Objective To investigate the action mechanism of IL-35 gene transfection ameliorating cardiac allograft rejection and prolonging allograft survival.Methods pEBI3-L-p35-Fc plasmid was amplified by polymerase chain reaction.In vitro plasmid DNA pEBI3-L-p35-Fc or pSec-L-Fc was,respectively,transfected into HEK293 cells using Lipofectamine 3000.At 48 and 72 h after transfection,IL-35 concentration in culture supernatant of transfected HEK293 cells was detected by ELISA.Balb/c and C57BL/6 splenocytes treated with mitomycin (MMC) served as the stimulators,those not treated with MMC as responders,and they were subjected to one-way mixed lymphocyte culture (MLC).In the presence or absence of IL-35,the percentage of CD4+ CD25+ Tregs was detected by flow cytometry.Abdominal heterotopic heart transplantation model was established by using inbred male Balb/c mice as donors and C57BL/6 as recipients respectively.In experimental group,recipients were intravenously administrated with IL-35 plasmid (50μg) on the day 1 to day 3 post-transplantation.The control mice were treated with normal saline.The IL-35 expression in the blood,CD4+ CD25+ Tregs proportion in the blood and spleen,and the survival and the histopathologic changes of the cardiac grafts were also observed.Results In vitro the transfected HEK293 cells expressed IL-35.IL-35 enhanced the proliferation of CD4+ CD25+ Tregs of MLC in vitro.The median survival time of the cardiac grafts in experimental group (16 days) was significantly longer than in control group (7 days) (P＜0.01).As compared with control group,CD4+ CD25+ Tregs proportion was significantly increased (P＜0.01),CD8+ T cells proportion was decreased (P＜0.01) and the proliferation of lymphocytes and monocytes infiltration was inhibited in the experimental group.Conclusion IL-35 could alleviate cardiac allograft rejection and prolong cardiac allograft survival via the induction of proliferation and differentiation of CD4+ CD25+ Tregs and inhibition of proliferation of CD8 + effector T cells.

Objective To observe the clinical efficacy and safety of 5% imiquimod cream in topical treatment of anogenital warts. Methods A randomized, double-blind, parallel placebo-controlled clinical study was conducted. Patients with anogenital warts were instructed to apply the test drug topically and then clean the drug with water 6 ~ 8 hours later, three times a week for 8 weeks. Patients whose warts cleared completely were followed up for one month to determine recurrence rates. Results Two hundred and thirty-one patients with anogenital warts were enrolled in this trial. One hundred and sixteen patients were randomly selected to receive 5% imiquimod cream; and the other receive placebo cream. For 2, 4, 6, 8 weeks, the cure rates were 8.41%, 30.84%, 49.53%, 61.68%, respectively in the study group, and 2.68%, 7.14%, 16.07%, 24.11%, respectively in the control group (P

Objective:To explore the effect and mechanism of interleukin-35 gene modified mesenchyma stem cells(MSC)on ameliorating cardiac allograft rejection and prolonging graft survival of transplanted heart in mice.Methods:In this study, IL-35-MSC secreting IL-35 continuously and steadily were successfully constructed in vitro. Abdominal heterotopic heart transplantation model was established successfully. And they were randomly divided into syngeneic control group; saline control group, MSC treatment group and IL-35-MSC experimental group(n=12 each). Six mice were randomly selected for sacrificing at Day 5 post-operation for detecting the related indicators in each group: Hematoxylin eosin staining was used for pathological examination. Enzyme-linked immunosorbent assay(ELISA)was employed for detecting the concentration of IL-35 in peripheral blood and the proportion of T lymphocyte subsets in spleen was analyzed by flow cytometry(FCM). Then the remaining mice were used for recording the graft survival.Results:The model of abdominal heterotopic heart transplantation in mice was successfully constructed. As compared with saline control group(6.50±0.55 d)and MSC treatment group(12.00±0.89 days), IL-35-MSC significantly alleviated rejection after transplantation and effectively prolonged the survival time of graft(18.50±1.64 days)(n=6, P<0.01). As compared with other groups, percentage of Th17 cells and Th1/Th2 ratio in spleen decreased significantly while the proportion of CD4 + Foxp3 + Treg increased significantly in IL-35-MSC experimental group at Day 5 post-transplantation(n=6, P<0.01). Conclusions:IL-35-MSC may alleviate cardiac allograft rejection and prolong graft survival. And cellular immunotherapy based upon IL-35-MSC may provide a new approach for inducing immune tolerance.

