Mucin and carbon nanotube-based biosensor for detection of glucose in human plasma.

This work reports an amperometric enzyme-electrode prepared with glucose oxidase, which have been immobilized by a cross-linking step with glutaraldehyde in a mixture containing albumin and a novel carbon nanotubes-mucin composite (CNT-muc). The obtained hydrogel matrix was trapped between two polycarbonate membranes and then fixed at the surface of a Pt working electrode. The developed biosensor was optimized by evaluating different compositions and the analytical properties of an enzymatic matrix with CNT-muc. Then, the performance of the resulting enzymatic matrix was evaluated for direct glucose quantification in human blood plasma. The novel CNT-muc composite provided a sensitivity of 0.44 ±â€¯0.01 mA M-1 and a response time of 28 ±â€¯2 s. These values were respectively 20% higher and 40% shorter than those obtained with a sandwich-type biosensor prepared without CNT. Additionally, CNT-muc based biosensor exhibited more than 3 orders of magnitude of linear dynamic calibration range and a detection limit of 3 µM. The short-term and long-term stabilities of the biosensors were also examined and excellent results were obtained through successive experiments performed within the first 60 days from their preparation. Finally, the storage stability was remarkable during the first 300 days.

Isolation of multipotent progenitor cells from human fetal liver capable of differentiating into liver and mesenchymal lineages.

Little is known about the differentiation capabilities of nonhematopoietic cells of the human fetal liver. We report the isolation and characterization of a human fetal liver multipotent progenitor cell (hFLMPC) population capable of differentiating into liver and mesenchymal cell lineages. Human fetal livers (74-108 days of gestation) were dissociated and maintained in culture. We treated the colonies with geneticin and mechanically isolated hFLMPCs, which were kept in an undifferentiated state by culturing on feeder layers. We derived daughter colonies by serial dilution, verifying monoclonality using the Humara assay. hFLMPCs, which have been maintained in culture for up to 100 population doublings, have a high self-renewal capability with a doubling time of 46 h. The immunophenotype is: CD34+, CD90+, c-kit+, EPCAM+, c-met+, SSEA-4+, CK18+, CK19+, albumin-, alpha-fetoprotein-, CD44h+, and vimentin+. Passage 1 (P1) and P10 cells have identical morphology, immunophenotype, telomere length, and differentiation capacity. Placed in appropriate media, hFLMPCs differentiate into hepatocytes and bile duct cells, as well as into fat, bone, cartilage, and endothelial cells. Our results suggest that hFLMPCs are mesenchymal-epithelial transitional cells, probably derived from mesendoderm. hFLMPCs survive and differentiate into functional hepatocytes in vivo when transplanted into animal models of liver disease. hFLMPCs are a valuable tool for the study of human liver development, liver injury, and hepatic repopulation.

Protection of human and murine hepatocytes against Fas-induced death by transferrin and iron.

Recent studies in a murine model show that transferrin (Tf) interferes with Fas-mediated hepatocyte death and liver failure by decreasing pro-apoptotic and increasing anti-apoptotic signals. We show here in vitro in murine and human hepatocyte cell lines and in vivo in mice that Fas-induced apoptosis is modulated by exogenous Tf and iron. The results obtained with iron-free Tf (ApoTf), iron-saturated Tf (FeTf), and the iron chelator salicylaldehyde isonicotinoyl hydrazone (SIH) in its iron-free and iron-saturated (FeSIH) forms indicate that apoptosis-modulating effects of Tf are not mediated by iron alone. Both the Tf molecule and iron affect multiple aspects of cell death, and the route of iron delivery to the cell may be critical for the final outcome of cellular Fas signaling. Survival of hepatocytes 'stressed' by Fas signals can be manipulated by Tf and iron and may be a target for prophylactic and therapeutic interventions.

Enhanced acetaminophen hepatotoxicity in transgenic mice overexpressing BCL-2.

