Objective:To establish and validate a fluorescence PCR with internal positive control for rapid Mycoplasma detection. Methods:A fluorescence PCR with internal positive control for Mycoplasma detection was developed and verified for its specificity, limit of detection, and robustness. A sample of fever with thrombocytopenia syndrome (SFTSV) virus strains was tested with this method, and the result was compared with those of culture method and indicator cell culture method. Results:The established fluorescence PCR had good specificity and could amplify 11 kinds of plasmids containing Mycoplasma 16S rRNA gene with high efficiency. There was no cross reaction with the genomic DNAs of Clostridium sporogenes, Clostridium acetobutylicum, Lactobacillus acidophilus, Streptococcus pneumoniae, Bacillus subtilis, Staphylococcus aureus, Salmonella enteritidis, Escherichia coli, Pseudomonas aeruginosa, Aspergillus niger, Candida albicans, Vero cells, RD cells, and SF9 cells. The amplification efficiency of the internal positive control was basically consistent with that of the target gene of Mycoplasma, suggesting that the internal positive control could be used to detect the presence of PCR inhibitors. The sensitivity of the established method was high, and the detection limit was 10 colony-forming unit (CFU)/ml for Mycoplasma fermentans, 5 CFU/ml for Mycoplasma arginine, 5 CFU/ml for Mycoplasma gallisepticum, 5 CFU/ml for Mycoplasma hyorhinis, 5 CFU/ml for Acholeplasma laidlawii, 5 CFU/ml for Mycoplasma orale, 5 CFU/ml for Mycoplasma pneumoniae, 5 CFU/ml for Mycoplasma synoviae, and 1 CFU/ml for Spiroplasma citri by 7500 Fast real-time PCR system. At the detection limit of each species, there was no significant difference in the positive detection rate using different thermal cycler types. The established fluorescence PCR, culture method, and indicator cell culture were performed to detect Mycoplasma in the sample of SFTSV virus strains, and the results all showed Mycoplasma contamination. Conclusions:The established fluorescence PCR has high specificity, sensitivity, and robustness, and can be used as an alternative method for rapid detection of Mycoplasma.

Objective To evaluate the role of preoperative serological indexes in predicting long-term survival and tumor recurrence of hepatocellular carcinoma (HCC) patients after liver transplantation, aiming to explore its significance in expanding the Milan criteria. Methods Clinical data of 669 recipients undergoing liver transplantation for HCC were retrospectively analyzed. The optimal cut-off value was calculated by the receiver operating characteristic (ROC) curve. The risk factors affecting the overall survival and recurrence-free survival rates of HCC patients after liver transplantation were identified by univariate and multivariate regression analyses. The correlation between preoperative serum liver enzymes and pathological characteristics in HCC patients was analyzed. The predictive values of alpha-fetoprotein (AFP) combined with γ -glutamyl transferase (GGT) and different liver transplant criteria for the survival and recurrence of HCC patients after liver transplantation were compared. Results Exceeded Milan criteria, total tumor diameter (TTD) > 8 cm, AFP > 200 ng/mL and GGT > 84 U/L were the independent risk factors for the overall survival and recurrence-free survival rates of HCC patients after liver transplantation (all P < 0.05). Correlation analysis showed that preoperative serum GGT level was correlated with TTD, number of tumor, venous invasion, microsatellite lesions, capsular invasion, tumor, node, metastasis (TNM) stage, Child-Pugh score and exceeded Milan criteria (all P < 0.05). Milan-AFP-GGT-TTD (M-AGT) criteria were proposed by combining Milan criteria, TTD with serum liver enzyme indexes (AFP and GGT). The 5-year overall survival and recurrence-free survival rates of HCC recipients who met the M-AGT criteria (111 cases of exceeded Milan criteria) were significantly higher than those who met Hangzhou criteria (both P < 0.05), whereas had no significant difference from their counterparts who met the University of California at San Francisco (UCSF) criteria (both P > 0.05). Conclusions Preoperative serological indexes of AFP and GGT could effectively predict the long-term survival and tumor recurrence of HCC patients after liver transplantation. Establishing the M-AGT criteria based on serological indexes contributes to expanding the Milan criteria, which is convenient and feasible.