To address the issues in the current lung nodule detection for tuberculosis where the existing object detection algorithms have limited precision for small nodules and often predict bounding box locations inaccurately,a lung nodule detection method based on YOLOv7 is presented for obtaining small lung nodules more effectively and realizing the continuous convergence of target detection box.Based on the framework of YOLOv7 network model,the improvements are made in the following 3 aspects.(1)The cross-channel information and target airspace information are obtained with the effective SimAM channel attention mechanism embed in the Head network,so as to highlight the target features and enable the model to identify the regions of interest more accurately.(2)SIOU boundary loss function is used to increase the angle cost on the original loss function,and redefine the distance cost and shape cost to improve the convergence rate and reduce the loss value.(3)SIOU-NMS is used to replace the non-maximum suppression algorithm for reducing the error suppression due to target occlusion.The results of experiments on a custom lung nodule dataset show that compared with the original YOLOv7,the proposed method improves accuracy and recall rate by 2.9%and 3.1%,and the mean average precision at a confidence threshold of 0.5 is increased by 3.7%.The model can effectively assist in the diagnosis of lung nodules.

Objective To observe the clinical efficacy and safety of5%imiquimod cream in the top-ical treatment of condyloma acuminatum(CA).Methods A randomized,double-blind,parallel placebo-controlled clinical study was conducted.The test drug was topically used in CA patients,three times a week for8weeks.Patients whose warts cleared completely were followed up for one month to determine recurrence rates.Results Two hundred fifty-eight patients with anogenital warts were enrolled into this trial.One hun-dred twenty-nine patients were randomly selected to receive5%imiquimod cream;129patients were ran-domly chosen to receive placebo cream.Results showed that the cure rates were12.30%,32.79%,50%,60.66%respectively in study group for2,4,6,8weeks and were4.88%,14.63%,19.51%,26.02%respec-tively in control group for2,4,6,8weeks(P

