Matrix Metalloproteinase 1 as a Marker of Tonsil-Derived Mesenchymal Stem Cells to Assess Bone Marrow Cell Migration

BACKGROUND@#To achieve optimal bone marrow engraftment during bone marrow transplantation, migration of donor bone marrow cells (BMCs) toward the recipient’s bone marrow is critical. Despite the enhanced engraftment of BMCs by co-administration of mesenchymal stem cells (MSCs), the efficiency can be variable depending on MSC donor. The purpose of this study is to examine the functional heterogeneity of tonsil-derived MSCs (TMSCs) and to identify a marker to evaluate efficacy for the enhancement of BMC migration. @*METHODS@#To examine the donor-to-donor variation of TMSCs in potentiating BMC migration, we isolated TMSCs from 25 independent donors. Transcriptome of TMSCs and proteome of conditioned medium derived from TMSC were analyzed. @*RESULTS@#Enhanced BMC migration by conditioned medium derived from TMSCs was variable depending on TMSC donor. The TMSCs derived from 25 donors showed distinct expression profiles compared with other cells, including fibroblasts, adipose-derived MSCs and bone marrow–derived MSCs. TMSCs were distributed in two categories: high- and low-efficacy groups for potentiating BMC migration. Transcriptome analysis of TMSCs and proteome profiles of conditioned medium derived from TMSCs revealed higher expression and secretion of matrix metalloproteinase (MMP) 1 in the high-efficacy group. MMP1 knockdown in TMSCs abrogated the supportive efficacy of conditioned medium derived from TMSC cultures in BMC migration. @*CONCLUSION@#These data suggest that secreted MMP1 can be used as a marker to evaluate the efficacy of TMSCs in enhancing BMC migration. Furthermore, the strategy of analyzing transcriptomes and proteomes of the MSCs may be useful to set the standard for donor variation.

Mesenchymal Stem Cell-Derived Exosomes Attenuate TLR7-Mediated Mast Cell Activation

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 tonsilderived 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.

Promotion of Platelet Production by Co-Transplantation of Mesenchymal Stem Cells in Bone Marrow Transplantation

BACKGROUND@#Therapeutic strategies that can promote platelet production are in demand to enhance clinical outcomes of bone marrow transplantation (BMT). Our research group has studied human tonsil-derived mesenchymal stem cells (TMSCs) and their effectiveness in promoting bone marrow (BM) engraftment. Here, we analyzed the effects of T-MSCs on platelet production and hemostasis. @*METHODS@#Donor BM cells (BMCs) were isolated from C57BL/6 mice and transplanted with or without T-MSCs to BALB/c recipient mice. Mice were sacrificed and blood cells were counted using an Auto Hematology Analyzer. Femur sections were stained with CD41 antibody to analyze megakaryocytes in the BM. Growth factor secretion from MSCs was analyzed using the Quantibody Array. Effects of T-MSC conditioned medium (CM) on megakaryopoiesis were investigated using the MegaCult assay. In a mouse model of BMT, T-MSC CM was injected with or without anti-placental growth factor (a-PlGF) blocking antibody, and blood cell numbers and coagulation were analyzed. @*RESULTS@#T-MSC co-transplantation increased percent survival of BMT mice. Platelet numbers were significantly lower in the BMC-only group, whereas T-MSC co-transplantation restored circulating platelets to levels similar to those of the control group. Significantly reduced numbers of CD41 ? megakaryocytes in Bu-Cy and BMC groups were increased by T-MSC co-transplantation. PlGF secretion from T-MSCs were detected and enhanced megakaryopoiesis, platelet production, and coagulation by T-MCS CM were disrupted in the presence of the a-PlGF blocking antibody. @*CONCLUSION@#We demonstrated the effectiveness of T-MSC co-transplantation in promoting platelet production and coagulation after BMT. These findings highlight the potential therapeutic relevance of T-MSCs for preventing thrombocytopenia after BMT.

Toll‐Like Receptor 7 (TLR7) Mediated Transcriptomic Changes on Human Mast Cells

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 upregulated 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.

Toll‐Like Receptor 7 (TLR7) Mediated Transcriptomic Changes on Human Mast Cells

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 upregulated 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.

