Objective:To construct a mutant strain of hypervirulent Klebsiella pneumoniae NTHU-K2044 with hfq gene deletion and to analyze its biological characteristics. Methods:The hfq gene of NTUH-K2044 was knocked out by homologous recombination technology to construct △ hfq mutant strain. Its biological characteristics including growth rate, environmental stress tolerance, biofilm formation, capsular polysaccharide synthesis, resistance to neutrophil phagocytosis and lethality to Galleria mellonella larvae were analyzed by comparing with the wild-type strain using phenotypic experiments. Results:The △ hfq mutant strain of hypervirulent Klebsiella pneumoniae NTHU-K2044 was successfully constructed. Phenotypic experiments showed that the △ hfq mutant strain had significantly slower growth rate, smaller colonies and decreased hypermucoviscosity. Its growth was significantly inhibited under different environmental stress conditions such as pH9, pH5.5, 0.7 mmol/L SDS, 5% NaCl, 0.1% H 2O 2 and high temperature of 50℃. In terms of virulence and pathogenicity, the △ hfq mutant strain showed decreased ability to form biofilm and capsule, significantly down-regulated expression of magA and rmpA genes required for capsule synthesis, lower survival rate in the neutrophil bactericidal test and obviously reduced lethality to Galleria mellonella larvae. Conclusions:As a RNA chaperone, Hfq protein could participate in post-transcriptional regulation and play an important role in regulating the physiology, environmental adaptability and virulence of hypervirulent Klebsiella pneumoniae. This study provided reference for further study on hypervirulent Klebsiella pneumoniae.

Objective:To explore the long-term preservation value and repair effect of normothermic machine perfusion (NMP) on clinically discarded kidneys.Methods:A case of clinical discarded donor kidney was collected, and NMP was carried out in vitro for 9 hours with recovered blood. The dynamic changes of renal appearance, blood gas and biochemistry analysis of perfusate and renal pathology were recorded. Results:In the second to fifth hour of NMP, the appearance of renal was pink and ex vivo normothermic perfusion assessment score (EVNP) was grade Ⅰ. While, the sixth hour and beyond of NMP, the appearance of kidney turned to dark red and EVNP was grade Ⅲ. The renal perfusion blood flow maintained above 150 ml/min in the first 6 hours and decreased significantly after that, and at the end, was only 50 ml/min. During the whole process of perfusion, urine output was maintained at about 100 ml/h. PO 2 remained above 100 mmHg in the first 5 hours of perfusion and from the 6th hour, was lower than 80 mmHg and continued to decline, and was close to 0 at the end of perfusion. The results showed that although the K + concentration changes in blood and urine in the first 5 hours of NMP had a good consistency, the lactic acid level had been rising. In addition, there was no significant change in the histopathology at the fourth hour of perfusion compared with that before zero-point puncture, and the fibrinous thrombus in glomeruli was improved compared with that before perfusion. However, at the sixth hour after perfusion and before the end of perfusion, the pathological changes of renal tissue were significantly worse. There were a large of thrombosis in glomerular blood vessels, renal tubular atrophy and acute tubular necrosis. Conclusions:NMP can realize the evaluation of extended criteria donors before transplantation, and it proves the feasibility and repair potential of NMP in kidney to a certain extent. At the same time, NMP also provides a new way to expand the source of donor kidney and to pre-treat organ in vitro.

Objective:To summarize the clinical characteristics of children with Mycoplasma pneumoniae pneumonia(MPP) complicated with pleural effusion, and explore the effect of mixed adenovirus infection on children with MPP complicated with pleural effusion.Methods:The clinical data of children with MPP complicated with pleural effusion diagnosed in Children′s Medical Center at the First Affiliated Hospital of Hunan Normal University (Hunan Provincial People′s Hospital) from January 2013 to December 2019 were collected.MPP cases were divided into single infection group and mixed infection group according to whether mixing adenovirus infection.The clinical characteristics were compared between two groups.Results:A total of 180 children with MPP complicated with pleural effusion were included, the male to female ratio was 1.22∶1 (99/81), the age was 66.13 (44.35, 83.98) months, and the most common cases were children over 5 years old (55.56%). The length of hospitalization was 9.00 (7.00, 12.00) days.Fever (93.33%) and cough (98.33%) were the most common clinical manifestations, and mild increases in C-reactive protein, erythrocyte sedimentation rate and D-dimer were the most common laboratory results.Among included children, right pleural effusion was the most common (54.44%), bilateral pleural effusion accounted for 26.67%, and left pleural effusion accounted for 18.89%.Compared with single infection group, the mixed infection group had a longer hospital stay, a higher proportion of oxygen intake, a higher proportion of gamma globulin use, and a higher value of lactate dehydrogenase and aspartate aminotransferase.The results of multivariate Logistic regression analysis showed that compared with single infection group, although the mixed infection group had a higher proportion of gamma globulin use (36.54% vs.10.93%, P<0.05), the length of hospital stay, clinical manifestations, laboratory examination, chest CT and fiberoptic bronchoscopy showed no statistically significant difference between two groups. Conclusion:MPP complicated with pleural effusion is more common in children over 5 years old, especially in the right side.Mild increases of C-reactive protein, erythrocyte sedimentation rate, and D-dimer are more common.The clinical features of MPP complicated with pleural effusion are similar between mixed adenovirus infection group and single infection group.

