Metabolism of Acetaminophen by Enteric Epithelial Cells Mitigates Hepatocellular Toxicity In Vitro.

The gut-liver axis is defined by dietary and environmental communication between the gut, microbiome and the liver with its redox and immune systems, the overactivation of which can lead to hepatic injury. We used media preconditioning to mimic some aspects of the enterohepatic circulation by treating the human Caco-2 intestinal epithelial cell line with 5, 10 and 20 mM paracetamol (N-acetyl-para-aminophenol; APAP) for 24 h, after which cell culture supernatants were transferred to differentiated human hepatic HepaRG cells for a further 24 h. Cell viability was assessed by mitochondrial function and ATP production, while membrane integrity was monitored by cellular-based impedance. Metabolism by Caco-2 cells was determined by liquid chromatography with tandem mass spectrometry. Caco-2 cell viability was not affected by APAP, while cell membrane integrity and tight junctions were maintained and became tighter with increasing APAP concentrations, suggesting a reduction in the permeability of the intestinal epithelium. During 24 h incubation, Caco-2 cells metabolised 64-68% of APAP, leaving 32-36% of intact starting compound to be transferred to HepaRG cells. When cultured with Caco-2-preconditioned medium, HepaRG cells also showed no loss of cell viability or membrane integrity, completely in contrast to direct treatment with APAP, which resulted in a rapid loss of cell viability and membrane integrity and, ultimately, cell death. Thus, the pre-metabolism of APAP could mitigate previously observed hepatotoxicity to hepatic tight junctions caused by direct exposure to APAP. These observations could have important implications for the direct exposure of hepatic parenchyma to APAP, administered via the intravenous route.

Oxidative Stress and Antioxidant Therapy in Pulmonary Hypertension.

Pulmonary hypertension (PH) is a progressive disease characterized by elevated artery pressures and pulmonary vascular resistance. Underlying mechanisms comprise endothelial dysfunction, pulmonary artery remodeling and vasoconstriction. Several studies have shown evidence of the critical role of oxidative stress in PH pathophysiology. Alteration of redox homeostasis produces excessive generation of reactive oxygen species, inducing oxidative stress and the subsequent alteration of biological molecules. Exacerbations in oxidative stress production can lead to alterations in nitric oxide signaling pathways, contributing to the proliferation of pulmonary arterial endothelial cells and smooth muscle cells, inducing PH development. Recently, antioxidant therapy has been suggested as a novel therapeutic strategy for PH pathology. However, the favorable outcomes observed in preclinical studies have not been consistently reproduced in clinical practice. Therefore, targeting oxidative stress as a therapeutic intervention for PH is an area that is still being explored. This review summarizes the contribution of oxidative stress to the pathogenesis of the different types of PH and suggests antioxidant therapy as a promising strategy for PH treatment.

Pancreas-derived mesenchymal stromal cells share immune response-modulating and angiogenic potential with bone marrow mesenchymal stromal cells and can be grown to therapeutic scale under Good Manufacturing Practice conditions.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) isolated from various tissues are under investigation as cellular therapeutics in a wide range of diseases. It is appreciated that the basic biological functions of MSCs vary depending on tissue source. However, in-depth comparative analyses between MSCs isolated from different tissue sources under Good Manufacturing Practice (GMP) conditions are lacking. Human clinical-grade low-purity islet (LPI) fractions are generated as a byproduct of islet isolation for transplantation. MSC isolates were derived from LPI fractions with the aim of performing a systematic, standardized comparative analysis of these cells with clinically relevant bone marrow-derived MSCs (BM MSCs). METHODS: MSC isolates were derived from LPI fractions and expanded in platelet lysate-supplemented medium or in commercially available xenogeneic-free medium. Doubling rate, phenotype, differentiation potential, gene expression, protein production and immunomodulatory capacity of LPIs were compared with those of BM MSCs. RESULTS: MSCs can be readily derived in vitro from non-transplanted fractions resulting from islet cell processing (i.e., LPI MSCs). LPI MSCs grow stably in serum-free or platelet lysate-supplemented media and demonstrate in vitro self-renewal, as measured by colony-forming unit assay. LPI MSCs express patterns of chemokines and pro-regenerative factors similar to those of BM MSCs and, importantly, are equally able to attract immune cells in vitro and in vivo and suppress T-cell proliferation in vitro. Additionally, LPI MSCs can be expanded to therapeutically relevant doses at low passage under GMP conditions. CONCLUSIONS: LPI MSCs represent an alternative source of GMP MSCs with functions comparable to BM MSCs.

