1.Efficacy of vitamin A adjuvant therapy on bronchial asthma in children and its influence on serum transforming growth factor-β1, eosinophils and interleukin-17 levels
Honghong HOU ; Yan SUN ; Jianli PAN ; Zhijuan LUO ; Shasha LI ; Ying LIU
Clinical Medicine of China 2025;41(1):20-26
Objective:To explore the clinical effect of vitamin A in the adjuvant treatment of bronchial asthma in children and its influence on serum transforming growth factor-β1 (TGF-β1), eosinophils (EOS) and interleukin-17 (IL-17) levels.Methods:A prospective study was conducted on 110 children with bronchial asthma who received treatment in Department of Pediatrics, Xi'an Central Hospital from January 2022 to December 2023. Based on the principle of balanced and comparable baseline characteristics between groups, they were randomly divided into a control group and an observation group, with 55 cases in each group, using a random number table method. The control group was treated with routine pediatric bronchial asthma therapy, while the observation group was added with vitamin A adjuvant therapy on the basis of the control group. After 15 days of continuous treatment, the scores of asthma control condition (Childhood-Asthma Control Test (C-ACT), Asthma Control Questionnaire (ACQ)) in the two groups were evaluated. The pulmonary ventilation function (forced expiratory volume in one second (FEV 1), forced expiratory volume in one second/forced vital capacity (FEV 1/FVC), peak expiratory flow (PEF)), serum inflammatory factors (TGF-β1, EOS, IL-17) and immune function indicators (T helper 17 cell (Th17), T helper 2 cell (Th2), regulatory T cell (Treg) ) were compared between groups of children before treatment and after 15 days of treatment. Measurement data with normal or approximate distribution were expressed as xˉ± s, and independent sample t test was used for comparison between groups. Enumeration data were expressed as percentage, and chi-square test was adopted for between-group comparison. Results:After 15 days of treatment, the C-ACT score with (16.20±3.14) points in observation group was higher than (14.80±2.62) points in control group while the ACQ score with (30.30±4.14) points was lower than (34.60±6.23) points in control group, with statistical differences between groups (t values were 2.54 and 4.26; P values were 0.012 and <0.001). The pulmonary ventilation function indicators in observation group and control group after 15 days of treatment (FEV 1: (1.76±0.34) L与(1.54±0.32) L, FEV 1/FVC:(76.89±5.76)%与(70.25±6.42)%, PEF(2.89±0.35) L/s与(2.68±0.39) L/s) were higher than those before treatment (FEV 1:(1.12±0.31) L与(1.20±0.33) L, FEV 1/FVC:(56.96±4.35)%与(58.12±3.48)%, PEF(2.15±0.66) L/s与(2.34±0.56) L/s), and the differences were statistically significant ( t values were 10.32, 5.49, 20.48, 10.43, 7.35, 3.70, respectively; all P<0.001), and the indicators in observation group were higher compared to control group, the differences were statistically significant ( t values were 3.49, 5.71, and 2.97; P values were 0.001, <0.001, and 0.004). After 15 days of treatment, the levels of serum inflammatory factors (TGF-β1:(6.32±1.36) ng/L与(8.75±1.81) ng/L, EOS:(3.56±0.65)%与(4.28±0.82)%, IL-17:(5.53±1.22) ng/L与(6.42±1.51) ng/L) and CD4 + T lymphocyte immune function indicators (Th17:(0.97±0.26) ng/L与(1.23±0.35) ng/L, Th2:(2.32±0.64) ng/L与(3.15±0.52) ng/L, Treg:(5.41±0.76) ng/L与(5.86±0.23) ng/L ) were lower in observation group and control group than those before treatment (TGF-β1: (14.35±2.23)与(15.26±3.05) ng/L, EOS: (6.32±1.33)%与(6.41±1.27)%, IL-17:(8.86±1.68)与(9.03±1.89) ng/L, Th17:(1.82±0.75)与(1.67±0.68) ng/L, Th2:(4.15±1.49)与(3.98±1.28) ng/L, Treg: (7.26±1.35)与(6.92±1.72) ng/L), and the differences were statistically significant ( t values were 22.80, 13.61, 13.83, 10.45, 11.90, 8.08, 7.94, 4.27, 8.37, 4.46, 8.86, 4.58, respectively; all P<0.001). However, the above indicators in the observation group were lower than those in the control group ( t values were 7.96, 5.10, 3.40, 4.42, 7.47, 4.20, and P-values were <0.001, <0.001, 0.001, <0.001, <0.001, and <0.001 respectively). Conclusion:Vitamin A adjuvant therapy is helpful to the control of bronchial asthma, and it can effectively improve the pulmonary function, reduce the inflammatory reaction and enhance the body's immunity.
