ABSTRACT
BACKGROUND: CD24+CK19+/CD24+SOX9+ resident liver cells are activated and expanded after chronic liver injury in a ductular reaction. However, the sources and functions of these cells in liver damage remain disputed. RESULTS: The current study combined genetic lineage tracing with in vitro small-molecule-based reprogramming to define liver progenitor cells (LPCs) derived from hepatic parenchymal and non-parenchymal tissues. tdTom+ hepatocytes were isolated from ROSA26tdTomato mice following AAV8-Tbg-Cre-mediated recombination, EpCAM+ biliary epithelial cells (BECs) from wild-type intrahepatic bile ducts and ALB/GFP-EpCAM- cells were isolated from AlbCreERT/R26GFP mice. A cocktail of small molecules was used to convert the isolated cells into LPCs. These in vitro cultured LPCs with CD24 and SOX9 expression regained the ability to proliferate. Transcriptional profiling showed that the in-vitro cultured LPCs derived from the resident LPCs in non-parenchymal tissues expressed Lipocalin-2 (Lcn2) at high levels. Accordingly, endogenous Cd24a+Lcn2+ LPCs were identified by integration of sc-RNA-sequencing and pathological datasets of liver dysfunction which indicates that LPCs produced by ductular reactions might also originate from the resident LPCs. Transplantation of in-vitro cultured Cd24a+Lcn2+ LPCs into CCl4-induced fibrotic livers exacerbated liver damage and dysfunction, possibly due to LCN2-dependent macrophage inflammatory response. CONCLUSIONS: CD24+LCN2+ LPCs constituted the expanding ductular reaction and contributed to macrophage-mediated inflammation in chronic liver damage. The current findings highlight the roles of LPCs from distinct origins and expose the possibility of targeting LPCs in the treatment of chronic hepatic diseases.
ABSTRACT
Globally, about two million people die from liver diseases every year. Liver transplantation is the only reliable therapy for severe end-stage liver disease, however, the shortage of organ donors is a huge limitation. Human hepatocytes derived liver progenitor-like cells (HepLPCs) have been reported as a novel source of liver cells for development of in vitro models, cell therapies, and tissue-engineering applications, but their functionality as transplantation donors is unclear. Here, a 3-dimensional (3D) co-culture system using HepLPCs and human umbilical vein endothelial cells (HUVECs) was developed. These HepLPC spheroids mimicked the cellular interactions and architecture of mature hepatocytes, as confirmed through ultrastructure morphology, gene expression profile and functional assays. HepLPCs encapsulated in alginate beads are able to mitigate liver injury in mice treated with carbon tetrachloride (CCL4), while alginate coating protects the cells from immune attack. We confirmed these phenomena due to HUVECs producing glial cell line-derived neurotrophic factor (GDNF) to promote HepLPCs maturation and enhance HepLPCs tight junction through MET phosphorylation. Our results display the efficacy and safety of the alginate microencapsulated spheroids in animal model with acute liver injury (ALF), which may suggest a new strategy for cell therapy.
ABSTRACT
Rationale: We developed a cocktail of soluble molecules mimicking the in vivo milieu supporting liver regeneration that could convert mature hepatocytes to expandable liver progenitor-like cells in vitro. This study aimed to induce endogenous liver progenitor cells by the administration of the soluble molecules to provide an alternative approach for the resolution of liver fibrosis. Methods:In vitro cultured hepatocyte-derived liver progenitor-like cells (HepLPCs) were transplanted into CCL4-treated mice to investigate the therapeutic effect against liver fibrosis. Next, we used HGF in combination with a cocktail of small molecules (Y-27632, A-83-01, and CHIR99021 (HACY)) to induce endogenous CD24+ liver progenitor cells and to inhibit the activation of hepatic stellate cells (HSCs) during CCL4-induced hepatic injury. RNA sequencing was performed to further clarify the features of HACY-induced CD24+ cells compared with CCL4-induced CD24+ cells and in vitro derived HepLPCs. Finally, we evaluated the expansion of HACY-induced CD24+ cells in human hepatocyte-spheroids from fibrotic liver tissues. Results: HepLPCs exhibited the capacity to alleviate liver fibrosis after transplantation into CCL4-treated mice. The in vivo administration of HACY not only induced the conversion of mature hepatocytes (MHs) to CD24+ progenitor cells but prevented the activation of HSCs, thus leading to enhanced improvement of liver fibrosis in CCL4-treated mice. Compared to CD24+ cells induced by CCL4 alone, HACY-induced CD24+ cells retained an enhanced level of hepatic function and could promote the restoration of liver function that exhibited comparable gene expression profiles with HepLPCs. CD24+ cells were also observed in human liver fibrotic tissues and were expanded in three-dimensional (3D) hepatic spheroids in the presence of HACY in vitro. Conclusions: Hepatocyte-derived liver progenitor-like cells are crucial for liver regeneration during chronic hepatic injuries. The administration of HACY, which allowed the induction of endogenous CD24+ progenitor cells and the inactivation of HSCs, exerts beneficial effects in the treatment of liver fibrosis by re-establishing a balance favoring liver regeneration while preventing fibrotic responses.
