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1.  MicroRNAs Associated with Shoulder Tendon Matrisome Disorganization in Glenohumeral Arthritis 
PLoS ONE  2016;11(12):e0168077.
The extracellular matrix (ECM) provides core support which is essential for the cell and tissue architectural development. The role of ECM in many pathological conditions has been well established and ECM-related abnormalities leading to serious consequences have been identified. Though much has been explored in regards to the role of ECM in soft tissue associated pathologies, very little is known about its role in inflammatory disorders in tendon. In this study, we performed microRNA (miRNA) expression analysis in the long head of the human shoulder biceps tendon to identify key genes whose expression was altered during inflammation in patients with glenohumeral arthritis. We identified differential regulation of matrix metalloproteinases (MMPs) that could be critical in collagen type replacement during tendinopathy. The miRNA profiling showed consistent results between the groups and revealed significant changes in the expression of seven different miRNAs in the inflamed tendons. Interestingly, all of these seven miRNAs were previously reported to have either a direct or indirect role in regulating the ECM organization in other pathological disorders. In addition, these miRNAs were also found to alter the expression levels of MMPs, which are the key matrix degrading enzymes associated with ECM-related abnormalities and pathologies. To our knowledge, this is the first report which identifies specific miRNAs associated with inflammation and the matrix reorganization in the tendons. Furthermore, the findings also support the potential role of these miRNAs in altering the collagen type ratio in the tendons during inflammation which is accompanied with differential expression of MMPs.
doi:10.1371/journal.pone.0168077
PMCID: PMC5161352  PMID: 27992561
2.  TREM-1, HMGB1 and RAGE in the Shoulder Tendon: Dual Mechanisms for Inflammation Based on the Coincidence of Glenohumeral Arthritis 
PLoS ONE  2016;11(10):e0165492.
Rotator cuff injury (RCI) is a major musculoskeletal disorder in the adult population where inflammation and pain are major contributing factors. Coincidence of other clinical conditions like glenohumeral arthritis aggravates inflammation and delays the healing response. The mechanism and signaling factors underlying the sustenance of inflammation in the rotator cuff joint are largely unknown. The present article aims to elucidate the involvement of inflammatory molecule, TREM-1 (Triggering Receptors Expressed on Myeloid cells-1), and danger-associated molecular patterns (DAMPs), including high mobility group protein 1 (HMGB-1) and RAGE (receptor for advanced glycation end products), in the setting of RCI with respect to the severity of glenohumeral arthritis. Biceps tendons (15 specimens) from the shoulder and blood (11 samples) from patients with glenohumeral arthritis (Group-1, n = 4) and without glenohumeral arthritis (Group-2, n = 11) after RCI surgery were obtained for the study. Molecular and morphological alterations between the groups were compared using histology, immunofluorescence, RT-PCR and flow cytometry. MRI and histomorphology assessment revealed severe inflammation in Group-1 patients while in Group-2 ECM disorganization was prominent without any hallmarks of inflammation. A significant increase in TREM-1 expression in circulating neutrophils and monocytes was observed. Elevated levels of TREM-1, HMGB-1 and RAGE in Group-1 patients along with CD68+ and CD16+ cells confirmed DAMP-mediated inflammation. Expression of TREM-1 in the tendon of Group-2 patients even in the absence of immune cells presented a new population of TREM-expressing cells that were confirmed by real-time PCR analysis and immunofluorescence. Expression of HMGB-1 and RAGE in the biceps tendon from the shoulder of patients without glenohumeral arthritis implied TREM-1-mediated inflammation without involving immune cells, whereas in patients with glenohumeral arthritis, infiltration and the activation of the immune cells, primarily macrophages, release mediators to induce inflammation. This could be the reason for ECM disorganization without the classical signs of inflammation in patients without glenohumeral arthritis.
doi:10.1371/journal.pone.0165492
PMCID: PMC5085056  PMID: 27792788
3.  Vitamin D attenuates inflammation, fatty infiltration, and cartilage loss in the knee of hyperlipidemic microswine 
Background
Osteoarthritis (OA) of the knee joint is a degenerative process resulting in cartilage loss. Recent evidence suggests that OA is not merely a disease of cartilage but a disease of the entire knee joint and that inflammation may play an important role. OA has been associated with vitamin D deficiency. Vitamin D as an immunomodulator and anti-inflammatory agent may attenuate inflammation in the knee. The aim of this study was to assess the anti-inflammatory effect of vitamin D on inflammation in the knee.
Methods
This study was conducted with 13 microswine on a high cholesterol diet categorized into three groups of vitamin D-deficient, vitamin D-sufficient, and vitamin D supplementation. After 1 year, microswine were killed, and their knee joint tissues were harvested. Histological and immunofluorescence studies were carried out on the tissue specimens to evaluate the effect of vitamin D status.
Results
Histological and immunofluorescence studies of the knee joint tissues showed (1) increased inflammation in the knee joint tissues, (2) fatty infiltration in quadriceps muscle, patellar tendon, and collateral ligaments, and (3) chondrocyte clustering in the vitamin D-deficient and vitamin D-sufficient groups compared with the vitamin D supplementation group. Architectural distortion of the quadriceps muscle, patellar tendon, and collateral ligaments was also seen in the areas of inflammatory foci and fatty infiltration in the vitamin D-deficient group.
