Helminthic therapy of experimental autoimmune encephalomyelitis by Dicrocoelium ova

Helminthic therapy of experimental autoimmune encephalomyelitis by Dicrocoelium ova | 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 Helminthic therapy of experimental autoimmune encephalomyelitis by Dicrocoelium ova Zahra Navi, Mozhdeh Jafari Rad, Abdolmajid Fata, Elham Moghaddas, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-17288/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Experimental Autoimmune Encephalomyelitis (EAE) is an animal model of Multiple Sclerosis (MS). This study was conducted to evaluate the efficacy of Dicrocoelium ova on Experimental Autoimmune Encephalomyelitis (EAE) treatment in C57BL6 mice. Methods Twenty-eight C57BL/6 mice were assigned in four groups as control(C), prophylaxis (P), treatment1 (T1), and treatment2 (T2). Prior to induction of EAE in prophylaxis group and on days 7 and 18 in T1 and T2 groups, respectively, Dicrocoelium eggs were injected to each mouse. Clinical score, weight changes, and incidence time of EAE were recorded. IFN- γ and IL-4 assay and histopathological study by (H&E) and Toluidine-Blue (TB), and Luxol Fast Blue (LFB) were done. Data were analyzed using SPSS software version 21. Results The disease score was significantly lower in P and T1 groups than the control group (p=0.01). IFN- γ was lower in P and T1 groups than the control group. The highest level of IL-4 was observed in the T1 group. The total number of Neuroglia cells of corpus callosum was similar in all groups, but density increased in T1 group compared to the control group (P = 0.03). Conclusions : The results of the study showed that, Dicrocoelium eggs have the great potential efficacy to stimulate immunomodulation toward treatment of EAE during the initial phase. Immunology Allergy & Immune Disorders Encephalomyelitis Autoimmune Experimental Dicrocoelium Th1-Th2 Balance Multiple Sclerosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background A dramatic increase has been observed in the prevalence of autoimmune diseases in populations who have born in developed countries during1980-1999 [1, 2]. Expansion of inflammatory diseases has been coincided with the improvement of vaccination and antibiotics consumption over such a short time [3]. These mentioned factors prevent the natural contact of individual’s immunity with infectious agents ,which contributed to the development of immune system disorders suggesting the popular hygiene hypothesis [4]. Multiple Sclerosis(MS) disease is mediated with autoimmune CD4 + Th 1 and Th 17 responses [5, 6]. There are documented evidences showing IFN-γ (Th 1 ) is abundant in CNS of patients suffering from MS. Moreover, it is responsible for aggravation of their signs and symptoms [7],[8]. Experimental Autoimmune Encephalomyelitis ( EAE) is an animal model of demyelinating disease so called MS in human. EAE extensively helps us to clarify the role of some specific cells in the pathology of MS. [9]. Helminths are known to be the dominant immune regulators with more than 20 different mechanisms for induction of immune regulation [10, 11]. Infections with helminth can shift the immune system toward Th 2 responses and suppress the Th 1, Th 17 immune responses and induce M 2 macrophage differentiation [12, 13].Especially, IL4 (Th 2 ) is mostly effective in suppression of Th 1 pro inflammatory cells and diminishes the production of TNFα and IL 1 [14, 15]. Helminths also develop the activity of Tregs consequently suppressing the increase in Th 1 inflammatory productions in autoimmune diseases. In spite of extensive researches, treatment of MS has been remained unsolved. In addition, long time drug use is ineffective, which might be followed by severe toxic side effects. There are controversial notions about helminth therapy of MS due to the existence of some limitations in treatment of patients with live worms, and it might be harmful for humans. Immunoregulation by helminths components could be more safe and reliable [13]. In this study, the effect of Dicrocoelium ova administration as crud antigens in treatment of EAE was investigated. This study was conducted because of high overall prevalence of MS (about 11.4%) in Iran [16] .Also, Dicrocoelium spp.is the most common liver fluke found in the ruminants in Iran [17]. Methods Animals Twenty-eight, 5-6 -week-old C57BL/6 female mice were purchased from Rouyan Institute of Iran (Tehran) and were housed in the pathogen-free animal lab, at 23±2 o C, with a relative humidity of 50±5 % and a 12-h light/dark cycle. Facilities regarding the access to food and water supply were provided for the paralyzed mice. This study was approved by the Committee for Animal Ethics of Mashhad University of Medical Sciences (Ethical code: IR – MUMS. FM. REC 1396. 203). Dicrocoelium spp. Eggs Preparation Livers of the slaughtered sheep infected with Dicrocoelium flukes were obtained from a slaughterhouse located in Mashhad (Iran). Dicrocoelium dendriticum adults were identified and were separated according to their microscopic characters, then were washed with sterile PBS. Eggs were washed with penicillin 500 U/mL- streptomycin 0.5 mg/mL (with a catalog number of XC-A4122/100). Then, the antibiotics were removed by10 times washing the eggs with sterile PBS. Finally, eggs were counted using Neobar slide, and were stored at -80 o C. Experimental Design 28 mice were randomly assigned in four different groups including prophylaxis (P), treatment 1 (T1), and treatment 2 (T2), each group had 8 mice and the control (C) group had 4 mice. 2 weeks before EAE induction, 10,000 Dicrocoelium eggs were injected intraperitoneally (IP) to the prophylaxis group. 4 days prior to EAE, a second dose of eggs was injected to prophylaxis group; induction was performed using 5000 eggs via IP and 5000 via SC injections. EAE induction was initiated, when all mice were 8-9 weeks old. Both T 1 & T 2 groups received 20000 D. Dendriticum ova on 7 th day after EAE induction and 18 th day after appearance of paralysis, respectively. The control group remained untreated. Mice in the control group received PBS. According to an approved protocol[ 18 ] , on 40 th day ,all mice were sacrificed ,and the spleen and brain of mice were removed for cytokine and histopathological assays, respectively. EAE Induction To perform the EAE induction, all mice were immunized with synthetic peptide of Myelin Oligodendrocyte Glycoprotein (MOG) amino acid residues 35–55 (MEVGWYRSPFSRVVHLYRNGK) (SBS Genetech Co. Ltd., Beijing, China). Induction was performed by preparing an emulsion of MOGp 35–55 (300 mg) in complete Freund’s adjuvant containing 5mg/ ml of heat- killed Mycobacterium tuberculosis (H37Ra strain; Sigma, St. Louis, MO); yielding a solution containing1mg of MOG/ml. Each mouse received 200 ml of this emulsion through SC injection into two sites on its flank (100 ml/ site). Each mouse also received 250ng of pertussis toxin (Sigma, Germany) through IP injection on the day of immunization and 48h later [ 19 ]. Animals were assessed clinically according to the standard criteria( 0: normal; 1: loss of tail tone; 2: hind limb weakness; 3: hind limb paralysis; 4: hind limb and forelimb paralysis; and 5: moribund or dead [ 18 ]. To evaluate the pre -immunization and efficacy of Dicrocoelium eggs on EAE, clinical score, weight changes, and incidence time of EAE were recorded in all groups each day. Methods of Euthanasia CO2 narcosis is done for euthanasia procedure, a method approved by the University of Connecticut Health Center Animal Care Committee (IACUC). Histologic Analysis Histopathologic evaluation was performed on 20 mice brains (6 P, 6 T1, 4 T2, and 4 C). Haematoxylin- Eosin (H&E) and Toluidine-Blue (TB) staining techniques were used to evaluate the neuroglia cells number in corpus callosum area. Also, brain sections were stained by Luxol Fast Blue (LFB) to evaluate the density. A total of 9 sections per mouse in each group were investigated and observed as blind. To perform the statistical analysis, Kruskal- Wallis test was performed. The p –value of < 0.05 was considered as statistically significant. RNA Extraction To compare the expression of Th 1/ Th 2 related cytokines between different groups, the mRNA gene expression levels of IFN- γ and IL-4 in mice splenocytes were investigated by Real -Time Polymerase Chain Reaction (RT- PCR) (Rotor Gene Q - CIAGEN). About 2 million cells were separated from the spleens and were stored at -20̊ C with 750 μl of TRIZOL (Invitrogen – Germany). Total RNA was isolated using TRIZOL (Invitrogen – Germany), and then the reverse transcription was performed according to easy cDNA synthesis kit (Pars-Tous, Mashhad, Iran). All the procedures were conducted according to the manufacturer’s instructions. The primer sequences are listed in Table 1. The relative expression of each gene was calculated as the ratio of gene expression to the housekeeping gene (B2 macroglobulin). Table1. Sequence of real-time primer sequences implemented in this study Sequence Primer 5′-CCAAGTTT GAGGTCAACA-3′5′-CTGGCAGAATTATTCTTATTGG-3′ IFN-gamma 5′- CCTGTATGCTATCCAGAA -3′ 5′- GTAGCAGTTCAGTATGTTC -3′ B2mG 5′CTGGATTCATCGATAAGC-3′ 5′-GATGCTCTTTAGGCTTTC-3′ IL4 Statistical Analysis Data were analyzed using SPSS software version 21. 