total of 24 patients (7 asymptomatic, 9 with mild disease, and 8 with severe disease) and 6 healthy volunteers were analyzed. SARS-CoV-2-specific OX40+CD137+CD4+ T cells and CD69+CD137+CD8+ T cells persisted at 8 months PSO. Also, antigen-specific cytokine-producing or polyfunctional CD4+ T cells were maintained for up to 8 months PSO. Memory CD4+ T-cell responses tended to be greater in patients who had severe illness than in those with mild or asymptomatic disease., Conclusions: Memory response to SARS-CoV-2, based on the frequency and functionality, persists for 8 months PSO. Further investigations involving its longevity and protective effect from reinfection are warranted., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Although tumor genomic profiling has identified small subsets of gastric cancer (GC) patients with clinical benefit from anti-PD-1 treatment, not all responses can be explained by tumor sequencing alone. We investigate epigenetic elements responsible for the differential response to anti-PD-1 therapy by quantitatively assessing the genome-wide chromatin accessibility of circulating CD8 + T cells in patients' peripheral blood. Using an assay for transposase-accessible chromatin using sequencing (ATAC-seq), we identify unique open regions of chromatin that significantly distinguish anti-PD-1 therapy responders from non-responders. GC patients with high chromatin openness of circulating CD8 + T cells are significantly enriched in the responder group. Concordantly, patients with high chromatin openness at specific genomic positions of their circulating CD8 + T cells demonstrate significantly better survival than those with closed chromatin. Here we reveal that epigenetic characteristics of baseline CD8 + T cells can be used to identify metastatic GC patients who may benefit from anti-PD-1 therapy.

Although several evidence has suggested the impact of exercise on the prevention of aging phenotypes, few studies have been conducted on the mechanism by which exercise alters the immune-cell profile, thereby improving metabolism in senile obesity. In this study, we confirmed that 4-week treadmill exercise sufficiently improved metabolic function, including increased lean mass and decreased fat mass, in 88-week-old mice. The expression level of the senescence marker p16 in the white adipose tissue (WAT) was decreased after 4-weeks of exercise. Exercise induced changes in the profiles of immune-cell subsets, including natural killer (NK) cells, central memory CD8 + T cells, eosinophils, and neutrophils, in the stromal vascular fraction of WAT. In addition, it has been shown through transcriptome analysis of WAT that exercise can activate pathways involved in the interaction between WAT and immune cells, in particular NK cells, in aged mice. These results suggest that exercise has a profound effect on changes in immune-cell distribution and senescent-cell scavenging in WAT of aged mice, eventually affecting overall energy metabolism toward a more youthful state.

