OBJECTIVE: To explore the clinical effect of acupuncture and the potential effect mechanism in patients with premature ovarian failure. METHODS: A total of 104 patients with premature ovarian failure were randomized into an acupuncture group and a western medication group, 52 cases in each one. In the western medication group, the conjugated estrogens tablets were prescribed for oral administration, 0.625 mg each time, once a day, consecutively for 21 days. On the 16th day of medication with conjugated estrogens tablets, the oral administration of medroxyprogesterone acetate tablets were supplemented, 10 mg each time, once a day, consecutively for 5 days, and then, these two kinds of western medication were discontinued for 1 week. A total of 3 cycles were required in treatment with 28 days as an artificial cycle. In the acupuncture group, acupuncture was applied. Two groups of acupoints were selected. The first group of acupoints were stimulated before ovulation and the acupoints were Guanyuan (CV 4), Guilai (ST 29), Taichong (LR 3), Taixi (KI 3), Xuehai (SP 10), Sanyinjiao (SP 6), Zigong (EX-CA 1), Yinlingquan (SP 9), Zusanli (ST 36), Shuidao (ST 28), Dahe (KI 12) and Tianshu (ST 25). The second group of acupoints were stimulated after ovulation and the acupoints included Ciliao (BL 32), Shiqizhui (EX-B 8), Ganshu (BL 18), Shenshu (BL 23), Geshu (BL 17) and Pishu (BL 20). The therapeutic effect was observed and compared in the patients between the two groups, as well as the expressions of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) and the levels of serum luteinizing hormone (LH), follicule stimulating hormone (FSH) and estradiol (E) before and after treatment. RESULTS: The total effective rate was 90.4% (47/52) in the acupuncture group, higher than 67.3% (35/62) in the western medication group (<0.05). After treatment, the expressions of IFN-γ and TNF-α in the acupuncture group were obviously lower than the western medication group (<0.05). Except for serum LH after treatment, at the end of treatment and in 30 days and 90 days after treatment, the levels of serum E in the acupuncture group were higher obviously than the western medication group and the levels of serum LH and FSH were lower obviously than the western medication group (all <0.05). CONCLUSION Acupuncture promotes the regular menstruation, effectively regulates the levels of serum LH, FSH and E and improves the pituitary gland and the ovary endocrine in the patients with premature ovarian failure. Such effect may be related to the the improvements in the expressions of IFN-γ and TNF-α, the inhibition of the apoptosis of ovarian granulosa cells, the recovery of ovarian function and the enhancement of reserve capacity.
Acupuncture Points ; Acupuncture Therapy ; Female ; Humans ; Interferon-gamma ; blood ; Primary Ovarian Insufficiency ; blood ; therapy ; Tumor Necrosis Factor-alpha ; blood

OBJECTIVETo investigate the effect of leukodepleted blood transfusions on peripheral blood Th1/Th2 cell balance in patients with acute lymphoblastic leukemia (ALL).

METHODSFifty-seven ALL patients in our hospital from March 2016 to August 2017 were selected, 31 of them received routine blood transfusion were enrolled in group A, and 26 patients received depleted-blood leukotransfusion were enrolled in group B, 36 cases in normal physical examination at the same period were enrolled in control group. And the basic clinical characteristics of patients were recorded; the ratio of Th1/Th2 cells in peripheral blood of patients was analyzed by flow cytometry;the serum levels of IL-2, IFN-&gamma;, IL-4, IL-10 were detected by ELISA method; the mRNA levels of T-bet and GATA-3 in lymphocytes were detected by RT-PCR;the protein levels of T-bet and GATA-3 in lymphocyte were detected by Western blot.

