Search

Bone marrow (BM) transplantation (BMT) represents a curative treatment for various hematological disorders. Prior to BMT, a large amount of the relevant anticancer drug needed to be administered to eliminate cancer cells. However, during this pre‑BMT cytotoxic conditioning regimen, hematopoietic stem cells in the BM and thymic epithelial cells were also destroyed. The T cell receptor (TCR) recognizes diverse pathogen, tumor and environmental antigens, and confers immunological memory and self‑tolerance. Delayed thymus reconstitution following pre‑BMT cytotoxic conditioning impedes de novo thymopoiesis and limits T cell‑mediated immunity. Several cytokines, such as RANK ligand, interleukin (IL)‑7, IL‑22 and stem cell factor, were recently reported to improve thymopoiesis and immune function following BMT. In the present study, it was found that the co‑transplantation of tonsil‑derived mesenchymal stromal cells (T‑MSCs) with BM‑derived cells (BMCs) accelerated the recovery of involuted thymuses in mice following partial pre‑BMT conditioning with busulfan‑cyclophosphamide treatment, possibly by inducing FMS‑like tyrosine kinase 3 ligand (FLT3L) and fibroblast growth factor 7 (FGF7) production in T‑MSCs. The co‑transplantation of T‑MSCs with BMCs also replenished the CD3+ cell population by inhibiting thymocyte apoptosis following pre‑BMT cytotoxic conditioning. Furthermore, T‑MSC co‑transplantation improved the recovery of the TCR repertoire and led to increased thymus‑generated T cell diversity.

Reactive oxygen species (ROS) generated by neutrophils provide a frontline defence against invading pathogens. We investigated the supportive effect of tonsil-derived mesenchymal stem cells (TMSCs) on ROS generation from neutrophils using promyelocytic HL-60 cells. Methods: Differentiated HL-60 (dHL-60) cells were cocultured with TMSCs isolated from 25 independent donors, and ROS generation in dHL-60 cells was measured using luminescence. RNA sequencing and real-time PCR were performed to identify the candidate genes of TMSCs involved in augmenting the oxidative burst of dHL-60 cells. Transcriptome analysis of TMSCs derived from 25 independent donors revealed high levels of procollagen C-endopeptidase enhancer 2 (PCOLCE2) in TMSCs, which were highly effective in potentiating ROS generation in dHL-60 cells. In addition, PCOLCE2 knockdown in TMSCs abrogated TMSC-induced enhancement of ROS production in dHL-60 cells, indicating that TMSCs increased the oxidative burst in dHL-60 cells via PCOLCE2. Furthermore, the direct addition of recombinant PCOLCE2 protein increased ROS production in dHL-60 cells. These results suggest that PCOLCE2 secreted by TMSCs may be used as a therapeutic candidate to enhance host defences by increasing neutrophil oxidative bursts. PCOLCE2 levels in TMSCs could be used as a marker to select TMSCs exhibiting high efficacy for enhancing neutrophil oxidative bursts.

Tonsil-derived mesenchymal stem cells (TMSCs) showed therapeutic effects on acute and chronic murine colitis models, owing to their immunomodulatory properties; therefore, we evaluated enhanced therapeutic effects of TMSCs on a murine colitis model using three-dimensional (3D) culture method. The expression of angiogenic factors, VEGF, and anti-inflammatory cytokines, IL-10, TSG-6, TGF-β, and IDO-1, was significantly higher in the 3D-TMSC-treated group than in the 2D-TMSC-treated group (P

BACKGROUND: Mast cells are immune sentinels in the skin that respond to a wide range of pathological and environmental stimuli; they owe their function to the expression of Toll-like receptors (TLRs). We previously found that tonsil-derived mesenchymal stem cells (T-MSCs) were able to effectively attenuate TLR7-mediated skin inflammation in mice, which was accompanied by an increase in mast cell number. The present study investigated whether T-MSC extracellular vesicles, such as exosomes, are able to regulate mast cell activation in response to TLR7 stimulation. METHODS: The HMC-1 human mast cell line was treated with a TLR7 agonist in the presence or absence of T-MSC exosomes, and the levels of expressed inflammatory cytokines were assessed. Additionally, mice were repeatedly injected with a TLR7 agonist with or without interval treatments with T-MSC exosomes and assessed dermal distribution of mast cells and related immune cells. RESULTS: We showed that T-MSC exosomes containing microRNAs that target inflammatory cytokines significantly reduced the expression of inflammatory cytokines in TLR7 agonist-treated HMC-1 cells. In addition, T-MSC exosomes inhibited the increase in the number of both dermal mast cells and CD14-positive cells in TLR7 agonist-treated mice. CONCLUSION: Our data suggest that T-MSC exosomes have regulatory effects on mast cell activation under inflammatory conditions, including TLR7 stimulation.

