Hongcui Cao

Reciprocal regulation of Akt and Oct4 promotes the self-renewal and survival of embryonal carcinoma cells.

Signaling via the Akt serine/threonine protein kinase plays critical roles in the self-renewal of embryonic stem cells and their malignant counterpart, embryonal carcinoma cells (ECCs). Here we show that in ECCs, Akt phosphorylated the master pluripotency factor Oct4 at threonine 235, and that the levels of phosphorylated Oct4 in ECCs correlated with resistance to apoptosis and tumorigenic potential. Phosphorylation of Oct4 increased its stability and facilitated its nuclear localization and its interaction with Sox2, which promoted the transcription of the core stemness genes POU5F1 and NANOG. Furthermore, in ECCs, unphosphorylated Oct4 bound to the AKT1 promoter and repressed its transcription. Phosphorylation of Oct4 by Akt resulted in dissociation of Oct4 from the AKT1 promoter, which activated AKT1 transcription and promoted cell survival. Therefore, a site-specific, posttranslational modification of the Oct4 protein orchestrates the regulation of its stability, subcellular localization, and transcriptional activities, which collectively promotes the survival and tumorigenicity of ECCs.

Clinical, Virological, and Histopathological Manifestations of Fatal Human Infections by Avian Influenza A(H7N9) Virus

BACKGROUND Systematic analysis of histopathological and serial virological changes of fatal influenza A(H7N9) cases is lacking. METHODS Patients with A(H7N9) infection admitted to our intensive care unit during 10-23 April 2013 were included. Viral loads in the respiratory tract, as inferred from the cycle threshold (Ct) value of reverse transcription polymerase chain reaction (RT-PCR), and the serum hemagglutination inhibition (HAI) antibody titer, were analyzed. Postmortem biopsies of the lung, liver, kidney, spleen, bone marrow, and heart were examined. RESULTS Twelve patients (6 deaths, 6 survivors) were included. Median viral load was higher in sputa than the nasopharyngeal swabs for fatal cases (median Ct, 23 vs 30.5; P = .08). RT-PCR for A(H7N9) was positive in stool samples (4/6 [67%]) of fatal cases and (2/6 [33%]) of survivors, but was negative in the cerebrospinal fluid, urine, or blood of all patients. Nosocomial bacterial infections were more common in patients who died than in survivors (83% vs 50%). HAI titers increased by ≥4-fold in those with convalescent sera. Postmortem biopsy for 3 patients showed acute diffuse alveolar damage. Patient 1, who died 8 days after symptom onset, had intra-alveolar hemorrhage. Patients 2 and 3, who died 11 days after symptom onset, had pulmonary fibroproliferative changes. Reactive hemophagocytosis in the bone marrow and lymphoid atrophy in splenic tissues were compatible with laboratory findings of leukopenia, lymphopenia, and thrombocytopenia. Hypoxic and fatty changes of kidney and liver tissues are compatible with impaired renal or liver function. CONCLUSIONS Fatal A(H7N9) infection was characterized by viral and secondary bacterial pneumonia with 67% having positive RT-PCR in stool.

Altered immune function of monocytes in different stages of patients with acute on chronic liver failure

The aim of this study was to investigate the characteristics of the immune function of monocytes in different stages of the patients with acute on chronic liver failure (ACLF). Human leucocyte antigen (HLA)‐DR and Toll‐like receptor 4 (TLR‐4) expression on monocytes in early and late stages of acute on chronic liver failure were detected by flow cytometry. The secretion function of monocytes was measured by cytometric bead array. Compared with healthy controls, the levels of HLA‐DR expression on monocytes in patients with chronic hepatitis B, liver cirrhosis and acute on chronic liver failure were gradually decreased, especially in the late stage of acute on chronic liver failure (P < 0·001). TLR‐4 expression on monocytes in patients with liver cirrhosis and acute on chronic liver failure were higher than the healthy controls. The concentrations of interleukin (IL)‐1β, tumour necrosis factor (TNF)‐α and IL‐12p70 in early‐stage ACLF were significantly higher compared with healthy controls and lower in late‐stage ACLF (P < 0·01, 0·05). However, a significantly lower amount of IL‐10 was found on monocytes in early‐stage ACLF than that of late‐stage ACLF and healthy controls (P < 0·01). Monocyte HLA‐DR expression in patients who died was significantly lower compared with patients who survived in the early and late stages of ACLF (P < 0·01). The dynamic detection of HLA‐DR expression or cytokines secreted from monocytes could contribute to the estimation of the status of the immune function of patients with acute on chronic liver failure.

