Ruthenium-induced corneal collagen crosslinking under visible light.

Corneal collagen crosslinking (CXL) is a commonly used minimally invasive surgical technique to prevent the progression of corneal ectasias, such as keratoconus. Unfortunately, riboflavin/UV-A light-based CXL procedures have not been successfully applied to all patients, and result in frequent complications, such as corneal haze and endothelial damage. We propose a new method for corneal crosslinking by using a Ruthenium (Ru) based water-soluble photoinitiator and visible light (430 nm). Tris(bipyridine)ruthenium(II) ([Ru(bpy)3]2+) and sodium persulfate (SPS) mixture covalently crosslinks free tyrosine, histidine, and lysine groups under visible light (400-450 nm), which prevents UV-A light-induced cytotoxicity in an efficient and time saving collagen crosslinking procedure. In this study, we investigated the effects of the Ru/visible blue light procedure on the viability and toxicity of human corneal epithelium, limbal, and stromal cells. Then bovine corneas crosslinked with ruthenium mixture and visible light were characterized, and their biomechanical properties were compared with the customized riboflavin/UV-A crosslinking approach in the clinics. Crosslinked corneas with a ruthenium-based CXL approach showed significantly higher young's modulus compared to riboflavin/UV-A light-based method applied to corneas. In addition, crosslinked corneas with both methods were characterized to evaluate the hydrodynamic behavior, optical transparency, and enzymatic resistance. In all biomechanical, biochemical, and optical tests used here, corneas that were crosslinked with ruthenium-based approach demonstrated better results than that of corneas crosslinked with riboflavin/ UV-A. This study is promising to be translated into a non-surgical therapy for all ectatic corneal pathologies as a result of mild conditions introduced here with visible light exposure and a nontoxic ruthenium-based photoinitiator to the cornea. STATEMENT OF SIGNIFICANCE: Keratoconus, one of the most frequent corneal diseases, could be treated with riboflavin and ultraviolet light-based photo-crosslinking application to the cornea of the patients. Unfortunately, this method has irreversible side effects and cannot be applied to all keratoconus patients. In this study, we exploited the photoactivation behavior of an organoruthenium compound to achieve corneal crosslinking. Ruthenium-based organic complex under visible light demonstrated significantly better biocompatibility and superior biomechanical results than riboflavin and ultraviolet light application. This study promises to translate into a new fast, efficient non-surgical therapy option for all ectatic corneal pathologies.

Investigation of Mitophagy Biomarkers in Corneal Epithelium of Keratoconus Patients.

PURPOSE: The pathological mechanisms of keratoconus (KC) have not been elucidated yet. Mitophagy is an important mechanism that eliminates damaged mitochondria under oxidative stress, and it could be one of the leading pathological causes of KC. This study aimed to find out the role of mitophagy in the keratoconic corneal epithelium. METHODS: The corneal epithelia were collected from the 103 progressive KC patients and the 46 control subjects. The real-time quantitative PCR was performed for PTEN-putative kinase-1 (PINK1), PARKIN, p62, and BNIP3 gene expressions in 31 KC and 9 control subjects. Western blot analyses were performed to investigate the protein expressions of PINK1, PARKIN, LC3B, ATG5, and BECLIN in the remaining 109 corneal epithelium samples from 72 patients and 37 control subjects. RESULTS: mRNA and protein expressions of PINK1 decreased significantly in the corneal epithelium of KC patients compared to the control subjects. No significant change was found in mRNA levels of PARKIN, p62, and BNIP3 in KC patients. The protein expression of PARKIN, LC3B, ATG5, and Beclin did not significantly differ between KC patients and control subjects. Gene expression levels of mitophagy biomarkers were not affected by the KC grade. CONCLUSIONS: PINK1/PARKIN-dependent mitophagy is affected in the keratoconic corneal epithelium. We found significant decreases in both mRNA and protein expressions of PINK1 in the keratoconic corneal epithelium. However, we did not observe any other significant change in mitophagy markers. Mitochondrial stress-related mitophagy pathways could be interrupted by the decreased levels of PINK1 in the keratoconic corneal epithelium, but solely PINK1 dysregulation is not likely to induce KC pathogenesis.

Mesenchymal stem cells derived from human dental follicle modulate the aberrant immune response in atopic dermatitis.

