Human Dental Mesenchymal Stem Cells Restorate Immune Response in Sera of Pemphigus Vulgaris Patients

Human Dental Mesenchymal Stem Cells Restorate Immune Response in Sera of Pemphigus Vulgaris Patients | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Human Dental Mesenchymal Stem Cells Restorate Immune Response in Sera of Pemphigus Vulgaris Patients Zuleyha Ozgen, Yazgul Duran, Tulin Ergun, Kamil Göker, Sabriye Senem, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4643043/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Pemphigus is an IgG-mediated autoimmune condition characterized by autoantibodies targeting desmogleins, leading to acantholysis. Current treatments, including systemic corticosteroids and immunosuppressive drugs, are associated with significant adverse effects. Mesenchymal stem cells (MSCs) offer a promising alternative due to their immunomodulatory properties and low immunogenicity. This study evaluates the immunomodulatory effects of Dental Follicle Mesenchymal Stem Cells (DF-MSCs) on Pemphigus Vulgaris (PV) patients and healthy controls by examining T-cell proliferation, apoptosis, cytokine levels, and anti-desmoglein 1/3 IgG profiles. Twenty-one symptomatic PV patients and eleven healthy volunteers were included. DF-MSCs were isolated, characterized, and differentiated into osteocytes, adipocytes, and chondrocytes. Peripheral Blood Mononuclear Cells (PBMCs) were co-cultured with DF-MSCs, and various assays were conducted to evaluate T-cell proliferation, apoptosis, regulatory T cells, cytokine expression, and autoantibody levels. Results showed that DF-MSC co-cultures significantly reduced lymphocyte proliferation (43.58–16.27%), IL-4 (38.06 ng/L to 32.26 ng/L), TNF-α (32.45 ng/L to 29.41 ng/L), and DSG1 (3.29 ng/ml to 3.00 ng/ml) and DSG3 (262.40 ng/ml to 245.08 ng/ml) levels in PV patients. An increase in regulatory T cells (1.22–3.75%), IL-10 (47.46 pg/ml to 54.94 pg/ml), and IFN-γ (12.39 ng/ml to 19.70 ng/ml) was also observed. No significant changes were noted in healthy controls. These findings suggest that DF-MSCs could potentially offer a curative approach for treating pemphigus by restoring immune balance. However, further clinical trials are necessary to confirm their efficacy. Pemphigus Mesenchymal stem cell DF-MSCs CD4 + CD25 + Foxp3 + Treg anti-desmoglein Figures Figure 1 Figure 2 Figure 3 INTRODUCTION Pemphigus comprises a set of IgG-mediated autoimmune conditions characterized by the presence of autoantibodies targeting desmogleins (desmoglein 1 and desmoglein 3). These proteins are part of the cadherin family of cell–cell adhesion molecules, crucial for upholding intercellular adhesion in stratified squamous epithelia, like the skin and oral mucosa [ 1 ]. Pemphigus involves intricate interactions between various pathways and cell types contributing to the disease pathogenesis. Within a self-perpetuating cycle, dysfunctional T lymphocytes trigger the reactivation of B cells, leading to the attack on self-tissues by B cells [ 2 ]. A crucial event in this process is the development of IgG autoantibodies targeting the extracellular domains of Desmogleins (Dsg 1 and 3), which are integral cell membrane proteins in keratinocytes, ultimately mediating acantholysis [ 3 ]. The precise role of cytokines in the pathogenesis of Pemphigus remains incompletely elucidated. Although definitive evidence is lacking, it is widely acknowledged that there is an elevation of T helper-2 (Th2) originated cytokines and a decrease in T helper-1 (Th1) induced cytokines [ 3 ]. The therapeutic armamentarium for pemphigus currently encompasses systemic corticosteroids, rituximab, and immunosuppressive medications, but these interventions are associated with an elevated risk of infections, vascular incidents, and other adverse events [ 3 ]. Consequently, an unmet need persists for the development of effective and safe treatment modalities. Mesenchymal stem cells (MSCs) represent a promising avenue in this context, as they are multipotent cells that can be obtained from various sources such as bone marrow, peripheral blood, adipose tissue, placental tissue, umbilical cord, Wharton’s jelly, and dental tissues. MSCs exhibit the remarkable capability to modulate immune responses, specifically by restoring a balance in the Th1 (associated with autoimmunity) and Th2 (related to allergic diseases) ratio. Emerging evidence has reported the efficacy of stem cell therapies in treating diverse autoimmune diseases, including pemphigus [ 4 ]. Notably, when administered intravenously, MSCs exhibit a propensity to selectively target sites of inflammation and participate in tissue repair and regeneration. During this process, MSCs undergo differentiation into various cell types and concurrently secrete multiple bioactive molecules, facilitating the recovery of injured cells and suppressing inflammatory processes [ 4 ]. Furthermore, a distinct advantage of MSCs lies in their limited immunogenicity, minimizing the potential for adverse immunological reactions. Though only limited case reports and small series of cases exist, both autologous and allogeneic hematopoietic stem cell therapies have shown promising outcomes in treating stubborn cases of pemphigus. However, there's a significant lack of research exploring the therapeutic benefits of Mesenchymal Stem Cells (MSCs) in treating human subjects [ 5 – 8 ]. Interestingly, the transplantation of MSCs derived from adipose tissue has shown a beneficial impact on refractory skin lesions associated with pemphigus foliaceus in a dog [ 8 ]. Dental Mesenchymal Stem Cells (DMSCs) are extracted from various sources within the oral cavity, including dental pulp, the periodontal ligament, dental follicle, apical papilla, and dental cementum. Although DMSCs share numerous similarities with other types of MSCs, they possess distinct characteristics as well. One of the advantages of DMSCs is that they can be effortlessly isolated from teeth that are usually discarded. Additionally, their high proliferation rate makes DMSCs particularly suitable for both research and therapeutic purposes [ 9 , 10 ]. Like other Mesenchymal Stem Cells, Dental Mesenchymal Stem Cells (DMSCs) possess immunomodulatory properties, primarily through the secretion of cytokines. These attributes potentially make them an effective tool in the treatment of immune-mediated diseases, including pemphigus. Because MSCs exhibit low immunogenicity, they can be safely utilized in treating a range of disorders. Even though there are concerns about the tumorigenic potential of MSCs, no cases of tumor development associated with MSC treatments have been reported in existing literature [ 11 – 13 ]. The purpose of this study is to evaluate the immunomodulatory impact of Dental Follicle Mesenchymal Stem Cells (DF-MSCs) on Pemphigus Vulgaris and healthy control subjects. This will be done by examining T-cell proliferation and viability, apoptosis, levels of inflammatory and regulatory cytokines, and profiles of anti-desmoglein 1/3 IgG. MATERIAL & METHOD Selection Criteria This study received ethical approval from a university's Ethics Committee (Protocol No: 09.2016.196/70737436-050.06.04). Twenty-one symptomatic PV patients (mean age 49.43 ± 13.25 years; 13 males, 8 females) treated at a university dermatology department were included. Diagnoses were confirmed via histopathological and direct immunofluorescence analysis. PDAI scores ranged from 7 to 35. Treatments varied: five were newly diagnosed, six received methylprednisolone (≤ 20 mg/day), six were on methotrexate, mycophenolate, or azathioprine, and four received Rituximab. No concurrent inflammatory or autoimmune diseases were present. The control group comprised eleven healthy volunteers (mean age 51.73 ± 5.92 years; 7 males, 4 females) with no history of PV or other autoimmune/inflammatory diseases (Table 1 ). Table 1 Demographic and Clinical Characteristics of Pemphigus Vulgaris (PV) Patients and Healthy Controls (HC) Patients Age Gender Duration of disease (month) PDAI score Concomittant treatment PV1 58 M 45 13 MTX + MP PV2 63 M 12 21 RTX PV3 49 F 22 7 < 20 mg MP PV4 64 M 1 14 - PV5 53 F 8 29 RTX PV6 47 F 22 9 < 20 mg MP PV7 37 F 19 11 < 20 mg MP PV8 72 M 62 9 < 20 mg MP PV9 47 M 11 17 RTX PV10 44 F 3 35 - PV11 53 F 7 27 RTX PV12 48 F 9 9 < 20 mg MP PV13 59 M 16 13 AZA PV14 59 F 11 11 MTX + MP PV15 45 M 2 7 - PV16 36 M 6 10 MTX + MP PV17 38 F 13 9 < 20 mg MP PV18 28 M 1 15 - PV19 21 F 2 31 - PV20 53 F 13 13 AZA + MP PV21 72 F 34 14 MTX HC1 48 F HC2 42 F HC3 53 M HC4 45 F HC5 51 M HC6 53 F HC7 63 M HC8 58 F HC9 49 M HC10 51 F HC11 56 F Dental Folicle Derived Mesenchymal Stem Cell (DF-MSC) Isolations Participants aged 18–25 years were selected from Marmara University's Faculty of Dentistry. Dental follicle tissues collected from these volunteers were physically segmented and enzymatically digested with Phosphate Buffered Saline (PBS; Gibco, Gaithersburg, MD) containing 3 mg/ml collagenase type I (Sigma, Grand Island, USA). Samples were collected under sterile conditions and transported in cold Dulbecco's phosphate-buffered saline (DPBS, Gibco, Grand Island, NY) with 1% penicillin/streptomycin (Gibco, USA). All laboratory studies were conducted in the Pediatric Allergy-Immunology department of our university. To digest the dental follicle tissues, 10 mL of Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin/streptomycin was added, followed by centrifugation at 1500 rpm for 10 minutes. The resulting cell pellets were obtained, and the supernatant was aspirated. Dental follicle-derived MSCs (DF-MSCs) were cultivated in T-25 flasks in a 5% CO 2 atmosphere at 37°C using a culture medium of DMEM, 10% FBS, and 1% penicillin/streptomycin. DF-MSCs were washed with DPBS and provided with fresh culture medium, which was changed every 3 to 4 days until confluence was reached. When