Platelet count reduction and outcomes in living liver donors.

BACKGROUND: Platelet count reduction in living donors after graft harvesting is very common. The mechanisms and the subsequent adverse consequences are not clear. The present study was to explore the mechanisms and the consequences of platelet count reduction in living donors. METHODS: We collected data from 231 living liver donor patients who donated at our transplant center between July 2002 and August 2009. Baseline and post-operative platelet counts were collected and analyzed. Multivariate logistic regression analysis was used to compare the risk factors for the persistent decrease in platelet counts. Complications and other post-operative recovery were compared between the donors. RESULTS: Platelet count decreased differently at each of the follow-up intervals, and the average reduction from baseline evaluation to year 3 was 18.2%. A concomitant decrease in white blood cells was observed with platelet count reduction. All of the splenic volumes at the post-operative follow-up time points were significantly higher than those at baseline (P<0.01). Multivariate logistic regression analysis indicated that the graft-to-donor weight ratio was a risk factor for low post-operative platelet counts in living donors at the three follow-up time points: one week (P=0.047), one month (P=0.034), and three months (P=0.047). At the one week follow-up time, 77 donor platelet counts were higher (group 1) and 151 donor platelet counts were lower (group 2) than baseline levels. Two hemorrhage events (1.3%) were observed in group 2, while three hemorrhage events (3.9%) were observed in group 1 (P=0.211). The overall complication rate was comparable between the two groups (P=0.972). CONCLUSION: An increase in harvesting graft may decrease platelet counts, but this reduction does not produce short- or long-term damage in living liver donors.

Expression and purification of recombinant human serum albumin fusion protein with VEGF165b in Pichia pastoris.

VEGF165b is an endogenous transcriptional splice variant of VEGF and has been shown to have a therapeutic potency as an anti-cancer agent. In this report, a fusion gene consisting of a human VEGF165b and a human albumin (HSA) gene was constructed and then inserted into a pPIC9k vector. The recombinant fusion protein, rhHSA-VEGF165b, was over expressed in the methylotrophic yeast Pichia pastoris under the control of AOX1 promoter. After induction with methanol, the expression level of rhHSA-VEGF165b was 275 mg/L in broth. The fusion protein rhHSA-VEGF165b was purified to more than 95% purity by using Blue Sepharose Fast Flow and SP Sepharose Fast Flow. Biological activity of the prepared rhHSA-VEGF165b was characterized by transwell migration assay, retaining about 9% of that of unmodified rhVEGF165b on a molar basis. Data from mice show that the serum half-life time of rhHSA-VEGF165b was nearly 20 times longer than that of rhVEGF165b.

Risk factors of SFSS in adult-to-adult living donor liver transplantation using the right liver: a single-center analysis of 217 cases.

BACKGROUND/AIMS: Small-for-size Syndrome (SFSS) in adult-adult living right lobe liver transplantation (A-ALRLT) remains the greatest limiting factor for the expansion of using segmental liver transplantation and the major cause of worse short-term prognoses after LDLT. The causes of SFSS are not clear, so in this study we approached the risk factors of the SFSS. METHODOLOGY: The study included 217 consecutive adult recipients that underwent living right lobe liver transplantation at our center. Cases were divided into two groups: 45 cases were determined as SFSS and 172 cases without SFSS within one month after transplantation. Preoperative factors like donor and recipient characteristics, operational data and postoperative factors were compared between the two groups. Factors possibly related to postoperative SFSS were also analyzed using logistic regression. RESULTS: After comparing the two groups, there was no significant difference in donor and recipient background characteristics and no differences were found between the two groups, except for portal inflow volume and MELD score which were much higher and GRWR and outflow volume were much lower in G1. Logistic regression analysis revealed four independent factors associated with SFSS development in right lobe graft: GRWR, MELD score, portal inflow volume and outflow capacity. CONCLUSIONS: Small GRWR, high MELD score, high portal inflow volume and low outflow capacity are four risk factors in A-ALRLT.

Graft size alone should not affect donors selection and be used to predict the prognosis of recipients after living donor liver transplantation.

