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The liver has been studied extensively due to the broad number of diseases affecting its vital functions. However, therapeutic advances have been hampered by the lack of knowledge concerning human hepatic development. Here, we addressed this limitation by describing the developmental trajectories of different cell types that make up the human liver at single-cell resolution. These transcriptomic analyses revealed that sequential cell-to-cell interactions direct functional maturation of hepatocytes, with non-parenchymal cells playing essential roles during organogenesis. We utilized this information to derive bipotential hepatoblast organoids and then exploited this model system to validate the importance of signalling pathways in hepatocyte and cholangiocyte specification. Further insights into hepatic maturation also enabled the identification of stage-specific transcription factors to improve the functionality of hepatocyte-like cells generated from human pluripotent stem cells. Thus, our study establishes a platform to investigate the basic mechanisms directing human liver development and to produce cell types for clinical applications.

IMPORTANCE: Colorectal cancer is a leading cause of cancer-related death, and nearly 70% of patients with this cancer have unresectable colorectal cancer liver metastases (CRLMs). Compared with chemotherapy, liver transplant has been reported to improve survival in patients with CRLMs, but in North America, liver allograft shortages make the use of deceased-donor allografts for this indication problematic. OBJECTIVE: To examine survival outcomes of living-donor liver transplant (LDLT) for unresectable, liver-confined CRLMs. DESIGN, SETTING, AND PARTICIPANTS: This prospective cohort study included patients at 3 North American liver transplant centers with established LDLT programs, 2 in the US and 1 in Canada. Patients with liver-confined, unresectable CRLMs who had demonstrated sustained disease control on oncologic therapy met the inclusion criteria for LDLT. Patients included in this study underwent an LDLT between July 2017 and October 2020 and were followed up until May 1, 2021. EXPOSURES: Living-donor liver transplant. MAIN OUTCOMES AND MEASURES: Perioperative morbidity and mortality of treated patients and donors, assessed by univariate statistics, and 1.5-year Kaplan-Meier estimates of recurrence-free and overall survival for transplant recipients. RESULTS: Of 91 evaluated patients, 10 (11%) underwent LDLT (6 [60%] male; median age, 45 years [range, 35-58 years]). Among the 10 living donors, 7 (70%) were male, and the median age was 40.5 years (range, 27-50 years). Kaplan-Meier estimates for recurrence-free and overall survival at 1.5 years after LDLT were 62% and 100%, respectively. Perioperative morbidity for both donors and recipients was consistent with established standards (Clavien-Dindo complications among recipients: 3 [10%] had none, 3 [30%] had grade II, and 4 [40%] had grade III; donors: 5 [50%] had none, 4 [40%] had grade I, and 1 had grade III). CONCLUSIONS AND RELEVANCE: This study’s findings of recurrence-free and overall survival rates suggest that select patients with unresectable, liver-confined CRLMs may benefit from total hepatectomy and LDLT.

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There is a tremendous focus on the application of nanomaterials for the treatment of cancer. Nonprimate models are conventionally used to assess the biomedical utility of nanomaterials. However, these animals often lack an intact immunological background, and the tumors in these animals do not develop spontaneously. We introduce a preclinical woodchuck hepatitis virus-induced liver cancer model as a platform for nanoparticle (NP)-based in vivoexperiments. Liver cancer development in these out-bred animals occurs as a result of persistent viral infection, mimicking human hepatitis B virus-induced HCC development. We highlight how this model addresses key gaps associated with other commonly used tumor models. We employed this model to (1) track organ biodistribution of gold NPs after intravenous administration, (2) examine their subcellular localization in the liver, (3) determine clearance kinetics, and (4) characterize the identity of hepatic macrophages that take up NPs using RNA-sequencing (RNA-seq). We found that the liver and spleen were the primary sites of NP accumulation. Subcellular analyses revealed accumulation of NPs in the lysosomes of CD14+cells. Through RNA-seq, we uncovered that immunosuppressive macrophages within the woodchuck liver are the major cell type that take up injected NPs. The woodchuck-HCC model has the potential to be an invaluable tool to examine NP-based immune modifiers that promote host anti-tumor immunity.