Subject(s)
Hepatic Stellate Cells/drug effects , Liver Cirrhosis/drug therapy , Liver/drug effects , Small Molecule Libraries/pharmacology , Stem Cells/drug effects , Amides/pharmacology , Animals , CD24 Antigen/metabolism , Carbon Tetrachloride/pharmacology , Cells, Cultured , Gene Expression/drug effects , Hepatic Stellate Cells/metabolism , Hepatocytes/drug effects , Hepatocytes/metabolism , Humans , Liver/metabolism , Liver Cirrhosis/metabolism , Liver Regeneration/drug effects , Male , Mice , Mice, Inbred C57BL , Pyridines/pharmacology , Pyrimidines/pharmacology , Stem Cells/metabolismABSTRACT
The purpose of this study was to evaluate the role of 2-D speckle tracking imaging in assessing left ventricular diastolic function in patients with connective tissue disease (CTD). A total of 98 CTD patients and 32 healthy controls were prospectively recruited. Early (E) and late (A) diastolic velocities of the transmitral flow were measured by pulsed Doppler echocardiography. Peak early diastolic myocardial velocity (E') was calculated on tissue Doppler echocardiography. The longitudinal strain rate (SR) was calculated as the average of three apical views, while circumferential and radial SRs were measured in three short-axis views. Pulmonary arterial hypertension (PAH) was defined as systolic pulmonary arterial pressure (sPAP) >36 mm Hg. Compared with the control group, CTD patients exhibited significant impairment of left ventricular diastolic function, manifested as lower global SR during early diastole (SRe) in the longitudinal deformation and higher E/SRe in both longitudinal and radial deformation. CTD-PAH patients had significantly lower SRe and higher E/SRe values in both the longitudinal and radial deformation compared with the patients with CTD without PAH. Pearson's correlation analysis revealed that sPAP levels correlated positively with E/E', longitudinal E/SRe, circumferential E/SRe and radial SRe, and it correlated negatively with septal E' and radial E/SRe. Receiver operating characteristic curve analysis suggested that E/E', longitudinal E/SRe and radial SRe could be used to predict PAH. The present study indicates that 2-D speckle tracking imaging is a useful method for evaluation of left ventricular diastolic function, and these derived parameters can serve as good predictors of PAH, but it may not be superior to the commonly used E/E' in CTD patients.
Subject(s)
Connective Tissue Diseases/physiopathology , Echocardiography/methods , Pulmonary Arterial Hypertension/physiopathology , Ventricular Dysfunction, Left/diagnostic imaging , Ventricular Dysfunction, Left/physiopathology , Adult , Blood Flow Velocity , Case-Control Studies , Connective Tissue Diseases/complications , Diastole , Female , Humans , Male , Middle Aged , Observer Variation , Prospective Studies , Pulmonary Arterial Hypertension/etiology , ROC Curve , Reproducibility of Results , Ventricular Dysfunction, Left/etiologyABSTRACT
Clinical advancement of the bioartificial liver is hampered by the lack of expandable human hepatocytes and appropriate bioreactors and carriers to encourage hepatic cells to function during extracorporeal circulation. We have recently developed an efficient approach for derivation of expandable liver progenitor-like cells from human primary hepatocytes (HepLPCs). Here, we generated immortalized and functionally enhanced HepLPCs by introducing FOXA3, a hepatocyte nuclear factor that enables potentially complete hepatic function. When cultured on macroporous carriers in an air-liquid interactive bioartificial liver (Ali-BAL) support device, the integrated cells were alternately exposed to aeration and nutrition and grew to form high-density three-dimensional constructs. This led to highly efficient mass transfer and supported liver functions such as albumin biosynthesis and ammonia detoxification via ureagenesis. In a porcine model of drug overdose-induced acute liver failure (ALF), extracorporeal Ali-BAL treatment for 3 hours prevented hepatic encephalopathy and led to markedly improved survival (83%, n = 6) compared to ALF control (17%, n = 6, P = 0.02) and device-only (no-cell) therapy (0%, n = 6, P = 0.003). The blood ammonia concentrations, as well as the biochemical and coagulation indices, were reduced in Ali-BAL-treated pigs. Ali-BAL treatment attenuated liver damage, ameliorated inflammation, and enhanced liver regeneration in the ALF porcine model and could be considered as a potential therapeutic avenue for patients with ALF.