Immunogenic photodynamic therapy (PDT) has the potential to moderate the shortfalls of cancer immunotherapy. However, its efficacy is severely limited particularly because of the lack of optimal photosensitizers and smart delivery processes and the inherent shortcomings of PDT (e.g., hypoxia resistance). Here, we demonstrate a clinically promising approach that utilizes a water-soluble phthalocyanine derivative (PcN4) concomitantly delivered with a hypoxia-activated prodrug (AQ4N) to amplify the effect of PDT and enhance cancer immunotherapy. After intravenous injection, PcN4 selectively interacted with endogenous albumin dimers and formed supramolecular complexes, providing a facile and green approach for tumor-targeted PDT. The concomitant delivery of AQ4N overcame the limitations of hypoxia in PDT and improved the antitumor activity of PDT. Treatment with PcN4-mediated and AQ4N-amplified PDT almost completely eradicated sizable primary tumors in a triple-negative breast cancer model and significantly activated CD8 + T cells. As the majority of tumor infiltrating CD8 + T cells were both PD-1- and TIM3-positive, additional combination therapy using PD-L1/PD-1 pathway blockade was warranted. After combination with immune checkpoint blockade treatment, an enhanced abscopal effect was achieved in both distant and metastatic tumors., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Effector memory (EM) CD8 + T cells expressing lower levels of IL-7R α (IL-7Rα low ) from healthy individuals are partly compromised in vitro, but the identity of these cells has remained unclear. In this study, we demonstrate that human IL-7Rα low EM CD8 + T cells are naturally occurring anergic cells in vivo and impaired in proliferation and IL-2 production but competent in IFN-γ and TNF-α production, a state that can be restored by IL-2 stimulation. IL-7Rα low EM CD8 + T cells show decreased expression of GATA3 and c-MYC and are defective in metabolic reprogramming toward glycolysis, a process required for the proliferation of T cells. However, IL-7Rα low EM CD8 + T cells can proliferate with TCR stimulation in the presence of IL-2 and IL-15, suggesting that these cells can be restored to normality or increased activity by inflammatory conditions and may serve as a reservoir for functional immunity., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Objectives: We hypothesized that immune response may contribute to progression of coronavirus disease-19 (COVID-19) at the second week of illness. Therefore, we compared cell-mediated immune (CMI) responses between severe and mild COVID-19 cases., Methods: We examined peripheral blood mononuclear cells of laboratory-confirmed COVID-19 patients from their first and third weeks of illness. Severe pneumonia was defined as an oxygen saturation ≤93% at room air. Expressions of molecules related to T-cell activation and functions were analyzed by flow cytometry., Results: The population dynamics of T cells at the first week were not different between the two groups. However, total numbers of CD4 + and CD8 + T cells tended to be lower in the severe group at the third week of illness. Expressions of Ki-67, PD-1, perforin, and granzyme B in CD4 + or CD8 + T cells were significantly higher in the severe group than in the mild group at the third week. In contrast to the mild group, the levels of their expression did not decrease in the severe group., Conclusions: Severe COVID-19 had a higher degree of proliferation, activation, and cytotoxicity of T-cells at the late phase of illness without cytotoxic T-cell contraction, which might contribute to the development of severe COVID-19., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Inhibition of immune checkpoint proteins like programmed death 1 (PD-1) is a promising therapeutic approach for several cancers, including non-small cell lung cancer (NSCLC). Although PD-1 ligand (PD-L1) expression is used to predict anti-PD-1 therapy responses in NSCLC, its accuracy is relatively less. Therefore, we sought to identify a more accurate predictive blood biomarker for evaluating anti-PD-1 response. We evaluated the frequencies of T cells, B cells, natural killer (NK) cells, polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), mononuclear myeloid-derived suppressor cells (M-MDSCs), and Lox-1 + PMN-MDSCs in peripheral blood samples of 62 NSCLC patients before and after nivolumab treatment. Correlation of immune-cell population frequencies with treatment response, progression-free survival, and overall survival was also determined. After the first treatment, the median NK cell percentage was significantly higher in responders than in non-responders, while the median Lox-1+ PMN-MDSC percentage showed the opposite trend. NK cell frequencies significantly increased in responders but not in non-responders. NK cell frequency inversely correlated with that of Lox-1 + PMN-MDSCs after the first treatment cycle. The NK cell-to-Lox-1 + PMN-MDSC ratio (NMR) was significantly higher in responders than in non-responders. Patients with NMRs ≥ 5.75 after the first cycle had significantly higher objective response rates and longer progression-free and overall survival than those with NMRs <5.75. NMR shows promise as an early predictor of response to further anti-PD-1 therapy.

Tissue-resident macrophages have unique tissue-specific functions in maintaining homeostasis and resolving inflammation. However, the repair role and relevant molecules of kidney-resident macrophages after ischemic injury remain unresolved. To this end, mice without kidney-resident R1 macrophages but containing infiltrating monocyte-derived R2 macrophages were generated using differential cellular kinetics following clodronate liposome treatment. When ischemia-reperfusion injury was induced in these mice, late phase repair was reduced. Transcriptomic and flow cytometric analyses identified that V-domain Ig suppressor of T cell activation (VISTA), an inhibitory immune checkpoint molecule, was constitutively expressed in kidney-resident R1 macrophages, but not in other tissue-resident macrophages. Here, VISTA functioned as a scavenger of apoptotic cells and served as a checkpoint to control kidney-infiltrating T cells upon T cell receptor-mediated stimulation. Together these functions improved the repair process after ischemia-reperfusion injury. CD14 + CD33 + mononuclear phagocytes of human kidney also expressed VISTA, which has similar functions to the mouse counterpart. Thus, VISTA is upregulated in kidney macrophages in a tissue-dependent manner and plays a repair role during ischemic injury., (Copyright © 2019 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