BACKGROUND/AIMS: Graft size is recognized as one of the most important factors that affect prognosis of the liver recipients. This study determines whether the graft to recipient weight ratio (GRWR) alone can be used to select the liver donor and as an outcome predictor before living donor liver transplantation (LDLT). METHODOLOGY: LDLT patients (202) were divided into three groups according to the GRWR: Group S (n=46, GRWR <0.8); Group M (n=83, GRWR 0.8-1.0); Group L (n=73, GRWR >1.0). Recovery of graft function, incidence of small-for-size syndrome and rate of complications were compared among the three groups. RESULTS: There were no significant differences in the baseline characteristics in both the donors and recipients, nor in the intensive care unit stay hours, re-operation rate, hospital stay after operation, Clavien System score and recovery of graft function after transplantation, among the three groups. The small-for-size syndrome rates were 13%, 7.23% and 11% in Groups S, M and B, and no significant difference was noted among the three groups. CONCLUSIONS: GRWR may not be the only factor affecting recipient prognosis after LDLT. Local graft dysfunction such as impaired venous outflow, severity of disease and portal hyperperfusion in the recipient, and fatty liver in donor may influence the graft and thus the prognosis of transplantation.

[Design and activity verification of human parathyroid hormone (1-34) mutant protein].

Through protein-protein BLAST of homologous sequences in different species in NCBI database and preliminary simulating molecular docking and molecular dynamics by computer software discovery studio 3.1, three amino acids R25K26K27 of natural human parathyroid hormone (1-34) with Q25E26L27 were mutated and the biological activity of the mutant peptide was evaluated. Result showed that: root mean superposition deviation RMSD value between PTH (1-34)-(RKK-QEL) and PTH (1-34) peptide main chain was 2.509 3, indicating that the differences between the two main chain structural conformation was relatively small; the interaction energy between PTH (1-34)-(RKK-QEL) and its receptor protein PTH1R had been enhanced by 7.5% compared to nature PTH (1-34), from -554.083 kcal x mol(-1) to -599.253 kcal x mol(-1); the number of hydrogen bonds was increased from 32 to 38; PTH (1-34)-(RKK-QEL) can significantly stimulate the RANKL gene expression (P < 0.01) while inhibiting the OPG gene expression (P < 0.01) in UAMS-32P cells; in the co-culture system of UAMS-32P cells and mouse primary femur bone marrow cells, PTH (1-34)-(RKK-QEL) stimulated the formation of osteoclasts (P < 0.01) and had a higher biological activity than PTH (1-34) standard reagents.

Optimization of fermentation medium for triterpenoid production from Antrodia camphorata ATCC 200183 using artificial intelligence-based techniques.

In this study, alteration in morphology of submergedly cultured Antrodia camphorata ATCC 200183 including arthroconidia, mycelia, external and internal structures of pellets was investigated. Two optimization models namely response surface methodology (RSM) and artificial neural network (ANN) were built to optimize the inoculum size and medium components for intracellular triterpenoid production from A. camphorata. Root mean squares error, R (2), and standard error of prediction given by ANN model were 0.31%, 0.99%, and 0.63%, respectively, while RSM model gave 1.02%, 0.98%, and 2.08%, which indicated that fitness and prediction accuracy of ANN model was higher when compared to RSM model. Furthermore, using genetic algorithm (GA), the input space of ANN model was optimized, and maximum triterpenoid production of 62.84 mg l(-1) was obtained at the GA-optimized concentrations of arthroconidia (1.78 × 105 ml(-1)) and medium components (glucose, 25.25 g l(-1); peptone, 4.48 g l(-1); and soybean flour, 2.74 g l(-1)). The triterpenoid production experimentally obtained using the ANN-GA designed medium was 64.79 ± 2.32 mg l(-1) which was in agreement with the predicted value. The same optimization process may be used to optimize many environmental and genetic factors such as temperature and agitation that can also affect the triterpenoid production from A. camphorata and to improve the production of bioactive metabolites from potent medicinal fungi by changing the fermentation parameters.

Identification of binding peptides of the ADAM15 disintegrin domain using phage display.

