Cell Transplantation, Vol. 23, Supplement 1, pp. S113–S122, 2014 Printed in the USA. All rights reserved. Copyright Ó 2014 Cognizant Comm. Corp.
0963-6897/14 $90.00 + .00 DOI: http://dx.doi.org/10.3727/096368914X685005 E-ISSN 1555-3892 www.cognizantcommunication.com
The Potential of Human Umbilical Cord-Derived Mesenchymal Stem Cells as a Novel Cellular Therapy for Multiple Sclerosis Jin-Feng Li,*†1 Da-Jin Zhang,‡1 Tongchao Geng,§ Lin Chen,§ Hongyun Huang,¶ Hong-Lei Yin,* Yu-zhen Zhang,* Ji-Yu Lou,# Bingzhen Cao,† and Yun-Liang Wang*# *The Neurology Department of the 148th Hospital, Zibo, P. R. China †General Hospital of Jinan Military Region, Jinan, P. R. China ‡Medical Research Center of Naval General Hospital, Beijing, P. R. China §Second Affiliated Hospital of Tsinghua University, Beijing, P. R. China ¶Beijing Hongtianji Neuroscience Academy, Beijing, P. R. China #The Neurology Department of The Second Affiliated Hospital of Zhengzhou University, Zhengzhou, P. R. China
Multiple sclerosis (MS) is a complex disease of neurological disability, affecting more than 300 out of every 1 million people in the world. The purpose of the study was to evaluate the therapeutic effects of human umbilical cord-derived mesenchymal stem cell (hUC-MSC) transplantation in MS patients. Twenty-three patients were enrolled in this study, and 13 of them were given hUC-MSC therapy at the same time as antiinflammatory treatment, whereas the control patients received the anti-inflammatory treatment only. Treatment schedule included 1,000 mg/kg of methylprednisolone intravenously (IV) daily for 3 days and then 500 mg/kg for 2 days, followed by oral prednisone 1 mg/kg/day for 10 days. The dosage of prednisone was then reduced by 5 mg every 2 weeks until reaching a 5-mg/day maintenance dosage. Intravenous infusion of hUC-MSCs was applied three times in a 6-week period for each patient. The overall symptoms of the hUC-MSC-treated patients improved compared to patients in the control group. Both the EDSS scores and relapse occurrence were significantly lower than those of the control patients. Inflammatory cytokines were assessed, and the data demonstrated a shift from Th1 to Th2 immunity in hUC-MSC-treated patients. Our data demonstrated a high potential for hUC-MSC treatment of MS. This manuscript is published as part of the International Association of Neurorestoratology (IANR) special issue of Cell Transplantation. Key words: Multiple sclerosis (MS); Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs); Stem cell transplantation; Cytokine; Th1 immunity: Th2 immunity
INTRODUCTION Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) that may result in severe disability and neurological defects (20). The onset of MS is usually between the ages of 20 and 40 and is twice as common in females than in males (12). It is characterized by damage of myelin sheaths, the protective material around nerve fibers in brain and spinal cord, and therefore results in disruption of normal conduction of nerve impulses. It is commonly believed that MS is an autoimmune disease; however, there is also evidence pointing to the loss of oligodendrocytes for myelin generation as an underlying mechanism for MS (7,12,19,20). Four subtypes of MS have been classified: relapsing– remitting MS (RRMS), secondary progressive MS (SPMS),
primary progressive MS (PPMS), and progressive-relapsing MS (PRMS). No therapeutic method that has been developed for MS is a cure. Nonetheless, anti-inflammatory or immunosuppressive agents are commonly used as treatment options for symptom alleviation. These medications often have severe adverse effects and fail to prevent disease progression in many circumstances (6,15). Emerging data suggested that stem cells have great potential in regenerative medicine, and such cell-based therapy may provide an alternative approach to the currently approved MS treatments (5,10,11). Mesenchymal stem cells (MSCs) are adult stem cells typically found in the bone marrow and in other tissues including umbilical cord (UC), fetal liver, and adipose tissue. MSCs are considered to be multipotent