Mitochondria play an important role in the cell death induced by many drugs, including hepatotoxicity from overdose of the popular analgesic, acetaminophen (APAP). To investigate mitochondrial alterations associated with APAP-induced hepatotoxicity, the subcellular distribution of proapoptotic BAX was determined. Based on the antiapoptotic characteristics of BCL-2, we further hypothesized that if a BAX component was evident then BCL-2 overexpression may be hepatoprotective. Mice, either with a human bcl-2 transgene (-/+) or wild-type mice (WT; -/-), were dosed with 500 or 600 mg/kg (i.p.) APAP or a nonhepatotoxic isomer, N-acetyl-m-aminophenol (AMAP). Immunoblot analyses indicated increased mitochondrial BAX-beta content very early after APAP or AMAP treatment. This was paralleled by disappearance of BAX-alpha from the cytosol of APAP treated animals and, to a lesser extent, with AMAP treatment. Early pathological evidence of APAP-induced zone 3 necrosis was seen in bcl-2 (-/+) mice, which progressed to massive panlobular necrosis with hemorrhage by 24 h. In contrast, WT mice dosed with APAP showed a more typical, and less severe, centrilobular necrosis. AMAP-treated bcl-2 (-/+) mice displayed only early microvesicular steatosis without progression to extensive necrosis. Decreased complex III activity, evident as early as 6 h after treatment, correlated well with plasma enzyme activities at 24 h (AST r(2) = 0.89, ALT r(2) = 0.87) thereby confirming a role for mitochondria in APAP-mediated hepatotoxicity. In conclusion, these data suggest for the first time that BAX may be an early determinant of APAP-mediated hepatotoxicity and that BCL-2 overexpression unexpectedly enhances APAP hepatotoxicity.

Expression of suppressors of cytokine signaling during liver regeneration.

The cytokines TNF and IL-6 play a critical role early in liver regeneration following partial hepatectomy (PH). Since IL-6 activates signal transducers and activators of transcription (STATs), we examined whether the suppressors of cytokine signaling (SOCS) may be involved in terminating IL-6 signaling. We show here that SOCS-3 mRNA is induced 40-fold 2 hours after surgery. SOCS-2 and CIS mRNA are only weakly induced, and SOCS-1 is not detectable. SOCS-3 induction after PH is transient and correlates with a decrease in STAT-3 DNA binding and a loss of tyrosine 705 phosphorylation. This response is markedly reduced in IL-6 knockout (KO) mice. TNF injection induces SOCS-3 mRNA in wild-type mice (albeit weakly compared with the increase observed after PH) but not in TNF receptor 1 or IL-6 KO mice. In contrast, IL-6 injection induces SOCS-3 in these animals, demonstrating a requirement for IL-6 in SOCS-3 induction. IL-6 injection into wild-type mice also induces SOCS-1, -2, and CIS mRNA, in addition to SOCS-3. Together, these results suggest that SOCS-3 may be a key component in downregulating STAT-3 signaling after PH and that SOCS-3 mRNA levels in the regenerating liver are regulated by IL-6.

Bcl-2 delays and alters hepatic carcinogenesis induced by transforming growth factor alpha.

Transgenic mice that overexpress transforming growth factor (TGF)-alpha develop liver tumors between 12 and 15 months of age. Tumor development is preceded by an overall increase in the rates of hepatocyte proliferation and cell death. To examine the role of apoptosis in the development of TGF-alpha-induced liver tumors, we generated TGF-alpha/Bcl-2 double transgenic mice by crossing TGF-alpha transgenic mice with Bcl-2 transgenic mice expressing a zinc-inducible Bcl-2 transgene. Overexpression of the Bcl-2 transgene protected hepatocytes from Fas-mediated apoptosis. We anticipated that hepatocytes in TGF-alpha/Bcl-2 double transgenic mice would be stimulated to proliferate but would fail to undergo apoptosis, leading to increased liver weights and accelerated tumorigenesis. At 4 weeks of age, both TGF-alpha single transgenic and TGF-alpha/Bcl-2 double transgenic mice had elevated hepatocyte proliferation and increased liver:body weight ratios. However, by 8 months, the liver:body weight ratios had normalized in both TGF-alpha single transgenic and TGF-alpha/Bcl-2 double transgenic mice. Furthermore, Bcl-2 functioned as a tumor suppressor, significantly decreasing the frequency and delaying the development of TGF-alpha-induced liver tumors, despite having comparable levels of TGF-alpha transgene expression in both single and double transgenic mice. Between 11 and 12 months of age, >80% of the TGF-alpha single transgenic mice had developed tumors, whereas only 54% of the double transgenic mice had developed tumors after 13 months of age. The tumors that eventually developed in the TGF-alpha/Bcl-2 double transgenic mice were histologically distinct and smaller in size and had lower hepatocyte mitotic activity than tumors from TGF-alpha single transgenic mice. Furthermore, delaying Bcl-2 expression until 8.5 months of age was sufficient to inhibit TGF-alpha-induced tumorigenesis. These results indicate that Bcl-2 inhibits tumor progression in the liver, possibly by interfering with hepatocyte proliferation.

