To explore the role of methotrexate (MTX) in regulating the number of regulatory T cells (Treg) and the mRNA expression of transcription factor Foxp3.
 Methods: 1) We analyzed the number of Treg and the mRNA expression of Foxp3 by flow cytometry (FCM) and quantitative real-time PCR (qRT-PCR) respectively in patients with psoriasis vulgaris, patients with psoriasis vulgaris after the 8-week treatment of MTX, and healthy people. 2) BALB/c female mice were smeared with imiquimod (IMQ) cream for 6 days. We recorded the change of the lesion in mice every day. The morphological changes of lesion in mice were evaluated by the psoriasis area and severity index (PASI) and HE staining. 3) The mouse model was randomly divided into a control group and an MTX group. The MTX group was treated with different doses of MTX (38.5 and 77.0 nmol/L) on the third day of this experiment. The morphological changes of lesion in mice were evaluated by PASI and HE staining. We tested the number of Treg and the expression level of Foxp3 mRNA in splenic lymphocytes.
 Results: 1) The number of Treg and the expression level of Foxp3 mRNA were lower in psoriasis vulgaris patients than those in the healthy control group (P<0.05). After 8-week treatment of MTX, the number of Treg was increased (P<0.05) and Foxp3 mRNA level was up-regulated (P<0.01). 2) Typical psoriasis-like skin lesions, such as red scaly skin plaque were found after topical application of IMQ. Both the number of Treg in the splenic lymphocytes of mice and the Foxp3 mRNA level of Treg were reduced by IMQ (P<0.01 and P<0.05). 3) Different doses of MTX for mice showed the ability to improve skin lesion, increase the number of Treg in the spleen of mice and Foxp3 mRNA level in psoriatic dermatitis of mice (P<0.05).
 Conclusion: MTX is able to regulate the number of Treg and Foxp3 mRNA expression in psoriasis.
Adjuvants, Immunologic ; pharmacology ; Aminoquinolines ; pharmacology ; Animals ; Case-Control Studies ; Female ; Forkhead Transcription Factors ; metabolism ; Humans ; Imiquimod ; Immunosuppressive Agents ; administration & dosage ; pharmacology ; Lymphocyte Count ; Methotrexate ; administration & dosage ; pharmacology ; Mice ; Mice, Inbred BALB C ; Psoriasis ; drug therapy ; immunology ; metabolism ; pathology ; RNA, Messenger ; metabolism ; Random Allocation ; Spleen ; cytology ; T-Lymphocytes, Regulatory ; cytology ; drug effects ; metabolism

OBJECTIVETo investigate whether Shen-Fu Injection (, SFI) reduces post-resuscitation immune dysfunction in a porcine model of cardiac arrest by modulating apoptosis of regulatory T lymphocytes (Treg) in the spleen.

METHODSAfter 8-min untreated ventricular fibrillation and 2-min basic life support, 24 pigs were divided into 3 groups with a random number table, i.e. SFI group, epinephrine (EP) group, and saline (SA) group (8 in each group), which received central venous injection of SFI (1.0 mL/kg), EP (0.02 mg/kg) and SA, respectively. The same procedure without CA initiation was achieved in the sham-operated (sham) group (n=6). After successful return of spontaneous circulation (ROSC), apoptosis rate of splenic Treg was detected by flow cytometry; and the mRNA expression of forkhead/winged helix transcription factor (Foxp3) of splenic Treg was detected by real time-polymerase chain reaction; and the levels of interleukin-4 (IL-4) and interferon-γ (IFN-γ) in porcine splenic Treg were detected by using enzyme-linked immunosorbent assay (ELISA).

RESULTSCompared with the sham group, the apoptosis rate of Treg was significantly decreased, and the levels of Foxp3 mRNA expression, IFN-γ, IL-4 and IFN-γ/IL-4 were increased in the SA group (P<0.05 or P<0.01). Compared with the EP and SA groups, SFI treatment increased the apoptosis rate of Treg and reduced the levels of Foxp3 mRNA expression, IFN-γ and IFN-γ/IL-4 (P<0.05).