These authors provided equal contribution to this work. Received January 25, 2014; final acceptance October 31, 2014. Online prepub date: November 5, 2014. Address correspondence to Bingzhen Cao, General Hospital of Jinan Military Region, 25 Shifan Road, Jinan 250031, P. R. China. Tel: +86 533-6552100; Fax: +86 533-6552137; E-mail: [email protected] or Yun-Liang Wang, The Neurology Department of the 148th Hospital, 20 Zhanbei Road, Zibo 255300, P. R. China. Tel: +86 533-6552100; Fax: +86 533-6552137; E-mail: [email protected]
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and have anti-inflammatory as well as regenerative properties (3,5,10). MSCs derived from the human umbilical cord (hUC-MSCs) are more primitive and possess multiple advantages including ethical agreeableness, a lessinvasive procedure for isolation, low immunogenicity, high proliferation capacity, and multilineage differentiation capability (13,15). Several studies have used hUCMSCs to modulate immune responses in autoimmune diseases including rheumatoid arthritis, type 1 diabetes, and encephalomyelitis (8,13,14). Therefore, it is reasonable to hypothesize that hUC-MSCs may have a significant therapeutic effect on MS. We reported a study of 23 MS patients treated in our hospital from January 2010 to December 2012. Thirteen patients received anti-inflammatory and immunosuppressive reagents combined with hUC-MSC therapy, while the other 10 only received anti-inflammatory and immunosuppressive therapy. The efficacy and toxicity of hUC-MSC therapy in MS patients were evaluated, and its possible mechanisms were explored. MATERIALS AND METHODS Subjects The study was approved by the Ethics Committees of the 148th Hospital, and written informed consent was obtained from both hUC-MSC donors and MS patients. A randomized, two-armed study was designed to assess the effect of hUC-MSC therapy in addition to the conventional anti-inflammatory and immunosuppressive remedy. The inclusion criteria were set as 1) RRMS or SPMS with an average Expanded Disability Status Scale (EDSS) of 5.0 (range 4.0–8.0) at the time of enrollment and clinical deterioration at 1.0 point or higher on EDSS over the past 12 months; 2) males or nonpregnant females with an age range from 25 to 55; 3) had two or more attacks in the last 2 years with a disease course of more than 2 years; 4) characteristic abnormalities on magnetic resonance imaging (MRI) scan. Patients were excluded from the study if they 1) were treated with cytotoxic medications (i.e., cyclophosphamide, mitoxantrone, and azathioprine) in the past 3 months before the trial; 2) had significant cardiac, renal, or hepatic failure or any other severe diseases that may interfere with result interpretation; 3) had an active infection; 4) showed severe cognitive decline; and 5) were unable to understand and sign the informed consent. The patients did not pay for their inclusion or treatment in this study. hUC-MSC Isolation, Expansion, and Characterization hUC-MSCs were isolated from human UCs and cultured as previously described (13). Fresh, smooth, flat, relatively thin, hUCs of uniform thickness were obtained from healthy donors (both genders) in our hospital after normal deliveries and were processed immediately. The
UCs were washed by phosphate-buffered saline (PBS; Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China), and blood vessels were removed. The remaining tissue was sectioned to approximately 2-mm pieces and transferred to a 25-cm2 tissue culture flask (Dongge Biotechnology Co., LTD, Beijing, China) containing Ham’s F12 media (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Biochrom AG, Berlin, Germany). Tissue was incubated at 37°C with 5% CO2 in a humidified chamber. The cultured cells were harvested within two to three passages and assessed for lineage markers via flow cytometry. Briefly, cells were fixed in neutralized 2% paraformaldehyde (PFA; Dongge Biotechnology Co., Ltd.) solution for 30 min. Fixed cells were washed twice with PBS and incubated for 30 min with fluorescein isothiocyanate (FITC) or phycoerythrin (PE)conjugated antibodies against cluster of differentiation 44 (CD44), CD29, CD105, CD31, CD45, and human leukocyte antigen (HLA)-DR (Abcam, Cambridge, UK; antibody concentration in accordance with the instructions) before being analyzed by flow cytometry (BD, Franklin