Thirty-five consecutive pediatric living donor liver transplantation: experiences and lessons learned from a single center.

BACKGROUND/AIMS: In the last 10 years, the early patient outcome of liver transplantation in children have significantly improved. Now the overall outcomes of pediatric LT are promising. METHODOLOGY: In this study, we review the outcome of all pediatric liver transplants performed at our center and analyze our experiences with pediatric liver transplant. Of the 34 liver transplant recipients, 26 were highly urgent (19.7%). RESULTS: Actuarial patient survival rates at 6, 12, and 36 months was 82.9%, 79.8% and 72.2%, respectively. Indications for liver transplant were biliary atresia (n = 22), Wilson's disease (n = 4), glycogen storage disease (n = 3), portal vein cavernous transformation (PVCT) (n = 3), fulminant liver failure (n = 1), and cryptogenic cirrhosis (n = 1). The main complications were surgical complications (including biliary complications, portal vein or arterial complications, intestinal perforation, postoperative bleeding, of which 20% required reoperation) and infections. Cyclosporine was the primary immunosuppressive agent used in 70.6% of patients, with a 26.5% incidence of acute allograft rejection within the first six months. One children underwent re-transplant as a result of hepatic artery thrombosis. Nine children died during followup. They were related to portal vein thrombosis (one), chronic rejection (one), sepsis (one), post-transplant lymphoproliferative disease (one) and so on. CONCLUSIONS: The overall outcomes of pediatric liver transplantation at our center are promising. Advances in post-transplant care and monitoring of the recipients, technical refinements enable these results.

Usage of 64-detector-row spiral computed tomography volumetry in preoperative volume prediction in living donor liver transplantation in children.

PURPOSE: To investigate the correlation between the graft volume calculated by 64-detector-row spiral computed tomography (CT) and the graft weight measured during the living donor liver transplantation (LDLT) operation, and try to get an equation to help determine the possible weight of graft before operation. METHODS: 23 donors with left lateral lobe LDLT were enrolled to undergo 64-detector-row spiral CT and the imaging data at the hepatic venous phase was used for whole and partial liver volumetric measurement on a dedicated image postprocessing workstation. The resected part of donor liver was weighed during the operation. Statistical analysis with SPSS15.0 was used to analyze the correlation between the estimated liver volume by CT and the actual graft weight. RESULTS: The graft volume calculated preoperatively by CT (293.35 ± 53.43 ml) was significantly larger than measured graft weight during the operation (252.82 ± 50.96 g) (P < 0.05). All corresponding pre- and intraoperative data correlated significantly (R = 0.885) (P < 0.001). Intraoperatively expected weight (W (intraop)) in grams and volume calculated preoperatively by CT (V (preop)) in milliliters can be calculated with the equation W (intraop) (g) = 0.844 × V (preop) (ml) + 5.271. CONCLUSION: Liver volume calculated by 64-detector-row spiral CT preoperatively can predict the actual graft weight, which is very useful in donor selection in LDLT.

[Retrospective analysis of maternal and infant birth features of hepatoblastoma patients].

OBJECTIVE: To explore the risk factors for hepatoblastoma. METHODS: A case-cohort study using Logistic regression multiple variables analysis of medical record data sets was conducted to examine infant and perinatal risk factors for hepatoblastoma. RESULTS: Birth weight less than 1,000 g was associated with a strongly increased risk of hepatoblastoma (odds risk, OR = 26.0, 95% confidence interval, CI: 14.0 to 65.7). After adjustment of birth weight, a moderately increased risk of hepatoblastoma was found for older maternal age ( > 35 years vs. 20 to 34 years: OR = 2.6, 95% CI: 0.9 to 5.9), maternal smoking (OR = 2.9, 95% CI: 1.1 to 4.2) and higher maternal pregnancy body mass index (OR = 3.2, 95% CI: 1.0 to 6.7). CONCLUSION: Very low birth weight and maternal characteristics including overweight, smoking are associated with hepatoblastoma risk.

[Living-related liver transplantation for cavernous transformation of portal vein: a clinical study of 3 cases].