Conclusions
Decreased inflammation and fatty infiltration in the vitamin D supplementation group suggest the potential role of vitamin D in attenuating inflammation and fatty infiltration as well as in protecting the architecture of the tissue in the knee joint.
Electronic supplementary material
The online version of this article (doi:10.1186/s13075-016-1099-6) contains supplementary material, which is available to authorized users.
doi:10.1186/s13075-016-1099-6
PMCID: PMC5022245  PMID: 27624724
Osteoarthritis; Vitamin D deficiency; Cartilage loss; Inflammation; Fatty infiltration; Vitamin D supplementation; Inflammation attenuation
4.  Vitamin D Supplementation Reduces Intimal Hyperplasia and Restenosis following Coronary Intervention in Atherosclerotic Swine 
PLoS ONE  2016;11(6):e0156857.
Vitamin D is a fat-soluble steroid hormone that activates vitamin D receptor to regulate multiple downstream signaling pathways and transcription of various target genes. There is an association between vitamin D deficiency and increased risk for cardiovascular disease. However, most of the studies are observational and associative in nature with limited data on clinical application. Thus, there is a need for more prospective randomized controlled studies to determine whether or not vitamin D supplementation provides cardiovascular protection. In this study, we examined the effects of the deficiency and supplementation of vitamin D on coronary restenosis following coronary intervention in atherosclerotic Yucatan microswine. Twelve Yucatan microswine were fed vitamin D-deficient (n = 4) or -sufficient (n = 8) high cholesterol diet for 6-months followed by coronary intervention. Post-intervention, swine in the vitamin D-sufficient high cholesterol diet group received daily oral supplementation of either 1,000 IU (n = 4) or 3,000 IU (n = 4) vitamin D3. Six months later, optical coherence tomography (OCT) was performed to monitor the development of intimal hyperplasia and restenosis. Animals were euthanized to isolate arteries for histomorphometric and immunohistochemical studies. Animals had graded levels of serum 25(OH)D; vitamin D-deficient (15.33 ± 1.45 ng/ml), vitamin D-sufficient + 1,000 IU oral vitamin D post-intervention (32.27 ± 1.20 ng/ml), and vitamin D-sufficient + 3,000 IU oral vitamin D post-intervention (51.00 ± 3.47 ng/ml). Findings from the OCT and histomorphometric studies showed a decrease in intimal hyperplasia and restenosis in vitamin D-supplemented compared to vitamin D-deficient swine. Vitamin D supplementation significantly decreased serum levels of TNF-α and IFN-γ, upregulated serum levels of IL-10, and had no effect on serum IL-6 levels. These findings suggest that vitamin D supplementation limits neointimal formation following coronary intervention in atherosclerotic swine and provide the support for vitamin D supplementation to protect against the development of coronary restenosis.
doi:10.1371/journal.pone.0156857
PMCID: PMC4894559  PMID: 27271180
5.  Immunological Basis in the Pathogenesis of Intrahepatic Cholestasis of Pregnancy 
Summary
Intrahepatic cholestasis of pregnancy poses a great risk to both maternal and fetal health. Despite extensive research, much of the pathogenesis of this disorder is unknown. The increase in bile acids observed in patients with intrahepatic cholestasis of pregnancy has been noted to cause a change in the immune system from the normally mediated TH2 response to one that is more oriented towards TH1. In this literature review, we have critically reviewed the current literature regarding the changes in the immune system and the potential effects of immunological changes in the management of the patient. The current treatment, ursodeoxycholic acid, is also discussed along with potential combination therapies and future directions for research.
doi:10.1586/1744666X.2016.1101344
PMCID: PMC4893793  PMID: 26469633
Intrahepatic cholestasis of pregnancy; Bile acids; S-adenosylmethionine; T-lymphocytes; Ursodeoxycholic acid
6.  Arterial Catheterization and Infection: Toll-like receptors in defense against microorganisms and therapeutic implications 
Radial artery catheterization has become a preferred route over femoral artery catheterization, in order to monitor the blood pressure of hemodynamically unstable patients or for repeated sampling of arterial blood gases. While the incidence of catheter-related infection is lower in the radial artery than the femoral artery, infection remains a major issue that requires attention. In this review of the literature, we discuss infectious complications of radial artery catheterization, with a focus on various risk factors and establishing the most common causative agents. We also critically review the role of the innate immune system involving Toll-like receptors (TLRs) in host-defense, with the goal of establishing a common pathway used by the innate immune system via TLRs to combat the pathogens that most commonly cause infection in radial artery catheterization. If this pathway can be therapeutically manipulated to preemptively attack pathogenic agents, immunomodulation may be an option in reducing the incidence of infection in this procedure.