A significant challenge to delivering therapeutic doses of nanoparticles to targeted disease sites is the fact that most nanoparticles become trapped in the liver. Liver-resident macrophages, or Kupffer cells, are key cells in the hepatic sequestration of nanoparticles. However, the precise role that the macrophage phenotype plays in nanoparticle uptake is unknown. Here, we show that the human macrophage phenotype modulates hard nanoparticle uptake. Using gold nanoparticles, we examined uptake by human monocyte-derived macrophages that had been driven to a “regulatory” M2 phenotype or an “inflammatory” M1 phenotype and found that M2-type macrophages preferentially take up nanoparticles, with a clear hierarchy among the subtypes (M2c > M2 > M2a > M2b > M1). We also found that stimuli such as LPS/IFN-γ rather than with more “regulatory” stimuli such as TGF-β/IL-10 reduce per cell macrophage nanoparticle uptake by an average of 40%. Primary human Kupffer cells were found to display heterogeneous expression of M1 and M2 markers, and Kupffer cells expressing higher levels of M2 markers (CD163) take up significantly more nanoparticles than Kupffer cells expressing lower levels of surface CD163. Our results demonstrate that hepatic inflammatory microenvironments should be considered when studying liver sequestration of nanoparticles, and that modifying the hepatic microenvironment might offer a tool for enhancing or decreasing this sequestration. Our findings also suggest that models examining the nanoparticle/macrophage interaction should include studies with primary tissue macrophages.

The liver and spleen are major biological barriers to translating nanomedicines because they sequester the majority of administered nanomaterials and prevent delivery to diseased tissue. Here we examined the blood clearance mechanism of administered hard nanomaterials in relation to blood flow dynamics, organ microarchitecture and cellular phenotype. We found that nanomaterial velocity reduces 1,000-fold as they enter and traverse the liver, leading to 7.5 times more nanomaterial interaction with hepatic cells relative to peripheral cells. In the liver, Kupffer cells (84.8 ± 6.4%), hepatic B cells (81.5 ± 9.3%) and liver sinusoidal endothelial cells (64.6 ± 13.7%) interacted with administered PEGylated quantum dots, but splenic macrophages took up less material (25.4 ± 10.1%) due to differences in phenotype. The uptake patterns were similar for two other nanomaterial types and five different surface chemistries. Potential new strategies to overcome off-target nanomaterial accumulation may involve manipulating intra-organ flow dynamics and modulating the cellular phenotype to alter hepatic cell interactions.

Pancreas transplant recipient obesity has been associated with increased risk of perioperative complications, graft failure, and death. The imperative to maximize organ utility must be balanced against the need to maintain equity of access, including for the increasing number of obese diabetic patients.

Controversy persists over the safety and efficacy of pancreas transplantation in patients with insulin-dependent diabetes mellitus who have received a prior kidney transplant.

Thymoglobulin (ATG) and basiliximab induction therapies are used by the majority of centers for pancreas transplantation today. Although both strategies have different mechanisms, there is a paucity of studies comparing them. We compared the efficacy and side effects of both methods in simultaneous pancreas-kidney (SPK) transplantation.

Tumor number, size, and macrovascular invasion (MacroVI) are the most widely used predictors of survival after liver transplantation (LT) for hepatocellular carcinoma (HCC). We analyzed all patients undergoing LT for HCC at our center to establish the accuracy of preoperative clinical staging and to determine which patients have a higher probability of being understaged.

The shortage of deceased organ donors has created a need for right lobe living donor liver transplantation (RLDLT) in adults. Concerns regarding donor safety, however, necessitate continuous assessment of donor acceptance criteria and documentation of donor morbidity. We report the outcomes of our first 101 donors who underwent right lobectomy between April 2000 and November 2004. The cohort comprised 58 men and 43 women with a median age of 37.8 years (range: 18.6–55 years); median follow-up is 24 months. The middle hepatic vein (MHV) was taken with the graft in 55 donors. All complications were recorded prospectively and stratified by grade according to Clavien's classification. Overall morbidity rate was 37%; all complications were either grade 1 or 2, and the majority occurred during the first 30 days after surgery. Removal of the MHV did not affect morbidity rate. There were significantly fewer complications in the later half of our experience. All donors are well and have returned to full activities. With careful donor selection and specialized patient care, low morbidity rates can be achieved after right hepatectomy for living donor liver transplantation.

