ABSTRACT
BACKGROUND & AIMS: Liver transplantation (LT) is the most effective treatment for patients with acute liver failure (ALF), but is limited by surgical risks and the need for life-long immunosuppression. Transplantation of microencapsulated human hepatocytes in alginate is an attractive option over whole liver replacement. The safety and efficacy of hepatocyte microbead transplantation have been shown in animal models. We report our experience of this therapy in children with ALF treated on a named-patient basis. METHODS: Clinical grade human hepatocyte microbeads (HMBs) and empty microbeads were tested in immunocompetent healthy rats. Subsequently, 8 children with ALF, who were awaiting a suitable allograft for LT, received intraperitoneal transplantation of HMBs. We monitored complications of the procedure, assessing the host immune response and residual function of the retrieved HMBs, either after spontaneous native liver regeneration or at the time of LT. RESULTS: Intraperitoneal transplantation of HMBs in healthy rats was safe and preserved synthetic and detoxification functions, without the need for immunosuppression. Subsequently, 8 children with ALF received HMBs (4 neonatal haemochromatosis, 2 viral infections and 2 children with unknown cause at time of infusion) at a median age of 14.5 days, range 1 day to 6 years. The procedure was well tolerated without complications. Of the 8 children, 4 avoided LT while 3 were successfully bridged to LT following the intervention. HMBs retrieved after infusions (at the time of LT) were structurally intact, free of host cell adherence and contained viable hepatocytes with preserved functions. CONCLUSION: The results demonstrate the feasibility and safety of an HMB infusion in children with ALF. LAY SUMMARY: Acute liver failure in children is a rare but devastating condition. Liver transplantation is the most effective treatment, but it has several important limitations. Liver cell (hepatocyte) transplantation is an attractive option, as many patients only require short-term liver support while their own liver recovers. Human hepatocytes encapsulated in alginate beads can perform the functions of the liver while alginate coating protects the cells from immune attack. Herein, we demonstrated that transplantation of these beads was safe and feasible in children with acute liver failure.
Subject(s)
Alginates , Cell Encapsulation/methods , Hepatocytes/transplantation , Liver Failure, Acute/surgery , Liver Transplantation/adverse effects , Liver Transplantation/methods , Microspheres , Animals , Cells, Cultured , Child , Child, Preschool , Feasibility Studies , Female , Humans , Infant , Infant, Newborn , Liver Regeneration , Male , Models, Animal , Rats , Tissue and Organ Procurement/methods , Transplantation, Heterologous/methods , Transplantation, Homologous/methods , Treatment OutcomeABSTRACT
Neonatal livers are a potential source of good-quality hepatocytes for clinical transplantation. We compared viability and function of neonatal hepatocytes (NHs) and adult hepatocytes (AHs) and report their clinical use both intraportally and in alginate microbeads. Following isolation from donor livers, hepatocyte function was assessed using albumin, alpha-1-antitrypsin, and factor VII. Metabolic function was investigated by measuring resorufin conjugation, ammonia metabolism, uridine diphosphate glucuronosyltransferase enzyme activity, and cytochrome P450 (CYP) function following induction. Activation of the instant blood-mediated inflammatory reaction by NHs and AHs was investigated using an in vitro blood perfusion model, and tissue factor expression was analyzed using real-time polymerase chain reaction (RT-PCR). Clinical hepatocyte transplantation (HT) was undertaken using standard protocols. Hepatocytes were isolated from 14 neonatal livers, with an average viability of 89.4% ± 1.8% (mean ± standard error of the mean) and average yield of 9.3 × 106 ± 2.0 × 106 cells/g. Hepatocytes were isolated from 14 adult livers with an average viability of 78.6% ± 2.4% and yield 2.2 × 106 ± 0.5 × 105 cells/g. NHs had significantly higher viability after cryopreservation than AHs, with better attachment efficiency and less plasma membrane leakage. There were no differences in albumin, alpha-1-antitrypsin, and factor VII synthesis between NHs and AHs (P > 0.05). Neonatal cells had inducible phase 1 enzymes as assessed by CYP function and functional phase 2 enzymes, in which activity was comparable to AHs. In an in vitro blood perfusion model, AHs elicited increased thrombus formation with a greater consumption of platelets and white cells compared with NHs (28.3 × 109 versus 118.7 × 109 and 3.3 × 109 versus 6.6 × 109 ; P < 0.01). Intraportal transplantation and intraperitoneal transplantation of alginate encapsulated hepatocytes was safe, and preliminary data suggest the cells may activate the immune response to a lesser degree than adult cells. In conclusion, we have shown NHs have excellent cell viability, function, and drug metabolism making them a suitable alternative source for clinical HT. Liver Transplantation 24 394-406 2018 AASLD.