Objective To investigate the effect of expression of Cullin 4B (CUL4B) on the prognosis of patients after liver transplantation for hepatocellular carcinoma (HCC).Methods The retrospective case-control study was conducted.The clinicopathological data of 79 patients who underwent liver transplantation for HCC in the First Affiliated Hospital of Sun Yat-sen University between January 1,2014 and June 30,2015 were collected.The specimens of HCC tissues were collected and embedded in paraffin,and then were detected by immunohistochemistry staining.Observation indicators:(1) expression of CUL4B in HCC tissues;(2) follow-up and survival;(3) prognostic factors analysis after liver transplantation;(4) association between expression of CUL4B and recurrence and metastasis of tumor after liver transplantation.Follow-up using outpatient examination and telephone interview was performed to detect tumor recurrence or metastasis and survival up to June 2018.Measurement data with normal distribution were represented as (x)±s.The comparison between groups of count data was done using the chi-square test.The survival curve drawn using the Kaplan-Meier method,and the survival analysis was done by Log-rank test.The univariate and multivariate analysis were respectively done using the COX regression model.The association analysis was done using the Pearson test.Results (1) Expression of CUL4B in HCC tissues:immunohistochemistry staining showed that CUL4B was mainly expressed in the cytoplasm,with a powerful brownish-yellow staining.The high expression and low expression of CUL4B in HCC tissues were detected in 64 and 15 patients,respectively.(2) Follow-up and survival:79 patients were followed up for 38-56 months,with an average time of 46 months.During the follow-up,37 patients had no tumor recurrence and 42 had tumor recurrence (32 with tumor extrahepatic metastasis and 10 with intrahepatic metastasis);36 had survival and 43 died;the 1-and 3-year overall survival rates were respectively 86.84％ and 63.25％,and 1-and 3-year tumorfree survival rates were respectively 62.31％ and 51.27％.(3) Prognostic factors analysis after liver transplantation:① Results of univariate analysis showed that preoperative alpha-fetoprotein (AFP),Child-Pugh score,maximum tumour dimension,capsular invasion,intravascular tumor thrombus,Edmonson pathological grading and expression of CUL4B were related factors affecting the 3-year overall survival rate of patients after liver transplantation for HCC [Hazard Ratio (HR) =2.17,3.36,3.66,2.43,2.19,3.36,2.84,95％ confidence interval(CI):1.17-4.04,1.53-7.42,2.10-6.42,1.33-4.17,1.08-9.04,1.58-7.59,1.17-6.32,P＜ 0.05].The preoperative alpha-fetoprotein (AFP),Child-Pugh score,maximum tumour dimension,capsular invasion,intravascular tumor thrombus,Edmonson pathological grading and expression of CUL4B were related factors affecting the 3-year tumor-free survival rate of patients after liver transplantation for HCC (HR =2.06,3.72,3.16,2.36,2.83,3.21,1.69,95％CI:1.34-4.85,1.72-8.63,1.79-7.31,1.46-4.86,1.19-8.63,1.19-7.92,1.06-4.87,P＜0.05).② Results of multivariate analysis showed that maximum tumour dimension,intravascular tumor thrombus and expression of CUL4B were independent factors affecting the 3-year overall survival rate of patients after liver transplantation for HCC [Odds ratio(OR) =3.43,3.69,2.81,95％CI:1.16-6.02,1.96-9.38,1.04-9.63,P＜0.05].The maximum tumour dimension,intravascular tumor thrombus and expression of CUL4B were independent factors affecting the 3-year tumor-free survival rate of patients after liver transplantation for HCC (OR=2.25,4.72,2.74,95％C1:1.16-4.02,1.98-9.47,1.03-7.10,P＜ 0.05).The 3-year overall survival rate in patients with high-and low-expressions of CUL4B was respectively 66.7％ and 32.8％,with a statistically significant difference (x2 =5.69,P＜0.05).The 3-year tumor-free survival rate in patients with high-and low-expressions of CUL4B was respectively 73.3％ and 18.6％,with a statistically significant difference (x2 =4.63,P＜0.05).(4) Association between expression of CUL4B and recurrence and metastasis of tumor after liver transplantation:results of Pearson test showed that expression of CUL4B was significantly associated with HCC recurrence and metastasis after liver transplantation (r =0.62,P＜0.05).The further analysis showed that expression of CUL4B was significantly associated with extrahepatic metastasis after liver transplantation (r=0.84,P ＜ 0.05).Conclusion The expression of CUL4B is associated with HCC recurrence after liver transplantation,and it can be as a predictor for HCC recurrence and distant metastasis after liver transplantation.

