The biological artificial liver(BAL) can offer reliable artificial liver support for the patients with hepatic failure.All BAL devices contain hepatocytes as their biological component,whose specific biological characteristics contribute to the function of the BAL.During the past two decades,various cells including human hepatocytes,heterogeneous hepatocytes and liver cell lines have been used and different culture methods have been studied to optimize the activity of the biological component.However,both functionality and safety of these cells should be improved before successful use in BAL. This paper summarizes the latest progress on it.

Recently a great number of new immunodepressants have emerged due to the side effects of steroids.Therefore,relatively more perfect steroid withdrawl regimens have been studied by many researchers at home and abroad.This article reviews the course of steroid withdrawal in liver transplantation,introduces and compares different protocols of steroid withdrawal.

Objective To investigate the prognosis of patients with solitary large hepatocellular carcinoma (SLHCC) and with small hepatocellular carcinoma (SHCC),and analyze the risk factors affecting the prognosis of patients with SLHCC.Methods The retrospective case-control study was conducted.The clinicopathological data of 856 patients with hepatitis B virus (HBV)-related HCC who were admitted to the Eastern Hepatobiliary Surgery Hospital of the Second Military Medical University from January 2008 to December 2008 were collected.Of 856 patients,693 HCC patients with tumor diameter ≤5 cm were allocated into the SHCC group and 163 HCC patients with tumor diameter ＞ 5 cm and with solitary,expansive growth and complete capsule tumors were allocated into the SLHCC group.Patients underwent preoperative antiviral therapy,laboratory and imaging examinations,and then surgical planning was determined based on the preoperative results.Observation indicators:(1) comparisons of clinicopathological features between the 2 groups:sex,age,Child-Pugh grade,HBeAg,serum level of HBV-DNA,platelet (PLT),albumin (Alb),total bilirubin (TBil),alpha-fetoprotein (AFP),tumor diameter,microvascular invasion,Edmondson-Steiner grade and liver cirrhosis;(2) treatment situations between the 2 groups:surgical procedures,operation time,volume of intraoperative blood loss,number of patients with blood transfusion and time of hepatic inflow occlusion;(3) survival analysis between the 2 groups;(4) prognostic analysis of patients with SLHCC.Follow-up using telephone interview and outpatient examination was performed once every 3 months within 2 years postoperatively and once every 6 months after 2 years postoperatively up to June 23,2014.Follow-up included tumor marker,liver function,serum level of HBV-DNA and abdominal B-ultrasound examination.The patients received reexamination of computed tomography (CT) or magnetic resonance imaging (MRI) once every 6 months or when there was suspicion of tumor recurrence or metastasis.Tumor recurrence or metastasis was confirmed through typical HCC imaging findings of CT and MRI,and PET/CT examination was conducted if necessary.Tumor-free survival time was from operation time to time of tumor recurrence,and overall survival time was from operation time to death or the last follow-up.Measurement data with normal distribution were represented as-x±s,and continuous variables were analyzed by the t test or Mann-Whitney U test.Measurement data with skewed distribution were described as M (range).Categorical variables were represented as count (percentage) and analyzed by the chi-square test or calibration chi-square test.The survival curve and survival rate were respectively drawn and calculated by the Kaplan-Meier method and Log-rank test.COX regression model was used for prognostic analysis.Results (1) Comparisons of clinicopathological features between the 2 groups:number of patients with PLT＜ 100× 109/L,with positive microvascular invasion and with liver cirrhosis and tumor diameter were 197,133,447,(3.1±1.1)cm in the SHCC group and 28,53,79,(8.9±3.3) cm in the SLHCC group,respectively,with significant differences between the 2 groups (x2=28.618,t =37.286,x2 =213.773,214.325,P ＜ 0.05).