2.Effect of tolerogenic dendritic cells on autophagy of synovial cells in collagen-induced arthritis rats
Honghong LI ; Xiufang WAN ; Rui YUAN ; Tiaoyu LONG ; Yan ZHOU ; Hongmei JIANG
Chinese Journal of Immunology 2025;41(9):2067-2073,2080
Objective:To investigate the effect of tolerogenic dendritic cells(tolDC)on autophagy of synovial cells in collagen-induced arthritis(CIA)rats.Methods:Bone marrow mononuclear cells of rats were extracted and induced into tolDC using IL-4,GM-CSF and NF-κB oligonucleotide decoy,and loaded with BⅡC to become BⅡC-tolDC.SD rats were randomly divided into normal control group,CIA model group and BⅡC-tolDC intervention group,with 3 rats in each group.Normal female SD rats were immunized with bovine type Ⅱ collagen solution to construct CIA model.Rats in BⅡC-tolDC intervention group were infused with BⅡC-tolDC via tail vein on the 21st day after initial immunization for two weeks,arthritis indexes were recorded weekly.On the 35th day,the rats were sacrificed,and synovial histopathology of ankle joint of rats in each group were observed by HE staining;the number of osteo-clasts in cartilage of rats in each group were observed by TRAP staining.The number of autophagic of ankle synovial cells of rats in each group were observed by transmission electron microscopy.Levels of serum TNF-α and IL-1β of rats in each group were detected by ELISA.LC3,Beclin-1 and ATG5 proteins of synovial cells of ankle joints of rats in each group were detected by Immunohistochemical staining.Results:CIA rats were constructed successfully by immunization with bovine type Ⅱ collagen.BⅡC-tolDC intervention re-duced the arthritis index of CIA rats,inhibited synovial inflammation and abnormal proliferation of synovial tissue,improved joint bone and cartilage injury,and reduced the number of osteoclasts in cartilage tissue and the number of autophagosomes in synovial cells.At the same time,reduced levels of serum TNF-α,IL-1β,and protein expressions of LC3,Beclin-1 and ATG5 of synovial cell of CIA rats.Conclusion:BⅡC-tolDC may alleviate arthritis lesions of CIA rats by inhibiting synovial cell autophagy of CIA rats.
3.Effect of tolerogenic dendritic cells on autophagy of synovial cells in collagen-induced arthritis rats
Honghong LI ; Xiufang WAN ; Rui YUAN ; Tiaoyu LONG ; Yan ZHOU ; Hongmei JIANG
Chinese Journal of Immunology 2025;41(9):2067-2073,2080
Objective:To investigate the effect of tolerogenic dendritic cells(tolDC)on autophagy of synovial cells in collagen-induced arthritis(CIA)rats.Methods:Bone marrow mononuclear cells of rats were extracted and induced into tolDC using IL-4,GM-CSF and NF-κB oligonucleotide decoy,and loaded with BⅡC to become BⅡC-tolDC.SD rats were randomly divided into normal control group,CIA model group and BⅡC-tolDC intervention group,with 3 rats in each group.Normal female SD rats were immunized with bovine type Ⅱ collagen solution to construct CIA model.Rats in BⅡC-tolDC intervention group were infused with BⅡC-tolDC via tail vein on the 21st day after initial immunization for two weeks,arthritis indexes were recorded weekly.On the 35th day,the rats were sacrificed,and synovial histopathology of ankle joint of rats in each group were observed by HE staining;the number of osteo-clasts in cartilage of rats in each group were observed by TRAP staining.The number of autophagic of ankle synovial cells of rats in each group were observed by transmission electron microscopy.Levels of serum TNF-α and IL-1β of rats in each group were detected by ELISA.LC3,Beclin-1 and ATG5 proteins of synovial cells of ankle joints of rats in each group were detected by Immunohistochemical staining.Results:CIA rats were constructed successfully by immunization with bovine type Ⅱ collagen.BⅡC-tolDC intervention re-duced the arthritis index of CIA rats,inhibited synovial inflammation and abnormal proliferation of synovial tissue,improved joint bone and cartilage injury,and reduced the number of osteoclasts in cartilage tissue and the number of autophagosomes in synovial cells.At the same time,reduced levels of serum TNF-α,IL-1β,and protein expressions of LC3,Beclin-1 and ATG5 of synovial cell of CIA rats.Conclusion:BⅡC-tolDC may alleviate arthritis lesions of CIA rats by inhibiting synovial cell autophagy of CIA rats.
