Recruited macrophages that colonize the post-inflammatory peritoneal niche convert into functionally divergent resident cells.

Inflammation generally leads to recruitment of monocyte-derived macrophages. What regulates the fate of these cells and to what extent they can assume the identity and function of resident macrophages is unclear. Here, we show that macrophages elicited into the peritoneal cavity during mild inflammation persist long-term but are retained in an immature transitory state of differentiation due to the presence of enduring resident macrophages. By contrast, severe inflammation results in ablation of resident macrophages and a protracted phase wherein the cavity is incapable of sustaining a resident phenotype, yet ultimately elicited cells acquire a mature resident identity. These macrophages also have transcriptionally and functionally divergent features that result from inflammation-driven alterations to the peritoneal cavity micro-environment and, to a lesser extent, effects of origin and time-of-residency. Hence, rather than being predetermined, the fate of inflammation-elicited peritoneal macrophages seems to be regulated by the environment.

Activation of stem cells in hepatic diseases.

The liver has enormous regenerative capacity. Following acute liver injury, hepatocyte division regenerates the parenchyma but, if this capacity is overwhelmed during massive or chronic liver injury, the intrinsic hepatic progenitor cells (HPCs) termed oval cells are activated. These HPCs are bipotential and can regenerate both biliary epithelia and hepatocytes. Multiple signalling pathways contribute to the complex mechanism controlling the behaviour of the HPCs. These signals are delivered primarily by the surrounding microenvironment. During liver disease, stem cells extrinsic to the liver are activated and bone-marrow-derived cells play a role in the generation of fibrosis during liver injury and its resolution. Here, we review our current understanding of the role of stem cells during liver disease and their mechanisms of activation.

Experimental hepatology applied to stem cells.

Transplantation is an accepted treatment today for many people suffering from organ failure. More and more patients are referred for transplant surgery, and the waiting lists are growing longer because not enough organs and tissues are donated for transplantation. This has led to several potentially viable alternatives being considered, including bio-artificial support devices, the transplantation of mature cells or stem/progenitor cells and the potential transplantation of xenogenic organs and cells [Burra P, Samuel D, Wendon J, Pietrangelo A, Gupta S. Strategies for liver support: from stem cells to xenotransplantation. J Hepatol 2004;41:1050-9]. Numerous investigators around the world are engaged in these investigations and the pace of discovery has begun to accelerate in recent years. To take stock of the achievements of recent years, the AISF sponsored a Single-Topic Conference, held in Padua on 26-27 May, 2006, with the participation of many leading investigators from various parts of Italy and Europe. This present paper summarizes the content of the Conference. Different issues were analysed, from the biology of stem cells to the possible use of gene therapy. The speakers were clinicians and scientists interested in diseases not only of the liver but also of other organs such as the kidney or heart. The fact that numerous specialties were represented helped the audience to understand the stem cell research area from different standpoints, and what research has achieved so far.

Liver repopulation with bone marrow derived cells improves the metabolic disorder in the Gunn rat.

BACKGROUND: Reversible ischaemia/reperfusion (I/R) liver injury has been used to induce engraftment and hepatic parenchymal differentiation of exogenous beta2-microglubulin(-)/Thy1(+) bone marrow derived cells. AIM: To test the ability of this method of hepatic parenchymal repopulation, theoretically applicable to clinical practice, to correct the metabolic disorder in a rat model of congenital hyperbilirubinaemia. METHODS AND RESULTS: Analysis by confocal laser microscopy of fluorescence labelled cells and by immunohistochemistry for beta2-microglubulin, 72 hours after intraportal delivery, showed engraftment of infused cells in liver parenchyma of rats with I/R, but not in control animals with non-injured liver. Transplantation of bone marrow derived cells obtained from GFP-transgenic rats into Lewis rats resulted in the presence of up to 20% of GFP positive hepatocytes in I/R liver lobes after one month. The repopulation rate was proportional to the number of transplanted cells. Infusion of GFP negative bone marrow derived cells into GFP positive transgenic rats resulted in the appearance of GFP negative hepatocytes, suggesting that the main mechanism underlying parenchymal repopulation was differentiation rather than cell fusion. Transplantation of wild type bone marrow derived cells into hyperbilirubinaemic Gunn rats with deficient bilirubin conjugation after I/R damage resulted in 30% decrease in serum bilirubin, the appearance of bilirubin conjugates in bile, and the expression of normal UDP-glucuronyltransferase enzyme evaluated by polymerase chain reaction. CONCLUSIONS: I/R injury induced hepatic parenchymal engraftment and differentiation into hepatocyte-like cells of bone marrow derived cells. Transplantation of bone marrow derived cells from non-affected animals resulted in the partial correction of hyperbilirubinaemia in the Gunn rat.