RESULTSThe Th1/Th2 ratio in peripheral blood of ALL patients significantly related with patient age and risk grade (P<0.05).After treatment,the change of Th1/Th2 ratio in group A showed no statistical difference from Th1/Th2 ratio before treatment (P>0.05), while the Th1/Th2 ratio in group B increased (P<0.05);the levels of IL-2, IFN-&gamma;, IL-4 and IL-10 secreted from Th1 and Th2 cells of ALL patients in A group were not changed significantly(P>0.05), while the levels of IL-2 and IFN-&gamma; secreted from Th1 cells of ALL patients in group B increased, the levels of IL-4 and IL-10 secreted from Th2 cells in group B decreased with statistical difference (P<0.05); the RT-PCR and Western blot showed that the expression levels of T-bet mRNA and T-bet protein in group A were lower than those in control group, while the expression levels of T-bet mRNA and T-bet protein in group B were higher than those in group A (P<0.05); the expression levels of mRNA and GATA-3 protein in group A were higher than those in control group, the expression levels of mRNA and GATA-3 protein in group B were lower than those in group A (P<0.05).

CONCLUSIONThe leukoreduced blood transfusion helps to improve the balance of Th1/Th2 cells in peripheral blood and improve the immune function of patients, which may closely relate with the expression levels of T-bet and GATA-3.

Blood Transfusion ; GATA3 Transcription Factor ; Humans ; Interferon-gamma ; Interleukin-4 ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; Th1 Cells ; Th2 Cells

Indoor animal husbandry environments are inevitably contaminated with endotoxins. Endotoxin exposure is associated with various inflammatory illnesses in animals. This cross-sectional study evaluated the relationship between the degree of endotoxin exposure and the cellular and humoral immune profiles of fattening pigs. Blood samples were taken from the jugular vein of 47 pigs from ten pig farms in Korea. Whole blood cell counts and plasma immunoglobulin (Ig) classes were determined. Peripheral-blood mononuclear cells were stimulated in vitro with concanavalin A for 48 h, and cytokines released into culture supernatants were measured. The barns in which the pigs lived were assessed for endotoxin levels in the total and respirable dust by using the limulus amebocyte lysate kinetic QCL method. Low and high endotoxin exposures were defined as ≤ 30 and > 30 EU/m³, respectively. Compared to pigs with low endotoxin exposure (n = 19), highly exposed pigs (n = 28) had higher circulating neutrophil and lymphocyte (particularly B cells) counts, IgG and IgE levels, interferon-gamma (IFNγ) and interleukin (IL)-4 productions, and lower IgA levels and tumor necrosis factor-alpha (TNFα) production. The IL-4, IFNγ, and TNFα levels significantly correlated with endotoxin level and/or pig age. Constant exposure of pigs to high levels of airborne endotoxins can lead to aberrant immune profiles.
Agriculture ; Animal Husbandry ; Animals ; Blood Cell Count ; Concanavalin A ; Cross-Sectional Studies ; Cytokines ; Dust ; Endotoxins ; Horseshoe Crabs ; Housing ; Immunity, Cellular ; Immunoglobulin A ; Immunoglobulin E ; Immunoglobulin G ; Immunoglobulins ; In Vitro Techniques ; Interferon-gamma ; Interleukin-4 ; Interleukins ; Jugular Veins ; Korea ; Lymphocytes ; Methods ; Neutrophils ; Plasma ; Swine ; Tumor Necrosis Factor-alpha

OBJECTIVETo observe the effects of recombinant fusion protein interleukin (IL)-18 on the expression of immune-inflammatory factors in the mice infected with Staphylococcus aureus (SA), and to investigate the mechanism of action of IL-18 in defense of SA infection in vivo.

METHODSA total of 40 specific pathogen-free female BLAB/c mice were randomly divided into four groups: control, SA infection, immunized, and intervention. A mouse model of SA infection was established by nasal inoculation with SA liquid. The immunized group and the intervention group were intranasally given IL-18 before SA modeling, and then the SA infection group and the intervention group received the nasal inoculation with SA liquid; the control group was treated with phosphate buffered saline instead. The levels of IL-4, interferon (IFN)-&gamma;, tumor necrosis factor (TNF), granulocyte colony-stimulating factor (G-CSF), IgM in the serum and bronchoalveolar lavage fluid (BALF) of mice were measured by enzyme-linked immunosorbent assay. The expression of macrophage inflammatory protein (MIP)-1α mRNA and MIP-2β mRNA in the lung tissue of mice were determined by real-time fluorescent quantitative PCR.