95% confidence interval and 5% significance level were considered in all tests. Normality of the data was evaluated by Kolmogorov-Smirnov test. Levenes̓ test was used to assess the homogeneity of variances. To describe the data, frequency, mean, standard deviation, or mean tables were used. ANOVA test was used to compare the means. Tukeys̓ post hoc test was used for multiple comparisons. Repeated -measures ANOVA was used to evaluate the differences in weight over time in each of four groups. Normality of residuals was evaluated by Shapiro-Wilk test. Mauchly’s test was applied to test the sphericity. Results There were statistically significant differences between group mean scores as determined by one-way ANOVA (F (3,210) = 10.08, p = .001). The results showed that, disease score was significantly lower in P and T 1 groups than the control group (p = 0.01). Mice in P and T 1 had trivial sign of paralysis which developed slowly, however, the scores over than 3 were not observed. No significant difference was found in clinical scores between T 2 and control groups (p > 0.05) (Fig. 1 ). Peaks of EAE were shorter and fewer in P and T1 groups than the control group. Otherwise, EAE developed faster and reached to score 5 in T 2 group, and we missed one mouse same as the control group. After injection of eggs on day 18 in T2 group, remission was observed compared to the control group, but signs and symptoms were presented very quickly after 1 week. In the control group, EAE was not seen in only one mouse. In T1 and P groups, just 2 mice from 8 mice in each group afflicted to the EAE. 50% decrease of cumulative incidence in the two mentioned groups was attributed to protective effect of Dicrocoelium eggs immunization and treatment. Dicrocoelium eggs immunization and treatment significantly reduced cumulative incidence rate in prophylaxis and treatment 1 groups (p = 0.0), and caused a delay of about 5 days in the onset of EAE in the mentioned groups. (Control n = 4), (Prophylaxis n = 8), (Treatment 1 n = 8), and (Treatment 2 n = 8). EAE developed in most mice in two groups (the control and treatment 2 groups) on day 10 [mean day of onset] but in prophylaxis and treatment1 groups, EAE developed just in 25% of mice on day 15 after induction (Fig. 2 ). There were no statistically significant differences between group mean weight changes at baseline as determined by one-way ANOVA (F (3,20) = 1.995, p = 0.147). Mauchly’s test indicated the violation in the assumption of sphericity, χ 2(35) = 93.578 p < .001, therefore degrees of freedom were corrected using the Greenhouse-Geisser estimates of sphericity (ε = 0.429). The result showed that, there was no significant difference in weight changes between the groups in four times of measurement (weight * groups), F (10.28, 65.1), p = .0.115. But, helminthic therapy with Dicrocoelium eggs could inhibit severe weight loss in prophylaxis and treatment1 groups. In the control group, after induction of EAE, mean weight of mice reduced dramatically from 19 gr on day 10 to 17.5 gr on day 20. During 10 days, the mean weight loss was about 1.5 gr. In treatment group 2, mean weight loss from 20.5 gr on day 5 reached to 17.8 gr on day 15. Approximately, a 2.8 -gr weight loss was observed in this group. However, the maximum weight loss was estimated lower than 0.5 gr after induction of EAE in other two groups (Prophylaxis, treatment1groups) (Fig. 3 ). Evaluation of Neuroglia Cell Number and Density in Corpus Callosum To evaluate the effect of Dicrocoelium egg administration on the number of neuroglia cells in corpus callosum, H&E and TB staining were investigated by light microscope. The neuroglia cells were counted in a Scale bar of 200 µm. The results demonstrated that, the quantity of neuroglia cells in corpus callosum is similar in 4 groups (P > 0.05; Fig. 4 ). The LFB stained sections showed more density in treatment 1 group compared to the control group (P = 0.03). No significant difference was observed between prophylaxis, treatment 2, and control groups (P > 0.05, Fig. 5 ). Brains from each mouse (collected on day 40 post-immunization) were fixed, and were embedded in paraffin, sections (5-µm) were prepared, and then the tissues were stained with H&E (Fig. 4 A) and TLB (Fig. 4 B) to count the number of neuroglia cells (as presented in the left side of Fig. 4 A and 4 B). The quantification of neuroglia cells is shown in the right side of Fig A and B. (Control n = 4), (Prophylaxis n = 6), (Treatment 1 n = 6), and (Treatment 2 n = 4). No significant difference was found between 4groups (P > 0.05). Brains from each mouse tissues were stained with LFB to assess the extent of density of brain tissue. Histologic features were scored semi quantitatively as presented in the right side of Fig. 5 (control, n = 4), (Prophylaxis, n = 6), (Treatment 1, n = 6), and (Treatment 2, n = 8). The quantification of brain tissue density is shown in the left side of Fig. 5 . No significant difference was found between prophylaxis, treatment 2, and control groups (P > 0.05). There was a significant difference in EAE incidence between treatment1 and control groups (P = 0.03, *p < 0.05). Evaluation of IFN- γ and IL-4 m RNA Expression There were no statistically significant differences between group means of IFN- γ and IL-4 as determined by one-way ANOVA (F (3, 19) =.90, p = .459) (F (3,19) =1.210, p = .333). In prophylaxis and treatment 1 groups, the treatment induced a great up-regulation of IL-4, compared to the control group. The highest level of IL-4 was observed in the treatment 1 group (RQ=2.65). The IL-4 mRNA gene expression level was lowest in the treatment 2group (RQ= 0.92), also, different levels of mRNA gene expression were observed in the IFN- γ. The mRNA gene expression level of IFN- γ was at highest copies in the control (RQ=8.09) and T2 (RQ= 5.91) groups, whereas the mRNA gene expression of IFN- γ was lower in the prophylaxis (RQ=4.03) and treatment 1 groups (RQ=0.37) than the control group (RQ=8.09) (Figure 6). Relative mRNA gene expression of genes was determined compared to the housekeeping gene, β2 microglobulin. Discussion Immunotherapy by helminth is considered as one of the effective therapeutic methods for treatment of multiple sclerosis and other autoimmune diseases. The evidence indicates that helminthic infections lead to the reduction of autoimmune paroxysm in animal models [20, 21] . In most studies, the prevalence of helminthic infections before deterioration of EAE not only plummeted EAE scores but also decreased the incidence rate in the infected animal groups [22–24] . Many studies have been conducted so far to investigate the immune therapy of EAE (animal model of Multiple Sclerosis) by different helminth species such as Schistosoma mansoni, Trichinella spiralis, Fasciola hepatica, Trichinella psudospiralis, Taenia crassiceps, Strongyloides venezuelensis, Schistosoma japonicum, and Trichuris suis [18, 20, 21, 23, 25–30] . Among all