CONCLUSIONSSFI has signifificant effects in attenuating post-resuscitation immune dysfunction by modulating apoptosis of Treg in the spleen.

Animals ; Apoptosis ; drug effects ; Cardiopulmonary Resuscitation ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; therapeutic use ; Forkhead Transcription Factors ; genetics ; metabolism ; Heart Arrest ; drug therapy ; immunology ; pathology ; physiopathology ; Hemodynamics ; drug effects ; Injections ; Interferon-gamma ; metabolism ; Interleukin-4 ; metabolism ; Lymphocyte Subsets ; drug effects ; metabolism ; Male ; Oxygen ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Spleen ; immunology ; Survival Analysis ; Swine ; Swine, Miniature ; T-Lymphocytes, Regulatory ; drug effects ; immunology

OBJECTIVETo study the dynamic changes in the percentage of Th17 cells/CD4CD25regulatory T cells after intervention with montelukast sodium, a leukotriene receptor antagonist, in asthmatic mice and the association between them.

METHODSBalb/c mice were randomly divided into blank group, asthma group, and montelukast sodium group. The asthmatic mouse model of airway remodeling was established by sensitization with intraperitoneal injection of chicken ovalbumin (OVA) and aluminum hydroxide suspension and aerosol inhalation of OVA. The mice in the blank group were given normal saline, and those in the montelukast sodium group were given montelukast sodium by gavage before aerosol inhalation. Eight mice were randomly sacrificed within 24 hours after 2, 4, and 8 weeks of aerosol inhalation. The pathological sections of lung tissue were used to observe the degree of airway remodeling. Flow cytometry was used to measure the percentages of Th17 cells and CD4CD25regulatory T cells in CD4T cells.

RESULTSThe asthma group and the montelukast sodium group had significantly higher bronchial wall thickness and smooth muscle thickness at all time points compared with the blank group (P<0.05). At 8 weeks of intervention, the montelukast sodium group had significantly greater improvements in the above changes compared with the asthma group (P<0.05). Compared with the blank group, the asthma group and the montelukast sodium group had significant increases in Th17 cells (positively correlated with airway remodeling) and significant reductions in CD4CD25regulatory T cells (negatively correlated to airway remodeling) at all time points (P<0.05). At 8 weeks of intervention, the montelukast sodium group had a significant reduction in the number of Th17 cells and a significant increase in the number of CD4CD25regulatory T cells compared with the asthma group (P<0.05).

CONCLUSIONSMontelukast sodium intervention can alleviate airway remodeling and achieve better improvements over the time of intervention. The possible mechanism may be related to the improvement of immunologic derangement of CD4CD25regulatory T cells and inhibition of airway inflammation.

Acetates ; pharmacology ; Airway Remodeling ; drug effects ; Animals ; Asthma ; drug therapy ; immunology ; Female ; Lung ; pathology ; Mice ; Mice, Inbred BALB C ; Quinolines ; pharmacology ; T-Lymphocytes, Regulatory ; immunology ; Th17 Cells ; immunology

