Objective To investigate the therapeutic effect of SPK1 gene transfected adipose derived mesenchymal stem cells(ADMSC)on experimental autoimmune encephalomyelitis mice and the effect on T helper cell 17(Th17)/regulatory T(Treg) cells balance. Methods EAE was induced by myelin oligodendrocyte glycoprotein 35-55 in mice.Totally 44 mice were randomly divided into four groups:normal control group(NC group),model group(EAE group),ADMSC group,and ADMSC-SPK1 group.Forty days after injection,the pathological changes of brain and spinal cord,Th17/Treg-related inflammatory markers in brain tissue,expressions of interleukin-17A(IL-17A)and forkhead box protein p3(Foxp3)in brain and spinal cord tissue,and flow cytometric results of spleen immune cells were detected. Results Forty days after the injection,serious inflammatory cell infiltration and demyelination occurred in the brain and spinal cord of EAE group,whereas demyelination and axonal injury were improved in ADMSC group and ADMSC-SPK1 group.Compared with EAE group,the ADMSC group and ADMSC-SPK1 group had significantly improved levels of IL-17A(
Adipose Tissue/cytology* ; Animals ; Cytokines ; Encephalomyelitis, Autoimmune, Experimental/therapy* ; Interleukin-17 ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stem Cells/cytology* ; Mice ; Mice, Inbred C57BL ; Phosphotransferases (Alcohol Group Acceptor)/genetics* ; T-Lymphocytes, Regulatory/cytology* ; Th17 Cells/cytology* ; Transfection

Astragalus membranaceus may be a potential therapy for childhood asthma but its driving mechanism remains elusive. The main components of A. membranaceus were identified by HPLC. The children with asthma remission were divided into two combination group (control group, the combination of budesonide and terbutaline) and A. membranaceus group (treatment group, the combination of budesonide, terbutaline and A. membranaceus). The therapeutic results were compared between two groups after 3-month therapy. Porcine peripheral blood mononuclear cells (PBMCs) were isolated from venous blood by using density gradient centrifugation on percoll. The levels of FoxP3, EGF-β, IL-17 and IL-23 from PBMCs and serum IgE were measured. The relative percentage of Treg/Th17 cells was determined using flow cytometry. The main components of A. membranaceus were calycosin-7-O-glucoside, isoquercitrin, ononin, calycosin, quercetin, genistein, kaempferol, isorhamnetin and formononetin, all of which may contribute to asthma therapy. Lung function was significantly improved in the treatment group when compared with a control group (P < 0.05). The efficacy in preventing the occurrence of childhood asthma was higher in the treatment group than the control group (P < 0.05). The levels of IgE, IL-17 and IL-23 were reduced significantly in the treatment group when compared with the control group, while the levels of FoxP3 and TGF-β were increased in the treatment group when compared with the control group (P < 0.05). A. membranaceus increased the percentage of Treg cells and reduced the percentage of Th17 cells. A. membranaceus is potential natural product for improving the therapeutic efficacy of combination therapy of budesonide and terbutaline for the children with asthma remission by modulating the balance of Treg/Th17 cells.
Animals ; Asthma ; drug therapy ; immunology ; Astragalus propinquus ; chemistry ; Budesonide ; administration & dosage ; Cells, Cultured ; Child ; Child, Preschool ; Cytokines ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; Female ; Humans ; Immunologic Factors ; administration & dosage ; pharmacology ; Leukocytes, Mononuclear ; drug effects ; metabolism ; Lung ; drug effects ; physiology ; Male ; Swine ; T-Lymphocytes, Regulatory ; cytology ; drug effects ; Terbutaline ; administration & dosage ; Th17 Cells ; cytology ; drug effects ; Treatment Outcome

