Preliminary Investigation on Efficacy and Safety of Substance P-Coated Stent for Promoting Re-Endothelialization: A Porcine Coronary Artery Restenosis Model.

BACKGROUND: Current polymer-based drug-eluting stents (DESs) have fundamental issues about inflammation and delayed re-endothelializaton of the vessel wall. Substance-P (SP), which plays an important role in inflammation and endothelial cells, has not yet been applied to coronary stents. Therefore, this study compares poly lactic-co-glycolic acid (PLGA)-based everolimus-eluting stents (PLGA-EESs) versus 2-methacryloyloxyethyl phosphorylcholine (MPC)-based SP-eluting stents (MPC-SPs) in in-vitro and in-vivo models. METHODS: The morphology of the stent surface and peptide/drug release kinetics from stents were evaluated. The in-vitro proliferative effect of SP released from MPC-SP is evaluated using human umbilical vein endothelial cell. Finally, the safety and efficacy of the stent are evaluated after inserting it into a pig's coronary artery. RESULTS: Similar to PLGA-EES, MPC-SP had a uniform surface morphology with very thin coating layer thickness (2.074 µm). MPC-SP showed sustained drug release of SP for over 2 weeks. Endothelial cell proliferation was significantly increased in groups treated with SP (n = 3) compared with the control (n = 3) and those with everolimus (n = 3) (SP: 118.9 ± 7.61% vs. everolimus: 64.3 ± 12.37% vs. the control: 100 ± 6.64%, p < 0.05). In the animal study, the percent stenosis was higher in MPC-SP group (n = 7) compared to PLGA-EES group (n = 7) (MPC-SP: 28.6 ± 10.7% vs. PLGA-EES: 16.7 ± 6.3%, p < 0.05). MPC-SP group showed, however, lower inflammation (MPC-SP: 0.3 ± 0.26 vs. PLGA-EES: 1.2 ± 0.48, p < 0.05) and fibrin deposition (MPC-SP: 1.0 ± 0.73 vs. PLGA-EES: 1.5 ± 0.59, p < 0.05) around the stent strut. MPC-SP showed more increased expression of cluster of differentiation 31, suggesting enhanced re-endothelialization. CONCLUSION: Compared to PLGA-EES, MPC-SP demonstrated more decreased inflammation of the vascular wall and enhanced re-endothelialization and stent coverage. Hence, MPC-SP has the potential therapeutic benefits for the treatment of coronary artery disease by solving limitations of currently available DESs.

Altered Frequency, Activation, and Clinical Relevance of Circulating Innate and Innate-Like Lymphocytes in Patients With Alcoholic Liver Cirrhosis.

Alcoholic liver cirrhosis (ALC) is caused by chronic alcohol overconsumption and might be linked to dysregulated immune responses in the gut-liver axis. However, there is a lack of comprehensive research on levels and functions of innate lymphocytes including mucosal-associated invariant T (MAIT) cells, NKT cells, and NK (NK) cells in ALC patients. Thus, the aim of this study was to examine the levels and function of these cells, evaluate their clinical relevance, and explore their immunologic roles in the pathogenesis of ALC. Peripheral blood samples from ALC patients (n = 31) and healthy controls (HCs, n = 31) were collected. MAIT cells, NKT cells, NK cells, cytokines, CD69, PD-1, and lymphocyte-activation gene 3 (LAG-3) levels were measured by flow cytometry. Percentages and numbers of circulating MAIT cells, NKT cells, and NK cells were significantly reduced in ALC patients than in HCs. MAIT cell exhibited increased production of IL-17 and expression levels of CD69, PD-1, and LAG-3. NKT cells displayed decreased production of IFN-γ and IL-4. NK cells showed elevated CD69 expression. Absolute MAIT cell levels were positively correlated with lymphocyte count but negatively correlated with C-reactive protein. In addition, NKT cell levels were negatively correlated with hemoglobin levels. Furthermore, log-transformed absolute MAIT cell levels were negatively correlated with the Age, Bilirubin, INR, and Creatinine score. This study demonstrates that circulating MAIT cells, NKT cells, and NK cells are numerically deficient in ALC patients, and the degree of cytokine production and activation status also changed. Besides, some of their deficiencies are related to several clinical parameters. These findings provide important information about immune responses of ALC patients.