doi:10.1111/cts.12320
PMCID: PMC4703511  PMID: 26271949
Arterial catheterization; Innate immunity; Radial artery; Toll-like receptors
7.  Diagnostic value of tumor antigens in malignant pleural effusion: A meta-analysis 
The diagnostic value of tumor markers, CEA, CA 15-3, CA 19-9, CA 125, CYFRA, and NSE in pleural fluid to differentiate between benign and malignant pleural effusion (MPE) has not yet been clearly established. A review of English language studies using human subjects was performed. Sensitivity and specificity values of the chosen tumor markers were pooled using a random effects model to generate hierarchical summary receiver-operator curves to determine the diagnostic performance of each tumor marker. A total of 49 studies were included in the final analysis. Pooled sensitivity and specificity values for chosen tumor markers for diagnosing MPE are as follows: CEA, 0.549/0.962; CA 15-3, 0.507/0.983; CA 19-9, 0.376/0.980; CA 125, 0.575/0.928; CYFRA, 0.625/0.932; NSE, 0.613/0.884. The use of individual tumor markers in diagnosing MPE has many benefits (cost, invasiveness, etc.). While these tumor markers exhibit high specificity, the low sensitivity of each marker limits the diagnostic value. We conclude that tumor markers used individually are of insufficient diagnostic accuracy for clinical use. Tumor markers used in various combinations or from serum may have some potential worth further investigation.
doi:10.1016/j.trsl.2015.04.006
PMCID: PMC4608857  PMID: 25953662
8.  Immune-mediated Adverse Effects of Anti-CTLA-4 Antibody Therapy in Metastatic Melanoma 
Ipilimumab, an antibody that blocks cytotoxic T lymphocyte-associated antigen-4 (CTLA-4; CD152), was approved by the Food and Drug Administration (FDA) in 2011 for the treatment of unresectable stage III or IV malignant melanoma. Although the addition of this particular immunotherapy has broadened treatment options, immune-related adverse events (irAEs) are associated with ipilimumab therapy, including dermatologic effects, colitis and diarrhea, endocrine effects, hepatotoxicity, ocular effects, renal effects, neurologic effects, and others. In this article, a critical evaluation of the underlying mechanisms of irAEs associated with anti-CTLA-4 therapy is presented. Additionally, potentially beneficial effects of combinational therapies to alleviate ipilimumab-induced irAEs in malignant melanoma are discussed. Future research is warranted to elucidate the efficacy of such combination therapies as well as specific biomarkers that would help to predict a clinical response to ipilimumab in patients with malignant melanoma.
doi:10.1016/j.trsl.2015.06.005
PMCID: PMC4609598  PMID: 26118951
Ipilimumab; CTLA-4; malignant melanoma; immune-related adverse events
9.  Vitamin D Protects Against Atherosclerosis via Regulation of Cholesterol Efflux and Macrophage Polarization in Hypercholesterolemic Swine 
Objective
Prevalence of vitamin D-deficiency and its association with the risk of cardiovascular disease prompted us to evaluate the effect of vitamin D status on lipid metabolism and atherosclerosis in hypercholesterolemic microswine.
Approach and Results
Yucatan microswine were fed with vitamin D-deficient (0IU/d), vitamin D-sufficient (1,000IU/d) or vitamin D-supplemented (3,000IU/d) high cholesterol diet for 48 weeks. Serum lipids and 25(OH)-cholecalciferol levels were measured biweekly. Histology and biochemical parameters of liver and arteries were analyzed. Effect of 1,25(OH)2D3 on cholesterol metabolism was examined in human HepG2 and THP-1 macrophage-derived foam cells. Vitamin D-deficiency decreased plasma HDL levels, expression of liver-X-receptors (LXRs), ATP binding cassette transporter A1 (ABCA1) and ABCG1, and promoted cholesterol accumulation and atherosclerosis in hypercholesterolemic microswine. Vitamin D promoted nascent HDL formation in HepG2 cells via ABCA1-mediated cholesterol efflux. CYP27B1 and VDR were predominantly present in the CD206 + M2 macrophage foam cell-accumulated cores in coronary artery plaques. 1,25(OH)2D3 increased the expression of LXRs, ABCA1, ABCG1, and promoted cholesterol efflux in THP-1 macrophage-derived foam cells. 1,25(OH)2D3 decreased intracellular free cholesterol and polarized macrophages to M2-phenotype with decreased expression of TNF-α, IL-1β, IL-6 under LPS-stimulation. 1,25(OH)2D3 markedly induced CYP27A1 expression via a VDR-dependent JNK1/2 signaling pathway and increased 27-hydroxycholesterol levels, which induced LXRs, ABCA1 and ABCG1 expression, stimulated cholesterol efflux that was inhibited by VDR antagonist and JNK1/2 signaling inhibitor in THP-1 macrophage-derived foam cell.
Conclusion
Vitamin D protects against atherosclerosis in hypercholesterolemic swine via controlling cholesterol efflux and macrophage polarization via increased CYP27A1 activation.
doi:10.1161/ATVBAHA.115.306132
PMCID: PMC4618721  PMID: 26381871
Atherosclerosis; Cholesterol efflux; Macrophage polarization; Vitamin D
10.  Atherogenic Cytokines Regulate VEGF-A-Induced Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells into Endothelial Cells 
Stem Cells International  2015;2015:498328.
