Objective To estimate the activity of the phosphatidylinositol3-kinase (PI3K) signaling pathway in peripheral blood T cells from patients with atopic dermatitis (AD),and to investigate its clinical significance.Methods T cells were isolated by using the Rosettsep T cell purification kit from the peripheral blood of 38 patients with AD and 38 healthy human controls,and classified into several groups to be treated with anti-CD3 monoclonal antibody,anti-CD28 monoclonal antibody,and LY294002 (an inhibitor of PI3K) respectively.The activity of PI3K signaling pathway in T cells was estimated by immunoprecipitation and enzyme-linked immunosorbent assay (ELISA).Western blot was performed to measure the expressions of total Akt and phosphorylated Akt in T cells,methyl thiazolyl tetrazolium (MTT) assay to examine the proliferation of T cells,and ELISA to determine the levels of interleukin 6 (IL-6) and IL-10.Results The activity of PI3K and Akt was significantly higher in freshly isolated patient-derived T cells than in control-derived T cells (both P ＜ 0.05).However,the difference in the activity of PI3K and Akt between patient-derived and control-derived T cells disappeared (both P ＞ 0.05) after 24-hour in vitro culture.The activity of PI3K and Akt in control-derived T cells was significantly increased after 24-hour incubation with sera from the patients with AD (both P ＜ 0.05).In addition,compared with patient-derived T cells treated with patients' sera or anti-CD3/CD28 monoclonal antibody alone,those treated with the combination of LY294002 and patients' sera or anti-CD3/CD28 monoclonal antibody showed a significant decrease in the proliferative activity (63％ ± 11％ vs.123％ ± 25％,125％ ± 22％ vs.195％ ± 28％,both P＜ 0.05),supematant levels of IL-6 ((168 ± 33) vs.(265 ± 46) ng/L,(431 ± 64) vs.(823 ± 128) ng/L,both P＜ 0.05) and IL-10 ((56 ± 14) vs.(98 ± 25) ng/L,(120 ± 21) vs.(213 ± 35) ng/L,both P＜ 0.05).Eczema area and severity index (EASI) was unassociated with the activity of PI3K or Akt in fresh T cells from patients with AD (both P ＞ 0.05).Conclusions The PI3K signaling pathway is abnormally activated in peripheral blood T cells from patients with AD,which is associated with the proliferation of,as well as secretion of cytokines by,T cells,suggesting that there exist serum factors activating this pathway in peripheral blood of patients with AD.

Objective To study the activation of alveolar macrophage β (AM) and the expression of co-stimulatory molecule CD40 in transfusion-related acute lung injury (TRALI) model in order to illustrate the pathogenesis of TRALI.Methods Sixty SD rats were randomly (random number) divided into normal control group (n =15) with sham operation using normal saline instead of LPS and plasma,positive control group (n =15) with ALI induced by intravenous infusion of 5 mg/kg lipopolysaccharide (LPS) in equivalent volume of whole blood drawn out),and TRALI group (n =15) treated by intra-peritoneal injection of 2 mg/kg LPS 2 h before the transfusion of human plasma (1 mL whole blood about 10％ of total blood volume drawn out and replaced with 1 mL plasma),LPS control group (n =15) treated by intra-peritoneal injection of 2 mg/kg LPS 2 h before saline infusion in equivalent volume of blood drawn out.The pathologic changes of rat lung tissue were observed by HE staining.The expression of TLR4 was examined by RT-PCR.The activation of NF-κB in AM was measured by electrophoresis mobility shift assay (EMSA).The expression of CD40 mRNA and CD40 molecule were analyzed by Northern blot and flow cytometry respectively.ELISA was performed to detect the concentration of TNF-α,MIP-2 and IL-1 β in broncho-alveolar lavage fluid (BALF).Results Broken alveolar septa,hyperemia,and massive infiltration of inflammatory cells including the neutrophils were observed in lung tissues of TRALI group.The expression of TLR4 gene was detected in activated macrophage phi (AMφ) of TRALI group rats.The activation of NF-κB was increased in TRALI group rats.The expression of CD40 in AMφ was higher in rats of TRALI group than that in rats of control group and LPS control group.The concentration of TNF-αt,MIP-2 and IL-1β were enhanced significantly in BALF of TRALI group rats.Conclusion The activation of AM and up-regulation of costimulatory molecule CD40 induced release of some inflammatory cytokines.It suggested that AM activation may play an important role in the pathogenesis of TRALI.