Background: Atopic dermatitis (AD) is an inflammatory cutaneous disorder. The advancements in the understanding of AD immunological pathogenesis have caused the development of therapies that suppress the dysregulated immune response. We aimed to evaluate the immunomodulatory effect of dental stem cells (dental follicle-mesenchymal stem cells [DF-MSCs]) on AD patients. Materials & methods: We investigated the immunoregulatory potential of DF-MSCs on T cell response in AD and compared them with psoriasis and healthy individuals and the underlying mechanisms. Results: DF-MSCs significantly reduced Fas, FasL and TNFR II frequency in T cells, increased naive T cell population while reducing memory T cell, decreased inflammatory cytokine levels and promoted Tregs frequency in the AD population. Conclusion: These results imply that DF-MSCs are modulating inflammation through decreasing T cell apoptosis, inducing Treg expansion and stabilizing cytokine levels.

Lay abstract Background: Atopic dermatitis (AD) is an inflammatory cutaneous disorder characterized by immune-mediated inflammation and epidermal barrier dysfunction. There is no definite solution for the treatment of AD. We aimed to evaluate the immunomodulatory and immunosuppressive effect of dental stem cells (dental follicle-mesenchymal stem cell [DF-MSCs]) on AD. Materials & methods: We investigated the immunoregulatory potential of DF-MSCs on inflammatory response in AD and compared them with psoriasis and healthy individuals and the mechanism underlying it. Results: DF-MSCs significantly reduced apoptosis-related markers in immune cells, decreased inflammatory cytokine levels and promoted Treg frequency in the AD. Conclusion: Our findings provide basic evidence for the potential role of DF-MSCs as a cellular therapy option in the treatment of AD and shed light on future clinical studies.

The Effect of Androgens on Proinflammatory Cytokine Secretion from Human Ocular Surface Epithelial Cells.

Purpose: The purpose of this study is to explore the effects of dihydrotestosterone (DHT) on lipopolysaccharide (LPS)-induced proinflammatory cytokine release in human ocular surface epithelial cells exposed to LPS and LPS-binding protein (LBP).Methods: Immortalized human corneal, conjunctival, and meibomian gland epithelial cells were cultured in keratinocyte-free medium. After confluency, they were exposed to a stratification medium Dulbecco's modified Eagle medium (DMEM)/F12 in the presence of fetal bovine serum and were exposed to vehicle, LPS + LBP, or DHT. Culture media were processed for multiplex-bead analysis of specific proinflammatory cytokines including interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-2, IL-4, IL-8, IL-6, IL-10, IL-1ß, vascular endothelial growth factor (VEGF)-A. Cytokine concentrations were compared by analysis of variance with Tukey post hoc testing. p < 0.05 was considered statistically significant.Results: The results are LPS + LBP-induced the secretion of IFN-γ, IL-6, IL-10, IL-1ß, VEGF-A cytokines in corneal epithelial cells; TNF-α, IL-2, IL-8, IL-6, IL-1ß, VEGF-A cytokines in conjunctival epithelial cells; and IL-8, IL-6, IL-1ß, VEGF-A cytokines in meibomian gland epithelial cells. When these LPS + LBP-stimulated cells were exposed to DHT for 2 days, it was found that DHT suppressed the secretion of IL-6, IL-10, IL-1ß, VEGF-A cytokines in corneal epithelial cells; TNF-α, IL-6, IL-1ß, VEGF-A cytokines in conjunctival epithelial cells; and IL-6, IL-1ß, VEGF-A cytokines in meibomian gland epithelial cells.Conclusion: LPS + LBP is shown to induce the secretion of certain proinflammatory cytokines from ocular surface and adnexal epithelial cells. DHT showed anti-inflammatory activity by suppressing some of those cytokines in these cell lines.

Human dental follicle mesenchymal stem cells alleviate T cell response in inflamed tissue of Crohn's patients.