The mechanism underlying CD4CD25Foxp3regulatory T cells (Tregs) promoting the development of colorectal cancer (CRC) was elucidated in the present study. Forty-eight cases of colorectal carcinomas, 22 cases of colon polyps and 21 cases of normal colorectal tissues were collected. The correlation among Foxp3, IL-10 and Stat3, and the clinical relevance of these three indexes were analyzed. The results showed that the levels of Foxp3 expressed in infiltrating CD4CD25Foxp3Tregs, and IL-10 and Stat3 in CRC tissues were all significantly higher than those in polypus tissues and normal colon tissues (P< 0.01). Pearson correlation analysis indicated that the expression level of Foxp3 was positively correlated with Stat3 at mRNA level (r=0.526, P=0.036), and was positively correlated with IL-10 at protein level (r=0.314, P=0.030). The Foxp3 expressed in CD4CD25Foxp3Tregs was correlated with the histological grade, lymph node metastasis and TNM stage of CRC (P<0.05 for all). The IL-10 expression was correlated with the histological grade and TNM stage (both P<0.05). The Stat3 expression was correlated with the lymph node metastasis and TNM stage (both P<0.05). It was concluded that CD4CD25Foxp3Tregs can inhibit tumor immunity in combination with some other related inhibitory cytokines and that Foxp3 expression in CD4CD25Foxp3Tregs correlates with CRC progression.
Adult ; Aged ; CD4-Positive T-Lymphocytes ; immunology ; Colorectal Neoplasms ; genetics ; immunology ; pathology ; Female ; Forkhead Transcription Factors ; biosynthesis ; genetics ; immunology ; Gene Expression Regulation, Neoplastic ; immunology ; Humans ; Immunity ; genetics ; Interleukin-10 ; biosynthesis ; immunology ; Interleukin-2 Receptor alpha Subunit ; immunology ; Lymphatic Metastasis ; Male ; Middle Aged ; STAT3 Transcription Factor ; biosynthesis ; immunology ; T-Lymphocytes, Regulatory ; immunology

OBJECTIVETo explore the role of Treg cells in the pathogenesis of idiopathic thrombocytopenic purpura (ITP).

METHODSThe ITP mouse model was established, the Treg cell ratio in peripheral blood and spleen was detected by flow cytometry, the CD4+ CD25+ T cells were sorted by immunomagnetic beads, the Treg cell associated transcription factors (Foxp3, Smad7, STAT5 and Akt-1) and cytokines (IL-10, TGF-β) in CD4+ CD25+ T cells were enriched from spleen mononuclear cells, and the mRNA expression of Treg cell was measured by real-time PCR.

RESULTSThe ratio of Tregs in peripheral blood and spleen decreased significantly in ITP mouse, as compared with the controls (P<0.01). In addition, the mRNA expression of IL-10, TGF-β and Foxp3 decreased significantly in spleen CD4+ CD25+ T cells (P<0.05). Expression of Smad 7 mRNA was higher than that of controls.

CONCLUSIONThe alteration in Treg frequency and function may be responsible for the immune dysfunction in ITP disease. It is also speculated that the lower mRNA expression of Foxp3 and higher mRNA expression of Smad 7 may inhibit the proliferation and differentiation of Treg cells.

Animals ; Flow Cytometry ; Forkhead Transcription Factors ; metabolism ; Interleukin-10 ; metabolism ; Mice ; Purpura, Thrombocytopenic, Idiopathic ; immunology ; pathology ; RNA, Messenger ; metabolism ; Real-Time Polymerase Chain Reaction ; Smad7 Protein ; metabolism ; Spleen ; cytology ; T-Lymphocytes, Regulatory ; cytology ; Transforming Growth Factor beta ; metabolism