Objective To investigate the effects of miRNA-146a on the differentiations and functions of CD4+ T lymphocytes in patients with psoriasis vulgaris.Metbods Thirty patients with psoriasis vulgaris and twenty heathy subjects were enrolled in this study.Quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of miRNA-146a in CD4+ T lymphocytes isolated from the peripheral blood samples.The levels of IFN-γ and IL-4 in serum samples were determined by using enzyme-linked immunosorbent assay (ELISA).Mononuclear cells were isolated from human peripheral blood samples by using Ficoll-Hypaque density-gradients centrifugation,from which the CD4+ T lymphocytes were separated by magnetic-activated cell sorting.The CD4+ T cells (2× 106/ml) were seeded in culture plates with 6 wells.The CD4+ T lymphocytes were divided into 3 groups including the control group,miRNA-146a group and miRNA-146a inhibitor group.The numbers of Th1 and Th2 cells were measured by flow cytometry analysis (FACS).The expression of IFN-γRα,T-bet and GATA-3 at mRNA and protein levels were measured by using RT-PCR and Western blot assay,respectively.The levels of IFN-γ and IL-4 in culture supernatants of CD4+ T lymphocytes were detected by using ELISA.Results In comparison with the normal control group,there were significant increases in the expression of miRNA-146a in CD4+ T lymphocytes and the level of IFN-γin serum samples from patients with psoriasis vulgaris [(2.43±0.94) vs (1.05±0.23),(27.69±7.64) ng/L vs (9.75±2.81) ng/L,all P＜0.01].A positive correlation between the expression of miRNA-146a and the level of IFN-γ in serum was observed (r=0.837,P＜0.01).Results of the in vitro culture of CD4+ T lymphocytes showed that the number of Th1 cells,the expression of T-bet at mRNA and protein levels and the level of IFN-γin culture supernatant were significantly increased,while the expression of IFN-γRα protein was decreased in the miRNA-146a group in comparison with those of the control group (all P＜0.01).No significant differences in the number of Th2 cells,the expression of GATA-3 protein,the expression of GATA-3 and IFN-γRα at mRNA level and the level of IL-4 in culture supernatants were found between the control and miRNA-146a groups (all P＞0.05).The miRNA-146a inhibitor could effectively attenuate the effects of miRNA-146a on Th1 cells.Conclusion The miRNA-146a could promote the differentiation and enhance the function of Th1 cells.It might play an important role in the pathogenesis of psoriasis vulgaris.