BACKGROUND/AIMS: Crohn's Disease (CD) is a chronic inflammatory condition characterized by various abnormalities that lead to overly aggressive T-cell responses. Our in vitro experiments aimed to investigate the potential use of Dental Follicle Mesenchymal Stem Cells (DF-MSCs) to suppress the exaggerated immune response in inflamed and non-inflamed tissue of Crohn's Disease (CD). MATERIAL AND METHODS: Dental follicle tissues were obtained from extracted third molar teeth of 3 healthy volunteers who have no abscess or inflammatory diseases. Eleven patients included the experiment who had been diagnosed with CD and not received steroid maintenance therapy for more than 1 month. Mononuclear Cells (MNCs) were isolated from inflamed and non-inflamed tissue of CD. Isolated cells were stimulated with anti-CD3/anti-CD28 monoclonal antibodies in the presence and absence of DF-MSCs and analyzed for lymphocytes proliferation capacity and viability, T lymphocyte subsets, CD4+IL22BP and CD4+CD25+Foxp3+ regulatory T cell (Tregs) frequencies and cytokine levels. RESULTS: A significant downregulation of lymphocyte proliferation and CD4+IL22BP T cell ratio were found in inflamed cultures with DF-MSCs (p<0,005). Also, the frequency of Tregs increased with DF-MSCs (p<0,05). Pro-inflammatory cytokine levels (TNF-α and IL-6) were decreased (p<0,05) and IL-10 levels were increased (p<0,05) in the supernatant of inflamed cultures. CONCLUSION: DF-MSCs reduced the inflammatory immune response, induced Tregs and downregulated CD4+IL22BP T cell ratio in inflamed samples of CD patients, which may be exploited for significant therapeutic use.

Immunomodulatory and Tissue-preserving Effects of Human Dental Follicle Stem Cells in a Rat Cecal Ligation and Perforation Sepsis Model.

BACKGROUND: Mesenchymal stem cells may be used for the treatment of sepsis. Dental follicle stem cells (DFSCs) are easily accessible but have not been studied in vivo or in clinical trials in sepsis models. AIM OF THE STUDY: We aim to elucidate DFSC effects on host immunological functions in a rat cecal ligation and perforation (CLP) sepsis model. METHODS: Adult male rats were categorized into group 1 (sham procedure SP), group 2 (SP + 1 × 106 DFSCs administered 0 h after SP), group 3 (CLP + saline), group 4 (CLP + 1 × 106 DFSCs administered 0 h after CLP), and group 5 (CLP + 1 × 106 DFSCs administered 4 h after CLP). Green fluorescent protein-labeled cells were used for imaging. Histopathological examination of ileal tissues was performed. RESULTS: A significant increase in the percentage of CD4+/CD25+/Foxp3+ Treg cells in groups 4 and 5 occurred compared with that in group 3. No significant changes in CD3+/CD4+ helper T-cells and CD3+/CD8+ cytotoxic T-cells were observed. Treatment with DFSCs at 4 h significantly decreased the level of TNF-α compared with that in group 3. No significant changes in IL-10 levels and lymphocyte proliferation suppression were observed. During histopathological examination, no high scoring (Chiu scores: 3 or 4) rats were observed in the curative treatment group (group 5). CONCLUSIONS: Treatment with DFSC after 4 h of sepsis induction downregulates tissue inflammatory responses by decreasing TNF-α levels and increasing Treg cell ratio. This also has a protective effect on intestinal tissues during sepsis.

Androgen Suppresses Hyperosmolarity-Induced Inflammatory Mediators in Human Corneal Epithelial Cells.

PURPOSE: To investigative the effects of sex steroids on hyperosmolar stress-induced proinflammatory cytokine expression of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-8, and IL-6, and on the mitogen-activated protein kinase pathway in immortalized human corneal epithelial cells (hCECs). METHODS: Immortalized hCECs were cultured with keratinocyte-free medium until reaching 80% confluency with either 10 M dihidrotestosteron (DHT) or 10 M 17-ß-estradiol, and then, the medium was changed to hyperosmolar for various time points. After hyperosmolar treatment, a real-time polymerase chain reaction was performed to show the TNF-α, IL-8, and IL-6 gene expression levels in hCECs. In addition, the treated cells were lysed, and Western blot analysis was applied for phosphorylated and nonphosphorylated forms of extracellular signal-regulated kinase 1/2 (ERK1/2), p38 kinase, and c-Jun N-terminal kinase 1/2 (JNK1/2). hCECs viability was measured with Annexin V/propidium iodide. RESULTS: Pretreatment with 10 M DHT or 17-ß-estradiol inhibited the high osmolarity-induced expression of TNF-α, IL-8, and IL-6. The upregulation of p-ERK, p-JNK, and p-p38 with high osmolarity was inhibited partially by DHT, but 17-ß-estradiol pretreatment only affected p-p38 for a short time interval. In addition, DHT increased cell viability of hCECs under hyperosmolar conditions. CONCLUSIONS: Our results demonstrated that DHT and 17-ß-estradiol decreased the proinflammatory cytokine gene expression levels which were stimulated by high osmolarity in immortalized hCECs. The mitogen-activated protein kinase signaling pathway is partially involved in the regulatory effects of DHT on hCECs. These findings may contribute to the etiologic role and therapeutic implications of sex steroids in certain ocular surface diseases.