OBJECTIVE: To review the outcomes of living-related liver transplantation (LRLT) in treating 3 cases of cavernous transformation of portal vein (CTPV) with severe portal hypertension. METHODS: Three children (two boys and one girl) were presented to our hospital with recurring esophageal variceal bleeding, decompensating ascites, splenomegaly and refractory anemia. CTPV was confirmed by intravenous computed tomographic portography using a helical computed tomography scanner and 3-dimensional image reconstruction. LRLT were performed in these 3 patients from July 2006 to January 2007. The evaluation of the outcomes was made by referring to their clinical features and laboratory and imaging examination findings. RESULTS: Although one patient died from early graft thrombosis, the other two patients showed excellent prognoses. They lived and stayed well during a follow-up period of 12-14 months. Following the transplantations, there had been no esophageal variceal hemorrhage, the ascites disappeared and the portal hypertension vanished. Their hemoglobin, blood platelets count, and serum albumin reached normal values. CONCLUSION: LRLT is an effective procedure in treating CTPV with severe portal hypertension. The reconstruction of the portal vein is the difficult part of the LRLT procedure.

BMP4 knockdown of NCSCs leads to aganglionosis in the middle embryonic stage.

Transplacental bone morphogenetic protein (BMP)4 RNA interference (RNAi) is a technique used to knockdown genes in embryos. BMP4 are essential for the development of nervous system in the differentiation of neural crest stem cells (NCSCs). The failure of differentiation and migration of NCSCs may lead to aganglionosis. In the present study, pregnant mice were divided into three groups: Ringer's group, pSES group and RNAi­BMP4 group. In order to silence the BMP4 gene in the first generation (F1), 11.5 day pregnant mice were injected with the small interfering RNA BMP4 plasmid, pSES or Ringer's solution via the tail vein. Semi­quantitative reverse transcriptase­polymerase chain reaction (RT­PCR)and western blotting were employed to ensure the downregulation of BMP4. Finally, X­rays were performed following a barium enema. Aganglionosis was diagnosed by general anatomy and immunohistochemistry. Compared with the control group, transplacental RNAi was able to downregulate the BMP4­Smad4 of 11.5 day embryos, as determined by semi­quantitative RT­PCR and western blotting. The megacolons of the mice were demonstrated by X­ray and confirmed by general anatomy. Aganglionosis of colonic mucosa and submucosa were diagnosed by pathology, and immunohistochemistry. Knockdown of BMP4 in pregnant mice at the middle embryonic stage led to aganglionosis. It was therefore demonstrated that BMP­Smad was essential to the NCSCs of middle stage embryos. BMP­Smad served important roles in the generation of aganglionosis. This technique of knockdown BMP4 gene may be used to establish an aganglionosis mouse model.

Chemoprotection effect of retroviral vector encoding multidrug resistance 1 gene to allow intensified chemotherapy in vivo.

Increasing the expression of human multidrug resistance (MDR) 1 gene in bone marrow cells to prevent or circumvent bone morrow toxicity from chemotherapy agent is a high priority of dose intensification protocols. In this study, we have used a tumor-bearing model to investigate the chemoprotection effect of MDR1 gene by transfecting retroviral vectors containing and expressing the MDR gene in vivo. Hematopoietic progenitor cells were served as target of MDR1 gene transferred by the mediation of retrovirus vector and engrafted into the BALB/c mice with 60Co-gamma ray exposure in advance. Doxorubicin (5, 10, and 20 mg/kg) suppressed tumor growth of the xenograft significantly in a dose-dependence mode if supported by suitable peripheral WBC. WBC count revealed that the mice that had received gene-transduced cells showed a significant increase in WBC count compared with their gene-transduced naive counterparts. The function and expression of MDR1 gene were detected by flow cytometry, RT-PCR, and immunohistochemistry (IC) method. MDRl mRNA expression could be detected in BM. Spleens contained measurable amounts of MDRl mRNA. Tail vein blood and tumor tissue detected MDRl DNA but no MDRl mRNA expression. FACS analysis of infected BM cells obtained 6 weeks later showed high levels of P-gp function. Based on these results we conclude that cytostatic drug resistance gene therapy may provide some degree of chemoprotection and so can increase the chemotherapy dose to kill tumor cells.