(2) Treatment situations between the 2 groups:all the 856 patients underwent hepatectomy,including 326 with hepatic segments of resection ≥ 3 and 530 with hepatic segments of resection ＜ 3.Operation time,volume of intraoperative blood loss,number of patients with intraoperative blood transfusion and with time of hepatic inflow occlusion ＞ 20 minutes were 90 minutes (range,60-200 minutes),200 mL (range,20-5 200 mL),47,125 in the SHCC group and 110 minutes (range,60-230 min),300 mL (range,50-3 200 mL),31,58 in the SLHCC group,respectively.(3) Survival analysis between the 2 groups:all the 856 patients were followed up for 32.5 months (range,1.O-72.3 months).The median survival time,median tumor-free survival time,1-,3-,5-year overall survival rates and 1-,3-,5-year tumor-free survival rates were 56.2 months (range,1.6-75.8 months),39.5 months(range,1.0-75.0 months),90％,71％,58％,70％,48％,38％ in the SHCC and 50.3 months (range,1.1-76.0 months),30.7 months (range,1.0-72.0 months),87％,59％,47％,65％,46％,33％ in the SLHCC group,respectively,with no significant difference in tumor-free survival between the 2 groups (x2=0.514,P＞0.05) and with a significant difference in overall survival between the 2 groups (x2=10.067,P＜0.05).Stratified analysis:there were 117 SLHCC patients with 5 cm ＜ tumor diameter ＜ 10 cm and 46 SLHCC patients with tumor diameter ＞ 10 cm.The 1-,3-,5-year overall survival rates and 1-,3-,5-year tumor-free survival rates were 91％,65％,53％,70％,48％,35％ in 117 SLHCC patients with 5 cm ＜ tumor diameter ＜ 10 cm,respectively,with no significant difference compared with SHCC group (x2=1.832,0.042,P＞0.05).The 1-,3-,5-year overall survival rates and 1-,3-,5-year tumor-free survival rates were 78％,46％,31％,49％,39％,30％ in 46 SLHCC patients with tumor diameter ＞ 10 cm,respectively,with significant differences compared with SHCC group (x2=21.136,4.097,P＜0.05).(4) Prognostic analysis of patients with SLHCC:results of univariate analysis showed that serum level of HBV-DNA,tumor diameter and microvascular invasion were risk factors affecting postoperative 5-year tumor-free survival rate of SLHCC patients (x2 =5.193,3.377,5.509,P＜0.05);sex,serum level of HBV-DNA,tumor diameter and microvascular invasion were risk factors affecting postoperative 5-year overall survival rate of SLHCC patients (x2=4.546,18.053,7.780,10.569,P＜0.05).Results of multivariate analysis showed that serum level of HBV-DNA ≥ 104 U/mL,tumor diameter ＞ 10 cm and positive microvascular invasion were independent risk factors affecting postoperative 5-year tumor-free survival rate of SLHCC patients [HR =2.77,1.85,1.86,95％ confidence interval (CI):1.74-4.40,1.16-2.94,1.17-2.96,P＜ 0.05] and affecting postoperative 5-year overall survival rate of SLHCC patients (HR=2.73,1.98,1.69,95％CI:1.72-4.33,1.23-3.17,1.04-2.72,P＜0.05).Conclusions There are similar prognosis between SLHCC patients with 5 cm ＜ tumor diameter ＜ 10 cm and SHCC patients,however,prognosis of SLHCC patients with tumor diameter ＞ 10 cm is worse than that of SHCC patients.Serum level of HBV-DNA ≥ 104 U/mL,tumor diameter ＞ 10 cm and positive microvascular invasion are independent risk factors affecting prognosis of SLHCC patients.

Objective To analyse the diagnosis and treatment of testicular torsion. Methods The clinical data of 66 cases of testicular torsion were retrospectively analysed. Results Among the 66 cases,32(48.5%) paid the first medical visit within 10 h,and 24(36.4%)were confirmed diagnosis at the first visit.False negative results occurred with color Doppler flow imaging(CDFI),and 8 testicles were damaged due to the false negative diagnosis.Thirty-three patients without prophylactic contralateral orchidopexy were followed up for 6 months to 20 years,and one experienced recurrent torsion. Conclusion The testicular torsion must be considered when a sudden acute scrotum pain is occurred.Testicular damage is closely related to the torsion time,and delayed medical intervention contributes to the testicular damage.Highly suspected cases should be performed surgical exploration timely due to the false negative results with CDFI.Prophylactic contralateral orchidopexy is recommended.