Objective To evaluate the effect of single injection of low dose zoledronic acid on bone resorption.Methods332 menopausal patients with bone deficiency treated in our hospital were selected.The patients were treated with zoledronic acid 1mg (1mg group), 2.5mg treatment group (2.5mg group), 5mg treatment group (5 mg group) and placebo treatment group (control group), each group of 83 patients.The patients of 1mg group, 2.5mg group and 5 mg group were treated with 1mg, 2.5mg and 5mg zoledronic acid alone.The patients in the control group were given intravenous infusion of placebo.Evaluated the lumbar spine (L1-L4) and total hip bone mineral density (BMD) and bone metabolic markers at baseline, 6, 12, 18, and 24 months in the four groups.The bone metabolic criteria included t β-Cterminal-telopeptide of type I collagen (β-CTX) and procollagen type-I N-terminal propeptide (P1NP).ResultsThe Lumbar spine BMD and the Total hip BMD were significantly higher in 1mg group than baseline value and Simultaneous valueand in the control group (P<0.05), The difference were statistically significant (all P<0.05).The values at 8 and 24 months decreased gradually.The value was significantly lower (P<0.05) compared with the control group, There were no statistically significant difference compared with the simultaneous value in control group.The lumbar BMD and the total hip BMD in 2.5mg and 5mg groups were significantly lower than the baseline values during the whole trial period (all P<0.05).The trend of β-CTX and P1NP was similar to that of BMD in each group.ConclusionIntravenous injection of 1 mg and 2.5 mg of zoledronic acid produces anti-bone resorption that can last for at least 1 year.After one year of treatment, The effect of single injection of 2.5 mg of zoledronic acid on bone is similar to that of single injection of 5 mg zoledronic acid.1 mg zoledronic acid produced by anti-bone resorption can last for 12 months, and then slowly disappear.

OBJECTIVETo explore the diagnosis, treatment and long-term outcome of late acute rejection (LAR) following adult orthotropic liver transplantation (OLT).

METHODSA total of 398 consecutive adult patients who underwent OLT in Organ Transplant Center, the First Affiliated Hospital, Sun Yat-sen University between January 2007 and December 2012 were reviewed retrospectively. There were 48 patients (12. 1%) developed to LAR, including 43 male patients and 5 female patients, with an average age of (52 ± 13) years(18 - 70 years). The mean body mass index was (22.1 ± 4. 5) kg/m2 (15. 4 - 30. 4 kg/m2). The indications of the liver transplantation recipients included 16 cases of end-staged liver cirrhosis after hepatitis B or C(33. 3%), 14 cases with severe hepatitis (29. 2%), 9 cases of primary liver cancer(18. 5%), 5 cases of alcoholic liver cirrhosis (10. 4%), 1 case with autoimmune liver disease (2. 1%) , the other 3 cases (6. 3%). They were followed up by outpatient service, telephone and other means. Survival curves were generated with the Kaplan-Meier method and Cox proportional hazards modeling was used for predictors of mortality. Statistically significant variables found by single factor regression analysis were put into the Cox proportional hazards regression model of multivariate analysis.

RESULTSThe time-to-event was 23. 6 months after OLT which were more common in the first year to the third year post-transplant (26/48,52. 4%). Thirty-five cases were assessed as mild, 11 cases were assessed as moderate, and 2 cases were assessed as severe ,based on the Banff schema. After adjustment to the immunosuppressive regimen, the overall recovery rate reached to 81. 3%. The rate of steroid-resistant acute rejection was 11. 8% (4/34). Inadequate immunosuppression and steroid pulsation were two independent risk factors affecting the prognosis of LAR (P = 0. 008, P = 0. 003, respectively).

CONCLUSIONSLAR is an uncommon complication after OLT. Inadequate immunosuppression and steroid pulsation are the major risk factors for prognosis of LAR. Improving patient compliance and strengthening blood concentration surveillance can increase the patient survival.