ADAM15 plays an important role in tumour development by interacting with integrins. In this study, we investigated the target peptides of the ADAM15 disintegrin domain. First, we successfully produced the recombinant human ADAM15 disintegrin domain (RADD) that could inhibit melanoma cell adhesion by using Escherichia coli. Second, four specific binding peptides (peptides A, B, C, and D) were selected using a phage display 12-mer peptide library. The screening protocol involved 4 rounds of positive panning on RADD and 2 rounds of subtractive selection with streptavidin. By using the BLAST software and a relevant protein database, integrin alpha v beta 3 was found to be homologous to peptide A. Synthetic peptide A had a highly inhibitory effect on RADD-integrin alpha v beta 3 binding. The results demonstrate the potential application of short peptides for disrupting high-affinity ADAM-integrin interactions.

Expression, purification, and characterization of recombinant human serum albumin fusion protein with two human glucagon-like peptide-1 mutants in Pichia pastoris.

Glucagon-like peptide-1 (GLP-1) is a 30-residue peptide hormone secreted by intestinal L-cells in response to nutrient ingestion. In the present study, overlapping PCR technology was employed to construct two GLP-1 mutants (GLP-1(A2G))2 and human albumin (HSA) genes in vitro without linker. The spliced gene, (GLP-1(A2G))2-HSA, was over expressed under the control of promoter AOX1 and Mat alpha signal peptide in Pichia pastoris. SDS-PAGE and Western blotting were applied to assay the recombinant fusion protein in the culture broth. The results demonstrated that the recombinant (GLP-1(A2G))2-HSA concentration in the broth could reach a level of 245.0 mg/L and the expressed fusion protein was capable of cross-reacting with anti-human GLP-1 and anti-human albumin antibody. The recombinant (GLP-1(A2G))2-HSA protein was purified by ultrafiltration, columns of Q-sepharose fast flow and Superdex 75 size-exclusion. The recombinant (GLP-1(A2G))2-HSA protein obtained could lower in vivo glucose concentration in blood and stimulate in vitro islet cell proliferation. In mouse model, the fusion protein was detectable in plasma even 308 h after a single subcutaneous dose of 1.25 mg/kg. The result showed that the terminal biological half-time of the protein was about 54.2 h which is 650-fold longer than that of GLP-1. The pharmacokinetic analysis of the protein suggests its promising application in clinical medicine.

Model of lymph node metastasis posterior to the right recurrent laryngeal nerve in papillary thyroid carcinoma.

BACKGROUND: Cervical lymph node metastasis (LNM) is a prognostic factor of papillary thyroid carcinoma (PTC). The way to deal with lymph node posterior to the right recurrent laryngeal nerve (LN-prRLN) is controversial. Nevertheless, if metastatic lymph nodes are not removed during the first operation, the subsequent salvage surgery of recurrent tumor in this area would entail high risk and complication. The purpose of this study was to develop a preoperative prediction model for LN-prRLN metastasis in PTC patients using clinicopathological characteristics. PATIENTS AND METHODS: We performed a prospective study of 595 patients with PTC who underwent LN-prRLN dissection from March 2014 to June 2017. The clinicopathological data were randomly divided into derivation (n=476) and validation sets (n=119). A predictive model was initially established based upon the data of the derivation set via multivariate analyses, and the accuracy of the model was then examined with data of the validation set. The discriminative power of this model was assessed in both sets. RESULTS: Metastases of the LN-prRLN were identified in 102 (17.14%) of 595 patients. Age (odds ratio [OR] 0.971, 95% CI, 0.949-0.994, p=0.013), tumor size (OR 2.163, 95% CI, 1.431-3.270, p<0.001), capsular invasion (OR 1.934, 95% CI, 1.062-3.522, p=0.031), and right LNM (OR 3.786, 95% CI, 2.012-7.123, p<0.001) were significantly associated with LN-prRLN metastasis. The areas under the curves were 0.790 for the derivation set (sensitivity 71.95%, specificity 78.68%) and 0.878 for the validation set (sensitivity 85.00%, specificity 78.79%). CONCLUSION: We developed and validated the first model to predict LN-prRLN metastases in patients with PTC based on clinicopathological parameters.

Hangzhou criteria for liver transplantation in hepatocellular carcinoma: a single-center experience.