RESULTSCompared with the control group, the SA infection group and the immunized group had significantly higher levels of IL-4, G-CSF, and IgM in the serum and BALF and expression of MIP-1α mRNA and MIP-2β mRNA in the lung tissue (P<0.05); the SA infection group had a significantly lower level of IFN-&gamma; and a significantly higher level of TNF in the serum and BALF (P<0.05); the immunized group had a significantly higher level of IFN-&gamma; in the serum and BALF (P<0.05). Compared with the SA infection group, the intervention group had significantly higher levels of IL-4, IFN-&gamma;, G-CSF, and IgM in the serum and BALF and expression of MIP-1α mRNA in the lung tissue. In contrast, the intervention group showed a significantly lower level of TNF in the serum and BALF and expression of MIP-2β mRNA in the lung tissue (P<0.05). All the above indicators in the intervention group were significantly higher than those in the control group (P<0.05), except the serum level of IFN-&gamma;.

CONCLUSIONSIn the mice infected with SA, the recombinant fusion protein IL-18 by mucosal immunity can affect inflammatory factors in the serum and BALF and the expression of MIP-1α mRNA and MIP-2β mRNA in the lung tissue to promote the anti-infective immune response and enhance the ability to clear pathogens.

Animals ; Chemokine CCL3 ; analysis ; Female ; Granulocyte Colony-Stimulating Factor ; blood ; Interferon-gamma ; blood ; Interleukin-18 ; therapeutic use ; Interleukin-4 ; blood ; Mice ; Mice, Inbred BALB C ; Recombinant Fusion Proteins ; pharmacology ; therapeutic use ; Staphylococcal Infections ; drug therapy ; immunology

OBJECTIVETo assess the association of IFNG gene polymorphisms with preeclampsia among pregnant woman from Shaanxi Province.

METHODSGenomic DNA was extracted from peripheral blood samples collected from 280 patients with preeclampsia and 344 healthy pregnant women. Five tag single nucleotide polymorphisms (SNPs) of the IFNG gene (rs2069705, rs2430561, rs1861493, rs2069718, and rs2193050) were genotyped with a SNaPshot method. Genotypic and allelic frequencies were evaluated with a Chi square test. Genotype data was corrected by Logistic regression for body mass index and age. The level of IFN-gamma was determined with an ELISA assay.

RESULTSThe distribution of five tag SNPs all conformed to Hardy-Weinberg equilibrium (P> 0.05). Significant association with preeclampsia was found with the T allele of rs2430561 (OR=1.54, 95% CI:1.15-2.09, P=6.99× 10), under a dominant model (OR=3.77, 95% CI: 1.09-13.29, P=0.029) and a recessive model (OR=1.53, 95% CI:1.09-2.15, P=0.018). For the patient group, the IFN-gamma level of those with a TT genotype for rs2430561 was significantly higher than those with an AA or AT genotype [(13.69± 0.79) pg/mL vs. (13.11± 1.56) pg/mL, P< 0.05].

CONCLUSIONPolymorphism of the rs2430561 locus of the IFNG gene is associated with increased risk for preeclampsia as well as serum level of IFN-gamma among pregnant woman from Shaanxi. The role of the IFNG gene in the regulation of preeclampsia requires further investigation.

Adult ; Female ; Genetic Predisposition to Disease ; Genotype ; Humans ; Interferon-gamma ; blood ; genetics ; Logistic Models ; Polymorphism, Single Nucleotide ; Pre-Eclampsia ; etiology ; genetics ; Pregnancy