of these studies, F. chiuso-minicucci et al. (2011) showed that, the infection with Strongyloides venezuelensis could not be effective in treatment of EAE. Yet, they emphasized that, their study alone cannot reject the hygiene hypothesis, and they recommended the conduction of more studies to determine the immune regulation role of other species of helminth [25]. In this study, the effect of Dicrocoelium ova immunization in EAE was evaluated for the first time. Clinical scores, IL4, and IFN-γ genes expression along with neuroglia cell counting and demyelination of brain cells in corpus callosum were evaluated and analyzed between the groups. The results illustrated that, pre-immunization of EAE in prophylaxis and treatment1 groups attenuated the scores, delayed onset day of EAE and reduced its incidence in the mentioned groups dramatically, as confirmed in the study by Swell et al. ( 2003 ) who investigated the treatment of EAE by Shistosoma mansoni so that, EAE helminthic therapy just could be effective in initial phase of EAE not in effective phase[18]. In addition, EAE suppression was accompanied with a dramatic increase in the density in brain of mice in T1 group. Considering similar count of neuroglia cells in 4 groups, a dramatic increase in the density observed in treatment1 group is not associated with the number of myelin-producing cells. Merrill JE. et al (1992) found that, the demyelination reduction in spinal cords was related to the reduction of inflammatory leukocytes and cytokines [22] . Zhiliang Wu et al (2010) showed that, the administration of 200 larvae of Trichinella psudospiralis (15 days before induction) attenuated EAE score and delayed the incidence of EAE. Also, demyelination in spinal cord decreased ,and inflammatory brain cells reduced [28] . La Flamma et al ( 2003) indicated that, the reduction of demyelination in spinal cord of EAE mice was rooted in the plummeted inflammatory macrophage [21]. In our study, the assessment of cytokines with Real Time PCR showed that, IFN- γ gene expression was lower in prophylaxis and treatment1 groups than the control and treatment2 groups. In contrast, IL-4 gene expression was more in prophylaxis and treatment1 groups than the control and treatment2 groups. So, slight down- regulation of IFN- γ along with up- regulation of IL-4 in T1 and P groups could not exclusively interpret significant preventable effect of Dicrocoelium eggs immunization. Previous studies showed that, Th1 lymphocytes are the critical cells causing the EAE. However, recent studies have demonstrated that, Th17 cells are more responsible for autoimmune diseases especially in case of multiple sclerosis. Briefly, remission of EAE relatively but not completely depends on the existence of balance between Th1/ Th2 [31, 32]. Cross immunomodulation of Th1/Th2 in helminthic therapy is disputable yet [33]. Due to variable functions of cytokines, it is difficult to obtain a definite conclusion and interpretation of the condition [34]. The suppression of EAE has been demonstrated to result from a decrease in IL12 in helminth therapy with Shistosoma mansoni . Also, the reduction of IFN- γ and increase of IL-4 production was not significant in the treated groups ,indicating that EAE attenuation was related to the suppressed IL12 ,and shift of immune response toward Th2 cytokines was not responsible for EAE amelioration [21] . However, Swell et al proved that, EAE improvement is merely related to predominant Th2 cytokines [18] .In addition, the role of innate immune system [35] such as macrophages, B lymphocytes, regulator cells of immune system CD4 CD8 Tregs is effective regarding mediation of most of the protective mechanisms against EAE and MS. Besides, [36] Th17 is the most effective element in EAE induction explaining that, why the mechanisms of autoimmune diseases such as MS and EAE cannot be exclusively interpreted via Th1/Th2 responses [37, 38]. Therefore, it is suggested to evaluate Th17 and Tregs cytokine and inflammation as well as assessing the infiltration of immune cells in spinal cord in future studies, which could be considered as a limitation in the present study. Conclusion The results of the current study showed that, the treatment with Dicrocoelium ova immunization attenuated EAE score and delayed the incidence of EAE especially in initial phase of diseases with down -regulation of IFN- γ and up -regulation of IL-4. Dicrocoelium egg did not have any effect on neuroglia cells number, so EAE amelioration alongside increase of density in the brain cells might be related to another factors. Hence, it can be concluded that, Dicrocoelium eggs have the potential to be considered as useful therapy for treatment of MS in initial phase in future studies especially in human clinical trial studies. Declarations Acknowledgments We thank the Animal lab employees for feeding and watering of the mouse and checking the room temperature daily. Authors' contributions Designed and performed experiments: E.M and M.M. Analyzed data and co-wrote the paper: A.F and N.M.A. Performed experiments: Z.N and M.J.R. Performed transporter experiments: M.R. Performed Statistical Analysis: M.M.B. Supervised the research: A.R.H and F.L.A. Histologic Analysis: S.S.N. Funding The authors gratefully acknowledge the support provided from the Student Research Committee (grant # 970556) and the Grants-in-Aid for Scientific Research (Grant # 951683) from Mashhad University of Medical Sciences (MUMS), Mashhad, Iran. Competing Interests The authors declare no conflicts of interest. 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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-17288","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":407505,"identity":"ff392cd9-09e0-4968-aefa-b19cfa6cd647","order_by":1,"name":"Zahra Navi","email":"","orcid":"","institution":"Mashhad University of Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zahra","middleName":"","lastName":"Navi","suffix":""},{"id":407506,"identity":"9bb4a693-94c5-4224-beb1-1350a0ed38c8","order_by":2,"name":"Mozhdeh Jafari Rad","email":"","orcid":"","institution":"Mashhad University of Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mozhdeh","middleName":"Jafari","lastName":"Rad","suffix":""},{"id":407507,"identity":"f06048c1-ab52-4abb-909f-7516d6761dbf","order_by":3,"name":"Abdolmajid Fata","email":"","orcid":"","institution":"Mashhad University of Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Abdolmajid","middleName":"","lastName":"Fata","suffix":""},{"id":407508,"identity":"c42a7b60-b08f-4ca6-b3d3-45cdc141feb2","order_by":4,"name":"Elham Moghaddas","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIiWNgGAWjYHACNiBiBtLMB+BCB3CpRdPClkCyFh4D4lyl295+7cGPMmt5+faer5t5c+oY+NsPMB6uwKPF7MyZcsOec+mGG86c3Xabd9thBokzCQwHz+DTciMnTYK37TDjBolckBagL24wMBxsIKBF8m/bYfv58988A2qpY5AnrCX9mDTQlsSGGzxsQC3MDAYEtZw5wyYtcy49ecOZNLObc7cd5jE8k9iAX8vx9meSb8qsbee3H3524+22Ojm544cPf8SnBSM6eBgYGPFrYGBgf0BAwSgYBaNgFIx4AABBNFVgKu7eBwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-7080-2768","institution":"Mashhad University of Medical Sciences","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Elham","middleName":"","lastName":"Moghaddas","suffix":""},{"id":407509,"identity":"e6ab7f45-089d-41cf-9545-247bdf7ee319","order_by":5,"name":"Mahmoud Mahmoudi","email":"","orcid":"","institution":"Mashhad University of Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mahmoud","middleName":"","lastName":"Mahmoudi","suffix":""},{"id":407510,"identity":"f7885cf3-9255-4fe8-b1e4-382675e39751","order_by":6,"name":"Maryam Rastin","email":"","orcid":"","institution":"Mashhad University of Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Maryam","middleName":"","lastName":"Rastin","suffix":""},{"id":407511,"identity":"4d49b6ed-f736-49b5-a576-80a05f90cadf","order_by":7,"name":"Mojtaba Mousavi Bazaz","email":"","orcid":"","institution":"Mashhad University of Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mojtaba","middleName":"Mousavi","lastName":"Bazaz","suffix":""},{"id":407512,"identity":"396c9d7a-b7dc-4d81-b8cc-a7144b49b1f4","order_by":8,"name":"Fahimeh Lavi