Impaired preneoplastic changes and liver tumor formation in tumor necrosis factor receptor type 1 knockout mice.

Hepatic stem cells (oval cells) proliferate within the liver after exposure to a variety of hepatic carcinogens and can generate both hepatocytes and bile duct cells. Oval cell proliferation is commonly seen in the preneoplastic stages of liver carcinogenesis, often accompanied by an inflammatory response. Tumor necrosis factor (TNF), an inflammatory cytokine, is also important in liver regeneration and hepatocellular growth. The experiments reported here explore the relationship among the TNF inflammatory pathway, liver stem cell activation, and tumorigenesis. We demonstrate that TNF is upregulated during oval cell proliferation induced by a choline-deficient, ethionine-supplemented diet and that it is expressed by oval cells. In TNF receptor type 1 knockout mice, oval cell proliferation is substantially impaired and tumorigenesis is reduced. Oval cell proliferation is impaired to a lesser extent in interleukin 6 knockout mice and is unchanged in TNF receptor type 2 knockout mice. These findings demonstrate that TNF signaling participates in the proliferation of oval cells during the preneoplastic phase of liver carcinogenesis and that loss of signaling through the TNF receptor type 1 reduces the incidence of tumor formation. The TNF inflammatory pathway may be a target for therapeutic intervention during the early stages of liver carcinogenesis.

Disruption of redox homeostasis in tumor necrosis factor-induced apoptosis in a murine hepatocyte cell line.

Tumor necrosis factor (TNF) is a mediator of the acute phase response in the liver and can initiate proliferation and cause cell death in hepatocytes. We investigated the mechanisms by which TNF causes apoptosis in hepatocytes focusing on the role of oxidative stress, antioxidant defenses, and mitochondrial damage. The studies were conducted in cultured AML12 cells, a line of differentiated murine hepatocytes. As is the case for hepatocytes in vivo, AML12 cells were not sensitive to cell death by TNF alone, but died by apoptosis when exposed to TNF and a small dose of actinomycin D (Act D). Morphological signs of apoptosis were not detected until 6 hours after the treatment and by 18 hours approximately 50% of the cells had died. Exposure of the cells to TNF+Act D did not block NFkappaB nuclear translocation, DNA binding, or its overall transactivation capacity. Induction of apoptosis was characterized by oxidative stress indicated by the loss of NAD(P)H and glutathione followed by mitochondrial damage that included loss of mitochondrial membrane potential, inner membrane structural damage, and mitochondrial condensation. These changes coincided with cytochrome C release and the activation of caspases-8, -9, and -3. TNF-induced apoptosis was dependent on glutathione levels. In cells with decreased levels of glutathione, TNF by itself in the absence of transcriptional blocking acted as an apoptotic agent. Conversely, the antioxidant alpha-lipoic acid, that protected against the loss of glutathione in cells exposed to TNF+Act D completely prevented mitochondrial damage, caspase activation, cytochrome C release, and apoptosis. The results demonstrate that apoptosis induced by TNF+Act D in AML12 cells involves oxidative injury and mitochondrial damage. As injury was regulated to a larger extent by the glutathione content of the cells, we suggest that the combination of TNF+Act D causes apoptosis because Act D blocks the transcription of genes required for antioxidant defenses.

Prevention of hepatic apoptosis and embryonic lethality in RelA/TNFR-1 double knockout mice.

Mice deficient in the nuclear factor kappaB (NF-kappaB)-transactivating gene RelA (p65) die at embryonic days 14-15 with massive liver apoptosis. In the adult liver, activation of the NF-kappaB heterodimer RelA/p50 can cause hepatocyte proliferation, apoptosis, or the induction of acute-phase response genes. We examined, during wild-type fetal liver development, the expression of the Rel family member proteins, as well as other proteins known to be important for NF-kappaB activation. We found these proteins and active NF-kappaB complexes in the developing liver from at least 2 days before the onset of lethality observed in RelA knockouts. This suggests that the timing of NF-kappaB activation is not related to the timing of lethality. We therefore hypothesized that, in the absence of RelA, embryos were sensitized to tumor necrosis factor (TNF) receptor 1 (TNFR-1)-mediated apoptosis. Thus, we generated mice that were deficient in both RelA and TNFR-1 to determine whether apoptotic signaling through TNFR-1 was responsible for the lethal phenotype. RelA/TNFR-1 double knockout mice survived embryonic development and were born with normal livers without evidence of increased hepatocyte apoptosis. These animals became runted shortly after birth and survived an average of 10 days, dying from acute hepatitis with an extensive hepatic infiltration of immature neutrophils. We conclude that neither RelA nor TNFR-1 is required for liver development and that RelA protects the embryonic liver from TNFR-1-mediated apoptotic signals. However, the absence of both TNFR-1 signaling and RelA activity in newborn mice makes these animals susceptible to endogenous hepatic infection.