cells reached 70–80% confluence, they were detached with 0.25% trypsin-EDTA (Gibco, USA). Adherent cells cultured to the third passage were characterized and analyzed for specific surface markers using flow cytometry [ 14 ]. Dental Folicle Derived Mesenchymal Stem Cell (DF-MSC) Characterisation Analysis: Flow Cytometry Analysis For cell surface antigen analysis, third passage DF-MSCs were incubated with antibodies for human CD73 allophycocyanin (APC), CD90 fluorescein isothiocyanate (FITC), CD44 phycoerythrin (PE), CD105 Peridinin-Chlorophyll-Protein (PerCP), CD4 FITC, CD19 (PerCP), CD25 APC, and HLA-DR PE at room temperature in the dark. Control antibodies were phycoerythrin-conjugated or fluorescein isothiocyanate-conjugated and allophycocyanin-conjugated mouse IgG1 and mouse IgG2 (BD Biosciences, San Diego, CA, USA). Flow cytometry results were analyzed using a Beckman Coulter Navios EX and the Kaluza software [ 14 ]. Dental Folicle Derived Mesenchymal Stem Cell (DF-MSC) Differentiation Analysis: Adipogenic, Osteogenic, Chondrogenetic The differentiation of DF-MSCs into osteocytes, adipocytes, and chondrocytes was carried out using the StemPro Osteogenesis Differentiation Kit, StemPro Adipogenesis Differentiation Kit, and StemPro Chondrogenesis Differentiation Kit (all from Gibco, Grand Island, NY, USA) following the manufacturer's instructions. Briefly, cells (5×10^4 per well) were seeded into 6-well plates, and differentiation media was introduced 21–28 days later. During cell growth, the culture medium was changed twice a week. Alizarin Red staining was used to detect osteogenesis, Oil Red O staining for adipogenesis, and Alcian Blue staining for chondrogenesis. Images of stained cells were captured using a light microscope [ 14 ]. Isolation of Peripheral Blood Mononuclear Cells (PBMC) Peripheral blood (10 ml) was collected from both patients and healthy controls into heparinized tubes. These samples were diluted 1:1 with sterile Phosphate Buffered Saline (PBS) and subjected to ficoll density gradient centrifugation at 2000 rpm for 25 minutes. The buffy coat layer was harvested and washed with PBS. Cell counts were determined, and the cells were prepared for co-culture experiments. The Peripheral Blood Mononuclear Cells (PBMCs) were then resuspended in a culture medium consisting of RPMI-1640, supplemented with 1% Penicillin/Streptomycin (P/S) and 10% Fetal Bovine Serum (FBS, Invitrogen) [ 15 ]. Co-cultivation of DF-MSCs and PBMCs Initially, DF-MSCs (5x10 4 ) were cultured for 48 hours prior to the introduction of PBMCs. Subsequently, 5x10 5 PBMCs were added to each well containing complete RPMI (cRPMI), both in the presence and absence of DF-MSCs, and co-cultivated for a period of 3 days. These cultures were then stimulated with 0.5ug/ml of anti-CD3 (eBioscience, San Diego, CA) and 0.5ug/ml of CD28 (eBioscience, San Diego, CA) [ 12 ]. CFSE Proliferation Assay To perform the Carboxyfluorescein Succinimidyl Ester (CFSE) proliferation assay, 1µl of CFSE (Invitrogen™, USA) was added to PBMCs suspended in 1ml of PBS at a concentration of 10 6 cells/mL and then incubated for a duration of 7 minutes. Following the incubation period, the cells were washed twice using cRPMI. Finally, the resuspended cells were transferred into culture plates. Apoptosis Assay After co-culturing for 72 hours, the cells were harvested and washed twice with 1X PBS. The cell pellet was then resuspended in 1X Binding Buffer. To this suspension, 5µl of Annexin V (BD Bioscience, USA) was added and incubated for 15 minutes at room temperature (25°C) in a dark environment. Following the incubation, the cells were washed again with 1X PBS. The pellet was subsequently resuspended in 1X Binding Buffer. 5µl of Propidium Iodide (PI, PE) was then added to the cells, which were subsequently analyzed using flow cytometry. Regulatory T Cell Assay CD4 + CD25 + FOXP3 + T regulatory cells (Tregs) were analyzed within the PBMCs after a 72-hour period. The cultures were examined by flow cytometry, utilizing a Human regulatory T cell kit as per the manufacturer's instructions (eBioscience, USA). In brief, cells were rinsed twice with 1X PBS. The cell pellet was stained with anti-CD4 and anti-CD25 antibodies for 20 minutes in the dark and then centrifuged at 1500 rpm for 5 minutes. Subsequently, the pellet was incubated with a fixation/permeabilization buffer for 30 minutes and washed twice with 1X permeabilization buffer. Following this, the FOXP3 antibody was applied. After incubation, the cells were rinsed twice with 1X permeabilization buffer and then centrifuged at 1500 rpm for 5 minutes. Cytokine Expression Profile Analysis The expression levels of cytokines (IL-4, IL-10, IFN-γ, TNFα) in the culture supernatants were evaluated using ELISA (BT-LAB, China). The standard solutions and wash buffer were prepared in accordance with the manufacturer's protocol. Initially, 50µl of the standard solution was introduced into the Standard wells, and 40µl of the culture supernatant was transferred to sample wells. This was followed by the addition of 10µl of the Human IL4 antibody to the sample wells. For detection, 50µl of streptavidin-HRP was added to both sample and standard wells. The plate was then sealed and incubated for 60 minutes at 37°C. Subsequently, it was washed five times with wash buffer and a mixture of 50µl each of substrate solutions A and B was added to each well. After a 10-minute incubation at 37°C in the dark, 50µl of Stop Solution was added to each well. The absorbance was then measured using a microplate reader at 450 nm. The results were determined as per the manufacturer's instructions. Evaluation of DSG1 and DSG3 Autoantibody Levels The levels of DSG1 and DSG3 autoantibodies in the culture supernatants were quantified using ELISA (EIAab Human DSG1 and DSG3 kit, China). The standard solutions, wash buffer, and Reagents A and B were prepared in line with the manufacturer's protocol. A volume of 100 µl of the standard solution was added to the standard wells, while 100 µl of culture supernatant was used as the sample. The plate was then sealed and incubated for 2 hours at 37°C. The fluid from each well was removed and replaced with 100 µl of Reagent A solution, followed by another round of sealing and incubation for 1 hour at 37°C. Each well was then aspirated and washed twice, after which 100 µl of Reagent B solution was added, and the plate was incubated for another hour at 37°C. The wells were once again aspirated and washed three times. A volume of 90 µl of Substrate Solution was added to each well and the plate was incubated for 15–30 minutes at 37°C in darkness. Finally, 50 µl of Stop Solution was added to each well, and the absorbance was measured using a microplate reader set to 450 nm. The results were computed as per the manufacturer's instructions. Statistical Evaluation The data were presented as the mean ± standard error of the mean (SEM). The statistical analysis was carried out utilizing GraphPad Prism 9 (GraphPad Software, La Jolla, CA, USA). For comparisons involving multiple groups, a one-way analysis of variance (ANOVA) and JAMOVI software coupled with Tukey's multiple comparison test was employed, while a two-tailed unpaired Student's t test was used for comparisons between two groups. A p-value of < 0.05 was deemed to indicate statistical significance. RESULTS PV patients displayed a significantly elevated baseline lymphocyte proliferation rate (43.58 ± 6.75%) in contrast to the HC group (33.40 ± 2.94%) (p 0.001). After 72 hours of co-culturing with DF-MSCs, only the pemphigus group experienced a significant reduction in the lymphocyte proliferation rate (PV: 16.46 ± 5.62%) (p0.0001) and not the HC group (30.68 ± 2.27%) (p > 0.05) (Fig. 2 A- 2 B). The annexin analysis revealed that the rate of lymphocyte apoptosis was significantly higher in PV (30.44 ± 4.97%) than in HC (11.74 ± 1.4%) (p < 0.0001). However, in co-culture with DF-MSC, the apoptosis rate decreased to 8.75 ± 2% in PV and 10.79 ± 1.3% in HC. Only the PV group displayed a statistically significant decrease (p 0.05) (Fig. 2 C- 2 D). The lymphocyte viability rate in the PV group increased from 34.32 ± 4.98 to 77.81 ± 4.91 (p 0.05) (Fig. 2 C- 2 D). The proportions of CD4 + CD25 + FOXP3 + (T regulatory-Treg) cells were significantly different between PV (1.22 ± 0.58) and HC (2.01 ± 0.98) groups (p = 0.007). DF-MSC increased the percentage of Treg cells to 3.75 ± 1.42 in the PV group but had no effect on the HCs (Fig. 2 E- 2 F). ELISA results revealed that the average DSG1 antibody levels (ng/ml) were higher in PV (3.29 ± 0.22) compared to HCs (0.72 ± 0.3) (p < 0.0001). Co-culturing with DF-MSC resulted in a significant reduction in anti-DSG1 level in PV (3.00 ± 0.22) (p = 0.0005), but not in HCs (0.67 ± 0.26) (p = 0.72) (Fig. 3 A). Similarly, average DSG3 antibody levels (ng/ml) were significantly elevated in PV (262.40 ± 10.97) compared to HCs (0.54 ± 0.23) (p < 0.0001). Co-culturing with DF-MSC led to a significant decrease in PV (245.08 ± 13.18) (p = 0.0003), but not in HCs (0.52 ± 12) (p = 0.84) (Fig. 3 B). In the PBMC supernatant, the average IL-4 (ng/L) level was significantly higher in PV (38.06 ± 5.91) than in HC (31.20 ± 6.69) (p = 0.01). Co-culturing with DF-MSC led to a decrease in IL-4 level in the PV group (32.26 ± 5.89) (p = 0.005), but no change in HCs (30.04 ± 5.84) (p = 0.70) (Fig. 3 C). The mean TNF-α (ng/L) levels were similar in PV (32.45 ± 2.38) and HC (33.08 ± 2.11) (p = 0.48). However, the level decreased to 29.41 ± 2.24 in the PV group (p = 0.005) after co-culturing with DF-MSC, with no significant change in HCs (32.93 ± 2.21) (p = 0.87) (Fig. 3 F). The average IL-10 (pg/ml) levels were significantly lower in PV (47.46 ± 5.64) than in HCs (96.08 ± 11.13) (p < 0.0001). After co-culturing with DF-MSC, IL-10 level increased in the PV group (54.94 ± 8.60) (p = 