Arab","email":"","orcid":"","institution":"Mashhad University of Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Fahimeh","middleName":"Lavi","lastName":"Arab","suffix":""},{"id":407513,"identity":"aca6156d-6159-49e9-8efa-afd2783336b9","order_by":9,"name":"Sajad Sahab Negah","email":"","orcid":"","institution":"Mashhad University of Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sajad","middleName":"Sahab","lastName":"Negah","suffix":""},{"id":407514,"identity":"c1ae435f-baea-4633-9e8f-7eb507c81297","order_by":10,"name":"Amir Reza Heidari","email":"","orcid":"","institution":"Mashhad University of Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Amir","middleName":"Reza","lastName":"Heidari","suffix":""},{"id":407515,"identity":"9fe484e5-eccb-4377-8876-3ba67ea77352","order_by":11,"name":"Naser Morgan Azghadi","email":"","orcid":"","institution":"Ferdowsi University of Mashhad Faculty of Veterinary Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Naser","middleName":"Morgan","lastName":"Azghadi","suffix":""}],"badges":[],"createdAt":"2020-03-11 16:54:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-17288/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-17288/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":653063,"identity":"8d3e785e-65bf-4fad-980c-ef9140c376b9","added_by":"auto","created_at":"2020-03-16 15:23:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":197885,"visible":true,"origin":"","legend":"Mean of clinical score in helminthic therapy of EAE mice with Dicrocoelium eggs in four groups (control, prophylaxis, treatment 1, and treatment 2)","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-17288/v1/1.png"},{"id":653064,"identity":"1ffc4c87-9b7c-46cb-bf9f-f2c480be2b5f","added_by":"auto","created_at":"2020-03-16 15:23:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":74108,"visible":true,"origin":"","legend":"Cumulative incidence in control, prophylaxis, treatment 1, and treatment 2 groups obtained as 75, 25, 25, and 62.5%, respectively.","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-17288/v1/2.png"},{"id":653065,"identity":"1f54871b-6a66-499f-a749-672dd9720bbe","added_by":"auto","created_at":"2020-03-16 15:23:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":126443,"visible":true,"origin":"","legend":"Mean weight in immunotherapy of EAE mice with Dicrocoelium eggs in four groups (control, prophylaxis, treatment 1, and treatment 2)","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-17288/v1/3.png"},{"id":653066,"identity":"594fc5a5-9e7b-41e4-975f-f338be62b504","added_by":"auto","created_at":"2020-03-16 15:23:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":198980,"visible":true,"origin":"","legend":"Effects of Dicrocoelium egg administration on the pathologies associated with EAE by H\u0026E (Fig 4A) and TLB (Fig 4B) to count the number of neuroglia cells (as presented in the left side of Fig 4A and 4B).","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-17288/v1/4.png"},{"id":653067,"identity":"a191bc36-7460-4608-b61b-1444520c55ed","added_by":"auto","created_at":"2020-03-16 15:23:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":133932,"visible":true,"origin":"","legend":"Effects of Dicrocoelium eggs administration on the pathologies associated with EAE","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-17288/v1/5.png"},{"id":653068,"identity":"90f6387b-f308-4359-b0a0-fc350d02dd54","added_by":"auto","created_at":"2020-03-16 15:23:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":106793,"visible":true,"origin":"","legend":"mRNA expression of IL4 and IFN- γ in EAE mice treated with Dicrocoelium eggs in four groups (control, prophylaxis, treatment 1, and treatment 2)","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-17288/v1/6.png"},{"id":13493753,"identity":"b5943f65-379f-4de2-be25-6fe3765d92dd","added_by":"auto","created_at":"2021-09-16 22:38:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":999170,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-17288/v1/12519825-b8e1-46e8-b2c2-49456e4b933e.pdf"},{"id":653062,"identity":"ddbe0af4-6711-4171-9677-8233356f9161","added_by":"auto","created_at":"2020-03-16 15:23:19","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":3516569,"visible":true,"origin":"","legend":"","description":"","filename":"NC3RsARRIVEGuidelines2013.docx","url":"https://assets-eu.researchsquare.com/files/rs-17288/v1/NC3Rs ARRIVE Guidelines 2013.docx"}],"financialInterests":"","formattedTitle":"Helminthic therapy of experimental autoimmune encephalomyelitis by Dicrocoelium ova","fulltext":[{"header":"Background","content":" \u003cp\u003eA dramatic increase has been observed in the prevalence of autoimmune diseases in populations who have born in developed countries during1980-1999 [1, 2]. Expansion of inflammatory diseases has been coincided with the improvement of vaccination and antibiotics consumption over such a short time [3]. These mentioned factors prevent the natural contact of individual\u0026rsquo;s immunity with infectious agents ,which contributed to the development of immune system disorders suggesting the popular hygiene hypothesis [4]. Multiple Sclerosis(MS) disease is mediated with autoimmune CD4\u003csup\u003e+\u003c/sup\u003e Th\u003csub\u003e1\u003c/sub\u003e and Th\u003csub\u003e17\u003c/sub\u003e responses [5, 6]. There are documented evidences showing IFN-γ (Th\u003csub\u003e1\u003c/sub\u003e) is abundant in CNS of patients suffering from MS. Moreover, it is responsible for aggravation of their signs and symptoms [7],[8]. Experimental Autoimmune Encephalomyelitis \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003e(\u003c/span\u003eEAE) is an animal model of demyelinating disease so called MS in human. EAE extensively helps us to clarify the role of some specific cells in the pathology of MS. [9]. Helminths are known to be the dominant immune regulators with more than 20 different mechanisms for induction of immune regulation [10, 11]. Infections with helminth can shift the immune system toward Th\u003csub\u003e2\u003c/sub\u003e responses and suppress the Th\u003csub\u003e1,\u003c/sub\u003e Th\u003csub\u003e17\u003c/sub\u003e immune responses and induce M\u003csub\u003e2\u003c/sub\u003e macrophage differentiation [12, 13].Especially, IL4 (Th\u003csub\u003e2\u003c/sub\u003e) is mostly effective in suppression of Th\u003csub\u003e1\u003c/sub\u003e pro inflammatory cells and diminishes the production of TNFα and IL\u003csub\u003e1\u003c/sub\u003e [14, 15]. Helminths also develop the activity of Tregs consequently suppressing the increase in Th\u003csub\u003e1\u003c/sub\u003e inflammatory productions in autoimmune diseases. In spite of extensive researches, treatment of MS has been remained unsolved. In addition, long time drug use is ineffective, which might be followed by severe toxic side effects.\u003c/p\u003e \u003cp\u003eThere are controversial notions about helminth therapy of MS due to the existence of some limitations in treatment of patients with live worms, and it might be harmful for humans. Immunoregulation by helminths components could be more safe and reliable [13]. In this study, the effect of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eDicrocoelium\u003c/span\u003e ova administration as crud antigens in treatment of EAE was investigated. This study was conducted because of high overall prevalence of MS (about 11.4%) in Iran [16] .Also, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eDicrocoelium\u003c/span\u003e spp.is the most common liver fluke found in the ruminants in Iran [17].\u003c/p\u003e "},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwenty-eight, 5-6 -week-old C57BL/6 female mice were purchased from Rouyan Institute of Iran (Tehran) and were housed in the pathogen-free animal lab, at 23\u0026plusmn;2\u003csup\u003eo\u003c/sup\u003e C, with a relative humidity of 50\u0026plusmn;5 % and a 12-h light/dark cycle. Facilities regarding the access to food and water supply were provided for the paralyzed mice. This study was approved by the Committee for Animal Ethics of Mashhad University of Medical Sciences (Ethical code: IR \u0026ndash; MUMS. FM. REC 1396. 