Liver regeneration.

The liver can precisely regulate its growth and mass. Surgical resection of hepatic lobes or hepatocyte loss caused by viral or chemical injury triggers hepatocyte replication while enlarged liver mass is corrected by apoptosis. Hepatocytes have a great replicative capacity and are capable of repopulating the liver. However, "stem-like" cells proliferate when hepatocyte replication is blocked or delayed. Detailed studies of the mechanisms that regulate liver growth have been done in animals subjected to partial hepatectomy or chemical injury. Substantial progress has been achieved using appropriate transgenic and knockout mouse models for this work. Gene expression in the regenerating liver can be divided into several phases, starting with expression of a large number of immediate early genes. Hepatocytes need to be primed before they can fully respond to the growth factors HGF (Hepatocyte Growth Factor), TGFalpha (Transforming Growth Factor Alpha), and EGF (Epidermal Growth Factor) in vitro. Priming requires the cytokines TNF and IL-6 in addition to other agents that prevent cytotoxicity. Reactive Oxygen Species and glutathione content can determine whether the TNF effect on hepatocytes is proliferative or apoptotic. At least four transcription factors, NFkappaB, STAT3 (which are strongly induced by TNF), AP-1 and C/EBPbeta play major roles in the initiation of liver regeneration. In addition, extensive remodeling of the hepatic extracellular matrix occurs shortly after partial hepatectomy. Progression through the cell cycle beyond the initiation phase requires growth factors. The expression of Cyclin D1 probably establishes the stage at which replication becomes growth factor-independent and autonomous. Knowledge about the mechanisms of liver regeneration can now be applied to correct clinical problems caused by deficient liver growth.

Lessons from genetically engineered animal models. V. Knocking out genes to study liver regeneration: present and future.

Studies utilizing knockout mice have contributed important new knowledge about the mechanisms that initiate liver regeneration. New mouse lines need to be established to address major questions about these mechanisms, targeting genes for which there is experimental evidence of their involvement in important pathways. Development of conditional, liver-specific knockout mice would be of great value for these studies.

Mouse liver tumorigenesis: models, mechanisms, and relevance to human disease.

Hepatocytes have a remarkable proliferative capacity, but are quiescent in normal liver. Cell cycle activation in hepatocarcinogenesis can be directly triggered by overexpression of single and combinations of genes or be initiated indirectly by compensatory proliferation in response to liver injury. Work with transgenic and knockout mice indicate that regardless of the initiating cause, constitutive hepatocyte proliferation accompanied by genomic damage are essential factors for liver tumor development. The carcinogenic process is best described as a continuum that involves unregulated hyperplasia, dysplasia, and adenoma formation. The critical steps required for the transition from regulated to constitutive hepatocyte proliferation and the mechanisms of genomic damage in proliferating cells are being investigated. This knowledge should be directly applicable to studies of human liver tumorigenesis.

Tumor necrosis factor induces DNA replication in hepatic cells through nuclear factor kappaB activation.