SARS-CoV-2 Infection and the Liver.

A novel strain of coronoviridae (SARS-CoV-2) was reported in Wuhan China in December 2019. Initially, infection presented with a broad spectrum of symptoms which typically included muscle aches, fever, dry cough, and shortness of breath. SARS-CoV-2 enters cells via ACE2 receptors which are abundant throughout the respiratory tract. However, there is evidence that these receptors are abundant throughout the body, and just as abundant in cholangiocytes as alveolar cells, posing the question of possible direct liver injury. While liver enzymes and function tests do seem to be deranged in some patients, it is questionable if the injury is due to direct viral damage, drug-induced liver injury, hypoxia, or microthromboses. Likely, the injury is multifactoral, and management of infected patients with pre-existing liver disease should be taken into consideration. Ultimately, a vaccine is needed to aid in reducing cases of SARS-CoV-2 and providing immunity to the general population. However, while considering the types of vaccines available, safety concerns, particularly of RNA- or DNA-based vaccines, need to be addressed.

Oxygen Plasma Substrate and Specific Nanopattern Promote Early Differentiation of HepaRG Progenitors.

Fully differentiated HepaRG™ cells are the hepatic cell line of choice for in vitro study in toxicology and drug trials. They are derived from a hepatoblast-like progenitor (HepaRG-P) that differentiates into a coculture of hepatocyte-like and cholangiocyte-like cells. This process that requires 2 weeks of proliferation followed by 2 weeks of differentiation using dimethyl sulfoxide (DMSO) can be time consuming and costly. Identifying a method to accelerate HepaRG-Ps toward a mature lineage would save both time and money. The ability to do this in the absence of DMSO would remove the possibility of confounding toxicology results caused by DMSO induction of CYP pathways. It has been shown that tissue culture substrates play an important role in the development and maturity of a cell line, and this is particularly important for progenitor cells, which retain some form of plasticity. Oxygen plasma treatment is used extensively to modify cell culture substrates. There is also evidence that patterned rather than planar surfaces have a positive effect on proliferation and differentiation. In this study, we compared the effect of standard tissue culture plastic (TCP), oxygen plasma coated (OPC), and nanopatterned substrates (NPS) on early differentiation and function of HepaRG-P cells. Since NPS were OPC we initially compared the effect of TCP and OPC to enable comparison between all three culture surfaces using OPC as control to asses if patterning further enhanced early differentiation and functionality. The results show that HepaRG-P's grown on OPC substrate exhibited earlier differentiation, proliferation, and function compared with TCP. Culturing HepaRG-P's on OPC with the addition of NPS did not confer any additional advantage. In conclusion, OPC surface appeared to enhance hepatic differentiation and functionality and could replace traditional methods of differentiating HepaRG-P cells into fully differentiated and functional HepaRGs earlier than standard methods. Impact statement We show significantly earlier differentiation and function of HepaRG progenitor cells when grown in dimethyl sulfoxide-free medium on oxygen plasma substrates versus standard tissue culture plastic. Further investigation showed that nanopatterning of oxygen plasma substrates did not confer any additional advantage over smooth oxygen plasma, although one pattern (DSQ120) showed comparable early differentiation and function.

Validation of Reference Genes for Gene Expression Studies by RT-qPCR in HepaRG Cells during Toxicity Testing and Disease Modelling.