BACKGROUND: The associations between hepatitis B virus (HBV) infection and natural killer cell activity (NKA) have been studied. However, there have been no studies on detection of NKA in HBV patients using NK Vue Gold kit (ATGen Co., Korea), which has been developed in Korea and can easily detect NKA in whole blood. METHODS: A patient group of 40 patients diagnosed with hepatitis B antigen-positive chronic hepatitis B and a control group of 40 people eligible for blood donation were selected for this study. Whole blood (1 mL) was collected in special tubes for analysis with NK Vue Gold kit. The concentration of interferon-γ (IFN-γ) was measured and considered to represent NKA. RESULTS: There was no significant difference in the median (value, range) of concentration of IFN-γ between the patient (2,000, 89–2,000 pg/mL) and control groups (1,786, 333–2,000 pg/mL). The concentration of IFN-γ was inversely correlated with leukocyte count in both patient (r=–0.390, P=0.022) and control (r=–0.339, P=0.032) groups. The concentration of IFN-γ in the patient group was inversely correlated with concentrations of alanine aminotransferase (r=–0.363, P=0.021), aspartate aminotransferase (r=–0.336, P=0.037), and bilirubin (r=–0.376, P=0.020). CONCLUSIONS: There was no significant difference in NKA between the patient and control group. Caution should be practiced when interpreting results of NKA, because leukocyte count can affect concentration of IFN-γ.
Alanine Transaminase ; Aspartate Aminotransferases ; Bilirubin ; Blood Donors ; Hepatitis B ; Hepatitis B virus ; Hepatitis B, Chronic* ; Hepatitis, Chronic* ; Humans ; Interferon-gamma ; Killer Cells, Natural ; Korea ; Leukocyte Count

OBJECTIVETo primarily evaluate the effects of ulinastatin on immune function of patients with severe burn injury.

METHODSForty patients with severe burn admitted to our ward from March 2013 to October 2015, conforming to the study criteria, were divided into conventional treatment group (CT, n=20) and ulinastatin treatment group (UT, n=20) according to the random number table and patient's consent. After admission, patients in group CT received antishock treatment, antibiotic treatment, debridement, skin grafting, and nutrition support, etc. On the basis of the above-mentioned treatment, patients in group UT received intravenous drip of ulinastatin from first day after admission twice a day, with a dosage of 8×10(5) U every time, for 7 days in addition. Peripheral venous blood samples were collected from patients in groups CT and UT on post treatment day (PTD) 1, 3, 5 and 7, respectively. Twenty healthy volunteer were selected as health control group (HC), and peripheral venous blood samples were collected on the first day of the study. Percentage of CD4(+) CD25(+) regulatory T lymphocytes (Tregs) was determined by flow cytometer. The proliferative activity of T lymphocytes was detected by microplate reader (denoted as absorbance value). Content of interleukin 2 (IL-2) in culture supernatant of T lymphocytes, and content of IL-4 and &gamma; interferon (IFN-&gamma;) in serum were detected by enzyme-linked immunosorbent assay. Expression of human leukocyte antigen-DR (HLA-DR) on CD14(+) monocytes was determined by flow cytometer. Data were processed with analysis of variance for repeated measurement, chi-square test, and LSD-t test.

RESULTS(1) Compared with that of volunteer in group HC, the percentage of CD4(+) CD25(+) Tregs of patients in group CT was significantly increased from PTD 1 to 7 (with t values from 13.303 to 26.043, P values below 0.01). Compared with that in group CT, the percentage of CD4(+) CD25(+) Tregs of patients in group UT was significantly decreased on PTD 5 and 7 (with t values respectively 8.317 and 15.071, P values below 0.01). (2) The proliferative activity of T lymphocytes of patients in group CT on PTD 1, 3, 5, and 7 was respectively 0.71±0.11, 0.61±0.15, 0.54±0.12, and 0.67±0.17, which was significantly lower than that in group HC (1.21±0.22, with t values from 8.686 to 11.957, P values below 0.01). The proliferative activity of T lymphocytes of patients in group UT on PTD 3, 5, and 7 were respectively 0.81±0.11, 0.85±0.14, and 1.08±0.13, which was significantly higher than that in group CT (with t values from 4.808 to 8.568, P values below 0.01). (3) Compared with those of volunteer in group HC, content of IL-2 in culture supernatant of T lymphocytes of patients in group CT was significantly decreased from PTD 1 to 7 (with t values from 8.073 to 9.288, P values below 0.01), content of IL-4 in serum of patients in group CT was significantly increased from PTD 1 to 7 (with t values from 18.926 to 41.451, P values below 0.01), and content of IFN-&gamma; in serum of patients in group CT was significantly decreased from PTD 1 to 7 (with t values from 4.543 to 27.659, P values below 0.01). Compared with those in group CT, content of IL-2 in culture supernatant of T lymphocytes of patients in group UT was significantly increased from PTD 3 to 7 (with t values from 6.507 to 8.869, P values below 0.01), content of IL-4 in serum of patients in group UT was significantly decreased from PTD 3 to 7 (with t values from 6.922 to 8.843, P values below 0.01), and content of IFN-&gamma; in serum of patients in group UT was significantly increased on PTD 5 and 7 (with t values respectively 5.369 and 13.521, P values below 0.01). (4) The percentages of CD14(+) monocytes with positive expression of HLA-DR of patients in group CT on PTD 1, 3, 5, and 7 were respectively (28±6)%, (25±7)%, (25±7)%, and (39±10)%, which were significantly lower than the percentage of volunteer in group HC [(87±8)%, with t values from 16.323 to 25.645, P values below 0.01]. The percentages of CD14(+) monocytes with positive expression of HLA-DR of patients in group UT on PTD 3, 5, and 7 were respectively (40±6)%, (42±9)%, and (49±10)%, which were significantly higher than those in group CT (with t values from 3.071 to 7.324, P values below 0.01).

