Objective To explore the change in EGF(epidermal growth factor)and EGFR(epidermal growth factor receptor)expression in recurrent aphthous ulcer. Methods The expression intensity and distribution of EGF and EGFR were examined with immunohistochemistry method in 8 cases of recurrent aphthous ulcer and 7 specimens of normal oral mucosa. Results In normal oral mucosa, immuno staining of EGF was mainly localized in basal layer cells and prickle layer cells. The expression intensity of EGF was higher in normal oral mucosa than that in aphthous ulcer ( P

ObjectiveTo identify the factors influencing the transfer of porcine endogenous retroviruses across the membrane of a new bioartificial liver (BAL),which is a necessary caution to consider for BALs carrying porcine hepatocytes.MethodsA novel porcine BAL composed of two circuits was constructed using a plasma filter with membrane pore size of 500 nm and a plasma component separator with membrane pore sizes 10 nm,20 nm,30 nm,and 35 nm.Co-cultured cells of porcine hepatocytes and mesenchymal stem cells or single porcine hepatocytes were incubated in the bioreactors.The BAL was continuously worked for 72 hours during which the supernatant was collected from the internal and external circuits every 12 hours.PERV RNA,reverse transcriptase (RT) activity,and in vitro infectivity from the supernatant were detected.ResultsIn the plasma filter group,the PERV RNAlevels were the same in both circuits,suggesting little to no effect of the plasma filter on the PERV RNA's crossing.With plasma component separators,PERV RNA was found in the external circuits at different times without positive RT activity and HEK293 cell infection,but its level was reduced significantly.The larger the membrane pore size was,the earlier and the more RNA was detected.ConclusionsThe membrane pore size,the treatment time and the viral level in the internal circuit are contributing factors influencing the transfer of PERV RNA across the membrane in a BAL.

Objective To investigate the influence of membrane molecular weight cut off in our bioartificial liver(BAL)system.Methods Beagle dogs were used for a model of acute liver failure through D-galactosamine administration.The acute liver failure Beagles were divided into two groups by the membrane molecular weight cut off.Group A was treated with BAL containing 200 kDa retention rating membrane.Group B was treated with BAL containing 1200 kDa retention rating membrane.Each group underwent two six-hour BAL treatments that were performed on day 1 and day 21.BAL medium were examined and levels of IgG,IgM,and complement hemolytic unit of 50％(CH50)antibodies were measured in all Beagles and.Results BAL treatment was associated with a significant decline in levels of CH50.1200 kDa group experienced a significant increase in levels of IgG and IgM after two BAL treatments.Significant levels of canine proteins were detected in BAL medium from 1200 kDa group.Conclusions Xenogeneic immune response in the BAL system was influenced by membrane molecular weight cut off.

ObjectiveTo study the effects of membrane molecular weight cut off on a novel bioartificial liver(BAL) system.MethodsHealthy beagles underwent 6-hour treatment with a BAL containing membrane with 200 kDa retention rating or 1200 kDa retention rating.The functional changes and cell viability were characterized.ResultsHepatocyte performance levels such as albumin secretion,urea synthesis and viability were significantly higher in the 200 kDa retention rating group when compared with the 1200 kDa retention rating group (P＜0.05).Significant levels of canine proteins were detected in the BAL medium from the 1200 kDa retention rating group.Fluorescence microscopy further verified that heavy deposition of canine IgG,IgM and complement (C3) on co-culture cells were obtained after BAL treatment in the 1200 kDa retention rating group.ConclusionsSmall membrane molecular weight cut off of BAL could reduce the transfer of xenoreactive antibodies into the BAL medium and improved the performance of the BAL.

Objective To investigate the safety and therapeutic effects ot a novel multi-layer flat-plate bioartificial liver (BAL) for patients with liver failure.Methods Thirty-eight patients with liver failure from Dec.2010 to Dec.2011 were treated with a novel BAL based on multi-layer flatplate bioreactor and the co-cultured cells of the porcine hepatocytes and mesenchymal stem cells.A total of 48 treatments was performed,4 h each time.The clinical signs and symptoms,liver function,ammonia,coagulation function and complete blood count were evaluated before,during and after the treatment.DNA in the collected PBMCs was extracted for PCR with PERV specific primers and the porcine specific primer Sus scrofa cytochrome B.The RT activity was detected as well.Levels of xenoantibodies (IgG,IgM) were determined by using ELISA kit. Titers of complement were quantified by CH50 kit.Results All treatment procedures were completed successfully without any adverse reaction. All samples presented negative PERV DNA and RT activity. The levels of antibodies were similar before and after treatment.Treatment was associated with a temporary decline in levels of complement,and then the levels were recovered quickly.The clinical symptoms such as acratia,anorexia and abdominal distension were improved.The stage of hepatic encephalopathy in 16 patients was decreased. The liver function and ammonia was reduced disproportionately. Seven patients in all were bridged to liver transplantation,2 patients died and 2 patients gave up the treatment,and the others were turned better.After the outcome judgment according to the standard developed by the Artificial Liver Group,and Chinese Association of Infectious and Parasitic Diseases,there were 9 patients with clinical healing,25 patients with improvement and 4 patients with no effect,and the cure-improvement rate was 89.5％.Conclusion The novel multi-layer flat-plate BAL could be used as a safe and effective therapy for patients with liver failure.