Subject(s)
Liver Failure, Acute , Liver, Artificial , Albumins , Animals , Hepatocytes , Humans , Liver , Liver Failure, Acute/therapy , SwineABSTRACT
OBJECTIVE: To explore the metabolic characteristics and metabolic markers of WBC-depleted RBCs in MAP preservation solution and to analyzed the metabolic profile of RBC in MAP preservation solution by using metabolomics. METHODS: The changes of metabolitcs in 10 U WBC-depleted RBC suspension at 3-different storage period (D 0, D 14 and D 35) were detected by using the UPLC-MS/MS, the charaeteristic ions and metabolic markers of RBC stored in preservation sblution for 0 d, 14 d and 35 days were analyzed by using the principal component analysis(PCA). RESULTS: The number of characteristic ions in RBC and supernatant extracts detected during the initial, middle and final storage could be clearly distinguiseed. The 5 metabolism-related substamces such as lact-c acid, nicotinamide, glucose, 5-htdroxyproline and malic acid showed statistically significant difference in 3 storage period. CONCLUSION: The UPLC-MS/MS method combined with statistical analysis of multivariate data can be used to study the metabolic characteristics of RBC, the different metabolites of RBC in different storage stages can be used as the potential markers for evaluation of guality of RBC in storag period. The results of this study provide a basis for studing the RBC guality changes in storage period.
Subject(s)
Blood Preservation , Tandem Mass Spectrometry , Chromatography, Liquid , Erythrocytes , MetabolomeABSTRACT
The study of pathophysiological mechanisms in human liver disease has been constrained by the inability to expand primary hepatocytes in vitro while maintaining proliferative capacity and metabolic function. We and others have previously shown that mouse mature hepatocytes can be converted to liver progenitor-like cells in vitro with defined chemical factors. Here we describe a protocol achieving efficient conversion of human primary hepatocytes into liver progenitor-like cells (HepLPCs) through delivery of developmentally relevant cues, including NAD + -dependent deacetylase SIRT1 signaling. These HepLPCs could be expanded significantly during in vitro passage. The expanded cells can readily be converted back into metabolically functional hepatocytes in vitro and upon transplantation in vivo. Under three-dimensional culture conditions, differentiated cells generated from HepLPCs regained the ability to support infection or reactivation of hepatitis B virus (HBV). Our work demonstrates the utility of the conversion between hepatocyte and liver progenitor-like cells for studying HBV biology and antiviral therapies. These findings will facilitate the study of liver diseases and regenerative medicine.
Subject(s)
Hepatitis B virus/physiology , Hepatitis B/pathology , Hepatocytes , Liver/pathology , Stem Cells , Animals , Cell Differentiation , Cells, Cultured , Hepatocytes/metabolism , Hepatocytes/pathology , Humans , Mice , Sirtuin 1/metabolism , Stem Cells/cytology , Stem Cells/pathologyABSTRACT
BACKGROUND: Daunorubicin is a traditional chemotherapeutic agent that plays a pivotal role in leukemia therapy. However, the dose-related toxicity remains a considerable challenge. The apoptosis-regulating gene, PDCD5, is downregulated in various tumors, including leukemias, and may provide a potential target for the diagnosis and treatment of leukemia. The purpose of this study was to construct a triple-regulated oncolytic adenovirus carrying a PDCD5 gene expression cassette (SG611-PDCD5) and explore the combined antitumor efficacy of SG611-PDCD5 in combination with low dose daunorubicin on leukemic cells. MATERIALS AND METHODS: A variety of leukemic cell lines, including K562, MEG-01, KG-1a, HL-60, SUP-B15, and BV-173, were cultured according to the providers' instructions. The insertion and orientation of all recombined plasmids were confirmed by restriction enzyme digestion and PCR. The tumor-selective replication of the constructed conditionally replicating SG611-PDCD5 and its antitumor efficacy in combination with daunorubicin were characterized in leukemic cell lines in vitro and in a nude mouse xenograft model. Cell viability was detected using cell-counting kit-8. Apoptosis was detected in whole living cells using flow cytometry and in paraffin-embedded tumor tissues using a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. RESULTS: The triple-regulated CRAd carrying SG611-PDCD5 and nude mouse xenograft models of K562 cells were successfully constructed. In vitro treatment with SG611-PDCD5 in combination with low-dose daunorubicin elicited more potent anti-proliferative and proapoptotic effects in leukemic cells in a dose-dependent manner. The Chou-Talalay analysis revealed synergistic anti-proliferative effects in all of the above cell lines. In the nude mice xenograft model, the tumor size in the control, daunorubicin, SG611-PDCD5, and combined treatment groups on day 10 were 170.1±47.8, 111.9±81.1, 60.7±12.3, and 33.2±17.5 mm3, respectively (all P<0.05). The results of the TUNEL assay showed significantly more apoptotic cells in the SG611-PDCD5 plus daunorubicin group than in the SG611-PDCD5 or daunorubicin groups alone (25±0.82, 12.5±2.27, and 7.8±2.67 apoptotic cells/field, respectively) (P<0.05). CONCLUSION: The findings suggest that combined treatment with SG611-PDCD5 and daunorubicin may be a promising strategy for enhancing chemosensitivity and thus lowering the dose-related toxicity of daunorubicin in leukemia therapy.