Subject(s)
Cryopreservation , Hepatocytes/transplantation , Liver Failure, Acute/surgery , Liver Transplantation/methods , Adult , Biomarkers/metabolism , Biotransformation , Blood Coagulation , Cell Adhesion , Cell Shape , Cell Survival , Cells, Cultured , Child, Preschool , Female , Hepatocytes/enzymology , Hepatocytes/immunology , Hepatocytes/pathology , Humans , Infant , Infant, Newborn , Liver Failure, Acute/blood , Liver Failure, Acute/diagnosis , Liver Failure, Acute/etiology , Male , Phenotype , Preliminary Data , Primary Cell Culture , Treatment OutcomeABSTRACT
UNLABELLED: Acetaminophen-induced acute liver failure (AALF) is characterized both by activation of innate immune responses and susceptibility to sepsis. Circulating monocytes and hepatic macrophages are central mediators of inflammatory responses and tissue repair processes during human AALF. Secretory leukocyte protease inhibitor (SLPI) modulates monocyte/macrophage function through inhibition of nuclear factor kappa B (NF-κB) signaling. The aims of this study were to establish the role of SLPI in AALF. Circulating levels of SLPI, monocyte cluster of differentiation 163 (CD163), human leukocyte antigen-DR (HLA-DR), and lipopolysaccharide (LPS)-stimulated levels of NF-κBp65, tumor necrosis factor alpha (TNF-α) and interleukin (IL)-6 were determined in patients with AALF, chronic liver disease, and healthy controls. Immunohistochemistry and multispectral imaging of AALF explant tissue determined the cellular sources of SLPI and hepatic macrophage phenotype. The phenotype and function of monocytes and macrophages was determined following culture with recombinant human (rh)-SLPI, liver homogenates, and plasma derived from AALF patients in the presence and absence of antihuman (α)SLPI. Hepatic and circulatory concentrations of SLPI were elevated in AALF and immunohistochemistry revealed SLPI expression in biliary epithelial cells and within hepatic macrophages (h-mψ) in areas of necrosis. H-mψ and circulating monocytes in AALF exhibited an anti-inflammatory phenotype and functional characteristics; typified by reductions in NF-κBp65, TNF-α, and IL-6 and preserved IL-10 secretion following LPS challenge. Culture of healthy monocytes with AALF liver homogenates, plasma, or rhSLPI induced monocytes with strikingly similar anti-inflammatory characteristics which were reversed by inhibiting the activity of SLPI. CONCLUSION: SLPI is a pivotal mediator of anti-inflammatory responses in AALF through modulation of monocyte/macrophage function, which may account for the susceptibility to sepsis in AALF.
Subject(s)
Acetaminophen/adverse effects , Chemical and Drug Induced Liver Injury/physiopathology , Inflammation/prevention & control , Inflammation/physiopathology , Macrophages/physiology , Monocytes/physiology , Secretory Leukocyte Peptidase Inhibitor/physiology , Adolescent , Adult , Antigens, CD/blood , Antigens, Differentiation, Myelomonocytic/blood , Case-Control Studies , Chemical and Drug Induced Liver Injury/blood , HLA-DR Antigens/blood , Humans , Inflammation/blood , Interleukin-6/blood , Middle Aged , NF-kappa B/blood , Phenotype , Receptors, Cell Surface/blood , Secretory Leukocyte Peptidase Inhibitor/blood , Signal Transduction/physiology , Tumor Necrosis Factor-alpha/blood , Young AdultABSTRACT
The BMP/SMAD4 pathway has major effects on liver hepcidin levels. Bone morphogenetic protein-binding endothelial cell precursor-derived regulator (Bmper), a known regulator of BMP signaling, was found to be overexpressed at the mRNA and protein levels in liver of genetically hypotransferrinemic mice (Trf(hpx/hpx)). Soluble BMPER peptide inhibited BMP2- and BMP6-dependent hepcidin promoter activity in both HepG2 and HuH7 cells. These effects correlated with reduced cellular levels of pSMAD1/5/8. Addition of BMPER peptide to primary human hepatocytes abolished the BMP2-dependent increase in hepcidin mRNA, whereas injection of Bmper peptide into mice resulted in reduced liver hepcidin and increased serum iron levels. Thus Bmper may play an important role in suppressing hepcidin production in hypotransferrinemic mice.