OBJECTIVE: Behcet's disease (BD) is an autoimmune disorder that causes most commonly mouth and genital ulcerations and erythema nodules of the skin and currently has limited options of therapeutic medicines. Metformin is recently reported to suppress immune reaction, and we hypothesized that metformin could be an option for treatment of BD. METHODS: Thirty patients with BD were enrolled in this perspective single-blinded, before-after study. We recorded the changes in the mucocutaneous activity index for BD (MAIBD), relapse frequency, C-reactive protein (CRP) level and erythrocyte sedimentation rate (ESR) after metformin treatment to assess the changes in the disease activity. We also analyzed the changes in the protein and mRNA expression levels of Foxp3, interleukin-35 (IL-35), transforming growth factor-β (TGF-β), Ror-γt, IL-17, and tumor necrosis factor- (TNF-) in these patients using ELISA and qRT-PCR. RESULTS: Of the 30 patients enrolled, 26 completed the trial. After the treatment, favorable responses were achieved in 88.46% (23/26) of the patients, and partial remission was obtained in 11.54% (4/26) of them. During the treatment, 8 patients complained of gastrointestinal side effects, for which 4 chose to withdraw from the study in the first week. Our results showed that metformin treatment decreased MAIBD and relapse frequency in the patients, and significantly lowered the clinical inflammatory indexes including CRP and ESR. The results of ELISA and qRT-PCR revealed that metformin treatment obviously increased Foxp3 and TGF-β expressions at both the protein and mRNA levels and significantly decreased the levels of ROR-γt, IL-17 and TNF- as well as IL-35 level in these patients. CONCLUSIONS Metformin treatment relieves the clinical symptoms, reduces the inflammatory reaction indexes and regulates the Treg/Th17 axis in patients with BD, suggesting the potential of metformin as a candidate medicine for treatment of BD.
Behcet Syndrome ; drug therapy ; metabolism ; Controlled Before-After Studies ; Forkhead Transcription Factors ; metabolism ; Humans ; Immunosuppressive Agents ; adverse effects ; therapeutic use ; Interleukin-17 ; metabolism ; Interleukins ; metabolism ; Metformin ; adverse effects ; therapeutic use ; Neoplasm Recurrence, Local ; Nuclear Receptor Subfamily 1, Group F, Member 3 ; metabolism ; RNA, Messenger ; metabolism ; Recurrence ; Single-Blind Method ; T-Lymphocytes, Regulatory ; cytology ; Th17 Cells ; cytology ; Transforming Growth Factor beta ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

Triptolide( TP) is isolated from the traditional Chinese medicine Tripterygium wilfordii,which exhibits notable immuneregulative effect. Th17 cells involve in inflammatory response and Treg cells contribute to immune tolerance. They both play an important role in immune response. Previous studies have investigated that TP induced hepatic Th17/Treg imbalance. However,the effect of TP on spleen Th17/Treg cells remains unclear. Therefore,the aim of present study was to investigate the effect of TP on Th17/Treg cells in spleen. In this study,the effect of TP on the proliferation of splenic lymphocyte was detected by cytotoxicity test in vitro. After different concentrations of TP( 2. 5,5,20,40 nmol·L~(-1)) were given to splenic lymphocyte,cytokines secreted from the supernatant of splenic lymphocyte were detected by cytometric bead array,and the expression of suppressor of cytokine signaling( SOCS) mRNA was detected by qRT-PCR. Female C57 BL/6 mice were continuously observed for 24 h after treatment of 500 μg·kg-1 TP. The effects of TP on the splenic tissue structure and the percentage of Th17/Treg cells were examined. The results showed that the IC50 of TP was19. 6 nmol·L~(-1) in spleen lymphocytes. TP inhibited the secretion of IL-2 and IL-10 and induced the expression of SOCS-1/3 mRNA in spleen lymphocytes at the dosage of 2. 5 and 5 nmol·L~(-1) after 24 h in vitro. Administration of TP at dosage of 500 μg·kg-1 had no significant spleen toxicity in vivo. TP treatment increased the percentage of Th17 cells after 12 h and inhibited the proportion of Treg cells after 12 and 24 h. In conclusion,TP reduced the secretion of IL-2 and IL-10 through SOCS-1/3 signaling pathway,thereby induced the percentage of Th17 cells and inhibited the percentage of Treg cells.
Animals ; Cytokines ; metabolism ; Diterpenes ; pharmacology ; Epoxy Compounds ; pharmacology ; Female ; Mice ; Mice, Inbred C57BL ; Phenanthrenes ; pharmacology ; Signal Transduction ; Spleen ; cytology ; drug effects ; Suppressor of Cytokine Signaling 1 Protein ; metabolism ; Suppressor of Cytokine Signaling 3 Protein ; metabolism ; T-Lymphocytes, Regulatory ; cytology ; Th17 Cells ; cytology