4.Efficacy of vitamin A adjuvant therapy on bronchial asthma in children and its influence on serum transforming growth factor-β1, eosinophils and interleukin-17 levels
Honghong HOU ; Yan SUN ; Jianli PAN ; Zhijuan LUO ; Shasha LI ; Ying LIU
Clinical Medicine of China 2025;41(1):20-26
Objective:To explore the clinical effect of vitamin A in the adjuvant treatment of bronchial asthma in children and its influence on serum transforming growth factor-β1 (TGF-β1), eosinophils (EOS) and interleukin-17 (IL-17) levels.Methods:A prospective study was conducted on 110 children with bronchial asthma who received treatment in Department of Pediatrics, Xi'an Central Hospital from January 2022 to December 2023. Based on the principle of balanced and comparable baseline characteristics between groups, they were randomly divided into a control group and an observation group, with 55 cases in each group, using a random number table method. The control group was treated with routine pediatric bronchial asthma therapy, while the observation group was added with vitamin A adjuvant therapy on the basis of the control group. After 15 days of continuous treatment, the scores of asthma control condition (Childhood-Asthma Control Test (C-ACT), Asthma Control Questionnaire (ACQ)) in the two groups were evaluated. The pulmonary ventilation function (forced expiratory volume in one second (FEV 1), forced expiratory volume in one second/forced vital capacity (FEV 1/FVC), peak expiratory flow (PEF)), serum inflammatory factors (TGF-β1, EOS, IL-17) and immune function indicators (T helper 17 cell (Th17), T helper 2 cell (Th2), regulatory T cell (Treg) ) were compared between groups of children before treatment and after 15 days of treatment. Measurement data with normal or approximate distribution were expressed as xˉ± s, and independent sample t test was used for comparison between groups. Enumeration data were expressed as percentage, and chi-square test was adopted for between-group comparison. Results:After 15 days of treatment, the C-ACT score with (16.20±3.14) points in observation group was higher than (14.80±2.62) points in control group while the ACQ score with (30.30±4.14) points was lower than (34.60±6.23) points in control group, with statistical differences between groups (t values were 2.54 and 4.26; P values were 0.012 and <0.001). The pulmonary ventilation function indicators in observation group and control group after 15 days of treatment (FEV 1: (1.76±0.34) L与(1.54±0.32) L, FEV 1/FVC:(76.89±5.76)%与(70.25±6.42)%, PEF(2.89±0.35) L/s与(2.68±0.39) L/s) were higher than those before treatment (FEV 1:(1.12±0.31) L与(1.20±0.33) L, FEV 1/FVC:(56.96±4.35)%与(58.12±3.48)%, PEF(2.15±0.66) L/s与(2.34±0.56) L/s), and the differences were statistically significant ( t values were 10.32, 5.49, 20.48, 10.43, 7.35, 3.70, respectively; all P<0.001), and the indicators in observation group were higher compared to control group, the differences were statistically significant ( t values were 3.49, 5.71, and 2.97; P values were 0.001, <0.001, and 0.004). After 15 days of treatment, the levels of serum inflammatory factors (TGF-β1:(6.32±1.36) ng/L与(8.75±1.81) ng/L, EOS:(3.56±0.65)%与(4.28±0.82)%, IL-17:(5.53±1.22) ng/L与(6.42±1.51) ng/L) and CD4 + T lymphocyte immune function indicators (Th17:(0.97±0.26) ng/L与(1.23±0.35) ng/L, Th2:(2.32±0.64) ng/L与(3.15±0.52) ng/L, Treg:(5.41±0.76) ng/L与(5.86±0.23) ng/L ) were lower in observation group and control group than those before treatment (TGF-β1: (14.35±2.23)与(15.26±3.05) ng/L, EOS: (6.32±1.33)%与(6.41±1.27)%, IL-17:(8.86±1.68)与(9.03±1.89) ng/L, Th17:(1.82±0.75)与(1.67±0.68) ng/L, Th2:(4.15±1.49)与(3.98±1.28) ng/L, Treg: (7.26±1.35)与(6.92±1.72) ng/L), and the differences were statistically significant ( t values were 22.80, 13.61, 13.83, 10.45, 11.90, 8.08, 7.94, 4.27, 8.37, 4.46, 8.86, 4.58, respectively; all P<0.001). However, the above indicators in the observation group were lower than those in the control group ( t values were 7.96, 5.10, 3.40, 4.42, 7.47, 4.20, and P-values were <0.001, <0.001, 0.001, <0.001, <0.001, and <0.001 respectively). Conclusion:Vitamin A adjuvant therapy is helpful to the control of bronchial asthma, and it can effectively improve the pulmonary function, reduce the inflammatory reaction and enhance the body's immunity.