CONCLUSIONSOn the basis of CT, additional ulinastatin intervention can decrease CD4(+) CD25(+) Tregs percentage, improve the immune function of T lymphocytes and T helper cells, and increase expression of HLA-DR on CD14(+) monocytes of patients with severe burn injury, thus improve the immune function of patients.

Burns ; drug therapy ; immunology ; Cells, Cultured ; Debridement ; Enzyme-Linked Immunosorbent Assay ; Glycoproteins ; therapeutic use ; Humans ; Interferon-gamma ; blood ; Interleukin-2 ; metabolism ; Interleukin-4 ; blood ; Monocytes ; immunology ; Skin Transplantation ; T-Lymphocytes, Regulatory ; immunology

OBJECTIVETo observe the effects of ulinastatin on immune function of splenic CD4(+) T lymphocytes and CD4(+) CD25(+) regulatory T lymphocytes (Tregs) and content of high mobility group box 1 (HMGB1) in peripheral blood of severely burned rats, and to analyze the possible mechanisms.

METHODSNinety-six male SD rats were divided into sham injury group, burn group, and ulinastatin group according to the random number table, with 32 rats in each group. Rats in sham injury group were sham injured on the back by immersing in 37 ℃ warm water for 12 s. Rats in burn group and ulinastatin group were inflicted with 30% total body surface area full-thickness scald (hereinafter referred to as burn) on the back by immersing in 94 ℃ hot water for 12 s. Immediately after injury, rats in each group were intraperitoneally injected with saline (40 mL/kg), meanwhile rats in ulinastatin group were intraperitoneally injected with ulinastatin (4×10(4) U/kg), once per 12 h, till post injury hour 72. Eight rats of each group were respectively selected on post injury day (PID) 1, 3, 5, and 7 to collect abdominal aortic blood samples. Serum content of HMGB1 was detected by enzyme-linked immunosorbent assay (ELISA). And then, rats of the 3 groups were sacrificed immediately to collect spleens and separate CD4(+) CD25(+) Tregs and CD4(+) T lymphocytes. Flow cytometer was used to detect positive expression rates of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and forkhead-winged helix transcription factor p3 (Foxp3) in CD4(+) CD25(+) Tregs. Content of IL-10 in culture supernatant of CD4(+) CD25(+) Tregs, and content of interleukin 2 (IL-2), IL-4, and &gamma; interferon (IFN-&gamma;) in culture supernatant of CD4(+) T lymphocytes was detected by ELISA. The proliferative activity of CD4(+) T lymphocytes was determined by microplate reader. The sample number of above-mentioned experiments was 8 at each time point in each group. Data were processed with analysis of variance of factorial design and LSD test.