Objective To investigate the effect of adipose mesenchymal stem cells(AMSCs) on the peripheral blood lymphocyte(PBL) in psoriasis vulgaris(PV) patients and the expression and secretion profiles of related inflammatory cytokines in the PBL.Methods AMSCs from three PV patients were co-cultured with PBL. Peripheral blood regulatory cells(Treg) and T helper cell 17(Th17)ratio was measured by flow cytometry. The anti- and pro-inflammatory cytokines expressed and secreted by PBL were detected by quantitative real-time polymerase chain reaction(qRT-PCR) and enzyme-linked immunosorbent assay(ELISA).Results The Treg/total lymphocyte ratio was significantly higher in the healthy people AMSCs+PBL co-culture group[(3.2±0.5)%;P=0.001],but AMSCs in patients had a tendency to promote the proliferation of Treg cells [(1.3±0.2)%],with no significant difference(P=0.485) when compared with the PBL culture alone group[(1.0±0.1)%]. qRT-PCR showed that the ability of PBL in expressing Treg transcription factor forkhead box p3 and transforming growth factor(TGF)-Β mRNA was significantly lower in psoriasis AMSCs+PBL co-culture group than in the healthy people AMSCs+PBL co-culture group(P=0.00,P=0.03),AMSCs had a tendency to promote the expression of interlukin(IL)-10 in peripheral blood lymphocytes,but there was no significant difference(P=0.09).ELISA showed the PBL in healthy people AMSCs+PBL co-culture group secreted the anti-inflammatory cytokine IL-10[(156.9±41.8) ng/Μl] and TGF-Β[(2774.1 ± 526.4) ng/Μl];in contrast,the abilities of PBL in PV patient AMSCs+PBL co-culture group in secreting the anti-inflammatory cytokines has a downward trend:IL-10[(90.4±28.8) ng/Μl] and TGF-Β[(1597.9±55.7) ng/Μl],although the differences were not statistically significant. After the co-culture,the proportion of Th17 cells in the psoriasis AMSCs+PBL co-culture group[(0.8±0.3)%] showed a decreasing trend when compared with the PBL culture alone group[(1.1±0.1)%],although the results were not statistically significant. Also,the proportion of Th17 cells showed no significant difference between PV patient AMSCs+PBL co-culture group and healthy people AMSCs+PBL co-culture group. Finally,both the psoriasis AMSCs+PBL co-culture group and the healthy people AMSCs+PBL co-culture group showed no obvious inhibitory effect on the expression and secretion of Th17 transcription factor retinoid-related orphan nuclear receptor Γt and pro-inflammatory cytokines IL-17 and IL-23 in PBL,and there was no significant difference between these two groups.Conclusions AMSCs in PV patients have decreased ability in regulating the anti-inflammatory function of peripheral blood Treg lymphocytes. However,they have no effect on the proinflammatory effect of peripheral blood Th17 lymphocytes.
Adipose Tissue ; cytology ; Cytokines ; immunology ; Forkhead Transcription Factors ; immunology ; Humans ; Inflammation ; immunology ; Mesenchymal Stem Cells ; cytology ; Psoriasis ; immunology ; T-Lymphocytes, Regulatory ; immunology ; Th17 Cells ; immunology

OBJECTIVETo evaluate the efficiency and safety of rituximab and dexamethasone combined with cyclophosphamide for treating patients with relapsed and refractory immune thrombocytopenia (ITP).

METHODSTwelve patients with relapsed and refractory immune thrombocytopenia were prospectively enrolled in this study, and received rituximab 375 mg/m(2) once a week for 4 weeks, dexamethasone 40 mg once a day for consecutive 4 days, and cyclophosphamide 500 mg/m(2) biweekly for 2 weeks. The levels of IFN-r and IL-4 in peripheral blood of patients were measured by enzyme-linked immunosorbent assay (ELISA), and the percentages of Breg, Treg and Th17 cells were detected by flow cytometry before and after treatment. Efficiency was evaluated according to platelet counts, and side effects were observed.

RESULTSSix out of 12 patients reached to complete remission and 4 patients reached to partial remission, with the total response rate 83.33%. The platelet counts [(115.42 ± 76.60) × 10(9)/L] after treatment were significantly higher than that before treatment [(115.42 ± 76.60) × 10(9)/L] (P < 0.001). The ratio of IFN-r/ IL4 after treatment (5.89 ± 2.30) was very significantly lower than that before treatment (7.00 ± 2.73) (P = 0.002). The percentage of Breg cells after treatment [(21.27 ± 4.28)%] were much significantly higher than that before treatment [(15.48 ± 1.67)%] (P < 0.001). The ratio of Treg/Th17 after treatment (3.07 ± 1.50) was significantly higher than that before treatment (0.98 ± 0.45) (P < 0.001). Infusion reaction was observed in 1 patient, secondary hypertension and hyperglycemia were in 1 patient, and pneumonia in 2 patients.

CONCLUSIONRituximab and dexamethasone combined with cyclophosphamide can improve the outcomes of patients with relapsed and refractory immune thrombocytopenia patients and they were well tolerated, its mechanism may be related with the balance between T cell sunsets and Treg cells.