Acute Disease ; Adolescent ; Adult ; Aged ; Female ; Graft Rejection ; diagnosis ; mortality ; therapy ; Hepatitis B ; Humans ; Immunosuppression ; Liver Cirrhosis ; Liver Neoplasms ; Liver Transplantation ; mortality ; Male ; Middle Aged ; Multivariate Analysis ; Prognosis ; Proportional Hazards Models ; Retrospective Studies ; Risk Factors ; Steroids ; Young Adult

Acute Disease ; Glucocorticoids ; Graft Rejection ; Heart Rate ; Humans ; Hyperglycemic Hyperosmolar Nonketotic Coma ; Liver Transplantation

Objective To explore the clinical efficacy of the upper abdomen organ transplantation in the treatment of end stage liver disease and type 2 diabetes mellitus (T2DM).Methods The clinical data of 7 cases receiving liver-duodeno-pancreatic organ cluster transplantation in patients with end-stage liver disease and T2DM were retrospectively analyzed.The pancreas and the whole digestive tract of the recipients were reserved during operation,simple liver excision was executed,and abdominal multiple organs including pancreas,duodenum and part of jejunum were transplanted.The liver and kidney functions,blood glucose,C peptide,infection,rejection,vascular complications,biliary complications and other indicators were monitored postoperatively.Results No insulin was used in all the patients 1-7 days after operations,the blood glucose levels returned to almost normal, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin became normal after 1 week,and C peptide levels reached the normal range in 1 to 2 weeks.Among 7 patients,one died of graft-versus-host disease 1 month after operation,one got acute renal failure,one suffered from pulmonary infection, one had pancreatic leakage but recovered after unobstructed drainage,and no obvious complications were observed in the remaining patients.So far 6 live recipients were followed up for 1-39 months,the functions of the liver and pancreatic grafts were normal without hepatitis B and tumor recurrence, and the quality of life in the patients was satisfactory.Conclusion Liver-duodeno-pancreatic organ cluster transplantation is technically feasible and an effective method for the treatment of end-stage liver disease with T2DM.

ObjectiveTo investigate a reasonable perioperative nutrition support therapy for combined ‘en bloc’ liver/pancreas transplants (CLPT).MethodThe clinical data of 10 patients,5 with gastrointestinal malignancy and liver metastasis and the other 5 with end-stage liver cirrhosis complicated with insulin-dependent type 2 diabetes mellitus ( T2DM),who had undergone CLPT in our center from May 2004 to September 2010 were retrospectively analyzed.All these patients received preioperative nutrition support,including normal food combined with nutrient solution before surgery,and total parenteral nutrition (TPN) to parenteral nutrition (PN) +enteral nutrition ( EN ) and to EN after surgery.The intestinal absorption,nutritional status,laboratory test results,and complications were recorded.ResultsAll recipients experienced a smooth recovery from the procedure,with normal or almost normal liver,pancreas,and duodenum graft functions.Three patients suffered from intestinal fistula,and all of them received TPN therapy; two patients died of multiple organ failure and one recovered from the complication.The remaining seven patients had smooth transition from TPN to EN.Of the 5 patients with malignancies,three died of multiple organ failure and 2 died of cancer recurrence.Of the 5 patients with liver cirrhosis and T2DM,four patients survived and 1 patient died of graft-verse-host disease.ConclusionRational perioperative nutrition support is important for the successful recovery after CLPT.

Objective To evaluate the diagnosis and treatment of early-stage hepatic artery thrombosis(HAT) after adult liver transplantation.Methods387 consecutive adult patients who underwent liver transplantation from June 2007 to October 2010 by the same surgery team in the Transplant Center,First Affiliated Hospital of Sun Yat-sen University were retrospectively studied.Hepatic arterial blood flow was monitored by color Doppler ultrasound (DUS) daily during the first week after transplantation.Ultrasonic contrast or hepatic artery angiography was performed on recipients with suspected HAT.Results10 patients developed HAT on 7(2-18)d after operation.The incidence of HAT was 2.6％ (10/387).Interventional therapy was performed in 2 patients with one patient who received a stent because of hepatic artery stricture.Three patients underwent emergent hepatic artery revascularization combined with intra-arterial urokinase thrombolysis treatment.One developed a rethrombosis and died.The remaining 2 patients received re-transplantation.Three patients died of liver failure and severe infection.The mortality rate was 40％ (10/387).ConclusionsIt is essential to diagnoses HAT by monitoring the artery flow by Doppler ultrasound screening in the early period after operation.Interventional therapy,emergent hepatic artery revascularization and re-transplantation are effective rescue treatments.Prevention of HAT is most important.