Hepatocyte growth factor gene transfer with naked plasmid DNA ameliorates dimethylnitrosamine-induced liver fibrosis in rats

Aim: Hepatocyte growth factor (HGF) has various biological properties, including antifibrogenic activity. In the present study, we tested the efficacy of HGF gene therapy using naked plasmid DNA in dimethylnitrosamine (DMN)-induced liver fibrosis in a rat model. Methods: Naked plasmid DNA encoding human HGF was injected once, together with a hypertonic solution, into the hepatic artery after DMN treatment on three consecutive days per week for 3 weeks. Naked plasmid DNA encoding beta-galactosidase was injected similarly in the DMN-treated control rats. DMN treatment was continued once weekly after gene transfer for additional 3 weeks. Results: The human HGF protein expression was detected in livers transfected with human HGF naked plasmid DNA, gradually decreasing by day 21. The expression of the endogenous rat HGF protein was also upregulated after human HGF gene transfer. Phosphorylation of c-Met, a HGF receptor, was detected only in livers transfected with human HGF plasmid DNA. Fibrosis was attenuated significantly in livers transfected with the human HGF plasmid. Attenuation wasaccompanied by decreased expression of alpha-smooth muscle actin. Increased portal vein pressure after treatment with DMN was suppressed significantly by HGF gene transfer. The upregulated hepatic protein expression of transforming growth factor-beta (TGF-beta) in response to DMN was markedly attenuated by HGF gene transfer accompanied by the increased protein expression for matrix metalloproteinases (MMP)-3 and -13. Conclusion: The hepatic arterial injection of human naked plasmid HGF DNA was effective in suppressing liver fibrosis induced in rats by DMN. The mechanisms by which HGF expression attenuated liver fibrosis may include the suppression of hepatic TGF-beta expression and the induction of MMP expression.     

三明治构型大鼠原代肝细胞长期培养及功能测定

目的观察三明治构型大鼠原代肝细胞长期培养的形态学变化,并对其功能进行测定。方法采用改良原位两步法门静脉胶原酶灌注分离单肝细胞,台盼蓝拒染实验观察细胞活力,利用三明治培养构型培养成年大鼠原代肝细胞,倒置显微镜下连续观察肝细胞的形态学变化,定期收集培养细胞上清液,检测所培养肝细胞的分泌及生物转化功能,并与单层胶原培养肝细胞比较。结果平均每个鼠肝可获取(2～3)×108个肝细胞,存活率为(93±3)%;体外肝细胞培养第3天,细胞活力、清蛋白分泌功能恢复到最佳状态;三明治构型培养第7天,地西泮24h代谢量达到最高峰。三明治构型培养的肝细胞形成肝索样结构,并逐渐形成胆小管网络;在培养的21d内,清蛋白分泌、地西泮代谢始终维持较高的水平;肝细胞形态维持可达28d以上。结论三明治构型肝细胞培养体系更接近于肝细胞体内生长环境,肝细胞可在较长时间内保持良好的形态结构和功能。三明治构型不仅可以应用于肝细胞的基础研究,而且为肝细胞移植和生物人工肝治疗肝衰竭奠定了一定的基础。     

Purification and in vitro toxicity of gamma amanitin

We aimed to obtain gamma amanitin with high purity through a purification process and compare toxic effects of alpha, beta, and gamma amanitin. Specific concentrations of the toxins (25, 10, 1, and 0.1?μg/mL) were applied to the C3A human hepatocytes. A MTT test was performed to determine the level of toxicity. Alpha amanitin showed a higher toxicity in 48?h while the lowest toxicity was observed in beta amanitin. The toxicity level of gamma amanitin was found between the alpha and beta amanitin toxicity. Our method is suitable for obtaining gamma amanitin with high purity (>99%) as well as for obtaining alpha amanitin and beta amanitin. Gamma amanitin has been shown to have equal responsibility for toxicity as alpha amanitin in amanita poisoning.     

Analysis of the functional integrity of cryopreserved human liver cells including xenografting in immunodeficient mice to address suitability for clinical applications.

BACKGROUND: The availability of well-characterized human liver cell populations that can be frozen and thawed will be critical for cell therapy. We addressed whether human hepatocytes can recover after cryopreservation and engraft in immunodeficient mice. METHODS: We isolated cells from discarded human livers and studied the properties of cryopreserved cells. The viability of thawed cells was established with multiple in vitro assays, including analysis of liver gene expression, ureagenesis, cytochrome P450 activity, and growth factor-induced cell proliferation. The fate of transplanted cells was analysed in immunodeficient NOD-SCID mice. RESULTS: After thawing, the viability of human hepatocytes exceeded 60%. Cells attached to culture dishes, proliferated following growth factor stimulation and exhibited liver-specific functions. After transplantation in NOD-SCID mice, cells engrafted in the peritoneal cavity, a heterologous site, as well as the liver itself, retained hepatic function and proliferated in response to liver injury. Transplanted hepatocytes were integrated in the liver parenchyma. Occasionally, transplanted cells were integrated in bile ducts. CONCLUSIONS: Cryopreserved human liver cell showed the ability to retain functional integrity and to reconstitute both hepatic and biliary lineages in mice. These studies offer suitable paradigms aimed at characterizing liver cells prior to transplantation in people.     