ABSTRACT
Despite their central function in tumor immunity, dendritic cells (DCs) can respond to inhibitory signals and become tolerogenic, curtailing T cell responses in vivo. Here, we provide the evidence for an inhibitory function of signal regulatory protein (SIRP) α in DC survival and activation. In tumors from human liver cancer patients, infiltrative DCs expressed elevated levels of SIRPα, which is correlated with the induction of immune tolerance within the tumors. Silencing of SIRPα resulted in a significant increase in the longevity of antigen-pulsed DCs in the draining lymph nodes. In addition, SIRPα controls the activation and output of DCs. Silencing of DC-expressed SIRPα induced spontaneous and enhanced production of IL12 and costimulatory molecules, resulting in more potent cytotoxic T lymphocyte responses, including the eradication of previously established solid tumors. SIRPα exerted such effects, at least in part, via the association and sequestration of p85 subunit of PI3K. Thus, SIRPα is a critical regulator of DC lifespan and activity, and its inhibition might improve the clinical efficacy of DC-based tumor vaccines.
ABSTRACT
V-set and transmembrane domain-containing 1 (VSTM1), which is downregulated in bone marrow cells from leukemia patients, may provide a diagnostic and treatment target. Here, a triple-regulated oncolytic adenovirus was constructed to carry a VSTM1 gene expression cassette, SG611-VSTM1, and contained the E1a gene with a 24-nucleotide deletion within the CR2 region under control of the human telomerase reverse transcriptase promoter, E1b gene directed by the hypoxia response element, and VSTM1 gene controlled by the cytomegalovirus promoter. Real-time quantitative PCR and Western blot analyses showed that SG611-VSTM1 expressed VSTM1 highly efficiently in the human leukemic cell line K562 compared with SG611. In Cell Counting Kit-8 and flow cytometric assays, SG611-VSTM1 exhibited more potent anti-proliferative and pro-apoptotic effects in leukemic cells compared with SG611 and exerted synergistic cytotoxicity with low-dose daunorubicin (DNR) in vitro. In xenograft models, SG611-VSTM1 intratumorally injected at a dose of 1 × 10(9) plaque forming units combined with intraperitoneally injected low-dose DNR displayed significantly stronger antitumor effects than either treatment alone. Histopathologic examination revealed that SG611-VSTM1 induced apoptosis of leukemic cells. These results implicate an important role for VSTM1 in the pathogenesis of leukemia, and SG611-VSTM1 may be a promising agent for enhancing chemosensitivity in leukemia therapy.