3D PLGA scaffolds improve differentiation and function of bone marrow mesenchymal stem cell-derived hepatocytes.

UNLABELLED Liver tissue engineering with hepatic stem cells provides a promising alternative to liver transplantation in patients with acute and chronic hepatic failure. In this study, a three-dimensional (3D) bioscaffold was introduced for differentiation of rat bone marrow mesenchymal stem cells (BMSCs) into hepatocytes. For hepatocyte differentiation, third passage BMSCs isolated from normal adult F344 rats were seeded into collagen-coated poly(lactic-co-glycolic acid) (C-PLGA) 3D scaffolds with hepatocyte differentiation medium for 3 weeks. Hepatogenesis in scaffolds was characterized by reverse transcript PCR, western blot, confocal laser scanning microscopy (CLSM), periodic acid-Schiff staining, histochemistry, and biochemical assays with hepatic-specific genes and markers. A monolayer culture system was used as a control differentiation group. The results showed that isolated cells possessed the basic features of BMSCs. Differentiated hepatocyte-like cells in C-PLGA scaffolds expressed hepatocyte-specific markers [eg, albumin (ALB), alpha-fetoprotein, cytokeratin 18, hepatocyte nuclear factor 4alpha, and cytochrome P450] at mRNA and protein levels. Most markers were expressed in C-PLGA group 1 week earlier than in the control group. Results of biocompatibility indicated that the differentiated hepatocyte-like cells grew more stably in C-PLGA scaffolds than that in controls during a 3-week differentiation period. The significantly higher metabolic functions in hepatocyte-like cells in the C-PLGA scaffold group further demonstrated the important role of the scaffold. CONCLUSION As the phenomenon of transdifferentiation is uncommon, our successful transdifferentiation rates of BMSCs to mature hepatocytes prove the superiority of the C-PLGA scaffold in providing a suitable environment for such a differentiation. This material can possibly be used as a bioscaffold for liver tissue engineering in future clinical therapeutic applications.

Immediate intraportal transplantation of human bone marrow mesenchymal stem cells prevents death from fulminant hepatic failure in pigs

The effectiveness of human bone marrow mesenchymal stem cell (hBMSC) transplantation to treat acute and chronic liver injury has been demonstrated in animal models and in a few nonrandomized clinical trials. However, no studies have investigated hBMSC transplantation in the treatment of fulminant hepatic failure (FHF), especially in large animal (pig) models. The aim of this study was to demonstrate the safety, effectiveness, and underlying mechanism of hBMSC transplantation for treating FHF in pigs through the intraportal route. Human BMSCs (3 × 107) were transplanted into pigs with FHF via the intraportal route or peripheral vein immediately after D‐galactosamine injection, and a sham group underwent intraportal transplantation (IPT) without cells (IPT, peripheral vein transplantation [PVT], and control groups, respectively, n = 15 per group). All of the animals in the PVT and control groups died of FHF within 96 hours. In contrast, 13 of 15 animals in the IPT group achieved long‐term survival (>6 months). Immunohistochemistry demonstrated that transplanted hBMSC‐derived hepatocytes in surviving animals were widely distributed in the hepatic lobules and the liver parenchyma from weeks 2 to 10. Thirty percent of the hepatocytes were hBMSC‐derived. However, the number of transplanted cells decreased significantly at week 15. Only a few single cells were scattered in the regenerated liver lobules at week 20, and the liver tissues exhibited a nearly normal structure. Conclusion: Immediate IPT of hBMSCs is a safe and effective treatment for FHF. The transplanted hBMSCs may quickly participate in liver regeneration via proliferation and transdifferentiation into hepatocytes during the initial stage of FHF. This method can possibly be used in future clinical therapy. (HEPATOLOGY 2012;56:1044–1052)

Fecal metabolome profiling of liver cirrhosis and hepatocellular carcinoma patients by ultra performance liquid chromatography-mass spectrometry.