5.Application of nursing cooperation method in pulmonary nodule localization using indocyanine green combined with methylene blue
Xiaojing ZHENG ; Bin GAN ; Zihong XIAO ; Minmin HUANG ; Honghong YAN ; Xiumei CHEN
Journal of Interventional Radiology 2024;33(8):898-901
Objective To discuss the nursing cooperation method in CT-guided pulmonary nodule localization using indocyanine green combined with methylene blue.Methods A total of 81 patients,who needed to receive pulmonary nodule localization before thoracoscopic resection,were enrolled in this study.The nursing cooperation measures,including the preparation and injection of indocyanine green combined with methylene blue,breathing mode coordination,position management,precise localization coordination,and psychological care,were analyzed.Results Successful nodule localization was accomplished in all patients.No statistically significant difference in the incidence of adverse events existed between the patients with different clinical characteristics(P>0.05).The gender,age,lesion site,nodule size,nodule-pleura distance,posture during puncturing and needle path route were not the factors influencing the occurrence of adverse reactions in patients receiving CT-guided pulmonary nodule localization using indocyanine green combined with methylene blue.Conclusion In performing CT-guided pulmonary nodule localization using indocyanine green combined with methylene blue,the nursing cooperation method carries generality and stability,which is suitable for patients with different clinical characteristics.
6.Correlation between serum HbA1c,LAG-3 and thyroid nodules in patients with type 2 diabetes mellitus
Xiaoyue YANG ; Honghong SONG ; Shaozhen HU ; Ying PAN ; Xiaoxue BAO ; Wenying YAN
International Journal of Laboratory Medicine 2024;45(2):160-164
Objective To explore the correlation between serum glycosylated hemoglobin(HbA1c),lym-phocyte activation gene-3(LAG-3)and thyroid nodules in patients with type 2 diabetes mellitus(T2DM).Methods A total of 120 T2DM patients with thyroid nodules admitted to the Third Hospital of Hebei Medi-cal University from July 2021 to July 2022 were included as the study group,and 100 simple T2DM patients(without thyroid nodules)were included as the control group during the same period.According to the patho-logical examination results of thyroid nodules,the study group was grouped into a benign nodule group(85 ca-ses)and a malignant nodule group(35 cases).Enzyme linked immunosorbent assay was applied to detect the serum LAG-3 level of all study subjects.Fully automated glycated hemoglobin analyzer was applied to detect HbA1c level in all study subjects.Spearman method was applied to analyze the correlation between serum HbA1c,LAG-3,and thyroid imaging report and data system(TI-RADS)scores in patients with T2DM and thyroid nodules.Multivariate Logistic regression was applied to analyze the influencing factors of T2DM with thyroid nodules.Receiver operating characteristic(ROC)curve was applied to analyze the diagnostic value of HbA1c and LAG-3 levels in T2DM with thyroid nodules.Results Compared with the control group,the level of HbA1c in the study group was obviously higher(P<0.05),while the level of LAG-3 was obviously lower(P<0.05).Compared with the benign nodule group,the serum LAG-3 level in the malignant nodule group was obviously lower(P<0.05),while the level of LAG-3 was obviously lower(P<0.05).Spearman analysis showed a positive correlation between HbA1c level and TI-RADS score in T2DM patients with thyroid nod-ules(r=0.378,P<0.001),while the serum LAG-3 level was negatively correlated with TI-RADS score(r=-0.472,P<0.001).The results of multivariate Logistic regression analysis showed that HbA1c was a risk factor for the occurrence of thyroid nodules in T2DM patients(P<0.05),and LAG-3 was a protective factor for the occurrence of thyroid nodules in T2DM patients(P<0.05).The combination of HbA1c and LAG-3 in the diagnosis of T2DM with thyroid nodules was superior to their individual diagnosis(Zcombination-HbA1c=2.542,P=0.011;Zcombination-LAG-3=3.098,P=0.002).Conclusion Patients with T2DM combined with thyroid nodules have obviously lower serum LAG-3 level and higher HbA1c level,and the two are related to the malignancy of thyroid nodules.