203).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDicrocoelium \u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003espp. Eggs Preparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLivers of the slaughtered sheep infected with \u003cem\u003eDicrocoelium \u003c/em\u003eflukes were obtained from a slaughterhouse located in Mashhad (Iran). \u003cem\u003eDicrocoelium\u003c/em\u003e dendriticum adults \u0026nbsp;\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ewere identified and were separated according to their microscopic characters, then were washed with sterile PBS. Eggs were washed with penicillin 500 U/mL- streptomycin 0.5 mg/mL (with a catalog number of XC-A4122/100). Then, the antibiotics were removed by10 times washing the eggs with sterile PBS. Finally, eggs were counted using Neobar slide, and were stored at -80\u003csup\u003e o\u003c/sup\u003e C.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eExperimental Design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e28 mice were randomly assigned in four different groups including prophylaxis (P), treatment 1 (T1), and treatment 2 (T2), each group had 8 mice and the control (C) group had 4 mice. 2 weeks before EAE induction, 10,000\u003cem\u003e Dicrocoelium\u003c/em\u003e eggs were injected intraperitoneally (IP) to the prophylaxis group. 4 days prior to EAE, a second dose of eggs was injected to prophylaxis group; induction was performed using 5000 eggs via IP and 5000 via SC injections. EAE induction was initiated, when all mice were 8-9 weeks old. \u0026nbsp;Both T\u003csub\u003e1\u003c/sub\u003e \u0026amp; T\u003csub\u003e2\u003c/sub\u003e groups received 20000 \u003cem\u003eD. Dendriticum\u003c/em\u003e ova on 7\u003csup\u003eth\u003c/sup\u003e day after EAE induction and 18\u003csup\u003eth\u003c/sup\u003e day after appearance of paralysis, respectively. The control group remained untreated. Mice in the control group received PBS. \u0026nbsp;According to an approved protocol[\u003ca href=\"#_ENREF_18\"\u003e18\u003c/a\u003e] , on 40\u003csup\u003eth \u003c/sup\u003eday ,all mice were sacrificed ,and the spleen and brain of mice were removed for cytokine and histopathological\u0026nbsp; assays, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEAE Induction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo perform the EAE induction, all mice were immunized with synthetic peptide of Myelin Oligodendrocyte Glycoprotein (MOG) amino acid residues 35\u0026ndash;55 (MEVGWYRSPFSRVVHLYRNGK) (SBS Genetech Co. Ltd., Beijing, China). Induction was performed by preparing an emulsion of MOGp 35\u0026ndash;55 (300 mg) in complete Freund\u0026rsquo;s adjuvant containing 5mg/ ml of heat- killed \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e (H37Ra strain; Sigma, St. Louis, MO); yielding a solution containing1mg of MOG/ml. Each mouse received 200 ml of this emulsion through SC injection into two sites on its flank (100 ml/ site). Each mouse also received 250ng of pertussis toxin (Sigma, Germany) through IP injection on the day of immunization and 48h later [\u003ca href=\"#_ENREF_19\"\u003e19\u003c/a\u003e]. \u003csup\u003e\u0026nbsp;\u003c/sup\u003eAnimals were assessed clinically according to the standard criteria( 0: normal; 1: loss of tail tone; 2: hind limb weakness; 3: hind limb paralysis; 4: hind limb and forelimb paralysis; and 5: moribund or dead [\u003ca href=\"#_ENREF_18\"\u003e18\u003c/a\u003e]. To evaluate the pre -immunization and efficacy of \u003cem\u003eDicrocoelium\u003c/em\u003e eggs on EAE, clinical score, weight changes, and incidence time of EAE were recorded in all groups each day.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods of Euthanasia\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCO2 narcosis is done for euthanasia procedure, a method approved by the University of Connecticut Health Center Animal Care Committee (IACUC).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistologic Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHistopathologic evaluation was performed on 20 mice brains (6 P, 6 T1, 4 T2, and 4 C). Haematoxylin- Eosin (H\u0026amp;E) and Toluidine-Blue (TB) staining techniques were used to evaluate the neuroglia cells number in corpus callosum area. Also, brain sections were stained by Luxol Fast Blue (LFB) to evaluate the density. A total of 9 sections per mouse in each group were investigated and observed as blind. To perform the statistical analysis, Kruskal- Wallis test was performed. The p \u0026ndash;value of \u0026lt; 0.05 was considered as statistically significant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA Extraction \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo compare the expression of Th\u003csub\u003e1/ \u003c/sub\u003eTh\u003csub\u003e2 \u003c/sub\u003erelated cytokines between different groups, the mRNA gene expression levels of IFN- \u0026gamma; and IL-4 in mice splenocytes were investigated by Real -Time Polymerase Chain Reaction (RT- PCR) (Rotor Gene Q - CIAGEN). About 2 million cells were separated from the spleens and were stored at -20̊ C with 750 \u0026mu;l of TRIZOL (Invitrogen \u0026ndash; Germany). Total RNA was isolated using TRIZOL (Invitrogen \u0026ndash; Germany), and then the reverse transcription was performed according to easy cDNA synthesis kit (Pars-Tous, Mashhad, Iran). All the procedures were conducted according to the manufacturer\u0026rsquo;s instructions. The primer sequences are listed in Table 1. The relative expression of each gene was calculated as the ratio of gene expression to the housekeeping gene (B2 macroglobulin).\u003c/p\u003e\n\u003cp style=\"margin: 0in 0in 10pt; line-height: 22px; font-size: 15px; font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 11px; line-height: 24px; font-family: Verdana, Geneva, sans-serif;\"\u003eTable1. Sequence of real-time primer sequences implemented in this study\u003c/span\u003e\u003c/p\u003e\n\u003cdiv align=\"left\"\u003e\n\u003ctable dir=\"rtl\" style=\"border-collapse: collapse; border: none;\" border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 275.6pt; border: 1pt solid #bfbfbf; padding: 0in 5.4pt; vertical-align: top;\" valign=\"top\" width=\"73.54709418837675%\"\u003e\n\u003cp dir=\"RTL\" style=\"margin: 0in 0in 0.0001pt; line-height: 22px; font-size: 15px; font-family: Calibri, sans-serif; text-align: center; text-indent: 17pt;\"\u003e\u003cspan style=\"font-family: Verdana, Geneva, sans-serif;\"\u003e\u003cspan style=\"font-size: 11px;\"\u003e\u003cstrong\u003e\u003cspan dir=\"LTR\" style=\"line-height: 24px;\"\u003eSequence\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 98.8pt; border-top-width: 1pt; border-bottom-width: 1pt; border-left-width: 1pt; border-style: solid none solid solid; border-top-color: #bfbfbf; border-bottom-color: #bfbfbf; border-left-color: #bfbfbf; padding: 0in 5.4pt; vertical-align: top;\" valign=\"top\" width=\"26.452905811623246%\"\u003e\n\u003cp dir=\"RTL\" style=\"margin: 0in 0in 0.0001pt; line-height: 22px; font-size: 15px; font-family: Calibri, sans-serif; text-align: center;\"\u003e\u003cspan style=\"font-family: Verdana, Geneva, sans-serif;\"\u003e\u003cspan style=\"font-size: 11px;\"\u003e\u003cstrong\u003e\u003cspan dir=\"LTR\" style=\"line-height: 24px;\"\u003ePrimer\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 275.6pt; border-right-width: 1pt; border-bottom-width: 1pt; border-left-width: 1pt; border-style: none solid solid; border-right-color: #bfbfbf; border-bottom-color: #bfbfbf; border-left-color: #bfbfbf; background-color: #f2f2f2; padding: 0in 5.4pt; vertical-align: top;\" valign=\"top\" width=\"73.54709418837675%\"\u003e\n\u003cp dir=\"RTL\" style=\"margin: 0in 0in 0.0001pt; line-height: 22px; font-size: 15px; font-family: Calibri, sans-serif; text-align: center; text-indent: 17pt;\"\u003e\u003cspan style=\"font-family: Verdana, Geneva, sans-serif;\"\u003e\u003cspan style=\"font-size: 11px;\"\u003e\u003cspan dir=\"LTR\" style=\"line-height: 24px;\"\u003e5\u0026prime;-CCAAGTTT GAGGTCAACA-3\u0026prime;5\u0026prime;-CTGGCAGAATTATTCTTATTGG-3\u0026prime;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 