Conditioned Medium from Tonsil-Derived Mesenchymal Stem Cells Relieves CCl₄-Induced Liver Fibrosis in Mice

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 (CCl₄) and investigated the therapeutic potential of conditionedmedium from tonsil-derivedMSCs (T-MSCCM). 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 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. CONCLUSION: 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 and Microbiome in Skin Immunity

The skin functions as a physical barrier against entry of pathogens while concomitantly supporting a myriad of commensal organisms. The characterization of these microbial communities has enhanced our knowledge of the ecology of organisms present in normal skin, and studies have begun to illuminate the intimate relationship between the host and resident microbes. The cutaneous innate and adaptive immune responses can modulate skin microbiota, while simultaneously, the microbiota educates the host immune system. A crucial element of the innate immune response is mast cells, which reside strategically in tissues that are commonly exposed to the external environment, such as the skin and mucosae. Mast cells are present on the frontline of defense against pathogens, suggesting they may play an important role in fostering the host-microbiota relationship. In this review, we highlight findings regarding the interaction between skin microbiota and mast cells and the resulting outcomes in skin homeostasis.

Mast Cells and Microbiome in Skin Immunity

The skin functions as a physical barrier against entry of pathogens while concomitantly supporting a myriad of commensal organisms. The characterization of these microbial communities has enhanced our knowledge of the ecology of organisms present in normal skin, and studies have begun to illuminate the intimate relationship between the host and resident microbes. The cutaneous innate and adaptive immune responses can modulate skin microbiota, while simultaneously, the microbiota educates the host immune system. A crucial element of the innate immune response is mast cells, which reside strategically in tissues that are commonly exposed to the external environment, such as the skin and mucosae. Mast cells are present on the frontline of defense against pathogens, suggesting they may play an important role in fostering the host-microbiota relationship. In this review, we highlight findings regarding the interaction between skin microbiota and mast cells and the resulting outcomes in skin homeostasis.

Mast Cells and Lipid Mediators

Human mast cells are potent effector cells in host defense mechanisms of innate and acquired immunity, including inflammatory diseases such as asthma and atherosclerosis. Mast cells originate from pluripotent hematopoietic progenitors in the bone marrow. Activation of mast cells by different stimuli triggers the release of a large range of mediators, including de novo synthesized eicosanoids which are highly biologically active lipid mediators. For the generation of lipid mediators, cytoplasmic lipid droplets have been shown to function as a major intracellular pool of arachidonic acid, the precursor for eicosanoids biosynthesis. The article summarizes current knowledge on mast cell biosynthesis of lipid mediator and the role in inflammation.

Effects of Interleukin-9 Blockade on Chronic Airway Inflammation in Murine Asthma Models

PURPOSE: Asthma is a chronic inflammatory disease of the airways associated with structural changes and airway remodeling. Interleukin (IL)-9 has pleiotropic effects on both inflammatory cells and airway structural cells, which are involved in asthma pathogenesis. We evaluated the effects of IL-9 blockade on chronic airway inflammation. METHODS: Acute airway inflammation was induced in Balb/c mice using aerosolized ovalbumin (OVA), whereas chronic asthma was induced by OVA exposure for 5 weeks with anti-IL-9 or isotype-matched antibody (Ab) treatment during the OVA challenge. Inflammatory cells in bronchoalveolar lavage fluid (BALF) were counted and lung tissues were stained to detect cellular infiltration, mucus deposition, and collagen accumulation. The levels of interferon (IFN)-gamma, IL-4, IL-5, IL-9, IL-17, and immunoglobulin E (IgE) in BALF were measured using enzyme linked immunosorbent assays, and profiles of inflammatory cells and subsets of T helper (Th) cells were analyzed using flow cytometry. RESULTS: IL-9, IL-17, and IFN-gamma levels were significantly increased in the chronic group compared to the acute asthma group. However, the number of IL-9-positive cells was not affected, with a decrease in Th17 cells in OVA-challenged caspase-1 knockout mice. Numbers of eosinophils, neutrophils, B cells, mast cells, and Th17 cells decreased after administration of anti-IL-9 Ab. Total IgE, IL-5, IL-9, and IL-17 levels were also lower in the anti-IL-9 group. CONCLUSIONS: Our results suggest that anti-IL-9 Ab treatment inhibits pulmonary infiltration of inflammatory cells and cytokine production, especially IL-17. These results provide a basis for the use of an anti-IL-9 Ab to combat IL-17-mediated airway inflammation.