Gene expression analysis by quantitative real-time polymerase chain reaction (RT-qPCR) is routinely used in biomedical studies. The reproducibility and reliability of the data fundamentally depends on experimental design and data interpretation. Despite the wide application of this assay, there is significant variation in the validation process of gene expression data from research laboratories. Since the validity of results depends on appropriate normalisation, it is crucial to select appropriate reference gene(s), where transcription of the selected gene is unaffected by experimental setting. In this study we have applied geNorm technology to investigate the transcription of 12 'housekeeping' genes for use in the normalisation of RT-qPCR data acquired using a widely accepted HepaRG hepatic cell line in studies examining models of pre-clinical drug testing. geNorm data identified a number of genes unaffected by specific drug treatments and showed that different genes remained invariant in response to different drug treatments, whereas the transcription of 'classical' reference genes such as GAPDH (glyceralde- hyde-3-phosphate dehydrogenase) was altered by drug treatment. Comparing data normalised using the reference genes identified by geNorm with normalisation using classical housekeeping genes demonstrated substantial differences in the final results. In light of cell therapy application, RT-qPCR analyses has to be carefully evaluated to accurately interpret data obtained from dynamic cellular models undergoing sequential stages of phenotypic change.

Human umbilical cord perivascular cells improve human pancreatic islet transplant function by increasing vascularization.

Islet transplantation is an efficacious therapy for type 1 diabetes; however, islets from multiple donor pancreata are required, and a gradual attrition in transplant function is seen. Here, we manufactured human umbilical cord perivascular mesenchymal stromal cells (HUCPVCs) to Good Manufacturing Practice (GMP) standards. HUCPVCs showed a stable phenotype while undergoing rapid ex vivo expansion at passage 2 (p2) to passage 4 (p4) and produced proregenerative factors, strongly suppressing T cell responses in the resting state and in response to inflammation. Transplanting an islet equivalent (IEQ):HUCPVC ratio of 1:30 under the kidney capsule in diabetic NSG mice demonstrated the fastest return to normoglycemia by 3 days after transplant: Superior glycemic control was seen at both early (2.7 weeks) and later stages (7, 12, and 16 weeks) versus ratios of 1:0, 1:10, and 1:50, respectively. Syngeneic islet transplantation in immunocompetent mice using the clinically relevant hepatic portal route with a marginal islet mass showed that mice transplanted with an IEQ:HUCPVC ratio of 1:150 had superior glycemic control versus ratios of 1:0, 1:90, and 1:210 up to 6 weeks after transplant. Immunodeficient mice transplanted with human islets (IEQ:HUCPVC ratio of 1:150) exhibited better glycemic control for 7 weeks after transplant versus islet transplant alone, and islets transplanted via the hepatic portal vein in an allogeneic mouse model using a curative islet mass demonstrated delayed rejection of islets when cotransplanted with HUCPVCs (IEQ:HUCPVC ratio of 1:150). The immunosuppressive and proregenerative properties of HUCPVCs demonstrated long-term positive effects on graft function in vivo, indicating that they may improve long-term human islet allotransplantation outcomes.

Application of Impedance-Based Techniques in Hepatology Research.

There are a variety of end-point assays and techniques available to monitor hepatic cell cultures and study toxicity within in vitro models. These commonly focus on one aspect of cell metabolism and are often destructive to cells. Impedance-based cellular assays (IBCAs) assess biological functions of cell populations in real-time by measuring electrical impedance, which is the resistance to alternating current caused by the dielectric properties of proliferating of cells. While the uses of IBCA have been widely reported for a number of tissues, specific uses in the study of hepatic cell cultures have not been reported to date. IBCA monitors cellular behaviour throughout experimentation non-invasively without labelling or damage to cell cultures. The data extrapolated from IBCA can be correlated to biological events happening within the cell and therefore may inform drug toxicity studies or other applications within hepatic research. Because tight junctions comprise the blood/biliary barrier in hepatocytes, there are major consequences when these junctions are disrupted, as many pathologies centre around the bile canaliculi and flow of bile out of the liver. The application of IBCA in hepatology provides a unique opportunity to assess cellular polarity and patency of tight junctions, vital to maintaining normal hepatic function. Here, we describe how IBCAs have been applied to measuring the effect of viral infection, drug toxicity /IC50, cholangiopathies, cancer metastasis and monitoring of the gut-liver axis. We also highlight key areas of research where IBCAs could be used in future applications within the field of hepatology.