FoxP3 reporter mice expressing green fluorescence protein (GFP) have been used as a very convenient tool to investigate the impact of regulatory T (Treg) cells on pathogenesis in autoimmune diseases. Here, we found that GFP-FoxP3 + knock-in (KI) mice showed alterations in the production of anti-nuclear autoantibodies (ANAs) and nephritis with different extent, depending on the presence or absence of lupus susceptibility gene locus 1 ( Sle1 ) and KI method: contrasting with B6. Sle1 .fGFP-FoxP3 mice, expressing GFP via N-terminal insertion, B6. Sle1 .iGFP-FoxP3, expressing GFP via bicistronic internal ribosome entry site-driven promotion, exhibited significantly lower penetrance of serum ANA, comparing to control B6. Sle1 mice. Moreover, B6.Sle1 .GFP-FoxP3 + mice reduced the Sle1 -induced splenomegaly and B-cell expansion independently of the KI method employed, mainly by reducing the numbers of transitional 1 (T1) B cells and CD21 - CD23 - B cells, including plasmablasts and plasma cells. The absolute numbers of both splenic CD4 + T cells and Treg cells from B6. Sle1 .GFP-FoxP3 KI mice were significantly reduced but their proportion was not changed, compared to B6. Sle1 mice. Although the glomerular basement membranes were thickened in both B6. Sle1 and B6. Sle1 .iGFP-FoxP3 mice, they were thinner in B6. Sle1 .fGFP-FoxP3 mice. The latter mice expressed more nephrophilic autoantibodies and deposited more complement component 3 in glomeruli compared to B6.iGFP-FoxP3 mice. FoxP3 + Treg cells may modulate B-cell tolerance in lupus-prone B6. Sle1 mice, presumably by modulating pathogenic, nephrophilic autoantibody production and nephritis.

Precise mechanisms underlying interleukin-7 (IL-7)-mediated tumor invasion remain unclear. Thus, we investigated the role of IL-7 in tumor invasiveness using metastatic prostate cancer PC-3 cell line derivatives, and assessed the potential of IL-7 as a clinical target using a Janus kinase (JAK) inhibitor and an IL-7-blocking antibody. We found that IL-7 stimulated wound-healing migration and invasion of PC-3 cells, increased phosphorylation of signal transducer and activator of transcription 5, Akt, and extracellular signal-regulated kinase. On the other hand, a JAK inhibitor and an IL-7-blocking antibody decreased the invasiveness of PC-3 cells. IL-7 increased tumor sphere formation and expression of epithelial-mesenchymal transition (EMT) markers. Importantly, lentiviral delivery of IL-7Rα to PC-3 cells significantly increased bone metastasis in an experimental murine metastasis model compared to controls. The gene expression profile of human prostate cancer cells from The Cancer Genome Atlas revealed that EMT pathways are strongly associated with prostate cancers that highly express both IL-7 and IL-7Rα. Collectively, these data suggest that IL-7 and/or IL-7Rα are promising targets of inhibiting tumor metastasis.

High expression of cluster of differentiation (CD)39 and CD73 has cardio-protective effects. We hypothesised that the expression of CD39 and CD73 would differ between propofol- and volatile anaesthetic-based anaesthesia in patients undergoing open heart surgery (OHS). The objective of this prospective randomized trial was to compare the changes in CD39 and CD73 levels in CD4 + T cells between propofol- and sevoflurane-based anaesthesia during OHS. The study randomly allocated 156 patients undergoing OHS to a propofol or sevoflurane group. Blood was obtained preoperatively and up to 48 hours after weaning from cardiopulmonary bypass (CPB). The expression levels of CD39 and CD73 in circulating CD4 + T cells, serum cytokines and other laboratory parameters were analysed. The primary outcome was the expression of CD39 and CD73 on CD4 + T cells. Demographic data and perioperative haemodynamic changes did not show significant differences between the two groups. The expression of CD39 and CD73 in the sevoflurane group was significantly lower than in the propofol group (P < 0.001). Other laboratory findings including cardiac enzymes and cytokine levels, did not show significant intergroup differences. Propofol attenuated the decrease in CD39 and CD73 in circulating CD4 + T cells compared to sevoflurane-based anaesthesia during OHS.

The voltage-gated potassium channel, Kv1.3, and the Ca 2+ -activated potassium channel, KCa3.1, regulate membrane potentials in T cells, thereby controlling T cell activation and cytokine production. However, little is known about the expression and function of potassium channels in human effector memory (EM) CD8 + T cells that can be further divided into functionally distinct subsets based on the expression of the interleukin (IL)-7 receptor alpha (IL-7Rα) chain. Herein, we investigated the functional expression and roles of Kv1.3 and KCa3.1 in EM CD8 + T cells that express high or low levels of the IL-7 receptor alpha chain (IL-7Rα high and IL-7Rα low , respectively). In contrast to the significant activity of Kv1.3 and KCa3.1 in IL-7Rα high EM CD8 + T cells, IL-7Rα low EM CD8 + T cells showed lower expression of Kv1.3 and insignificant expression of KCa3.1. Kv1.3 was involved in the modulation of cell proliferation and IL-2 production, whereas KCa3.1 affected the motility of EM CD8 + T cells. The lower motility of IL-7Rα low EM CD8 + T cells was demonstrated using transendothelial migration and motility assays with intercellular adhesion molecule 1- and/or chemokine stromal cell-derived factor-1α-coated surfaces. Consistent with the lower migration property, IL-7Rα low EM CD8 + T cells were found less frequently in human skin. Stimulating IL-7Rα low EM CD8 + T cells with IL-2 or IL-15 increased their motility and recovery of KCa3.1 activity. Our findings demonstrate that Kv1.3 and KCa3.1 are differentially involved in the functions of EM CD8 + T cells. The weak expression of potassium channels in IL-7Rα low EM CD8 + T cells can be revived by stimulation with IL-2 or IL-15, which restores the associated functions. This study suggests that IL-7Rα high EM CD8 + T cells with functional potassium channels may serve as a reservoir for effector CD8 + T cells during peripheral inflammation.