Objective To evaluate changes of Rho kinase (ROK)activity in peripheral blood T lymphocytes from patients with atopic dermatitis (AD), and to analyze their clinical significance. Methods Eight milliliters of heparin-anticoagulated blood samples were collected from 60 patients with AD and 60 healthy human controls followed by separation of T lymphocytes and sera from these blood samples as well as culture of isolated T lymphocytes with 10% fetal bovine serum for 24 hours. Both patient- and control-derived T lymphocytes were classified into two groups to be cultured with patient- or control-derived sera. In addition, some patient-derived T lymphocytes were classified into 4 groups:Y27632 group treated with the Rho kinase-specific inhibitor Y2763, CD3/CD28 group treated with anti-CD3/anti-CD28 monoclonal antibodies, Y27632 + CD3/CD28 group treated with Y27632 and anti-CD3/anti-CD28 monoclonal antibodies, and control group treated with patient-derived sera. Subsequently, Western-blot analysis was performed to evaluate ROK activity in cells, methyl thiazolyl tetrazolium(MTT)assay to evaluate proliferative activity of T lymphocytes, and ELISA to measure interleukin 6 (IL-6)and IL-10 levels in supernatants of T lymphocytes. Results ROK activity was significantly lower in fresh T lymphocytes from patients than in those from healthy controls (2.47% ± 0.89% vs. 0.65% ± 0.35%, t =2.729, P < 0.05). After 24-hour culture with 10% fetal bovine serum in vitro, ROK activity was significantly decreased in patient-derived T lymphocytes compared with those before culture (0.70% ± 0.38% vs. 2.47% ± 0.89%, t = 2.658, P <0.05), but no significant difference was observed between patient- and control-derived T lymphocytes(0.70% ± 0.38% vs. 0.63% ± 0.32%, t = 1.010, P > 0.05). Compared with T lymphocytes cultured with control-derived sera, those cultured with patient-derived sera showed significantly increased ROK activity (F = 8.22, P < 0.001). Concretely speaking, ROK activity was significantly higher in patient-derived T lymphocytes cultured with patient-derived sera than in those cultured with control-derived sera (2.41% ± 0.87% vs. 0.76% ± 0.41%, P < 0.05), and higher in control-derived T lymphocytes cultured with patient-derived sera than in those cultured with control-derived sera(2.17% ± 0.85% vs. 0.64% ± 0.33%, P< 0.05)at 24 hours. Y27632 could significantly inhibit the proliferation of as well as secretion of IL-6 (F = 18.68, 22.95, respectively, both P < 0.001)by patient-derived T lymphocytes, but had insignificant effects on secretion of IL-10. The cellular proliferative activity and IL-6 supernatant level were significantly lower in the Y27632 group than in the control group, and lower in the Y27632 + CD3/CD28 group than in the CD3/CD28 group (all P < 0.05). Conclusion Aberrant activation of ROK exists in T lymphocytes from patients with AD, which may play a certain role in the pathogenesis of AD.

Objective To evaluate regulatory effects of miRNA-146a on peripheral blood CD4 + T lymphocytes from patients with psoriasis vulgaris, and to investigate the role of miRNA-146a in the pathogenesis of psoriasis vulgaris. Methods Totally, 30 patients with psoriasis vulgaris and 30 healthy human controls were enrolled into this study. Venous blood samples were obtained from these subjects, and CD4 + T lymphocytes were isolated from these samples by using magnetic activated cell sorting (MACS). Real-time quantitative PCR (RT-PCR)was performed to measure the expression of miRNA-146a in peripheral blood CD4+ T lymphocytes, and enzyme-linked immunosorbent assay(ELISA)to determine plasma levels of interferon-γ(IFN-γ)and interleukin 4(IL-4). Some CD4+ T lymphocytes were divided into 3 groups to be transfected with 50 nmol/L negative control miRNA (control group), miRNA-146a mimics(miRNA-146a group)or miRNA-146a inhibitor (miRNA-146a inhibitor group). After 24-hour additional culture, flow cytometry was conducted to determine the number of Th1 and Th2 cells, Western-blot analysis and RT-PCR were performed to measure the protein and mRNA expressions of IFN-γ receptor α (IFN-γRα), T-box expressed in T cells (T-bet)and GATA-binding protein-3 (GATA-3)respectively, and ELISA was carried out to determine the levels of IFN-γ and IL-4 in supernatants of CD4 + T lymphocytes. Results Compared with the healthy control group, the patient group showed significantly increased miRNA-146a expression in peripheral blood CD4 + T lymphocytes (243.81% ± 94.32% vs. 105.74% ± 22.93%, t = 6.653, P < 0.01)and plasma IFN-γ level (27.69 ± 7.64 ng/L vs. 9.75 ± 2.81 ng/L, t = 4.237, P <0.01). Moreover, miRNA-146a expression was positively correlated with plasma IFN-γ level in the patients(r = 0.837, P <0.01). After 24-hour culture in vitro, there was a significant increase in the number of Th1 cells, protein and mRNA expressions of T-bet, and supernatant level of IFN-γ, but a significant decrease in the protein expression of IFN-γRα in the miRNA-146a group compared with the control group (all P < 0.01). However, no significant differences were observed in the number of Th2 cells, mRNA or protein expressions of GATA-3, or supernatant level of IL-4 among the control group,miRNA-146a group and miRNA-146a inhibitor group (all P > 0.05). Conclusion miRNA-146a may play an important role in the pathogenesis of psoriasis vulgaris by participating in the regulation of peripheral blood CD4+ T lymphocytes via affecting Th1 cell differentiation and function.