Effect of Spa Spring Water on Cytokine Expression in Human Keratinocyte HaCaT Cells and on Differentiation of CD4+ T Cells

BACKGROUND: Skin acts as the first line of defense against any foreign materials outside of our body. In inflammatory skin disease, the pathogenesis is due to an immune reaction in the keratinocytes, immune cells and soluble mediators. Balneotherapy is widely used for the treatment of inflammatory skin disease, but the mechanisms are only partly understood by immune regulation. Balneotherapy in dermatologic disease can affect the secretion of pro-inflammatory cytokines, IL-1alpha and tumor necrosis factor from keratinocytes, and possibly affect the T cell differentiation. OBJECTIVE: In this study, we evaluated the effect of spa spring water from Yong-gung oncheon on the cells, and investigated the skin immune reaction. METHODS: We investigated the immunomodulatory or anti-inflammatory effect of thermal spring water on the expression of pro-inflammatory cytokines in the HaCaT cells under Toll-like receptor (TLR) stimulation, as well as the effect on the differentiation of CD4+ T cells under spring water. RESULTS: The treatment of spa spring water from Yong-gung oncheon decreased the expression of proinflammatory cytokines under TLR stimulation to the HaCaT cells and antigen presenting cells. In addition, spa spring water attenuated the differentiation process of subsets of CD4+ T cells, i.e., Th1, Th2 and Th17 cells. All these immune parameters can be used to evaluate the efficacy of spa spring water in Korea, in terms of the immune modulatory effect. CONCLUSION: Spa spring water treatment suppressed the inflammatory cytokines production and also modulated the differentiation of CD4+ T cells into Th1, Th2, and Th17 cells, but not the Tregs cells.

Interleukin-17 and Interleukin-22 Induced Proinflammatory Cytokine Production in Keratinocytes via Inhibitor of Nuclear Factor kappaB Kinase-alpha Expression

BACKGROUND: The pathogenesis of psoriasis may involve the interleukin (IL)-23 and Th17-mediated immune responses. Th17 cells secret IL-17 and IL-22, which mediates dermal inflammation and acanthosis. OBJECTIVE: As inhibitor of nuclear factor kappaB kinase-alpha (IKKalpha) has been previously identified as a primary regulator of keratinocyte differentiation and proliferation, we proposed that IL-17 and IL-22 might affect keratinocyte differentiation by changing the expression of IKKalpha. METHODS: We employed HaCaT cells maintained culture medium at a low calcium concentration (0.06 mM) and induced differentiation by switching to the high concentration (2.8 mM) media with IL-17 or IL-22, then compared the IKKalpha expression and the cell cycle. We employed reconstituted human epidermal skin (Neoderm) and mice ears for the in vivo studies. RESULTS: Elevated calcium concentration induced IKKalpha expression and terminal differentiation with cell cycle arrest in HaCaT cell cultures. Moreover, IL-17 and IL-22 treatment also induced IKKalpha in HaCaT cells and reconstituted human epidermis. IKKalpha induction was also noted, following the injection of IL-17 and IL-22 into mice ears. CONCLUSION: Although the induction of IKKalpha was accompanied by keratinocyte differentiation, IL-17 and IL-22 did not affect calcium-mediated differentiation or the cell cycle. Rather, IL-17 and IL-22 appear to contribute to the inflammation occurring via the induction of IKKalpha from keratinocytes or skin layers.

Effect on Cell Cycle Progression by N-Myc Knockdown in SK-N-BE(2) Neuroblastoma Cell Line and Cytotoxicity with STI-571 Compound / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Neuroblastoma is a common tumor in childhood, and generally exhibits heterogeneity and a malignant progression. MYCN expression and amplification profiles frequently correlate with therapeutic prognosis. Although it has been reported that MYCN silencing causes differentiation and apoptosis in human neuroblastoma cells, MYCN expression influences the cytotoxic potential of chemotherapeutic drugs via the deregulation of the cell cycle. STI-571 may constitute a promising therapeutic agent against neuroblastoma, particularly in cases in which c-Kit is expressed preferentially in MYCN-amplified neuroblastoma. MATERIALS AND METHODS: To determine whether STI-571 exerts a synergistic effect on cytotoxicity with MYCN expression, we assessed apoptotic cell death and cell cycle distribution after 72 h of exposure to STI-571 with or with out treatment of SK-N-BE(2) neuroblastoma cells with MYCN siRNA. RESULTS: MYCN siRNA-treated SK-N-BE(2) cells did not affect apoptosis and cells were arrested in G0/G1 phase after STI-571 treatment. CONCLUSIONS: siRNA therapy targeted to MYCN may not be effective when administered in combination with STI-571 treatment in cases of neuroblastoma. Therefore, chemotherapeutic drugs that target S or G2-M phase may prove ineffective when applied to cells arrested in the G0/1 phase as the result of MYCN knockdown and STI-571 treatment.