98.8pt; border-style: none none solid solid; border-left-width: 1pt; border-left-color: #bfbfbf; border-bottom-width: 1pt; border-bottom-color: #bfbfbf; background-color: #f2f2f2; padding: 0in 5.4pt; vertical-align: top;\" valign=\"top\" width=\"26.452905811623246%\"\u003e\n\u003cp dir=\"RTL\" style=\"margin: 0in 0in 0.0001pt; line-height: 22px; font-size: 15px; font-family: Calibri, sans-serif; text-align: center;\"\u003e\u003cspan style=\"font-family: Verdana, Geneva, sans-serif;\"\u003e\u003cspan style=\"font-size: 11px;\"\u003e\u003cspan dir=\"LTR\" style=\"line-height: 24px;\"\u003eIFN-gamma\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 275.6pt; border-right-width: 1pt; border-bottom-width: 1pt; border-left-width: 1pt; border-style: none solid solid; border-right-color: #bfbfbf; border-bottom-color: #bfbfbf; border-left-color: #bfbfbf; padding: 0in 5.4pt; vertical-align: top;\" valign=\"top\" width=\"73.54709418837675%\"\u003e\n\u003cp dir=\"RTL\" style=\"margin: 0in 0in 0.0001pt; line-height: 22px; font-size: 15px; font-family: Calibri, sans-serif; text-align: center; text-indent: 17pt;\"\u003e\u003cspan style=\"font-family: Verdana, Geneva, sans-serif;\"\u003e\u003cspan style=\"font-size: 11px;\"\u003e\u003cspan dir=\"LTR\" style=\"line-height: 24px;\"\u003e5\u0026prime;-\u0026nbsp;CCTGTATGCTATCCAGAA\u0026nbsp;-3\u0026prime;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp dir=\"RTL\" style=\"margin: 0in 0in 0.0001pt; line-height: 22px; font-size: 15px; font-family: Calibri, sans-serif; text-align: center; text-indent: 17pt;\"\u003e\u003cspan style=\"font-family: Verdana, Geneva, sans-serif;\"\u003e\u003cspan style=\"font-size: 11px;\"\u003e\u003cspan dir=\"LTR\" style=\"line-height: 24px;\"\u003e5\u0026prime;-\u0026nbsp;GTAGCAGTTCAGTATGTTC\u0026nbsp;-3\u0026prime;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 98.8pt; border-style: none none solid solid; border-left-width: 1pt; border-left-color: #bfbfbf; border-bottom-width: 1pt; border-bottom-color: #bfbfbf; padding: 0in 5.4pt; vertical-align: top;\" valign=\"top\" width=\"26.452905811623246%\"\u003e\n\u003cp dir=\"RTL\" style=\"margin: 0in 0in 0.0001pt; line-height: 22px; font-size: 15px; font-family: Calibri, sans-serif; text-align: center;\"\u003e\u003cspan style=\"font-family: Verdana, Geneva, sans-serif;\"\u003e\u003cspan style=\"font-size: 11px;\"\u003e\u003cspan dir=\"LTR\" style=\"line-height: 24px;\"\u003eB2mG\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 275.6pt; border-right-width: 1pt; border-bottom-width: 1pt; border-left-width: 1pt; border-style: none solid solid; border-right-color: #bfbfbf; border-bottom-color: #bfbfbf; border-left-color: #bfbfbf; padding: 0in 5.4pt; vertical-align: top;\" valign=\"top\" width=\"73.54709418837675%\"\u003e\n\u003cp dir=\"RTL\" style=\"margin: 0in 0in 0.0001pt; line-height: 22px; font-size: 15px; font-family: Calibri, sans-serif; text-align: center; text-indent: 17pt;\"\u003e\u003cspan style=\"font-family: Verdana, Geneva, sans-serif;\"\u003e\u003cspan style=\"font-size: 11px;\"\u003e\u003cspan dir=\"LTR\" style=\"line-height: 24px;\"\u003e5\u0026prime;CTGGATTCATCGATAAGC-3\u0026prime;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp dir=\"RTL\" style=\"margin: 0in 0in 0.0001pt; line-height: 22px; font-size: 15px; font-family: Calibri, sans-serif; text-align: center; text-indent: 17pt;\"\u003e\u003cspan style=\"font-family: Verdana, Geneva, sans-serif;\"\u003e\u003cspan style=\"font-size: 11px;\"\u003e\u003cspan dir=\"LTR\" style=\"line-height: 24px;\"\u003e5\u0026prime;-GATGCTCTTTAGGCTTTC-3\u0026prime;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 98.8pt; border-style: none none solid solid; border-left-width: 1pt; border-left-color: #bfbfbf; border-bottom-width: 1pt; border-bottom-color: #bfbfbf; padding: 0in 5.4pt; vertical-align: top;\" valign=\"top\" width=\"26.452905811623246%\"\u003e\n\u003cp dir=\"RTL\" style=\"margin: 0in 0in 0.0001pt; line-height: 22px; font-size: 15px; font-family: Calibri, sans-serif; text-align: center;\"\u003e\u003cspan style=\"font-size: 11px; font-family: Verdana, Geneva, sans-serif;\"\u003e\u003cspan dir=\"LTR\" style=\"line-height: 24px;\"\u003eIL4\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData were analyzed using SPSS software version 21. 95% confidence interval and 5% significance level were considered in all tests. Normality of the data was evaluated by Kolmogorov-Smirnov test. Levenes̓ test was used to assess the homogeneity of variances. To describe the data, frequency, mean, standard deviation, or mean tables were used. ANOVA test was used to compare the means. Tukeys̓ post hoc test was used for multiple comparisons. Repeated -measures ANOVA was used to evaluate the differences in weight over time in each of four groups. Normality of residuals was evaluated by Shapiro-Wilk test. Mauchly\u0026rsquo;s test was applied to test the sphericity.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThere were statistically significant differences between group mean scores as determined by one-way ANOVA (F (3,210)\u0026thinsp;=\u0026thinsp;10.08, p\u0026thinsp;=\u0026thinsp;.001). The results showed that, disease score was significantly lower in P and T\u003csub\u003e1\u003c/sub\u003e groups than the control group (p\u0026thinsp;=\u0026thinsp;0.01). Mice in P and T\u003csub\u003e1\u003c/sub\u003e had trivial sign of paralysis which developed slowly, however, the scores over than 3 were not observed. No significant difference was found in clinical scores between T\u003csub\u003e2\u003c/sub\u003e and control groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Peaks of EAE were shorter and fewer in P and T1 groups than the control group. Otherwise, EAE developed faster and reached to score 5 in T\u003csub\u003e2\u003c/sub\u003e group, and we missed one mouse same as the control group. After injection of eggs on day 18 in T2 group, remission was observed compared to the control group, but signs and symptoms were presented very quickly after 1 week.\u003c/p\u003e\n\u003cp\u003eIn the control group, EAE was not seen in only one mouse. In T1 and P groups, just 2 mice from 8 mice in each group afflicted to the EAE. 50% decrease of cumulative incidence in the two mentioned groups was attributed to protective effect of \u003cspan class=\"Italic\"\u003eDicrocoelium\u003c/span\u003e eggs immunization and treatment. \u003cspan class=\"Italic\"\u003eDicrocoelium\u003c/span\u003e eggs immunization and treatment significantly reduced cumulative incidence rate in prophylaxis and treatment 1 groups (p\u0026thinsp;=\u0026thinsp;0.0), and caused a delay of about 5 days in the onset of EAE in the mentioned groups. (Control n\u0026thinsp;=\u0026thinsp;4), (Prophylaxis n\u0026thinsp;=\u0026thinsp;8), (Treatment 1 n\u0026thinsp;=\u0026thinsp;8), and (Treatment 2 n\u0026thinsp;=\u0026thinsp;8). EAE developed in most mice in two groups (the control and treatment 2 groups) on day 10 [mean day of onset] but in prophylaxis and treatment1 groups, EAE developed just in 25% of mice on day 15 after induction (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThere were no statistically significant differences between group mean weight changes at baseline as determined by one-way ANOVA (F (3,20)\u0026thinsp;=\u0026thinsp;1.995, p\u0026thinsp;=\u0026thinsp;0.147). Mauchly\u0026rsquo;s test indicated the violation in the assumption of sphericity, \u0026chi; 2(35)\u0026thinsp;=\u0026thinsp;93.578 p\u0026thinsp;\u0026lt;\u0026thinsp;.001, therefore degrees of freedom were corrected using the Greenhouse-Geisser estimates of sphericity (\u0026epsilon;\u0026thinsp;=\u0026thinsp;0.429). The result showed that, there was no significant difference in weight changes between the groups in four times of measurement (weight * groups), F (10.28, 65.1), p\u0026thinsp;=\u0026thinsp;.0.115. But, helminthic therapy with \u003cspan class=\"Italic\"\u003eDicrocoelium\u003c/span\u003e eggs could inhibit severe weight loss in prophylaxis and treatment1 groups. In the control group, after induction of EAE, mean weight of mice reduced dramatically from 19 gr on day 10 to 17.5 gr on day 20. During 10 days, the mean weight loss was about 1.5 gr. In treatment group 2, mean weight loss from 20.5 gr on day 5 reached to 17.8 gr on day 15. Approximately, a 2.8 -gr weight loss was observed in this group. However, the maximum weight loss was estimated lower than 0.5 gr after induction of EAE in other two groups (Prophylaxis, treatment1groups) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan class=\"Bold\"\u003eEvaluation of Neuroglia Cell Number and Density in Corpus Callosum\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the effect of \u003cspan class=\"Italic\"\u003eDicrocoelium egg administration\u003c/span\u003e on the number of neuroglia cells in corpus callosum, H\u0026amp;E and TB staining were investigated by light microscope. The neuroglia cells were counted in a Scale bar of 200\u0026nbsp;\u0026micro;m.