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

Eupatilin is the main active component of DA-9601, an extract from Artemisia. Recently, eupatilin was reported to have anti-inflammatory properties. We investigated the anti-arthritic effect of eupatilin in a murine arthritis model and human rheumatoid synoviocytes. DA-9601 was injected into collagen-induced arthritis (CIA) mice. Arthritis score was regularly evaluated. Mouse monocytes were differentiated into osteoclasts when eupatilin was added simultaneously. Osteoclasts were stained with tartrate-resistant acid phosphatase and then manually counted. Rheumatoid synoviocytes were stimulated with TNF-alpha and then treated with eupatilin, and the levels of IL-6 and IL-1beta mRNA expression in synoviocytes were measured by RT-PCR. Intraperitoneal injection of DA-9601 reduced arthritis scores in CIA mice. TNF-alpha treatment of synoviocytes increased the expression of IL-6 and IL-1beta mRNAs, which was inhibited by eupatilin. Eupatilin decreased the number of osteoclasts in a concentration dependent manner. These findings, showing that eupatilin and DA-9601 inhibited the expression of inflammatory cytokines and the differentiation of osteoclasts, suggest that eupatilin and DA-9601 is a candidate anti-inflammatory agent.
Animals ; Anti-Inflammatory Agents/pharmacology/*therapeutic use ; Arthritis, Experimental/chemically induced/*drug therapy ; Arthritis, Rheumatoid/drug therapy/pathology ; Cell Differentiation/*drug effects ; Cells, Cultured ; Collagen Type II ; Cytokines/biosynthesis ; Disease Models, Animal ; Drugs, Chinese Herbal/therapeutic use ; Female ; Flavonoids/pharmacology/*therapeutic use ; Humans ; Inflammation/drug therapy/immunology ; Interleukin-1beta/genetics/metabolism ; Interleukin-6/genetics/metabolism ; Lymph Nodes/cytology ; Mice ; Mice, Inbred DBA ; Monocytes/cytology ; Osteoclasts/*cytology ; Plant Extracts/pharmacology ; RNA, Messenger/biosynthesis ; Synovial Membrane/cytology ; T-Lymphocytes, Regulatory/cytology/immunology ; Tumor Necrosis Factor-alpha/pharmacology

PURPOSE: Cutaneous lymphocyte-associated antigen (CLA)-expressing CD8+T cells have been known to play an important role in the pathogenesis of atopic dermatitis (AD). However, the mechanisms underlying the loss of self-tolerance remain unclear. Regulatory T cells (Tregs) play a key role in the development of homeostasis in the immune system. We, therefore, hypothesized that a reduced ability of Tregs to inhibit autologous CD8+CLA+T cells might be underlying mechanism in AD. MATERIALS AND METHODS: CD8+CLA+T cells and Tregs were obtained from the peripheral blood of AD patients and control volunteers. The frequencies of CD8+CLA+T cells were evaluated. The proliferative responses of CD8+CLA+T cells were assessed by flow cytometry, and the levels of transforming growth factor-beta1 (TGF-beta1) and interleukin-10 (IL-10) in culture supernatants were detected by enzyme-linked immunosorbent assay. RESULTS: Our results revealed higher frequency and increased expression of perforin and granzyme-B in peripheral CD8+CLA+T cells in AD, and lower inhibitory ability of Tregs on proliferation of CD8+CLA+T cells in AD. Meanwhile, the levels of TGF-beta1 produced by Tregs were significantly lower in AD, and anti-TGF-beta1 abolished such suppression. CONCLUSION: The attenuated inhibitory ability of Tregs on hyper-activated autologous CD8+CLA+T cells, mediated by TGF-beta1, plays an important role in the pathogenesis of AD.
Adult ; Aged ; CD8-Positive T-Lymphocytes/drug effects/*immunology ; Case-Control Studies ; Cell Proliferation ; Cell Separation ; Dermatitis, Atopic/*immunology/pathology ; Female ; Granzymes/metabolism ; Humans ; Interleukin-10/metabolism ; Lymphocyte Count ; Male ; Perforin/metabolism ; Skin/*immunology/pathology ; T-Lymphocytes, Cytotoxic/drug effects/immunology ; T-Lymphocytes, Regulatory/drug effects/*immunology ; Transforming Growth Factor beta1/pharmacology

OBJECTIVETo investigate the changes of CD4 + CD25 + Foxp3 + regulatory T cells in the peripheral blood mononuclear cells (PBMC) and their association with insulin resistance in different stages of prostate cancer (PCa).

METHODSUsing flow cytometry, we counted the CD4+ CD25 + Foxp3 + regulatory T cells in the PBMCs of 62 PCa patients (5 cases of TNM stage I, 16 cases of stage II, 21 cases of stage III, and 20 cases of stage IV) and 42 normal healthy controls, and calculated their proportion in the CD4+ T-lymphocytes. We determined the levels of fast blood glucose (FBG) and fast insulin (FINS) for the insulin resistance index (HOMA-IR), obtained the serum IGF-1 level by ELISA, and analyzed the relationship of the count and proportion of CD4+ CD25+ Foxp3+ regulatory T cells with insulin resistance by comparison between the PCa patients and normal healthy controls.