Lakes, NJ, USA). Microorganisms or endotoxin in hUC-MSCs were tested in the clinical laboratory of our hospital. In order to detect whether there was bacterial contamination, the hUC-MSC medium was cultured overnight on nutrient agar plates (Haibo Biological Technology Co. Ltd., Shanghai, China). The chromogenic end-point tachypleus amebocyte lysate (CE-TAL) assay (Ruicheng Biotechnology Co., Ltd, Shanghai, China) was used to detect cytotoxins. Treatment Baseline data were collected from each patient prior to any treatment, including serological samples. At acute relapsing phase, all patients were given anti-inflammatory and immunomodulation reagent methylprednisolone (Jin-yao Pharmaceutical Co., Ltd, Tianjin, China) intravenously 1,000 mg/kg daily for 3 days, 500 mg/kg for 2 days, followed by oral prednisone (Xian Ju Pharmaceutical Co., Ltd, Zhejiang, China) 1 mg/kg/day for 10 days. Subsequent dosage of prednisone was reduced by 5 mg every 2 weeks until reaching a 5-mg/day maintenance dosage. Serum g-globulin (KangBao Biological Products Co., Ltd, Shanxi, China) was simultaneously supplemented at 400 mg/kg/ day for 5 days. Thirteen patients were randomly selected for hUC-MSC therapy (intravenous infusion, hUC-MSCs suspended in 100 ml normal saline) at a dosage of 4 × 106 cells/kg once every 2 weeks for three consecutive times. The control group did not receive a saline infusion. All patients continued the maintenance dosage of anti-inflammatory and immunosuppressive therapy during the follow-up stage. Safety surveillance included physical examination, adverse event documentation, and quarterly brain MRI (Siemens, Munich, Germany).
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Figure 1. Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) and evaluation of their membrane proteins by flow cytometry with CellQuest software. (A) Morphology of hUC-MSCs in culture. Scale bar: 100 μm. (B): Evaluation of hUC-MSC membrane proteins by flow cytometry with CellQuest software. Acquisitions are shown in histograms of flow cytometry; the number of acquired events was 20,000 in each acquisition. The hUC-MSCs were positive for CD29 (94.20 ± 1.56%), CD44 (95.63 ± 1.23%), and CD105 (90.03 ± 0.64%), and were negative for CD31 (6.89 ± 0.09%), CD45 (5.07 ± 0.068%), and HLA-DR (0.33 ± 0.04%). IgG1-FITC and IgG1-PE antibodies were utilized as isotype controls. FSC, forward scatter; SSC, side scatter.
Endpoint Evaluation The EDSS was used to assess the status of neurological function. The scores range from 0 to 10, representing normal to death, respectively (2). Patient scores ranged from 5 to 8 at enrollment, indicating impairment of daily activities and degrees of disability ranging from inability to walk longer than 200 m to restriction to a wheelchair. Scheduled clinical evaluations were performed before
hUC-MSC treatment (baseline) and at 1, 2, 3, 6, 9, and 12 months after treatment. Two neurologists who had no knowledge of the study evaluated every case independently to minimize bias. Detection of Cytokines With ELISA Serum of every patient at enrollment and 1, 2, 3, 6, 9, and 12 months posttreatment was collected. Cytokines
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hepatocyte growth factor (HGF), interleukin-4 (IL-4), IL-10, interferon-g (IFN-g), IL-17, and tumor necrosis factor-a (TNF-a) were detected with ELISA kits (R&D System, Minneapolis, MN, USA) according to the manufacturer’s instructions. Statistical Analysis Data analysis was performed by SPSS v19.0 software (IBM, Armonk, NY, USA). Normality of the data was confirmed by Kolmogorov–Smirnov and Shapiro–Wilk tests. In order to compare data at different time points, chi-square test and analysis of covariance were applied. Mann–Whitney and Wilcoxon tests were used when a nonnormal distribution of data was determined. Statistical significance in serum targets between the control and experimental group at every time point were assessed by Student’s t test. The relapse occurrences of patients in both control and experimental group in a 1-year period were assessed by the Student’s t test (and confirmed by Mann–Whitney test). The data of ELISA results and relapse occurrences were expressed as mean ± standard deviation (SD). It was considered to be statistically significant when the value of p