Coronary artery stenting or angioplasty procedures frequently result in long-term endothelial dysfunction or loss and complications including arterial thrombosis and myocardial infarction. Stem cell-based therapies have been proposed to support endothelial regeneration. Mesenchymal stem cells (MSCs) differentiate into endothelial cells (ECs) in the presence of VEGF-A in vitro. Application of VEGF-A and MSC-derived ECs at the interventional site is a complex clinical challenge. In this study, we examined the effect of atherogenic cytokines (IL-6, TNFα, and Ang II) on EC differentiation and function. MSCs (CD44+, CD73+, CD90+, CD14−, and CD45−) were isolated from the bone marrow of Yucatan microswine. Naïve MSCs cultured in differentiation media containing VEGF-A (50 ng/mL) demonstrated increased expression of EC-specific markers (vWF, PECAM-1, and VE-cadherin), VEGFR-2 and Sox18, and enhanced endothelial tube formation. IL-6 or TNFα caused a dose-dependent attenuation of EC marker expression in VEGF-A-stimulated MSCs. In contrast, Ang II enhanced EC marker expression in VEGF-A-stimulated MSCs. Addition of Ang II to VEGF-A and IL-6 or TNFα was sufficient to rescue the EC phenotype. Thus, Ang II promotes but IL-6 and TNFα inhibit VEGF-A-induced differentiation of MSCs into ECs. These findings have important clinical implications for therapies intended to increase cardiac vascularity and reendothelialize coronary arteries following intervention.
doi:10.1155/2015/498328
PMCID: PMC4464597  PMID: 26106428
11.  Effects of matrix metalloproteinases on the fate of mesenchymal stem cells 
Mesenchymal stem cells (MSCs) have great potential as a source of cells for cell-based therapy because of their ability for self-renewal and differentiation into functional cells. Moreover, matrix metalloproteinases (MMPs) have a critical role in the differentiation of MSCs into different lineages. MSCs also interact with exogenous MMPs at their surface, and regulate the pericellular localization of MMP activities. The fate of MSCs is regulated by specific MMPs associated with a key cell lineage. Recent reports suggest the integration of MMPs in the differentiation, angiogenesis, proliferation, and migration of MSCs. These interactions are not fully understood and warrant further investigation, especially for their application as therapeutic tools to treat different diseases. Therefore, overexpression of a single MMP or tissue-specific inhibitor of metalloproteinase in MSCs may promote transdifferentiation into a specific cell lineage, which can be used for the treatment of some diseases. In this review, we critically discuss the identification of various MMPs and the signaling pathways that affect the differentiation, migration, angiogenesis, and proliferation of MSCs.
doi:10.1186/s13287-016-0393-1
PMCID: PMC5016871  PMID: 27612636
Mesenchymal stem cells; Matrix metalloproteinases; Extracellular matrix; Osteogenic differentiation; Adipogenic differentiation; Chondrogenic differentiation; Migration; Angiogenesis; Proliferation
12.  Vitamin D machinery and metabolism in porcine adipose-derived mesenchymal stem cells 
Background
Vitamin D, a hormone once thought to have a role limited to calcium homeostasis and bone mineralization, has pleiotropic effects on different types of cells. Vitamin D receptors are reported in vascular smooth muscle cells, endothelial cells, and cardiomyocytes. Adipose-derived MSCs (ADMSCs) are multipotent cells with the capacity to differentiate into cells of different lineages. To our knowledge, the presence of vitamin D machinery on porcine ADMSCs has not yet been examined. In this study, we investigated the presence of vitamin D machinery and metabolism in ADMSCs by analyzing the expression levels of vitamin D receptor (VDR), vitamin D metabolizing enzymes (CYP24A1 and CYP27B1) after in vitro stimulation with active vitamin D, calcitriol.
Methods and results
ADMSCs isolated from porcine adipose tissue were characterized by positive staining for ADMSC markers, CD44, CD73, and CD90, and negative staining for macrophage marker CD11b and hematopoietic stem cell markers CD34 and CD45, and trilineage differentiation to osteocytes, chondrocytes, and adipocytes. No cytotoxicity was observed when MSCs were stimulated with 0.1–10 nM calcitriol. The ADMSCs were analyzed for mRNA and protein expression of CYP24A1, CYP27B1, and VDR by immunostaining, qPCR, and ELISA. A significant increase (p <0.01) in the mRNA expression of CYP24A1, CYP27B1, and VDR was observed after stimulation of ADMSCs with calcitriol (10 nM). The in vitro time-dependent effect of calcitriol (10 nM) on the components of vitamin D machinery in cultured MSCs was determined by qPCR. The VDR and CYP27B1 expression peaked at 3 h and CYP24A1 at 24 h, respectively. The in vitro biosynthesis of 1, 25(OH)2D3 by ADMSCs was analyzed by ELISA and Western blot. The levels of the active form of vitamin D were significantly decreased once the CYP enzymes were inhibited (p <0.01), demonstrating the ability of ADMSCs to convert inactive vitamin D into active vitamin D for cellular action.