Subject(s)
Antimicrobial Cationic Peptides/blood , Carrier Proteins/metabolism , Iron/blood , Liver/metabolism , Transferrin/metabolism , Up-Regulation , Animals , Antimicrobial Cationic Peptides/genetics , Bone Morphogenetic Protein 2/antagonists & inhibitors , Bone Morphogenetic Protein 2/genetics , Bone Morphogenetic Protein 2/metabolism , Carrier Proteins/genetics , Hep G2 Cells , Hepcidins , Humans , Mice , Mice, Transgenic , Peptides/pharmacology , Smad Proteins/genetics , Smad Proteins/metabolism , Transferrin/geneticsABSTRACT
OBJECTIVE: To evaluate the role of hepatocellular and extrahepatic apoptosis during the evolution of acetaminophen-induced acute liver failure. DESIGN AND SETTING: A prospective observational study in two tertiary liver transplant units. PATIENTS: Eighty-eight patients with acetaminophen-induced acute liver failure were recruited. Control groups included patients with nonacetaminophen-induced acute liver failure (n = 13), nonhepatic multiple organ failure (n = 28), chronic liver disease (n = 19), and healthy controls (n = 11). MEASUREMENTS: Total and caspase-cleaved cytokeratin-18 (M65 and M30) measured at admission and sequentially on days 3, 7, and 10 following admission. Levels were also determined from hepatic vein, portal vein, and systemic arterial blood in seven patients undergoing transplantation. Protein arrays of liver homogenates from patients with acetaminophen-induced acute liver failure were assessed for apoptosis-associated proteins, and histological assessment of liver tissue was performed. MAIN RESULTS: Admission M30 levels were significantly elevated in acetaminophen-induced acute liver failure and non-acetaminophen induced acute liver failure patients compared with multiple organ failure, chronic liver disease, and healthy controls. Admission M30 levels correlated with outcome with area under receiver operating characteristic of 0.755 (0.639-0.885, p < 0.001). Peak levels in patients with acute liver failure were seen at admission then fell significantly but did not normalize over 10 days. A negative gradient of M30 from the portal to hepatic vein was demonstrated in patients with acetaminophen-induced acute liver failure (p = 0.042) at the time of liver transplant. Analysis of protein array data demonstrated lower apoptosis-associated protein and higher catalase concentrations in acetaminophen-induced acute liver failure compared with controls (p < 0.05). Explant histological analysis revealed evidence of cellular proliferation with an absence of histological evidence of apoptosis. CONCLUSIONS: Hepatocellular apoptosis occurs in the early phases of human acetaminophen-induced acute liver failure, peaking on day 1 of hospital admission, and correlates strongly with poor outcome. Hepatic regenerative/tissue repair responses prevail during the later stages of acute liver failure where elevated levels of M30 are likely to reflect epithelial cell death in extrahepatic organs.