0.003), but remained unchanged in HCs(100.04 ± 11.68) (p = 0.44) (Fig. 3 D). The mean IFN-γ (ng/ml) levels were also significantly lower in PV (12.39 ± 5.59) compared to HCs (20.06 ± 3.69) (p = 0.0007). However, after co-culturing with DF-MSC, IFN-γ levels increased in the PV group (19.70 ± 13.74) (p = 0.04), but were stable in HCs (20.07 ± 3.43) (p = 0.99) (Fig. 3 E). DISCUSSION In this study we examined the influence of DF-MSCs on a variety of factors, including T-cell proliferation, regulatory T cell counts, T lymphocyte apoptosis, as well as the levels of IL-4, IL-10, IFN-γ, TNFα, and DSG antibodies. Our results showed that in the PV group, the co-cultures with DF-MSCs resulted in a significant decline in lymphocyte proliferation, IL-4, TNFα, and DSG1 and DSG3 levels. Simultaneously, we noted an increase in regulatory T cell numbers, IL-10, and IFN-γ. DF-MSCs have recently garnered considerable interest due to their capacity to fine-tune immune responses and their high safety profile. Our study, to the best of our knowledge, is the first to scrutinize the potential role of DF-MSCs in PV treatment. A case report in veterinary medicine supports our findings, which showed improvement in a dog suffering from refractory pemphigus foliaceous after MSC treatment derived from adipose tissue [ 8 ]. The precise role of cytokines in the pathogenesis of pemphigus remains unclear, though many studies implicate high levels of Th2-associated cytokines (like IL-4 and IL-10) and low levels of Th1-related cytokines (like IFN-γ). In alignment with previous research, our study also noted elevated IL-4 levels. However, IL-10 results are inconsistent across studies, with some reflecting our findings of decreased IL-10 levels in pemphigus. Our study also demonstrated a reduction in IFN-γ levels in pemphigus patients' sera, consistent with the majority of the research. TNF-α, a multifunctional and predominantly Th1-related cytokine, contributes to inflammation and is produced by several cell types, not just Th cells. Despite variable findings in different studies, our results did not show a significant difference in TNF-α levels between PV/PF patients and healthy controls, which is in line with some other research [ 16 , 17 ]. T regulatory (Treg) cells play a vital role in immune tolerance, and their deficiency can lead to various autoimmune diseases, including pemphigus. Our study corroborates findings of diminished Treg levels in pemphigus. As the elevation of Treg levels is being explored as a possible treatment for autoimmune diseases, it's encouraging to note the increased Treg levels in our pemphigus patients' sera after co-culture with DF-MSC. Anti-desmoglein 1 and 3 auto-antibody levels were significantly higher in the PV group, but these levels decreased following co-culture with DF-MSC, implying that MSCs could specifically address the pathological process of pemphigus. This substantial reduction of autoantibodies targeting desmogleins bolsters the belief that DF-MSCs could potentially treat pemphigus at its root. Our findings indicate that DF-MSC can restore the equilibrium of immune cells, cytokines, and auto-antibodies in pemphigus, without altering normal responses, emphasizing their potential to specifically target abnormal immune pathways. This paints a promising picture for potential curative approaches in treating pemphigus, given the excellent safety profile of DF-MSC. Nonetheless, our study's in vitro design is a limitation, and we need well-structured clinical trials to confirm the efficacy and safety of MSCs in a real-life context. In our laboratory research, we explored the effects of DF-MSCs on T-cell proliferation, the counts of regulatory T cells, T lymphocyte apoptosis, and levels of IL-4, IL-10, IFN-γ, TNFα, and DSG antibodies. Our findings show that DF-MSCs co-cultures led to a substantial reduction in lymphocyte proliferation, IL-4, TNFα, and DSG1 and DSG3 levels in the PV group. On the contrary, we observed an increase in regulatory T cell numbers, IL-10, and IFN-γ. Recent research has placed considerable emphasis on MSCs due to their unique ability to fine-tune the immune response while maintaining a high safety profile. As far as we are aware, this is the first detailed study to evaluate the potential role of DF-MSCs in treating PV. A case study in veterinary medicine, which documents the improvement of a dog suffering from refractory pemphigus foliaceous following treatment with adipose tissue-derived MSC, lends further credibility to our research [ 8 ]. The exact roles of different cytokines in the pathogenesis of pemphigus are not entirely understood. Despite some inconsistent results, most research, including our study, finds increased levels of Th2-related cytokines (e.g., IL-4 and IL-10) and decreased levels of Th1-related cytokines (e.g., IFN-γ). IL-4, a signature cytokine of Th2 immune response, has consistently shown increased levels in multiple studies. The levels of regulatory cytokine IL-10 differ across studies; however, some limited studies, including ours, showed decreased levels in pemphigus [ 16 , 17 ]. Consistent with most studies, our research found decreased levels of Th1-related cytokine IFN-γ in the sera of pemphigus patients. TNF-α is a multifunctional cytokine typically categorized under Th1-related cytokines, which can be produced by various cell types and plays a significant role in inflammation. In pemphigus patients, serum and blister cytokine levels have been found to be either high or normal [ 18 ]. Our study, similar to some others, did not find a difference in TNF-α levels between PV/PF patients and healthy controls. To date, no study has reported a decrease in these cytokines [ 17 ]. Regulatory T (Treg) cells are a crucial subgroup of T cells that play a vital role in maintaining immune tolerance. A deficiency in Treg cells may precipitate various autoimmune diseases, including pemphigus. Our study corroborates earlier findings of reduced Treg numbers in pemphigus. While many studies aim to treat autoimmune diseases by increasing Treg levels, our study revealed that co-culture with DF-MSC improved Treg levels in the pemphigus group without affecting Treg levels in the healthy control (HC) group. We found significantly elevated levels of anti-desmoglein 1 and 3 auto-antibodies in the PV group. However, these levels decreased following co-culture with DF-MSC, demonstrating that MSCs specifically target and ameliorate the pathological process. This marked reduction of pathogenic auto-antibodies aimed at desmogleins paves the way for the use of DF-MSCs to tackle pemphigus at its core. Our study demonstrates that DF-MSCs can restore the balance of immune cells, cytokines, and auto-antibodies in pemphigus. Furthermore, we found that DF-MSCs adjust the parameters involved in the pathological process, either by increasing or decreasing their levels, without affecting normal responses. This points to the specific ability of DF-MSCs to target abnormal immune pathways. Given the excellent safety profile of DF-MSCs, these promising results raise hopes for new, potentially curative strategies for pemphigus. However, our study's in vitro design presents a limitation, and there is a pressing need for well-structured clinical trials to assess the efficacy and safety of MSCs in real-life scenarios. Declarations Acknowledgements Author Contributions: All authors contributed to the conceptualization, data curation, writing, review, editing, methodology, formal analysis, supervision, validation, and visualization of this study. All authors reviewed and approved the final manuscript. A funding statement: Supported by Marmara University with project number: SAG-A-120917-0497 Data Availability: The data presented in this study are available on request from the corresponding author. Statement on any prior presentation Contents of the manuscript have not been previously published and are not currently submitted else­where. Conflict of interest: None declared. Ethics: This study was approved by Ethics Committee of the Marmara University School of Medicine, Istanbul, Turkey (Protocol No: 09.2016.196/70737436-050.06.04). Written informed consent was obtained from all participants. References Kridin K. Pemphigus group: overview, epidemiology, mortality, and comorbidities. 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Cell biology and translational medicine, volume 10: stem cells in tissue regeneration , 167-176. Tuncer Budanur, D., Zibandeh, N., Genç, D., Gökalp, M., Kaşali, K., Akkoç, T., & Sepet, E. (2018). Effect of c DMEM media containing Ectoine on human periodontal ligament mesenchymal stem cell survival and differentiation. Dental Traumatology , 34 (3), 188-200. Zibandeh, N., Genc, D., Ozgen, Z., Duran, Y., Kasap, N., Goker, K., ... & Akkoc, T. (2019). Effect of dental follicle mesenchymal stem cell on Th1 and Th2 derived naive T cells in atopic dermatitis patients. Clinical and Experimental Health Sciences , 9 (3), 220-227. Tavakolpour S, Mahmoudi H, Mirzazadeh A, Balighi K, Darabi-Monadi S, Hatami S, GhasemiAdl M, Daneshpazhooh M. Pathogenic and protective roles of cytokines in pemphigus: A systematic review. Cytokine. 2020 May;129:155026. doi: 10.1016/j.cyto.2020.155026. Epub 2020 Feb 10. Khozeimeh F, Savabi O, Esnaashari M. Evaluation of interleukin-1α, interleukin-10, tumor necrosis factor-α and transforming growth factor-β in the serum of patients with pemphigus vulgaris. J Contemp Dent Pract. 2014 Nov 1;15(6):746-9. doi: 10.5005/jp-journals-10024-1610. Sonmez Kaplan, S., Sazak Ovecoglu, H., Genc, D., & Akkoc, T. (2023). TNF-α, IL-1B and IL-6 affect the differentiation ability of dental pulp stem cells. BMC Oral Health , 23 (1), 555. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 30 Jul, 2024 Reviews received at journal 27 Jul, 2024 Reviews received at journal 25 Jul, 2024 Reviewers agreed at journal 19 Jul, 2024 Reviewers agreed at journal 19 Jul, 2024 Reviewers agreed at journal 17 Jul, 2024 Reviewers agreed at journal 17 Jul, 2024 Reviewers invited by journal 17 Jul, 2024 Editor assigned by journal 27 Jun, 2024 Submission checks completed at journal 27 Jun, 2024 First submitted to journal 26 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4643043","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":328778375,"identity":"ce5e0c71-d04a-4139-aabb-3f77533d3e72","order_by":0,"name":"Zuleyha Ozgen","email":"","orcid":"","institution":"Marmara University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zuleyha","middleName":"","lastName":"Ozgen","suffix":""},{"id":328778376,"identity":"abbe9721-926c-42a6-aff8-62a913234c6f","order_by":1,"name":"Yazgul Duran","email":"","orcid":"","institution":"Marmara University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yazgul","middleName":"","lastName":"Duran","suffix":""},{"id":328778378,"identity":"f6d8a9bb-db39-4914-8720-d6adfb2db061","order_by":2,"name":"Tulin Ergun","email":"","orcid":"","institution":"Marmara University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Tulin","middleName":"","lastName":"Ergun","suffix":""},{"id":328778379,"identity":"b996820d-8f75-445d-b098-cab91b9f1ae3","order_by":3,"name":"Kamil Göker","email":"","orcid":"","institution":"Marmara University School of Dentistry","correspondingAuthor":false,"prefix":"","firstName":"Kamil","middleName":"","lastName":"Göker","suffix":""},{"id":328778381,"identity":"bc844f62-0393-4e42-ad91-a58b46d86ad1","order_by":4,"name":"Sabriye Senem","email":"","orcid":"","institution":"Marmara University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Sabriye","middleName":"","lastName":"Senem","suffix":""},{"id":328778382,"identity":"8517d173-3c18-45c2-b17e-768e2c4b4672","order_by":5,"name":"Tunç AKKOÇ","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYNCCAwkMDOwNQIaBBWHFPHAtPAdAWiRI0SKRAGIRocVeIvfghx9n0uT4Zz6/uuFHgQQDf3t3An5bJPKSJXtu5BhL3M4pu9kDdJjEmbMbCGjJMWPg+VCRuEE6J+0GD1CLgUQuYS2Mf0BaJM+k3fxDrBZmnhs5iRsk2I/dJs6WM2+MpWXOpBlLnMlhuy1jIMFD0C/s7TmGH98cS5bjbz/+7OabPzZARi9+LcgWGoBJYpWDLXxAiupRMApGwSgYQQAA5BpFi1Kty/oAAAAASUVORK5CYII=","orcid":"","institution":"Marmara University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Tunç","middleName":"","lastName":"AKKOÇ","suffix":""}],"badges":[],"createdAt":"2024-06-26 13:23:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4643043/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4643043/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60709510,"identity":"df657b80-3996-44ff-b4d1-5d3e8bd3dec2","added_by":"auto","created_at":"2024-07-19 19:56:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4743649,"visible":true,"origin":"","legend":"\u003cp\u003eA. Microscopic images of isolated DF-MSCs at different passages (P0, P1, P2, P3). Scale bar: 1000 µm. B: Flow cytometry analysis of DF-MSC surface markers. Positive markers: CD29 PE (97.6%), CD105 FITC (96.1%), CD73 APC (99.3%), CD90 PerCP (98.7%). Negative markers: CD14 PE (1.3%), CD34 APC (1.0%), CD45 PerCP (1.9%), HLA-DR FITC (0.7%). C: Differentiation potential of DF-MSCs into three lineages (adipogenic, osteogenic, chondrogenic). Adipogenic differentiation (Oil Red O staining, yellow arrows), Osteogenic differentiation (Alizarin Red S staining, yellow arrows), Chondrogenic differentiation (Alcian Blue staining, yellow arrows). Scale bar: 1000 µm.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4643043/v1/b59b1022064308adcffc4d98.png"},{"id":60710386,"identity":"e00b7614-c994-4002-ba95-943215c5fe1a","added_by":"auto","created_at":"2024-07-19 20:04:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1235532,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Lymphocyte proliferation rates in PV and healthy controls after co-culture with DF-MSCs. \u003cstrong\u003eB.\u003c/strong\u003e Apoptosis rates in PV and healthy controls after co-culture with DF-MSCs. \u003cstrong\u003eC.\u003c/strong\u003e Proportion of CD4+CD25+FOXP3+ regulatory T cells in PV and healthy controls after co-culture with DF-MSCs.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4643043/v1/e89f2e2a9ee6f7adaf50523c.png"},{"id":60710385,"identity":"a5fb91c4-28d5-4dde-9e37-a05d60616055","added_by":"auto","created_at":"2024-07-19 20:04:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":162217,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e IL-4 levels in PV and healthy controls after co-culture with DF-MSCs. \u003cstrong\u003eB.\u003c/strong\u003e TNF-α levels in PV and healthy controls after co-culture with DF-MSCs. \u003cstrong\u003eC.\u003c/strong\u003e IL-10 levels in PV and healthy controls after co-culture with DF-MSCs. \u003cstrong\u003eD.\u003c/strong\u003e IFN-γ levels in PV and healthy controls after co-culture with DF-MSCs. \u003cstrong\u003eE\u003c/strong\u003e. DSG1 antibody levels in PV and healthy controls after co-culture with DF-MSCs. \u003cstrong\u003eF.\u003c/strong\u003e DSG3 antibody levels in PV and healthy controls after co-culture with DF-MSCs.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4643043/v1/bd5265e69540747763b8be50.png"},{"id":60710814,"identity":"5b868b2e-445a-449e-a893-82ca49786861","added_by":"auto","created_at":"2024-07-19 20:12:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9010250,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4643043/v1/9fdea2b9-c258-45e0-9c95-a4279f518370.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Human Dental Mesenchymal Stem Cells Restorate Immune Response in Sera of Pemphigus Vulgaris Patients","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003ePemphigus comprises a set of IgG-mediated autoimmune conditions characterized by the presence of autoantibodies targeting desmogleins (desmoglein 1 and desmoglein 3). These proteins are part of the cadherin family of cell\u0026ndash;cell adhesion molecules, crucial for upholding intercellular adhesion in stratified squamous epithelia, like the skin and oral mucosa [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePemphigus involves intricate interactions between various pathways and cell types contributing to the disease pathogenesis. Within a self-perpetuating cycle, dysfunctional T lymphocytes trigger the reactivation of B cells, leading to the attack on self-tissues by B cells [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. A crucial event in this process is the development of IgG autoantibodies targeting the extracellular domains of Desmogleins (Dsg 1 and 3), which are integral cell membrane proteins in keratinocytes, ultimately mediating acantholysis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe precise role of cytokines in the pathogenesis of Pemphigus remains incompletely elucidated. Although definitive evidence is lacking, it is widely acknowledged that there is an elevation of T helper-2 (Th2) originated cytokines and a decrease in T helper-1 (Th1) induced cytokines [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe therapeutic armamentarium for pemphigus currently encompasses systemic corticosteroids, rituximab, and immunosuppressive medications, but these interventions are associated with an elevated risk of infections, vascular incidents, and other adverse events [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Consequently, an unmet need persists for the development of effective and safe treatment modalities. Mesenchymal stem cells (MSCs) represent a promising avenue in this context, as they are multipotent cells that can be obtained from various sources such as bone marrow, peripheral blood, adipose tissue, placental tissue, umbilical cord, Wharton\u0026rsquo;s jelly, and dental tissues. MSCs exhibit the remarkable capability to modulate immune responses, specifically by restoring a balance in the Th1 (associated with autoimmunity) and Th2 (related to allergic diseases) ratio. Emerging evidence has reported the efficacy of stem cell therapies in treating diverse autoimmune diseases, including pemphigus [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNotably, when administered intravenously, MSCs exhibit a propensity to selectively target sites of inflammation and participate in tissue repair and regeneration. During this process, MSCs undergo differentiation into various cell types and concurrently secrete multiple bioactive molecules, facilitating the recovery of injured cells and suppressing inflammatory processes [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Furthermore, a distinct advantage of MSCs lies in their limited immunogenicity, minimizing the potential for adverse immunological reactions.\u003c/p\u003e \u003cp\u003eThough only limited case reports and small series of cases exist, both autologous and allogeneic hematopoietic stem cell therapies have shown promising outcomes in treating stubborn cases of pemphigus. However, there's a significant lack of research exploring the therapeutic benefits of Mesenchymal Stem Cells (MSCs) in treating human subjects [\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Interestingly, the transplantation of MSCs derived from adipose tissue has shown a beneficial impact on refractory skin lesions associated with pemphigus foliaceus in a dog [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDental Mesenchymal Stem Cells (DMSCs) are extracted from various sources within the oral cavity, including dental pulp, the periodontal ligament, dental follicle, apical papilla, and dental cementum. Although DMSCs share numerous similarities with other types of MSCs, they possess distinct characteristics as well. One of the advantages of DMSCs is that they can be effortlessly isolated from teeth that are usually discarded. Additionally, their high proliferation rate makes DMSCs particularly suitable for both research and therapeutic purposes [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLike other Mesenchymal Stem Cells, Dental Mesenchymal Stem Cells (DMSCs) possess immunomodulatory properties, primarily through the secretion of cytokines. These attributes potentially make them an effective tool in the treatment of immune-mediated diseases, including pemphigus. Because MSCs exhibit low immunogenicity, they can be safely utilized in treating a range of disorders. Even though there are concerns about the tumorigenic potential of MSCs, no cases of tumor development associated with MSC treatments have been reported in existing literature [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe purpose of this study is to evaluate the immunomodulatory impact of Dental Follicle Mesenchymal Stem Cells (DF-MSCs) on Pemphigus Vulgaris and healthy control subjects. This will be done by examining T-cell proliferation and viability, apoptosis, levels of inflammatory and regulatory cytokines, and profiles of anti-desmoglein 1/3 IgG.