The shortage of deceased organ donors has created a need for right lobe living donor liver transplantation (RLDLT) in adults. Concerns regarding donor safety, however, necessitate continuous assessment of donor acceptance criteria and documentation of donor morbidity. We report the outcomes of our first 101 donors who underwent right lobectomy between April 2000 and November 2004. The cohort comprised 58 men and 43 women with a median age of 37.8 years (range: 18.6–55 years); median follow‐up is 24 months. The middle hepatic vein (MHV) was taken with the graft in 55 donors. All complications were recorded prospectively and stratified by grade according to Clavien's classification. Overall morbidity rate was 37%; all complications were either grade 1 or 2, and the majority occurred during the first 30 days after surgery. Removal of the MHV did not affect morbidity rate. There were significantly fewer complications in the later half of our experience. All donors are well and have returned to full activities. With careful donor selection and specialized patient care, low morbidity rates can be achieved after right hepatectomy for living donor liver transplantation.

Whether peritoneal dialysis is a risk factor for the development of intra-abdominal infection (IAI) after simultaneous pancreas kidney (SPK) transplantation is controversial.

The value of magnetic resonance cholangiography (MRC) in assessing potential adult-to-adult living liver transplant (ALDLT) donors remains poorly defined. The purpose of this study is to determine the accuracy of MRC in assessing biliary anatomy with intraoperative confirmation.

During the past decade, polyoma virus (PV) infection has emerged as an important cause of graft dysfunction and failure in kidney transplant recipients. Establishing the correct diagnosis can be difficult, however, because the histologic appearance of PV infection can resemble acute cellular rejection. We report a kidney-pancreas transplant recipient with PV infection, in whom both vascular and cellular rejection were dominant histologic features in a renal biopsy specimen. The patient was successfully managed by tapering immunosuppressive therapy, and continues to have good graft function 3 years after diagnosis of PV infection. This case highlights the spectrum of inflammatory changes associated with PV infection, and the need for caution when such changes are attributed to rejection.

During the past decade, polyoma virus (PV) infection has emerged as an important cause of graft dysfunction and failure in kidney transplant recipients. Establishing the correct diagnosis can be difficult, however, because the histologic appearance of PV infection can resemble acute cellular rejection. We report a kidney-pancreas transplant recipient with PV infection, in whom both vascular and cellular rejection were dominant histologic features in a renal biopsy specimen. The patient was successfully managed by tapering immunosuppressive therapy, and continues to have good graft function 3 years after diagnosis of PV infection. This case highlights the spectrum of inflammatory changes associated with PV infection, and the need for caution when such changes are attributed to rejection.

The expression of tissue factor (TF) by monocytes that have transmigrated across the endothelium to sites of extravascular inflammation acts both to focus and amplify the inflammatory response. Because clustering of the integrins responsible for endothelial adhesion and transmigration induces tyrosine phosphorylation and activation of the mitogen-activated protein (MAP) kinases, we postulated that transmigration might lead to monocyte activation and TF production. Monocytes were migrated across TNF-primed ECV304 cells grown on fibronectin-coated Transwell chambers in response to FMLP (10−8M). After transmigration, monocytes showed a time-dependent increase in surface TF expression and biological procoagulant activity. TF expression was dependent on monocyte adhesion to ECV304 cells. Specifically, TF was not induced by FMLP treatment of suspended monocytes, by migration across fibronectin alone, or by soluble factors induced during migration, whereas monocyte-ECV304 adhesion was sufficient to stimulate TF. Antibodies against CD29 (1integrin), but not against CD18 (2integrin) or CD31 (PECAM-1), inhibited TF expression. Monocyte adhesion to ECV304 cells induced tyrosine phosphorylation of cellular proteins and specifically of the ERK and p38 MAP kinases. Tyrosine kinase inhibition with genistein (10 g/mL) blocked transmigration, whereas selective ERK inhibition with PD98059 (50 M) or p38 inhibition with SB203580 (20 M) did not. However, both ERK and p38 inhibition dose dependently abolished TF expression. These studies suggest that an extravascular focus of infection or inflammation can promote both intravascular thrombosis and extravascular fibrin deposition during the process of adhesion and transmigration across the endothelial barrier. The selective inhibition of the mitogen-activated protein kinases may offer a novel therapeutic means of modulating this inflammatory sequence.