Subject(s)
Acetaminophen/toxicity , Analgesics, Non-Narcotic/toxicity , Apoptosis/physiology , Chemical and Drug Induced Liver Injury/physiopathology , Critical Illness , APACHE , Adult , Aged , Female , Humans , Keratin-18/blood , Liver , Liver Failure, Acute/physiopathology , Liver Function Tests , Male , Middle Aged , Multiple Organ Failure/physiopathology , Peptide Fragments/blood , Prospective Studies , Time FactorsABSTRACT
UNLABELLED: Acetaminophen-induced acute liver failure (AALF) is associated with innate immunity activation, which contributes to the severity of hepatic injury and clinical outcome. A marked increase in hepatic macrophages (h-mφ) is observed in experimental models of AALF, but controversy exists regarding their role, implicating h-mφ in both aggravation and resolution of liver injury. The role of h-mφ in human AALF is virtually unexplored. We sought to investigate the role of chemokine (C-C motif) ligand 2 (CCL2) in the recruitment of circulating monocytes to the inflamed liver and to determine how the h-mφ infiltrate and liver microenvironment may contribute to tissue repair versus inflammation in AALF. We evaluated circulating monocytes, their chemokine (C-C motif) receptor 2 (CCR2) expression, and serum CCL2 levels in patients with AALF. Cell subsets and numbers of circulation-derived (MAC387+) or resident proliferating (CD68/Ki67+) h-mφ in hepatic immune infiltrates were determined by immunohistochemistry. Inflammatory cytokine levels were determined in whole and laser microdissected liver tissue by proteome array. In AALF, circulating monocytes were depleted, with the lowest levels observed in patients with adverse outcomes. CCL2 levels were high in AALF serum and hepatic tissue, and circulating monocyte subsets expressed CCR2, suggesting CCL2-dependent hepatic monocyte recruitment. Significant numbers of both MAC387+ and CD68+ h-mφ were found in AALF compared with control liver tissue with a high proportion expressing the proliferation marker Ki67. Levels of CCL2, CCL3, interleukin (IL)-6, IL-10, and transforming growth factor-ß1 were significantly elevated in AALF liver tissue relative to chronic liver disease controls. CONCLUSION: In AALF, the h-mφ population is expanded in areas of necrosis, both through proliferation of resident cells and CCL2-dependent recruitment of circulating monocytes. The presence of h-mφ within an anti-inflammatory/regenerative microenvironment indicates that they are implicated in resolution of inflammation/tissue repair processes during AALF.
Subject(s)
Acetaminophen/poisoning , Chemical and Drug Induced Liver Injury/pathology , Liver Failure, Acute/chemically induced , Liver Failure, Acute/pathology , Macrophages/pathology , Adult , Analgesics, Non-Narcotic/poisoning , Antigens, CD/metabolism , Antigens, Differentiation, Myelomonocytic/metabolism , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , Chemical and Drug Induced Liver Injury/immunology , Chemokine CCL2/metabolism , Chemokine CCL3/metabolism , Chemotaxis/immunology , Female , Humans , Interleukin-10/metabolism , Interleukin-8/metabolism , Ki-67 Antigen/metabolism , Liver Failure, Acute/immunology , Macrophages/immunology , Macrophages/metabolism , Male , Middle Aged , Monocytes/immunology , Monocytes/metabolism , Monocytes/pathology , Receptors, CCR2/metabolism , Transforming Growth Factor beta1/metabolismABSTRACT
Over the past 35 years, the outlook for a patient presenting with acute liver failure (ALF) has changed beyond all recognition. A patient presenting in 1984 had an 80 % likelihood of succumbing to intracranial hypertension. Today due to dramatic improvements in intensive care in dedicated liver transplant units, this has been reduced to just 20 %. Prompt fluid resuscitation, empirical treatment for sepsis and standardised management protocols that include early intubation and high flow hemofiltration for ammonia removal, limit the numbers of patients who die from the sequelae of cerebral edema and ALF. With the evolution and development of bedside prognostic markers that will include personalised genomic, metabonomic and immune profiling, rationalisation of grafts to those who are not predicted to survive is likely to further minimise the number of grafts utilised. Furthermore, in those patients with a dismal prognosis, the use of plasmapheresis, immunomodulatory therapies, biological liver support systems and hepatocyte transplantation offer a potential bridge until the injured liver can begin to regenerate avoiding transplantation and life-long immunosuppressant therapy.
Subject(s)
Brain Diseases/therapy , Liver Failure, Acute/complications , Brain Diseases/etiology , Hepatocytes/transplantation , Humans , Intracranial Hypertension/therapy , Randomized Controlled Trials as Topic , Stem Cell TransplantationABSTRACT
BACKGROUND & AIMS: Hepatitis C virus (HCV) causes progressive liver disease and is a major risk factor for the development of hepatocellular carcinoma (HCC). However, the role of infection in HCC pathogenesis is poorly understood. We investigated the effect(s) of HCV infection and viral glycoprotein expression on hepatoma biology to gain insights into the development of HCV associated HCC. METHODS: We assessed the effect(s) of HCV and viral glycoprotein expression on hepatoma polarity, migration and invasion. RESULTS: HCV glycoproteins perturb tight and adherens junction protein expression, and increase hepatoma migration and expression of epithelial to mesenchymal transition markers Snail and Twist via stabilizing hypoxia inducible factor-1α (HIF-1α). HIF-1α regulates many genes involved in tumor growth and metastasis, including vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGF-ß). Neutralization of both growth factors shows different roles for VEGF and TGFß in regulating hepatoma polarity and migration, respectively. Importantly, we confirmed these observations in virus infected hepatoma and primary human hepatocytes. Inhibition of HIF-1α reversed the effect(s) of infection and glycoprotein expression on hepatoma permeability and migration and significantly reduced HCV replication, demonstrating a dual role for HIF-1α in the cellular processes that are deregulated in many human cancers and in the viral life cycle. CONCLUSIONS: These data provide new insights into the cancer-promoting effects of HCV infection on HCC migration and offer new approaches for treatment.