1,25-Dihydroxyvitamin D3 Restrains CD4+ T Cell Priming Ability of CD11c+ Dendritic Cells by Upregulating Expression of CD31.

Dendritic cells (DC) are specialized sentinel cells that bridge the innate and adaptive immune response and play a crucial role in shaping the adaptive immune response. Vitamin D, a known epidemiological risk factor for the development of several autoimmune diseases, influences the development of dendritic cells. Consequently, vitamin D metabolites are frequently used in protocols to develop therapeutic dendritic cell therapies for autoimmune diseases. However, the mechanisms by which vitamin D modulates DC function remain poorly understood. We investigated the effects of vitamin D on murine CD11c+ bone marrow derived DC (BMDC) function by analyzing global gene expression in CD11c+ BMDC generated in the presence (VitD-CD11c+BMDC) or absence (Veh-CD11c+BMDC) of the active vitamin D metabolite, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Seven genes were significantly increased in expression in both immature and LPS-matured VitD-CD11c+BMDC, one of which was CD31, a member of the immunoglobulin superfamily. Gene knockdown of CD31 enhanced the ability of VitD-CD11c+BMDC to prime naïve CD4+ T cells in vitro; conversely, increased expression of CD31 on vehicle treated CD11c+BMDC restrained their T cell priming abilities. Time-lapse imaging of BMDC and CD4+ T cells during in vitro priming revealed that CD31 reduced the BMDC-T cell interaction time. Finally, we confirmed a similar effect of 1,25(OH)2D3 on human CD34+ cell-derived CD11c+DC, whereby DC generated in the presence of 1,25(OH)2D3 had increased CD31 expression. In summary, we show that both mouse and human DC generated in the presence of 1,25(OH)2D3 upregulate CD31 expression, resulting in a reduced ability to prime CD4+ T cells by impairing a stable cell-cell contact.

Chlorpromazine toxicity is associated with disruption of cell membrane integrity and initiation of a pro-inflammatory response in the HepaRG hepatic cell line.

Chlorpromazine (CPZ) is a neuroleptic drug and prototype compound used to study intrahepatic cholestasis. The exact mechanisms of CPZ induced cholestasis remain unclear. Rat hepatocytes, or a sandwich culture of rat and human hepatocytes, have been the most commonly used models for studying CPZ toxicity in vitro. However, to better predict outcomes in pre-clinical trials where cholestasis may be an unwanted consequence, a human in vitro model, based on human HepaRG cells, capable of real-time, non-invasive and label free monitoring, alongside molecular investigations would be beneficial. To address this we used the human hepatic HepaRG cell line, and established concentrations of CPZ ranging from sub-toxic, 25 µM and 50 µM, to toxic 100 µM and compared them with untreated control. To assess the effect of this range of CPZ concentrations we employed electrical cell-substrate impedance sensing (ECIS) to measure viability and cell membrane interactions alongside traditional viability assays, immunocytostaining and qRT-PCR to assess genes of interest within adaptive and inflammatory pathways. Using these methods, we show a concentration dependant response to CPZ involving pro-inflammatory pathway, loss of tight junctions and membrane integrity, and an adaptive response mediated by Cytochrome P450 (CYP) enzyme activation and up-regulation of membrane phospholipid and xenobiotic transporters. In conclusion, structural changes within the membrane caused by sub-toxic and toxic concentrations of CPZ negatively impact the function of the cellular membrane. Damage to efflux transport proteins caused by CPZ induce cholestasis alongside downstream inflammation, which activates compensatory responses for cell survival. LAY SUMMARY: Chlorpromazine is a drug used to treat patients with schizophrenia, which has a known association with liver damage. Here we show that it causes inflammation and alters the cell membranes in liver and bile duct cells similar to what is seen within a human population. The initiation of the inflammatory response and changes to cellular structure may provide insight into the damage and disease process and inform medical treatment.