Activation and increased production of interleukin-17 and tumour necrosis factor-α of mucosal-associated invariant T cells in patients with periodontitis.

AIM: Mucosal-associated invariant T (MAIT) cells are known to be resident in oral mucosal tissue, but their roles in periodontitis are unknown. This study aimed to examine the level and function of MAIT cells in periodontitis patients. MATERIALS AND METHODS: Frequency, activation, and function of MAIT cells from 28 periodontitis patients and 28 healthy controls (HCs) were measured by flow cytometry. RESULTS: Circulating MAIT cells were numerically reduced in periodontitis patients. Moreover, they exhibited higher expression of CD69 and annexin V, together with more increased production of interleukin (IL)-17 and tumour necrosis factor (TNF)-α, in periodontitis patients than in HCs. Interestingly, periodontitis patients had higher frequencies of MAIT cells in gingival tissue than in peripheral blood. In addition, circulating MAIT cells had elevated expression of tissue-homing chemokine receptors such as CCR6 and CXCR6, and the corresponding chemokines (i.e., CCL20 and CXCL16) were more strongly expressed in inflamed gingiva than in healthy gingiva. CONCLUSIONS: This study demonstrates that circulating MAIT cells are numerically deficient with an activated profile toward the production of IL-17 and TNF-α in periodontitis patients. Furthermore, circulating MAIT cells have the potential to migrate to inflamed gingival tissues.

Altered distribution, activation and increased IL-17 production of mucosal-associated invariant T cells in patients with acute respiratory distress syndrome.

OBJECTIVE: Mucosal-associated invariant T (MAIT) cells are a subset of innate-like T cells that are engaged in a number of diseases, but their roles in acute respiratory distress syndrome (ARDS) are not fully examined yet. This study aimed to examine levels and functions of MAIT cells in patients with ARDS. METHODS: Peripheral blood samples from patients with ARDS (n=50) and healthy controls (HCs, n=50) were collected. Levels of MAIT cells, cytokines, CD69, programmed cell death-1 (PD-1) and lymphocyte-activation gene 3 (LAG-3) were measured by flow cytometry. RESULTS: Circulating MAIT cell levels were significantly reduced in patients with ARDS than in HCs. MAIT cell levels were inversely correlated with disease severity and mortality. Cytokine production profiles in MAIT cells showed that percentages of interleukin (IL)-17 producing MAIT cell were significantly higher in patients with ARDS than in HCs. Patients with ARDS exhibited higher expression levels of CD69, PD-1 and LAG-3 in circulating MAIT cells. Moreover, levels of MAIT cells and expression levels of CD69, PD-1 and IL-17 in MAIT cells were higher in bronchoalveolar lavage fluid samples than in peripheral blood samples. Our in vitro experiments showed that MAIT cells triggered macrophages to produce proinflammatory cytokines such as tumour necrosis factor-α, IL-1ß and IL-8. CONCLUSIONS: This study demonstrates that circulating MAIT cells are numerically deficient in patients with ARDS. In addition, MAIT cells were found to be activated, migrate into lung, secrete IL-17 and then stimulate macrophages. These findings suggest that MAIT cells contribute to the worsening of inflammation in the lung of patients with ARDS.

Circulating Plasmacytoid and Conventional Dendritic Cells Are Numerically and Functionally Deficient in Patients With Scrub Typhus.