Background Co-transplantation of bone marrow cells (BMCs) and mesenchymal stem cells (MSCs) is used as a strategy to improve the outcomes of bone marrow transplantation. Tonsil-derived MSCs (TMSCs) are a promising source of MSCs for co-transplantation. Previous studies have shown that TMSCs or conditioned media from TMSCs (TMSC-CM) enhance BMC engraftment. However, the factors in TMSCs that promote better engraftment have not yet been identified. Methods Mice were subjected to a myeloablative regimen of busulfan and cyclophosphamide, and the mRNA expression in the bone marrow was analyzed using an extracellular matrix (ECM) and adhesion molecule-targeted polymerase chain reaction (PCR) array. Nano-liquid chromatography with tandem mass spectrometry, real-time quantitative PCR, western blots, and enzyme-linked immunosorbent assays were used to compare the expression levels of metalloproteinase 3 (MMP3) in MSCs derived from various tissues, including the tonsils, bone marrow, adipose tissue, and umbilical cord. Recipient mice were conditioned with busulfan and cyclophosphamide, and BMCs, either as a sole population or with control or MMP3-knockdown TMSCs, were co-transplanted into these mice. The effects of TMSC-expressed MMP3 were investigated. Additionally, Enzchek collagenase and Transwell migration assays were used to confirm that the collagenase activity of TMSC-expressed MMP3 enhanced BMC migration. Results Mice subjected to the myeloablative regimen exhibited increased mRNA expression of collagen type IV alpha 1/2 (Col4a1 and Col4a2). Among the various extracellular matrix-modulating proteins secreted by TMSCs, MMP3 was expressed at higher levels in TMSCs than in other MSCs. Mice co-transplanted with BMCs and control TMSCs exhibited a higher survival rate, weight recovery, and bone marrow cellularity compared with mice co-transplanted with BMCs and MMP3-knockdown TMSCs. Control TMSC-CM possessed higher collagenase activity against collagen IV than MMP3-knockdown TMSC-CM. TMSC-CM also accelerated BMC migration by degrading collagen IV in vitro. Conclusions Collectively, these results indicate that TMSCs enhance BMC engraftment by the secretion of MMP3 for the modulation of the bone marrow extracellular matrix.

Located near the oropharynx, the tonsils are the primary mucosal immune organ. Tonsil tissue is a promising alternative source for the high-yield isolation of adult stem cells, and recent studies have reported the identification and isolation of tonsil-derived stem cells (T-SCs) from waste surgical tissue following tonsillectomies in relatively young donors (i.e., under 10 years old). As such, T-SCs offer several advantages, including superior proliferation and a shorter doubling time compared to bone marrow-derived mesenchymal stem cells (MSCs). T-SCs also exhibit multi-lineage differentiation, including mesodermal, endodermal (e.g., hepatocytes and parathyroid-like cells), and even ectodermal cells (e.g., Schwann cells). To this end, numbers of researchers have evaluated the practical use of T-SCs as an alternative source of autologous or allogenic MSCs. In this review, we summarize the details of T-SC isolation and identification and provide an overview of their application in cell therapy and regenerative medicine.

Animal studies using chronic social defeat stress (CSDS) in mice showed that brain-derived neurotrophic factor (BDNF) signaling in the mesolimbic dopamine (DA) circuit is important for the development of social aversion. However, the downstream molecular targets after BDNF release from ventral tegmental area (VTA) DA terminals are unknown. Here, we show that depressive-like behaviors induced by CSDS are mediated in part by Gadd45b downstream of BDNF signaling in the nucleus accumbens (NAc). We show that Gadd45b mRNA levels are increased in susceptible but not resilient mice. Intra-NAc infusion of BDNF or optical stimulation of VTA DA terminals in NAc enhanced Gadd45b expression levels in the NAc. Importantly, Gadd45b downregulation reversed social avoidance in susceptible mice. Together, these data suggest that Gadd45b in NAc contributes to susceptibility to social stress. In addition, we investigated the function of Gadd45b in demethylating CpG islands of representative gene targets, which have been associated with a depressive phenotype in humans and animal models. We found that Gadd45b downregulation changes DNA methylation levels in a phenotype-, gene-, and locus-specific fashion. Together, these results highlight the contribution of Gadd45b and changes in DNA methylation in mediating the effects of social stress in the mesolimbic DA circuit.

Background: The skin is a dynamic body organ that can be activated by both central and local hypothalamic-pituitary-adrenal axis systems. This phenomenon might be the crucial explanation why stress can cause relapse of chronic inflammatory skin diseases, such as psoriasis. Here, we determined the effects of mast cells on keratinocyte proliferation under stress hormone stimulation. Methods: We subcutaneously injected dexamethasone on the shaved back of mice and evaluated histological changes and keratinocyte growth factor (KGF) expression on dermal mast cells. Further, human mast cell line (HMC-1) and keratinocyte cell line (HaCaT) cells were treated with dexamethasone in vitro to observe the extent of proliferation and the expression of KGF. Finally, the supernatants of HMC-1 cells treated with dexamethasone were used for the culture of HaCaT cells to investigate the effect on proliferation. Results: We observed epidermal thickening in dexamethasone-injected mice, accompanied by an increase in the number of KGF-expressing dermal mast cells. Similar to mouse dermal mast cells, KGF was highly expressed in the human mast cell line HMC-1 following stimulation with dexamethasone. Further, dexamethasone-treated mast cells promoted keratinocyte proliferation in vitro. However, the effects of mast cells on keratinocytes were significantly diminished in the presence of anti-KGF-blocking antibodies. Conclusion: Taken together, our results show that a stressful environment may disturb skin barrier homeostasis through mast cell-derived KGF expression.

The liver is an organ with remarkable regenerative capacity; however, once chronic fibrosis occurs, liver failure follows, with high mortality and morbidity rates. Continuous exposure to proinflammatory stimuli exaggerates the pathological process of liver failure; therefore, immune modulation is a potential strategy to treat liver fibrosis. Mesenchymal stem cells (MSCs) with tissue regenerative and immunomodulatory potential may support the development of therapeutics for liver fibrosis. Here, we induced hepatic injury in mice by injecting carbon tetrachloride (CCl4) and investigated the therapeutic potential of conditioned medium from tonsil-derived MSCs (T-MSC CM). In parallel, we used recombinant human IL-1Ra, which, as we have previously shown, is secreted exclusively from T-MSCs and resolves the fibrogenic activation of myoblasts. Hepatic inflammation and fibrosis were determined by histological analyses using H&E and Picro-Sirius Red staining. The results demonstrated that T-MSC CM treatment significantly reduced inflammation as well as fibrosis in the CCl4-injured mouse liver. IL-1Ra injection showed effects similar to T-MSC CM treatment, suggesting that T-MSC CM may exert anti-inflammatory and anti-fibrotic effects via the endogenous production of IL-1Ra. The expression of genes involved in fibrosis was evaluated, and the results showed significant induction of alpha-1 type I collagen, transforming growth factor beta, and tissue inhibitor of metalloproteases 1 upon CCl4 injection, whereas treatment with T-MSC CM or IL-1Ra downregulated their expression. Taken together, these data support the therapeutic potential of T-MSC CM and/or IL-1Ra for the alleviation of liver fibrosis, as well as in treating diseases involving organ fibrosis.