Low-dose acetaminophen induces early disruption of cell-cell tight junctions in human hepatic cells and mouse liver.

Dysfunction of cell-cell tight junction (TJ) adhesions is a major feature in the pathogenesis of various diseases. Liver TJs preserve cellular polarity by delimiting functional bile-canalicular structures, forming the blood-biliary barrier. In acetaminophen-hepatotoxicity, the mechanism by which tissue cohesion and polarity are affected remains unclear. Here, we demonstrate that acetaminophen, even at low-dose, disrupts the integrity of TJ and cell-matrix adhesions, with indicators of cellular stress with liver injury in the human hepatic HepaRG cell line, and primary hepatocytes. In mouse liver, at human-equivalence (therapeutic) doses, dose-dependent loss of intercellular hepatic TJ-associated ZO-1 protein expression was evident with progressive clinical signs of liver injury. Temporal, dose-dependent and specific disruption of the TJ-associated ZO-1 and cytoskeletal-F-actin proteins, correlated with modulation of hepatic ultrastructure. Real-time impedance biosensing verified in vitro early, dose-dependent quantitative decreases in TJ and cell-substrate adhesions. Whereas treatment with NAPQI, the reactive metabolite of acetaminophen, or the PKCα-activator and TJ-disruptor phorbol-12-myristate-13-acetate, similarly reduced TJ integrity, which may implicate oxidative stress and the PKC pathway in TJ destabilization. These findings are relevant to the clinical presentation of acetaminophen-hepatotoxicity and may inform future mechanistic studies to identify specific molecular targets and pathways that may be altered in acetaminophen-induced hepatic depolarization.

Human Hepatic HepaRG Cells Maintain an Organotypic Phenotype with High Intrinsic CYP450 Activity/Metabolism and Significantly Outperform Standard HepG2/C3A Cells for Pharmaceutical and Therapeutic Applications.

Conventional in vitro human hepatic models for drug testing are based on the use of standard cell lines derived from hepatomas or primary human hepatocytes (PHHs). Limited availability, interdonor functional variability and early phenotypic alterations in PHHs restrict their use, whilst standard cell lines such as HepG2 lack a substantial and variable set of liver-specific functions such as CYP450 activity. Alternatives include the HepG2-derivative C3A cells selected as a more differentiated and metabolically active hepatic phenotype. Human HepaRG cells are an alternative organotypic co-culture model of hepatocytes and cholangiocytes reported to maintain in vivo-like liver-specific functions, including intact Phase I-III drug metabolism. In this study, we compared C3A and human HepaRG cells using phenotypic profiling, CYP450 activity and drug metabolism parameters to assess their value as hepatic models for pre-clinical drug testing or therapeutics. Compared with C3As, HepaRG co-cultures exhibit a more organotypic phenotype, including evidence of hepatic polarity with the strong expression of CYP3A4, the major isoform involved in the metabolism of over 60% of marketed drugs. Significantly greater CYP450 activity and expression of CYP1A2, CYP2E1 and CYP3A4 genes in HepaRG cells (comparable with that of human liver tissue) was demonstrated. Moreover, HepaRG cells also preferentially expressed the hepatic integrin α5 ß1 - an important modulator of cell behaviour including growth and survival, differentiation and polarity. Drug metabolite profiling of phenacetin (CYP1A2) and testosterone (CYP3A4) using LC-MS/MS and HPLC, respectively, revealed that HepaRGs had more intact (Phase I-II) metabolism profile. Thus, HepaRG cells significantly outperform C3A cells for the potential pharmaceutical and therapeutic applications.