0963-6897/14 $90.00 + .00 DOI: http://dx.doi.org/10.3727/096368914X685005 E-ISSN 1555-3892 www.cognizantcommunication.com
The Potential of Human Umbilical Cord-Derived Mesenchymal Stem Cells as a Novel Cellular Therapy for Multiple Sclerosis Jin-Feng Li,*†1 Da-Jin Zhang,‡1 Tongchao Geng,§ Lin Chen,§ Hongyun Huang,¶ Hong-Lei Yin,* Yu-zhen Zhang,* Ji-Yu Lou,# Bingzhen Cao,† and Yun-Liang Wang*# *The Neurology Department of the 148th Hospital, Zibo, P. R. China †General Hospital of Jinan Military Region, Jinan, P. R. China ‡Medical Research Center of Naval General Hospital, Beijing, P. R. China §Second Affiliated Hospital of Tsinghua University, Beijing, P. R. China ¶Beijing Hongtianji Neuroscience Academy, Beijing, P. R. China #The Neurology Department of The Second Affiliated Hospital of Zhengzhou University, Zhengzhou, P. R. China
Multiple sclerosis (MS) is a complex disease of neurological disability, affecting more than 300 out of every 1 million people in the world. The purpose of the study was to evaluate the therapeutic effects of human umbilical cord-derived mesenchymal stem cell (hUC-MSC) transplantation in MS patients. Twenty-three patients were enrolled in this study, and 13 of them were given hUC-MSC therapy at the same time as antiinflammatory treatment, whereas the control patients received the anti-inflammatory treatment only. Treatment schedule included 1,000 mg/kg of methylprednisolone intravenously (IV) daily for 3 days and then 500 mg/kg for 2 days, followed by oral prednisone 1 mg/kg/day for 10 days. The dosage of prednisone was then reduced by 5 mg every 2 weeks until reaching a 5-mg/day maintenance dosage. Intravenous infusion of hUC-MSCs was applied three times in a 6-week period for each patient. The overall symptoms of the hUC-MSC-treated patients improved compared to patients in the control group. Both the EDSS scores and relapse occurrence were significantly lower than those of the control patients. Inflammatory cytokines were assessed, and the data demonstrated a shift from Th1 to Th2 immunity in hUC-MSC-treated patients. Our data demonstrated a high potential for hUC-MSC treatment of MS. This manuscript is published as part of the International Association of Neurorestoratology (IANR) special issue of Cell Transplantation. Key words: Multiple sclerosis (MS); Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs); Stem cell transplantation; Cytokine; Th1 immunity: Th2 immunity
INTRODUCTION Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) that may result in severe disability and neurological defects (20). The onset of MS is usually between the ages of 20 and 40 and is twice as common in females than in males (12). It is characterized by damage of myelin sheaths, the protective material around nerve fibers in brain and spinal cord, and therefore results in disruption of normal conduction of nerve impulses. It is commonly believed that MS is an autoimmune disease; however, there is also evidence pointing to the loss of oligodendrocytes for myelin generation as an underlying mechanism for MS (7,12,19,20). Four subtypes of MS have been classified: relapsing– remitting MS (RRMS), secondary progressive MS (SPMS),
primary progressive MS (PPMS), and progressive-relapsing MS (PRMS). No therapeutic method that has been developed for MS is a cure. Nonetheless, anti-inflammatory or immunosuppressive agents are commonly used as treatment options for symptom alleviation. These medications often have severe adverse effects and fail to prevent disease progression in many circumstances (6,15). Emerging data suggested that stem cells have great potential in regenerative medicine, and such cell-based therapy may provide an alternative approach to the currently approved MS treatments (5,10,11). Mesenchymal stem cells (MSCs) are adult stem cells typically found in the bone marrow and in other tissues including umbilical cord (UC), fetal liver, and adipose tissue. MSCs are considered to be multipotent