Effects of Type 2 Diabetes Mellitus on Gene Expressions of Mouse Meibomian Glands.

Purpose: Type 2 Diabetes mellitus (DM) is a major health problem and its ocular complications like orbital infections, cataract and diabetic retinopathy cause blindness. Meibomian gland (MG) dysfunction and dry eye disease are also important ocular complications of type 2 DM but not enough research has been conducted on these complications. Our hypothesis suggests type 2 DM can alter significant gene expressions of MG. In our study, MGs of leptin-deficient spontaneous diabetic and non-diabetic mice were extracted, and gene expression profiles were analyzed with microarray technology.Methods: Mice were divided into two groups; nine Lep b/ob spontaneous diabetic mice as type 2 DM group and nine non-diabetic Balb/c mice as controls. Blood glucose levels, tearfilm break-up time and fluorescein scores were measured in both two groups for 12 weeks. MGs were dissected and RNAs were isolated for microarray gene expression analysis. We filtered probes with standard deviation of more than 0.1 and we used 40452 of 45281 probes for processing. We performed fold change analysis and identified which genes are affected, and we analyzed the impact of genes on proteins, pathways and gene ontologies by using various databases.Results: We observed 172 up-regulated and 118 down-regulated genes in type 2 diabetic mice when compared to non-diabetic mice. Interestingly, expression of collagen type I, integrin beta-I binding protein-I, pyruvate dehydrogenase kinase, TNF receptor genes up-regulated with DM; on the other hand, IL-33, cholecystokinin, plasminogen activator, IL-1 and serine peptidase inhibitor genes down-regulated significantly. Also, we have seen a significant decrease in WNT signaling and pentose phosphate pathways-related genes.Conclusion: Our data show these changes in gene expression caused by endocrine and immune mechanisms of type 2 DM which result disrupted homeostasis of epithelial cells of MG. Increased expressions of apoptosis and inflammation-related genes and their effects on related pathways have proven that MGs were negatively affected by type-2 DM.

IFN-γ stimulated dental follicle mesenchymal stem cells regulate activated lymphocyte response in rheumatoid arthritis patients in vitro.

BACKGROUND: Multipotent mesenchymal stem cells (MSCs) have been investigated in autoimmune diseases such as rheumatoid arthritis (RA) due to their immunomodulatory and regenerative properties. In this study, their immunosuppressive effects on peripheral blood mononuclear cells (PBMC) of RA patients were studied. METHODS: Dental follicle stem cells (DFSCs) were isolated from follicle tissue in the orofacial region. Characterization and multipotency analyses were performed. Lymphocytes were isolated from peripheral venous blood of RA patients (n = 5) and healthy individuals (n = 5). DFSCs were preincubated with IFN-γ for 48 h. PBMCs of RA patients and healthy individuals were separately cultured with or without DFSCs for 72 h. After culture period, lymphocyte proliferation and viability, the frequency of CD4+ CD25+FoxP3+ T regulatory cells, IL-10 and TNF-α levels in the culture supernatants were measured via flow cytometry. RESULTS: Our results demonstrated that DFSCs suppressed proliferation of T lymphocytes by increasing the number of FoxP3 expressing CD4+CD25+ T regulatory cells and suppressed lymphocyte apoptosis in RA patients. Also, DFSCs reduced TNF-α cytokine secretion and upregulated IL-10 secreting cells. DISCUSSION: Such cells could potentially be a source for future immunomodulatory treatments of RA patients.

Mesenchymal stem cells suppress hepatic fibrosis accompanied by expanded intrahepatic natural killer cells in rat fibrosis model.