Objective:To evaluate the factors affecting the incidence of intrahepatic venovenous shunt (IVVS) in patients with cirrhosis and its impact on hepatic venous pressure gradient (HVPG).Methods:A retrospective analysis was performed on the data of patients with liver cirrhosis who received HVPG measurement in Nanjing Drum Tower Hospital from April 2013 to March 2022. Univariate and multivariate regression analyses were used to investigate the incidence rate and risk factors of IVVS and its impact on HVPG. The t-test and rank-sum test were used for the measurement data, and the χ2 test was used for the count data. Results:A total of 242 cases with cirrhosis were included in the statistical analysis, including 54 (22.3%) with IVVS and 188 (77.7%) without IVVS. There was a statistically significant difference ( P<0.05) in prothrombin time (PT), HVPG, and splenectomy history between the two groups of patients' baseline data (all P<0.05). The multiple logistic regression analysis results showed that PT was an independent risk factor for the occurrence of IVVS ( P<0.05), and patients combined with IVVS had lower HVPG values [(17.58±5.57) mmHg vs. (11.92±5.38) mmHg, 1 mmHg=0.133 kPa; t=6.623, P<0.001]. Conclusions:Patients with liver cirrhosis have a high incidence rate of IVVS, which is closely associated with a low prothrombin time. Additionally, patients combined with IVVS have low HVPG values, which affect its accuracy.

ObjectiveTo investigate the clinical features and causes of death after transjugular intrahepatic portosystemic shunt (TIPS) in patients with hepatic sinus obstruction syndrome (HSOS), as well as the prevention and treatment measures to further improve the survival rate of such patients. MethodsA retrospective analysis was performed for 293 patients with HSOS who were admitted to Nanjing Drum Tower Hospital from January 2013 to December 2019, among whom 20 patients died after TIPS. General information, laboratory examination, and clinical treatment regimen were analyzed, and clinical indices and complications were compared at different stages of the disease. The paired t-test was used for comparison of normally distributed continuous data between groups, and the Wilcoxon rank-sum test was used for comparison of non-normally distributed continuous data between groups. ResultsThe mean survival time was 15.15±4.21 weeks for the 20 patients who died, among whom there were 15 male patients and 5 female patients, with a mean age of 67.60±7.01 years; there were 17 patients (85%) aged ≥60 years, and more than 90% of the patients had abdominal distention and oliguria. Among the 20 patients who died, 9 (45%) had chronic underlying diseases, and 5 (25%) had more than two underlying diseases. Portal venous pressure decreased from 21.67±5.15 mm Hg before surgery to 8.17±4.98 mm Hg after surgery (t=10.318,P＜0.05). The levels of total bilirubin, direct bilirubin, and D-dimer were significantly higher than the normal values before surgery, and there were significant increases in these levels on day 5 after surgery (Z=3.823,3.823,2.756, all P＜0.05); the hemoglobin level, platelet count, and creatinine level tended to decrease on day 5 after surgery (t=4.979,t=2.147,Z=-3.125, all P＜005). Three patients had hepatic encephalopathy before surgery, while 10 patients (50%) had hepatic encephalopathy after surgery. Causes of death included acute liver failure, infectious shock, and multiple organ failure syndrome (MODS). ConclusionThe possible risk factors for death after TIPS in HSOS patients include underlying diseases, high bilirubin, and complications such as hepatic encephalopathy and renal dysfunction. Causes of death mainly include acute liver failure and MODS. Ultrasound and laboratory markers should be reexamined during anticoagulation therapy to identify the patients with progression to severe diseases as early as possible, and in case of progressive deterioration of indices, TIPS should be selected as early as possible to improve the survival rate and prognosis of such patients. In addition, hemobilia should be observed during and after surgery, and intervention measures should be adopted in time to further reduce mortality rate.