BACKGROUND AND AIM: The inclusion criteria for liver transplantation (LT) in hepatocellular carcinoma (HCC) are being expanded, and the Hangzhou criteria are the most accepted criteria in China. The aim of our study was to evaluate the Hangzhou criteria for LT in HCC with respect to the Milan criteria. METHODS: We retrospectively collected data of 298 cases of LT in HCC in our center from August 2000 to December 2010, and then divided these patients into three groups according to the tumor characteristics: the Milan criteria group (n=97), the Hangzhou criteria group (n=172), and the out of Hangzhou criteria group (n=126). We compared the baseline characteristics and outcome of these three groups of patients. RESULTS: Baseline patient characteristics showed no significant difference among the three groups, except for younger age in the out of Hangzhou group (P<0.05). Overall the 1-, 3-, and 5-year survival rates were 91.8, 88.7, and 86.6%, respectively, for the Milan criteria group; 86.6, 76.7, and 73.8% for the Hangzhou criteria group; and 76.2, 57.1, and 56.3% for the out of Hangzhou criteria group (P<0.05). The 1-, 3-, and 5-year tumor-free survival rates were 88.7, 86.6, and 86.6%, respectively, for the Milan criteria group; 83.7, 73.8, and 73.3% for the Hangzhou criteria group (P=0.014); and 63.5, 48.4, and 48.4% for the out of Hangzhou group, which was significantly lower than the rates in the other two groups (P=0.000). CONCLUSION: Although the Hangzhou criteria lead to lower overall survival and tumor-free survival rates compared with the Milan criteria, the Hangzhou criteria indicate more HCC patients for LT and are associated with a considerable long-term outcome. The Hangzhou criteria should be accepted as the inclusion criteria for LT in HCC.

Up-to-seven criteria for hepatocellular carcinoma liver transplantation: a single center analysis.

AIM: To detect whether the up-to-seven should be used as inclusion criteria for liver transplantation for hepatocellular carcinoma. METHODS: Between April 2002 and July 2008, 220 hepatocellular carcinoma (HCC) patients who were diagnosed with HCC and underwent liver transplantation (LT) at our liver transplantation center were included. These patients were divided into three groups according to the characteristics of their tumors (tumor diameter, tumor number): the Milan criteria group (Group 1), the in up-to-seven group (Group 2) and the out up-to-seven group (Group 3). Then, we compared long-term survival and tumor recurrence of these three groups. RESULTS: The baseline characteristics of transplant recipients were comparable among these three groups, except for the type of liver graft (deceased donor liver transplant or live donor liver transplantation). There were also no significant differences in the pre-operative α-fetoprotein level. The 1-, 3-, and 5-year overall survival and tumor-free survival rate for the Milan criteria group were 94.8%, 91.4%, 89.7% and 91.4%, 86.2%, and 86.2% respectively; in the up-to-seven criteria group, these rates were 87.8%, 77.8%, and 76.6% and 85.6%, 75.6%, and 75.6% respectively (P < 0.05). However, the advanced HCC patients' (in the group out of up-to-seven criteria) overall and tumor-free survival rates were much lower, at 75%, 53.3%, and 50% and 65.8%, 42.5%, and 41.7%, respectively (P < 0.01). CONCLUSION: Considering that patients in the up-to-seven criteria group exhibited a considerable but lower survival rate compared with the Milan criteria group, the up-to-seven criteria should be used carefully and selectively.

Transplantation vs resection for hepatocellular carcinoma with compensated liver function after downstaging therapy.