\u003c/p\u003e"},{"header":"MATERIAL \u0026 METHOD","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSelection Criteria\u003c/h2\u003e \u003cp\u003e This study received ethical approval from a university's Ethics Committee (Protocol No: 09.2016.196/70737436-050.06.04). Twenty-one symptomatic PV patients (mean age 49.43\u0026thinsp;\u0026plusmn;\u0026thinsp;13.25 years; 13 males, 8 females) treated at a university dermatology department were included. Diagnoses were confirmed via histopathological and direct immunofluorescence analysis. PDAI scores ranged from 7 to 35. Treatments varied: five were newly diagnosed, six received methylprednisolone (\u0026le;\u0026thinsp;20 mg/day), six were on methotrexate, mycophenolate, or azathioprine, and four received Rituximab. No concurrent inflammatory or autoimmune diseases were present. The control group comprised eleven healthy volunteers (mean age 51.73\u0026thinsp;\u0026plusmn;\u0026thinsp;5.92 years; 7 males, 4 females) with no history of PV or other autoimmune/inflammatory diseases (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDemographic and Clinical Characteristics of Pemphigus Vulgaris (PV) Patients and Healthy Controls (HC)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatients\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGender\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDuration of disease (month)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePDAI score\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eConcomittant treatment\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePV1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMTX\u0026thinsp;+\u0026thinsp;MP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePV2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRTX\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePV3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;20 mg MP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePV4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePV5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRTX\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePV6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;20 mg MP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePV7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;20 mg MP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePV8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;20 mg MP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePV9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRTX\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePV10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePV11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRTX\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePV12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;20 mg MP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePV13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAZA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePV14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMTX\u0026thinsp;+\u0026thinsp;MP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePV15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePV16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMTX\u0026thinsp;+\u0026thinsp;MP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePV17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;20 mg MP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePV18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePV19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePV20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAZA\u0026thinsp;+\u0026thinsp;MP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePV21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMTX\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHC1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHC2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHC3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHC4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHC5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHC6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHC7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHC8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHC9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHC10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHC11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eDental Folicle Derived Mesenchymal Stem Cell (DF-MSC) Isolations\u003c/h2\u003e \u003cp\u003eParticipants aged 18\u0026ndash;25 years were selected from Marmara University's Faculty of Dentistry. Dental follicle tissues collected from these volunteers were physically segmented and enzymatically digested with Phosphate Buffered Saline (PBS; Gibco, Gaithersburg, MD) containing 3 mg/ml collagenase type I (Sigma, Grand Island, USA). Samples were collected under sterile conditions and transported in cold Dulbecco's phosphate-buffered saline (DPBS, Gibco, Grand Island, NY) with 1% penicillin/streptomycin (Gibco, USA). All laboratory studies were conducted in the Pediatric Allergy-Immunology department of our university.\u003c/p\u003e \u003cp\u003eTo digest the dental follicle tissues, 10 mL of Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin/streptomycin was added, followed by centrifugation at 1500 rpm for 10 minutes. The resulting cell pellets were obtained, and the supernatant was aspirated. Dental follicle-derived MSCs (DF-MSCs) were cultivated in T-25 flasks in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere at 37\u0026deg;C using a culture medium of DMEM, 10% FBS, and 1% penicillin/streptomycin. DF-MSCs were washed with DPBS and provided with fresh culture medium, which was changed every 3 to 4 days until confluence was reached. When cells reached 70\u0026ndash;80% confluence, they were detached with 0.25% trypsin-EDTA (Gibco, USA). Adherent cells cultured to the third passage were characterized and analyzed for specific surface markers using flow cytometry [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eDental Folicle Derived Mesenchymal Stem Cell (DF-MSC) Characterisation Analysis: Flow Cytometry Analysis\u003c/h2\u003e \u003cp\u003eFor cell surface antigen analysis, third passage DF-MSCs were incubated with antibodies for human CD73 allophycocyanin (APC), CD90 fluorescein isothiocyanate (FITC), CD44 phycoerythrin (PE), CD105 Peridinin-Chlorophyll-Protein (PerCP), CD4 FITC, CD19 (PerCP), CD25 APC, and HLA-DR PE at room temperature in the dark. Control antibodies were phycoerythrin-conjugated or fluorescein isothiocyanate-conjugated and allophycocyanin-conjugated mouse IgG1 and mouse IgG2 (BD Biosciences, San Diego, CA, USA). Flow cytometry results were analyzed using a Beckman Coulter Navios EX and the Kaluza software [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eDental Folicle Derived Mesenchymal Stem Cell (DF-MSC) Differentiation Analysis: Adipogenic, Osteogenic, Chondrogenetic\u003c/h2\u003e \u003cp\u003eThe differentiation of DF-MSCs into osteocytes, adipocytes, and chondrocytes was carried out using the StemPro Osteogenesis Differentiation Kit, StemPro Adipogenesis Differentiation Kit, and StemPro Chondrogenesis Differentiation Kit (all from Gibco, Grand Island, NY, USA) following the manufacturer's instructions. Briefly, cells (5\u0026times;10^4 per well) were seeded into 6-well plates, and differentiation media was introduced 21\u0026ndash;28 days later. During cell growth, the culture medium was changed twice a week. Alizarin Red staining was used to detect osteogenesis, Oil Red O staining for adipogenesis, and Alcian Blue staining for chondrogenesis. Images of stained cells were captured using a light microscope [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eIsolation of Peripheral Blood Mononuclear Cells (PBMC)\u003c/h2\u003e \u003cp\u003ePeripheral blood (10 ml) was collected from both patients and healthy controls into heparinized tubes. These samples were diluted 1:1 with sterile Phosphate Buffered Saline (PBS) and subjected to ficoll density gradient centrifugation at 2000 rpm for 25 minutes. The buffy coat layer was harvested and washed with PBS. Cell counts were determined, and the cells were prepared for co-culture experiments. The Peripheral Blood Mononuclear Cells (PBMCs) were then resuspended in a culture medium consisting of RPMI-1640, supplemented with 1% Penicillin/Streptomycin (P/S) and 10% Fetal Bovine Serum (FBS, Invitrogen) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCo-cultivation of DF-MSCs and PBMCs\u003c/h2\u003e \u003cp\u003eInitially, DF-MSCs (5x10\u003csup\u003e4\u003c/sup\u003e) were cultured for 48 hours prior to the introduction of PBMCs. Subsequently, 5x10\u003csup\u003e5\u003c/sup\u003e PBMCs were added to each well containing complete RPMI (cRPMI), both in the presence and absence of DF-MSCs, and co-cultivated for a period of 3 days. These cultures were then stimulated with 0.5ug/ml of anti-CD3 (eBioscience, San Diego, CA) and 0.5ug/ml of CD28 (eBioscience, San Diego, CA) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCFSE Proliferation Assay\u003c/h2\u003e \u003cp\u003eTo perform the Carboxyfluorescein Succinimidyl Ester (CFSE) proliferation assay, 1\u0026micro;l of CFSE (Invitrogen\u0026trade;, USA) was added to PBMCs suspended in 1ml of PBS at a concentration of 10\u003csup\u003e6\u003c/sup\u003e cells/mL and then incubated for a duration of 7 minutes. Following the incubation period, the cells were washed twice using cRPMI. Finally, the resuspended cells were transferred into culture plates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eApoptosis Assay\u003c/h2\u003e \u003cp\u003eAfter co-culturing for 72 hours, the cells were harvested and washed twice with 1X PBS. The cell pellet was then resuspended in 1X Binding Buffer. To this suspension, 5\u0026micro;l of Annexin V (BD Bioscience, USA) was added and incubated for 15 minutes at room temperature (25\u0026deg;C) in a dark environment. Following the incubation, the cells were washed again with 1X PBS. The pellet was subsequently resuspended in 1X Binding Buffer. 