OBJECTIVETo investigate the significance of mutation and single nucleotide polymorphism (SNP) of class III receptor tyrosine kinases such as PDGFRbeta and SHIP in acute myeloid leukemia (AML) patients.

METHODSScreening of the mutation and SNP of PDGFRbeta and SHIP by genomic PCR, RT-PCR, directly sequencing and Mass-ARRAY system was carried out in 273 AML patients.

RESULTSThe mutations of PDGFRbeta R685C and SHIP Q1153L were detected for the first time in AML patients. The positivity ratio was 0.73% and 0.36% respectively.

CONCLUSIONThe mutations of PDGFRbeta R685C and SHIP Q1153L may contribute to leukemogenesis of AML.

Humans ; Inositol Phosphates ; genetics ; Leukemia, Myeloid, Acute ; genetics ; Mass Spectrometry ; Mutation ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide ; Receptor, Platelet-Derived Growth Factor beta ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVEAcute monocytic leukemia (AML)-M5 is the common type of acute myeloid leukemias in children. Studies have shown that there are abundant lipopolysaccharide (LPS) receptor (designated as CD14) molecules on the cell membrane of M5 cells and they play an important role in the diagnosis of M5, since they can be recognized and bound by mouse-anti-human CD14 monoclonal antibody (McAb). However, mouse-originated antibodies are largely not suitable for clinical application due to the severe side effects, thus "humanized antibody" is desired. As the first step for developing humanized antibody, the construction and expression of single chain antibody (scFv) with functional protein are necessary. The present study aimed to express ZCH-7-2F9 ScFv (scFv(2F9)) in eukaryotic cells and obtain the scFv(2F9) protein with a high biological activity.

METHODSFour primers were synthesized to construct the eukaryotic expressional vector, which included SfiI and EcoRI enzyme cleaving site, 6 x His and stop code TGA sequences. scFv(2F9) gene was amplified through splicing by overlap extension (SOE) using the high fidelity Taq polymerase. Positive recombinants (pSectag2A/scFv(2F9)) were identified through enzyme cleaving and sequenced before expression and were transformed into Chinese hamster ovary (CHO) cells for expression. Western-Blot and flow cytometry (FCM) were carried out to determine the relative molecular mass (Mr) and binding activity of scFv(2F9).

RESULTSThe cloned scFv(2F9) gene was identified to be functional by sequencing and expressing. The interested protein could be detected in the culture supernatant of transformed CHO cells with an Mr of 31 000. The blocking test showed that the positive cell percentages, the mean fluorescence intensity (MFI) and the peak of channel (peak Ch) were reduced by 90.02%, 63.30% and 63.38%, respectively after blocking with scFv(2F9), while those were 4.55%, 10.09% and 5.02% after blockage using the supernatant from the CHO cells transfected with blanked vector pSectag2A.

CONCLUSIONSThe scFv(2F9) against human CD14 antigen was successfully expressed in eukaryotic cells and showed a high biological activity, which may be useful for the further studies on its humanized antibodies.

Amino Acid Sequence ; Animals ; Antibodies, Monoclonal ; genetics ; isolation & purification ; Base Sequence ; CHO Cells ; Child ; Cloning, Molecular ; Cricetinae ; Cricetulus ; Humans ; Lipopolysaccharide Receptors ; immunology ; Molecular Sequence Data ; Single-Chain Antibodies ; genetics ; isolation & purification

OBJECTIVETo analyze the influence of pancreatic fistula in middle segmental pancreatic resection and summarize the experience in dealing with the stump.

METHODSThe data of 40 cases undergoing middle pancreatectomy were reviewed retrospectively to analyze the curative effect and pancreatic fistula between April 2003 and December 2009. Of these, 36 patients with benign cases outcomes were compared with 2 separate control groups, 44 pancreaticoduodenectomy (PD) and 26 extended distal pancreatectomy (EDP).