Background: Dendritic cells (DCs) are specialized antigen-presenting cells known to bridge innate and adaptive immune reactions. However, the relationship between circulating DCs and Orientia tsutsugamushi infection is unclear. Therefore, this study aimed to examine the level and function of plasmacytoid DCs (pDCs) and conventional DCs (cDCs), two subsets of circulating DCs, in scrub typhus patients. Methods: The study included 35 scrub typhus patients and 35 healthy controls (HCs). pDC and cDC levels, CD86 and CD274 expression, and cytokine levels were measured using flow cytometry. Results: Circulating pDC and cDC levels were found to be significantly reduced in scrub typhus patients, which were correlated with disease severity. The patients displayed increased percentages of CD86+ pDCs, CD274+ pDCs, and CD274+ cDCs in the peripheral blood. The alterations in the levels and surface phenotypes of pDCs and cDCs were recovered in the remission state. In addition, the production of interferon (IFN)-α and tumor necrosis factor (TNF)-α by circulating pDCs, and interleukin (IL)-12 and TNF-α by circulating cDCs was reduced in scrub typhus patients. Interestingly, our in vitro experiments showed that the percentages of CD86+ pDCs, CD274+ pDCs, and CD274+ cDCs were increased in cultures treated with cytokines including IFN-γ, IL-12, and TNF-α. Conclusions: This study demonstrates that circulating pDCs and cDCs are numerically deficient and functionally impaired in scrub typhus patients. In addition, alterations in the expression levels of surface phenotypes of pDCs and cDCs could be affected by pro-inflammatory cytokines.

Activation, Deficiency, and Reduced IFN-γ Production of Mucosal-Associated Invariant T Cells in Patients with Inflammatory Bowel Disease.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that can activate either in response to T-cell receptor (TCR) engagement or through activating cytokines and play an important role in autoimmune disorders. The study examined the level and function of MAIT cells in patients with inflammatory bowel disease (IBD). Circulating MAIT cell levels were significantly reduced in IBD patients. This MAIT cell deficiency was correlated with IBD disease activity grades, hemoglobin, and CRP. IFN-γ production of circulating MAIT cells in response to both MHC class 1b-like related protein (MR1)-dependent and -independent stimulations was decreased in IBD patients, which was partially associated with reduced activation of nuclear factor of activated T cells 1 (NFAT1) transcription factor, a main regulator of IFN-γ production. Expression levels of CD69, programmed death-1 (PD-1), and annexin V in MAIT cells were elevated in IBD patients. CCL20, CXCL10, CXCL16, and CCL25 were expressed higher in inflamed intestinal tissues than in noninflamed tissues. This study demonstrates that circulating MAIT cells are activated and numerically and functionally deficient in IBD patients. Furthermore, activated MAIT cells have the potential to migrate to inflamed tissues. These findings suggest an important role of MAIT cells in mucosal immunity in IBD.

Natural Killer T Cells Are Numerically and Functionally Deficient in Patients with Trauma.

Natural killer T (NKT) cells rapidly produce Th1 and Th2 cytokines such as interferon-γ (IFN-γ) and interleukin (IL)-4. This study examined the frequency and function of NKT cells in trauma patients. Frequencies, proliferative responses to α-galactosylceramide (α-GalCer), and Th1/Th2 cytokine secretion levels of NKT cells in peripheral blood mononuclear cells from trauma patients and healthy controls (HC) were measured by flow cytometry. Circulating NKT cell levels were significantly reduced in trauma patients. Proliferation and IFN-γ production of circulating NKT cells in response to α-GalCer were markedly decreased in trauma patients. CD69 expression levels produced by NKT cells were significantly upregulated in trauma patients compared to those in HC. In addition, annexin V+ NKT cells were profoundly increased in trauma patients after α-GalCer stimulation. Trauma patients had higher plasma levels of IL-6, IL-8, and TNF-α compared to HC. In particular, the proliferative response of NKT cells to α-GalCer was significantly decreased in the presence of these cytokines. Such decrease was partially recovered after treatment with blocking antibodies against these cytokines. This study demonstrates that circulating NKT cells are numerically deficient and functionally impaired in IFN-γ production in trauma patients. These findings provide an important insight into the trauma-related innate immune response.

Altered distribution and enhanced osteoclastogenesis of mucosal-associated invariant T cells in gouty arthritis.