The heterogeneity of microglial functions have either beneficial or detrimental roles in specific physiological or pathological environments. However, the details of what transcriptional mechanisms induce microglia to take beneficial phenotypes remain unknown. Here, we report that Foxp3 is essential for beneficial outcome of the microglial response and depends upon signalling by the immunoglobulin CD200 through its receptor (CD200R). Foxp3 expression was up-regulated in microglia activated by excitotoxicity-induced hippocampal neuroinflammation. Suppression of CD200R prevented anti-inflammatory phenotype of microglia, but over-expression of Foxp3 enhanced it. Phosphorylation of STAT6, a downstream effector of CD200R, modulated transcription of Foxp3. Finally, CD200R/Foxp3-mediated signalling enhanced hippocampal neuronal viability and conferred a degree of neuroprotection, presumably by counteracting inducible nitric oxide synthase. We conclude that enhancement of Foxp3 through CD200R could be neuroprotective by targeting the microglia.

Objective: We investigated whether the frequency, phenotype, and suppressive function of CD4+ FOXP3+ regulatory T cells (Tregs) are altered in young TS patients with the 45,X karyotype compared to age-matched controls., Design and Methods: Peripheral blood mononuclear cells from young TS patients (n = 24, 17.4-35.9 years) and healthy controls (n = 16) were stained with various Treg markers to characterize their phenotypes. Based on the presence of thyroid autoimmunity, patients were categorized into TS (-) (n = 7) and TS (+) (n = 17). Tregs sorted for CD4+ CD25bright were co-cultured with autologous CD4+ CD25- target cells in the presence of anti-CD3 and -CD28 antibodies to assess their suppressive function., Results: Despite a lower frequency of CD4+ T cells in the TS (-) and TS (+) patients (mean 30.8% and 31.7%, vs. 41.2%; P = 0.003 and P < 0.001, respectively), both groups exhibited a higher frequency of FOXP3+ Tregs among CD4+ T cells compared with controls (means 1.99% and 2.05%, vs. 1.33%; P = 0.029 and P = 0.004, respectively). There were no differences in the expression of CTLA-4 and the frequency of Tregs expressing CXCR3+, and CCR4+ CCR6+ among the three groups. However, the ability of Tregs to suppress the in vitro proliferation of autologous CD4+ CD25- T cells was significantly impaired in the TS (-) and TS (+) patients compared to controls (P = 0.003 and P = 0.041). Meanwhile, both the TS (-) and TS (+) groups had lower frequencies of naïve cells (P = 0.001 for both) but higher frequencies of effector memory cells (P = 0.004 and P = 0.002) than did the healthy control group., Conclusions: The Tregs of the TS patients could not efficiently suppress the proliferation of autologous effector T cells, despite their increased frequency in peripheral CD4+ T cells.