Understanding liver regeneration to bring new insights to the mechanisms driving cholangiocarcinoma.

Cancer frequently arises in epithelial tissues subjected to repeated cycles of injury and repair. Improving our understanding of tissue regeneration is, therefore, likely to reveal novel processes with inherent potential for aberration that can lead to carcinoma. These highly conserved regenerative mechanisms are increasingly understood and in the liver are associated with special characteristics that underlie the organ's legendary capacity for restoration of size and function following even severe or chronic injury. The nature of the injury can determine the cellular source of epithelial regeneration and the signalling mechanisms brought to play. These observations are shaping how we understand and experimentally investigate primary liver cancer, in particular cholangiocarcinoma; a highly invasive malignancy of the bile ducts, resistant to chemotherapy and whose pathogenesis has hitherto been poorly understood. Interestingly, signals that drive liver development become activated in the formation of cholangiocarcinoma, such as Notch and Wnt and may be potential future therapeutic targets. In this review, we summarise the work which has led to the current understanding of the cellular source of cholangiocarcinoma, how the tumour recruits, sustains and is educated by its supporting stromal environment, and the tumour-derived signals that drive the progression and invasion of the cancer. With few current treatments of any true efficacy, advances that will improve our understanding of the mechanisms driving this aggressive malignancy are welcome and may help drive therapeutic developments.

Patients with the worst outcomes after paracetamol (acetaminophen)-induced liver failure have an early monocytopenia.

BACKGROUND: Acute liver failure (ALF) is associated with significant morbidity and mortality. Studies have implicated the immune response, especially monocyte/macrophages as being important in dictating outcome. AIM: To investigate changes in the circulating monocytes and other immune cells serially in patients with ALF, relate these with cytokine concentrations, monocyte gene expression and patient outcome. METHODS: In a prospective case-control study in the Scottish Liver Transplant Unit, Royal Infirmary Edinburgh, 35 consecutive patients admitted with paracetamol-induced liver failure (POD-ALF), 10 patients with non-paracetamol causes of ALF and 16 controls were recruited. The peripheral blood monocyte phenotype was analysed by flow cytometry, circulating cytokines quantified by protein array and monocyte gene expression array performed and related to outcome. RESULTS: On admission, patients with worst outcomes after POD-ALF had a significant monocytopenia, characterised by reduced classical and expanded intermediate monocyte population. This was associated with reduced circulating lymphocytes and natural killer cells, peripheral cytokine patterns suggestive of a 'cytokine storm' and increased concentrations of cytokines associated with monocyte egress from the bone marrow. Gene expression array did not differentiate patient outcome. At day 4, there was no significant difference in monocyte, lymphocyte or natural killer cells between survivors and the patients with adverse outcomes. CONCLUSIONS: Severe paracetamol liver failure is associated with profound changes in the peripheral blood compartment, particularly in monocytes, related with worse outcomes. This is not seen in patients with non-paracetamol-induced liver failure. Significant monocytopenia on admission may allow earlier clarification of prognosis, and it highlights a potential target for therapeutic intervention.