Therapeutic efficacy and bone marrow protection of the mdr1 gene and over-dose chemotherapy with doxorubicin for rabbits with VX2 hepatocarcinoma.

BACKGROUND: Malignant tumors are common diseases threatening to the health and life of human being. Clinically, the multidrug resistance of tumor cells and bone marrow depression caused by chemotherapeutic agents are the main obstacles to the treatment of tumors, and both are related to the mdr1 gene. The over expression of the mdr1 gene in tumor cells contributes to the multidrug resistance of malignant tumor cells. With little expression of the mdr1 gene, bone marrow cells particularly susceptible to multidrug resistance-sensitive agents, which cause serious toxicity in bone marrow. This study was undertaken to assess therapeutic efficacy of transplantation of bone marrow mononuclear cells transferred with the mdr1 gene and over-dose chemotherapy with doxorubicin for VX2 hepatocarcinoma of rabbits. METHODS: The mdr1 gene was transferred into the bone marrow mononuclear cells of rabbits, which was co-cultured with retroviral vector-containing supernatant, and the cells were autotransplanted into a rabbit model with VX2 hepatocarcinoma. After chemotherapy with doxorubicin, the protective effects of the mdr1 gene and therapeutic efficacy of over-dose chemotherapy were observed. RESULTS: The mdr1 gene was transferred successfully into the bone marrow mononuclear cells, with a transduction efficiency of 35%. After autotransplantation, the mdr1 gene was expressed functionally in bone marrow with a positive rate of 8%, indicating that the gene played an important role in bone marrow protection. The rabbits with VX2 hepatocarcinoma, which had received the mdr1 gene-transduced cells, survived after chemotherapy with a 3-fold dose of adriamycin, and their white blood cell counts were (4.26 +/- 1.03) x 10(4)/L. Since hepatocarcinoma cells were eradicated, the survival time (97.00 +/- 46.75 d) of the rabbits was extended (P<0.05) and the healing rate of the tumor was increased (P<0.05). CONCLUSIONS: The transferring of the mdr1 gene into bone marrow mononuclear cells could confer chemoprotection to bone marrow, and over-dose chemotherapy could be prescribed for the treatment of malignant tumors.

[Establishment and identification of bone morrow specific transgenic mouse model with tumorigenesis by mutant Myc retrovirus infection.].

OBJECTIVE: To establish a novel Myc gene transgenic mouse model for spontaneously forming B-lymphoma and assessing its tumorigenesis potential. METHODS: Freshly isolated hematopoietic progenitor cells served as the target for Myc gene transfer mediated by a retrovirus vector. These cells were engrafted into C57BL/6 mice with (60)Co-gamma ray radiation in advance. Tumor latency was measured and the tumor loaded mice were followed for survival time. Tumor was identified with histology and immunostaining. The exogenous Myc gene was detected by Western blot (in liver, spleen, tumor tissue) and flow cytometry (FCM) \[in bone marrow (BM)\]. RESULTS: Mice BM-infected with mutant Myc gene more readily gave rise to B-cell lymphomas than those infected with wild type Myc gene did Myc gene was expressed highly in BM and tumor tissues but not in liver and spleen. CONCLUSION: Our model will be a tool in assessing the transforming potential of Myc mutants and in studying cooperation between Myc and other oncogenes. Mutant Myc is more effective than wild-type Myc in promoting B cell lymphomagenesis in mice.

Screening and cloning of multi-drug resistant genes in HL-60/MDR cells.

OBJECTIVE: To screen and clone multi-drug resistance (MDR) related genes in MDR acute myeloid leukemia cells (HL-60/MDR). METHODS: HL-60/MDR was established using All-Trans Retinoic Acid. With the HL-60 cells as "tester" and HL60/MDR as "driver", the cDNA library of HL-60/MDR was established by suppression subtractive hybridization. Then 12 of the resulting subtracted cDNA clones were selected for DNA sequencing and homology analysis. The obtained expressed sequence tags (ESTs) were analyzed with the GenBank BLASTN program to identify sequence homologies to known genes. RESULTS: The HL-60/MDR cells had different multi-drug resistance to six kinds of chemotherapeutic drugs. The 211 positive gene clones in differential cDNA library of HL-60/MDR cells were amplified with PCR and 46 gene clones exhibited differential expression (ratio >3). Twelve gene clones with significant differential expression (ratio >5) were screened out to homology analysis. Of these, 11 matched known genes and the rest 1 showed no significant homology to human or non-human known sequences. It was named as gene clone HA117. CONCLUSIONS: This effort provides the partial list of genes differential expressed in HL-60/MDR cells and a novel gene HA117 was found to be related to MDR. Identification of these genes contributes to our understanding of MDR development, and potentially provides candidate target genes to overcome MDR.