Objective:To explore the role of serum pyrrole-protein-adduct (PPA) in evaluating the severity and predicting the anticoagulant efficacy in patients with pyrrolidine alkaloid-related hepatic sinusoidal obstruction syndrome (PA-HSOS).Methods:From April 2018 to December 2019, the data of 48 patients with PA-HSOS admitted and treated at Drum Tower Hospital, Affiliated Medical College of Nangjing University were collected, which included PPA level, portal vein velocity (PVV), ascites grading, PA-HSOS severity grading (according to the new severity grading criteria for suspected hepatic sinusoidal obstruction syndrome in adults by the European Society of Blood and Bone Marrow Transplantation and adjusted) and the outcome of anticoagulation. Patients with acute onset (onset of symptoms within 1 month after consuming pyrrolizidine alkaloid-containing plants) were taken as research subjects. The combination of PPA with PVV or with ascites classification of PA-HSOS severity assessment model was fitted by logistic regression, and the logit values of 2 combination models were calculated, the formula was logit 1=0.034×PPA(nmol/L)+ 0.055×PVV(cm/s)-3.287, logit 2=0.039×PPA(nmol/L)-2.712×ascites grade 2 (Yes=1, No=0)-0.388×ascites grade 3 (Yes=1, No=0)-0.899. The patients received initial anticoagulation therapy at Drum Tower Hospital, Affiliated Medical College of Nanjing University were selected as research subjects. The anticoagulant efficacy prediction model of combination of PPA with serum creatinine (SCR) and with hepatic venous pressure gradient (HVPG) was fitted by logistic regression, and the logit value was calculated, the formula was logit 3=0.013×PPA(nmol/L)+ 0.064×SCR (mol/L)+ 0.542×HVPG (mmHg, 1 mmHg=0.133 kPa)-16.005. The predictive value of PPA in evaluating the severity of PA-HSOS and anticoagulant efficacy was evaluated. Receiver operating characteristic curve analysis was performed for statistical analysis. Results:The serum PPA level of 48 patients was 10.81 nmol/L (3.91 nmol/L, 32.04 nmol/L). Among them, 33 cases (68.8%) were mild PA-HSOS, 3 cases (6.2%) were moderate PA-HSOS, no severe PA-HSOS case and 12 cases (25.0%) were very severe PA-HSOS. Among 23 patients received initial anticoagulant therapy at Drum Tower Hospital, Affiliated Medical College of Nanjing University and with complete data, 8 patients responded and survived, and 15 patients did not respond (5 patients died, 1 patient relieved after continue anticoagulant therapy, and 9 patients survived after switching to anticoagulant therapy and transjugular intrahepatic portosystemic shunt (TIPS) treatment). One patient without initial anticoagulant therapy, survived after TIPS treatment because of the progress of the disease. Area under the curve (AUC) of PPA to assess the severity of acute onset PA-HSOS was 0.75, 95% confidence interval ( CI) was 0.52 to 0.98 ( P=0.047). When PPA≥45.519 nmol/L, the specificity and sensitivity in evaluating severe and very severe PA-HSOS was 100.0% and 57.1%, respectively. AUC of combination of PPA and PVV to assess the severity of PA-HSOS was 0.77, 95% CI was 0.55 to 1.00 ( P=0.032). When the logit of combination model≥0.180, the specificity and sensitivity in evaluating severe and very severe PA-HSOS was 71.4% and 81.8%, respectively. AUC of combination of PPA and ascites grade (grade 1, 2 or 3) to assess the severity of PA-HSOS was 0.85, 95% CI was 0.63 to 1.00 ( P=0.005). When the logit of combination model≥0.347, the specificity and sensitivity in evaluating severe and very severe PA-HSOS was 85.7% and 92.0%, respectively. AUC of combination of PPA, SCR and HVPG to predict anticoagulation efficacy was 0.85, 95% CI was 0.69 to 1.00 ( P=0.009). When the logit≥0.393, the specificity and sensitivity in predicting anticoagulation efficacy was 62.5% and 91.7%, respectively. Conclusions:PPA can be used to assess the severity of acute onset PA-HSOS patients, and combined with ascites grading can significantly improve its efficiency. PPA combined with SCR and HVPG can better predict anticoagulant efficacy.