(1) Background: six mammalian ceramide synthases (CerS1–6) determine the acyl chain length of sphingolipids (SLs). Although ceramide levels are increased in murine allergic asthma models and in asthmatic patients, the precise role of SLs with specific chain lengths is still unclear. The role of CerS2, which mainly synthesizes C22–C24 ceramides, was investigated in immune responses elicited by airway inflammation using CerS2 null mice. (2) Methods: asthma was induced in wild type (WT) and CerS2 null mice with ovalbumin (OVA), and inflammatory cytokines and CD4 (cluster of differentiation 4)+ T helper (Th) cell profiles were analyzed. We also compared the functional capacity of CD4+ T cells isolated from WT and CerS2 null mice. (3) Results: CerS2 null mice exhibited milder symptoms and lower Th2 responses than WT mice after OVA exposure. CerS2 null CD4+ T cells showed impaired Th2 and increased Th17 responses with concomitant higher T cell receptor (TCR) signal strength after TCR stimulation. Notably, increased Th17 responses of CerS2 null CD4+ T cells appeared only in TCR-mediated, but not in TCR-independent, treatment. (4) Conclusions: altered Th2/Th17 immune response with higher TCR signal strength was observed in CerS2 null CD4+ T cells upon TCR stimulation. CerS2 and very-long chain SLs may be therapeutic targets for Th2-related diseases such as asthma.

Background: Mast cells are skin immune sentinels located in the upper dermis, where wheal formation and sensory nerve stimulation take place. Skin inflammation is occasionally accompanied by mast cell-driven responses with wheals, angioedema, or both. Immunoglobulin E (IgE) antibodies are regarded as typical stimuli to drive mast cell activation. However, various causative factors, including microbial infections, can drive IgE-independent mast cell response. When infected, the innate immunity orchestrates an immune response by activating receptor signaling via Toll-like receptors (TLRs). Objective: In this study, we determined the effect of TLR7 stimulation on mast cells to investigate the possible mechanism of IgE-independent inflammatory response. Methods: Human mast cell (HMC) line, HMC-1 cells were treated with TLR7 agonist and the morphologic alteration was observed in transmission electron microscopy. Further, TLR7 agonist treated HMC-1 cells were conducted to RNA sequencing to compare transcriptomic features. Results: HMC-1 cells treated with TLR7 agonist reveals increase of intracellular vesicles, lipid droplets, and ribosomes. Also, genes involved in pro-inflammatory responses such as angiogenesis are highly expressed, and Il12rb2 was the most highly up-regulated gene. Conclusion: Our data suggest that TLR7 signaling on mast cells might be a potential therapeutic target for mast cell-driven, IgE-independent skin inflammation. (Ann Dermatol 33(5) 402∼408, 2021)

Lymphatic vessels serve as conduits through which immune cells traffic. Because lymphatic vessels are also involved in lipid transport, their function is vulnerable to abnormal metabolic conditions such as obesity and hyperlipidemia. Exactly how these conditions impact immune cell trafficking, however, is not well understood. Here, we found higher numbers of LYVE-1-positive lymphatic endothelial cells and CD3-positive T cells in the lymph nodes of mice fed high-cholesterol or high-fat diets compared with those of mice fed a normal chow diet. To confirm the effect of fat content on immune cell trafficking, the lymphatic endothelial SVEC4-10 cell line was treated with palmitic acid at a 100 μM concentration. After 24 h, palmitic acid-treated cells exhibited increased expression of podoplanin and vascular growth-associated molecules (VEGFC, VEGFD, VEGFR3, and NRP2) and enhanced tube formation. Microarray analysis showed an increase in pro-inflammatory cytokine and chemokine transcription after palmitic acid treatment. Finally, transwell migration assay confirmed that T cell line moved toward medium previously cultured with palmitic acid-treated SVEC4-10 cells. Together, our results suggest that hyperlipidemia drives lymphatic vessel remodeling and T cell migration toward lymphatic endothelial cells.

Cotransplantation of mesenchymal stem cells (MSCs) with hematopoietic stem cells (HSCs) has been widely reported to promote HSC engraftment and enhance marrow stromal regeneration. The present study aimed to define whether MSC conditioned medium could recapitulate the effects of MSC cotransplantation. Mouse bone marrow (BM) was partially ablated by the administration of a busulfan and cyclophosphamide (Bu&ndash, Cy)-conditioning regimen in BALB/c recipient mice. BM cells (BMCs) isolated from C57BL/6 mice were transplanted via tail vein with or without tonsil-derived MSC conditioned medium (T-MSC CM). Histological analysis of femurs showed increased BM cellularity when T-MSC CM or recombinant human pleiotrophin (rhPTN), a cytokine readily secreted from T-MSCs with a function in hematopoiesis, was injected with BMCs. Microstructural impairment in mesenteric and BM arteriole endothelial cells (ECs) were observed after treatment with Bu&ndash, Cy-conditioning regimen, however, T-MSC CM or rhPTN treatment restored the defects. These effects by T-MSC CM were disrupted in the presence of an anti-PTN antibody, indicating that PTN is a key mediator of EC restoration and enhanced BM engraftment. In conclusion, T-MSC CM administration enhances BM engraftment, in part by restoring vasculature via PTN production. These findings highlight the potential therapeutic relevance of T-MSC CM for increasing HSC transplantation efficacy.