Conclusions
Porcine ADMSCs possess vitamin D hydrolases and VDR to metabolize and respond to vitamin D. Hence, in vivo circulating 25-hydroxy vitamin D levels may have a significant role in regulating the differentiation of ADMSCs into different lineages, which might assist in stem cell-based therapy.
doi:10.1186/s13287-016-0382-4
PMCID: PMC4988022  PMID: 27530414
Vitamin D3; Calcitriol; Adipose-derived mesenchymal stem cell; CYP24A; CYP27B1; Vitamin D receptor
13.  Epigenetics in the Pathogenesis of Esophageal Adenocarcinoma 
Epigenetic influences, such as DNA methylation, histone acetylation and upregulation/downregulation of genes by microRNAs, change the genetic makeup of an individual without affecting DNA base pair sequences. Indeed, epigenetic changes play an integral role in the progression from normal esophageal mucosa to Barrett’s esophagus to esophageal adenocarcinoma via dysplasia- metaplasia-neoplasia sequence. Many genes involved in esophageal adenocarcinoma display hypermethylation, leading to their downregulation. The classes of these genes include cell cycle control, DNA and growth factor repair, tumor suppressors, anti-metastasis, WNT-related genes, and pro-apoptotic genes. Histone acetylation in the pathophysiology of esophageal diseases has not been thoroughly investigated, and its critical role in the development of esophageal adenocarcinoma is less defined. Many microRNAs have been associated with the development of Barrett’s esophagus and esophageal adenocarcinoma. Here, we critically addressed the specific steps most closely influenced by microRNAs in the progression from Barrett’s esophagus to esophageal adenocarcinoma. However, microRNAs can target up to hundreds of genes, making it difficult to correlate directly with a given phenotype of the disease. Esophageal adenocarcinoma progressing from pre-malignant condition of Barrett’s esophagus carries an extremely poor prognosis. Risk stratification for patients based on their epigenetic profiles may be useful in providing more targeted and directed treatment to patients.
doi:10.1111/cts.12242
PMCID: PMC4429045  PMID: 25388215
DNA methylation; epigenetics; Barrett’s esophagus; esophageal adenocarcinoma; esophageal squamous cell carcinoma; histone acetylation; miRNA
14.  Functional Constituents of a Local Serotonergic System, Intrinsic to the Human Coronary Artery Smooth Muscle Cells 
Molecular biology reports  2015;42(8):1295-1307.
Human coronary artery smooth muscle cells (HCASMCs) play an important role in the pathogenesis of coronary atherosclerosis and coronary artery diseases (CAD). Serotonin is a mediator known to produce vascular smooth muscle cell (VSMC) mitogenesis and contribute to coronary atherosclerosis. We hypothesize that the human coronary artery smooth muscle cell possesses certain functional constituents of the serotonergic system such as: tryptophan hydroxylase and serotonin transporter. Our aim was to examine the presence of functional tryptophan hydroxylase-1 (TPH1) and serotonin transporter (SERT) in HCASMCs. The mRNA transcripts by qPCR and protein expression by Western blot of TPH1 and SERT were examined. The specificity and accuracy of the primers were verified using DNA gel electrophoresis and sequencing of qPCR products. The functionality of SERT was examined using a fluorescence dye-based serotonin transporter assay. The enzymatic activity of TPH was evaluated using UPLC. The HCASMCs expressed both mRNA transcripts and protein of SERT and TPH. The qPCR showed a single melt curve peak for both transcripts and in sequence analysis the amplicons were aligned with the respective genes. SERT and TPH enzymatic activity was present in the HCASMCs. Taken together, both TPH and SERT are functionally expressed in HCASMCs. These findings are novel and represent an initial step in examining the clinical relevance of the serotonergic system in HCASMCs and its role in the pathogenesis of coronary atherosclerosis and CAD.
doi:10.1007/s11033-015-3874-x
PMCID: PMC4506849  PMID: 25861735
Atherosclerosis; Coronary artery disease; Serotonin transporter; Tryptophan hydroxylase; Vascular smooth muscle cell
15.  Chloride Channel 3 Channels in the Activation and Migration of Human Blood Eosinophils in Allergic Asthma 
Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is responsible for respiratory burst in immune cells. Chloride channel 3 (CLC3) has been linked to the respiratory burst in eosinophils and neutrophils. The effect of cytokines and the involvement of CLC3 in the regulation of NADPH-dependent oxidative stress and on cytokine-mediated migration of eosinophils are not known. Human peripheral blood eosinophils were isolated from healthy individuals and from individuals with asthma by negative selection. Real-time PCR was used to detect the expression of NADPH oxidases in eosinophils. Intracellular reactive oxygen species (ROS) measurement was done with flow cytometry. Superoxide generation was measured with transforming growth factor (TGF)-β, eotaxin, and CLC3 blockers. CLC3 dependence of eosinophils in TGF-β– and eotaxin-induced migration was also examined. The messenger RNA (mRNA) transcripts of NADPH oxidase (NOX) 2, dual oxidase (DUOX) 1, and DUOX2 were detected in blood eosinophils, with very low expression of NOX1, NOX3, and NOX5 and no NOX4 mRNA. The level of NOX2 mRNA transcripts increased with disease severity in the eosinophils of subjects with asthma compared with healthy nonatopic volunteers. Change in granularity and size in eosinophils, but no change in intracellular ROS, was observed with phorbol myristate acetate (PMA). PMA, TGF-β, and eotaxin used the CLC3-dependent pathway to increase superoxide radicals. TGF-β and eotaxin induced CLC3-dependent chemotaxis of eosinophils. These findings support the requirement of CLC3 in the activation and migration of human blood eosinophils and may provide a potential novel therapeutic target to regulate eosinophil hyperactivity in allergic airway inflammation in asthma.