The unique pathophysiology of patients with end-stage liver disease has important implications for their critical care treatment, particularly in the postoperative state. To gauge hemodynamic parameters and responses, each patient must be carefully evaluated for their place in the clinical spectrum of cirrhosis and portal hypertension. Although the data are limited, the biology of the consequences of liver disease is emphasized by novel treatments of hepatorenal syndrome, portopulmonary hypertension, and hepatopulmonary syndrome. These issues become more relevant with increased adult-to-adult living donor liver transplantation, in which technical considerations may further complicate the general treatment of the postoperative transplant patient.

Plasmodium falciparum is the most lethal form of malaria and is increasing both in incidence and in its resistance to antimalarial agents. An improved understanding of the mechanisms of malarial clearance may facilitate the development of new therapeutic interventions. We postulated that the scavenger receptor CD36, an important factor in cytoadherence of P falciparum–parasitized erythrocytes (PEs), might also play a role in monocyte- and macrophage-mediated malarial clearance. Exposure of nonopsonized PEs to Fc receptor–blocked monocytes resulted in significant PE phagocytosis, accompanied by intense clustering of CD36 around the PEs. Phagocytosis was blocked 60% to 70% by monocyte pretreatment with monoclonal anti-CD36 antibodies but not by antibodies to avß3, thrombospondin, intercellular adhesion molecule-1, or platelet/endothelial cell adhesion molecule-1. Antibody-induced CD36 cross-linking did result in the early increase of surface CD11b expression, but there was no increase in, or priming for, tumor necrosis factor (TNF)-a secretion following either CD36 cross-linking or PE phagocytosis. CD36 clustering does support intracellular signaling: Antibody-induced cross-linking initiated intracellular tyrosine phosphorylation as well as extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) phosphorylation. Both broad-spectrum tyrosine kinase inhibition (genistein) and selective ERK and p38 MAPK inhibition (PD98059 and SB203580, respectively) reduced PE uptake to almost the same extent as CD36 blockade. Thus, CD36-dependent binding and signaling appears to be crucial for the nonopsonic clearance of PEs and does not appear to contribute to the increase in TNF-a that is prognostic of poor outcome in clinical malaria.

Plasmodium falciparumis the most lethal form of malaria and is increasing both in incidence and in its resistance to antimalarial agents. An improved understanding of the mechanisms of malarial clearance may facilitate the development of new therapeutic interventions. We postulated that the scavenger receptor CD36, an important factor in cytoadherence of P falciparum–parasitized erythrocytes (PEs), might also play a role in monocyte- and macrophage-mediated malarial clearance. Exposure of nonopsonized PEs to Fc receptor–blocked monocytes resulted in significant PE phagocytosis, accompanied by intense clustering of CD36 around the PEs. Phagocytosis was blocked 60% to 70% by monocyte pretreatment with monoclonal anti-CD36 antibodies but not by antibodies to αvβ3, thrombospondin, intercellular adhesion molecule-1, or platelet/endothelial cell adhesion molecule-1. Antibody-induced CD36 cross-linking did result in the early increase of surface CD11b expression, but there was no increase in, or priming for, tumor necrosis factor (TNF)-α secretion following either CD36 cross-linking or PE phagocytosis. CD36 clustering does support intracellular signaling: Antibody-induced cross-linking initiated intracellular tyrosine phosphorylation as well as extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) phosphorylation. Both broad-spectrum tyrosine kinase inhibition (genistein) and selective ERK and p38 MAPK inhibition (PD98059 and SB203580, respectively) reduced PE uptake to almost the same extent as CD36 blockade. Thus, CD36-dependent binding and signaling appears to be crucial for the nonopsonic clearance of PEs and does not appear to contribute to the increase in TNF-α that is prognostic of poor outcome in clinical malaria.