Fecal metabolome of healthy humans and patients suffering from liver cirrhosis and hepatocellular carcinoma (HCC) were studied using ultra performance liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOF MS). Metabolic features detected by the method were then statistically treated using partial least squares to latent structure-discriminant analysis (PLS-DA) models to discriminate between healthy and diseased states. PLS-DA was also used to discriminate between cirrhosis and HCC stressed fecal metabolomes and to identify potential biomarkers for cirrhosis and HCC that are expressed at significantly different amounts in fecal metabolomes. Score plots of pattern recognition analysis distinguished liver cirrhosis and HCC patients from healthy humans. Based on the variable of importance in the project (VIP) values and S-plots, six metabolites were considered as potential biomarkers with a strong increase in lysophosphatidylcholines and a dramatic decrease in bile acids and bile pigments in patients with liver cirrhosis and HCC in comparison with healthy humans. Results demonstrate the potential of UPLC-MS as an efficient and convenient method that can be applied to screen fecal samples and aid in the early diagnosis of cirrhosis and hepatocellular carcinoma.

Therapeutic potential of transplanted placental mesenchymal stem cells in treating Chinese miniature pigs with acute liver failure.

BackgroundStem cell-based therapy to treat liver diseases is a focus of current research worldwide. So far, most such studies depend on rodent hepatic failure models. The purpose of this study was to isolate mesenchymal stem cells from human placenta (hPMSCs) and determine their therapeutic potential for treating Chinese experimental miniature pigs with acute liver failure (ALF).MethodshPMSCs were isolated and analyzed for their purity and differentiation potential before being employed as the donor cells for transplantation. ALF models of Chinese experimental miniature pigs were established and divided into four groups: no cell transplantation; hPMSCs transplantation via the jugular vein; X-ray-treated hPMSCs transplantation via the portal vein; and hPMSCs transplantation via the portal vein. The restoration of biological functions of the livers receiving transplantation was assessed via a variety of approaches such as mortality rate determination, serum biochemical analysis, and histological, immunohistochemical, and genetic analysis.ResultshPMSCs expressed high levels of CD29, CD73, CD13, and CD90, had adipogenic, osteogenic, and hepatic differentiation potential. They improved liver functions in vivo after transplantation into the D-galactosamine-injured pig livers as evidenced by the fact that ALT, AST, ALP, CHE, TBIL, and TBA concentrations returned to normal levels in recipient ALF pigs. Meanwhile, histological data revealed that transplantation of hPMSCs via the portal vein reduced liver inflammation, decreased hepatic denaturation and necrosis, and promoted liver regeneration. These ameliorations were not found in the other three groups. The result of 7-day survival rates suggested that hPMSCs transplantation via the portal vein was able to significantly prolong the survival of ALF pigs compared with the other three groups. Histochemistry and RT-PCR results confirmed the presence of transplanted human cells in recipient pig livers (Groups III, IV).ConclusionsOur data revealed that hPMSCs could not only differentiate into hepatocyte-like cells in vitro and in vivo, but could also prolong the survival time of ALF pigs. Regarding the transplantation pathways, the left branch of the portal vein inside the liver was superior to the jugular vein pathway. Thus, hPMSCs transplantation through the portal vein by B-ultrasonography may represent a superior approach for treating liver diseases.