REpeated AutoLogous Infusions of STem cells In Cirrhosis (REALISTIC): a multicentre, phase II, open-label, randomised controlled trial of repeated autologous infusions of granulocyte colony-stimulating factor (GCSF) mobilised CD133+ bone marrow stem cells in patients with cirrhosis. A study protocol for a randomised controlled trial.

INTRODUCTION: Liver disease mortality and morbidity are rapidly rising and liver transplantation is limited by organ availability. Small scale human studies have shown that stem cell therapy is safe and feasible and has suggested clinical benefit. No published studies have yet examined the effect of stem cell therapy in a randomised controlled trial and evaluated the effect of repeated therapy. METHODS AND ANALYSIS: Patients with liver cirrhosis will be randomised to one of three trial groups: group 1: Control group, Standard conservative management; group 2 treatment: granulocyte colony-stimulating factor (G-CSF; lenograstim) 15 µg/kg body weight daily on days 1-5; group 3 treatment: G-CSF 15 µg/kg body weight daily on days 1-5 followed by leukapheresis, isolation and aliquoting of CD133+ cells. Patients will receive an infusion of freshly isolated CD133+ cells immediately and frozen doses at days 30 and 60 via peripheral vein (0.2×10(6) cells/kg for each of the three doses). Primary objective is to demonstrate an improvement in the severity of liver disease over 3 months using either G-CSF alone or G-CSF followed by repeated infusions of haematopoietic stem cells compared with standard conservative management. The trial is powered to answer two hypotheses of each treatment compared to control but not powered to detect smaller expected differences between the two treatment groups. As such, the overall α=0.05 for the trial is split equally between the two hypotheses. Conventionally, to detect a relevant standardised effect size of 0.8 point reduction in Model for End-stage Liver Disease score using two-sided α=0.05(overall α=0.1 split equally between the two hypotheses) and 80% power requires 27 participants to be randomised per group (81 participants in total). ETHICS AND DISSEMINATION: The trial is registered at Current Controlled Trials on 18 November 2009 (ISRCTN number 91288089, EuDRACT number 2009-010335-41). The findings of this trial will be disseminated to patients and through peer-reviewed publications and international presentations.

Review article: gene therapy in gastroenterology and hepatology.

Gene therapy for diseases of the gastrointestinal tract is an exciting prospect because of the fundamental cure that is potentially available. The gastrointestinal system, and especially the liver, is an area that will be central to the development of gene therapy. Techniques for gene replacement include homologous recombination and gene augmentation. For the treatment of cancer antisense strategy, pro-drug activation systems and gene immunotherapy are being investigated. Gene-carrying vectors divide into viral- and non-viral-based vectors, each with advantages and limitations. The accurate delivery of these vectors to sufficient numbers of target cells in vivo is still a major barrier to clinical use. Diseases that may be helped by gene therapy include: gastrointestinal malignancies, viral hepatitis, the haemophilias, hypercholesterolaemia, alpha 1-antitrypsin deficiency, and metabolic diseases of the liver and cystic fibrosis. In this review we will outline the principles of gene therapy, delivery vectors under investigation, diseases that may benefit from this technology and some of the remaining problems to be overcome.

Systematic review: the effects of autologous stem cell therapy for patients with liver disease.

BACKGROUND: As morbidity and mortality from liver disease continues to rise, new strategies are necessary. Liver transplantation is not only an expensive resource committing the patient to lifelong immunosuppression but also suitable donor organs are in short supply. Against this background, autologous stem cell therapy has emerged as a potential treatment option. AIM: To evaluate if it is possible to make a judgement on the safety, feasibility and effect of autologous stem cell therapy for patients with liver disease. METHODS: MEDLINE and EMBASE were searched up until July 2013 to identify studies where autologous stem cell therapy was administered to patients with liver disease. RESULTS: Of 1668 studies identified, 33 were eligible for inclusion evaluating a median sample size of 10 patients for a median follow-up of 6 months. Although there was marked heterogeneity between studies with regards to type, dose and route of delivery of stem cell, the treatment was shown to be safe and feasible largely when a peripheral route of administration was used. Of the studies which also looked at biochemical outcome, statistically significant improvement in liver function tests was seen in 16 studies post-treatment. CONCLUSION: Although autologous stem cell therapy is a much needed possibility in the treatment of liver disease, further robust clinical trials and collaborative protocols are required.