BACKGROUND: Microvascular thrombosis with intravascular fibrin deposition is a characteristic pathologic alteration during endotoxic shock. This effect is predominantly mediated by expression of the cellular procoagulant tissue factor by endothelial cells and cells of monocyte or macrophage lineage, resulting in acceleration of the coagulation cascade and fibrin deposition. OBJECTIVE: To determine whether modulation of this response by treatment with an antitissue factor antibody might have beneficial effects. DESIGN: A polyclonal antibody to murine tissue factor was prepared by injecting rabbits with a synthesized peptide sequence of murine tissue factor. To determine the activity of the antibody, elicited murine peritoneal macrophages were treated for 4 hours with 10-μg/mL lipopolysaccharide (LPS), and procoagulant activity was determined via a clotting assay (milliunits of activity per 106 macrophages). RESULTS: The tissue factor antibody abrogated LPS-induced macrophage procoagulant activity, confirming activity of the antibody (macrophages, 236±28 mU/106 macrophages; macrophages/LPS, 3801±190* mU/106 macrophages; macrophages/LPS/α-tissue factor, 753±92* mU/106 macrophages; n=3; the asterisk indicates P<.05 by an analysis of variance). Additionally, antibody-protein affinity was confirmed by Western blot analysis. Having determined the activity of the antibody in vitro, we tested its efficacy in vivo in a lethal endotoxemia model. Mice were immunized with 200 μL of antiserum intraperitoneally 2 hours before injection with 250 μg of LPS intraperitoneally and 24 hours later. Control animals received 200 μL of saline solution. All animals initially exhibited lethargy and piloerection, characteristic of the predicted response to LPS. However, immunized animals had a significantly (P<.05) reduced mortality compared with control animals. Fibrinogen levels were significantly (P<.05) higher in the immunized mice, suggesting decreased consumption of coagulation factors, a finding consistent with an antitissue factor effect. Further, plasma tumor necrosis factor levels 90 minutes after LPS injection were similar in both groups, suggesting normal induction of the cytokine cascade. CONCLUSIONS: Modulation of microvascular fibrin deposition by abrogating tissue factor—mediated coagulation significantly (P<.05) improved survival in this model without attenuating the initiation of the cytokine cascade. These findings suggest a pathogenic role for coagulation in the induction of acute organ injury during sepsis.Arch Surg. 1996;131:1273-1279

Abstract. Activation of the coagulation cascade during the inflammatory response is an essential component of the host response to infection. Coagulation represents a double-edged sword. Necessary for hemostasis and the acute containment of an infective focus, it also amplifies the inflammatory response, decreases bacterial clearance, and in the critically ill patient contributes to end-organ damage and death. Evidence for these concepts is found in the fact that critically ill and septic patients consistently demonstrate marked abnormalities in their biochemical indices of coagulation. Moreover, there is excellent experimental evidence to suggest that replacement or inhibition of individual coagulation factors, either consumed or up-regulated during the inflammatory response, can attenuate both local and systemic injury and protect animals from otherwise fatal outcomes. Based on these facts, novel therapeutic approaches to the treatment of the systemic response to infection have been developed and tested in intensive care settings. Although promising results have been obtained, much work remains to be done in defining exactly which coagulation factors (or combination of factors) should be inhibited or replaced during treatment of the septic, critically ill patient. The experimental and clinical evidence linking the coagulation cascade to local and systemic inflammation is reviewed, and the status of the clinical trials performed to date is overviewed.:

The development of the adult respiratory distress syndrome ARDS in the critically ill patient is associated with a significant morbidity and mortality. The pulmonary dysfunction in ARDS is largely secondary to neutrophilmediated oxidant injury. The purpose of these studies is to examine the effect of the antioxidant Nacetyl cysteine NAC on a rodent model of lung injury. We postulated that NAC might attenuate lung injury following intratracheal challenge with endotoxin lipopolysaccharide LPS. Male SpragueDawley rats were administered NAC systemically either before or after intratracheal administration of LPS. Lung injury was assessed by measuring the transpulmonary leakage of 125llabeled albumin, pulmonary myeloperoxidase content, bronchoalveolar lavage fluid cell counts, pulmonary lipid peroxidation and histology. NAC administration significantly attenuated the LPSinduced increases in lung permeability LPS .24 ± .08 vs. LPS NAC .12 ± .03, p< .05 and reduced the LPSdependent increase in lipid peroxidation. However, total and differential bronchoalveolar lavage cell counts and myeloperoxidase content were not affected by NAC pretreatment. Although neutrophil influx was unaffected, neutrophil activation as assessed by surface CD11b expression and chemiluminescence was significantly downregulated by NAC. Importantly, NAC administration up to 2 h after endotoxin challenge was still able to significantly ameliorate LPSinduced lung injury. Our data suggests that the attenuation of acute lung injury by NAC in our rodent model is related to free radical scavenging and inhibition of the neutrophil oxidative burst, rather than by an effect on inflammatory cell migration. These results suggest novel approaches for therapeutic interventions in acute lung injury.

The development of the adult respiratory distress syndrome ARDS in the critically ill patient is associated with a significant morbidity and mortality. The pulmonary dysfunction in ARDS is largely secondary to neutrophilmediated oxidant injury. The purpose of these studies is to examine the effect of the antioxidant Nacetyl cysteine NAC on a rodent model of lung injury. We postulated that NAC might attenuate lung injury following intratracheal challenge with endotoxin lipopolysaccharide LPS. Male SpragueDawley rats were administered NAC systemically either before or after intratracheal administration of LPS. Lung injury was assessed by measuring the transpulmonary leakage of 125llabeled albumin, pulmonary myeloperoxidase content, bronchoalveolar lavage fluid cell counts, pulmonary lipid peroxidation and histology. NAC administration significantly attenuated the LPSinduced increases in lung permeability LPS .24 ± .08 vs. LPS NAC .12 ± .03, p< .05 and reduced the LPSdependent increase in lipid peroxidation. However, total and differential bronchoalveolar lavage cell counts and myeloperoxidase content were not affected by NAC pretreatment. Although neutrophil influx was unaffected, neutrophil activation as assessed by surface CD11b expression and chemiluminescence was significantly downregulated by NAC. Importantly, NAC administration up to 2 h after endotoxin challenge was still able to significantly ameliorate LPSinduced lung injury. Our data suggests that the attenuation of acute lung injury by NAC in our rodent model is related to free radical scavenging and inhibition of the neutrophil oxidative burst, rather than by an effect on inflammatory cell migration. These results suggest novel approaches for therapeutic interventions in acute lung injury.

Activation of the coagulation cascade during the inflammatory response is an essential component of the host response to infection. Coagulation represents a double-edged sword. Necessary for hemostasis and the acute containment of an infective focus, it also amplifies the inflammatory response, decreases bacterial clearance, and in the critically ill patient contributes to end-organ damage and death. Evidence for these concepts is found in the fact that critically ill and septic patients consistently demonstrate marked abnormalities in their biochemical indices of coagulation. Moreover, there is excellent experimental evidence to suggest that replacement or inhibition of individual coagulation factors, either consumed or up-regulated during the inflammatory response, can attenuate both local and systemic injury and protect animals from otherwise fatal outcomes. Based on these facts, novel therapeutic approaches to the treatment of the systemic response to infection have been developed and tested in intensive care settings. Although promising results have been obtained, much work remains to be done in defining exactly which coagulation factors (or combination of factors) should be inhibited or replaced during treatment of the septic, critically ill patient. The experimental and clinical evidence linking the coagulation cascade to local and systemic inflammation is reviewed, and the status of the clinical trials performed to date is overviewed.