Subject(s)
Carcinoma, Hepatocellular/physiopathology , Cell Movement/physiology , Hepacivirus/physiology , Hypoxia-Inducible Factor 1, alpha Subunit/physiology , Liver Neoplasms/physiopathology , Virus Replication/physiology , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cell Polarity/physiology , Disease Progression , Glycoproteins/physiology , Hepatitis C/pathology , Hepatitis C/physiopathology , Humans , Liver Neoplasms/pathology , Tight Junctions/physiology , Transforming Growth Factor beta/physiology , Vascular Endothelial Growth Factor A/physiologyABSTRACT
We investigated the phenotype of hepatocellular carcinoma (HCC) in livers removed during transplantation after local ablation therapy by transarterial chemoembolization (TACE). This study involved 80 HCC nodules (40 treated with TACE and 40 not treated with local ablation before transplantation) observed in 64 explanted livers and included clinicopathological evaluations as well as single and double immunohistochemistry and reverse-transcription polymerase chain reaction (RT-PCR) for cytokeratin 19 (CK19), epithelial cell adhesion molecule (EpCAM), neural cell adhesion molecule (NCAM), and CD133. HCCs with complete necrosis post-TACE without viable tumors were excluded from the analysis. Cholangiolar, glandular, or spindle cell areas suggestive of a mixed hepatocholangiocellular phenotype were seen in 14 post-TACE HCCs and in none of the non-TACE HCCs (P < 0.001). According to single-epitope immunohistochemistry of post-TACE HCCs, CD133, CK19, EpCAM, and NCAM were expressed in 14 (35%), 8 (20%), 12 (30%), and 8 (20%), respectively. Only EpCAM was detected in 4 non-TACE HCC cases (10%). RT-PCR experiments using tissues obtained by laser microdissection showed that 4 of 5 investigated post-TACE HCCs expressed at least 1 of the markers, which were coexpressed in 3 of 5 tumors, whereas CD133 and EpCAM were individually expressed in 2 non-TACE HCCs. Double immunostaining showed that CD133(+) cells frequently coexpressed CK19, EpCAM, or NCAM. Interestingly, the recurrence rate for patients with CD133(+) post-TACE HCC was significantly higher than the rate for patients with CD133(-) post-TACE HCC (P = 0.025). In conclusion, HCC with the combined hepatocholangiocellular phenotype appears to be more frequent in post-TACE HCC versus untreated HCC. Further studies are needed to investigate the potential relationships between TACE and HCC subpopulations with a chemoembolization-resistant phenotype and their clinical significance.
Subject(s)
Carcinoma, Hepatocellular/therapy , Chemoembolization, Therapeutic/methods , Liver Neoplasms/therapy , Liver Transplantation/methods , AC133 Antigen , Adult , Aged , Antigens, CD/biosynthesis , Antigens, Neoplasm/metabolism , Carcinoma, Hepatocellular/etiology , Cell Adhesion Molecules/metabolism , Epithelial Cell Adhesion Molecule , Female , Glycoproteins/biosynthesis , Humans , Keratin-19/biosynthesis , Liver Neoplasms/etiology , Liver Transplantation/adverse effects , Male , Middle Aged , Neural Cell Adhesion Molecules/metabolism , Peptides , Phenotype , Reverse Transcriptase Polymerase Chain Reaction/methods , Treatment OutcomeABSTRACT
Cryopreservation of human hepatocytes is important for their use in hepatocyte transplantation. On thawing, cryopreserved hepatocytes often have reduced viability and metabolic function in comparison with fresh cells. The aim of this study was to modify the different steps in the standard cryopreservation procedure in an attempt to improve the overall outcome. Human hepatocytes with a viability of 69% +/- SD 16% were isolated from donor livers with a collagenase perfusion technique. Different cell densities, concentrations, rates, and methods of addition of dimethyl sulfoxide were tested for the freezing solution. Modified controlled-rate freezer programs were tested to obtain a linear decrease in the temperature. Once they were frozen, the storage time and thawing method for hepatocytes were investigated. The effects on thawed cell viability and attachment, lactate dehydrogenase release, cytochrome P450 1A1/2 activity, and albumin synthesis were determined. The results were used to produce an improved cryopreservation protocol suitable for good manufacturing practice conditions. With a cell density of 10(7) cells/mL in University of Wisconsin solution containing 300 mM glucose, 10% (vol/vol) dimethyl sulfoxide was added dropwise over 5 minutes, and was immediately frozen. Thawing was done rapidly at 37 degrees C, and dilution was performed with Eagle's minimum essential medium containing 300 mM glucose and 4% human serum albumin. Hepatocytes could be stored at -140 degrees C without significant further loss of function for up to 3 years. With this protocol, hepatocytes had a viability of 52% +/- 9%, an attachment efficiency of 48% +/- 8%, and lactate dehydrogenase leakage of 17% +/- 4%. This protocol is currently in use to cryopreserve hepatocytes for use in cell transplantation at our center.