doi:10.1165/rcmb.2014-0300OC
PMCID: PMC4566046  PMID: 25514499
airway inflammation; allergic asthma; CLC3 channels; eosinophils; NADPH oxidase
16.  Intermediate-conductance Calcium-activated Potassium Channel KCa3.1 and Chloride Channel Modulate Chemokine Ligand (CCL19/CCL21)-induced Migration of Dendritic Cells 
The role of ion channels is largely unknown in chemokine-induced migration in non-excitable cells such as dendritic cells. Here, we examined the role of KCa3.1 and chloride channels in lymphatic chemokines-induced migration of dendritic cells. The amplitude and kinetics of CCL19/21-induced Ca2+ influx were associated with CCR7 expression levels, extracellular free Ca2+ and Cl−, and independent of extracellular K+. Chemokines, CCL19 and CCL21, and KCa3.1 activator, 1-EBIO, induced plasma membrane hyperpolarization and K+ efflux, which was blocked by TRAM-34, suggesting that KCa3.1 carried larger conductance than the inward CRAC. Blockade of KCa3.1, low Cl− in the medium, and low dose of DIDS impaired CCL19/CCL21-induced Ca2+ influx, cell volume change, and DC migration. High doses of DIDS completely blocked DC migration possibly by significantly disrupting mitochondrial membrane potential. In conclusion, KCa3.1 and chloride channel are critical in human DC migration by synergistically regulating membrane potential, chemokine-induced Ca2+ influx, and cell volume.
doi:10.1016/j.trsl.2014.11.010
PMCID: PMC4458411  PMID: 25583444
17.  Immunomodulatory Role of Vitamin D in the Pathogenesis of Preeclampsia 
Expert review of clinical immunology  2015;11(9):1055-1063.
Summary
Worldwide, preeclampsia is a significant health risk to both pregnant women and their unborn children. Despite scientific advances, the exact pathogenesis of preeclampsia is not yet fully understood. Meanwhile, the incidence of preeclampsia is expected to increase. A series of potential etiologies for preeclampsia have been identified, including endothelial dysfunction, immunological dysregulation, and trophoblastic invasion. In this literature review, we have critically reviewed existing literature regarding the research findings that link the role of vitamin D to the pathogenesis and immunoregulation of preeclampsia. The relationship of vitamin D with the suspected etiologies of preeclampsia underscores its clinical potential in the diagnosis and treatment of preeclampsia.
doi:10.1586/1744666X.2015.1056780
PMCID: PMC4829935  PMID: 26098965
Cathelicidin; Endothelial dysfunction; Immunomodulation; Preeclampsia; Vitamin D
18.  Vitamin D and the immunomodulation of rotator cuff injury 
Tendon-to-bone healing after rotator cuff repair surgery has a failure rate of 20%–94%. There has been a recent interest to determine the factors that act as determinants between successful and unsuccessful rotator cuff repair. Vitamin D level in patients is one of the factors that have been linked to bone and muscle proliferation and healing, and it may have an effect on tendon-to-bone healing. The purpose of this article is to critically review relevant published research that relates to the effect of vitamin D on rotator cuff tears and subsequent healing. A review of the literature was conducted to identify all studies that investigate the relationship between vitamin D and tendon healing, in addition to its mechanism of action. The data were then analyzed in order to summarize what is currently known about vitamin D, rotator cuff pathology, and tendon-to-bone healing. The activated metabolite of vitamin D, 1α,25-dihydroxyvitamin D3, affects osteoblast proliferation and differentiation. Likewise, vitamin D plays a significant role in the tendon-to-bone healing process by increasing the bone mineral density and strengthening the skeletal muscles. The 1α,25-dihydroxyvitamin D3 binds to vitamin D receptors on myocytes to stimulate growth and proliferation. The form of vitamin D produced by the liver, calcifediol, is a key initiator of the myocyte healing process by moving phosphate into myocytes, which improves function and metabolism. Investigation into the effect of vitamin D on tendons has been sparse, but limited studies have been promising. Matrix metalloproteinases play an active role in remodeling the extracellular matrix (ECM) of tendons, particularly deleterious remodeling of the collagen fibers. Also, the levels of transforming growth factor-β3 positively influence the success of the surgery for rotator cuff repair. In the tendon-to-bone healing process, vitamin D has been shown to successfully influence bone and muscle healing, but more research is needed to delve into the mechanisms of vitamin D as a factor in skeletal tendon health and healing.
doi:10.2147/JIR.S106206
PMCID: PMC4913983  PMID: 27366101
bone; calcium; 1,25-dihydroxyvitamin D; matrix metalloproteinases; muscle; rotator cuff tear; tendon
19.  Dendritic Cells Expressing Triggering Receptor Expressed on Myeloid Cells-1 Correlate with Plaque Stability in Symptomatic and Asymptomatic Patients with Carotid Stenosis 
PLoS ONE  2016;11(5):e0154802.