\u003c/p\u003e\n\u003cp\u003eThe results demonstrated that, the quantity of neuroglia cells in corpus callosum is similar in 4 groups (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). The LFB stained sections showed more density in treatment 1 group compared to the control group (P\u0026thinsp;=\u0026thinsp;0.03). No significant difference was observed between prophylaxis, treatment 2, and control groups (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eBrains from each mouse (collected on day 40 post-immunization) were fixed, and were embedded in paraffin, sections (5-\u0026micro;m) were prepared, and then the tissues were stained with H\u0026amp;E (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA) and TLB (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB) to count the number of neuroglia cells (as presented in the left side of Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). The quantification of neuroglia cells is shown in the right side of Fig A and B. (Control n\u0026thinsp;=\u0026thinsp;4), (Prophylaxis n\u0026thinsp;=\u0026thinsp;6), (Treatment 1 n\u0026thinsp;=\u0026thinsp;6), and (Treatment 2 n\u0026thinsp;=\u0026thinsp;4). No significant difference was found between 4groups (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003cp\u003eBrains from each mouse tissues were stained with LFB to assess the extent of density of brain tissue. Histologic features were scored semi quantitatively as presented in the right side of Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e (control, n\u0026thinsp;=\u0026thinsp;4), (Prophylaxis, n\u0026thinsp;=\u0026thinsp;6), (Treatment 1, n\u0026thinsp;=\u0026thinsp;6), and (Treatment 2, n\u0026thinsp;=\u0026thinsp;8). The quantification of brain tissue density is shown in the left side of Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e. No significant difference was found between prophylaxis, treatment 2, and control groups (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). There was a significant difference in EAE incidence between treatment1 and control groups (P\u0026thinsp;=\u0026thinsp;0.03, *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of \u003c/strong\u003e\u003cstrong\u003eIFN- \u0026gamma; and IL-4 m RNA Expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere were no statistically significant differences between group means of IFN- \u0026gamma; and IL-4 as determined by one-way ANOVA (F (3, 19) =.90, p = .459) (F (3,19) =1.210, p = .333). \u0026nbsp;In prophylaxis and treatment 1 groups, the treatment induced a great up-regulation of IL-4, compared to the control group. The highest level of IL-4 was observed in the treatment 1 group (RQ=2.65). The IL-4 mRNA gene expression level was lowest in the treatment 2group (RQ= 0.92), also, different levels of mRNA gene expression were observed in the IFN- \u0026gamma;. The mRNA gene expression level of IFN- \u0026gamma; was at highest copies in the control (RQ=8.09) and T2 (RQ= 5.91) groups, whereas the mRNA gene expression of IFN- \u0026gamma; was lower in the prophylaxis (RQ=4.03) and treatment 1 groups (RQ=0.37) than the control group (RQ=8.09) (Figure 6).\u003c/p\u003e\n\u003cp\u003eRelative mRNA gene expression of genes was determined compared to the housekeeping gene, \u0026beta;2 microglobulin.\u003c/p\u003e"},{"header":"Discussion","content":" \u003cp\u003eImmunotherapy by helminth is considered as one of the effective therapeutic methods for treatment of multiple sclerosis and other autoimmune diseases. The evidence indicates that helminthic infections lead to the reduction of autoimmune paroxysm in animal models \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003e[20, 21]\u003c/span\u003e. In most studies, the prevalence of helminthic infections before deterioration of EAE not only plummeted EAE scores but also decreased the incidence rate in the infected animal groups \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003e[22\u0026ndash;24]\u003c/span\u003e. Many studies have been conducted so far to investigate the immune therapy of EAE (animal model of Multiple Sclerosis) by different helminth species such as \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSchistosoma mansoni, Trichinella spiralis, Fasciola hepatica, Trichinella psudospiralis, Taenia crassiceps, Strongyloides venezuelensis, Schistosoma japonicum, and Trichuris suis\u003c/span\u003e \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003e[18, 20, 21, 23, 25\u0026ndash;30]\u003c/span\u003e. Among all of these studies, F. chiuso-minicucci et al. (2011) showed that, the infection with \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eStrongyloides venezuelensis\u003c/span\u003e could not be effective in treatment of EAE. Yet, they emphasized that, their study alone cannot reject the hygiene hypothesis, and they recommended the conduction of more studies to determine the immune regulation role of other species of helminth [25].\u003c/p\u003e \u003cp\u003eIn this study, the effect of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eDicrocoelium\u003c/span\u003e ova immunization in EAE was evaluated for the first time. Clinical scores, IL4, and IFN-γ genes expression along with neuroglia cell counting and demyelination of brain cells in corpus callosum were evaluated and analyzed between the groups. The results illustrated that, pre-immunization of EAE in prophylaxis and treatment1 groups attenuated the scores, delayed onset day of EAE and reduced its incidence in the mentioned groups dramatically, as confirmed in the study by Swell et al. ( 2003 ) who investigated the treatment of EAE by \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eShistosoma mansoni\u003c/span\u003e so that, EAE helminthic therapy just could be effective in initial phase of EAE not in effective phase[18].\u003c/p\u003e \u003cp\u003eIn addition, EAE suppression was accompanied with a dramatic increase in the density in brain of mice in T1 group. Considering similar count of neuroglia cells in 4 groups, a dramatic increase in the density observed in treatment1 group is not associated with the number of myelin-producing cells. Merrill JE. et al (1992) found that, the demyelination reduction in spinal cords was related to the reduction of inflammatory leukocytes and cytokines \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003e[22]\u003c/span\u003e. Zhiliang Wu et al (2010) showed that, the administration of 200 larvae of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eTrichinella psudospiralis\u003c/span\u003e (15 days before induction) attenuated EAE score and delayed the incidence of EAE. Also, demyelination in spinal cord decreased ,and inflammatory brain cells reduced \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003e[28]\u003c/span\u003e. La Flamma et al ( 2003) indicated that, the reduction of demyelination in spinal cord of EAE mice was rooted in the plummeted inflammatory macrophage [21].