// Kyung-Ah Cho 1 , Minhwa Park 1 , Yu-Hee Kim 1 , Kyung-Ha Ryu 2 and So-Youn Woo 1 1 Department of Microbiology, College of Medicine, Ewha Womans University, Seoul, Republic of Korea 2 Department of Pediatrics, College of Medicine, Ewha Womans University, Seoul, Republic of Korea Correspondence to: So-Youn Woo, email: // Keywords : mesenchymal stem cells, osteoprotegerin, Th17 cells, osteoclasts, psoriasis, Immunology and Microbiology Section, Immune response, Immunity Received : February 20, 2017 Accepted : September 05, 2017 Published : September 28, 2017 Abstract Th17 cells play a critical role in several autoimmune diseases, including psoriasis and psoriatic arthritis (PsA). Psoriasis is a chronic inflammatory skin disease associated with systemic inflammation and comorbidities, such as PsA. PsA develops in nearly 70% of patients with psoriasis, and osteoclasts associated bone erosion is a hallmark of the disease. Thus far, the effect of Th17 cells on osteoclastogenesis via direct cell-to-cell interactions is less understood. In this study, we observed that Th17 cells directly promote osteoclast differentiation and maturation via expression of receptor activator of nuclear factor-κ β ligand (RANKL) in vitro . We investigated the impact of conditioned medium obtained from human palatine tonsil-derived mesenchymal stem cells (T-CM) on the interactions between osteoclasts and Th17 cells. T-CM effectively blunted the RANK-RANKL interaction between the osteoclast precursor cell line RAW 264.7 and Th17 cells via osteoprotegerin (OPG) activity. The frequency of tartrate-resistant acid phosphatase (TRAP)-positive cells in the bone marrow of an imiquimod (IMQ)-induced psoriasis mouse model was decreased following T-CM injection. Therefore, our data provide novel insight into the therapeutic potential of tonsil-derived mesenchymal stem cell-mediated therapy ( via OPG production) for the treatment of pathophysiologic processes induced by osteoclasts under chronic inflammatory conditions such as psoriasis.

Although mast cells are traditionally thought to function as effector cells in allergic responses, they have increasingly been recognized as important regulators of various immune responses. Mast cells mature locally; thus, tissue-specific influences are important for promoting mast cell accumulation and survival in the skin and the gastrointestinal tract. In this study, we determined the effects of keratinocytes on mast cell accumulation during Th17-mediated skin inflammation. We observed increases in dermal mast cells in imiquimod-induced psoriatic dermatitis in mice accompanied by the expression of epidermal stem cell factor (SCF), a critical mast cell growth factor. Similar to mouse epidermal keratinocytes, SCF was highly expressed in the human HaCaT keratinocyte cell line following stimulation with IL−17. Further, keratinocytes promoted mast cell proliferation following stimulation with IL−17 in vitro . However, the effects of keratinocytes on mast cells were significantly diminished in the presence of anti−CD117 (stem cell factor receptor) blocking antibodies. Taken together, our results revealed that the Th17-mediated inflammatory environment promotes mast cell accumulation through keratinocyte-derived SCF.

Mesenchymal stem cells (MSCs) are often considered to be a good source for the development of regenerative medicine. Previously, we reported that tonsil‑derived MSC conditioned medium (T‑MSC CM) produces visceral fat reducing effects. As reduced visceral adiposity is closely associated with an increase in circulating adiponectin, the present study investigated the effects of T‑MSC CM on adiponectin production. T‑MSC CM was collected from previously isolated and characterized T‑MSCs and injected into senescence‑accelerated mouse prone 6 mice, which exhibit characteristics of aging and obesity. The results demonstrated a reduction in mouse weight and epididymal adipose tissue (eAT) mass following injection of T‑MSC CM. Significant increases in adiponectin expression in the eAT, and total and high molecular weight (HMW) adiponectin in the circulation were observed in the T‑MSC CM‑injected mice compared with control mice using reverse transcription‑quantitative polymerase chain reaction, western blot analysis and ELISA. In 3T3‑L1 adipocytes, T‑MSC CM treatment increased adiponectin secretion and multimerization, as detected using western blotting under non‑reducing and non‑heat‑denaturing conditions. Furthermore, glucose oxidase was used to induce oxidative stress in 3T3‑L1 adipocytes and it was observed that T‑MSC CM reduced reactive oxygen species production and the expression of certain oxidative stress markers. In addition, the results also demonstrated that the production of HMW adiponectin was increased, which indicates that T‑MSC CM may enhance adiponectin multimerization via amelioration of oxidative stress. Further studies are required to elucidate anti‑oxidant molecules secreted from T‑MSCs, and these results highlight the potential therapeutic relevance of T‑MSC CM for the treatment of obesity or obesity‑associated diseases.

It has been established that mesenchymal stem cells (MSCs) can have a suppressive effect on T cells, yet much remains unknown about the underlying mechanisms that support this effect. The T cell co-stimulatory pathway involving the programmed death-1 (PD-1) receptor and its ligand PD-L1 regulates T cell activation, tolerance, and subsequent immune-mediated tissue damage. In this study, human palatine tonsil-derived MSCs (T-MSCs) constitutively expressed PD-L1 and exhibited a suppressive activity that specifically targeted murine Th17 differentiation. Additionally, polyinosinic-polycytidylic acid (poly I:C), a Toll-like receptor 3 (TLR3) ligand, increased PD-L1 expression on T-MSCs. The elevated PD-L1 levels enhanced the suppressive functions of T-MSCs on Th17 differentiation. Therefore, pre-stimulation of T-MSCs with poly I:C may serve as an effective therapeutic priming step for modulating Th17-dominant immune responses.