Atherosclerosis is a chronic inflammatory disease with atherosclerotic plaques containing inflammatory cells, including T-lymphocytes, dendritic cells (DCs) and macrophages that are responsible for progression and destabilization of atherosclerotic plaques. Stressed cells undergoing necrosis release molecules that act as endogenous danger signals to alert and activate innate immune cells. In atherosclerotic tissue the number of DCs increases with the progression of the lesion and produce several inflammatory cytokines and growth factors. Triggering receptor expressed on myeloid cells (TREM)-1 plays a crucial role in inflammation. However, relationship of DCs and the role of TREM-1 with the stability of atherosclerotic plaques have not been examined. In this study, we investigated the heterogeneity of the plaque DCs, myeloid (mDC1 and mDC2) and plasmacytoid (pDCs), and examined the expression of TREM-1 and their co-localization with DCs in the plaques from symptomatic (S) and asymptomatic (AS) patients with carotid stenosis. We found increased expression of HLA-DR, fascin, and TREM-1 and decreased expression of TREM-2 and α-smooth muscle actin in S compared to AS atherosclerotic carotid plaques. Both TREM-1 and fascin were co-localized suggesting increased expression of TREM-1 in plaque DCs of S compared to AS patients. These data were supported by increased mRNA transcripts of TREM-1 and decreased mRNA transcripts of TREM-2 in carotid plaques of S compared to AS patients. There was higher density of both CD1c+ mDC1 and CD141+ mDC2 in the carotid plaques from AS compared to S patients, where as the density of CD303+ pDCs were higher in the carotid plaques of S compared to AS patients. These findings suggest a potential role of pDCs and TREM-1 in atherosclerotic plaque vulnerability. Thus, newer therapies could be developed to selectively block TREM-1 for stabilizing atherosclerotic plaques.
doi:10.1371/journal.pone.0154802
PMCID: PMC4858252  PMID: 27148736
20.  Sry-type HMG box 18 Contributes to the differentiation of Bone Marrow-derived Mesenchymal Stem Cells to Endothelial Cells 
Objective
Mesenchymal stem cells (MSC) have shown therapeutic potential to engraft and either differentiate into or support differentiation of vascular endothelial cells (EC), smooth muscle cells and cardiomyocytes in animal models of ischemic heart disease. Following intracoronary or transendocardial delivery of MSCs, however, only a small fraction of cells engraft and the majority of those persist as an immature cell phenotype. The goal of the current study was to decipher the molecular pathways and mechanisms that control MSC differentiation into ECs. Vascular endothelial growth factor (VEGF-165) treatment is known to enhance in vitro differentiation of MSCs into ECs. We tested the possible involvement of the Sry-type HMG box (Sox) family of transcription factors in this process.
Method and Results
MSCs were isolated from the bone marrow of Yucatan microswine and underwent a 10 day differentiation protocol. VEGF-165 (50 ng/ml) treatment of MSCs in vitro induced a significant increase in the protein expression of VEGFR-2, Sox9 and Sox18, in addition to the EC markers PECAM-1, VE-cadherin and vWF, as determined by Western blot or flow cytometry. siRNA-mediated knockdown of Sox18, as opposed to Sox9, in MSCs prevented VEGF-165-mediated induction of EC markers and capillary tube formation. Inhibition of VEGFR-2 signaling (SC-202850) reduced Sox18 and reduced VEGF-165-induced differentiation of MSCs to ECs.
Conclusion
Here we demonstrate that VEGF-165 mediates MSC differentiation into ECs via VEGFR-2-dependent induction of Sox18, which ultimately coordinates the transcriptional upregulation of specific markers of the EC phenotype.
doi:10.1016/j.diff.2015.03.003
PMCID: PMC4479266  PMID: 25913202
Differentiation; Endothelial cell; Mesenchymal stem cell; Sox9; Sox18; Growth Factor
21.  Mesenchymal stem cells and cutaneous wound healing: novel methods to increase cell delivery and therapeutic efficacy 
Mesenchymal stem cells (MSCs) (also known as multipotent mesenchymal stromal cells) possess the capacity for self-renewal and multi-lineage differentiation, and their ability to enhance cutaneous wound healing has been well characterized. Acting via paracrine interactions, MSCs accelerate wound closure, increase angiogenesis, promote resolution of wound inflammation, favorably regulate extracellular matrix remodeling, and encourage regeneration of skin with normal architecture and function. A number of studies have employed novel methods to amplify the delivery and efficacy of MSCs. Non-traditional sources of MSCs, including Wharton’s jelly and medical waste material, have shown efficacy comparable to that of traditional sources, such as bone marrow and adipose tissue. The potential of alternative methods to both introduce MSCs into wounds and increase migration of MSCs into wound areas has also been demonstrated. Taking advantage of the associations between MSCs with M2 macrophages and microRNA, methods to enhance the immunomodulatory capacity of MSCs have shown success. New measures to enhance angiogenic capabilities have also exhibited effectiveness, often demonstrated by increased levels of proangiogenic vascular endothelial growth factor. Finally, hypoxia has been shown to have strong wound-healing potential in terms of increasing MSC efficacy. We have critically reviewed the results of the novel studies that show promise for the continued development of MSC-based wound-healing therapies and provide direction for continued research in this field.