RESULTSThe mean operating time of group MSP was 222 min, which was significantly shorter than that of group PD. The mean blood loss of group MSP was 316 ml, which was less than that of others. Otherwise, the postoperative nutritional status and blood sugar control in group MSP was superior to the other 2 groups. Through long-term follow-up, the patients in group MSP retained endocrine and exocrine function better. Only 1 patient developed new-onset diabetes mellitus after operation, and no patient required enzyme substitution. No lesion recurred. The rate of pancreatic fistula was highest (42%), but didn't result in the significant deference of overall discharge time with group PD and EDP. The pancreatic fistula level and the mean postoperative time in hospital didn't differ significantly from the other 2 groups.

CONCLUSIONSMiddle segmental pancreatectomy is a safe and feasible technique that is indicated for selected patients with benign or low malignant lesion in the neck and body of the pancreas. Though the rate of pancreatic fistula is higher, the risk of which is reduced by the marked curative effect. It is very important to deal with the stump reasonably.

Adult ; Female ; Follow-Up Studies ; Humans ; Male ; Middle Aged ; Pancreatectomy ; adverse effects ; methods ; Pancreatic Fistula ; etiology ; prevention & control ; Pancreatic Neoplasms ; surgery ; Postoperative Complications ; etiology ; prevention & control ; Retrospective Studies ; Treatment Outcome

OBJECTIVETo construct a prokaryotic vector of ZCH-7-2F9 single chain antibody (ScFv2F9) and to obtain the ScFv2F9 protein with biological activity for further studies.

METHODSPrimers were synthesized according to the gene sequence of ScFv2F9, four tandem glycin and one serine (G4S) 3linker and multiple cloning site(MCS) of pIVEX2.3-MCS vector, which included NdeI and SmaI enzyme cleaving sites. ScFv2F9 gene was amplified through splicing by overlap extension (SOE) using the high fidelity Taq polymerase. Then the gene was cloned to pGEM-T easy and pIVEX2.3-MCS vectors. Positive recombinants (pIVEX2.3-MCS/ScFv2F9) were identified through enzyme cleaving and sequenced before expression. The recombinant plasmids were transfected into E.coli BL21star(DE3)plysS for expression. After purification with Ni+resin and renaturation in vitro, the relative molecular mass (Mr) and the binding activity of the interesting protein were determined by SDS-PAGE and flow cytometry (FCM), respectively.

RESULTThe cloned ScFv2F9 gene was identified to be functional by sequencing and expressing. The interesting protein was detected in inclusion body with a Mr of 31 000. The blocking test showed that the positive cell percentage, the mean fluorescence intensity (MFI) and the peak of channel (peak Ch) were reduced by 11.73%, 11.96% and 31.57%, respectively after once blockage by scFv2F9 protein, and 26.44 %, 21.75 % and 42.11 % after blockage twice.

CONCLUSIONThe ScFv2F9 against human CD14 antigen has been successfully expressed in prokaryotic cells with partial biological activity, which lays the foundation for further studies on its immunotoxin and other kinds of engineered antibodies.

Antibodies, Monoclonal ; Biotechnology ; methods ; Cells, Cultured ; Cloning, Molecular ; Gene Expression ; Genetic Vectors ; Humans ; Lipopolysaccharide Receptors ; immunology ; Prokaryotic Cells ; metabolism ; Recombinant Fusion Proteins ; genetics