\u003c/p\u003e \u003cp\u003eIn our study, the assessment of cytokines with Real Time PCR showed that, IFN- γ gene expression was lower in prophylaxis and treatment1 groups than the control and treatment2 groups. In contrast, IL-4 gene expression was more in prophylaxis and treatment1 groups than the control and treatment2 groups. So, slight down- regulation of IFN- γ along with up- regulation of IL-4 in T1 and P groups could not exclusively interpret significant preventable effect of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eDicrocoelium\u003c/span\u003e eggs immunization. Previous studies showed that, Th1 lymphocytes are the critical cells causing the EAE. However, recent studies have demonstrated that, Th17 cells are more responsible for autoimmune diseases especially in case of multiple sclerosis. Briefly, remission of EAE relatively but not completely depends on the existence of balance between Th1/ Th2 [31, 32]. Cross immunomodulation of Th1/Th2 in helminthic therapy is disputable yet [33]. Due to variable functions of cytokines, it is difficult to obtain a definite conclusion and interpretation of the condition [34]. The suppression of EAE has been demonstrated to result from a decrease in IL12 in helminth therapy with \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eShistosoma mansoni\u003c/span\u003e. Also, the reduction of IFN- γ and increase of IL-4 production was not significant in the treated groups ,indicating that EAE attenuation was related to the suppressed IL12 ,and shift of immune response toward Th2 cytokines was not responsible for EAE amelioration \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003e[21]\u003c/span\u003e. However, Swell et al proved that, EAE improvement is merely related to predominant Th2 cytokines\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003e[18]\u003c/span\u003e.In addition, the role of innate immune system [35] such as macrophages, B lymphocytes, regulator cells of immune system CD4 CD8 Tregs is effective regarding mediation of most of the protective mechanisms against EAE and MS. Besides, [36] Th17 is the most effective element in EAE induction explaining that, why the mechanisms of autoimmune diseases such as MS and EAE cannot be exclusively interpreted via Th1/Th2 responses [37, 38]. Therefore, it is suggested to evaluate Th17 and Tregs cytokine and inflammation as well as assessing the infiltration of immune cells in spinal cord in future studies, which could be considered as a limitation in the present study.\u003c/p\u003e "},{"header":"Conclusion","content":" \u003cp\u003eThe results of the current study showed that, the treatment with \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eDicrocoelium\u003c/span\u003e ova immunization attenuated EAE score and delayed the incidence of EAE especially in initial phase of diseases with down -regulation of IFN- γ and up -regulation of IL-4. \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eDicrocoelium\u003c/span\u003e egg did not have any effect on neuroglia cells number, so EAE amelioration alongside increase of density in the brain cells might be related to another factors. Hence, it can be concluded that, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eDicrocoelium\u003c/span\u003e eggs have the potential to be considered as useful therapy for treatment of MS in initial phase in future studies especially in human clinical trial studies.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Animal lab employees for feeding and watering of the mouse and checking the room temperature daily.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDesigned and performed experiments: E.M and M.M. Analyzed data and co-wrote the paper: A.F and N.M.A. Performed experiments: Z.N and M.J.R. Performed transporter experiments: M.R. Performed Statistical Analysis: M.M.B. Supervised the research: A.R.H and F.L.A. Histologic Analysis: S.S.N.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge the support provided from the Student Research Committee (grant # 970556) and the Grants-in-Aid for Scientific Research (Grant # 951683) from Mashhad University of Medical Sciences (MUMS), Mashhad, Iran.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest. The authors alone are responsible for the content and writing of the paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAnandan, C., et al., \u003cem\u003eIs the prevalence of asthma declining? Systematic review of epidemiological studies.\u003c/em\u003e Allergy, 2010. \u003cstrong\u003e65\u003c/strong\u003e(2): p. 152-167.\u003c/li\u003e\n\u003cli\u003eOkada, H., et al., \u003cem\u003eThe \u0026lsquo;hygiene hypothesis\u0026rsquo; for autoimmune and allergic diseases: an update.\u003c/em\u003e Clin. Exp. Immunol, 2010. \u003cstrong\u003e160\u003c/strong\u003e(1): p. 1-9.\u003c/li\u003e\n\u003cli\u003eBrooks, C., N. Pearce, and J. Douwes, \u003cem\u003eThe hygiene hypothesis in allergy and asthma: an update.\u003c/em\u003e Current opinion in allergy and clinical immunology, 2013. \u003cstrong\u003e13\u003c/strong\u003e(1): p. 70-77.\u003c/li\u003e\n\u003cli\u003eGreenwood, B., E.M. Herrick, and A. 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Farez, \u003cem\u003eAssociation between parasite infection and immune responses in multiple sclerosis.\u003c/em\u003e Ann Neurol, 2007. \u003cstrong\u003e61\u003c/strong\u003e(2): p. 97-108.\u003c/li\u003e\n\u003cli\u003eHofstetter, H.H., et al., \u003cem\u003eTherapeutic efficacy of IL-17 neutralization in murine experimental autoimmune encephalomyelitis.\u003c/em\u003e Cell Mol Immunol 2005. \u003cstrong\u003e237\u003c/strong\u003e(2): p. 123-130.\u003c/li\u003e\n\u003cli\u003eKomiyama, Y., et al., \u003cem\u003eIL-17 plays an important role in the development of experimental autoimmune encephalomyelitis.\u003c/em\u003e J. Immunol, 2006. \u003cstrong\u003e177\u003c/strong\u003e(1): p. 566-573.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Encephalomyelitis, Autoimmune, Experimental, Dicrocoelium, Th1-Th2 Balance, Multiple Sclerosis","lastPublishedDoi":"10.21203/rs.3.rs-17288/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-17288/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground Experimental Autoimmune Encephalomyelitis (EAE) is an animal model of Multiple Sclerosis (MS). This study was conducted to evaluate the efficacy of Dicrocoelium ova on Experimental Autoimmune Encephalomyelitis (EAE) treatment in C57BL6 mice.\u003c/p\u003e\u003cp\u003eMethods Twenty-eight C57BL/6 mice were assigned in four groups as control(C), prophylaxis (P), treatment1 (T1), and treatment2 (T2). Prior to induction of EAE in prophylaxis group and on days 7 and 18 in T1 and T2 groups, respectively, Dicrocoelium eggs were injected to each mouse. Clinical score, weight changes, and incidence time of EAE were recorded. IFN- γ and IL-4 assay and histopathological study by (H\u0026amp;E) and Toluidine-Blue (TB), and Luxol Fast Blue (LFB) were done. Data were analyzed using SPSS software version 21.\u003c/p\u003e\u003cp\u003eResults The disease score was significantly lower in P and T1 groups than the control group (p=0.01). IFN- γ was lower in P and T1 groups than the control group. The highest level of IL-4 was observed in the T1 group. The total number of Neuroglia cells of corpus callosum was similar in all groups, but density increased in T1 group compared to the control group (P = 0.03).\u003c/p\u003e\u003cp\u003eConclusions : The results of the study showed that, Dicrocoelium eggs have the great potential efficacy to stimulate immunomodulation toward treatment of EAE during the initial phase.\u003c/p\u003e","manuscriptTitle":"Helminthic therapy of experimental autoimmune encephalomyelitis by Dicrocoelium ova","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-03-16 15:23:17","doi":"10.21203/rs.3.rs-17288/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"50857784-9465-46c7-8d00-6d1c0bb23f74","owner":[],"postedDate":"March 16th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":70433,"name":"Immunology"},{"id":70434,"name":"Allergy \u0026 Immune Disorders"}],"tags":[],"updatedAt":"2020-04-06T13:36:25+00:00","versionOfRecord":[],"versionCreatedAt":"2020-03-16 15:23:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-17288","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-17288","identity":"rs-17288","version":["v1"]},"buildId":"ApUGefWb6u5IBVtyqm6d5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}