Effect of telbivudine therapy on the cellular immune response in chronic hepatitis B

Weak T-cell reactivity to the hepatitis B virus (HBV) is believed to be the dominant cause of chronic HBV infection. Several lines of experimental evidence suggest that treatment with telbivudine increases the rate of HBV e antigen (HBeAg) loss, undetectable HBV DNA, and normalization of serum alanine aminotransferase (ALT) in chronic hepatitis B patients (CHB). However, it is still unclear how early antiviral therapy affects cellular immune responses during sustained telbivudine treatment. In order to investigate this issue, we measured detailed prospective clinical, virological, and biochemical parameters, and we examined the frequency of T cell subgroups as well as the ability of peripheral blood mononuclear cells (PBMC) to respond to stimuli at five protocol time points for 51 CHB patients who received telbivudine therapy for one year. The preliminary data from this study revealed that effective-treated patients showed an increased frequency of peripheral blood CD4(+)T lymphocytes, an augmented proliferative response of HBV-specific T-cells to the hepatitis B core antigen (HBcAg), and the induction of cytokines, such as interferon gamma (IFN-γ), tumour necrosis factor alpha (TNF-α) release at the site of infection compared to non-responsive patients. Enhanced HBV-specific T-cell reactivity to telbivudine therapy, which peaked at treatment week 12, was confined to a subgroup of effective-treated patients who achieved greater viral suppression.

Establishment of Highly Differentiated Immortalized Human Hepatocyte Line With Simian Virus 40 Large Tumor Antigen for Liver Based Cell Therapy

Acute liver failure and metabolic liver disorder animal models have demonstrated that hepatocytes transplanted into the liver or spleen survive and participate in the liver repopulation process, and recent studies have documented the usefulness of hepatocyte transplantation in humans. However, despite the promising cell therapy, there are still many restrictions, such as the shortage of donor human livers and the limited lifespan and the functional insufficiency of primary cultured hepatocytes. The immortalized and highly differentiated human hepatocyte could provide an unlimited supply of transplantable cells. In this study, we established an efficient and highly differentiated immortalized human hepatocyte line for bioartificial liver and hepatocyte transplantation research. Hepatocytes isolated from the liver of a 25 year old, brain dead male were transfected with pcDNA3.1(-) recombinant plasmid containing the genes encoding simian virus 40 (SV40) large tumor antigen. One of the hepatocyte clones, HepLL, displayed highly differentiated liver functions with immortalized characteristics and was selected with a 700–300 &mgr;g/ml of G418 technique in 42 days. To characterize this immortalized cell line for cell therapy in the near future, HepLL cells were studied with immunohistochemistry, reverse transcription-polymerase chain reactions, immunoblotting, and tumorigenicity tests. The results revealed that HepLL cells displayed morphologic characteristics of liver parenchymal cells, secreted albumin, synthesized urea and glycogen, and expressed liver enriched functional markers, but there were no tumorigenic qualities after transplantation into severe combined immunodeficiency mice. Thus this immortalized human hepatocyte line is expected to be a useful tool for studying the functions of differentiated human hepatocyte and a promising strategy to resolve the shortages of donor organs and the limits of primary human hepatocyte for transplantation and bioartificial liver support systems.

Angiotensin II plasma levels are linked to disease severity and predict fatal outcomes in H7N9-infected patients

A novel influenza A (H7N9) virus of avian origin emerged in eastern China in the spring of 2013. This virus causes severe disease in humans, including acute and often lethal respiratory failure. As of January 2014, 275 cases of H7N9-infected patients had been reported, highlighting the urgency of identifying biomarkers for predicting disease severity and fatal outcomes. Here, we show that plasma levels of angiotensin II, a major regulatory peptide of the renin–angiotensin system, are markedly elevated in H7N9 patients and are associated with disease progression. Moreover, the sustained high levels of angiotensin II in these patients are strongly correlated with mortality. The predictive value of angiotensin II is higher than that of C-reactive protein and some clinical parameters such as the PaO2/FiO2 ratio (partial pressure of arterial oxygen to the fraction of inspired oxygen). Our findings indicate that angiotensin II is a biomarker for lethality in flu infections. Supplementary information The online version of this article (doi:10.1038/ncomms4595) contains supplementary material, which is available to authorized users.