RESULTS(1) Compared with that in sham injury group, serum content of HMGB1 of rats in burn group was significantly increased from PID 1 to 7 (with P values below 0.01). Compared with that in burn group, serum content of HMGB1 of rats in ulinastatin group was significantly decreased from PID 1 to 7 (with P values below 0.01). (2) Compared with those in sham injury group, the positive expression rates of CTLA-4 and Foxp3 in CD4(+) CD25(+) Tregs and content of IL-10 in culture supernatant of CD4(+) CD25(+) Tregs of rats in burn group were significantly increased from PID 1 to 7 (with P values below 0.01), peaking on PID 3 [(65±10)%, (76±10)%, and (28.2±4.4) pg/mL respectively]. These 3 indexes of rats in sham injury group on PID 3 were (45±7)%, (46±7)%, and (11.2±2.3) pg/mL respectively. Compared with those in burn group, the positive expression rates of CTLA-4 and Foxp3 in CD4(+) CD25(+) Tregs and content of IL-10 in culture supernatant of CD4(+) CD25(+) Tregs of rats in ulinastatin group were significantly decreased from PID 1 to 7 (P<0.05 or P<0.01), reaching the nadir on PID 7 [(43±6)%], PID 1 [(50±8)%], and PID 7 [(12.4±3.4) pg/mL] respectively. These 3 indexes of rats in burn group on PID 7, 1, and 7 were (58±8)%, (71±9)%, and (19.7±2.8) pg/mL respectively. (3) Compared with those in sham injury group, the content of IL-2 and IFN-&gamma; in culture supernatant of CD4(+) T lymphocytes of rats was significantly decreased, while the content of IL-4 in culture supernatant of CD4(+) T lymphocytes of rats was significantly increased in burn group from PID 1 to 7, with P values below 0.01. Compared with that in burn group, the content of IL-2 and IFN-&gamma; in culture supernatant of CD4(+) T lymphocytes of rats was significantly increased, while the content of IL-4 in culture supernatant of CD4(+) T lymphocytes of rats was significantly decreased in ulinastatin group from PID 1 to 7, P<0.05 or P<0.01. (4) Compared with that in sham injury group, the proliferative activity of CD4(+) T lymphocytes of rats in burn group was significantly decreased from PID 1 to 7 (with P values below 0.01). Compared with that in burn group, the proliferative activity of CD4(+) T lymphocytes of rats in ulinastatin group was significantly increased from PID 1 to 7 (with P values below 0.01).

CONCLUSIONSUlinastatin can weaken the immunosuppressive function mediated by splenic CD4(+) CD25(+) Tregs in severely burned rats, and improve proliferative function and secretory function of splenic CD4(+) T lymphocytes, which may be attributed to the inhibiting effect of ulinastatin on the release of HMGB1 in large amount.

Animals ; Burns ; drug therapy ; CTLA-4 Antigen ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Forkhead Transcription Factors ; metabolism ; Glycoproteins ; pharmacology ; HMGB1 Protein ; blood ; Interferon-gamma ; metabolism ; Interleukin-10 ; metabolism ; Interleukin-2 ; metabolism ; Interleukin-4 ; metabolism ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Spleen ; drug effects ; T-Lymphocytes, Regulatory ; cytology ; drug effects

OBJECTIVETo study the expression levels of annexin A1 (ANXA1), GATA-3, and T-bet in T lymphocytes of peripheral blood in burned mice with sepsis at early stage, and to analyze their immune regulatory mechanisms.