Acetaminophen cytotoxicity is ameliorated in a human liver organotypic co-culture model.

Organotypic liver culture models for hepatotoxicity studies that mimic in vivo hepatic functionality could help facilitate improved strategies for early safety risk assessment during drug development. Interspecies differences in drug sensitivity and mechanistic profiles, low predictive capacity, and limitations of conventional monocultures of human hepatocytes, with high attrition rates remain major challenges. Herein, we show stable, cell-type specific phenotype/cellular polarity with differentiated functionality in human hepatocyte-like C3A cells (enhanced CYP3A4 activity/albumin synthesis) when in co-culture with human vascular endothelial cells (HUVECs), thus demonstrating biocompatibility and relevance for evaluating drug metabolism and toxicity. In agreement with in vivo studies, acetaminophen (APAP) toxicity was most profound in HUVEC mono-cultures; whilst in C3A:HUVEC co-culture, cells were less susceptible to the toxic effects of APAP, including parameters of oxidative stress and ATP depletion, altered redox homeostasis, and impaired respiration. This resistance to APAP is also observed in a primary human hepatocyte (PHH) based co-culture model, suggesting bidirectional communication/stabilization between different cell types. This simple and easy-to-implement human co-culture model may represent a sustainable and physiologically-relevant alternative cell system to PHHs, complementary to animal testing, for initial hepatotoxicity screening or mechanistic studies of candidate compounds differentially targeting hepatocytes and endothelial cells.

Efficient episomal reprogramming of blood mononuclear cells and differentiation to hepatocytes with functional drug metabolism.

The possibility of converting cells from blood mononuclear cells (MNC) to liver cells provides promising opportunities for the study of diseases and the assessment of new drugs. However, clinical applications have to meet GMP requirements and the methods for generating induced pluripotent cells (iPCs) have to avoid insertional mutagenesis, a possibility when using viral vehicles for the delivery of reprogramming factors. We have developed an efficient non-integration method for reprogramming fresh or frozen blood MNC, maintained in an optimised cytokine cocktail, to generate induced pluripotent cells. Using electroporation for the effective delivery of episomal transcription factors (Oct4, Sox2, Klf4, L-Myc, and Lin28) in a feeder-free system, without any requirement for small molecules, we achieved a reprogramming efficiency of up to 0.033% (65 colonies from 2×10(5) seeded MNC). Applying the same cytokine cocktail and reprogramming methods to cord blood or fetal liver-derived CD34(+) cells, we obtained 148 iPS colonies from 10(5) seeding cells (0.148%). The iPS cell lines we generated maintained typical characteristics of pluripotent cells and could be successfully differentiated into hepatocytes with drug metabolic function.

WNT signaling drives cholangiocarcinoma growth and can be pharmacologically inhibited.

Cholangiocarcinoma (CC) is typically diagnosed at an advanced stage and is refractory to surgical intervention and chemotherapy. Despite a global increase in the incidence of CC, little progress has been made toward the development of treatments for this cancer. Here we utilized human tissue; CC cell xenografts; a p53-deficient transgenic mouse model; and a non-transgenic, chemically induced rat model of CC that accurately reflects both the inflammatory and regenerative background associated with human CC pathology. Using these systems, we determined that the WNT pathway is highly activated in CCs and that inflammatory macrophages are required to establish this WNT-high state in vivo. Moreover, depletion of macrophages or inhibition of WNT signaling with one of two small molecule WNT inhibitors in mouse and rat CC models markedly reduced CC proliferation and increased apoptosis, resulting in tumor regression. Together, these results demonstrate that enhanced WNT signaling is a characteristic of CC and suggest that targeting WNT signaling pathways has potential as a therapeutic strategy for CC.

A novel malaria vaccine candidate antigen expressed in Tetrahymena thermophila.