Stem cell mobilization and collection in patients with liver cirrhosis.

BACKGROUND: Bone marrow-derived stem cells (BMSC) and granulocyte colony-stimulating factor (G-CSF) have been proved to contribute to tissue regeneration after liver injury. AIMS: To test the safety of G-CSF and define the exact dose capable of mobilizing BMSC in the majority of patients with liver cirrhosis; and to assess the feasibility of leukapheresis to collect BMSC from peripheral blood. METHODS: In this study, we treated 18 patients affected by liver cirrhosis with increasing doses of G-CSF to mobilize CD34(+) and CD133(+) BMSC into the peripheral blood. RESULTS: The dose-finding phase demonstrated that 15 microg/kg/day of G-CSF is the optimal dose to mobilize both CD34(+) and CD133(+) stem cells. Circulating BMSC were collected by a single step leukapheresis in three patients and the mean number of CD34(+) and CD133(+) cells cryopreserved was 1.3 +/- 0.7 and 1.2 +/- 0.5 x 10(6)/kg, respectively. No severe adverse events were observed during the drug administration and stem cell collection. Noteworthy is, none of the patients showed a significant modification of liver function. CONCLUSIONS: Our study demonstrates that G-CSF administration and BMSC collection from the peripheral blood is possible and safe in patients with liver cirrhosis. The optimal dose to mobilize BMSC in cirrhotics is 15 microg/kg/day. At this dose, G-CSF does not seem to modify the residual liver function in cirrhotic patients.

Permanent partial phenotypic correction and tolerance in a mouse model of hemophilia B by stem cell gene delivery of human factor IX.

Immune responses against an introduced transgenic protein are a potential risk in many gene replacement strategies to treat genetic disease. We have developed a gene delivery approach for hemophilia B based on lentiviral expression of human factor IX in purified hematopoietic stem cells. In both normal C57Bl/6J and hemophilic 129/Sv recipient mice, we observed the production of therapeutic levels of human factor IX, persisting for at least a year with tolerance to human factor IX antigen. Secondary and tertiary recipients also demonstrate long-term production of therapeutic levels of human factor IX and tolerance, even at very low levels of donor chimerism. Furthermore, in hemophilic mice, partial functional correction of treated mice and phenotypic rescue is achieved. These data show the potential of a stem cell approach to gene delivery to tolerize recipients to a secreted foreign transgenic protein and, with appropriate modification, may be of use in developing treatments for other genetic disorders.

Hepatic and renal differentiation from blood-borne stem cells.

The recognition that adult bone marrow stem cells (BMSCs) can traffic into the liver and kidney and differentiate into a variety of cell types such as epithelial cells, endothelial cells and myofibroblasts has caused excitement. This has expanded our knowledge of how these organs regenerate following damage and provides new opportunities for therapeutic exploitation. BMSC transplants have already been used to correct a murine model of metabolic liver disease. Bone marrow stem cells that transdifferentiate into long-lasting cells within the liver and kidney are proposed as suitable targets for gene therapy and may be used in the correction of single gene defects, or the delivery of antiviral and anti-inflammatory genes to the liver and kidney. There is growing evidence that BMSCs can repopulate the endothelium of transplanted livers and kidneys and thus may potentially be manipulated to induce graft tolerance within solid organ transplants. However, there are technical barriers to be overcome before the theoretical benefits of this exiting new area becomes a practical prospect.

Feasibility of an air motor-driven centrifugal blood-pumping system.