Subject(s)
Cryopreservation/methods , Cryoprotective Agents/pharmacology , Dimethyl Sulfoxide/pharmacology , Hepatocytes/cytology , Hepatocytes/transplantation , Adenosine/pharmacology , Allopurinol/pharmacology , Cell Count , Cell Survival , Cryopreservation/standards , Glutathione/pharmacology , Humans , Insulin/pharmacology , Organ Preservation Solutions/pharmacology , Raffinose/pharmacology , TemperatureABSTRACT
UNLABELLED: Interface hepatitis, the histological lesion typical of autoimmune hepatitis (AIH), is composed of CD4 and CD8 T lymphocytes and of innate immunity cells, particularly monocytes. Studies in AIH have focused on autoreactive CD4 and CD8 T cells and impairment of CD4+CD25+ regulatory T cells (T-regs), whereas little is known about the role of monocytes and their relationship with T-regs. We have investigated 51 patients with autoimmune liver disease (AILD) and 27 healthy subjects, finding that monocytes were higher in number (P = 0.044), had a more vigorous spontaneous migration (P < 0.0005 in patients with inactive disease [ID], and P < 0.001 in those with active disease [AD]), displayed a higher tumor necrosis factor alpha (TNF-alpha) over interleukin (IL)-10 production (P = 0.07 in ID and P = 0.0005 in AD), and expressed higher levels of Toll-like receptor (TLR) 4 (P = 0.048 in ID and P = 0.03 in AD). Addition of conventional T-regs (cT-regs) in AILD enhanced monocyte migration (P = 0.05 in ID and P = 0.08 in AD), magnified TNF-alpha over IL-10 production (P = 0.0005 in ID and P = 0.006 in AD), and markedly increased TLR4 expression levels (P = 0.01 in ID and P = 0.004 in AD), whereas in normal subjects it either restrained or left unchanged monocyte function. Because a CD127-negative subpopulation within CD4+CD25+ T cells exerts the strongest regulatory activity, we performed additional experiments using purified CD4+CD25+CD127- T cells (true T-regs [tT-regs]). Addition of tT-regs to monocytes decreased monocyte migration (P = 0.03) and promoted IL-10 production (P = 0.009), leaving unchanged TLR4 expression in healthy subjects, whereas in patients with AILD it induced only a marginal increase in IL-10 production (P = 0.045 in ID and P = 0.13 in AD). CONCLUSION: Monocyte overactivation and inability of cT-regs and tT-regs to restrain it may contribute to the loss of immune tolerance and perpetuation of the autoimmune attack in AILD.