BACKGROUND: We have previously shown that the thioloxidizing agent diethyl maleate prevents lipopolysaccharide (LPS)-induced up-regulation of endothelial cell intercellular adhesion molecule–1 (ICAM-1) in vitro. OBJECTIVE: To determine the effect of glutathione depletion on the development of local skin inflammation in vivo, a model known to be dependent on ICAM-1. DESIGN: Swiss Webster mice were injected with intradermal LPS (30 μg) or isotonic saline solution. INTERVENTION: Mice were pretreated for 1 hour with intraperitoneal diethyl maleate (6 mmol/kg) or corn oil vehicle. MAIN OUTCOME MEASURES: Injection sites were harvested after 12 and 24 hours and evaluated for changes in vascular permeability and histological characteristics. To determine the mechanism underlying our findings, we evaluated skin ICAM-1 immunohistochemistry, levels of ICAM-1 protein and messenger RNA (mRNA), and neutrophil CD11b expression at the 24-hour point. RESULTS: Diethyl maleate significantly decreased the skin permeability index in a dose-dependent fashion at 24 hours but not at 12 hours. Skin histological examination under light microscopy showed a marked LPSinduced neutrophil infiltration at 24 hours, which was inhibited with diethyl maleate pretreatment. Immunohistochemical examination showed that diethyl maleate reduced ICAM-1 expression. In keeping with the hypothesized mechanism, diethyl maleate attenuated the LPS-induced up-regulation of ICAM-1 mRNA by 44%. Diethyl maleate also slightly but insignificantly reduced CD11b expression in vivo. CONCLUSIONS: Diethyl maleate markedly attenuates LPS-induced dermal inflammation, primarily through a reduction in ICAM-1 protein and mRNA expression. These data suggest that manipulation of the intracellular redox state may have a beneficial role in neutrophil-mediated inflammation.Arch Surg. 1997;132:1165-1170

The clinical syndrome of acute liver failure produced by fulminant viral hepatitis can be reproduced in mice by infection with murine hepatitis virus strain 3 (MHV-3). Although it is clear that MHV-3-induced hepatitis depends upon macrophage activation and the expression of a specific prothrombinase,fgl-2, the signaling pathways involved in virally stimulated cell activation are unclear. Since we had previously found that MHV-3 induces the tyrosine phosphorylation of cellular proteins, we investigated the roles of the mitogen-activated protein kinase (MAPK) proteins. In a series of Western blots, immunoprecipitation and in vitrokinase assay studies, we found that both the extracellular signal-related kinase (ERK) and p38 MAPK proteins are tyrosine-phosphorylated and activated following exposure of murine peritoneal exudative macrophages (PEM) to MHV-3. Although p38 phosphorylation and activity are induced soon after MHV-3 exposure, peaking by 1–5 min, ERK phosphorylation and activity increase more gradually, peaking at 20–30 min and gradually fading thereafter. Interestingly, whereas selective p38 inhibition with SB203580 (1–20 μm) abolished the virally stimulated induction of fgl-2mRNA, protein, and functional activity, selective ERK inhibition with PD98059 (1–50 μm) limitedfgl-2functional activity but had little to no effect onfgl-2mRNA or protein levels. Moreover, whereas inhibition of ERK had no effect on p38 activity, p38 inhibition consistently increased MHV-3-induced ERK activity. To ensure that these pathways were relevant in vivo, MHV-3 was injected intraperitoneally, and peritoneal exudative macrophages were collected. Again, MHV-3 exposure led to increased p38 and ERK tyrosine phosphorylation. These data argue that MHV-3 induces tightly interconnected ERK and p38 MAPK cascades in the macrophage bothin vitroand in vivo. Although the ERK and p38 MAPK proteins have discordant effects at the level of fgl-2expression, both converge at the level of its activity, suggesting that targeted MAPK inhibition may ultimately be useful in the modulation of viral hepatitis.

Liver sinusoidal endothelial cells (LSECs) form a highly specialized microvasculature that plays a critical role in liver function and disease. To better understand this role, we developed a strategy to generate LSECs from human pluripotent stem cells (hPSCs) by first optimizing the specification of arterial and venous angioblasts and derivative endothelial populations. Induction of a LSEC-like fate by hypoxia, cyclic AMP (cAMP) agonism, and transforming growth factor β (TGF-β) inhibition revealed that venous endothelial cells responded more rapidly and robustly than the arterial cells to upregulate LSEC markers and functions in vitro. Upon intrahepatic transplantation in neonates, venous angioblasts engrafted the liver and generated mature, fenestrated LSECs with scavenger functions and molecular profiles of primary human LSECs. When transplanted into the liver of adult mice, angioblasts efficiently gave rise to mature LSECs with robust factor VIII (FVIII) production. Humanization of the murine liver with hPSC-derived LSECs provides a tractable system for studying the biology of this key liver cell type.