doi:10.1186/s13287-016-0303-6
PMCID: PMC4784457  PMID: 26960535
22.  Immunological Basis in the Pathogenesis and Treatment of Bladder Cancer 
The pathogenesis and transition of normal urothelium into bladder carcinoma are multifactorial processes. Chronic inflammation causes initiation and progression of the underlying pathophysiology of invasive and metastatic cancer. A dichotomy is observed in the role of immune cells in bladder cancer. While the immune response defends the host by suppressing neoplastic growth, several immune cells, including neutrophils, macrophages, and T-lymphocytes, promote tumor development and progression. The levels of human neutrophil peptide-1, -2, and -3, produced by neutrophils, increase in bladder cancer and might promote tumor angiogenesis and growth. The effect of macrophages is primarily mediated by pro-inflammatory cytokines, IL-6 and TNF-α. Additionally, the underlying immunological mechanisms of two treatments, BCG and cytokine gene-modified tumor vaccines, and future directions are critically discussed.
doi:10.1586/1744666X.2015.983082
PMCID: PMC4637163  PMID: 25391391
BCG; Bladder cancer; dendritic cells; macrophages; neutrophils; T cells; Urothelial cell carcinoma; Tumor vaccines
23.  Immunological Effects and Therapeutic Role of C5a in Cancer 
The specific role of C5a in cancer, especially in melanoma, has yet to be determined. Differential effects of C5a could be cancer-specific. In the host defense system, C5a functions to protect the body from harmful entities via a plethora of mechanisms. Yet, C5a may also serve to potentiate cancerous process. C5a facilitates cellular proliferation and regeneration by attracting myeloid-derived suppressor cells and supporting tumor promotion. In this article, we critically reviewed the properties, mechanisms of action, and functions of C5a, with particular emphasis on cancer inhibition and promotion, and clinical application of such knowledge in better management of cancer patients. Outstanding questions and future directions in regard to the function of C5a in melanoma and other cancers are discussed.
doi:10.1586/1744666X.2015.983081
PMCID: PMC4637164  PMID: 25387724
C5a; Cancer; Complement; Chronic inflammation; Immunosuppression; Melanoma
24.  Immunobiology of Interleukin-37: Mechanism of Action and Clinical Perspectives 
Expert review of clinical immunology  2014;10(12):1703-1709.
This article provides an overview of the biological function of a recently discovered cytokine, interleukin-37 (IL-37), formerly referred to as IL-1F7, and its role in chronic inflammation and autoimmune disease. Much has been discovered about IL-37 in the last decade, including its ability to down-regulate systemic and local inflammation by lowering levels of pro-inflammatory molecules. Here, we critically reviewed the published reports. Future research is necessary in order to understand the receptor-dependent effects of IL-37, its intracellular and extracellular functions in both normal and diseased states, and its potential role as a biomarker and pharmacological target in human disease.
doi:10.1586/1744666X.2014.971014
PMCID: PMC4596230  PMID: 25327443
Adaptive immunity; Autoimmune disease; Inflammation; Innate immunity; IL-37
25.  Triggering receptor expressed on myeloid cells (TREM) receptor family modulators: a patent review 
Expert opinion on therapeutic patents  2014;24(12):1383-1395.
Introduction
Triggering receptor expressed on myeloid cells (TREM) receptors and TREM-like transcript (TLT; or TREML) receptors of the immunoglobulin superfamily are known as key modulators of host immune responses. TREM-1 (CD354) and TREM-2 share the transmembrane adaptor DNAX-activation protein of 12 kDa (DAP12), but they possess separate stimulatory and inhibitory functional roles, especially in myeloid cells.
Areas covered
This review covers findings related to TREMs and TLTs published in patent applications from their discovery in 2000 to the present. New roles for TREM-1, TREM-2, TLT-1 and TLT-2 in maladies ranging from acute and chronic inflammatory disorders to cardiovascular diseases and cancers are discussed. Putative endogenous ligands and novel synthetic peptide blockers are also discussed.
Expert opinion
So far, therapeutic use of activators/blockers specific for TREMs and TLTs has been limited to pre-clinical animal models. TREM-1 is an immediate therapeutic target for acute and chronic inflammatory conditions, especially sepsis. Certain mutations in DAP12 and TREM-2 manifest into a disorder named polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL), and newly identified TREM-2 variants confer a significant increase in risk of developing Alzheimer’s disease. This makes TREM-2 an attractive therapeutic target for neurodegenerative diseases.
doi:10.1517/13543776.2014.977865
PMCID: PMC4596234  PMID: 25363248
CD177; DNAX activation protein of 12 kDa; Heat shock protein 60; High-mobility group box 1; Immunomodulator; Peptidoglycan recognition protein 1; Toll-like receptor; Triggering receptor expressed on myeloid cells; TREM-like transcript

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