Osteoarthritis (OA), in which M1 macrophage polarization in the synovium exacerbates disease progression, is a major cause of cartilage degeneration and functional disabilities. Therapeutic strategies of OA designed to interfere with the polarization of macrophages have rarely been reported. Here, we report that SHP099, as an allosteric inhibitor of src-homology 2-containing protein tyrosine phosphatase 2 (SHP2), attenuated osteoarthritis progression by inhibiting M1 macrophage polarization. We demonstrated that M1 macrophage polarization was accompanied by the overexpression of SHP2 in the synovial tissues of OA patients and OA model mice. Compared to wild-type (WT) mice, myeloid lineage conditional Shp2 knockout (cKO) mice showed decreased M1 macrophage polarization and attenuated severity of synovitis, an elevated expression of cartilage phenotype protein collagen II (COL2), and a decreased expression of cartilage degradation markers collagen X (COL10) and matrix metalloproteinase 3 (MMP3) in OA cartilage. Further mechanistic analysis showed thatSHP099 inhibited lipopolysaccharide (LPS)-induced Toll-like receptor (TLR) signaling mediated by nuclear factor kappa B (NF-κB) and PI3K-AKT signaling. Moreover, intra-articular injection of SHP099 also significantly attenuated OA progression, including joint synovitis and cartilage damage. These results indicated that allosteric inhibition of SHP2 might be a promising therapeutic strategy for the treatment of OA.

Background/Aims: Acute-on-chronic liver failure (ACLF) is a catastrophic illness. Few studies investigated the prognostic value of vitamin D-binding protein (VDBP) for hepatitis B virus (HBV)-related ACLF (HBV-ACLF) resulted in conflicting results. Methods: Two prospective HBV-ACLF cohorts (n=287 and n=119) were enrolled to assess and validate the prognostic performance of VDBP. Results: VDBP levels in the non-survivors were significantly lower than in the survivors (P<0.001). Multivariate Cox regression demonstrated that VDBP was an independent prognostic factor for HBV-ACLF. The VDBP level at admission gradually decreased as the number of failed organs increased (P<0.001), and it was closely related to coagulation failure. The areas under the receiver operating characteristic curve (AUCs) of the Child-Pugh-VDBP and chronic liver failuresequential organ failure assessment (CLIF–SOFA)-VDBP scores were significantly higher than those of Child-Pugh (P<0.001) and CLIF-SOFA (P=0.0013). The AUCs of model for end-stage liver disease (MELD)-VDBP were significantly higher than those of MELD (P= 0.0384) only in the case of cirrhotic HBV-ACLF patients. Similar results were validated using an external multicenter HBV-ACLF cohort. By longitudinal observation, the VDBP levels gradually increased in survivors (P=0.026) and gradually decreased in non-survivors (P<0.001). Additionally, the VDBP levels were found to be significantly decreased in the deterioration group (P=0.012) and tended to be decreased in the fluctuation group (P=0.055). In contrast, they showed a significant increase in the improvement group (P=0.036). Conclusions The VDBP was a promising prognostic biomarker for HBV-ACLF. Sequential measurement of circulating VDBP shows value for the monitoring of ACLF progression.

Objective To analyze the clinical efficacy and prognosis of living donor liver transplantation (LDLT) in children with biliary atresia (BA).Methods The clinical data of 306 cases of BA patients who received LDLT from June 2013 to December 2017 in the Department of Pediatric Liver Transplantation of Tianjin First Center Hospital were retrospectively analyzed.The incidence of post-LDLT complications was summarized and different factors influencing long-term survival of the recipients were analyzed.Results The median age of recipients at transplantation was 7 (6,9) months,and 88.9％ of the recipients received left lateral lobes.The surgical-related complications mainly included lymphatic leakage (30.7％),bile duct stricture (7.8％) and portal vein stenosis (6.9％).The non-surgical-related complications were mainly EBV infection (57.8％) and CMV infection (36.6％).The incidence of pulmonary infection and acute rejection was 18.6％ and 13.7％,respectively.The 1-,3-,and 5-year survival rates of recipients and grafts were 97.2％,97.2％,97.2％ and 97.2％,96.4％,and 94.6％,respectively.A total number of 8 patients died after LDLT,mainly due to the complications of cardio-pulmonary system.Two patients underwent retransplantation due to graft dysfunction caused by antibody-mediated rejection.Recipient age,PELD scores,GRWR,previous surgical history and matching of ABO blood group between donors and recipients did not affect the long-term survival rates of recipients (P＞0.05).Conclusions Children with biliary atresia who received LDLT can obtain satisfactory clinical results.