Subject(s)
Autoimmune Diseases/immunology , Leukocytes, Mononuclear/physiology , Liver Diseases/immunology , T-Lymphocytes, Regulatory/physiology , Adolescent , Child , Child, Preschool , Humans , Immune System PhenomenaABSTRACT
BACKGROUND: With the alarming growth in prevalence of paediatric nonalcoholic fatty liver disease (NAFLD), there is a need for noninvasive methods of stratifying disease severity. Our aim was to evaluate a combination of serum biomarkers as a measure of disease activity in paediatric NAFLD. PATIENTS AND METHODS: Forty-five children with biopsy-proven NAFLD were enrolled. Caspase-cleaved CK18 fragments (CK18 M30), hyaluronic acid, leptin, and adiponectin were measured in serum using enzyme-linked immunosorbent assays and high-sensitivity C-reactive protein using a colorimetric assay. RESULTS: Median age was 12.7 years (55% boys). Median body mass index z score was 1.7. CK18 M30 levels were significantly higher in patients with NAFLD versus controls, median 288 IU/L versus 172 IU/L (P < 0.001), and in those with steatohepatitis, median 347 IU/L versus simple steatosis (NAFLD activity score < 3), median 191 IU/L (P = 0.006). Significant fibrosis (≥F2) could be differentiated from no/minimal fibrosis (Subject(s)
Keratin-18/blood
, Leptin/blood
, Adiponectin/blood
, Biomarkers/blood
, C-Reactive Protein
, Child
, Colorimetry
, Enzyme-Linked Immunosorbent Assay
, Fatty Liver/blood
, Fatty Liver/diagnosis
, Fatty Liver/pathology
, Female
, Fibrosis
, Humans
, Hyaluronic Acid/blood
, Male
, Non-alcoholic Fatty Liver Disease
, Predictive Value of Tests
, Severity of Illness Index
ABSTRACT
Introduction: Hepatocyte transplantation (HT) is a promising alternative to liver transplantation for the treatment of liver-based metabolic diseases and acute liver failure (ALF). However, shortage of good-quality liver tissues, early cell loss post-infusion, reduced cell engraftment and function restricts clinical application.Areas covered: A comprehensive literature search was performed to cover pre-clinical and clinical HT studies. The review discusses the latest developments to address HT limitations: cell sources from marginal/suboptimal donors to neonatal livers, differentiating pluripotent stem cells into hepatocyte-like cells, in vitro expansion, prevention of immune response to transplanted cells by encapsulation or using innate immunity-inhibiting agents, and enhancing engraftment through partial hepatectomy or irradiation.Expert opinion: To date, published data are highly encouraging specially the alginate-encapsulated hepatocyte treatment of children with ALF. Hepatocyte functions can be further improved through co-culturing with mesenchymal stromal cells. Moreover, ex-vivo genetic correction will enable the use of autologous cells in future personalized medicine.
Subject(s)
Cell Transplantation/standards , Hepatocytes/transplantation , Liver Diseases/therapy , Cell Transplantation/methods , Cells, Cultured/transplantation , Humans , Liver Diseases/metabolism , Liver Diseases/surgery , Liver TransplantationABSTRACT
Protocols for isolation of human hepatocytes have been developed. The isolated cells can be used not only in research but also for transplantation in patients with liver disease, especially acute liver failure and liver-based metabolic/synthetic conditions. The aim of hepatocyte transplantation is to correct the missing liver function(s) and allow either the recovery of the liver or buy the patient time until a suitable donor liver is available for transplantation.
Subject(s)
Hepatocytes/cytology , Cell Separation/methods , Hepatocytes/metabolism , Humans , Liver/cytology , Liver/metabolism , Urea/metabolismABSTRACT
Cell transplantation is a promising approach to improve the life of patients with liver disease. At present, however, techniques to track and visualise transplanted cells in patients are fairly limited and further development of non-invasive imaging technology is needed to advance the monitoring of liver cell grafts. Magnetic resonance imaging (MRI) is a non-invasive imaging technology that already allows the visualisation of particular cell fractions in the liver by using MR contrast agents. The use of contrast agents to pre-label liver cells prior to transplantation will potentially provide a method to identify, track and study the integration of engrafted cells non-invasively by MRI. Before this technique can find its clinical application, in vitro and pre-clinical in vivo studies need to be conducted to determine the safety and specificity of this approach.