Natural killer (NK) cells have antifibrotic effects. We have evaluated the influence of rat bone marrow-mesenchymal stem cell (BM-MSC) treatment on liver histology, biochemical liver function tests, systemic immunoregulatory state and NK cell distribution in liver and peripheral blood in rat model of common bile duct (CBD) ligation and compared the results with the control group. Rats were divided into three groups: (1) CBD ligated (CBDL) rats received phosphate-buffered saline (CBDL + PBS group) or (2) MSC (CBDL + MSC group) and sham-operated rats received MSC (healthy + MSC group). We found significantly decreased fibrosis scores with BM-MSC treatment in CBDL rats compared to the control (CBDL + PBS) group while no fibrosis developed in sham operated (healthy + MSC) group. BM-MSC treatment has decreased the inflammation as reflected by the significantly decreased T cell proliferation and inflammatory cytokine concentrations from splenocyte culture and liver tissue itself compared to CBDL + PBS. NK cells both in parenchyme and portal areas decreased significantly in liver and blood in CBDL + PBS compared to healthy + MSC while they were found to be increased in CBDL + MSC compared to CBDL + PBS rats. In conclusion, BM-MSCs may suppress hepatic fibrosis accompanied by expanded intrahepatic NK cells in CBDL rats. Thus, our animal study shows that MSC treatment holds great promise for treatment of patients with end-stage liver diseases through a possible mechanism by adopting the NK cell population and new studies investigating the role of NK cells and clinical fibrosis are warranted.Trial registration number: Marmara University Animal Care and Use Committee approval code: 73.2013.mar.

Intranasal ovalbumin immunotherapy with mycobacterial adjuvant promotes regulatory T cell accumulation in lung tissues.

Allergen-specific immunotherapy to induce T regulatory cells in the periphery has been used to treat allergic diseases. Mycobacteria can be used as an adjuvant for inducing T regulatory cells. However, it is unclear whether intranasal immunotherapy in combination with Mycobacteria adjuvant induces regulatory T cell differentiation and attenuates allergic responses in vivo. To investigate the role of intranasal ovalbumin (OVA) treatment alone and in combination with Mycobacteria vaccae, proportions of FoxP3+ regulatory T cells and anti-inflammatory responses were evaluated in a murine model of asthma that was established in three groups of bicistronic Foxp3EGFP reporter BALB/c mice. Before establishment of the asthma model, two groups of mice received intranasal OVA immunotherapy and one also received simultaneous s.c. M. vaccae. Expression of CD4+ CD25+ Foxp3+EGFP+ T cells in the lung and spleen was analyzed by flow cytometry and the cytokine profiles of allergen-stimulated lung and spleen lymphocytes assessed. The intranasal OVA immunotherapy group showed greater expression of CD4+ CD25+ Foxp3+EGFP+ T cells in the spleen whereas in the group that also received M. vaccae such greater expression was demonstrated in the lung. Additionally, the proportion of IL-10 and IFN-γ-secreting splenocytes was greater in the intranasal OVA + M. vaccae group. CD25 neutralization decreased CD4+ Foxp3+ cells more than other groups. In parallel with this finding, production of IL-10 and IFN-γ was down-regulated. Mucosal administration of OVA antigen results in a greater proportion of CD4+ Foxp3+ T cells in the spleen. IL-10 and IFN-γ induced by intranasal OVA immunotherapy and M. vaccae administration is down-regulated after CD25 neutralization.

Effect of cDMEM media containing Ectoine on human periodontal ligament mesenchymal stem cell survival and differentiation.

BACKGROUND/AIM: Ectoine is an amino acid that can preserve osmotic balance and has more preservative activity than other osmoregulators. There are no published reports on the osmoregulators' effects on viability or differentiation of dental stem cells. The aim of this study was to investigate the effect of Ectoine as a storage media on the viability and differentiation potential of human periodontal ligament mesenchymal stem cells (hPDLMSCs). MATERIALS AND METHODS: hPDLMSCs were obtained from impacted third molar teeth. The cells were isolated, submitted to trilineage differentiation, and characterized by flow cytometer (FC). hPDLMSCs were incubated with different media containing Ectoine, complete DMEM (cDMEM), Ectoine+cDMEM, milk, and tap water for 2, 6, 12, and 24 h. The cells were analyzed by FC for viability, early-apoptosis, late apoptosis, and necrosis rates. Osteogenic and fibroblastic differentiation in hPDLMSCs were investigated by Alizarin red stain and vimentin expression. RESULTS: All treated groups showed significant decreases in cell viability after 2 h. Significant increases were detected in the number of dead cells between 2 and 12 h in all groups except the Ectoine+cDMEM group. The deposition of mineral matrix nodules was significantly higher in cells cultured with Ectoine+cDMEM compared with the other media. Higher vimentin expressions were detected in cells cultured with cDMEM and Ectoine+cDMEM media, respectively. CONCLUSIONS: Ectoine added to cDMEM media, promoted cell survival plus osteogenic and fibroblastic differentiation of hPDLMSCs.

Effects of Ankaferd BloodStopper on dermal healing in diabetic rats.