Objective:To investigate the relationship between hepatic venous pressure gradient (HVPG) and parameters of Doppler ultrasound in patients with pyrroidine alkaloid-related hepatic sinusoidal obstruction syndrome (PA-HSOS).Methods:From February 17, 2017 to April 22, 2020, the clinical data of 68 patients with PA-HSOS who underwent HVPG manometry and Doppler ultrasound examination at Drum Tower Hospital, the Affiliated Medical College of Nanjing University were retrospectively analyzed, which included HVPG, Drum Tower severity scoring (DTSS), time from PA-HSOS related symptoms appeared to diagnosis after taking pyrroidine alkaloid (hereinafter referred to as diagnosis time), and parameters of Doppler ultrasound induding portal vein trunk diameter (PD), peak portal vein velocity (PPV), splenic vein trunk diameter (SD) and peak splenic vein velocity (PSV). Receiver operating characteristic curve (ROC) was used to determine the optimal cut-off value of HVPG for predicting non-response to anticoagulation therapy. Binary logistic regression was used to analyze the independent risk factors for non-response to anticoagulation therapy, and Kaplan-Meier survival curve was used to analyze the prognostic survival rate of patients with different HVPG levels. Unitary linear regression was applied to analyze the correlation of HVPG with PD, PPV, SD and PSV in patients with different HVPG levels, patients with mild, moderate and severe DTSS, and patients with diagnosis time >1 month or ≤ 1 month. Chi-square test was used for statistical analysis.Results:The results of ROC analysis showed that the optimal cut-off value of HVPG for predicting non-response to anticoagulant therapy was 20.165 mmHg(1 mmHg=0.133 kPa). The result of multivariate analysis indicated that high HVPG (HVPG>20.165 mmHg) was an independent risk factor for predicting non-response to anticoagulant therapy ( OR (95% confidence interval)=6.039(1.466 to 24.869), P=0.013). Kaplan-Meier survival curve demonstrated that prognostic survival rate of patients with high HVPG was lower than that of patients with low HVPG (HVPG≤20.165 mmHg) (78.4% vs.96.8%), and the difference was statistically significant( χ2=4.74, P=0.030). The results of unitary linear regression analysis showed that there was a negative correlation between HVPG and PPV in 68 patients with PA-HSOS( r=-0.330, P=0.006); HVPG was positively correlated with PD and SD in patients with high HVPG ( r=0.540 and 0.341, P=0.001 and 0.039); there was a negative correlation between HVPG and PSV in patients with mild DTSS ( r=-0.519, P=0.019), HVPG was negatively correlated with PPV in patients with moderate DTSS ( r=-0.400, P=0.014). In patients with diagnosis time ≤1 month, there was a negative correlation between HVPG and PPV ( r=-0.391, P=0.010). Conclusions:HVPG can assist in judging the response to anticoagulation therapy and the prognosis of patients with PA-HSOS. Parameters of Doppler ultrasound can help to assess the degree of HVPG elevation in patients with PA-HSOS under certain conditions.

Objective:To explore pyrrolizidine alkaloid-induced hepatic sinusoidal obstruction syndrome (PA-HSOS) severity grading to predict the prognostic value for PA-HSOS patients treated with transjugular intrahepatic portosystemic shunt (TIPS).Methods:Clinical data of patients with PA-HSOS who were critically ill or had ineffective drug treatment and underwent TIPS treatment from December 2013 to September 2019 were retrospectively analyzed. PA-HSOS severity grading criteria in adult was quoted, revised and defined from the European Group for Blood and Marrow Transplantation (EBMT). The survival time, the rate of shunt dysfunction and the incidence of postoperative hepatic encephalopathy in different severity groups after TIPS were compared. Univariate Cox or Binomial Logistic regression analysis was used to evaluate the impact of each variable. Variables with P < 0.1 were regarded as statistically significant variables for the prognosis, and were introduced into Cox or Binomial Logistic regression hierarchical regression analysis as controlled covariates. PA-HSOS severity grading was analyzed as dummy variables.Results:A total of 102 patient data were collected, and the median follow-up time was 14.52 months. The difference in survival time of patients with different severity levels was statistically significant ( P = 0.023). The mortality risk in moderate patients was 1.575 times higher than that of mild patients (95% CI: 0.216-11.457, P = 0.654). The mortality risk of severe and very severe patients was 7.424 times higher than that of mild patients (95% CI: 1.612-34.197, P = 0.010). There was no statistically significant difference in postoperative hepatic encephalopathy recurrence rate and shunt dysfunction rate ( P > 0.05). Conclusion:PA-HSOS severity grading has prognostic value for PA-HSOS patients receiving TIPS treatment, and can be used as an important reference for guiding the timing of TIPS intervention.