Antibodies, Monoclonal, Murine-Derived ; B-Lymphocytes, Regulatory ; cytology ; Cyclophosphamide ; therapeutic use ; Dexamethasone ; therapeutic use ; Enzyme-Linked Immunosorbent Assay ; Humans ; Interferon-gamma ; blood ; Interleukin-4 ; blood ; Platelet Count ; Prospective Studies ; Purpura, Thrombocytopenic, Idiopathic ; drug therapy ; Remission Induction ; Rituximab ; therapeutic use ; T-Lymphocytes, Regulatory ; cytology ; Th17 Cells ; cytology

OBJECTIVETo observe the dynamic changes of Th17/Treg balance in patients following surgical intervention for intracranial aneurysm rupture.

METHODSThe percentage of Th cells and the intracellular IL-17 level, Treg cell percentage and transforming growth factor -β1 (TGF-β1) levels were examined in 73 patients with rupture of aneurysms before and at 24 h, 72 h and 1 week after operation, with 62 patients with unruptured aneurysms and 65 healthy volunteers as the control. The correlations among the immune cells, cytokines and clinical characteristics of the patients (NIHSS, ADL and hospitalization stay) were analyzed.

RESULTSTh17 percentage and intracellular IL-17 levels were significantly higher in the patients with ruptured and unruptured aneurysms than in the healthy volunteers, and were significantly higher in patients with ruptured aneurysms than in those with unruptured aneurysms. Treg cell percentage and TGF-β1 level were significantly lower in patients with aneurysms than in the healthy volunteers, and were lower in patients with ruptured aneurysms than in those with uruptured aneurysms (P<0.05). Patients with intracranial aneurysm rupture showed significantly increased Th17 cell percentage and IL-17 level but significantly lowered Treg cell percentage and TGF-β1 at 24 h following the surgery (P<0.05); these changes were reversed significantly at 72 h and 1 week after the surgery. Th17 cell percentage and IL-17 level were positively correlated with NIHSS and the length of postoperative hospital stay but inversely correlated with ADL; Treg cell percentage and TGF-β1 were inversely correlated with NIHSS and hospital stay but positively with ADL (P<0.05).

CONCLUSIONIn patients with intracranial aneurysms, the systemic immune inflammatory response is highlighted by excessive Th17 cells and insufficient Treg cells, which are closely related with the outcomes of the patients following surgical intervention. Evaluation of Th17/Treg balance and the cytokine levels can help to assess the prognosis of patients with aneurysm rupture.

Aneurysm, Ruptured ; immunology ; Flow Cytometry ; Humans ; Interleukin-17 ; blood ; Intracranial Aneurysm ; immunology ; Postoperative Period ; Prognosis ; T-Lymphocytes, Regulatory ; cytology ; Th17 Cells ; cytology ; Transforming Growth Factor beta1 ; blood

The effect of hydroxychloroquine (HCQ) on dry eye has not been fully determined. This study aimed to compare the 12-week efficacy of HCQ medication with that of a placebo in the management of dry eye in primary Sjögren's syndrome (pSS). A double-blind, randomized control study was conducted in 39 pSS subjects from May 2011 through August 2013. pSS was diagnosed based on the classification criteria of the American-European Consensus Group. Subjects received 300 mg of HCQ or placebo once daily for 12 weeks and were evaluated at baseline, 6, and 12 weeks, with a re-visit at 16 weeks after drug discontinuance. The fluorescein staining score, Schirmer test score, tear film break-up time (TBUT), and ocular surface disease index (OSDI) were measured, and tears and blood were collected for ESR, IL-6, IL-17, B-cell activating factor (BAFF), and Th17 cell analysis. Color testing was performed and the fundus was examined to monitor HCQ complications. Twenty-six subjects completed the follow-up. The fluorescein staining score and Schirmer test score did not differ significantly. The OSDI improved with medication in the HCQ group but was not significantly different between the groups. TBUT, serum IL-6, ESR, serum and tear BAFF, and the proportion of Th17 cells did not change in either group. HCQ at 300 mg daily for 12 weeks has no apparent clinical benefit for dry eye and systemic inflammation in pSS (ClinicalTrials.gov. NCT01601028).
Aged ; B-Cell Activating Factor/analysis/blood ; Blood Sedimentation ; Double-Blind Method ; Drug Administration Schedule ; Dry Eye Syndromes/complications/*drug therapy ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Hydroxychloroquine/*therapeutic use ; Interleukin-16/analysis/blood ; Interleukin-17/analysis/blood ; Male ; Middle Aged ; Placebo Effect ; Prospective Studies ; Sjogren's Syndrome/*complications/diagnosis ; Th17 Cells/cytology/immunology ; Treatment Outcome