Objective To study the quantity change and significance of myeloid‐derived suppressor cells(MDSCs) in patients withpost‐traumaticmultipleorgandysfunctionsyndrome(MODS).Methods 66patientswithMODS,34patientswithnon‐system‐ic inflammatory response syndrome(SIRS)and 37 healthy volunteers were enrolled in this study .Peripheral blood was collected and CD14-CD11b+ CD33+ were used as markers of MDSCs .The percentage of MDSCs was determined by flow cytometry and serum interleukin‐10(IL‐10) and tumor necrosis factor‐α(TNF‐α) levels were determined by ELISA .The MODS scoring system was used to assess patients′disease severity .The relationship was analyzed between MDSCs and TNF‐αand MODS score .Results The per‐centage of MDSCs in peripheral blood of healthy volunteers was(1 .18 ± 0 .22)% .after MODS ,the percentage of MDSCs in periph‐eral blood was(11 .84 ± 2 .18)% and(6 .52 ± 0 .37)% in patients with non‐MODS ,the percentages of MDSCs in three groups showed significant differences(P<0 .05) .Serum IL‐10 and TNF‐αin patients with MODS group and non‐MODS group were signif‐icant differences(P<0 .05) .The correlation was found between MDSCs percentage in peripheral blood and MODS score and TNF‐α(r=0 .342 6 ,0 .387 9 respectively ,P<0 .05) .Conclusion The increase proportion of MDSCs in peripheral blood correlates with the onset of infection in patients with MODS ,indicating that the expansion of MDSCs in peripheral blood may play important roles in immune dysfunction after MODS .

Objective To investigate the expression of miRNA-203 in skin lesions of patients with psoriasis vulgaris,and to explore its effect on the proliferation of a human keratinocyte cell line HaCaT.Methods Lesional skin and adjacent non-lesional skin tissues were obtained from 23 patients with psoriasis vulgaris from 2014 to 2016.Fluorescence-based quantitative PCR was performed to determine the expression of miRNA-203 in these skin tissues.Targeted miRNA in skin tissues was in situ hybridized by using 5'and 3'digoxigenin-labelled probes,so as to localize the expression of miRNA-203 in skin tissues.Cultured HaCaT cells were divided into 3 groups:miRNA-203 mimic group and negative control group transfected with miRNA-203 mimics and negative control miRNA-203 respectively,and blank control group receiving no treatment.Methyl thiazolyl tetrazolium (MTT) assay,flow cytometry and Western blot analysis were performed to investigate changes in cellular proliferative activity,cell cycle and its related proteins Cyclin D1 and Cyclin B1 in HaCaT cells respectively.Results MiRNA-203 was specifically expressed in epidermal keratinocytes.Besides the cell nuclei,it could be expressed in the cytoplasm.In the patients with psoriasis vulgaris,the expression of miRNA-203 was significantly higher in lesional skin tissues than in non-lesional skin tissues (1.35 ± 0.28 vs.0.52 ± 0.09,t =6.76,P =0.012).The transfection with miRNA-203 mimics could significantly inhibit the proliferation of HaCaT cells (F =9.36,P =0.007).Additionally,the blank control group,negative control group and miRNA-203 mimic group all showed a gradual increase in proliferative activity of HaCaT cells over time (F =18.68,P ＜ 0.001).HaCaT cells were arrested in G2/M phase in the miRNA-203 mimic group with the percentage of cells in G2/M phase being 31.33％ ± 4.56％,compared to 17.02％ ± 3.53％ in the negative control group (P ＜ 0.05) and 16.67％ ± 3.32％ in the blank control group (P ＜ 0.05).Moreover,the miRNA-203 mimic group showed significantly higher protein expression of Cyclin D1 (1.15 ± 0.13),but significantly lower protein expression of Cyclin B1 (0.43 ± 0.08),compared with the negative control group (0.52 ± 0.05,0.93 ± 0.16,respectively,both P ＜ 0.05) and blank control group (0.56 ± 0.07,0.91 ± 0.0.15,respectively,both P ＜ 0.05).Conclusion MiRNA-203 may participate in the occurrence and development of psoriasis vulgaris.