Effector B cells are central contributors to the development of autoimmune disease by activating autoreactive T cells, producing pro-inflammatory cytokines and organizing ectopic lymphoid tissue. Conversely, IL-10-producing regulatory B (Breg) cells have pivotal roles in maintaining immunological tolerance and restraining excessive inflammation in autoinflammatory disease. Thus, regulating the equilibrium between antibody-producing effector B cells and Breg cells is critical for the treatment of autoimmune disease. In this study, we investigated the effect of human palatine tonsil-derived mesenchymal stem cells (T-MSCs) on estradiol (E2)-induced B-cell responses in vivo and in vitro. Transplantation of T-MSC into E2-treated mice alleviated B-cell-mediated immune responses and increased the population of IL-10-producing Breg cells. T-MSCs regulated the B-cell populations by producing Epstein–Barr virus (EBV)-induced 3 (EBI3), one of the two subunits of IL-35 that is the well-known inducer of Breg cells. We demonstrate a critical role of EBI3 (IL-35) in vitro by depleting EBI3 in T-MSCs and by adding exogenous IL-35 to the culture system. Taken together, our data suggest that IL-35-secreting MSCs may become an attractive therapeutic to treat B-cell-mediated autoimmune diseases via expanding Breg cells.

The current study characterizes a cohort of limb-girdle muscular dystrophy (LGMD) in the United States using whole-exome sequencing. Fifty-five families affected by LGMD were recruited using an institutionally approved protocol. Exome sequencing was performed on probands and selected parental samples. Pathogenic mutations and cosegregation patterns were confirmed by Sanger sequencing. Twenty-two families (40%) had novel and previously reported pathogenic mutations, primarily in LGMD genes, and also in genes for Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital myopathy, myofibrillar myopathy, inclusion body myopathy and Pompe disease. One family was diagnosed via clinical testing. Dominant mutations were identified in COL6A1, COL6A3, FLNC, LMNA, RYR1, SMCHD1 and VCP, recessive mutations in ANO5, CAPN3, GAA, LAMA2, SGCA and SGCG, and X-linked mutations in DMD. A previously reported variant in DMD was confirmed to be benign. Exome sequencing is a powerful diagnostic tool for LGMD. Despite careful phenotypic screening, pathogenic mutations were found in other muscle disease genes, largely accounting for the increased sensitivity of exome sequencing. Our experience suggests that broad sequencing panels are useful for these analyses because of the phenotypic overlap of many neuromuscular conditions. The confirmation of a benign DMD variant illustrates the potential of exome sequencing to help determine pathogenicity.

The application of mesenchymal stem cells (MSCs) for treating bone-related diseases shows promising outcomes in preclinical studies. However, cells that are isolated and defined as MSCs comprise a heterogeneous population of progenitors. This heterogeneity can produce variations in the performance of MSCs, especially in applications that require differentiation potential in vivo, such as the treatment of osteoporosis. Here, we aimed to identify genetic markers in tonsil-derived MSCs (T-MSCs) that can predict osteogenic potential. Using a single-cell cloning method, we isolated and established several lines of nondifferentiating (ND) or osteoblast-prone (OP) clones. Next, we performed transcriptome sequencing of three ND and three OP clones that maintained the characteristics of MSCs and determined the top six genes that were upregulated in OP clones. Upregulation of WNT16 and DCLK1 expression was confirmed by real-time quantitative PCR, but only WNT16 expression was correlated with the osteogenic differentiation of T-MSCs from 10 different donors. Collectively, our findings suggest that WNT16 is a putative genetic marker that predicts the osteogenic potential of T-MSCs. Thus, examination of WNT16 expression as a selection criterion prior to the clinical application of MSCs may enhance the therapeutic efficacy of stem cell therapy for bone-related complications, including osteoporosis.

Type 2 diabetes mellitus (T2DM) is a prevalent chronic metabolic disorder accompanied by high blood glucose, insulin resistance, and relative insulin deficiency. Endoplasmic reticulum (ER) stress induced by high glucose and free fatty acids has been suggested as one of the main causes of &beta, cell dysfunction and death in T2DM. Stem cell-derived insulin-secreting cells were recently suggested as a novel therapy for diabetes. In the present study, we demonstrate the therapeutic potential of tonsil-derived mesenchymal stem cells (TMSCs) to treat high-fat diet (HFD)-induced T2DM. To explore whether TMSC administration can alleviate T2DM, TMSCs were intraperitoneally injected in HFD-induced T2DM mice once every 2 weeks. TMSC injection markedly improved glucose tolerance and glucose-stimulated insulin secretion and prevented HFD-induced pancreatic &beta, cell hypertrophy and cell death. In addition, TMSC injection relieved the ER-stress response and preserved gene expression related to glucose sensing and insulin secretion. Moreover, administration of TMSC-derived conditioned medium induced similar therapeutic outcomes, suggesting paracrine effects. Finally, proteomic analysis revealed high secretion of insulin-like growth factor-binding protein 5 by TMSCs, and its expression was critical for the protective effects of TMSCs against HFD-induced glucose intolerance and ER-stress response in pancreatic islets. TMSC administration can alleviate HFD-induced-T2DM via preserving pancreatic islets and their function. These results provide novel evidence of TMSCs as an ER-stress modulator that may be a novel, alternative cell therapy for T2DM.