Objective:To observe the changes of serum micro-inflammatory and nutritional status in elderly peritoneal dialysis patients after treatment with bifidobacterium triple viable bacteria, and the impact of Bifidobacterium triple viable bacteria treatment on the endpoint.Methods:Totally 180 elderly patients receiving peritoneal dialysis were divided into control group and observation group, with 90 cases in each.Both groups were treated on the basis of the routine treatment, the observation group was treated with oral Bifidobacterium triple viable bacteria for twelve months.Before treatment, 6 months and 12 months after treatment, fasting venous blood from the patients in the two groups were collected in the morning.The levels of serum micro-inflammatory indexes[tumor necrosis factor-α(TNF-α), interleukin-6(IL-6), interleukin-8(IL-8), C-reactive protein(CRP)]were detected by ELISA.The nutritional status and dialysis adequacy indexes[nutritional status: albumin(ALB), hemoglobin(Hb), transferrin(TRF), prealbumin(PA), calcium ion, phosphorus ion; indicators of dialysis adequacy: serum creatinine(Scr), blood uric acid(BUA), blood urea nitrogen(BUN), cystatin C(Cys-c)levels]were detected by automatic blood biochemical analyzer.After 24 months of follow-up, the occurrence of endpoint events(peritonitis, abdominal pain, malnutrition, abdominal infection, cardiovascular and cerebrovascular accidents)in the two groups was recorded.Results:After 24 months of treatment, the levels of TNF-α, IL-6, IL-8 and CRP in the two groups were lower than those before treatment, and the observation group was lower than control group[(25.7±4.0)μg/L vs.(33.6±6.0)μg/L, (2.9±0.7)ng/L vs.(4.9±1.2)ng/L, (17.0±7.2)ng/L vs.(22.8±7.9)ng/L, (4.6±0.7)mg/L vs.(6.9±1.2)mg/L]( t=10.272, 13.134, 5.040, 15.575, respectively, P<0.05 for all). After 24 months of treatment, the levels of ALB, Hb, TRF, PA and calcium ion of the two groups were higher than before treatment, and the levels of phosphorus ion were lower than before treatment, and the changes of the above indicators in observation group were more obvious than those in the control group[(45.7±5.2)g/L vs.(39.8±4.9)g/L, (72.7±8.0)g/L vs.(68.6±9.0)g/L, (4.3±1.0)g/L vs.(3.0±0.6)g/L, (321.5±29.0)mg/L vs.(297.6±25.1)mg/L, (4.9±1.3)mmol/L vs.(2.9±0.9)mmol/L, (1.3±0.9)/L vs.(1.8±0.3 mmol/L)]( t=7.737, 3.213, 9.880, 5.9 00, 11.937, 4.415, P<0.05 for all). There was no significant difference in intestinal flora between the two groups before treatment( P>0.05). After 24 months, an increase was observed in Lactobacilli and Bifidobacteria in both groups, while Enterobacteria and Enterococcus in both groups were decreased, and the changes of the above indicators in the observation group were also more obvious than those in the control group[(8.4±0.9)IgCFU/g ratio(6.4±0.9)IgCFU/g, (8.8±1.3)IgCFU/g ratio(7.9±1.3)IgCFU/g, (7.1±0.9)IgCFU/g ratio(8.0±1.1)IgCFU/g, (5.4±0.7)IgCFU/g ratio(6.9±0.9)IgCFU/g]( t=14.248, 4.339, 5.825, 12.753, P<0.05 for all). There was no significant difference in dialysis adequacy index between the two groups before treatment( P>0.05 for all). After 24 months of treatment, the levels of Scr, BUA, BUN and Cys-C in both groups decreased, and those of the observation group were lower than those of the control group[(471.5±50.5)μmol/L vs.(623.3±62.6)μmol/L, (17.5±0.5)mmol/L vs.(20.6±1.8)mmol/L, (16.4 ± 3.0)mmol/L vs.(22.5±2.0)mmol/L, (1.9±0.5)mg/L vs.(3.0±0.7)mg/L]( t=17.877, 14.976, 15.842, 11.749, P<0.05 for all). The incidence of endpoint events in the observation group was significantly lower than that in the control group(12.2% vs.2.2%, t=6.574, P<0.05 for all). Conclusions:After the treatment of Bifidobacterium triple viable bacteria in elderly peritoneal dialysis patients, the micro-inflammatory state of the patients was reduced, the nutritional status was improved, and the incidence of endpoint events was low, and has high clinical promotion and application value.