7.Neuroprotective therapy for acute ischemic stroke in the era of endovascular therapy
Mengmeng WANG ; Zhaoyang YAN ; Lingyu ZHANG ; Honghong JI ; Pengfei WANG
International Journal of Cerebrovascular Diseases 2024;32(1):56-61
In recent years, endovascular therapy has become the most important progress in the field of the treatment of acute ischemic stroke caused by large vessel occlusion. However, the vascular recanalization shown by imaging after endovascular treatment cannot fully translate into effective tissue reperfusion and functional outcome, a phenomenon known as "futile recanalization". Combined neuroprotective therapy after vascular recanalization is expected to reduce the occurrence of futile recanalization and improve the outcome of patients. This article briefly summarizes the main application progress of commonly used neuroprotective therapies in clinical practice (edaravone dexborneol, glucocorticoids, hypothermia, and remote ischemic conditioning). It explores the trend and direction of combining endovascular therapy and neuroprotective therapy for patients with acute ischemic stroke caused by large vessel occlusion, and provides further reference and suggestions for intervention measures after endovascular therapy.
8.Compound cottonrose hibiscus leaf gel plaster of optimal"Xiaozhongsan"formulation for knee synovitis
Wei YAN ; Bo KONG ; Xiaobing XI ; Yong XU ; Youji JIA ; Beite RUAN ; Jiahui ZHANG ; Honghong MA ; Zhongwei LI
Chinese Journal of Tissue Engineering Research 2024;28(10):1580-1585
BACKGROUND:Previous studies have confirmed that the new compound cottonrose hibiscus leaf gel plaster has a good effect in the treatment of acute soft tissue swelling. OBJECTIVE:To observe the clinical efficacy of compound cottonrose hibiscus leaf gel plaster in the treatment of synovitis of the knee joint. METHODS:Seventy-two patients with knee synovitis were selected from Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine from December 2019 to May 2021.These patients were randomly divided into a trial group and a control group,with 36 cases in each group.The trial group was treated with compound cottonrose hibiscus leaf gel plaster,once a day,12 hours each time,while the control group was treated with Diclofenac Diethylamine Emulgel,twice a day.After 28 days of treatment,visual analog scale score,WOMAC Osteoarthritis Index score,quality of life score(SF-36),thickness of knee synovium and comprehensive curative effect were compared between the two groups. RESULTS AND CONCLUSION:(1)Visual analog scale scores after treatment were lower than those before treatment(P<0.05).Visual analog scale scores in the trial group after 7,14 and 28 days of treatment were lower than those in the control group(P<0.05).The WOMAC Osteoarthritis Index scores of the two groups after treatment were lower than those before treatment(P<0.05),and the WOMAC Osteoarthritis Index scores in the trial group after 7,14 and 28 days of treatment were lower than those in the control group(P<0.05).(3)The SF-36 quality of life score in the two groups after 28 days of treatment was higher than that before treatment(P<0.05).SF-36 quality of life score in the trial group after 28 days of treatment was higher than that in the control group(P<0.05).(4)After 28 days of treatment,the thickness of knee synovium in the trial group was less than that in the control group(P<0.05),and the effective rate in the trial group was higher than that in the control group(P<0.05).(5)These findings indicate that compared with Diclofenac Diethylamine Emulgel,the compound cottonrose hibiscus leaf gel plaster can better relieve knee pain,enhance knee joint function,reduce synovial hyperplasia,and elevate the overall quality of life of patients.