OBJECTIVE: This study was designed to investigate the role of mucosal-associated invariant T (MAIT) cells in gouty arthritis (GA) and their effects on osteoclastogenesis. METHODS: Patients with GA (n = 61), subjects with hyperuricaemia (n = 11) and healthy controls (n = 30) were enrolled in this study. MAIT cells, cytokines, CD69, programmed death-1 (PD-1) and lymphocyte-activation gene 3 (LAG-3) levels were measured by flow cytometry. In vitro osteoclastogenesis experiments were performed using peripheral blood mononuclear cells in the presence of M-CSF and RANK ligand. RESULTS: Circulating MAIT cell levels were significantly reduced in GA patients. However, their capacities for IFN-γ, IL-17 and TNF-α production were preserved. Expression levels of CD69, PD-1 and LAG-3 in MAIT cells were found to be elevated in GA patients. In particular, CD69 expression in circulating MAIT cells was increased by stimulation with MSU crystals, suggesting that deposition of MSU crystals might contribute to MAIT cell activation. Interestingly, MAIT cells were found to be accumulated in synovial fluid and infiltrated into gouty tophus tissues within joints. Furthermore, activated MAIT cells secreted pro-resorptive cytokines (i.e. IL-6, IL-17 and TNF-α) and facilitated osteoclastogenesis. CONCLUSION: This study demonstrates that circulating MAIT cells are activated and numerically deficient in GA patients. In addition, MAIT cells have the potential to migrate to inflamed tissues and induce osteoclastogenesis. These findings provide an important role of MAIT cells in the pathogenesis of inflammation and bone destruction in GA patients.

Activation and Impaired Tumor Necrosis Factor-α Production of Circulating Mucosal-Associated Invariant T Cells in Patients with Trauma.

Mucosal-associated invariant T (MAIT) cells rapidly produce proinflammatory cytokines in an innate-like manner and play an important role in controlling the host immune response. This study examined the function of MAIT cells in trauma patients. The expression of cytokines in peripheral blood MAIT cells was measured by flow cytometry. MAIT cells in trauma patients displayed impaired tumor necrosis factor (TNF)-α production, together with elevated CD69 expression. The expression of CD69 was negatively correlated with MAIT cell frequency. These patients had higher plasma levels of interleukin (IL)-12 and IL-18. In particular, CD69 expression of MAIT cells was increased by stimulation with IL-18 in synergy with other proinflammatory cytokines or plasma of trauma patients. The production of TNF-α by MAIT cells was characterized by an initial burst and rapid decline, in contrast to delayed and sustained production of interferon (IFN)-γ. Activated MAIT cells showed a functional defect in the production of TNF-α upon restimulation. This study demonstrates that circulating MAIT cells are activated and functionally impaired in TNF-α production in patients with trauma. The activation and dysfunction of MAIT cells was mediated by proinflammatory cytokines. These findings provide important information underlying the innate immune response of patients with trauma.

Dysfunction of Circulating Natural Killer T Cells in Patients With Scrub Typhus.

Background: Human natural killer T (NKT) cells are known to serve as regulatory and/or effector cells in infectious diseases. However, little is known about the role of NKT cells in Orientia tsutsugamushi infection. Accordingly, the objective of this study was to examine the level and function of NKT cells in patients with scrub typhus. Methods: This study included 62 scrub typhus patients and 62 healthy controls (HCs). NKT cell level and function in peripheral blood samples were measured by flow cytometry. Results: Proliferation of NKT cells and their ability to produce interferon-γ and interleukin-4 (IL-4) were significantly lower in scrub typhus patients compared to those in HCs. However, circulating NKT cell levels were comparable between patients and HCs. Expression levels of CD69, programmed death-1 (PD-1), lymphocyte activation gene-3 (LAG-3), and T-cell immunoglobulin domain and mucin domain-containing molecule-3 (TIM-3) were significantly increased in scrub typhus patients. Elevated expression of CD69, PD-1, LAG-3, and TIM-3, impaired proliferation, and decreased IL-4 production by NKT cells were recovered in the remission phase. Conclusions: This study demonstrates that circulating NKT cells are numerically preserved but functionally impaired in scrub typhus patients. In addition, NKT cell dysfunction is recovered in the remission phase.