Objective: The mechanism by which IL-1β and thapsigargin (TG)-induced endoplasmic reticulum (ER) stress modulate the receptor activator of nuclear factor kappa-B ligand (RANKL)-mediated osteoclastogenesis remains elusive. Thus, we investigated the osteoclast-specific and ER signals in osteoclastogenesis of bone marrow-derived cells., Methods: Bone marrow cells (BMCs) were obtained from 5-week-old male ICR mice and cultured to be differentiated into osteoclasts with M-CSF and RANKL in the presence or absence of IL-1β, TG, or 4-phenylbutyric acid (PBA), an ER stress-reducing drug. The formation of osteoclasts was evaluated by tartrate-resistant acid phosphatase (TRAP) staining and resorption pit assay with a dentine slice. The molecular mechanism of IL-1β and ER stress in osteoclastogenesis was investigated in BMCs transfected with siRNA for GRP78, PERK and IRE1 using reverse transcription-polymerase chain reaction and immunoblotting for osteoclast-specific and ER stress signaling molecules., Results: IL-1β and ER stress induced by TG-augmented the formation of osteoclasts, which was significantly inhibited by PBA and was mediated with osteoclast-specific signals, including c-Fos, NFATc1, and ER stress- associated signaling pathways, such as PERK, IRE1, GRP78, and eIF2α. siRNA-mediated knockdown of ER stress signals inhibited the expression of NFATc1 and c-Fos, thus reducing IL-1β and/or TG-induced formation of osteoclasts., Conclusions: Osteoclastogenesis by IL-1β and/or ER stress is mainly associated with upregulation of eIF2α, GRP78, PERK and IRE1. These results suggest that the signaling pathway of ER stress-induced osteoclast formation might be a new therapeutic target to prevent inflammatory and destructive arthritic disease such as RA and diverse osteoporosis., (Copyright © 2014 Société française de rhumatologie. Published by Elsevier SAS. All rights reserved.)

Clinical characteristics of antitumor necrosis factor (TNF) agents-related tuberculosis (TB) in ankylosing spondylitis (AS) are not well described. The aim was to compare the incidences and the characteristics of TB in AS and rheumatoid arthritis (RA) during TNF inhibitor treatment. AS (n = 1,322) and RA (n = 3,154) patients who received medical care between January 2001 and August 2011 were enrolled. The incidence of TB in patients treated, or not, with TNF inhibitors and the clinical features associated with TB were explored. Seven patients with AS and seven with RA developed TB while receiving TNF inhibitor therapy, resulting in an incidence rate of 600.2/100,000 person-years (PYs) (95 % confidence interval (CI), 241.3-1236.3) for those with AS and 771.6/100,000 PYs (95 % CI, 310.2-1589.9) for those with RA. Incidence rate ratios for TNF inhibitor-treated vs. untreated patients were 4.87 for AS (95 % CI, 1.50-15.39; p < 0.001) and 3.61 for RA (95 % CI, 1.38-8.07; p < 0.001). Low body mass index was identified as a significant risk factor for TB in the AS group (odds ratio (OR), 13.0; p = 0.002). Extrapulmonary TB was predominant at 85.7 % during TNF inhibitor treatment. Three (42.8 %) of the AS patients, but none of the RA patients, developed TB with concomitant isoniazid. All AS patients recovered from TB whereas two of seven RA patients died. Treatment with TNF inhibitors significantly increases the risk of extrapulmonary TB in AS. Symptoms of infection should warrant clinicians to evaluate for TB during TNF inhibitor therapy in AS patients.

Vitamin C has been found to stimulate dendritic cells (DCs) to secrete more IL-12 and thereby drive naïve CD4(+) T cells to differentiate into Th1 cells. In the present study, we evaluated the effect of these vitamin C-treated DCs on CD8(+) T cell differentiation both in vitro and in vivo. Mouse bone marrow-derived DCs were prepared in the presence of GM-CSF and IL-15. With vitamin C treatment, these DCs, when LPS-stimulated, secreted more IL-12p70 and IL-15 than did untreated DCs. And when co-cultured with T cells, they yielded a higher frequency of IFN-γ(+) CD8(+) T cells. Moreover, we found that administering vitamin C-treated and tumor lysate-loaded DCs into mice yielded a higher frequency of CD44(high) CD62L(low) CD8(+) effector and effector memory T cells, which showed an increased ex vivo killing effect of the tumor cells. These DCs also elicited enhanced protective effects against inoculated tumor cells, most probably by way of the increased cytotoxic T cells, as was revealed by the decreased growth of the inoculated tumor cells in these mice. This ex vivo vitamin C treatment effect on DCs can be considered as a strategy for boosting DC vaccination potency against tumors., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Transglutaminase 2 (TG2) has been reported to play a role in dendritic cell activation and B-cell differentiation after immunization. Its presence and role in T cells, however, has not been explored. In the present study, we determined the expression of TG2 on mouse T cells, and evaluated its role by comparing the behaviours of wild-type and TG2(-/-) T cells after activation. In our results, naive T cells minimally expressed TG2, expression of which was increased after activation. T-cell proliferation, expression of activation markers such as CD69 and CD25, and secretions of interleukin-2 and interferon-γ were suppressed in the absence of TG2, presumably due, in part, to diminished nuclear factor-κB activation. These effects on T cells seemed to be reflected in the in vivo immune response, the contact hypersensitivity reaction elicited by 2,4-dinitro-1-fluorobenzene, with lowered peak responses in the TG2(-/-) mice. When splenic T cells from mice immunized with tumour lysate-loaded wild-type dendritic cells were re-challenged ex vivo with the same antigen, the profile of surface markers including CD44, CD62L, and CD127 strongly indicated lesser generation of memory CD8(+) T cells in TG2(-/-) mice. In the TG2(-/-)  CD8(+) T cells, moreover, Eomes expression was markedly decreased. These results indicate possible roles of TG2 in CD8(+) T-cell activation and CD8(+) memory T-cell generation., (© 2014 John Wiley & Sons Ltd.)