The use of cardiopulmonary bypass (CPB) is extending out of the cardiac surgery operating room into new venues. The long-term goal of this project is the development of a completely disposable temporary-use CPB system that could be economically distributed to all of the units where it might be needed. Centrifugal blood pumps have demonstrated successful and widespread use. However, they are not as widely available as might be desired because they require a large and expensive console. An inexpensive, small, lightweight, disposable unit, in contrast, could be widely distributed for emergency care of patients and would be logistically practical for patient transportation between the presenting institution and a major cardiac care facility equipped for definitive treatment. An air motor might be an approach to such a device. The current research project underway at the University of Akron in conjunction with the Cleveland Clinic Foundation has focused on the following key feasibility issues: air consumption, air motor noise, and sealing the rotating shaft. Prototypes have been constructed from commercially available vane and turbine motors. Early studies have demonstrated favorable results with regard to air consumption and shaft sealing and directions for handling air motor noise.

Update on hepatic stem cells.

The liver, like most organs in an adult healthy body, maintains a perfect balance between cell gain and cell loss. Though normally proliferatively quiescent, simple hepatocyte loss such as that caused by partial hepatectomy, uncomplicated by virus infection or inflammation, invokes a rapid regenerative response to restore liver mass. This restoration of moderate cell loss and 'wear and tear' renewal is largely achieved by hepatocyte self-replication. Furthermore, cell transplant models have shown that hepatocytes can undergo significant clonal expansion. Such observations indicate that hepatocytes are the functional stem cells of the liver. More severe liver injury activates a facultative stem cell compartment located within the intrahepatic biliary tree, giving rise to cords of biliary epithelia within the lobules before these cells differentiate into hepatocytes. A third population of stem cells with hepatic potential resides in the bone marrow; these haematopoietic stem cells can contribute to the albeit low renewal rate of hepatocytes, make a more significant contribution to regeneration, and even completely restore normal function in a murine model of hereditary tyrosinaemia. How these three stem cell populations integrate to achieve a homeostatic balance is not understood. This review focuses on three aspects of liver stem cell biology: 1) the hepatic stem cell candidates; 2) models of cell transplantation into the liver; and 3) the therapeutic potential of hepatic stem cells.

The new stem cell biology: something for everyone.

The ability of multipotential adult stem cells to cross lineage boundaries (transdifferentiate) is currently causing heated debate in the scientific press. The proponents see adult stem cells as an attractive alternative to the use of embryonic stem cells in regenerative medicine (the treatment of diabetes, Parkinson's disease, etc). However, opponents have questioned the very existence of the process, claiming that cell fusion is responsible for the phenomenon. This review sets out to provide a critical evaluation of the current literature in the adult stem cell field.

Tri-iodothyronine and a deleted form of hepatocyte growth factor act synergistically to enhance liver proliferation and enable in vivo retroviral gene transfer via the peripheral venous system.

Retroviral vectors integrate into the target cell genome in a stable manner and therefore offer the potential for permanent correction of the genetic diseases that affect the liver. These vectors, however, usually require cell division to occur in order to allow provirus entry into the nucleus. We have explored clinically acceptable methods to improve the efficiency of retroviral gene transfer to the liver, which avoid the need for liver damage. Tri-iodothyronine (T3) and recombinant hepatocyte growth factor have previously been used to induce hepatocyte proliferation in rat livers and allow in vivo retroviral gene transfer. We investigated the combined effects of these growth factors, with their differing mechanisms of action, on hepatocyte proliferation in vivo and assessed their effectiveness in priming cells for retroviral gene transfer. During the phase of hepatocyte proliferation retrovirus was administered via either the portal or tail vein. Acting synergistically, T3 and a truncated form of recombinant hepatocyte growth factor (dHGF) induced 30% of hepatocytes in normal rat liver to enter DNA synthesis at 24 h. This increased proliferation enabled the liver to be transduced in vivo by retroviral vectors via either the portal or peripheral venous system, achieving transduction efficiencies of 6.9 +/- 1.6% and 4.3 +/- 0.4% respectively. Thus, the liver can be simply and conveniently transduced in vivo with integrating vectors, introduced via the peripheral venous system during a wave of growth factor-induced proliferation, pointing the way to clinically applicable gene transfer techniques.