Relationship between matrix metalloproteinase 2 and lung cancer progression.

BACKGROUND AND OBJECTIVE: Matrix metalloproteinases (MMPs) play an important role in several steps of cancer development. MMP2 and MMP9 have previously been implicated in lymphatic and vascular invasion of lung cancer; however, the expression and prognostic significance of MMP2 and MMP9 is not fully clarified. This study was designed to investigate the significance of MMP2 and MMP9 in lung cancer tissue or serum, and their correlation with lung cancer prognosis. METHODS: Immunohistochemical analysis was performed to determine MMP2 and MMP9 staining in human nonsmall cell lung cancer (NSCLC). Serum MMP2 and MMP9 protein levels in patients after surgery were measured using the ELISA method. The correlation between MMP2 and MMP9 serum levels and clinicopathological features of NSCLC were analyzed by survival analysis. We also performed reverse transcriptase (RT)-PCR assays to detect messenger RNA (mRNA) expression to further confirm the activity of MMP2 and MMP9 in human lung cancer. RESULTS: Increased MMP2 immunostaining and MMP2 serum level correlated with advanced tumor stage and the presence of distant metastasis (Pearson's chi(2) test and ANOVA, p < 0.05). However, for MMP9, only serum level showed a correlation with advanced tumor stage. No significant correlation was observed between MMP2 or MMP9 immunostaining expression and tumor histologic features (Pearson's chi(2) test, p = 0.061 and p = 0.087, respectively). A high densitometry value of MMP2 and MMP9 PCR products (i.e. mRNA expression level) was related to poor differentiation grade, distant metastasis, and small cell carcinoma histologic type (ANOVA, p < 0.05). CONCLUSIONS: Our results suggest that MMP2 is a more sensitive predictor than MMP9 of lung cancer progression, metastasis, and survival. Serum MMP2 levels may be a valuable prognosis variable and could help to stratify lung cancer patients into low- and high-risk groups.

[Effect of fibronectin-thrombopoietin gene modification on human bone marrow mesenchymal stem cells].

OBJECTIVE: To observe the effect of Fn-TPO gene modification on human bone marrow mesenchymal stem cells (MSCs). METHODS: Retroviral vector containing Fn-TPO gene was constructed and bone marrow MSCs was modified by this vector. The transcription of Fn-TPO gene in MSCs was observed. The proliferation capacities, hematopoietic cells adhering capacities and TPO secretion capacities of gene modified MSCs were assayed respectively. Cord blood CD34 cells were seeded on the gene modified MSCs layers and several essential growth factors were added. After co-culturing in vitro for 7 days, the number of CD34 cells and their colony forming capacities were assayed by flow cytometry and semisolid culture assay. RESULTS: Retroviral vector containing Fn-TPO gene was successfully constructed and bone marrow MSCs were modified by this vector. Fn-TPO gene was expressed by bone marrow MSCs after gene modification. The viability of MSCs had no significant difference between pre- and post-gene-modification [(7.18 +/- 0.89) 10(4)/ml vs. (6.92 +/- 0.77) 10(4)/ml, P > 0.05]. The hematopoietic cells adhering ability of gene modified bone marrow MSCs was reinforced(0. 188 +/- 0.018 vs. 0.167 +/- 0.017, P < 0.01). The concentration of TPO in the MSCs culture supernatant raised from (5.58 +/- 0.37) ng/ml to (7.46 +/- 0.59) ng/ml (P < 0.01) and did not significantly decline in a short-time period, but influenced by the growth status of MSCs. After co-culturing with gene modified MSCs for 7 days, the absolute number of nucleated cells, the percentage of CD34+ cells and the colony numbers of BFU-E, CFU-GM, CFU-GEMM were (29.9 +/- 2.7) x 10(4), (33.3 +/- 2.8)% , 109.3 +/- 4.1, 163.7 +/- 7.1, 13.3 +/- 1.5, respectively, being significantly higher than that co-cultured with non-modified MSCs. CONCLUSIONS: Fn-TPO gene modification can improve the capacity of human bone marrow MSCs for hematopoietic cells adhering, TPO secretion and cord blood CD34 cells amplification.