Increased level and interferon-γ production of circulating natural killer cells in patients with scrub typhus.

BACKGROUND: Natural killer (NK) cells are essential immune cells against several pathogens. Not much is known regarding the roll of NK cells in Orientia tsutsugamushi infection. Thus, this study aims to determine the level, function, and clinical relevance of NK cells in patients with scrub typhus. METHODOLOGY/PRINCIPAL FINDINGS: This study enrolled fifty-six scrub typhus patients and 56 health controls (HCs). The patients were divided into subgroups according to their disease severity. A flow cytometry measured NK cell level and function in peripheral blood. Circulating NK cell levels and CD69 expressions were significantly increased in scrub typhus patients. Increased NK cell levels reflected disease severity. In scrub typhus patients, tests showed their NK cells produced higher amounts of interferon (IFN)-γ after stimulation with interleukin (IL)-12 and IL-18 relative to those of HCs. Meanwhile, between scrub typhus patients and HCs, the cytotoxicity and degranulation of NK cells against K562 were comparable. CD69 expressions were recovered to the normal levels in the remission phase. CONCLUSIONS: This study shows that circulating NK cells are activated and numerically increased, and they produced more IFN-γ in scrub typhus patients.

Deficiencies of Circulating Mucosal-associated Invariant T Cells and Natural Killer T Cells in Patients with Multiple Trauma.

Mucosal-associated invariant T (MAIT) cells and natural killer T (NKT) cells are known to play important roles in autoimmunity, infectious diseases and cancers. However, little is known about the roles of these invariant T cells in multiple trauma. The purposes of this study were to examine MAIT and NKT cell levels in patients with multiple trauma and to investigate potential relationships between these cell levels and clinical parameters. The study cohort was composed of 14 patients with multiple trauma and 22 non-injured healthy controls (HCs). Circulating MAIT and NKT cell levels in the peripheral blood were measured by flow cytometry. The severity of injury was categorised according to the scoring systems, such as Acute Physiology and Chronic Health Evaluation (APACHE) II score, Simplified Acute Physiology Score (SAPS) II, and Injury Severity Score (ISS). Circulating MAIT and NKT cell numbers were significantly lower in multiple trauma patients than in HCs. Linear regression analysis showed that circulating MAIT cell numbers were significantly correlated with age, APACHE II, SAPS II, ISS category, hemoglobin, and platelet count. NKT cell numbers in the peripheral blood were found to be significantly correlated with APACHE II, SAPS II, and ISS category. This study shows numerical deficiencies of circulating MAIT cells and NKT cells in multiple trauma. In addition, these invariant T cell deficiencies were found to be associated with disease severity. These findings provide important information for predicting the prognosis of multiple trauma.

Biomarkers for discrimination between latent tuberculosis infection and active tuberculosis disease.