Objective To explore the clinical pathological characteristics of breast solid papillary carcino-ma(SPC). Methods The clinical manifestation,pathology morphology,immunohistochemical characteristics and prognosis of 23 cases with SPC was reviewed. Results There were 16 cases with nipple discharge as the chief com-plaint while 7 cases were mass. 10 cases of ultrasonic examination showed 6 cases(60%)were above BI-RADS grade 4 while 8/13 in X-ray examination. In 8 cases of SPC with invasion,5 cases were luminal A and 3 cases were lumi-nal B. There were no significant differences in the mean age,mean diameter of the mass,neuroendocrine markers (CgA and Syn)and proliferation marker Ki67 between in situ SPC group and invasive SPC group(P > 0.05). The difference between P63 and CK5/6 was statistically significant(P = 0.001,P = 0.019). No recurrence was found in 21 patients. Conclusions SPC is a rare type of breast cancer with good prognosis. Imaging and ductosco-py are easy to make under-diagnosis while pathology is likely to make misdiagnosis,therefore clinical pathologists should pay more attention so as to treat it more accurately.

Objective To establish a stable model of reduced-size liver transplantations with rejection in rats.Methods Inbred adult male Lewis rats (weighing 220-250 g,8-10 weeks old) served as donors in liver transplantation,and inbred adult male Brown Norway (BN) rats (weighing 220-250 g,8-10 weeks old) served as recipients.Orthotopic group,50％ reduced-size liver transplantation rejection group (50％ group) and 30％ reduced-size liver transplantation rejection group (30％ group)were developed.The 1-,3-,7-and 14-survival rate was observed.Besides,the levels of ALT,AST and TBIL in the peripheral blood plasma were determined.The liver tissues were obtained,and HE staining was used to examine the pathological changes and rejection severity of the liver specimens.Results No significant difference in the operation-related indicators was found among the three groups.The 1-,3-,7-and 14-day survival rate in orthotopic group was as follows:91.20％,86.50％,81.10％ and 75.70％;85.00％,80.00％,62.50％ and 45.00％;65.00％,50.00％,35.00％ and 17.50％,respectively.The 1-,3-,7-and 14-survival rate in 30％ group was significantly lower than in other groups (P＜0.05),and showed no statistical difference between 50％ group and orthotopic group (P＞0.05).The ALT,AST and TBIL levels were significantly higher in 30％ group than in control group at all time points (P＜0.05),but there was no statistical difference between 50％ group and orthotopic group (P ＞ 0.05).All groups were the models of liver transplantations with rejection in rats,and rejection activity index score was increased with time delay.Conclusion Lewis→BN rats are chosen to establish liver transplant rejection model,and by comparing the survival rate and pathological changes among the groups,finally 50％ group is selected to be the model for a follow-up study.

Objective To assess the effects of the risk factors of grafts from donors after cardiac death (DCD) on the prognosis of liver transplantation (LT).Methods In this retrospectively study,215 cases of LT using DCD donor grafts were performed at our institution from September 2013 to January 2017.Due to the loss to follow-up in 4 cases,211 cases were enrolled in the study.The following DCD donor data were collected:gender,age,primary disease,ABO blood type,body mass index (BMI),medical history (fatty liver,hypertension),ICU hospitalization time,mechanical ventilation time,warm ischemia time,cold ischemia time,and indexes of routine laboratory test before donation.Statistical analyses using the Kaplan-Meier method,log-rank test,multivariate step-wise Cox regression were performed.Results Of the 211 donors,univariate analysis showed that the overall 6-month,1-,and 3-year survival rate after DCD LT was 88％,84％,and 82％,respectively.Univariate analysis showed that donor serum sodium level ＜136 mmol/L (P =0.018) and cold ischemia time ＞9 h (P =0.013) were all significant risk factors affecting overall survival after DCD LT.Additionally,donor BMI ＞30 kg/m2 (P =0.011) and donor age ＞60 years (P =0.025) were significantly associated with postoperative complications.Multivariate analysis showed that donor serum sodium level (P=0.025) was an independent risk factor of survival after DCD LT.Conclusion To select suitable DCD liver allografts and control risk factors of donor can help to improve outcomes of recipients.