Oral Tolerance Increased the Proportion of CD8+ T Cells in Mouse Intestinal Lamina Propria

BACKGROUND: Oral tolerance is defined by the inhibition of immune responsiveness to a protein previously exposed via the oral route. Protein antigens exposed via the oral route can be absorbed through the mucosal surfaces of the gastrointestinal tract and can make physical contact with immune cells residing in the intestinal lamina propria (LP). However, the mechanisms of oral tolerance and immune regulation in the intestines currently remain to be clearly elucidated. METHODS: In order to determine the effect of oral protein antigen intake (ovalbumin, OVA) on the intestinal LP, we assessed the expression profile of the T cell receptor and the co-receptors on the cells from the intestines of the tolerant and immune mouse groups. RESULTS: We determined that the proportion of OVA-specific B cells and gamma delta T cells had decreased, but the CD8alpha beta and CD8alpha alpha T cells were increased in the LP from the tolerant group. The proportion of CD8+ T cells in the spleen did not evidence any significant differences between treatment groups. CONCLUSION: These results indicate that CD8+ T cells in the intestinal LP may perform a regulatory role following antigen challenge via the oral route.

Topical Glyceryl Trinitrate (GTN) Versus Conservative Treatment (CT) in Chronic Anal Fissure: Prospective and Randomized Study

PURPOSE: This study was designed to compare the effect of treatment using glyceryl trinitrate (GTN) ointment with that of conservative treatment (CT) on chronic anal fissure. METHODS: As a preliminary study, maximal resting pressures of the anal canal were checked in 13 patients having chronic anal fissure before and 10 minutes after application of 0.2% GTN ointment. As the study groups, 59 patients having chronic anal fissure were randomly allocated to the GTN and the placebo groups. All the patients in both groups were given oral analgesics, sedatives, and bulk-forming agents. They had applied 0.2% GTN ointment or a placebo ointment three times a day to their perianal skin. Maximal resting pressures of the anal canal were checked at the beginning and at the endpoint of the treatment period which continued for 6 weeks. If there was complete healing of the fissure in the middle of the treatment, the treatment was stopped. Sixteen patients were lost during the study. RESULTS: Among the rest, 22 and 21 patients were included in the GTN group and the placebo group, respectively. The maximal resting pressure decreased significantly in all groups (p0.05). CONCLUSION: The effect of GTN on the symptomatic relief and results of treatment in patients having chronic anal fissure is not superior to that of conservative treatment.

Adrenal Hemorrhage in a Neonate

Neonatal adrenal hemorrhage is frequently associated with birth trauma or perinatal hypoxia. Hemorrhagic necrosis of the adrenal glands is often found at autopsy and many small lesions are usually asymptomatic. A palpable abdominal mass and jaundice are the usual presenting signs. Ultrasound is very useful in the diagnosis of this lesion; however, if the mass has mixed echoic pattern, magnetic resonance imaging (MRI) is helpful for the differential diagnosis from neuroblastoma. We present the case of a female newborn who was found to have a abdominal mass on physical examination. The patient showed anemia and hyperbilirubinemia. An ultrasonogram disclosed a 3.8 × 3.0 cm suprarenal mass with mixed echoic pattern. The mass was initially suspected to be neuroblastoma. An abdominal computed tomogram was not able to differentiate the mass. Magnetic resonance imaging revealed markedly increased signal intensity on T1 and T2-weighted sequences. This findLl1g was consistent with adrenal hemorrhage. Serial sonogram demonstrated the mass that resolved completely by 12 weeks of age.