Understanding of the interaction between human MG63 osteoblast-like cells and surfaces is necessary in the field of tissue engineering and biomaterials. Various titanium surfaces are widely used as not only implant materials, but also as miniscrews in orthodontics. Our goal was to assess the proteomic response of MG63 osteoblast-like cells to different titanium surfaces. MG63 osteoblast-like cells were cultured on three different titanium surfaces: a smooth surface (S), a sandblasted with large grit and acid-etched surface (SLA), and a surface coated with a thin layer of hydroxyapatite (HA). Cells grown on the rougher surfaces (SLA and HA) exhibited downregulated cell proliferation and morphological changes. In the proteomic analysis, cells grown on the SLA surface showed upregulated expression of protocadherin-β3 precursor, kinase insert domain receptor, fibroblast growth factor receptor-3, and insulin-like growth factor I, while the expression levels of cell adhesion kinase, collagen α-1(I) chain precursor, collagen type XI α2, and cadherin-11 were upregulated in cells grown on the HA surface. These proteins are known to be involved in osteoblast adhesion, growth, and differentiation. Thus, the surface properties of dental materials can influence the expression of proteins involved in osseointegration-related processes. Proteomic analysis may reveal changes in novel proteins that explain why osseointegration varies depending on surface properties.

Given the involvement of oxidative stress in liver-disease- or hepato-toxicant-induced hepatic damage and fibrosis, antioxidants are an effective preventive and therapeutic tool. The beneficial results of vitamin C, one of the physiological antioxidants, have been observed both in experimental animals and in humans. However, most of these studies have been concerned with supplementary vitamin C; the effects of under vitamin C insufficiency, which humans sometimes confront, have not been substantially investigated. In the present study, we established a vitamin C-insufficient animal model (half-to-normal serum vitamin C concentration) with gulo(-/-) mice that cannot synthesize vitamin C, and induced hepatotoxicity by means of thioacetamide (TAA) injections twice a week for 18 weeks. Additionally, we explored the direct effects of vitamin C both on immortalized human hepatic stellate LX-2 cells and on rat primary hepatic stellate cells. Vitamin C insufficiency resulted in a decreased survival rate and increased serum markers for hepatocyte damage, such as alanine aminotransferase and aspartate aminotransferase. Concomitantly, the levels of reactive oxygen species (ROS) and lipid peroxides in the liver were increased. Histological examinations of the vitamin C-insufficient liver revealed increases in collagen fiber deposition and activated-hepatic-stellate-cell number. Vitamin C, when directly applied to the LX-2 cells as well as the rat primary hepatic stellate cells, suppressed not only proliferation but hydrogen peroxide-induced collagen expression as well. In conclusion, vitamin C insufficiency exacerbated TAA-induced hepatotoxicity. These effects seem to be mainly from insufficient scavenging of ROS in the liver, and possibly in part, by directly affecting hepatic stellate cells., (© 2013 Elsevier Inc. All rights reserved.)

It is thought that vitamin C has protective roles on stress-induced heart damage and the development of cardiovascular diseases, but its precise role and mechanisms are unclear. In the present study, we investigated the specific mechanisms by which vitamin C leads to protecting the heart from stress-induced damage in the Gulo(-/-) mice which cannot synthesize vitamin C like humans. By exposure to stress (1h/day), the heartbeat and cardiac output in vitamin C-insufficient Gulo(-/-) mice were definitely decreased, despite a significant increase of adrenaline (ADR) and noradrenaline (NA) production. A change of cardiac structure caused by the death of cardiomyocytes and an increased expression of matrix metalloprotease (MMP)-2 and -9 were also found. Moreover, lipid peroxidation and the production of tumor necrosis factor-alpha (TNF-α) in the heart were increased. Finally, all vitamin C-insufficient Gulo(-/-) mice were expired within 2 weeks. Interestingly, all of the findings in vitamin C-insufficient Gulo(-/-) mice were completely prevented by the supplementation of a sufficient amount of vitamin C. Taken together, vitamin C insufficiency increases the risk of stress-induced cardiac damage with structural and functional changes arising from the apoptosis of cardiomyocytes., (Crown Copyright © 2013 Published by Elsevier Inc. All rights reserved.)