Subject(s)
Adenoviridae/genetics , Antineoplastic Agents/administration & dosage , Daunorubicin/administration & dosage , Leukemia/therapy , Oncolytic Viruses/genetics , Receptors, Immunologic/genetics , Adenoviridae/physiology , Animals , Apoptosis/drug effects , Cell Line, Tumor , Combined Modality Therapy , Female , Genetic Therapy , Genetic Vectors/genetics , Genetic Vectors/metabolism , Humans , Leukemia/drug therapy , Leukemia/physiopathology , Leukemia/virology , Mice , Mice, Inbred BALB C , Mice, Nude , Oncolytic Virotherapy , Oncolytic Viruses/physiology , Receptors, Immunologic/metabolismABSTRACT
BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) develops in response to chronic hepatic injury. Although induced cell death is regarded as the major component of p53 tumor-suppressive activity, we recently found that sustained p53 activation subsequent to DNA damage promotes inflammation-associated hepatocarcinogenesis. Here we aim at exploring the mechanism linking p53 activation and hepatic inflammation during hepatocarcinogenesis. METHODS: p53(-/-) hepatocytes expressing inducible p53 and primary wild type hepatocytes were treated to induce p53 expression. The supernatants were collected and analyzed for the presence of released inflammatory cytokines. Ethyl pyruvate was used in a rat model of carcinogen-induced hepatocarcinogenesis to examine its effect on p53-dependent chronic hepatic injury, inflammation, and tumorigenesis. RESULTS: Here we show that cytoplasmic translocation and circulating levels of potent inflammatory molecule high-mobility group protein 1 (HMGB1) were greater in wild type rats than in p53(+/-) rats following carcinogen administration. Restoration of p53 expression in p53-null hepatocytes or induction of endogenous p53 in wild type hepatocytes gives rise to the release of HMGB1. Administration of the HMGB1 release inhibitor ethyl pyruvate, which does not affect p53-mediated hepatic apoptosis, substantially prevented carcinogen-induced cirrhosis and tumorigenesis in rat livers. CONCLUSIONS: These results suggest that although p53 is usually regarded as a tumor suppressor, its constant activation can promote pro-tumorigenic inflammation, at least in part, via inducing HMGB1 release. Application of HMGB1 inhibitors when restoring p53 in cancer therapy might protect against pro-tumorigenic effects while leaving p53-mediated clearance of malignant cells intact.
Subject(s)
Genes, p53 , HMGB1 Protein/metabolism , Liver Neoplasms, Experimental/etiology , Animals , Cell Line , Diethylnitrosamine/toxicity , Gene Knockout Techniques , Hepatitis, Chronic/etiology , Hepatitis, Chronic/metabolism , Hepatitis, Chronic/pathology , Inflammation/etiology , Inflammation/metabolism , Inflammation/pathology , Inflammation Mediators/metabolism , Liver Neoplasms, Experimental/metabolism , Liver Neoplasms, Experimental/pathology , Rats , Transcriptional Activation , Tumor Suppressor Protein p53/metabolismABSTRACT
The p53 tumor suppressor gene is highly mutated in human cancers. Individuals who inherit one p53 mutant allele are susceptible to a wide range of tumor types, including breast cancer and sarcoma. We recently generated p53 knockout rats through gene targeting in embryonic stem cells. Here we show that rats homozygous for the null allele are prone to early onset spontaneous sarcomas and lymphoma with high incidence of metastases. Heterozygous rats are also highly predisposed to cancer, but with a delayed onset and a wider spectrum of tumor types compared with homozygotes. Importantly, up to 20% of female heterozygotes developed breast cancer and about 70% of the tumors were positive for estrogen receptor. Exposing p53-deficient rats to a low dose of the carcinogen diethylnitrosamine dramatically decreased the latency for sarcoma development and survival time compared with equivalently treated wild-type rats. These unique features make this knockout line a valuable model for investigating human malignancy and in vivo carcinogenicity of chemicals and therapeutic compounds.
Subject(s)
Gene Knockout Techniques , Genes, p53 , Models, Animal , Alleles , Animals , Breast Neoplasms/genetics , Cell Transformation, Neoplastic/genetics , Female , Heterozygote , Homozygote , Lymphoma/genetics , Quinolines , Rats , Sarcoma/geneticsABSTRACT
OBJECTIVE: To construct an adenovirus containing a mifepristone (RU486)-inducible regulation system for NRF2 gene, express the product in H460 cell and verify whether the mentioned system can control the gene expression and assess its efficiency. METHODS: A RU486-inducible regulation system for Nrf2 gene was introduced into an adenovirus. The confirmation was performed through the LUC and Dsred genes. And the expression pattern of Nrf2 at the viral level was examined by Western blot and RT-PCR (reverse transcription-polymerase chain reaction). RESULTS: The expressions of LUC and Dsred showed a rising trend with the incremental dose of RU486. After the transfection H460 cell with Ad-RUNrf2, the results of RT-PCR and Western blot demonstrated that the expression of Nrf2 was elevated with a rising dose of RU486. After the removal of RU486, the expression of Nrf2 was reduced. CONCLUSION: The construction of an adenovirus carrying Nrf2 gene regulated by a RU486-inducible system is successful, and RU486 can adjust the cellular expression of Nrf2 factor. The LUC and the Dsred expression assumes the dosage dependence along with RU486 to increase; after the Ad-RUNrf2 infects the H460 cell, through RTPCR and Western the Blot result demonstrated that the expression of Nrf2 increases along with the RU486 dosage increases, after removing RU486, the Nrf2 expression is weaken. Showing the construction of the adenovirus carrying Nrf2 gene regulated by the mifepristone (RU486)-inducible system is successful, and RU486 can adjust the Nrf2 factor in the cell the expression.