These authors provided equal contribution to this work. Received January 25, 2014; final acceptance October 31, 2014. Online prepub date: November 5, 2014. Address correspondence to Bingzhen Cao, General Hospital of Jinan Military Region, 25 Shifan Road, Jinan 250031, P. R. China. Tel: +86 533-6552100; Fax: +86 533-6552137; E-mail: [email protected] or Yun-Liang Wang, The Neurology Department of the 148th Hospital, 20 Zhanbei Road, Zibo 255300, P. R. China. Tel: +86 533-6552100; Fax: +86 533-6552137; E-mail: [email protected]
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and have anti-inflammatory as well as regenerative properties (3,5,10). MSCs derived from the human umbilical cord (hUC-MSCs) are more primitive and possess multiple advantages including ethical agreeableness, a lessinvasive procedure for isolation, low immunogenicity, high proliferation capacity, and multilineage differentiation capability (13,15). Several studies have used hUCMSCs to modulate immune responses in autoimmune diseases including rheumatoid arthritis, type 1 diabetes, and encephalomyelitis (8,13,14). Therefore, it is reasonable to hypothesize that hUC-MSCs may have a significant therapeutic effect on MS. We reported a study of 23 MS patients treated in our hospital from January 2010 to December 2012. Thirteen patients received anti-inflammatory and immunosuppressive reagents combined with hUC-MSC therapy, while the other 10 only received anti-inflammatory and immunosuppressive therapy. The efficacy and toxicity of hUC-MSC therapy in MS patients were evaluated, and its possible mechanisms were explored. MATERIALS AND METHODS Subjects The study was approved by the Ethics Committees of the 148th Hospital, and written informed consent was obtained from both hUC-MSC donors and MS patients. A randomized, two-armed study was designed to assess the effect of hUC-MSC therapy in addition to the conventional anti-inflammatory and immunosuppressive remedy. The inclusion criteria were set as 1) RRMS or SPMS with an average Expanded Disability Status Scale (EDSS) of 5.0 (range 4.0–8.0) at the time of enrollment and clinical deterioration at 1.0 point or higher on EDSS over the past 12 months; 2) males or nonpregnant females with an age range from 25 to 55; 3) had two or more attacks in the last 2 years with a disease course of more than 2 years; 4) characteristic abnormalities on magnetic resonance imaging (MRI) scan. Patients were excluded from the study if they 1) were treated with cytotoxic medications (i.e., cyclophosphamide, mitoxantrone, and azathioprine) in the past 3 months before the trial; 2) had significant cardiac, renal, or hepatic failure or any other severe diseases that may interfere with result interpretation; 3) had an active infection; 4) showed severe cognitive decline; and 5) were unable to understand and sign the informed consent. The patients did not pay for their inclusion or treatment in this study. hUC-MSC Isolation, Expansion, and Characterization hUC-MSCs were isolated from human UCs and cultured as previously described (13). Fresh, smooth, flat, relatively thin, hUCs of uniform thickness were obtained from healthy donors (both genders) in our hospital after normal deliveries and were processed immediately. The
UCs were washed by phosphate-buffered saline (PBS; Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China), and blood vessels were removed. The remaining tissue was sectioned to approximately 2-mm pieces and transferred to a 25-cm2 tissue culture flask (Dongge Biotechnology Co., LTD, Beijing, China) containing Ham’s F12 media (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Biochrom AG, Berlin, Germany). Tissue was incubated at 37°C with 5% CO2 in a humidified chamber. The cultured cells were harvested within two to three passages and assessed for lineage markers via flow cytometry. Briefly, cells were fixed in neutralized 2% paraformaldehyde (PFA; Dongge Biotechnology Co., Ltd.) solution for 30 min. Fixed cells were washed twice with PBS and incubated for 30 min with fluorescein isothiocyanate (FITC) or phycoerythrin (PE)conjugated antibodies against cluster of differentiation 44 (CD44), CD29, CD105, CD31, CD45, and human leukocyte antigen (HLA)-DR (Abcam, Cambridge, UK; antibody concentration in accordance with the instructions) before being analyzed by flow cytometry (BD, Franklin Lakes, NJ, USA). Microorganisms or endotoxin in hUC-MSCs were tested in the clinical laboratory of our