Development of effective malaria vaccines is hampered by the problem of producing correctly folded Plasmodium proteins for use as vaccine components. We have investigated the use of a novel ciliate expression system, Tetrahymena thermophila, as a P. falciparum vaccine antigen platform. A synthetic vaccine antigen composed of N-terminal and C-terminal regions of merozoite surface protein-1 (MSP-1) was expressed in Tetrahymena thermophila. The recombinant antigen was secreted into the culture medium and purified by monoclonal antibody (mAb) affinity chromatography. The vaccine was immunogenic in MF1 mice, eliciting high antibody titers against both N- and C-terminal components. Sera from immunized animals reacted strongly with P. falciparum parasites from three antigenically different strains by immunofluorescence assays, confirming that the antibodies produced are able to recognize parasite antigens in their native form. Epitope mapping of serum reactivity with a peptide library derived from all three MSP-1 Block 2 serotypes confirmed that the MSP-1 Block 2 hybrid component of the vaccine had effectively targeted all three serotypes of this polymorphic region of MSP-1. This study has successfully demonstrated the use of Tetrahymena thermophila as a recombinant protein expression platform for the production of malaria vaccine antigens.

Antibody responses to a novel Plasmodium falciparum merozoite surface protein vaccine correlate with protection against experimental malaria infection in Aotus monkeys.

The Block 2 region of the merozoite surface protein-1 (MSP-1) of Plasmodium falciparum has been identified as a target of protective immunity by a combination of seroepidemiology and parasite population genetics. Immunogenicity studies in small animals and Aotus monkeys were used to determine the efficacy of recombinant antigens derived from this region of MSP-1 as a potential vaccine antigen. Aotus lemurinus griseimembra monkeys were immunized three times with a recombinant antigen derived from the Block 2 region of MSP-1 of the monkey-adapted challenge strain, FVO of Plasmodium falciparum, using an adjuvant suitable for use in humans. Immunofluorescent antibody assays (IFA) against erythrocytes infected with P. falciparum using sera from the immunized monkeys showed that the MSP-1 Block 2 antigen induced significant antibody responses to whole malaria parasites. MSP-1 Block 2 antigen-specific enzyme-linked immunosorbent assays (ELISA) showed no significant differences in antibody titers between immunized animals. Immunized animals were challenged with the virulent P. falciparum FVO isolate and monitored for 21 days. Two out of four immunized animals were able to control their parasitaemia during the follow-up period, whereas two out of two controls developed fulminating parasitemia. Parasite-specific serum antibody titers measured by IFA were four-fold higher in protected animals than in unprotected animals. In addition, peptide-based epitope mapping of serum antibodies from immunized Aotus showed distinct differences in epitope specificities between protected and unprotected animals.

Haematopoietic differentiation is inhibited when Notch activity is enhanced in FLK1(+) mesoderm progenitors.

Notch signalling has been implicated during haematopoietic development in vivo and in the differentiation of haematopoietic cells from pluripotent cells in vitro. However interpretation of data from many of these studies has been complicated by the heterogeneous nature of cell populations under study and by the fact that the Notch pathway is active during embryogenesis prior to the development of the haematopoietic system. To define the role of Notch signalling in more precise cell populations during the early stages of haematopoietic development within the aorta-gonad-mesonephros (AGM) microenvironment we co-cultured differentiating ESCs on a stromal cell line derived from this region of the embryo. Our co-culture system had no effect on the production of FLK1(+) mesoderm progenitor cells but promoted their subsequent haematopoietic differentiation. We assessed the role of Notch signalling on haematopoietic differentiation of isolated FLK1(+) cells. Notch activity is dynamic and drops to basal levels as FLK1(+) cells commit to a haematopoietic fate. Further reduction of Notch activity by the inducible expression of dominant negative MAML had no functional consequences. In contrast, induction of Notch activity using an inducible NotchIC expression system had an inhibitory effect on haematopoietic differentiation. We used a Cre-mediated recombination strategy whereby NotchIC-expressing cells were marked with the hCD2 receptor and observed a reduction in the number of multi-lineage and myeloid colonies derived from NotchIC(+) compared to NotchIC(-) FLK1(+) cells isolated from the same culture. We believe that our culture system represents a good model for haematopoietic development within the AGM microenvironment and our data suggest that haematopoietic commitment of FLK1(+) cells in this setting occurs when Notch activity is below a specific threshold.