Objective:To study the effects of different calcium ion concentrations on epithelial mesenchymal transformation (EMT) of human peritoneal mesothelial cell (HPMC) via endoplasmic reticulum stress (ERS).Methods:HPMC cell line HMrSV5 was cultured in vitro and treated in groups. The cells in the control group, high calcium group 1, and high calcium group 2 were treated with medium containing calcium ion concentrations of 1.25, 1.75, and 2.25 mmol/L, respectively. The solvent control group was treated with medium containing 1.25 mmol/L physiological calcium ion concentration and 0.1% dimethyl sulfoxide (DMSO), the high calcium+solvent group was treated with medium containing 2.25 mmol/L calcium ion concentration and 0.1% DMSO, the high calcium+4-phenylbutyric acid (4-PBA) group was treated with medium containing 2.25 mmol/L calcium ion concentration and 1 mmol/L ERS inhibitor 4-PBA, and each group was treated for 48 hours. Morphological changes of cells in each group were observed under light microscope. The expressions of epithelial cell phenotype marker zonula occluden-1 (ZO-1) and mesenchymal cell phenotype marker α-smooth muscle actin (α-SMA) in the cells were observed by immunofluorescence staining. The expressions of EMT marker genes E-cadherin, ZO-1, α-SMA and Vimentin were detected by fluorescence quantitative polymerase chain reaction (PCR). The expressions of ERS marker proteins phosphorylated protein kinase R-like endoplasmic reticulum kinase (p-PERK), phosphorylated eukaryotic initiation factor 2α (p-eIF2α), transcription activating factor 4 (ATF4) and C/EBP homologous protein (CHOP) were detected by Western blotting. Results:Compared with the control group, the morphology of HMrSV5 cells became slender and fibrotic, the fluorescence intensity of ZO-1 increased, and the fluorescence intensity of α-SMA decreased in high calcium 1 and high calcium 2 groups, indicating that the cells transformed from epithelial cells to mesenchyme cells. The mRNA expressions of E-cadherin and ZO-1 were significantly decreased, while the mRNA expressions of α-SMA and Vimentin and the protein expressions of p-PERK, p-eIF2α, ATF4 and CHOP were significantly increased, moreover, the expressions of the above marker genes or proteins in the high calcium 2 group was more obvious than those in the high calcium 1 group [E-cadherin mRNA (2 -ΔΔCt): 0.53±0.05 vs. 0.75±0.09, ZO-1 mRNA (2 -ΔΔCt): 0.42±0.06 vs. 0.69±0.06, α-SMA mRNA (2 -ΔΔCt): 1.81±0.16 vs. 1.32±0.14, Vimentin mRNA (2 -ΔΔCt): 2.05±0.22 vs. 1.48±0.16, p-PERK protein (p-PERK/β-actin): 0.81±0.09 vs. 0.59±0.06, p-eIF2α protein (p-eIF2α/β-actin): 0.87±0.10 vs. 0.50±0.06, ATF4 protein (ATF4/β-actin): 0.93±0.10 vs. 0.72±0.06, CHOP protein (CHOP/β-actin): 0.79±0.09 vs. 0.46±0.04, all P < 0.05]. Compared with the solvent control group, the morphological changes of cells, the expressions of EMT marker genes and ERS marker proteins after high calcium ion concentration of 2.25 mmol/L were consistent with those in the high calcium 2 group than control group. Compared with the high calcium+solvent group, the cell morphology recovered the characteristics of polygonal and pebble-like epithelial cells in the high calcium+4-PBA group, the fluorescence intensity of ZO-1 increased, the fluorescence intensity of α-SMA decreased, and the mRNA expressions of E-cadherin and ZO-1 in the cells were significantly increased [E-cadherin mRNA (2 -ΔΔCt): 0.86±0.09 vs. 0.57±0.04, ZO-1 mRNA (2 -ΔΔCt): 0.81±0.06 vs. 0.48±0.05, both P < 0.05], the mRNA expressions of α-SMA and Vimentin and the protein expressions of p-PERK, p-eIF2α, ATF4 and CHOP were significantly decreased [α-SMA mRNA (2 -ΔΔCt): 1.21±0.13 vs. 1.77±0.15, Vimentin mRNA (2 -ΔΔCt): 1.30±0.14 vs. 1.94±0.20, p-PERK protein (p-PERK/β-actin): 0.38±0.04 vs. 0.92±0.11, p-eIF2α protein (p-eIF2α/β-actin): 0.34±0.05 vs. 1.05±0.13, ATF4 protein (ATF4/β-actin): 0.57±0.06 vs. 0.97±0.11, CHOP protein (CHOP/β-actin): 0.51±0.04 vs. 0.90±0.12, all P < 0.05]. Conclusion:High calcium ion concentrations of 1.75 mmol/L and 2.25 mmol/L promote EMT of HPMC via activating ERS.

OBJECTIVE： To establish the method for content determination of eugenol in Syzygium aromaticum oil dropping pills， and to optimize the preparation technology. METHODS： The content of eugenol in S. aromaticum oil dropping pills was determined by UV spectrophotometry. Based on single factor test， using the percentage of drugs in total amount， liquid temperature， falling distance of condensate， liquid drop distance as factors， taking the roundness， weight and hardness difference and comprehensive score as factors， L9（34） orthogonal design test was adopted to optimize the preparation process. RESULTS： The linear range of eugenol was 15.15-45.45 μg/mL（r＝0.999 6）； RSDs of precision， stability and reproducibility tests were all lower than 1％； the recoveries were 97.41％-100.59％（RSD＝1.35％， n＝6）. The optimal preparation technology included that the percentage of drugs in total amount was 5％； liquid temperature was 80 ℃； falling distance of condensate was 13 cm； liquid drop distance was 6 cm. The dropping pills had smooth appearance， good roundness and moderate hardness； the average content of engenol was 4.073％（RSD＝0.35％，n＝6）. CONCLUSIONS： The established method is simple， and can be used for the content determination of eugenol in S. aromaticum oil dropping pills. The optimal preparation technology is stable and feasible.