In order to provide the evidences for CD19 as a better antibody targeting molecule for B lineage acute leukemias than CD20 through the multi-parameter flow-cytometry analysis of leukemia cells, the samples from 321 patients with acute leukemia (AL) were immunophenotyped by multi-color flow cytometry and CD45/SSC gating strategy followed by the analysis of CD19 and CD20 expression. The results showed that the positive rate of CD19 (115/116, 99.1%) in 116 cases with B lineage acute lymphoblastic leukemia (B lineage ALL) was significantly higher than that of CD20 (33/116, 28.4%) (P < 0.01); in 17 patients with B lineage/Myeloid (B/My) acute mixed lineage leukemia (AMLL), the former positive rate (17/17, 100%) was also higher than the latter (5/17, 29.4%) (P < 0.01). Both of the two antigens were negative in 29 patients with acute T lymphoblastic leukemia and 7 patients with T/My AMLL. The positive rates of CD19 and CD20 in 152 patients with acute myeloid leukemia (AML) were 7.2% and 2.0%, respectively. The difference of the fluorescence intensity between the two antigens on the cells from each patient with B lineage ALL or B/My AMLL was statistically significant (t = 20.68, P < 0.001). The specificity of CD19 and CD20 in B lymphocytic lineage was 92.3% (132/143) and 92.7% (38/41), respectively, while the sensitivity was 99.2% (132/133) and 28.6% (38/133), respectively, the former sensitivity was significantly higher than the latter (chi(2) = 144.018, P = 0.001). It is concluded that CD19 continuously and steadily express on almost all subtypes of B lineage leukemic cells with homogeneous pattern while only a small number of leukemias express CD20. Both the specificity and sensitivity of CD19 were very high with a much broader reaction pattern than that of CD20 on this group of diseases. These indicate that CD19 may be a better antibody targeting molecule than CD20 for patients with B-lineage acute leukemia.
Acute Disease ; Adolescent ; Adult ; Antigens, CD19 ; biosynthesis ; Antigens, CD20 ; biosynthesis ; Bone Marrow Cells ; immunology ; metabolism ; pathology ; Cell Lineage ; Child ; Child, Preschool ; Female ; Flow Cytometry ; Humans ; Immunophenotyping ; Infant ; Leukemia ; blood ; immunology ; pathology ; Leukocytes, Mononuclear ; immunology ; metabolism ; pathology ; Male ; Middle Aged ; Tumor Cells, Cultured

The aim of this study was to detect the presence of human AML leukemia stem cells (LSC) in childhood patients with acute leukemia (AL) and analyze the correlation between LSC concentrations and minimal residual disease (MRD) levels in AML cases after remission. The multi-parameter flow cytometry (FCM) and a panel of monoclonal antibody combination were used to detect the AML LSC or AML LSC immunophenotype-identical cell (AML LSC-IPIC) concentrations in childhood AML or ALL leukemia both at new diagnosis and at remission and correlated AML LSC to the MRD levels at different time points after remission. The results indicated that the AML LSC or AML LSC-IPIC concentrations [in average 166 (range 14 - 1459)/100 000 mononuclear cells (MNCs)] in AML at initial diagnosis were significantly higher than those in ALL [7 (range 0 - 560)/100 000 MNCs, p < 0.017] and control [0 (range 0 - 6)/100 000 MNCs, p < 0.017], respectively. The AML LSC concentrations in AML at non-CR were in average 36 (range 5 - 224)/100 000 MNCs. No statistical difference (p > 0.05) was found between the AML LSC or AML LSC-IPIC concentrations in AML (in average 6 (range 0 - 41)/100, 000 MNCs) and ALL [10 (range 0 - 105)/100, 000 MNCs] after CR. The significantly negative correlation was noticed between AML LSC concentrations and MRD levels. It is concluded that the AML LSCs exist in newly diagnosed AML, which are significantly reduced when complete remission has achieved, but the low levels of these populations still remain. The phenotypically similar (CD34(+)CD38⁻CD123(+)) AML LSC populations have also been found in the bone marrow from ALL patients, but their concentrations are not significantly different when CR has achieved. The significantly negative correlation between AML LSC concentrations and MRD levels is observed in AML patients after remission.
Adolescent ; Child ; Child, Preschool ; Female ; Flow Cytometry ; Humans ; Immunophenotyping ; Leukemia, Myeloid, Acute ; immunology ; metabolism ; Male ; Neoplasm, Residual ; immunology ; metabolism ; Neoplastic Stem Cells ; cytology ; Tumor Stem Cell Assay