Objective:To explore the effect of decision-making assistance during the implantation selection of breast augmentation patients to provide a reference for improving decision-making aids.Methods:From June 2018 to June 2020, the decision-making assistance programs were used in the Department of Plastic and Reconstructive Surgery, Shanghai Ninth People＇s Hospital for prosthesis selection in 51 breast augmentation female patients, aged 23 to 42 years, with an average of 31 years old. The BREAST-Q scale was applied to assess the postoperative results of augmentation patients. Postoperative breast satisfaction was also evaluated by the physicians.Results:With decision-making assistance, breast augmentation patients＇ satisfaction with breasts was (80.27±11.45) points, satisfaction with surgical results (83.41±12.29) points, social and psychological status scores (87.24±7.62) points, and sexual life status scores (85.49±7.90) points, physical condition score (73.94±8.98) points. There was no statistical difference in the scores of breast size between physicians and patients ( P>0.05). In the satisfaction score and total score of breast shape and feeling, physicians＇ scores were higher than patients＇ self-report scores, and the difference was statistically significant ( P<0.05). Conclusions:The patient self-reported postoperative outcomes are at a high level under the application of decision aid program. We can further improve the decision aid program for breast augmentation patients, adjust patient＇s surgical expectation, and realize shared decision making.

OBJECTIVETo evaluate the changes of CD(4)(+) IL-17+T (Th17) and CD(4)(+)Foxp3+regulatory T (Treg) cells in peripheral blood and bronchoalveolar lavage fluid (BALF) , and therefore to explore the role of Th17 and Treg in acrolein exposure airway inflammation in rats.

METHODSForty male Wistar rats were randomly divided into 4 groups: a 2 wk acrolein exposure group, a 4 wk acrolein exposure group, a 2 wk control group and a 4 wk control group (n=10 each). Cells in BALF were collected and analyzed by absolute and differential cell counts.IL-17 and IL-6 levels in serum and BALF were tested by enzyme linked immunosorbent assay (ELISA). The proportion of CD(4)(+)IL-17+T and CD(4)(+) Foxp3+Treg in peripheral blood and BALF were determined by flow cytometry.The mRNA expressions of IL-17 and Foxp3 were measured by real-time PCR. Comparisons of the data between different groups were performed using one-way ANOVA, and SNK and Games-Howell test were used for comparison between 2 groups.

RESULTSLevels of IL-17 were remarkable increased in the 2 wk acrolein exposure group and the 4 wk acrolein exposure group in serum [(52.64 ± 1.89) ng/L, (76.73 ± 5.57) ng/L], and BALF [(79.07 ± 5.67) ng/L, (96.61 ± 6.44) ng/L] compared with the 2 wk control group [(40.05 ± 3.12) ng/L, (56.75 ± 4.37) ng/L] and the 4 wk control group [(38.75 ± 3.23) ng/L, (53.27 ± 4.48) ng/L], all P<0.01. IL-6 was increased in the 2 wk and the 4 wk acrolein exposure group [ (33.28 ± 2.27) ng/L, (46.24 ± 3.16) ng/L] compared with the 2 wk and the 4 wk control group [ (16.37 ± 1.49) ng/L, (17.02 ± 1.43) ng/L] in BALF.Ratio of Th17 was higher in the 2 wk and the 4 wk acrolein exposure groups in peripheral blood (1.82 ± 0.18) %, (3.75 ± 0.48) % and BALF [(7.23 ± 0.27) %, (8.12 ± 0.38) %] compared with the 2 wk [(0.96 ± 0.07) %, (5.64 ± 0.63) %] and the 4 wk control group [(1.01 ± 0.08) %, (5.86 ± 0.57) %]. Ratio of Treg in BALF was higher in the acrolein exposure groups [ (8.83 ± 0.52) %, (12.05 ± 0.74) %] compared with the control groups [(4.37 ± 0.27) %, (5.01 ± 0.37) %]. The level of IL-17 mRNA was increased in the 2 wk and the 4 wk acrolein exposure group in peripheral blood [(25.78 ± 2.31), (34.69 ± 2.01) ] and in BALF [(23.04 ± 1.78), (34.56 ± 3.12)] compared with the 2 wk [(11.04 ± 2.53), (11.08 ± 2.05)] and the 4 wk [(12.03 ± 2.34), (12.69 ± 2.69)] control groups. Foxp3 mRNA was increased in the acrolein exposure groups [ (26.37 ± 3.24), (33.19 ± 2.98)] (24.4 ± 2.7), (30.3 ± 2.7) compared with the control groups [(12.37 ± 2.56), (13.12 ± 3.08)]. Th17 in acrolein exposure groups was positively correlated with counts of total cells and macrophages (r=0.5126, 0.5437, all P<0.01).