Graft-vs.-host disease (GVHD) is a severe and potentially life-threatening complication of hematopoietic stem cell transplantation. Approximately 50% of patients exhibiting GVHD will not benefit from conventional steroid treatment. Although several second‑line treatments are available for these patients, their prognoses remain poor due to the increased risk of infection, immunosuppression-mediated toxicity and incomplete GVHD remission, which occurs in the majority of cases. Mesenchymal stem cells (MSCs), a multipotent cell population, possess broad immunosuppressive activity and are a reportedly effective treatment of GVHD. However, the therapeutic effects of conditioned medium from MSCs on GVHD have not been demonstrated. In the present study, the efficacy of conditioned medium from human palatine tonsil‑derived MSCs (T‑MSC‑CM) was validated against GVHD in mice. The suppressive function of T‑MSC‑CM on immune cell chemotaxis was confirmed in vitro. A systemic infusion of T‑MSC‑CM in mice with GVHD resulted in prolonged survival, rapid recovery from weight loss and reduced pathological damage in numerous GVHD‑targeted organs. Furthermore, lymphocyte gene expression was significantly downregulated in GVHD mice administered T‑MSC‑CM. These results indicate that T‑MSC‑CM is a promising cellular agent to prevent or treat transplantation‑associated complications such as GVHD.

Next-generation sequencing is commonly used to screen for pathogenic mutations in families with Mendelian disorders, but due to the pace of discoveries, gaps have widened for some diseases between genetic and pathophysiological knowledge. We recruited and analyzed 16 families with limb-girdle muscular dystrophy (LGMD) of Arab descent from Saudi Arabia and Sudan who did not have confirmed genetic diagnoses. The analysis included both traditional and next-generation sequencing approaches. Cellular and metabolic studies were performed on Pyroxd1 siRNA C2C12 myoblasts and controls. Pathogenic mutations were identified in eight of the 16 families. One Sudanese family of Arab descent residing in Saudi Arabia harbored a homozygous c.464A>G, p.Asn155Ser mutation in PYROXD1, a gene recently reported in association with myofibrillar myopathy and whose protein product reduces thiol residues. Pyroxd1 deficiency in murine C2C12 myoblasts yielded evidence for impairments of cellular proliferation, migration, and differentiation, while CG10721 (Pyroxd1 fly homolog) knockdown in Drosophila yielded a lethal phenotype. Further investigations indicated that Pyroxd1 does not localize to mitochondria, yet Pyroxd1 deficiency is associated with decreased cellular respiration. This study identified pathogenic mutations in half of the LGMD families from the cohort, including one in PYROXD1. Developmental impairments were demonstrated in vitro for Pyroxd1 deficiency and in vivo for CG10721 deficiency, with reduced metabolic activity in vitro for Pyroxd1 deficiency.

The author would like to correct the fifth, seventh and eighth co-authors’ name to Kyung-Ah Cho, Han Su Kim, Bo Young Park in the online published article.

INTRODUCTION The genetic causes of limb-girdle muscular dystrophy (LGMD) have been studied in numerous countries, but such investigations have been limited in Egypt. METHODS A cohort of 30 families with suspected LGMD from Assiut, Egypt, was studied using immunohistochemistry, homozygosity mapping, Sanger sequencing, and whole exome sequencing. RESULTS Six families were confirmed to have pathogenic mutations, 4 in SGCA and 2 in DMD. Of these, 3 families harbored a single nonsense mutation in SGCA, suggesting that this may be a common mutation in Assiut, Egypt, originating from a founder effect. CONCLUSIONS The Assiut region in Egypt appears to share at least several of the common LGMD genes found in other parts of the world. It is notable that 4 of the 6 mutations were ascertained by means of whole exome sequencing, even though it was the last approach adopted. This illustrates the power of this technique for identifying causative mutations for muscular dystrophies. Muscle Nerve 54: 690-695, 2016.

Osteoporosis is a disease that affects 35% women and 20% men aged more than 65 years. Reduction in bone formation and increased bone resorption are known factors that drive osteoporosis, but recent studies suggest a positive correlation between bone marrow adipose tissue (MAT) and osteoporosis. Previously, we have observed that tonsil-derived mesenchymal stem cells (T-MSCs) reduce MAT in a mouse model of bone marrow depletion. That prompted us to investigate on the senile osteoporosis to characterize the bone-forming effect, as well as MAT-reducing effect of T-MSCs. In a mouse model of senescence-accelerated mouse prone 6 (SAMP6), we injected T-MSCs or T-MSC conditioned medium (CM) through tail vein and examined changes in bone microstructure using micro-CT scan and hematoxylin & eosin (H&E) staining. Biochemical markers of osteoporosis, deoxypyridinoline (DPD) and osteocalcin, were examined by ELISA. Results demonstrated attenuation in the progression of osteoporosis, in part, by sustaining osteocalcin production and by blocking MAT accumulation. Increase in matrix mineralization was determined using in vitro culture of murine preosteoblast cell line by treatment of T-MSC CM. Interestingly, T-MSC CM induced continuous weight loss and selectively reduced visceral adipose tissue mass. Finally, antiadipogenic effects of T-MSC CM were determined in vitro. In conclusion, regulation of bone together with MAT could be considered as a new therapeutic option for the treatment of senile osteoporosis and this report may provide a framework for future cell therapy using T-MSCs.

Background and Purpose— Most patients with cerebral infarction die of brain edema because of the breakdown of the blood–brain barrier (BBB) in ischemic tissue. Caveolins (a group of proteins) are key modulators of vascular permeability; however, a direct role of caveolin-1 (Cav-1) in the regulation of BBB permeability during ischemic injury has yet to be identified. Methods— Cav-1 expression was measured by immunoblotting after photothrombotic ischemia. A direct functional role of Cav-1 in cerebral edema and BBB permeability during cerebral ischemia was investigated by genetic manipulation (gene disruption and re-expression) of Cav-1 protein expression in mice. Results— There was a significant correlation between the extent of BBB disruption and the Cav-1 expression. In Cav-1–deficient (Cav-1 −/− ) mice, the extent of BBB disruption after cerebral ischemia was increased compared with wild-type (Cav-1 +/+ ) mice, whereas the increase in cerebral edema volume was ameliorated by lentiviral-mediated re-expression of Cav-1. Furthermore, Cav-1 −/− mice had significantly higher degradation of tight junction proteins and proteolytic activity of matrix metalloproteinase than Cav-1 +/+ mice. Conversely, re-expression of Cav-1 in Cav-1 −/− mice restored tight junction protein expression and reduced matrix metalloproteinase proteolytic activity. Conclusions— These results indicate that Cav-1 is a critical determinant of BBB permeability. Strategies for regulating Cav-1 represent a novel therapeutic approach to controlling BBB disruption and subsequent neurological deterioration during cerebral ischemia.