OBJECTIVE: To investigate the expression levels of Th9, Th17 and Treg cells in peripheral blood of patients with chronic rhinosinusitis with nasal polyps (CRSwNP), and explore the role of Th9, Th17 and Treg cells in the progression of CRSwNP. METHOD: Forty-six cases with CRSwNP served as an experimental group, while 22 cases with simple nasal bleeding or nasal septum deviation served as a control group. The peripheral blood of patients in both groups was collected and analyzed. (1) Using flow cytometry (FCM) to detect the expression rates of Th9, Th17 and Treg cells in peripheral blood. (2) Using qRT-PCR to detect the expression of relevant transcription factor of Th9, Th17 and Treg cells (IL-9mRNA, PU. 1, IRF-4, RoRc, and Foxp3). (3) Using SPSS16.0 to analyse the differentiations and the revelance among these three cells. RESULT: (1) The expression rates of Th9 and Th17 cells in patients with CRSwNP (1.29% ± 0.18%, 4.03% ± 0.69%) was higher than the control group (0.45% ± 0.14%, 1.35% ± 0.26%). But the expression rates of Treg cells in the experimental group (2.98% ± 0.13%) was significantly lower than the control group (5.44% ± 0.57%). The differences were statistically significant (P < 0.05). (2) The expression of revelant transcription factor (IL-9mRNA, PU.1, IRF-4, RoRc) in NP group was also higher than the control group. The expression of Foxp3 in the control group was higher than NP, the differences both were statistically significant (P < 0.05). (3) The difference between Th9 and Th17 in patients with NP was not significant (P > 0.05), and the negative correlation was found between Th17 and Treg (r = -0.549, P < 0.05). CONCLUSION The high expression level of Th9 and Th17 cells might promote the development of NP, whereas the low expression level of Treg cells might further aggravate the occurrence of NP. The main function of the imbalance of Th17/Treg cells may be immune regulation in the pathogenesis of nasal polys.
Case-Control Studies ; Cell Differentiation ; Disease Progression ; Epistaxis ; Flow Cytometry ; Forkhead Transcription Factors ; metabolism ; Humans ; Nasal Polyps ; immunology ; pathology ; Nasal Septum ; abnormalities ; Rhinitis ; immunology ; pathology ; Sinusitis ; immunology ; pathology ; T-Lymphocytes, Regulatory ; cytology ; Th17 Cells ; cytology ; Transcription Factors ; metabolism

OBJECTIVE: To explore the mechanisms for an increase in susceptibility of asthma induced by respiratory syncytial virus (RSV), to observe the expression of interleukin-8 (IL-8) in human bronchial epithelial cells (HBECs) after RSV infection and to invesigate the regulatory effect of IL-8 on Th17/Treg differentiation.
 METHODS: HBECs were divided into a control group and a RSV infected group. The RSVE-infected model of HBECs was established and examined. The expression of IL-8 mRNA was detected by real-time PCR, and the levels of IL-8 were measured by ELISA. Peripheral blood lymphocytes in healthy people were extracted and divided into a control group and an IL-8 treatment group. Based on concentration of IL-8 in RSV-infected HBECs, lymphocytes were treated by a matched concentration of human recombinant IL-8 for 24 h. The distribution of Th17 and Treg subsets in lymphocytes were examined by flow cytometry.
 RESULTS: The RSV-infected HBECs model was successfully established. The infected HBECs were still able to split and passage. The RSV could be detected in every passage in the infected cells. Virus particles indicated by bright yellow green fluorescence were seen under fluorescence microscope. Edema of mitochondrias, expansion of endoplasmic reticulum, fissure around nucleus and intracellular virus particles were all observed under electron microscope. The expression IL-8 mRNA were significantly enhanced in the RSV-infected group, and the level of IL-8 in the RSV-infected group was higher than that in the control group (P<0.05). After IL-8 treatment for 24 h, the ratio of Th17 subsets in lymphocytes were dramatically increased compared to the control group (P<0.05), but there was no difference in the ratio of Treg subsets between the 2 groups (P>0.05).
 CONCLUSION Over-secretion of IL-8 by the RSV-infected HBECs may promote the differentiation of Th17 subsets and maintain the Th17/Tred imbalance.
Cell Differentiation ; Cells, Cultured ; Epithelial Cells ; drug effects ; virology ; Flow Cytometry ; Humans ; Interleukin-8 ; immunology ; pharmacology ; Real-Time Polymerase Chain Reaction ; Recombinant Proteins ; pharmacology ; Respiratory Syncytial Virus Infections ; immunology ; Respiratory Syncytial Viruses ; T-Lymphocytes, Regulatory ; cytology ; Th17 Cells ; cytology