The genetic abnormality in the beta cell determines the response to an oral glucose load

Aims/hypothesis: We assessed how the role of genes genetic causation in causing maturity-onset diabetes of the young (MODY) alters the response to an oral glucose tolerance test (OGTT). Methods: We studied OGTT in 362 MODY subjects, from seven European centres; 245 had glucokinase gene mutations and 117 had Hepatocyte Nuclear Factor –1 alpha (HNF-1α) gene mutations. Results: BMI and age were similar in the genetically defined groups. Fasting plasma glucose (FPG) was less than 5.5 mmol/l in 2 % glucokinase subjects and 46 % HNF-1 α subjects (p < 0.0001). Glucokinase subjects had a higher FPG than HNF-1 α subjects ([means ± SD] 6.8 ± 0.8 vs 6.0 ± 1.9 mmol/l, p < 0.0001), a lower 2-h value (8.9 ± 2.3 vs 11.2 ± 5.2 mmol/l, p < 0.0001) and a lower OGTT increment (2-h – fasting) (2.1 ± 2.3 vs 5.2 ± 3.9 mmol/l, p < 0.0001). The relative proportions classified as diabetic depended on whether fasting (38 % vs 22 %, glucokinase vs HNF-1 α) or 2-h values (19 % vs 44 %) were used. Fasting and 2-h glucose values were not correlated in the glucokinase subjects (r = –0.047, p = 0.65) but were strongly correlated in HNF-1 α subjects (r = 0.8, p < 0.001). Insulin concentrations were higher in the glucokinase subjects throughout the OGTT. Conclusion/interpretation: The genetic cause of the beta-cell defect results in clear differences in both the fasting glucose and the response to an oral glucose load and this can help diagnostic genetic testing in MODY. OGTT results reflect not only the degree of hyperglycaemia but also the underlying cause. [Diabetologia (2002) 45: 427–435] Received: 13 September 2001 and in revised form: 26 November 2001     

An in vitro model for the study of phagocytosis of damaged hepatocytes by rat Kupffer cells

Abstract: Aims/Background: One function of Kupffer cells is the phagocytosis of nonviable hepatocytes. Our aims were to develop a model for phagocytosis of damaged hepatocytes by rat Kupffer cells in vitro, and to characterise prostaglandin E₂ (PGE₂), prostacyclin (PGI), and tumour necrosis factor-α (TNF) production in this model. Methods: Kupffer cells were incubated alone or with damaged hepatocytes for up to 18 h, then washed and cultured for up to 66 h. To compare mediator responses produced during inert particle phagocytosis, Kupffer cells were also incubated with latex beads. Results: Phagocytic uptake of hepatocyte debris was confirmed in at least 50% of Kupffer cells. A dissociation between TNF and PGI responses was found for both latex beads and damaged hepatocytes, such that a TNF secretory response was not triggered by either stimulus whereas PGI production was increased for both. Although phagocytosis of beads increased PGE₂ production, phagocytosis of hepatocytes did not. Conclusions: Phagocytosis of damaged hepatocytes by Kupffer cells results in the production of PGI but not PGE₂ or TNF.     

Bioengineered AAV Capsids with Combined High Human Liver Transduction In Vivo and Unique Humoral Seroreactivity

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Clinical significance of vascular endothelial growth factor and hepatocyte growth factor in multiple myeloma

Angiogenesis is a crucial process in the progression of multiple myeloma (MM). Vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) are multifunctional cytokines that potently stimulate angiogenesis including tumour neovascularization. Serum levels of VEGF and HGF were measured in 52 patients with MM by enzyme-linked immunosorbent assay (ELISA). Serum levels of VEGF and HGF were elevated in MM patients compared with healthy controls (VEGF: mean 0.31 ng/ml and 0.08 ng/ml respectively, P < 0.01; HGF: mean 2.17 ng/ml and 0.45 ng/ml, respectively, P < 0.001). In serial samples taken after chemotherapy, serum VEGF and HGF levels were correlated with M-protein levels. Serum levels of VEGF were higher in patients with extramedullary plasmacytomas than in patients without them (P < 0.05). They were also significantly higher in a group of patients who showed poor response to chemotherapy (P < 0.01). Serum levels of HGF were higher in patients with complications such as anaemia, hypercalcaemia and amyloidosis than in patients without these complications (P < 0.01, P < 0.05, P < 0.05 respectively). Both serum VEGF and HGF levels were significant predictors of mortality (P = 0.01, P = 0.02, respectively, log-rank test). The present study demonstrated that serum levels of VEGF and HGF are significantly elevated and dependent on the severity of MM, suggesting that measurement of VEGF and HGF may be useful for assessing disease progression and for predicting the response to chemotherapy in MM patients.