Skin forms a physical barrier that protects the body against outside agents. The deepest layer of the skin, the stratum basale, contains two cell types: agent-sensing keratinocytes, and melanin-producing melanocytes. Keratinocytes can sense both harmless commensal organisms and harmful pathogens via Toll-like receptors (TLRs), and keratinocytes subsequently drive immune responses. Activation of TLR3 is required for barrier repair because it stimulates essential genes, including tight junction genes, and inflammatory cytokines. Within the basal layer of the skin, resident melanocytes use their dendritic processes to connect with approximately 30-40 neighboring keratinocytes. Most studies have focused on the transfer of melanin-synthesizing melanosomes from melanocytes to keratinocytes, but the potential regulation of melanogenesis by soluble factor(s) produced by keratinocytes remains to be explored. Studying such regulation in vivo is challenging because of the keratinocyte:melanocyte ratio in the epidermis and the location of the cells within the skin. Therefore, in this study, we investigated whether keratinocytes affected melanocyte melanogenesis in vitro under normal or inflammatory conditions. We found that polyinosinic-polycytidylic acid [poly(I:C)] stimulation induced PD-L1 secretion from HaCaT cells and that poly(I:C)-induced PD-L1 inhibited melanin production by B16F10 cells. These data provide key evidence that keratinocytes can alter melanocyte melanogenesis via the production of soluble factors under inflammatory conditions.

BACKGROUND: The liver is an organ with remarkable regenerative capacity; however, once chronic fibrosis occurs, liver failure follows, with high mortality and morbidity rates. Continuous exposure to proinflammatory stimuli exaggerates the pathological process of liver failure; therefore, immune modulation is a potential strategy to treat liver fibrosis. Mesenchymal stem cells (MSCs) with tissue regenerative and immunomodulatory potential may support the development of therapeutics for liver fibrosis. METHODS: Here, we induced hepatic injury in mice by injecting carbon tetrachloride (CCl(4)) and investigated the therapeutic potential of conditioned medium from tonsil-derived MSCs (T-MSC CM). In parallel, we used recombinant human IL-1Ra, which, as we have previously shown, is secreted exclusively from T-MSCs and resolves the fibrogenic activation of myoblasts. Hepatic inflammation and fibrosis were determined by histological analyses using H&E and Picro-Sirius Red staining. RESULTS: The results demonstrated that T-MSC CM treatment significantly reduced inflammation as well as fibrosis in the CCl(4)-injured mouse liver. IL-1Ra injection showed effects similar to T-MSC CM treatment, suggesting that T-MSC CM may exert anti-inflammatory and anti-fibrotic effects via the endogenous production of IL-1Ra. The expression of genes involved in fibrosis was evaluated, and the results showed significant induction of alpha-1 type I collagen, transforming growth factor beta, and tissue inhibitor of metalloproteases 1 upon CCl(4) injection, whereas treatment with T-MSC CM or IL-1Ra downregulated their expression. CONCLUSIONS: Taken together, these data support the therapeutic potential of T-MSC CM and/or IL-1Ra for the alleviation of liver fibrosis, as well as in treating diseases involving organ fibrosis.

Mast cells are central regulators of allergic inflammation that function by releasing various proallergic inflammatory mediators, including histamine, eicosanoids and proinflammatory cytokines. Occasionally, bacterial infections may initiate or worsen allergic inflammation. A number of studies have indicated that activation of lipoxygenase in mast cells positive regulates allergic inflammatory responses by generating leukotrienes and proinflammatory cytokines. In the present study, the effects of benzoxazole derivatives on the lipopolysaccharide (LPS)‑induced expression of proinflammatory cytokines, production of histamine and surface expression of co‑stimulatory molecules on bone marrow-derived mast cells (BMMCs) were studied. The benzoxazole derivatives significantly reduced the expression of interleukin (IL)‑1β, IL‑6, IL‑13, tumor necrosis factor‑α, perilipin (PLIN) 2, and PLIN3 in BMMCs treated with LPS. Furthermore, histamine production was suppressed in BMMCs treated with LPS, or treated with phorbol-12-myristate-13-acetate/ionomycin. Benzoxazole derivatives marginally affected the surface expression of cluster of differentiation (CD)80 and CD86 on BMMCs in the presence of LPS, although LPS alone did not increase the expression of those proteins. Therefore, benzoxazole derivatives inhibited the secretion of proinflammatory cytokines in mast cells and may be potential candidate anti‑allergic agents to suppress mast cell activation.

Human mesenchymal stem cells (MSCs) are adult multipotent cells that have plasticity and inhabit the stroma of diverse tissues. The potential utility of MSCs has been heavily investigated in the fields of regenerative medicine and cell therapy. However, MSCs represent diverse populations that may depend on the tissue of origin. Thus, the ability to identify specific MSC populations has remained difficult. Using RNA sequencing, we analyzed the whole transcriptomes of bone marrow-derived MSCs (BMs), adipose tissue-derived MSCs (AMs), and tonsil-derived MSCs (TMs). We categorized highly regulated genes from these MSC groups according to functional gene ontology (GO) classification. AMs and TMs showed higher expression of genes encoding proteins that function in protein binding, growth factor, or cytokine activity in extracellular compartments than BMs. Interestingly, TM were highly enriched for genes coding extracellular, protein-binding proteins compared with AMs. Functional Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis also showed differentially enriched signaling pathways between the three MSC groups. Further, we confirmed surface antigens expressed in common and in a tissue-specific manner on BMs, AMs, and TMs by flow cytometry analysis. This study provides comprehensive characteristics of MSCs derived from different tissues to better understand their cellular and molecular biology.