5\u0026micro;l of Propidium Iodide (PI, PE) was then added to the cells, which were subsequently analyzed using flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eRegulatory T Cell Assay\u003c/h2\u003e \u003cp\u003eCD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e+\u003c/sup\u003eFOXP3\u003csup\u003e+\u003c/sup\u003e T regulatory cells (Tregs) were analyzed within the PBMCs after a 72-hour period. The cultures were examined by flow cytometry, utilizing a Human regulatory T cell kit as per the manufacturer's instructions (eBioscience, USA). In brief, cells were rinsed twice with 1X PBS. The cell pellet was stained with anti-CD4 and anti-CD25 antibodies for 20 minutes in the dark and then centrifuged at 1500 rpm for 5 minutes. Subsequently, the pellet was incubated with a fixation/permeabilization buffer for 30 minutes and washed twice with 1X permeabilization buffer. Following this, the FOXP3 antibody was applied. After incubation, the cells were rinsed twice with 1X permeabilization buffer and then centrifuged at 1500 rpm for 5 minutes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCytokine Expression Profile Analysis\u003c/h2\u003e \u003cp\u003eThe expression levels of cytokines (IL-4, IL-10, IFN-γ, TNFα) in the culture supernatants were evaluated using ELISA (BT-LAB, China). The standard solutions and wash buffer were prepared in accordance with the manufacturer's protocol. Initially, 50\u0026micro;l of the standard solution was introduced into the Standard wells, and 40\u0026micro;l of the culture supernatant was transferred to sample wells. This was followed by the addition of 10\u0026micro;l of the Human IL4 antibody to the sample wells. For detection, 50\u0026micro;l of streptavidin-HRP was added to both sample and standard wells. The plate was then sealed and incubated for 60 minutes at 37\u0026deg;C. Subsequently, it was washed five times with wash buffer and a mixture of 50\u0026micro;l each of substrate solutions A and B was added to each well. After a 10-minute incubation at 37\u0026deg;C in the dark, 50\u0026micro;l of Stop Solution was added to each well. The absorbance was then measured using a microplate reader at 450 nm. The results were determined as per the manufacturer's instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of DSG1 and DSG3 Autoantibody Levels\u003c/h2\u003e \u003cp\u003eThe levels of DSG1 and DSG3 autoantibodies in the culture supernatants were quantified using ELISA (EIAab Human DSG1 and DSG3 kit, China). The standard solutions, wash buffer, and Reagents A and B were prepared in line with the manufacturer's protocol. A volume of 100 \u0026micro;l of the standard solution was added to the standard wells, while 100 \u0026micro;l of culture supernatant was used as the sample. The plate was then sealed and incubated for 2 hours at 37\u0026deg;C. The fluid from each well was removed and replaced with 100 \u0026micro;l of Reagent A solution, followed by another round of sealing and incubation for 1 hour at 37\u0026deg;C. Each well was then aspirated and washed twice, after which 100 \u0026micro;l of Reagent B solution was added, and the plate was incubated for another hour at 37\u0026deg;C. The wells were once again aspirated and washed three times. A volume of 90 \u0026micro;l of Substrate Solution was added to each well and the plate was incubated for 15\u0026ndash;30 minutes at 37\u0026deg;C in darkness. Finally, 50 \u0026micro;l of Stop Solution was added to each well, and the absorbance was measured using a microplate reader set to 450 nm. The results were computed as per the manufacturer's instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Evaluation\u003c/h2\u003e \u003cp\u003eThe data were presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). The statistical analysis was carried out utilizing GraphPad Prism 9 (GraphPad Software, La Jolla, CA, USA). For comparisons involving multiple groups, a one-way analysis of variance (ANOVA) and JAMOVI software coupled with Tukey's multiple comparison test was employed, while a two-tailed unpaired Student's t test was used for comparisons between two groups. A p-value of \u0026lt;\u0026thinsp;0.05 was deemed to indicate statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003ePV patients displayed a significantly elevated baseline lymphocyte proliferation rate (43.58\u0026thinsp;\u0026plusmn;\u0026thinsp;6.75%) in contrast to the HC group (33.40\u0026thinsp;\u0026plusmn;\u0026thinsp;2.94%) (p 0.001). After 72 hours of co-culturing with DF-MSCs, only the pemphigus group experienced a significant reduction in the lymphocyte proliferation rate (PV: 16.46\u0026thinsp;\u0026plusmn;\u0026thinsp;5.62%) (p0.0001) and not the HC group (30.68\u0026thinsp;\u0026plusmn;\u0026thinsp;2.27%) (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe annexin analysis revealed that the rate of lymphocyte apoptosis was significantly higher in PV (30.44\u0026thinsp;\u0026plusmn;\u0026thinsp;4.97%) than in HC (11.74\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4%) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). However, in co-culture with DF-MSC, the apoptosis rate decreased to 8.75\u0026thinsp;\u0026plusmn;\u0026thinsp;2% in PV and 10.79\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3% in HC. Only the PV group displayed a statistically significant decrease (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), not the HCs (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eThe lymphocyte viability rate in the PV group increased from 34.32\u0026thinsp;\u0026plusmn;\u0026thinsp;4.98 to 77.81\u0026thinsp;\u0026plusmn;\u0026thinsp;4.91 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) after co-culturing with DF-MSC, whereas the rate remained unchanged in the HC group (74.23\u0026thinsp;\u0026plusmn;\u0026thinsp;2.75 to 76.03\u0026thinsp;\u0026plusmn;\u0026thinsp;3.76) (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eThe proportions of CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e+\u003c/sup\u003eFOXP3\u003csup\u003e+\u003c/sup\u003e (T regulatory-Treg) cells were significantly different between PV (1.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58) and HC (2.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98) groups (p\u0026thinsp;=\u0026thinsp;0.007). DF-MSC increased the percentage of Treg cells to 3.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.42 in the PV group but had no effect on the HCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eELISA results revealed that the average DSG1 antibody levels (ng/ml) were higher in PV (3.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22) compared to HCs (0.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Co-culturing with DF-MSC resulted in a significant reduction in anti-DSG1 level in PV (3.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22) (p\u0026thinsp;=\u0026thinsp;0.0005), but not in HCs (0.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26) (p\u0026thinsp;=\u0026thinsp;0.72) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSimilarly, average DSG3 antibody levels (ng/ml) were significantly elevated in PV (262.40\u0026thinsp;\u0026plusmn;\u0026thinsp;10.97) compared to HCs (0.