Alloferon is a novel immunomodulatory peptide originally isolated from infected insects. It has anti-viral and anti-tumor effects via the activation of NK cells. However, specific mechanisms leading to NK cell activation and anti-tumor responses yet to be clarified. In this study, we demonstrate that alloferon increases killing activity of NK cells to cancer cells via the up-regulation of the expression of NK-activating receptors, 2B4. In addition, the production of IFN-γ and TNF-α and granule exocytosis from NK cells against cancer cell were increased by alloferon. Lastly, the anti-tumor effect of alloferon was confirmed in vivo to demonstrate effective retardation of tumor growth in the human-to-mouse xenograft model. All taken together, these results suggest that alloferon has anti-tumor effects through up-regulation of NK-activating receptor 2B4 and the enhancement of granule exocytosis from NK cells., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Maximum engraftment of transplanted islets is essential for the clinical application of a subcutaneous site. Significant barriers to the current approaches are associated with their low effectiveness, complexity and unproven biosafety. Here, we evaluated and optimized a fibrin-islet composite for effective glycemic control in a subcutaneous site whose environment is highly hypoxic due to low vascularization potential. In the setting of xenogeneic porcine islet transplantation into the subcutaneous space of a diabetic mouse, the in vivo islet functions were greatly affected by the concentrations of fibrinogen and thrombin. The optimized hydrogel-type fibrin remarkably reduced the marginal islet mass to approximately one tenth that of islets without fibrin. This marginal islet mass was comparable to that in the setting of the subcapsular space of the kidney, which is a highly vascularized organ. Highly vascularized structures were generated inside and on the outer surface of the grafts. A hydrogel-type fibrin-islet composite established early diabetic control within an average of 3.4days after the transplantation. In the mechanistic studies, fibrin promoted local angiogenesis, enhanced islet viability and prevented fragmentation of islets into single cells. In conclusion, in situ application of hydrogel-type fibrin-islet composite may be a promising modality in the clinical success of subcutaneous islet transplantation., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Little is known about the cellular characteristics of CD8(+) T cells in rheumatoid arthritis (RA). We addressed this by investigating whether the frequency of the CD8(+) T cell subsets and their phenotypic characteristics are altered in the peripheral blood and synovial fluid (SF) from patients with RA. In this study, CD8(+) T cells, mainly CD45RA(-) effector memory (EM) CD8(+) T cells, were increased significantly in the SF, but not in the peripheral blood from RA patients, compared with healthy controls. The synovial EM CD8(+) T cells were activated phenotypes with high levels of CD80, CD86, and PD-1, and had a proliferating signature in vivo upon Ki-67 staining, whereas the Fas-positive cells were prone to apoptosis. In addition, EM CD8(+) T cells in the SF were less cytotoxic, as they expressed less perforin and granzyme B. In particular, the proportions of synovial fluid mononuclear cells that were CCR4(+)CD8(+) T cells and IL-4-producing CD8(+) T cells (i.e., Tc2 cells) were significantly higher than those in peripheral blood mononuclear cells of patients with RA and healthy controls. In addition, the number of IL-10-producing CD8(+) suppressor T (Ts) cells increased significantly in the SF of RA patients. Especially, CD8(+) T cells were inversely correlated with disease activity. These findings strongly suggest that EM CD8(+) T cells in the SF are increased, likely because of inflammation, and they may be involved in modulating inflammation, thereby affecting the development and progression of RA.

Cytokeratin 20 (CK20) is an intermediate filament that is known to be a prognostic marker in several types of cancer. However, little is known about CK20 expression and tumor metastasis in tamoxifen-resistant MCF-7 (TRM-7) breast cancer cells. TRM-7 cells overexpress CK20, resulting in enhanced invasiveness in vitro. CK20 silencing reduced the invasiveness of TRM-7 cells. Moreover, CK20 expression in MCF-7 cells was regulated by peroxisome proliferator-activated receptor γ (PPARγ). Our findings suggest that PPARγ-dependent CK20 expression enhances the metastatic potential of MCF-7 breast cancer cells and may be a potential therapeutic target in tamoxifen-resistant breast cancer.