OBJECTIVE: We aimed to determine whether combinations of multiplex cytokine responses could differentiate Mycobacterium tuberculosis (Mtb) infection states. METHODS: Mtb-specific antigen-induced and unstimulated cytokines were measured by Luminex assay in supernatants of QuantiFERON® Gold In-Tube assay (QFT) in 48 active pulmonary TB patients (TB), 15 latent TB infection subjects (LTBI), and 13 healthy controls (HCs). RESULTS: Among the 29 cytokines, eight Mtb antigen-specific biomarkers (GM-CSF, IFN-γ, IL-1RA, IL-2, IL-3, IL-13, IP-10, and MIP-1ß) in the Mtb-infected group were significantly different from those of the HCs. Five Mtb-specific biomarkers (EGF, GM-CSF, IL-5, IL-10, and VEGF), two unstimulated biomarkers (TNF-α[Nil] and VEGF[Nil]), and one Mtb-specific biomarker ratio (IL-2/IFN-γ) showed significant differences between active TB and LTBI. Three unstimulated biomarkers (IL-8[Nil], IL-13[Nil], and VEGF[Nil]) and 5 Mtb-specific biomarkers (IFN-γ, IL-2, IL-3, IP-10, and VEGF) were significantly different between active TB and non-active TB groups. Combinations of three cytokine biomarkers resulted in the accurate prediction of 92.1-93.7% of Mtb-infected cases and 92.3-100% of HCs, respectively. Moreover, combinations of five biomarkers accurately predicted 90.9-100% of active TB cases and 80-100% of LTBI subjects, respectively. In discriminating between active TB and non-active TB regardless of QFT results, combinations of six biomarkers predicted 79.2-95.8% of active TB cases and 67.9-89.3% of non-active TB subjects. CONCLUSIONS: Taken together, our data suggest that combinations of whole blood Mtb antigen-dependent cytokines could serve as biomarkers to determine TB disease states. Especially, VEGF is highlighted as a key biomarker for reflecting active TB, irrespective of stimulation.

Clinical relevance of circulating mucosal-associated invariant T cell levels and their anti-cancer activity in patients with mucosal-associated cancer.

Mucosal-associated invariant T (MAIT) cells are an antimicrobial MR1-restricted T cell subset and play an important role in immune defense response to bacteria. However, little is known about the role of MAIT cells in cancer. The aims of this study were to examine the level and function of MAIT cells in cancer patients and to evaluate the clinical relevance of MAIT cell levels. Ninety-nine patients with cancer and 20 healthy controls were included in this study. Circulating MAIT cell levels were significantly reduced in patients with mucosal-associated cancers (MACs), such as gastric, colon and lung cancers, but their capacities for IFN-γ, IL-17, or TNF-α production were preserved. This MAIT cell deficiency was significantly correlated with N staging and carcinoembryonic antigen level. Percentages of MAIT cells were significantly higher in cancer tissue than in peripheral blood and immunofluorescent labeling showed MAIT cell infiltration into colon cancer tissues. Circulating MAIT cells exhibited high levels of CCR6 and CXCR6, and their corresponding chemokines, such as CCL20 and CXCL16, were strongly expressed in colon cancer tissues. Activated MAIT cells not only had lymphokine-activated killer activity, but they also had direct cytotoxicity on K562 cells via degranulation of granzyme B and perforin. This study primarily demonstrates that circulating MAIT cells are reduced in MAC patients due to migration to mucosal cancer tissues and they have the potential to kill cancer cells. In addition, this circulating MAIT cell deficiency is related to the degree of cancer progression in mucosal tissues.

Activation, Impaired Tumor Necrosis Factor-α Production, and Deficiency of Circulating Mucosal-Associated Invariant T Cells in Patients with Scrub Typhus.

BACKGROUND: Mucosal-associated invariant T (MAIT) cells contribute to protection against certain microorganism infections. However, little is known about the role of MAIT cells in Orientia tsutsugamushi infection. Hence, the aims of this study were to examine the level and function of MAIT cells in patients with scrub typhus and to evaluate the clinical relevance of MAIT cell levels. METHODOLOGY/PRINCIPAL FINDINGS: Thirty-eight patients with scrub typhus and 53 health control subjects were enrolled in the study. The patients were further divided into subgroups according to disease severity. MAIT cell level and function in the peripheral blood were measured by flow cytometry. Circulating MAIT cell levels were found to be significantly reduced in scrub typhus patients. MAIT cell deficiency reflects a variety of clinical conditions. In particular, MAT cell levels reflect disease severity. MAIT cells in scrub typhus patients displayed impaired tumor necrosis factor (TNF)-α production, which was restored during the remission phase. In addition, the impaired production of TNF-α by MAIT cells was associated with elevated CD69 expression. CONCLUSIONS: This study shows that circulating MAIT cells are activated, numerically deficient, and functionally impaired in TNF-α production in patients with scrub typhus. These abnormalities possibly contribute to immune system dysregulation in scrub typhus infection.