Subject(s)
Adenoviridae/genetics , Genetic Vectors , Mifepristone/pharmacology , NF-E2-Related Factor 2/genetics , Adenoviridae/drug effects , Cell Line , Gene Expression , Gene Expression Regulation , Mifepristone/metabolism , Promoter Regions, Genetic , TransfectionABSTRACT
The expression levels of programmed cell death 5 (PDCD5) are down-regulated in many malignancies. SG611-pdcd5, a recombinant conditionally replicative adenovirus carrying pdcd5 gene expression cassette, can evidently kill the leukemic cells and protect selectively the normal cells. The purpose of this study was to investigate the synergistic killing effect of SG611-pdcd5 and low-dose etoposide (VP-16) on K562 cells. K562 cells were treated with different concentrations of VP-16 or different multiplicities of infection (MOI) of SG611-pdcd5. After 48 hours of incubation the cell viability was determined by using MTT assay. The results showed that the cell viability of SG611-pdcd5 (MOI = 40) plus VP-16 (0.5 µg/ml) group significantly decreased as compared with single SG611-pdcd5 (MOI = 40) treatment group or single VP-16(0.5 µg/ml) treatment group (42.00 ± 5.75% vs 59.45 ± 4.12%; 42.00 ± 5.75% vs 82.91 ± 3.41%, respectively, both p < 0.05). The synergistic killing effect of SG611-pdcd5 plus VP-16 was higher than that of PDCD5 protein plus VP-16 or that of non-replicating adenovirus carrying pdcd5 (Ad-pdcd5) plus VP-16 (both p < 0.05). The cell viability of VP-16 (4.0 µg/ml) plus SG611-pdcd5 (MOI = 40) group, VP-16 (4.0 µg/ml) plus proPDCD5 (40 µg/ml) group and VP-16 (4.0 µg/ml) plus Ad-pdcd5 (MOI = 80) group was 37.09 ± 1.89%, 52.36 ± 1.64% and 73.64 ± 4.33%, respectively. It is concluded that SG611-pdcd5 can promote K562 cell death induced by low-dose VP-16. The combination of SG611-pdcd5 and VP-16 can enhance the killing effect on leukemic cells.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis Regulatory Proteins/genetics , Apoptosis , Etoposide/pharmacology , Neoplasm Proteins/genetics , Adenoviridae/genetics , Cell Survival , Genetic Vectors , Humans , K562 CellsABSTRACT
UNLABELLED: Increasing evidence suggests that the presence of endotoxemia is of substantial clinical relevance to patients with cirrhosis, but it is unclear whether and how gut-derived LPS amplifies the tumorigenic response of the liver. We found that the circulating levels of LPS were elevated in animal models of carcinogen-induced hepatocarcinogenesis. Reduction of LPS using antibiotics regimen in rats or genetic ablation of its receptor Toll-like receptor 4 (TLR4) in mice prevented excessive tumor growth and multiplicity. Additional investigation revealed that TLR4 ablation sensitizes the liver to carcinogen-induced toxicity via blocking NF-κB activation and sensitizing the liver to reactive oxygen species (ROS)-induced toxicity, but lessens inflammation-mediated compensatory proliferation. Reconstitution of TLR4-expressing myeloid cells in TLR4-deficient mice restored diethylnitrosamine (DEN)-induced hepatic inflammation and proliferation, indicating a paracrine mechanism of LPS in tumor promotion. Meanwhile, deletion of gut-derived endotoxin suppressed DEN-induced cytokine production and compensatory proliferation, whereas in vivo LPS pre-challenge promotes hepatocyte proliferation. CONCLUSION: Our data indicate that sustained LPS accumulation represents a pathological mediator of inflammation-associated hepatocellular carcinoma (HCC) and manipulation of the gut flora to prevent pathogenic bacterial translocation and endotoxin absorption may favorably influence liver function in patients with cirrhosis who are at risk of developing HCC.