Background: Vitamin C is an essential nutrient for maintaining human life. Vitamin C insufficiency in the plasma is closely related with the development of scurvy. However, in vivo kinetics of vitamin C regarding its storage and consumption is still largely unknown., Methods: We used Gulo(-/-) mice, which cannot synthesize vitamin C like human. Vitamin C level in plasma and organs from Gulo(-/-) mice was examined, and it compared with the level of wild-type mice during 5 weeks., Results: The significant weight loss of Gulo(-/-) mice was shown at 3 weeks after vitamin C withdrawal. However, there was no differences between wild-type and vitamin C-supplemented Gulo(-/-) mice (3.3 g/L in drinking water). The concentration of vitamin C in plasma and organs was significantly decreased at 1 week after vitamin C withdrawal. Vitamin C is preferentially deposited in adrenal gland, lymph node, lung, and brain. There were no significant changes in the numbers and CD4/CD8 ratio of splenocytes in Gulo(-/-) mice with vitamin C withdrawal for 4 weeks. And the architecture of spleen in Gulo(-/-) mice was disrupted at 5 weeks after vitamin C withdrawal., Conclusion: The vitamin C level of Gulo(-/-) mice was considerably decreased from 1 week after vitamin C withdrawal. Vitamin C is preferentially stored in some organs such as brain, adrenal gland and lung.

Scrub typhus is caused by the obligate intracellular rickettsia Orientia tsutsugamushi (previously called Rickettsia tsutsugamushi). The bacterium is maternally inherited in trombicuid mites and transmitted to humans by feeding larvae. We report here the 2,127,051-bp genome of the Boryong strain, which represents the most highly repeated bacterial genome sequenced to date. The repeat density of the scrub typhus pathogen is 200-fold higher than that of its close relative Rickettsia prowazekii, the agent of epidemic typhus. A total of 359 tra genes for components of conjugative type IV secretion systems were identified at 79 sites in the genome. Associated with these are >200 genes for signaling and host-cell interaction proteins, such as histidine kinases, ankyrin-repeat proteins, and tetratrico peptide-repeat proteins. Additionally, the O. tsutsugamushi genome contains >400 transposases, 60 phage integrases, and 70 reverse transcriptases. Deletions and rearrangements have yielded unique gene combinations as well as frequent pseudogenization in the tra clusters. A comparative analysis of the tra clusters within the genome and across strains indicates sequence homogenization by gene conversion, whereas complexity, diversity, and pseudogenization are acquired by duplications, deletions, and transposon integrations into the amplified segments. The results suggest intragenomic duplications or multiple integrations of a massively proliferating conjugative transfer system. Diversifying selection on host-cell interaction genes along with repeated population bottlenecks may drive rare genome variants to fixation, thereby short-circuiting selection for low complexity in bacterial genomes.

Objective: To determine whether CD40 ligation of rheumatoid arthritis synovial fibroblasts (RASFs) is able to induce RANKL expression and osteoclastogenesis in RASFs, and to identify its mechanism of action in patients with RA., Methods: CD40 of RASFs was ligated with CD40 ligand (CD40L)-transfected L cells or activated T cells. The formation of osteoclasts in cocultures of CD40-ligated RASFs and T lymphocyte-depleted peripheral blood mononuclear cells was evaluated by tartrate-resistant acid phosphatase staining, detection of calcitonin receptor, and resorption pit formation assay. The expression of NF-kappaB, IkappaB alpha, ERK-1/2, phospho-ERK-1/2, p38, phospho-p38, and RANKL was examined by immunoblotting and/or semiquantitative reverse transcription-polymerase chain reaction., Results: CD40 ligation of RASFs by CD40L-transfected L cells or activated T cells induced RANKL expression and enhanced osteoclastogenesis. CD40 ligation of RASFs also induced activation of ERK-1/2, p38 MAPK, and NF-kappaB and up-regulation of CD40 ligation-induced RANKL expression, whereas osteoclastogenesis was reduced in RASFs transfected with a dominant-negative mutant of IkappaB alpha or by an NF-kappaB inhibitor. However, specific inhibitors of MAPK/ERK-1/2 and p38 MAPK partially blocked the induction of RANKL expression and osteoclastogenesis. Monoclonal antibodies against interleukin-1 and tumor necrosis factor alpha partially inhibited CD40 ligation-mediated osteoclastogenesis., Conclusion: These results indicate that CD40 ligation of RASFs induces RANKL expression mainly via NF-kappaB activation and also results in enhanced osteoclast formation, both of which might play important roles in bone and cartilage destruction in RA. Inhibition of the CD40-CD40L interaction is a potential strategy for the prevention of bone damage in RA.