Subject(s)
Contrast Media/pharmacokinetics , Hepatocytes/metabolism , Liver/metabolism , Magnetic Resonance Imaging/methods , Staining and Labeling/methods , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Evaluation Studies as Topic , Hepatocytes/cytology , Hepatocytes/physiology , Humans , Liver/cytology , Liver/physiologyABSTRACT
OBJECTIVE: The aim of the study was to investigate the in vitro effects of sera from children with acute liver failure (ALF) who had received N-acetylcysteine (NAC) on the metabolic and synthetic activity of cryopreserved human hepatocytes. MATERIALS AND METHODS: Cryopreserved human hepatocytes were plated on collagen-coated culture plates and incubated in cell culture medium containing pooled sera at 20% (v/v) obtained from children with ALF (ALF) who received treatment with NAC (ALF + NAC), no treatment with NAC and from normal controls (normal sera [NS]). The effects of the sera on cell metabolic functions were assessed using methylthiazolyldiphenyltetrazolium bromide, [14C]-leucine incorporation, and cytochrome P-450 (CYP1A1/2) activity assays. RESULTS: The overall hepatocyte metabolic activity was lower with ALF sera than with NS and ALF + NAC sera. [14C]-leucine incorporation was higher with both ALF sera (ALF and ALF + NAC) than with NS sera. There was a slightly higher activity of cytochrome P450 (CYP1A1/2 activity) in cultures treated with ALF and ALF + NAC than with normal sera treated hepatocyte cultures. CONCLUSIONS: Sera from children with ALF who received NAC did not impair the overall cell metabolic activity of cryopreserved human hepatocyte in vitro, which is encouraging for the use of hepatocytes transplantation in these patients.
Subject(s)
Acetylcysteine/pharmacology , Cryopreservation , Free Radical Scavengers/pharmacology , Hepatocytes/drug effects , Liver Failure, Acute/metabolism , Serum/metabolism , Adolescent , Cells, Cultured , Child , Child, Preschool , Cytochrome P-450 CYP1A1/metabolism , Cytochrome P-450 CYP1A2/metabolism , Cytochrome P-450 Enzyme System/metabolism , Female , Hepatocytes/metabolism , Hepatocytes/transplantation , Humans , Infant , Liver Failure, Acute/therapy , Male , Proteins/metabolismABSTRACT
Recent advancements in the production of hepatocytes from human pluripotent stem cells (hPSC-Heps) afford tremendous possibilities for treatment of patients with liver disease. Validated current good manufacturing practice (cGMP) lines are an essential prerequisite for such applications but have only recently been established. Whether such cGMP lines are capable of hepatic differentiation is not known. To address this knowledge gap, we examined the proficiency of three recently derived cGMP lines (two hiPSC and one hESC) to differentiate into hepatocytes and their suitability for therapy. hPSC-Heps generated using a chemically defined four-step hepatic differentiation protocol uniformly demonstrated highly reproducible phenotypes and functionality. Seeding into a 3D poly(ethylene glycol)-diacrylate fabricated inverted colloid crystal scaffold converted these immature progenitors into more advanced hepatic tissue structures. Hepatic constructs could also be successfully encapsulated into the immune-privileged material alginate and remained viable as well as functional upon transplantation into immune competent mice. This is the first report we are aware of demonstrating cGMP-compliant hPSCs can generate cells with advanced hepatic function potentially suitable for future therapeutic applications. Stem Cells Translational Medicine 2019;8:124&14.
Subject(s)
Cell- and Tissue-Based Therapy/standards , Hepatocytes/cytology , Pluripotent Stem Cells/cytology , Animals , Cell Culture Techniques/standards , Cell Differentiation/physiology , Cell Line , Humans , Liver/cytology , MiceABSTRACT
There are limited data regarding donor hepatocyte engraftment into recipient liver after human hepatocyte transplantation (HHTx). We reviewed the explant livers of seven children with metabolic disorders [ornithine-transcarbamylase deficiency (one), coagulation factor VII deficiency (three), Crigler-Najjar syndrome (one), progressive familial intrahepatic cholestasis type 2 (PFIC-2) deficiency (two)] who received allograft hepatocytes by intraportal infusion with improvement in phenotype, although all later underwent liver transplantation (LT). Immunohistochemistry for bile salt export protein (BSEP) in the PFIC-2 patients and genetic typing following laser capture microdissection (LCM) of liver cells in the others were used to identify donor hepatocytes in recipient explant livers. Explant livers usually showed a preserved lobular architecture. In one patient, hepatocytes were identified inside portal vein thrombi. No donor hepatocytes in liver cell plates were identified immunohistochemically or by genetic typing. HHTx was generally followed by partial recovery of metabolic function; the procedure was well tolerated; any increase in portal vein pressure was transient. Hepatocytes were identified in portal vein thrombi, even months after portal vein infusion. Further studies are needed to monitor donor hepatocytes in vivo, to quantify better the efficacy of the procedure and to find ways of improving engraftment and function.