Objective To analyse our clinical experience in pediatric living donor liver transplantation (PLDLT).Methods The clinical data of 45 patients who underwent PLDLT in our hospital from April 2005 to April 2014 were retrospectively studied and their preoperative,intraoperative and postoperative data were analyzed.Results All donors recovered well.The graft to recipient weight ratio (GRWR) ranged from 1.0％ ～ 6.4％ (2.5％ ± 1.2％).Size reduction of graft were performed in 2 patients.An interposition venous conduit from the confluence of the native right and left portal vein (PV) to the graft PV was carried out in 1 patient,venous grafts for revascularization of the tributaries of the middle hepatic vein from segment Ⅴ and Ⅷ were used in 3 patients,and a venous patch for revascularization of the left hepatic vein was used in 2 patients.Hepatic artery re-reconstruction was performed in 3 patients after hypoperfusion was detected on intraoperative Doppler ultrasound.The postoperative complications included acute rejection (n =2),vascular complications (n =7),biliary complications (n =11),and infectious complications (n =27).The 1-,2-and 5-year survival rates were all 84.4％.Seven of 45 recipients died within one year post transplantation,with 3 patients who died of vascular complications,and 4 patients who died of infection.The differences in age [(50.8 ± 49.8) months vs (12.6 ± 14.9) months],body weight [(16.2 ± 10.5) kg vs (7.3 ± 1.7) kg],serum total bilirubin [(177.0 ± 126.5) μmol/L vs (301.9 ± 110.6)μmol/L],Pediatric end-stage liver disease (PELD) score (16.1 ± 12.1 vs 26.2 ± 11.3) and GRWR (2.2％ ± 0.8％ vs 4.2％ ± 1.6％) between the survival and the dead groups were significant (P ＜ 0.05).Conclusions PLDLT is an effective method to treat children with end-stage liver disease.Using a multidisciplinary approach in the preoperative management,excellent surgical techniques,and proper postoperative management are extremely helpful to improve postoperative survival rate.

Objective To investigate the relationship between HLA antibodies strength and complement-binding ability in sensitized renal patients waiting for renal transplantation.Method Serum samples of 31 sensitized renal patients waiting for renal transplantation were retrospectively analyzed by single-antigen bead array (SAB) to identify HLA antibodies and in parallel by C1q-SAB to determine the complement binding of HLA antibodies.Result C1q-positive HLA antibodies had significantly higher MFI than C1q-negative HLA antibody (for Class Ⅰ,11052 ± 3291 vs.4506 ± 2960,P＜0.05;for Class Ⅱ,13347 ± 4076 vs.4448 ± 3602,P＜0.05).The mean fluorescence intensities (MFI) of IgG-SAB were correlated with the MFI of C1q-SAB for the same antibodies (Spearman correlation; Class Ⅰ,r =0.665,P ＜ 0.01 ; Class Ⅱ,r =0.761,P ＜ 0.01).Receiver operating characteristics (ROC) curve analysis showed that the MFIs of HLA antibodies by IgG-SAB could predict their C1q-binding abilities [area under the curve (AUC)Class Ⅰ =0.917; AUCclass Ⅱ =0.927).Using MFI cut-off value of 8238 and 6754 for HLA Class Ⅰ and Class Ⅱ antibodies,respectively,the sensitivity and specificity for C1q binding were 82.4％ and 87.4％ for Class Ⅰ antibodies,and 90.9％ and 82％ for Class Ⅱ antibodies,respectively.Conclusion The MFI of HLA antibodies by IgG-SAB can predict the C1q binding capability at a certain extent before transplantation.