[Protection of mdr1 transfected cord blood mononuclear cell graft against anticancer agents in vivo].

OBJECTIVE: To explore the myelo-protection effect of mdr1 transfected cord blood cells (CBMNCs) graft against high-dose homoharringtonine leukemia-bearing severe combined immunodeficient (SCID) mice model. METHODS: Multidrug resistant (mdr1)gene was transferred into CBMNCs by a retrovirus vector, containing full-length cDNA of human mdr1 gene. CBMNCs and high-titer retrovirus supernatant were cocultured with cytokine combinations for 5 - 6 days. The SCID mouse models bearing human HL-60 cell leukemia were divided into three groups. Group A received tail vein injection of 2 x 10(6) mdr1 gene transduced CBMNCs at day 1 and 3, groups B and C 2 x 10(6) un-transduced CBMNCs and same volume of normal saline, respectively. The 3 groups of the mouse model were treated with weekly escalated doses of homoharringtonine. The peripheral white blood cell (WBC) counts, the human leukemia cells percentage in peripheral blood, the histological findings of main organs were assayed. The CD33 positive HL-60 cells in bone marrow were determined by flow cytometry. The function and expression of mdr1 gene were examined by PCR, immunochemistry (IC) and DNR extrusion test in vivo. RESULTS: (1) mdr1 gene was transferred into CBMNCs successfully and the transfection frequency was 30%. (2) Leukemia SCID mice were xenotransplanted with mdr1-transfected BMMNCs by a programmed procedure and could be used as a valuable model for in vivo evaluating myelo-protection effects. (3) The transfected mice could tolerate homoharringtonine 5 approximately 6 folds higher than conventional dose and kept peripheral WBC count at a mean of 3 x 10(9)/L, with the peripheral human myeloid leukemia cells percentage decreasing to less than 5%. Histological examination showed that there was no leukemia infiltration in the main organs, the CD33 positive HL-60 cells in bone marrow were less than 5%. (4) The repopulation frequency of the transfected CBMNs in marrow were 9.13%. DNR extrusion test confirmed that the P-gp product maintained its biological function in the marrow. CONCLUSION: mdr1 transferred-human CBMNC can xenotransplanted and repopulated in leukemia-bearing SCID mouse and are protected from chemotherapy-induced myelosuppression.

[Study on the collagen constitution of hyperplastic scar in different ages and its influencing factors].

OBJECTIVE: To investigate the collagen constitution of hyperplastic scar (HS) in different ages and the change of relative factors. METHODS: Thirty cases with HS were divided into two groups according to patients' age: group 1 (1 - 19 years, A) and group 2 (20 - 50 years, B). The normal skin (NS) from corresponding age of volunteers was employed as control group. The changes in TGFbeta1, collagenase (MMP-1) and tissue inhibitor of metalloproteinase (TIMP-1beta) and the collagen ratio were observed by means of in situ hybridization technique and SABC (Strept-Avidin-Biotin complex) immunohistochemistry and image analysis. RESULTS: The ratio of type I to type III collagen in A group was 6.48 in average and 3.76 in B group, but there was no evident difference in the ratio during the disease process in both groups. The expression of TGFbeta1 in A group was much higher than that in B group (P < 0.01). The TIMP-1 mRNA expression showed no difference among all age groups in HS patients, but it was much higher than that in NS group. The MMP-1 expression was evidently lower than TIMP-1 expression, and there was no difference in MMP-1 expression compared with NS group. CONCLUSION: (1) The TGFbeta1 expression in HS patients was negatively correlated with age, and the increased expression of TGFbeta1 produced an increase ratio of type I to type III collagen. (2) High level expression of TIMP-1 led to the formation of HS by inhibiting MMP-1 expression, and the expression was not related to age.