CONCLUSIONSA changed expression of Th17 and Treg cells and an vary of inflammatory cytokines were evident in airway inflammation of acrolein exposed rats, suggesting that Treg was involved in the immunological regulation and Th17 was associated with the persistent inflammation in acrolein induced airway inflammation in rats.

Acrolein ; toxicity ; Animals ; Bronchoalveolar Lavage Fluid ; cytology ; Cytokines ; metabolism ; Forkhead Transcription Factors ; metabolism ; Inflammation ; metabolism ; Male ; Rats ; Rats, Wistar ; T-Lymphocytes, Regulatory ; cytology ; Th17 Cells ; cytology

OBJECTIVETo investigate the transduction pathway of triggering receptor-1 expressed on myeloid cells (TREM-1) in acute lung injure induced by paraquat in rats through the activating or blocking TREM-1, to observe the effect of signal transduction pathway in the acute lung injure induced-paraquat.

METHODS80 SD rats were randomly divided into normal saline control group (n=20) , PQ poisoning group (n=20) , antibody group (n=20) , and LP17 group (n=20). poisoning group, antibody group and LP17 group were given saline diluting PQ 80 mg/kg of disposable lavage after 2 h, a single set of intraperitoneal injection of anti-TREM-1 mAb (250 g/kg) , tail intravenous LP17 group synthetic peptide (3.5 mg/kg) , poisoning group was given equal normal saline intraperitoneal injection, control group given normal saline 1 mg/kg after 2 h after lavage, given the amount of intraperitoneal injection of saline solution. The expression of NF-κB in lung tissue was determined by immunohistochemistry.The levels of TNF-a, IL-10, TREM-1, and soluble TREM-1 (sTREM-1) in lung tissue and serum were measured by ELISA. Pathology changes of lung were observed under light microscope, and lung score of pathology was compared.

RESULTSAdministration of anti-TREM-1 mAb after PQ poisoning modeling significantly increased the NF-κB expression in lung tissue at 48 h, resulting in a large number of pro-inflammatory cytokines releasing in the lung tissue and serum and lung pathology injury score increasing.Administration of LP17 after modeling significantly down-·regulated the expressions of NF-κB and proinflammatory cytokines, while led to a slight increase of anti-inflammatory cytokines and a decline of lung pathology injury score.

CONCLUSIONTREM-1 may involve in inflammatory response by promoting the generation of inflammatory factors via NF-κB pathway, thus lead to lung pathological changes.

Acute Lung Injury ; chemically induced ; metabolism ; Animals ; Interleukin-10 ; metabolism ; NF-kappa B ; metabolism ; Paraquat ; toxicity ; Rats ; Rats, Sprague-Dawley ; Receptors, Immunologic ; metabolism ; Signal Transduction ; Triggering Receptor Expressed on Myeloid Cells-1 ; Tumor Necrosis Factor-alpha ; metabolism