The constituents and mechanisms of generation of 'endothelial cell--colony forming units'.

AIMS: The formation of endothelial cell-colony forming units (EC-CFUs) is increased by vascular injury, although their function in vivo is unclear. We, therefore, examined the constituents of EC-CFUs and the mechanisms of their generation. METHODS AND RESULTS: We performed immunohistochemical characterization of EC-CFUs and their mononuclear precursors. Using fluorescent-activated cell sorting, we evaluated the capacity of mononuclear subpopulations to generate EC-CFUs, and monitored their migratory behaviour when co-incubated with EC-CFUs. Time-lapse microscopy was used to observe colony maturation. Cellular proliferation within EC-CFUs was assessed using bromodeoxyuridine (BrdU) and anti-proliferative agents. EC-CFUs exhibited typical endothelial characteristics; however, several endothelial markers were weakly expressed or absent. Macrophage and lymphocyte antigens were intensely expressed. EC-CFUs readily incorporated BrdU, and failed to develop in the presence of anti-proliferative agents (P < 0.01; n = 12). Time-lapse microscopy demonstrated that the characteristic EC-CFU 'spindle cells' are not EC-CFU progeny, but are mononuclear cells migrating towards, and incorporating into colonies. Only CD14(+) monocytes were necessary for EC-CFU formation. CD14 expression was progressively down-regulated during colony maturation (P < 0.001; n = 6). Although unable to generate EC-CFUs directly, CD34(+) cells could differentiate into CD14(+) cells and potentiate EC-CFU formation. CONCLUSIONS: EC-CFUs exhibit endothelial characteristics, but are predominantly CD14(+) derived macrophages and are a potent stimulus for lymphocyte migration. Proliferation is necessary for EC-CFU generation; however, colony growth also occurs as a result of leucocyte migration. Although confirmatory in vivo studies are required, EC-CFU formation likely reflects leucocyte activation as a reparatory response to vascular denudation or tissue ischaemia.

11ß-hydroxysteroid dehydrogenase type 1 deficiency in bone marrow-derived cells reduces atherosclerosis.

11ß-Hydroxysteroid dehydrogenase type-1 (11ß-HSD1) converts inert cortisone into active cortisol, amplifying intracellular glucocorticoid action. 11ß-HSD1 deficiency improves cardiovascular risk factors in obesity but exacerbates acute inflammation. To determine the effects of 11ß-HSD1 deficiency on atherosclerosis and its inflammation, atherosclerosis-prone apolipoprotein E-knockout (ApoE-KO) mice were treated with a selective 11ß-HSD1 inhibitor or crossed with 11ß-HSD1-KO mice to generate double knockouts (DKOs) and challenged with an atherogenic Western diet. 11ß-HSD1 inhibition or deficiency attenuated atherosclerosis (74-76%) without deleterious effects on plaque structure. This occurred without affecting plasma lipids or glucose, suggesting independence from classical metabolic risk factors. KO plaques were not more inflamed and indeed had 36% less T-cell infiltration, associated with 38% reduced circulating monocyte chemoattractant protein-1 (MCP-1) and 36% lower lesional vascular cell adhesion molecule-1 (VCAM-1). Bone marrow (BM) cells are key to the atheroprotection, since transplantation of DKO BM to irradiated ApoE-KO mice reduced atherosclerosis by 51%. 11ß-HSD1-null macrophages show 76% enhanced cholesterol ester export. Thus, 11ß-HSD1 deficiency reduces atherosclerosis without exaggerated lesional inflammation independent of metabolic risk factors. Selective 11ß-HSD1 inhibitors promise novel antiatherosclerosis effects over and above their benefits for metabolic risk factors via effects on BM cells, plausibly macrophages.