Subject(s)
Apoptosis/drug effects , Carcinogens/pharmacology , Carcinoma, Hepatocellular/etiology , Lipopolysaccharides/pharmacology , Liver Neoplasms/etiology , Toll-Like Receptor 4/physiology , Animals , Carcinoma, Hepatocellular/pathology , Cell Proliferation/drug effects , Diethylnitrosamine/toxicity , Intestines/microbiology , Lipopolysaccharides/blood , Liver Cirrhosis/complications , Male , Mice , NF-kappa B/antagonists & inhibitors , Neomycin/pharmacology , Polymyxin B/pharmacology , Rats , Reactive Oxygen Species/pharmacologyABSTRACT
OBJECTIVE: To establish a human gallbladder carcinoma cell line derived from a metastatic gallbladder carcinoma and identify its biological characteristics. METHODS: Tissue samples were separated from the surgical specimen obtained from a patient with metastatic carcinoma and single-cell suspension was prepared. Then the cells were cultured in DMEM medium supplemented with 15% fetal bovine serum. The morphology of tumor cells was observed under an electron microscope. The cell growth curve was plotted. The tumorigenicity of the cell line was studied by subcutaneous inoculation in SCID mice. The cells were infected by lentiviral vector carrying fluorescent report genes (lenti-GFP and lenti-Red2) separately for expressions of GFP and Red2, respectively. RESULTS: A novel metastatic gallbladder carcinoma cell line was successfully established and named "EH-GB1". It could be passaged for over 20 generations with typical malignant epithelial morphology and a stable growth cycle of 24 h. Tumors were formed in all of the 10 SCID mice inoculated with EH-GB1 cells subcutaneously, and the tumor cells were tumor marker CA19-9-positive. Continuous expressions of fluorescent report genes were observed in EH-GB1 cells infected by lenti-GFP and lenti-Red2. CONCLUSION: EH-GB1 cells might be the first stable cell line of human gallbladder carcinoma established from a metastatic focus of gallbladder carcinoma. This cell line with continuous expressions of GFP and Red2 might be a novel and perfect experimental model for clinical and basic research on gallbladder carcinoma.
Subject(s)
Abdominal Neoplasms/secondary , Adenocarcinoma/pathology , Cell Line, Tumor/pathology , Gallbladder Neoplasms/pathology , Abdominal Neoplasms/metabolism , Abdominal Neoplasms/pathology , Abdominal Wall , Adenocarcinoma/metabolism , Adenocarcinoma/secondary , Animals , CA-19-9 Antigen/metabolism , Cell Line, Tumor/metabolism , Female , Gallbladder Neoplasms/metabolism , Genes, Reporter , Green Fluorescent Proteins/metabolism , Humans , Mice , Mice, Nude , Mice, SCID , Middle Aged , Neoplasm TransplantationABSTRACT
At a Li density of approximately 10(13) cm(-3), the lithium vapor was irradiated in a five-arm stainless steel heat pipe oven containing Li and H2 with pulses of radiation from a N2-laser-pumped dye laser, populating Li(2P) state by the Li(2S-->2P) resonance transition at 670.8 nm. Typical operating pressure of H2 was 60-300 Pa. The cross section for Li(2P)+H2-->LiH+H reaction was measured using method of atomic fluorescence. The decay signal of the time-resolved fluorescence from the 2P-->2S transition was monitored. The decay curve of the Li(2P) can be treated as a single exponential function. The effective lifetimes of the 2P state was obtained. According to the Stern-Volmer equation, a plot of reciprocal of effective lifetimes of the 2P state quenched by H2 against its densities yielded a slope that indicated the total cross section for deactivation and an intercept (at which the H2 pressure is zero) that provided the information about the radiative lifetime of the state. The total quenching (reactive+nonreactive) cross section for deactivation of the 2P state by means of collisions with H2 is (25.1 +/- 4.0) x 10(-16) cm2. The reactive cross section could be obtained using results of the recording of the fluorescence signals with rapid rise in transient regime (<10 ns) Li(2P-->2S) at the different H2 densities. The authors fitted a two-state rate equation model to obtain the cross section sigma (Li(2P)+H2-->LiH+H) = (0.2 +/- 0.1) x 10(-16) cm2. The authors' results imply that reactive collisions occur on average 1/125 as often as quenching collisions. The cross section for reaction is small but not negligible.