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Co-culturing with DF-MSC led to a significant decrease in PV (245.08\u0026thinsp;\u0026plusmn;\u0026thinsp;13.18) (p\u0026thinsp;=\u0026thinsp;0.0003), but not in HCs (0.52\u0026thinsp;\u0026plusmn;\u0026thinsp;12) (p\u0026thinsp;=\u0026thinsp;0.84) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eIn the PBMC supernatant, the average IL-4 (ng/L) level was significantly higher in PV (38.06\u0026thinsp;\u0026plusmn;\u0026thinsp;5.91) than in HC (31.20\u0026thinsp;\u0026plusmn;\u0026thinsp;6.69) (p\u0026thinsp;=\u0026thinsp;0.01). Co-culturing with DF-MSC led to a decrease in IL-4 level in the PV group (32.26\u0026thinsp;\u0026plusmn;\u0026thinsp;5.89) (p\u0026thinsp;=\u0026thinsp;0.005), but no change in HCs (30.04\u0026thinsp;\u0026plusmn;\u0026thinsp;5.84) (p\u0026thinsp;=\u0026thinsp;0.70) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eThe mean TNF-α (ng/L) levels were similar in PV (32.45\u0026thinsp;\u0026plusmn;\u0026thinsp;2.38) and HC (33.08\u0026thinsp;\u0026plusmn;\u0026thinsp;2.11) (p\u0026thinsp;=\u0026thinsp;0.48). However, the level decreased to 29.41\u0026thinsp;\u0026plusmn;\u0026thinsp;2.24 in the PV group (p\u0026thinsp;=\u0026thinsp;0.005) after co-culturing with DF-MSC, with no significant change in HCs (32.93\u0026thinsp;\u0026plusmn;\u0026thinsp;2.21) (p\u0026thinsp;=\u0026thinsp;0.87) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eThe average IL-10 (pg/ml) levels were significantly lower in PV (47.46\u0026thinsp;\u0026plusmn;\u0026thinsp;5.64) than in HCs (96.08\u0026thinsp;\u0026plusmn;\u0026thinsp;11.13) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). After co-culturing with DF-MSC, IL-10 level increased in the PV group (54.94\u0026thinsp;\u0026plusmn;\u0026thinsp;8.60) (p\u0026thinsp;=\u0026thinsp;0.003), but remained unchanged in HCs(100.04\u0026thinsp;\u0026plusmn;\u0026thinsp;11.68) (p\u0026thinsp;=\u0026thinsp;0.44) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eThe mean IFN-γ (ng/ml) levels were also significantly lower in PV (12.39\u0026thinsp;\u0026plusmn;\u0026thinsp;5.59) compared to HCs (20.06\u0026thinsp;\u0026plusmn;\u0026thinsp;3.69) (p\u0026thinsp;=\u0026thinsp;0.0007). However, after co-culturing with DF-MSC, IFN-γ levels increased in the PV group (19.70\u0026thinsp;\u0026plusmn;\u0026thinsp;13.74) (p\u0026thinsp;=\u0026thinsp;0.04), but were stable in HCs (20.07\u0026thinsp;\u0026plusmn;\u0026thinsp;3.43) (p\u0026thinsp;=\u0026thinsp;0.99) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this study we examined the influence of DF-MSCs on a variety of factors, including T-cell proliferation, regulatory T cell counts, T lymphocyte apoptosis, as well as the levels of IL-4, IL-10, IFN-γ, TNFα, and DSG antibodies. Our results showed that in the PV group, the co-cultures with DF-MSCs resulted in a significant decline in lymphocyte proliferation, IL-4, TNFα, and DSG1 and DSG3 levels. Simultaneously, we noted an increase in regulatory T cell numbers, IL-10, and IFN-γ.\u003c/p\u003e \u003cp\u003eDF-MSCs have recently garnered considerable interest due to their capacity to fine-tune immune responses and their high safety profile. Our study, to the best of our knowledge, is the first to scrutinize the potential role of DF-MSCs in PV treatment. A case report in veterinary medicine supports our findings, which showed improvement in a dog suffering from refractory pemphigus foliaceous after MSC treatment derived from adipose tissue [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe precise role of cytokines in the pathogenesis of pemphigus remains unclear, though many studies implicate high levels of Th2-associated cytokines (like IL-4 and IL-10) and low levels of Th1-related cytokines (like IFN-γ). In alignment with previous research, our study also noted elevated IL-4 levels. However, IL-10 results are inconsistent across studies, with some reflecting our findings of decreased IL-10 levels in pemphigus. Our study also demonstrated a reduction in IFN-γ levels in pemphigus patients' sera, consistent with the majority of the research. TNF-α, a multifunctional and predominantly Th1-related cytokine, contributes to inflammation and is produced by several cell types, not just Th cells. Despite variable findings in different studies, our results did not show a significant difference in TNF-α levels between PV/PF patients and healthy controls, which is in line with some other research [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eT regulatory (Treg) cells play a vital role in immune tolerance, and their deficiency can lead to various autoimmune diseases, including pemphigus. Our study corroborates findings of diminished Treg levels in pemphigus. As the elevation of Treg levels is being explored as a possible treatment for autoimmune diseases, it's encouraging to note the increased Treg levels in our pemphigus patients' sera after co-culture with DF-MSC.\u003c/p\u003e \u003cp\u003eAnti-desmoglein 1 and 3 auto-antibody levels were significantly higher in the PV group, but these levels decreased following co-culture with DF-MSC, implying that MSCs could specifically address the pathological process of pemphigus. This substantial reduction of autoantibodies targeting desmogleins bolsters the belief that DF-MSCs could potentially treat pemphigus at its root.\u003c/p\u003e \u003cp\u003eOur findings indicate that DF-MSC can restore the equilibrium of immune cells, cytokines, and auto-antibodies in pemphigus, without altering normal responses, emphasizing their potential to specifically target abnormal immune pathways. This paints a promising picture for potential curative approaches in treating pemphigus, given the excellent safety profile of DF-MSC. Nonetheless, our study's in vitro design is a limitation, and we need well-structured clinical trials to confirm the efficacy and safety of MSCs in a real-life context.\u003c/p\u003e \u003cp\u003eIn our laboratory research, we explored the effects of DF-MSCs on T-cell proliferation, the counts of regulatory T cells, T lymphocyte apoptosis, and levels of IL-4, IL-10, IFN-γ, TNFα, and DSG antibodies. Our findings show that DF-MSCs co-cultures led to a substantial reduction in lymphocyte proliferation, IL-4, TNFα, and DSG1 and DSG3 levels in the PV group. On the contrary, we observed an increase in regulatory T cell numbers, IL-10, and IFN-γ.\u003c/p\u003e \u003cp\u003eRecent research has placed considerable emphasis on MSCs due to their unique ability to fine-tune the immune response while maintaining a high safety profile. As far as we are aware, this is the first detailed study to evaluate the potential role of DF-MSCs in treating PV. A case study in veterinary medicine, which documents the improvement of a dog suffering from refractory pemphigus foliaceous following treatment with adipose tissue-derived MSC, lends further credibility to our research [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe exact roles of different cytokines in the pathogenesis of pemphigus are not entirely understood. Despite some inconsistent results, most research, including our study, finds increased levels of Th2-related cytokines (e.g., IL-4 and IL-10) and decreased levels of Th1-related cytokines (e.g., IFN-γ). IL-4, a signature cytokine of Th2 immune response, has consistently shown increased levels in multiple studies. The levels of regulatory cytokine IL-10 differ across studies; however, some limited studies, including ours, showed decreased levels in pemphigus [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Consistent with most studies, our research found decreased levels of Th1-related cytokine IFN-γ in the sera of pemphigus patients.\u003c/p\u003e \u003cp\u003eTNF-α is a multifunctional cytokine typically categorized under Th1-related cytokines, which can be produced by various cell types and plays a significant role in inflammation. In pemphigus patients, serum and blister cytokine levels have been found to be either high or normal [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Our study, similar to some others, did not find a difference in TNF-α levels between PV/PF patients and healthy controls. To date, no study has reported a decrease in these cytokines [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRegulatory T (Treg) cells are a crucial subgroup of T cells that play a vital role in maintaining immune tolerance. A deficiency in Treg cells may precipitate various autoimmune diseases, including pemphigus. Our study corroborates earlier findings of reduced Treg numbers in pemphigus. While many studies aim to treat autoimmune diseases by increasing Treg levels, our study revealed that co-culture with DF-MSC improved Treg levels in the pemphigus group without affecting Treg levels in the healthy control (HC) group.\u003c/p\u003e \u003cp\u003eWe found significantly elevated levels of anti-desmoglein 1 and 3 auto-antibodies in the PV group. However, these levels decreased following co-culture with DF-MSC, demonstrating that MSCs specifically target and ameliorate the pathological process. This marked reduction of pathogenic auto-antibodies aimed at desmogleins paves the way for the use of DF-MSCs to tackle pemphigus at its core.\u003c/p\u003e \u003cp\u003eOur study demonstrates that DF-MSCs can restore the balance of immune cells, cytokines, and auto-antibodies in pemphigus. Furthermore, we found that DF-MSCs adjust the parameters involved in the pathological process, either by increasing or decreasing their levels, without affecting normal responses. This points to the specific ability of DF-MSCs to target abnormal immune pathways. Given the excellent safety profile of DF-MSCs, these promising results raise hopes for new, potentially curative strategies for pemphigus. However, our study's in vitro design presents a limitation, and there is a pressing need for well-structured clinical trials to assess the efficacy and safety of MSCs in real-life scenarios.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eAll authors contributed to the conceptualization, data curation, writing, review, editing, methodology, formal analysis, supervision, validation, and visualization of this study. All authors reviewed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA funding statement:\u0026nbsp;\u003c/strong\u003eSupported by Marmara University with project number: SAG-A-120917-0497\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability:\u0026nbsp;\u003c/strong\u003eThe data presented in this study are available on request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatement on any prior presentation\u0026nbsp;\u003c/strong\u003eContents of the manuscript have not been previously published and are not currently submitted else\u0026shy;where.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u0026nbsp;\u003c/strong\u003eNone declared.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics:\u0026nbsp;\u003c/strong\u003eThis study was\u0026nbsp;approved by\u0026nbsp;Ethics Committee of the Marmara University School of Medicine, Istanbul, Turkey (Protocol No:\u0026nbsp;09.2016.196/70737436-050.06.04).\u0026nbsp;Written informed consent was obtained from all participants.