Tumor necrosis factor (TNF) signaling through TNF receptor 1 (TNFR1) with downstream participation of nuclear factor kappaB (NFkappaB), interleukin 6 (IL-6), and signal transducers and activators of transcription 3 (STAT3) is required for initiation of liver regeneration. It is not known whether the proliferative effect of TNF on hepatocytes is direct or requires the participation of Kupffer cells, the liver resident macrophages. Moreover, it has not been determined whether NFkappaB activation is an essential step in TNF-induced proliferation. To answer these questions, we conducted studies in LE6 cells, a rat liver epithelial cell line with hepatocyte progenitor capacity. We report that TNF induces DNA replication in growth-arrested LE6 cells and that its effect involves the activation of NFkappaB and STAT3 and an increase in c-myc and IL-6 mRNAs. All of these effects, which mimic the events that initiate liver regeneration in vivo, are blocked if NFKB activation is inhibited by expression of a dominant-inhibitor IkappaBalpha mutant (deltaN-IkappaBalpha). Although NFkappaB blockage by deltaN-IkappaBalpha causes caspase activation and massive death of cells stimulated by TNF, inhibition of NFkappaB and STAT3 binding by the serine protease inhibitor N-tosyl-L-phenylalanine chloromethyl ketone results in G0-G1 cell cycle arrest without death. We conclude that NFkappaB is an essential component of the TNF proliferative pathway and that TNF-induced changes in IL-6 mRNA, STAT3, and c-myc mRNA are dependent on NFkappaB activation. Blockage of NFkappaB inhibits TNF-induced proliferation but does not necessarily cause cell death.

Generation of hepatocytes from oval cell precursors in culture.

Although there is experimental evidence supporting the involvement of hepatic stem cells in the pathogenesis of liver cancers, the detection and isolation of these cells remains elusive. A logical approach to detecting these cells would take advantage of their ability to differentiate (or to give rise to cells that differentiate) into hepatocytes. This approach requires an assay system that is conducive to hepatocytic differentiation. Here, we report the development of an in vitro system consisting of a three-dimensional collagen gel matrix and a fibroblast feeder layer that supports hepatocytic differentiation from precursor epithelial (oval) cell lines. The LE/2 and LE/6 oval cell lines used in this study are nontumorigenic cells that are derived from the livers of adult rats fed a choline-deficient diet containing 0.1% ethionine for 2 and 6 weeks, respectively. These lines consist of small cells that are phenotypically immature with few cytoplasmic organelles and a high nuclear-to-cytoplasmic ratio. After 4 weeks in the three-dimensional culture system, these cells acquired typical hepatocytic morphology. By electron microscopy, the cells formed canalicular structures that are typical of hepatocytes and were organelle rich, displaying peroxisomes, abundant mitochondria, and rough endoplasmic reticulum. The cells produced albumin and displayed a cytokeratin (CK) pattern typical of hepatocytes (CK 8 and CK 18-positive and CK 19-negative). The presence of a mesenchymal cell feeder layer was essential for supporting hepatocytic differentiation. Without a feeder layer but in the presence of hepatocyte growth factor and/or keratinocyte growth factor, the precursor cells formed ductal structures, suggestive of differentiation along the bile duct lineage. The three-dimensional system described provides direct proof of the lineage generation capacity of oval cells. It offers a model to study factors that may be important for hepatocytic differentiation from precursor cells and a means to assay cell populations for their ability to give rise to normal and transformed hepatocytes.

Tumor necrosis factor primes hepatocytes for DNA replication in the rat.

Signaling through tumor necrosis factor receptor type 1 (TNFR-1) using a pathway that involves nuclear factor kappaB (NF-kappaB), interleukin-6 (IL-6), and STAT3 is required for the initiation of liver regeneration. We have proposed that TNF primes hepatocytes to respond to the mitogenic effect of growth factors, but so far, there has been no experimental demonstration that TNF enhances growth factor responses of hepatocytes. To test this hypothesis, we infused hepatocyte growth factor (HGF) and transforming growth factor (TGF-) (40 microgram/24 h) directly into the portal vein of rats for 24 hours using osmotic pumps and determined whether TNF injection (5 microgram per rat) would significantly increase hepatocyte DNA labeling in these animals. All rats received 5-bromo-2'-deoxyuridine (BrdU) by intraperitoneal delivery during a 48-hour period (i.e., BrdU infusion continued for 24 hours after the end of growth factor administration). BrdU labeling in the liver was measured by both immunohistochemistry and flow cytometry, and the results obtained by these methods showed excellent concordance. The results demonstrate that TNF transiently activates NF-kappaB and STAT3 and increases the proliferative response of hepatocytes to HGF or TGF- by fourfold. Priming effects on hepatocyte DNA replication were also obtained with injection of lipopolysaccharide (LPS) and gadolinium chloride (GdCl3), agents that release TNF in the liver. Similarly to TNF, GdCl3 injection caused the activation of NF-kappaB and STAT3, reaching a maximum 8 to 12 hours after the injection. The results show that TNF acts as a primer to sensitize hepatocytes to the proliferative effects of growth factors and offers a mechanism to explain the initiation and progression phases of liver regeneration after partial hepatectomy (PH).