RESULTSCompared with the control group, the PCa patients showed significantly increased HOMA-IR (3.68 ± 1.42 vs 6.68 ± 1.66), decreased level of serum IGF-1 ([164.56 ± 30.58] vs [96.39 ± 21.21] ng/ml), and elevated count ([1.99 ± 0.78 ] x 10(7) vs [3.55 ± 0.29] x 10(7)) and proportion ([5.33 ± 0.65] vs [13.88 ± 0.96]%) of CD4 + CD25 + Foxp3 regulatory T cells in the PBMCs. The TNM stage was correlated positively with the count and percentage of CD4 + CD25+ Foxp3 + regulatory T cells and HOMA-IR, but negatively with the level of serum IGF-1. Meanwhile, the count and percentage of CD4 + CD25 + Foxp3 + regulatory T cells were found to have a positive correlation with HOMA-IR (r = 0.722 and 0.689, P < 0.01) but a negative correlation with the level of serum IGF-1 (r = -0.747 and -0.896, P < 0.01).

CONCLUSIONThe count and proportion of CD4+ CD25 + Foxp3 + regulatory T cells in the peripheral blood and insulin resistance increase with the elevated stage of PCa. CD4 + CD25 + Foxp3 + regulatory T cells may be involved in the occurrence and progression of PCa by regulating insulin resistance.

Aged ; CD8-Positive T-Lymphocytes ; Case-Control Studies ; Disease Progression ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Humans ; Hyperinsulinism ; Insulin ; blood ; Insulin Resistance ; Insulin-Like Growth Factor I ; metabolism ; Leukocytes, Mononuclear ; Lymphocyte Count ; Male ; Prostatic Neoplasms ; blood ; immunology ; pathology ; T-Lymphocytes, Regulatory

OBJECTIVETo study the dynamic changes in Th17 cells and CD4⁺ CD25⁺ regulatory T cells (Treg) in the spleen and to analyze their relationship with airway remodeling.

METHODSA total of 48 female specific pathogen-free Balb/c mice were randomly divided into control and asthmatic groups. To establish the asthmatic airway remodeling model, the mice were sensitized to ovalbumin (OVA) through intraperitoneal injection of OVA and aluminum hydroxide suspension and challenged by inhalation of aerosol OVA. The matched control group was treated with normal saline instead. In 24 hours after 2-week, 4-week, and 8-week aerosol inhalation, 8 mice were randomly selected from each group and sacrificed. Then histopathological examination of the left lung was performed to measure the degree of airway remodeling. The percentages of Th17 and CD4⁺ CD25⁺ Treg cells in total CD4(+) cells from the spleen were determined by flow cytometry.

RESULTSIn the asthmatic group, the ratios of total bronchial wall area to bronchial basement membrane perimeter (WAt/Pbm) and bronchial smooth muscle area to bronchial basement membrane perimeter (WAm/Pbm) significantly increased as the challenge proceeds (P<0.01). The percentage of Th17 cells derived from the cell suspension of the spleen gradually increased and it was positively correlated with the degree of asthmatic airway remodeling (P<0.01). The percentage of CD4⁺ CD25⁺ Treg cells from the suspension gradually decreased and it was negatively correlated with the degree of asthmatic airway remodeling (P<0.01).

CONCLUSIONSIn mice with asthma, as the challenge proceeds, the airway remodeling becomes more severe, the percentage of Th17 cells increases, and the percentage of CD4⁺ CD25⁺ Treg cells decreases. The immunological imbalance is possibly one of the important factors inducing airway remodeling.

Airway Remodeling ; Animals ; Asthma ; immunology ; pathology ; Disease Models, Animal ; Female ; Lung ; pathology ; Mice ; Mice, Inbred BALB C ; T-Lymphocytes, Regulatory ; immunology ; Th17 Cells ; immunology