Mucosal-Associated Invariant T Cell Deficiency in Chronic Obstructive Pulmonary Disease.

Mucosal-associated invariant T (MAIT) cells have been reported to play an important role in mucosal immunity. However, little is known about the roles of MAIT cells in chronic obstructive pulmonary disease (COPD). The aims of this study were to examine the levels of circulating MAIT cells and their subsets in COPD patients and to investigate the potential relationship between clinical parameters and MAIT cell levels. Forty-five COPD patients and 57 healthy control subjects were enrolled in the study. Circulating MAIT cells and their subset levels in the peripheral blood were measured by flow cytometry. Disease grades were classified according to the GOLD criteria for the assessment of severity of COPD. Circulating MAIT cell levels were found to be significantly reduced in COPD patients. In particular, this MAIT cell deficiency was more prominent in CD8+ and double-negative T cell subsets. Interestingly, elevated serum C-reactive protein level and reduced FEV1/FVC ratio were associated with MAIT cell deficiency in COPD patients. Furthermore, the circulating MAIT levels were found to be significantly lower in patients with moderate to severe COPD than in patients with mild COPD. Our data shows that MAIT cells are numerically deficient in the peripheral blood of patients with COPD. In addition, this MAIT cell deficiency was found to reflect inflammatory activity and disease severity. These findings provide important information for monitoring the changes in MAIT cell levels and for predicting the prognosis during the disease course.

Micro-Ribonucleic Acid Profiles From Microarray in Ankylosing Spondylitis.

OBJECTIVES: This study aims to detect candidate micro-ribonucleic acids (miRNAs) from microarray within peripheral blood mononuclear cells and synovial fluid mononuclear cells in patients with ankylosing spondylitis (AS). PATIENTS AND METHODS: Samples from three AS patients (3 males, mean age 37.3±2.5 years; range 35 to 40 years) and three healthy controls (3 males, mean age 39.0±2.6 years; range 37 to 42 years) were obtained for miRNA microarray. The microarray experiment proceeded only when the quality of total RNAs were considered to have "passed", and their integrity was good by total RNA quality control using Agilent Bioanalyzer 2100. Hierarchical clustering was performed to understand the impact of the storage condition on the miRNA expression profiles. MiScript primer assays were used for semiquantitative determination of the expression of human miRNAs to validate results from miRNA microarray. RESULTS: A total of 887 miRNAs were screened by microarray among groups. After normalization of the raw data, we noted that the expression of five miRNAs was significantly lower (fold change ≤0.5 and p≤0.05) and only hsa-miR-424-5p was significantly higher in AS peripheral blood mononuclear cell (fold change ≥2 and p≤0.05). In AS synovial fluid mononuclear cells, we identified that expressions of 16 miRNAs were significantly down regulated whereas only hsa-miR-424-5p was significantly upregulated (fold change ≥2 and p≤0.05). All above-mentioned miRNAs were reevaluated for further validation. Finally, significantly increased hsa-miR-424-5p and decreased hsa-miR-377 were found in synovial fluid mononuclear cells from AS patients compared with healthy controls. Based on target prediction programs and published papers, potential target genes and its pathways were screened. CONCLUSION: miR-424-5p was increased and miR-377 was decreased in synovial fluid mononuclear cells from patients with AS. These two miRs might have functional roles in patients with arthritis via different pathways.

Dysregulated osteoclastogenesis is related to natural killer T cell dysfunction in rheumatoid arthritis.