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKridin K. Pemphigus group: overview, epidemiology, mortality, and comorbidities. Immunol Res. 2018 Apr;66(2):255-270. doi: 10.1007/s12026-018-8986-7. \u003c/li\u003e\n\u003cli\u003eShah AA, Seiffert-Sinha K, Sirois D, Werth VP, Rengarajan B, Zrnchik W, Attwood K, Sinha AA. Development of a disease registry for autoimmune bullous diseases: initial analysis of the pemphigus vulgaris subset. Acta Derm Venereol. 2015 Jan;95(1):86-90. doi: 10.2340/00015555-1854. \u003c/li\u003e\n\u003cli\u003eKasperkiewicz M, Ellebrecht CT, Takahashi H, Yamagami J, Zillikens D, Payne AS, Amagai M. Pemphigus. Nat Rev Dis Primers. 2017 May 11;3:17026. doi: 10.1038/nrdp.2017.26. \u003c/li\u003e\n\u003cli\u003eBirant, S., Duran, Y., Akkoc, T., \u0026amp; Seymen, F. (2022). Cytotoxic effects of different detergent containing children\u0026apos;s toothpastes on human gingival epithelial cells. \u003cem\u003eBMC Oral Health\u003c/em\u003e, \u003cem\u003e22\u003c/em\u003e(1), 66. \u003c/li\u003e\n\u003cli\u003eOyama Y, Parker ER, Brieva J, Guitart J, Statkute L, Verda L, Burt RK. High-dose immune suppression and autologous hematopoietic stem cell transplantation in refractory pemphigus foliaceus. Bone Marrow Transplant. 2004 Dec;34(12):1097-8. doi: 10.1038/sj.bmt.1704679. \u003c/li\u003e\n\u003cli\u003eSuslova IM, Theodoropoulos DS, Cullen NA, Tetarnikova MK, Tetarnikov AS, Kolchak NA. Pemphigus vulgaris treated with allogeneic hematopoietic stem cell transplantation following non-myeloablative conditioning. Eur Rev Med Pharmacol Sci. 2010 Sep;14(9):785-8.\u003c/li\u003e\n\u003cli\u003eVanikar AV, Trivedi HL, Patel RD, Kanodia KV, Modi PR, Shah VR. Allogenic hematopoietic stem cell transplantation in pemphigus vulgaris: a single-center experience. Indian J Dermatol. 2012 Jan;57(1):9-11. doi: 10.4103/0019-5154.92667. \u003c/li\u003e\n\u003cli\u003eHan SM, Kim HT, Kim KW, Jeon KO, Seo KW, Choi EW, Youn HY. CTLA4 overexpressing adipose tissue-derived mesenchymal stem cell therapy in a dog with steroid-refractory pemphigus foliaceus. BMC Vet Res. 2015 Mar 6;11:49. doi: 10.1186/s12917-015-0371-3. \u003c/li\u003e\n\u003cli\u003eZibandeh, N., Gen\u0026ccedil;, D., Duran, Y., Banzragch, M., Sokwala, S., G\u0026ouml;ker, K., ... \u0026amp; Akko\u0026ccedil;, T. (2020). Human dental follicle mesenchymal stem cells alleviate T cell response in inflamed tissue of Crohn\u0026rsquo;s patients. \u003cem\u003eThe Turkish Journal of Gastroenterology\u003c/em\u003e, \u003cem\u003e31\u003c/em\u003e(5), 400.\u003c/li\u003e\n\u003cli\u003eZibandeh, N., Genc, D., Ozgen, Z., Duran, Y., Goker, K., Baris, S., ... \u0026amp; Akkoc, T. (2021). Mesenchymal stem cells derived from human dental follicle modulate the aberrant immune response in atopic dermatitis. \u003cem\u003eImmunotherapy\u003c/em\u003e, \u003cem\u003e13\u003c/em\u003e(10), 825-840.\u003c/li\u003e\n\u003cli\u003eUlusoy, C., Zibandeh, N., Yıldırım, S., Trakas, N., Zisimopoulou, P., K\u0026uuml;\u0026ccedil;\u0026uuml;kerden, M., ... \u0026amp; Akko\u0026ccedil;, T. (2015). Dental follicle mesenchymal stem cell administration ameliorates muscle weakness in MuSK-immunized mice. \u003cem\u003eJournal of Neuroinflammation\u003c/em\u003e, \u003cem\u003e12\u003c/em\u003e, 1-12.\u003c/li\u003e\n\u003cli\u003eGenc, D., Zibandeh, N., Nain, E., Arığ, \u0026Uuml;., G\u0026ouml;ker, K., Aydıner, E. K., \u0026amp; Akko\u0026ccedil;, T. U. N. \u0026Ccedil;. (2019). IFN-\u0026gamma; stimulation of dental follicle mesenchymal stem cells modulates immune response of CD4+ T lymphocytes in Der p1+ asthmatic patients in vitro. \u003cem\u003eAllergologia et Immunopathologia\u003c/em\u003e, \u003cem\u003e47\u003c/em\u003e(5), 467-476.\u003c/li\u003e\n\u003cli\u003eAkkoc, T. (2020). COVID-19 and mesenchymal stem cell treatment; mystery or not. \u003cem\u003eCell biology and translational medicine, volume 10: stem cells in tissue regeneration\u003c/em\u003e, 167-176.\u003c/li\u003e\n\u003cli\u003eTuncer Budanur, D., Zibandeh, N., Gen\u0026ccedil;, D., G\u0026ouml;kalp, M., Kaşali, K., Akko\u0026ccedil;, T., \u0026amp; Sepet, E. (2018). Effect of c DMEM media containing Ectoine on human periodontal ligament mesenchymal stem cell survival and differentiation. \u003cem\u003eDental Traumatology\u003c/em\u003e, \u003cem\u003e34\u003c/em\u003e(3), 188-200.\u003c/li\u003e\n\u003cli\u003eZibandeh, N., Genc, D., Ozgen, Z., Duran, Y., Kasap, N., Goker, K., ... \u0026amp; Akkoc, T. (2019). Effect of dental follicle mesenchymal stem cell on Th1 and Th2 derived naive T cells in atopic dermatitis patients. \u003cem\u003eClinical and Experimental Health Sciences\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e(3), 220-227.\u003c/li\u003e\n\u003cli\u003eTavakolpour S, Mahmoudi H, Mirzazadeh A, Balighi K, Darabi-Monadi S, Hatami S, GhasemiAdl M, Daneshpazhooh M. Pathogenic and protective roles of cytokines in pemphigus: A systematic review. Cytokine. 2020 May;129:155026. doi: 10.1016/j.cyto.2020.155026. Epub 2020 Feb 10.\u003c/li\u003e\n\u003cli\u003eKhozeimeh F, Savabi O, Esnaashari M. Evaluation of interleukin-1\u0026alpha;, interleukin-10, tumor necrosis factor-\u0026alpha; and transforming growth factor-\u0026beta; in the serum of patients with pemphigus vulgaris. J Contemp Dent Pract. 2014 Nov 1;15(6):746-9. doi: 10.5005/jp-journals-10024-1610. \u003c/li\u003e\n\u003cli\u003eSonmez Kaplan, S., Sazak Ovecoglu, H., Genc, D., \u0026amp; Akkoc, T. (2023). TNF-\u0026alpha;, IL-1B and IL-6 affect the differentiation ability of dental pulp stem cells. \u003cem\u003eBMC Oral Health\u003c/em\u003e, \u003cem\u003e23\u003c/em\u003e(1), 555.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-dermatological-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Archives of Dermatological Research](https://www.springer.com/journal/403)","snPcode":"403","submissionUrl":"https://submission.nature.com/new-submission/403/3","title":"Archives of Dermatological Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Pemphigus, Mesenchymal stem cell, DF-MSCs, CD4 + CD25 + Foxp3 + Treg, anti-desmoglein","lastPublishedDoi":"10.21203/rs.3.rs-4643043/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4643043/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePemphigus is an IgG-mediated autoimmune condition characterized by autoantibodies targeting desmogleins, leading to acantholysis. Current treatments, including systemic corticosteroids and immunosuppressive drugs, are associated with significant adverse effects. Mesenchymal stem cells (MSCs) offer a promising alternative due to their immunomodulatory properties and low immunogenicity. This study evaluates the immunomodulatory effects of Dental Follicle Mesenchymal Stem Cells (DF-MSCs) on Pemphigus Vulgaris (PV) patients and healthy controls by examining T-cell proliferation, apoptosis, cytokine levels, and anti-desmoglein 1/3 IgG profiles. Twenty-one symptomatic PV patients and eleven healthy volunteers were included. DF-MSCs were isolated, characterized, and differentiated into osteocytes, adipocytes, and chondrocytes. Peripheral Blood Mononuclear Cells (PBMCs) were co-cultured with DF-MSCs, and various assays were conducted to evaluate T-cell proliferation, apoptosis, regulatory T cells, cytokine expression, and autoantibody levels. Results showed that DF-MSC co-cultures significantly reduced lymphocyte proliferation (43.58\u0026ndash;16.27%), IL-4 (38.06 ng/L to 32.26 ng/L), TNF-α (32.45 ng/L to 29.41 ng/L), and DSG1 (3.29 ng/ml to 3.00 ng/ml) and DSG3 (262.40 ng/ml to 245.08 ng/ml) levels in PV patients. An increase in regulatory T cells (1.22\u0026ndash;3.75%), IL-10 (47.46 pg/ml to 54.94 pg/ml), and IFN-γ (12.39 ng/ml to 19.70 ng/ml) was also observed. No significant changes were noted in healthy controls. These findings suggest that DF-MSCs could potentially offer a curative approach for treating pemphigus by restoring immune balance. However, further clinical trials are necessary to confirm their efficacy.\u003c/p\u003e","manuscriptTitle":"Human Dental Mesenchymal Stem Cells Restorate Immune Response in Sera of Pemphigus Vulgaris Patients","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-19 19:56:08","doi":"10.21203/rs.3.rs-4643043/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-30T21:09:50+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-27T10:25:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-25T15:59:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"194170862248479585658525047806134113393","date":"2024-07-20T03:00:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"13446457039524487000801515690579198387","date":"2024-07-19T13:33:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"60616988983467197440051053389643782814","date":"2024-07-17T23:31:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"2271011600061455740432013071699726415","date":"2024-07-17T17:31:50+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-17T13:05:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-27T11:03:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-27T11:02:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Dermatological Research","date":"2024-06-26T13:22:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-dermatological-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Archives of Dermatological Research](https://www.springer.com/journal/403)","snPcode":"403","submissionUrl":"https://submission.nature.com/new-submission/403/3","title":"Archives of Dermatological Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6574bc0a-c4e6-4974-965b-7605fa992078","owner":[],"postedDate":"July 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-08-26T23:48:46+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-19 19:56:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4643043","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4643043","identity":"rs-4643043","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}