ABSTRACT
At a Cs density higher than 9 x 10(14) cm(-3), cesium vapor was irradiated in a glass fluorescence cell with pulses of radiation from an YAG-laser-pumped OPO laser, populating 6D(5/2) state by two-photon absorption. Energy transfer in Cs6(D(5/2)) + Cs (6S) collisions was studied using methods of atomic fluorescence. At the different Cs densities, we have measured the time-integrated intensities of the components and fitted a three-state rate equation model to obtain the cross sections. The experimental points were fitted to a straight line very well. The authors converted the gradient and intercept into cross sections. The cross section for 6D(5/2)-->6D(3/2) transfer is (2.1 +/- 0.4) x 10(14) cm2. The cross section for excitation transfer out of the 6D doublet is sigmaQ = (1.6 +/- 0.4) x 10(-14) cm2. The cross section on contains information on reverse energy pooling collisions [i.e., Cs(6D(3/2)) + Cs (6S(1/2))-->Cs(6P) + 6Cs(P)] and contribution from mining in 6Dj-->7P(J'), This latter contribution could be subtracted out using the results of a second experiment. At a Cs density lower than 6.0 x 10(12) cm3, the laser was used to pump the 6D(3/2) and 6D(5/2) states, respectively. The resulting fluorescence included the direct component emitted in the decay of the 6D(J) state and the sensitized component arising from the collisionally populated 7P(J') state. Relative intensities of the components yielded the cross sections. The cross-sections for the processes Cs(6D(5/2)) + Cs(6S(1/2))-->Cs(7P(J')) + Cs(6S(1/2)) are (1.6 +/- 0.5) x 10(-15) cm2. for J'= 3/2 and (6.5 +/- 2.1) x 10(-16) cm2, for J' = 1/2, respectively. The cross-sections for the processes Cs(6D(3/2) + Cs(6S(1/2))-->Cs (7P(J')) + Cs(6S(1/2)) are (1.9 +/- 0.6) x 10(-15) cm2. for J' = 3/2 and (7.6 + 2.4) x 10(-16) cm2, for J' = 1/2, respectively. The 6D(J) -->7P(J'), energy transfer rate coefficient is small. The total quenching rate coefficient out of the 6D(J) state is much larger. Evidence suggests that the quenching of the 6D(J) state is caused predominantly by reverse energy-pooling process. The cross section for this process, i.e., Cs(6D(3/2))+Cs(6S(1/2))-->Cs(6P) + Cs(6P) is (1.3 +/- 0.4) x 10(-14) cm2.
ABSTRACT
Energy pooling (EP) was observed in Rb vapor following pulsed optical excitation to the 5P1/2 state. The 5P3/2 state was populated by the energy transfer process: Rb(5P1/2)+Rb(5S1/2) --> Rb(5P3/2)+Rb(5Sl/2). The resulting densities of Rb atoms at the 5P1/2 level were obtained from the absorption of narrow spectral line from a Rb hollow cathode lamp, connecting the 5P1/2 state to 7S state. Since the effective lifetimes of the 5P1/2 and 5P3/2 states are approximately equal, the densities of the 5P3/2 level were obtained from the D2 to D1 fluorescence ratios where D1 and D2 are lines of the 5P1/2 --> 5S1/2 and 5P3/2 --> 5S1/2 transition. Because the time of the fine structure exchanging is much shorter than the lifetime of the 5D state, the fluorescence originating from the 5D state produced by the 5P1/2 + 5P3/2 and 5P3/2 + 5P3/2 processes follows the instantaneous production rate of the 5P1/2 + 5P1/2 process. It is clear that 5P/2 + 5P3/2 and 5P3/2 + 5P3/2 collisions can significantly influence the results obtained for the 5P1/2 + 5P1/2 rate since the energy defect for 5D state is much smaller for 5P1/2 + 5P3/2 and 5P3/2 + 5P3/2 collisions than for 5P1/2 + 5P1/2 collisions. Effective lifetimes of the 5P levels were calculated using radiation trapping theory. The time-integrated populations and signals were studied and analyzed. The resulting fluorescence included the direct component emitted in the decay of the optically excited 5P1/2 state and the sensitized component arising from the collisions for populating 5D state at different cell temperature. These relative intensities were combined with the measured excited atom densities to yield absolute energy-pooling rate coefficients. The cross sections (in units of 10(-14) cm2) for the energy-pooling collisions [i. e. , 5P1/2 + 5P1/2, 5P1/2 + 5P3/2, 5P3/2 + 5P3/2] are 0.78, 2.9 and 3.1, respectively. The dependence of the rates upon energy defect deltaE was examined, but the 5D3/2 level was approximately equally populated in 5P3/2 + 5P3/2 (deltaE = 68 cm(-1)) and 5P3/2 + 5P1/2 (deltaE = 306 cm(-1)) collisions. The 5P1/2 + 5P3/2 collisions are as efficient as 5P3/2 + 5P3/2 for populating 5D3/2 state.