METHODSSeven-hundred and eighty male mice of clean grade were divided into sham injury group (n=60, sham injured on the back by immersing in 37 ℃ warm water for 10 s), burn group (n=240, inflicted with 20% TBSA deep partial- thickness burn on the back by immersing in 100 ℃ hot water for 10 s), sepsis group (n=240, intraperitoneally injected with 6 mg/kg lipopolysaccharide), and burn+ sepsis group (n=240) according to the random number table. Mice of burn+ sepsis group were treated as that in burn group at first, and then they were treated as that in sepsis group. (1) Immediately after injury, six mice in sham injury group were selected to collect lymphocyte suspension of peripheral blood (1 tube each mouse) according to the random number table. According to the random number table, 6 mice of each of the other three groups were respectively selected at post injury hour (PIH) 12, 24, 48, and 72 for the collection of lymphocyte suspension from peripheral blood (1 tube each mouse). Each tube of cell suspension was equally divided into two parts. Fluorescein isothiocyanate (FITC)-labeled human anti-mouse CD4 monoclonal antibody and phycoerythrin (PE)-labeled human anti-mouse interferon-&gamma; monoclonal antibody were added to one part of cell suspension to mark helper T lymphocyte 1 (Th1). FITC-labeled human anti-mouse CD4 monoclonal antibody and PE-labeled human anti-mouse interleukin-4 (IL-4) monoclonal antibody were added to the other part of cell suspension to mark Th2. The percentages of Th1 and Th2 were determined with flow cytometer, and the ratio of Th1 to Th2 was calculated. (2) According to the random number table, 18 mice in sham injury group were selected immediately after injury for the collection of lymphocyte suspension of peripheral blood (1 tube each mouse), and 18 mice of each of the other 3 groups were respectively selected at PIH 12, 24, 48, and 72 to collect the lymphocyte suspension of peripheral blood (1 tube each mouse). The mRNA expression levels of ANXA1, GATA-3, and T-bet were determined by real-time fluorescent quantitative reverse transcription-PCR. (3) Immediately after injury, 36 mice in sham injury group were selected to collect lymphocyte suspension of peripheral blood (1 tube each mouse) according to the random number table, and then 36 tubes of cell suspension were divided into 6 batches (6 tubes each batch). Each one of 6 kinds of antibody combinations: antibodies for labeling Th1 and Th2 in combination with PE-anthocyanin 7 labeled human anti-mouse ANXA1 monoclonal antibody, PE-anthocyanin 7 labeled human anti-mouse GATA-3 monoclonal antibody, and PE-anthocyanin 7 labeled human anti-mouse T-bet monoclonal antibody was added to 1 tube of cell suspension at each batch. According to the random number table, 36 mice of each of the other 3 groups were respectively selected at PIH 12, 24, 48, and 72 for the collection of lymphocyte suspension of peripheral blood (1 tube each mouse), and then 36 tubes of cell suspension at each time point were divided into 6 batches for marking with 3 kinds of surface markers of Th1 and Th2 (6 tubes each batch). Each one of above-mentioned 6 kinds of antibodies was added to 1 tube of cell suspension at each time point for each batch. The percentages of ANXA1, GATA-3, and T-bet positive cells in Th1 and Th2 were determined with flow cytometer. Data were processed with one-way analysis of variance, analysis of variance of factorial design, and SNK test. The relationship between the percentages of ANXA1 positive cell and the percentages of GATA-3 positive cell in Th1 and Th2, and mRNA expression level of ANXA1 and mRNA expression level of GATA-3 in lymphocytes were assessed by linear correlation analysis.

RESULTS(1) Compared with those in sham injury group immediately after injury, the percentages of Th1 and Th2 and the ratio of Th1 to Th2 of mice in burn group were significantly decreased from PIH 24 on, with P values below 0.05; the percentages of Th1 and Th2 and the ratios of Th1 to Th2 of mice in sepsis group and burn+ sepsis group were significantly decreased from PIH 12 on, with P values below 0.05. (2) Compared with those in sham injury group immediately after injury, the mRNA expression levels of ANXA1 and GATA-3 in lymphocyte of mice in burn group were significantly decreased from PIH 24 on, with P values below 0.05; the mRNA expression level of T-bet was significantly decreased at PIH 24 but significantly increased at PIH 48 and 72, with P values below 0.05. Compared with those in sham injury group immediately after injury, the mRNA expression levels of ANXA1 and GATA-3 in lymphocytes of mice in sepsis group were significantly decreased from PIH 12 on, and the mRNA expression level of T-bet was increased significantly from PIH 12 on, with P values below 0.05; the mRNA expression levels of ANXA1, GATA-3, and T-bet in lymphocytes of mice in burn+ sepsis group were significantly decreased from PIH 12 on, with P values below 0.05, reaching the nadir at PIH 72 (0.50±0.04, 0.45±0.03, 0.21±0.05, respectively). (3) A significant positive correlation was observed between ANXA1 mRNA expression level and GATA-3 mRNA expression level in lymphocytes of peripheral blood (r=0.862, P<0.05). (4) Compared with those in sham injury group immediately after injury, the percentages of ANXA1 and GATA-3 positive cellsin Th1 and Th2 of mice in burn group were significantly lowered from PIH 24 on, and the percentage of T-bet positive cells was significantly decreased at PIH 24, but it was increased from PIH 48 on, with P values below 0.05. The percentages of ANXA1 and GATA-3 positive cells in Th1 and Th2 of mice in sepsis group were continuously decreased from PIH 12 on, which were lower at most time points than those in sham injury group immediately after injury, with P values below 0.05. The percentages of T-bet positive cells in Th1 and Th2 of mice in sepsis group were significantly increased since PIH 12 as compared with those in sham injury group immediately after injury, with P values below 0.05. The percentages of ANXA1, GATA-3, and T-bet positive cells in Th1 and Th2 of mice in burn+ sepsis group were continuously lowered from PIH 12, with significantly statistical differences at most time points as compared with those in sham injury group immediately after injury, with P values below 0.05. (5) The percentages of GATA-3 positive cells in Th1 and Th2 were significantly positively correlated with those of ANXA1 (with r values respectively 0.747 and 0.787, P values below 0.05).