Synergistic growth factors enhance rat liver proliferation and enable retroviral gene transfer via a peripheral vein.

BACKGROUND & AIMS: Genetic diseases reflecting abnormal hepatocyte function are potentially curable through gene therapy. Retroviral vectors offer the potential for permanent correction of such conditions. These vectors generally require cell division to occur to allow provirus entry into the nucleus, initiated in many experimental protocols by partial hepatectomy. We have explored methods to improve the efficiency of retroviral gene transfer that avoid the need for liver damage. METHODS: Triiodothyronine (T3) and keratinocyte growth factor (KGF) were used to induce hepatic proliferation in rats. The effects of intraportal and peripheral administration of a modified retrovirus that encoded the Lac Z gene during growth factor-induced liver hyperplasia were analyzed. RESULTS: T3 initiated hepatocyte proliferation midzonally; after KGF, proliferation was more diffuse. Optimal concentrations of T3 and KGF acted synergistically to induce proliferation in 61% of hepatocytes in the intact liver. This enabled in vivo hepatocyte transduction, leading to gene expression by up to 7.3% of hepatocytes after intraportal retroviral vector administration and 7. 1% after peripheral venous administration. CONCLUSIONS: T3 and KGF act synergistically to induce hepatocyte proliferation in undamaged liver. The liver can be simply transduced with integrating vectors via the peripheral venous system during a wave of growth factor-induced proliferation.

Retroviral gene transfer to the liver in vivo during tri-iodothyronine induced hyperplasia.

The liver is an important target organ for gene therapy but its mitotic quiescence makes it resistant to integrative gene transfer. Retrovirus-based vectors integrate into liver cells in vivo but require the liver to be primed before transduction; experimentally a 70% hepatectomy is commonly used to stimulate regeneration, rendering the liver susceptible to transduction during the resulting wave of cell proliferation. Our aim was to develop a clinically acceptable method of inducing hepatocyte replication before in vivo retroviral gene transfer which is both simple and effective. We have used the physiological hormone tri-iodothyronine (T3) to stimulate hepatocyte replication. A single dose of T3 (400 micrograms/100 g bw) was given subcutaneously to euthyroid rats. This produced a labelling index of 31.7% in the hepatocyte population without histological or biochemical evidence of preceding liver damage. Following T3 administration the rat livers were transfected in vivo with an amphotropic retrovirus, TELCeB/AF-7 which encodes the beta-galactosidase reporter gene together with a nuclear localisation signal. Transgene expression was noted only within the liver where 1.3% of hepatocytes expressed the beta-galactosidase enzyme. This compared to 5.2% of hepatocytes transduced following a 70% hepatectomy, and 0.02% in animals receiving neither T3 nor partial hepatic resection before transduction. T3 administration is a simple way to prime the liver before in vivo retroviral vector-based gene transfer.

Bioequivalency of oral suspension formulations of cefixime.

A study was performed in 24 healthy male subjects to establish that two suspension formulations of cefixime were bioequivalent to each other and to a reference oral solution. A single 400 mg oral dose of the drug was given in a randomized three-way crossover design as two suspensions (a research suspension (RS) used during clinical trials and a suspension intended for marketing (MS] and a reference oral solution (SOL). Each dose was separated from the other by a 3-day washout period. Mean peak serum concentrations (Cmax) were 4.67, 4.10, and 4.27 micrograms ml-1 after the MS, RS, and SOL, respectively. Although comparison (ANOVA) of the mean pharmacokinetic parameters for cefixime found significant differences (p less than 0.05) in Cmax, the time to Cmax, and area under the serum concentration time curve (AUC 0----infinity) values among the three formulations, the mean differences were less than 20 per cent. No significant differences (p greater than 0.05) were found in either the elimination half-life or renal clearance of unchanged drug. Overall, with a 98 per cent power to detect a 20 per cent difference in AUC0----infinity or urinary recovery values between the formulations tested, the results show that the MS was bioequivalent to the RS and that both suspensions were bioequivalent to the SOL.