OBJECTIVE: To investigate the role played by natural killer T (NKT) cells in osteoclastogenesis and their effects on inflammatory bone destruction. METHODS: Patients with rheumatoid arthritis (RA) (n = 25) and healthy controls (n = 12) were enrolled in this study. In vitro osteoclastogenesis experiments were performed using peripheral blood mononuclear cells (PBMCs) in the presence of macrophage colony-stimulating factor and RANKL. PBMCs were cultured in vitro with α-galactosylceramide (αGalCer), and proliferation indices of NKT cells were estimated by flow cytometry. In vivo effects of αGalCer-stimulated NKT cells on inflammation and bone destruction were determined in mice with collagen-induced arthritis. RESULTS: In vitro osteoclastogenesis was found to be significantly inhibited by αGalCer in healthy controls but not in RA patients. Proliferative responses of NKT cells and STAT-1 phosphorylation in monocytes in response to αGalCer were impaired in RA patients. Notably, αGalCer-stimulated NKT cells inhibited osteoclastogenesis mainly via interferon-γ production in a cytokine-dependent manner (not by cell-cell contact) and down-regulated osteoclast-associated genes. Mice treated with αGalCer showed less severe arthritis and reduced bone destruction. Moreover, proinflammatory cytokine expression in arthritic joints was found to be reduced by αGalCer treatment. CONCLUSION: This study primarily demonstrates that αGalCer-stimulated NKT cells have a regulatory effect on osteoclastogenesis and a protective effect against inflammatory bone destruction. However, it also shows that these effects of αGalCer are diminished in RA patients and that this is related to NKT cell dysfunction. These findings provide important information for those searching for novel therapeutic strategies to prevent bone destruction in RA.

Deficiencies of Circulating Mucosal-associated Invariant T Cells and Natural Killer T Cells in Patients with Acute Cholecystitis.

Mucosal-associated invariant T (MAIT) cells and natural killer T (NKT) cells are known to play crucial roles in a variety of diseases, including autoimmunity, infectious diseases, and cancers. However, little is known about the roles of these invariant T cells in acute cholecystitis. The purposes of this study were to examine the levels of MAIT cells and NKT cells in patients with acute cholecystitis and to investigate potential relationships between clinical parameters and these cell levels. Thirty patients with pathologically proven acute cholecystitis and 47 age- and sex-matched healthy controls were enrolled. Disease grades were classified according to the revised Tokyo guidelines (TG13) for the severity assessment for acute cholecystitis. Levels of MAIT and NKT cells in peripheral blood were measured by flow cytometry. Circulating MAIT and NKT cell numbers were significantly lower in acute cholecystitis patients than in healthy controls, and these deficiencies in MAIT cells and NKT cell numbers were associated with aging in acute cholecystitis patients. Notably, a reduction in NKT cell numbers was found to be associated with severe TG13 grade, death, and high blood urea nitrogen levels. The study shows numerical deficiencies of circulating MAIT and NKT cells and age-related decline of these invariant T cells. In addition, NKT cell deficiency was associated with acute cholecystitis severity and outcome. These findings provide an information regarding the monitoring of these changes in circulating MAIT and NKT cell numbers during the course of acute cholecystitis and predicting prognosis.

Mucosal-associated invariant T cells are numerically and functionally deficient in patients with mycobacterial infection and reflect disease activity.

Mucosal-associated invariant T (MAIT) cells contribute to protection against certain microorganism infections. The aims of this study were to examine the levels of MAIT cells in pulmonary tuberculosis (TB) and nontuberculous mycobacteria (NTM) lung disease patients, to evaluate the clinical relevance of MAIT cell levels, and to investigate the functions of MAIT cells. Patients with pulmonary TB (n = 35), NTM (n = 29), and healthy controls (n = 75) were enrolled in the study. MAIT cell levels and functions were measured by flow cytometry. Circluating MAIT cell levels were found to be reduced in TB and NTM patients. MAIT cell deficiency reflects a variety of clinical conditions. In particular, MAIT cell numbers were significantly correlated with sputum AFB positivity, extent of disease, hemoglobin levels, lymphocyte counts, CRP and ESR levels. MAIT cells in TB patients failed to produce interferon-γ irrespective of the mode of stimulation, whereas NTM patients displayed a defect in MR1-dependent signaling pathway. Notably, an elevated expression of programmed death-1 was also associated with MAIT cell deficiency in TB. This study shows that MAIT cells are numerically and functionally deficient in TB and NTM patients and these deficiencies could contribute to immune system dysreguation in mycobacterial infection.