hospital. In order to detect whether there was bacterial contamination, the hUC-MSC medium was cultured overnight on nutrient agar plates (Haibo Biological Technology Co. Ltd., Shanghai, China). The chromogenic end-point tachypleus amebocyte lysate (CE-TAL) assay (Ruicheng Biotechnology Co., Ltd, Shanghai, China) was used to detect cytotoxins. Treatment Baseline data were collected from each patient prior to any treatment, including serological samples. At acute relapsing phase, all patients were given anti-inflammatory and immunomodulation reagent methylprednisolone (Jin-yao Pharmaceutical Co., Ltd, Tianjin, China) intravenously 1,000 mg/kg daily for 3 days, 500 mg/kg for 2 days, followed by oral prednisone (Xian Ju Pharmaceutical Co., Ltd, Zhejiang, China) 1 mg/kg/day for 10 days. Subsequent dosage of prednisone was reduced by 5 mg every 2 weeks until reaching a 5-mg/day maintenance dosage. Serum g-globulin (KangBao Biological Products Co., Ltd, Shanxi, China) was simultaneously supplemented at 400 mg/kg/ day for 5 days. Thirteen patients were randomly selected for hUC-MSC therapy (intravenous infusion, hUC-MSCs suspended in 100 ml normal saline) at a dosage of 4 × 106 cells/kg once every 2 weeks for three consecutive times. The control group did not receive a saline infusion. All patients continued the maintenance dosage of anti-inflammatory and immunosuppressive therapy during the follow-up stage. Safety surveillance included physical examination, adverse event documentation, and quarterly brain MRI (Siemens, Munich, Germany).
hUC-MSC THERAPY FOR MULTIPLE SCLEROSIS
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Figure 1. Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) and evaluation of their membrane proteins by flow cytometry with CellQuest software. (A) Morphology of hUC-MSCs in culture. Scale bar: 100 μm. (B): Evaluation of hUC-MSC membrane proteins by flow cytometry with CellQuest software. Acquisitions are shown in histograms of flow cytometry; the number of acquired events was 20,000 in each acquisition. The hUC-MSCs were positive for CD29 (94.20 ± 1.56%), CD44 (95.63 ± 1.23%), and CD105 (90.03 ± 0.64%), and were negative for CD31 (6.89 ± 0.09%), CD45 (5.07 ± 0.068%), and HLA-DR (0.33 ± 0.04%). IgG1-FITC and IgG1-PE antibodies were utilized as isotype controls. FSC, forward scatter; SSC, side scatter.
Endpoint Evaluation The EDSS was used to assess the status of neurological function. The scores range from 0 to 10, representing normal to death, respectively (2). Patient scores ranged from 5 to 8 at enrollment, indicating impairment of daily activities and degrees of disability ranging from inability to walk longer than 200 m to restriction to a wheelchair. Scheduled clinical evaluations were performed before
hUC-MSC treatment (baseline) and at 1, 2, 3, 6, 9, and 12 months after treatment. Two neurologists who had no knowledge of the study evaluated every case independently to minimize bias. Detection of Cytokines With ELISA Serum of every patient at enrollment and 1, 2, 3, 6, 9, and 12 months posttreatment was collected. Cytokines
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hepatocyte growth factor (HGF), interleukin-4 (IL-4), IL-10, interferon-g (IFN-g), IL-17, and tumor necrosis factor-a (TNF-a) were detected with ELISA kits (R&D System, Minneapolis, MN, USA) according to the manufacturer’s instructions. Statistical Analysis Data analysis was performed by SPSS v19.0 software (IBM, Armonk, NY, USA). Normality of the data was confirmed by Kolmogorov–Smirnov and Shapiro–Wilk tests. In order to compare data at different time points, chi-square test and analysis of covariance were applied. Mann–Whitney and Wilcoxon tests were used when a nonnormal distribution of data was determined. Statistical significance in serum targets between the control and experimental group at every time point were assessed by Student’s t test. The relapse occurrences of patients in both control and experimental group in a 1-year period were assessed by the Student’s t test (and confirmed by Mann–Whitney test). The data of ELISA results and relapse occurrences were expressed as mean ± standard deviation (SD). It was considered to be statistically significant when the value of p