BACKGROUND/AIM: Diabetes mellitus inhibits wound-induced angiogenesis, impairs the wound healing process, and leads to the development of chronic wounds. Ankaferd BloodStopper (ABS) is a new and promising local haemostatic agent. Although the mechanism of ABS-mediated haemostasis is well established, little is known about the associated histological and biochemical tissue reactions. The aim of this study was to evaluate the effects of this new-generation local haemostatic agent on short-term soft-tissue healing in streptozotocin (STZ)-treated rats. MATERIALS AND METHODS: The 24 Wistar albino rats used in this study were divided into STZ-treated (STZ, n = 12) and nontreated groups (control, n = 12). Four days prior to surgery, rats in the STZ group were subcutaneously administered 60 mg/kg STZ intraperitoneally, while rats in the control group were administered 1 mL saline/kg. An incision was made in the dorsal dermal tissue of all rats, and either ABS or no haemostatic agent (NHAA) was applied to the wound before suturing. All of the rats were euthanised on postoperative day 4. Blood and skin samples were evaluated biochemically and histologically. RESULTS: The results showed that STZ treatment impaired soft-tissue healing, assessed by measuring glutathione and lipid peroxidation levels. Moreover, while good histological results were obtained in the control group treated with ABS, there were fewer benefits in the STZ-treated group. CONCLUSION: ABS's benefits in the control group seemed to lose their effectiveness under STZ medication.

The Comparison of the Immunologic Properties of Stem Cells Isolated from Human Exfoliated Deciduous Teeth, Dental Pulp, and Dental Follicles.

Aim. To compare the effects of various mesenchymal stem cells, those isolated from human exfoliated deciduous teeth (SHEDs), dental pulp stem cells (DPSCs), and dental follicle stem cells (DFSCs), on human peripheral blood mononuclear cells (PBMCs). Method. Mesenchymal stem cells were isolated from three sources in the orofacial region. Characterization and PCR analyses were performed. Lymphocytes were isolated from healthy peripheral venous blood. Lymphocytes were cocultured with stem cells in the presence and absence of IFN-γ and stimulated with anti-CD2, anti-CD3, and anti-CD28 for 3 days. Then, lymphocyte proliferation, the number of CD4(+)FoxP3(+) T regulatory cells, and the levels of Fas/Fas ligand, IL-4, IL-10, and IFN-γ in the culture supernatant were measured. Results. The DFSCs exhibited an enhanced differentiation capacity and an increased number of CD4(+)FoxP3(+) T lymphocytes and suppressed the proliferation and apoptosis of PBMCs compared with SHEDs and DPSCs. The addition of IFN-γ augmented the proliferation of DFSCs. Furthermore, the DFSCs suppressed IL-4 and IFN-γ cytokine levels and enhanced IL-10 levels compared with the other cell sources. Conclusion. These results suggest that IFN-γ stimulates DFSCs by inducing an immunomodulatory effect on the PBMCs of healthy donors while suppressing apoptosis and proliferation and increasing the number of CD4(+)FoxP3(+) cells.

Dental follicle mesenchymal stem cell administration ameliorates muscle weakness in MuSK-immunized mice.

BACKGROUND: Myasthenia gravis (MG) is an antibody-mediated autoimmune disease of the neuromuscular junction (NMJ), mostly associated with acetylcholine receptor (AChR) antibodies. Around 5-10 % of MG patients show antibodies to muscle-specific tyrosine kinase (MuSK). Mesenchymal stem cell (MSC) administration has been shown to ameliorate muscle weakness in the experimental autoimmune myasthenia gravis (EAMG) model induced by AChR immunization. METHODS: To investigate the efficacy of stem cell treatment in MuSK-related EAMG, clinical and immunological features of MuSK-immunized mice with or without dental follicle MSC (DFMSC) treatment were compared. RESULTS: MuSK-immunized mice intravenously treated with DFMSC after second and third immunizations showed significantly lower EAMG incidence and severity and reduced serum anti-MuSK antibody, NMJ IgG, and C3 deposit levels and CD11b+ lymph node cell ratios. Moreover, lymph node cells of DFMSC-administered mice showed reduced proliferation and IL-6 and IL-12 production responses to MuSK stimulation. By contrast, proportions of B and T cell populations and production of a wide variety of cytokines were not affected from DFMSC treatment. CONCLUSIONS: Our results suggest that DFMSC treatment shows its beneficial effects mostly through suppression of innate immune system, whereas other immune functions appear to be preserved. Stem cell treatment might thus constitute a specific and effective treatment method in MuSK-associated MG.