9.Analysis of gut target microbiota and species difference in patients with obstructive sleep apnea based on 16S rRNA sequencing
Jiwei ZHU ; Manlu LU ; Qianqian JIAO ; Yunliang SUN ; Lu LIU ; Honghong DING ; Yan YU ; Lei PAN
Journal of Southern Medical University 2024;44(1):146-155
Objective To explore the difference in gut microbiota composition between patients with obstructive sleep apnea(OSA)and healthy individuals and the role of gut microbiota in the pathogenesis of OSA.Methods Thirty-nine patients with OSA admitted to our hospital between May and December,2022 and 20 healthy individuals were enrolled in this study.Stool samples were collected from all the participants for analysis of microbiome composition using 16S rRNA high-throughput sequencing analysis.The alpha diversity,beta diversity,and species difference were determined between the two groups and marker species analysis and metabolic pathway function prediction analysis were performed.Results The species diversity(Shannon and Simpson)indexes,richness(observed species)and evenness(Pielou)of gut microbiota were significantly lower in OSA patients than in the healthy individuals(P<0.05).The OSA patients had also a significantly lowered community diversity(P<0.05)with different gut microbial communities from those of the healthy individuals shown by increased relative abundance of potentially pathogenic bacteria such as Pseudomonas and Monocytogenes(P<0.05).LEfSe analysis showed that the abundance of 23 species of gut microbiota differed significantly between the two groups and the OSA patients had significant increases in the abundance of Pseudomonas,Meganomonas,and Fusobacterium(P<0.05).The differential marker flora affected host homeostasis.Random Forest and ROC curve analyses confirmed that Pseudomonas could be used as important biomarkers for a differential diagnosis.Metabolic pathway function prediction analysis showed that biosynthesis function had the greatest contribution to maintaining gut microbiota homeostasis,and Pseudomonas affected the occurrence and progression of OSA by participating in aromatic bioamine degradation and ketogluconic acid metabolic pathway.Conclusion OSA patients have obvious gut microbiota disturbances,and Pseudomonas may affect the development of OSA by participating in substance metabolism to serve as the potential target gut bacteria for OSA treatment.
10.Analysis of gut target microbiota and species difference in patients with obstructive sleep apnea based on 16S rRNA sequencing
Jiwei ZHU ; Manlu LU ; Qianqian JIAO ; Yunliang SUN ; Lu LIU ; Honghong DING ; Yan YU ; Lei PAN
Journal of Southern Medical University 2024;44(1):146-155
Objective To explore the difference in gut microbiota composition between patients with obstructive sleep apnea(OSA)and healthy individuals and the role of gut microbiota in the pathogenesis of OSA.Methods Thirty-nine patients with OSA admitted to our hospital between May and December,2022 and 20 healthy individuals were enrolled in this study.Stool samples were collected from all the participants for analysis of microbiome composition using 16S rRNA high-throughput sequencing analysis.The alpha diversity,beta diversity,and species difference were determined between the two groups and marker species analysis and metabolic pathway function prediction analysis were performed.Results The species diversity(Shannon and Simpson)indexes,richness(observed species)and evenness(Pielou)of gut microbiota were significantly lower in OSA patients than in the healthy individuals(P<0.05).The OSA patients had also a significantly lowered community diversity(P<0.05)with different gut microbial communities from those of the healthy individuals shown by increased relative abundance of potentially pathogenic bacteria such as Pseudomonas and Monocytogenes(P<0.05).LEfSe analysis showed that the abundance of 23 species of gut microbiota differed significantly between the two groups and the OSA patients had significant increases in the abundance of Pseudomonas,Meganomonas,and Fusobacterium(P<0.05).The differential marker flora affected host homeostasis.Random Forest and ROC curve analyses confirmed that Pseudomonas could be used as important biomarkers for a differential diagnosis.Metabolic pathway function prediction analysis showed that biosynthesis function had the greatest contribution to maintaining gut microbiota homeostasis,and Pseudomonas affected the occurrence and progression of OSA by participating in aromatic bioamine degradation and ketogluconic acid metabolic pathway.Conclusion OSA patients have obvious gut microbiota disturbances,and Pseudomonas may affect the development of OSA by participating in substance metabolism to serve as the potential target gut bacteria for OSA treatment.

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