CONCLUSIONSThe expression levels of ANXA1, GATA-3, and T-bet were continuously lowered in burned mice with sepsis, and it may play an important role in Th1/Th2 balance switching to Th2 bias and immunosuppressive process.

Animals ; Biomarkers ; Burns ; immunology ; metabolism ; GATA3 Transcription Factor ; biosynthesis ; genetics ; Humans ; Interferon-gamma ; biosynthesis ; genetics ; Interleukin-4 ; metabolism ; Male ; Mice ; RNA, Messenger ; Real-Time Polymerase Chain Reaction ; Sepsis ; blood ; T-Lymphocytes ; metabolism ; Transcription Factors ; biosynthesis ; genetics

BACKGROUNDChronic obstructive pulmonary disease (COPD) is characterized by progressive loss of lung function and local and systemic inflammation, in which CD8+ T-cells are believed to play a key role. Activated CD8+ T-cells differentiate into distinct subpopulations, including interferon-&gamma; (IFN-&gamma;)-producing Tc1 and interleukin (IL)-17-producing Tc17 cells. Recent evidence indicates that Tc17 cells exhibit considerable plasticity and may convert into IL-17/IFN-&gamma;-double producing (Tc17/IFN-&gamma;) cells when driven by inflammatory conditions. The aim of this study was to investigate the Tc17/IFN-&gamma; subpopulation in peripheral blood of patients with COPD and to evaluate their potential roles in this disease.

METHODSPeripheral blood samples were collected from 15 never-smokers, 23 smokers with normal lung function, and 25 patients with COPD (Global Initiative for Chronic Obstructive Lung Disease 2-4). Proportions of the IL-17/IFN-&gamma;-double expressing subpopulation were assessed using flow cytometry. Plasma concentrations of cytokines favoring Tc17/IFN-&gamma; differentiation were measured by enzyme-linked immunosorbent assay.

RESULTSPatients with COPD had higher proportions of Tc17 cells and Tc17/IFN-&gamma; cells in the peripheral blood than smokers and never-smokers. The plasticity of Tc17 cells was higher than that of Th17 cells. The percentages of Tc17 cells and Tc17/IFN-&gamma; cells showed negative correlations with forced expiratory volume in 1 s % predicted value (r = -0.418, P = 0.03; r = -0.596, P = 0.002, respectively). The plasma concentrations of IL-6, transforming growth factor-β1, and IL-12 were significantly higher in patients with COPD compared with smokers and never-smokers.

CONCLUSIONSPeripheral Tc17 cells are increased and more likely to convert to Tc17/IFN-&gamma; cells in COPD, suggesting that Tc17 cell plasticity may be involved in persistent inflammation of the disease.

Aged ; Female ; Forced Expiratory Volume ; Humans ; Interferon-gamma ; biosynthesis ; Interleukin-12 ; blood ; Interleukin-6 ; blood ; Lung ; physiopathology ; Male ; Middle Aged ; Pulmonary Disease, Chronic Obstructive ; immunology ; physiopathology ; Th17 Cells ; immunology