AIM: Our study aimed to compare the results of liver transplantation (LT) and liver resection (LR) in patients with hepatocellular carcinoma (HCC) that met the Milan criteria after successful downstaging therapy. METHODS: From February 2004 to August 2010, a consecutive series of 102 patients were diagnosed with advanced-stage HCC that met the modified UCSF down-staging protocol inclusion criteria. All of the patients accepted various down-staging therapies. The types and numbers of treatments were tailored to each patient according to the tumor characteristics, location, liver function and response. After various downstaging therapies, 66 patients had tumor characteristics that met the Milan criteria; 31 patients accepted LT in our center, and 35 patients accepted LR. The baseline characteristics, down-staging protocols, postoperative complications, overall survival and tumor free survival rate, and tumor recurrence rate were compared between the two groups. Kaplan-Meier analyses were used to estimate the long-term overall survival and tumor-free survival rate. Meanwhile, a Cox proportional hazards model was used for the multivariate analyses of overall survival and disease-free survival rate. RESULTS: No significant difference was observed between the LT and LR groups with respect to the down-staging protocol, target tumor characteristics, and baseline patient characteristics. Fifteen patients suffered various complications after LT, and 8 patients had complications after LR. The overall complication rate for the LT group was 48.4%, which was significantly higher than the LR group (22.9%) (P = 0.031). The overall in-hospital mortality in hospital for the LT group was 12.9% vs 2.9% for the LR group (P = 0.172). The overall patient survival rates at 1-, 3- and 5-years were 87.1%, 80.6% and 77.4%, respectively, after LT and 91.4%, 77.1% and 68.6%, respectively, after LR (P = 0.498). The overall 1-, 3- and 5-year tumor recurrence-free rates were also comparable (P = 0.656). Poorer tumor differentiation (P = 0.041) and a higher post-downstage alpha-fetoprotein (AFP) level (> 400 ng/mL) (P = 0.015) were the two independent risk factors for tumor recurrence in the LT and LR patients who accepted successful down-staging therapy. CONCLUSION: Due to the higher postoperative morbidity and similar survival and tumor recurrence-free rates, LR might offer better or similar outcome over LT, but a larger number and further randomized studies may be needed in the future for drawing any positive conclusions.

"Metroticket" predictor for assessing liver transplantation to treat hepatocellular carcinoma: a single-center analysis in mainland China.

AIM: To validate the "Metroticket" predictor using a large cohort of liver transplantation (LT) patients with hepatocellular carcinoma (HCC) in China. METHODS: In total, 230 cases of LT for HCC treatment at our center, from July 2000 to August 2008, were included in the present study. The predicted 1-, 3- and 5-year post-LT survival rates were calculated using the Metroticket model (http://89.96.76.14/metroticket/calculator/). The predicted and observed long-term survival rates were then compared and analyzed. RESULTS: The predicted survival rates for all 230 cases, as calculated by the Metroticket model, were 64.7% and 56.2% at 3 and 5 years, respectively, and the observed survival rates for these patients were 71.3% and 57.8%, respectively. For the 23 cases with macrovascular invasion, the predicted 5-year survival rate was 43.5%, whereas the observed 5-year survival rate was only 8.7%. For the 42 cases with microvascular invasion but an absence of macrovascular invasion, the predicted 5-year survival rate was 44.9%, and the observed 5-year survival rate was 50%. For the remaining 165 patients without any vascular invasion, the predicted 5-year survival rate was 65.8%, and the observed 5-year survival rate was 66.7%. CONCLUSION: The Metroticket model can be used to accurately predict survival in HCC-related LT cases with an absence of macrovascular invasion.

A sandwich ELISA for assessment of pharmacokinetics of HSA-(BNP)2 fusion protein in mouse plasma.

Brain natriuretic peptide (BNP) is a circulating hormone of cardiac origin that plays an important role in the regulation of intravascular blood volume and vascular tone. HSA-(BNP)(2), derived from the joining of human BNP to the C-terminus of human serum albumin (HSA), has been developed to prolong the BNP pharmacodynamic action. For the analysis of pharmacokinetics of the new drug, a novel sandwich enzyme-linked immunosorbent assay (ELISA) was established and validated to quantify HSA-(BNP)(2) fusion protein in mouse plasma. The ELISA method was calibrated with 1:10 and 1:100 dilutions of blank mouse plasma spiked with HSA-(BNP)(2) standard and validated with respect to parallelism, precision (intra- and inter-assay variation), accuracy (recovery), specificity and stability. The practical working range was estimated to be 31.2-2000ng/ml with the limit of detection was 7.8ng/ml. Recoveries ranged from 80.5 to 108.4%, while the intra- and inter-assay precisions were <2.73% and <4.32%, respectively. The terminal half-life of HSA-(BNP)(2) was 2.14h, which had extended more than 40 times compared to 3.1min half-life of BNP monomer in mouse.