Impaired lipid metabolism and inflammatory pathways have individually been implicated in the development of insulin resistance in skeletal muscle; however, little evidence is available to date linking the two in this context. In this study, we explored a potential molecular mechanism underlying insulin resistance in myoblasts mediated by the crosstalk between lipid accumulation and inflammatory pathways. We examined the influence of perilipin 2 (PLIN2), one of the most highly expressed lipid droplet-associated proteins in skeletal muscle, on glucose uptake and on the nucleotide‑binding domain, leucine‑rich repeat containing protein 3 (NLRP3) inflammasome in vitro. PLIN2 overexpression in C2C12 cells led to an increased expression of NLRP3, caspase‑1 and interleukin (IL)‑1β, along with an impaired insulin‑induced glucose uptake. This defect was remedied by the RNAi‑mediated knockdown of NLRP3 expression. We also found that insulin receptor substrate‑1 (IRS‑1), a component of insulin signaling, was negatively regulated by NLRP3 and IL‑1β, and that IL‑1β inhibited insulin‑induced glucose uptake in myoblasts. These results suggest that PLIN2 inhibits insulin‑induced glucose uptake by activating NLRP3, caspase‑1 and IL‑1β, leading to a decreased IRS‑1 expression. This study provides in vitro evidence supporting an association between lipid metabolism and inflammatory pathways in the pathogenesis of insulin resistance in skeletal muscle, and suggests potential therapeutic targets that warrant further investigation.

Our knowledge of the immunomodulatory role of mesenchymal stem cells (MSCs) in both the innate and adaptive immune systems has dramatically expanded, providing great promise for treating various autoimmune diseases. However, the contribution of MSCs to Th17-dominant immune disease, such as psoriasis and its underlying mechanism remains elusive. In this study, we demonstrated that human palatine tonsil-derived MSCs (T-MSCs) constitutively express both the membrane-bound and soluble forms of programmed death-ligand 1 (PD-L1), which enables T-MSCs to be distinguished from MSCs originating from other organs (i.e. bone marrow or adipose tissue). We also found that T-MSC-derived PD-L1 effectively represses Th17 differentiation via both cell-to-cell contact and a paracrine effect. Further, T-MSCs increase programmed death-1 (PD-1) expression on T-cells by secreting IFN-β, which may enhance engagement with PD-L1. Finally, transplantation of T-MSCs into imiquimod-induced psoriatic skin inflammation in mice significantly abrogated disease symptoms, mainly by blunting the Th17 response in a PD-L1-dependent manner. This study suggests that T-MSCs might be a promising cell source to treat autoimmune diseases such as psoriasis, via its unique immunoregulatory features. Copyright © 2017 John Wiley & Sons, Ltd.

Mutations in MEGF10 cause early onset myopathy, areflexia, respiratory distress, and dysphagia (EMARDD), a rare congenital muscle disease, but the pathogenic mechanisms remain largely unknown. We demonstrate that short hairpin RNA (shRNA)-mediated knockdown of Megf10, as well as overexpression of the pathogenic human p.C774R mutation, leads to impaired proliferation and migration of C2C12 cells. Myoblasts from Megf10-/- mice and Megf10-/-/mdx double knockout (dko) mice also show impaired proliferation and migration compared to myoblasts from wild type and mdx mice, whereas the dko mice show histological abnormalities that are not observed in either single mutant mouse. Cell proliferation and migration are known to be regulated by the Notch receptor, which plays an essential role in myogenesis. Reciprocal co-immunoprecipitation studies show that Megf10 and Notch1 interact via their respective intracellular domains. These interactions are impaired by the pathogenic p.C774R mutation. Megf10 regulation of myoblast function appears to be mediated at least in part via interactions with key components of the Notch signaling pathway, and defects in these interactions may contribute to the pathogenesis of EMARDD.

Saturated free fatty acids (FFAs) act as lipid mediators and induce insulin resistance in skeletal muscle. Specifically, in obesity‐related diseases such as type 2 diabetes, FFAs directly reduce insulin sensitivity and glucose uptake in skeletal muscle. However, the knowledge of how FFAs mediate inflammation and subsequent tissue disorders, including fibrosis in skeletal muscle, is limited. FFAs are a natural ligand for toll‐like receptor 2 (TLR2) and TLR4, and induce chronic low‐grade inflammation that directly stimulates skeletal muscle tissue. However, persistent inflammatory stimulation in tissues could induce pro‐fibrogenic processes that ultimately lead to perturbation of the tissue architecture and dysfunction. Therefore, blocking the link between inflammatory primed skeletal muscle tissue and connective tissue might be an efficient therapeutic option for treating obesity‐induced muscle inactivity. In this study, we investigated the impact of conditioned medium obtained from human palatine tonsil‐derived mesenchymal stem cells (T‐MSCs) on the interaction between skeletal muscle cells stimulated with palmitic acid (PA) and fibroblasts. We found that PA‐treated skeletal muscle cells actively secreted interleukin‐1β (IL‐1β) and augmented the migration, proliferation and expression of fibronectin in L929 fibroblasts. Furthermore, T‐CM inhibited the skeletal muscle cell‐derived pro‐fibrogenic effect via the production of the interleukin‐1 receptor antagonist (IL‐1Ra), which is an inhibitor of IL‐1 signalling. Taken together, our data provide novel insights into the therapeutic potential of T‐MSC‐mediated therapy for the treatment of pathophysiological processes that occur in skeletal muscle tissues under chronic inflammatory conditions.