Characterization of diseased primary human hepatocytes in an all-human cell-based triculture system.

Liver diseases, including NAFLD, are a growing worldwide health concern. Currently, there is a lack of suitable in vitro models that sustain basic primary human hepatocyte (PHH) morphology and functionality while supporting presentation of disease-associated phenotypic characteristics such as lipid accumulation and inflammasome activation. In TruVivo, an all-human triculture system (hTCS), basic metabolic functions were characterized in PHHs isolated from normal or diseased livers during two-weeks of culture. Decreases in albumin and urea levels and CYP3A4 activity were seen in diseased-origin PHHs compared to normal PHHs along with higher CYP2E1 expression. Positive expression of the macrophage markers CD68 and CD163 were seen in the diseased PHH preparations. Elevated levels of the pro-inflammatory cytokines IL-6 and MCP-1 and the fibrotic markers CK-18 and TGF-ß were also measured. Gene expression of FASN, PCK1, and G6PC in the diseased PHHs was decreased compared to the normal PHHs. Further characterization revealed differences in lipogenesis and accumulation of intracellular lipids in normal and diseased PHHs when cultured with oleic acid and high glucose. TruVivo represents a promising new platform to study lipogenic mechanisms in normal and diseased populations due to the preservation of phenotypic differences over a prolonged culture period.

An All-Human Hepatic Culture System for Drug Development Applications.

Finding a long-term, human-relevant culture model for primary human hepatocytes (PHHs) for pharmacological and toxicological studies remains a challenge. Current in vitro model platforms are often inconvenient and complex, lack phenotypic stability over time, and do not support multiple PHH lots, lacking experimental reproducibility and flexibility. Here, we provide a detailed protocol for the thawing, plating, and maintenance of an all-human 2D+ hepatic system (TV2D+), which takes advantage of standard two-dimensional (2D) culture techniques and equipment while maintaining the longevity and phenotypic stability over time that typically accompany more complex three-dimensional (3D) systems. The results show attachment and percent plateability in TV2D+ as a function of PHH seeding density, as well as stable functionality for at least 2 weeks in culture. A range of PHH seeding densities are assessed to achieve a successful long-term culture. When established properly, the PHHs in TV2D+ organize into hepatocyte colonies, express a hepatic-specific marker, and maintain viability, architectural integrity, and physiologically relevant levels of albumin and urea. This unique combination of attributes makes the TV2D+ system a suitable hepatic model for a variety of pharmacological and toxicological applications.

The morphology, functionality, and longevity of a novel all human hepatic cell-based tri-culture system.

There remains a significant need for a convenient, phenotypically stable long-term culture platform for primary human hepatocytes (PHHs) for use in pharmacological and toxicological applications. Conventional in vitro models are often inconvenient, burdensome to use, and unable to support a multitude of donor lots or maintain PHH structural and functional properties over extended time. To address these limitations, an all-human cell-based hepatic tri-culture system (HTCS) has been developed comprised of frozen vials of PHHs and feeder cells. Qualified PHHs exhibited healthy morphological characteristics for ≥30 days. Extensive anastomosing networks of bile canaliculi with tight and gap junctions were established early and remained stable and functional throughout the culture period. After 5 culture days, albumin, urea, and basal Phase 1 and Phase 2 metabolic functions were stable for at least 2 weeks and significantly higher in the HTCS PHHs compared to sandwich monoculture PHHs. Induction of CYP functional activity by prototypical receptor agonists was stable after 4 days for at least 2 weeks. Gene expression of Alb and various CYPs in the HTCS PHHs was significantly higher compared to sandwich monoculture PHHs. The HTCS represents a convenient, phenotypically stable, all-human PHH culture platform for pharmacological and toxicological applications.

An increase in inflammation and islet dysfunction is a feature of prediabetes.

AIMS: Type 2 diabetes (T2D) is a global health problem that will be diagnosed in almost 300 million people by 2025 according to the World Health Organization. Before being diagnosed with T2D, individuals may have glucose levels above normal but below the diabetic range. This condition is known as prediabetes. Studies showed that people with prediabetes had an increase in several pro-inflammatory cytokines in their serum and in their fasting glucose levels. The answer remains unclear when inflammation begins in the pancreas and islets, and what is the extent of this inflammation. METHODS: Subjects with haemoglobin A1c levels from 5.7% to 6.4% were classified as pre-diabetic. Sections of pancreas and isolated islets from normal donors and donors with prediabetes were tested for markers of inflammation and glucose-stimulated insulin secretion (GSIS). RESULTS: Gene and protein expression of the inflammatory markers resistin, interleukin-1 beta, tumour necrosis factor-alpha, interleukin-6, and monocyte chemoattractant protein-1 increased in donors with prediabetes compared to normal donors. GSIS response was significantly decreased in pre-diabetic islets compared to normal islets. Donors with prediabetes also had decreased expression of CD163+ cells but not CD68+ cells. CONCLUSIONS: Based on our findings, inflammation and islet dysfunction may be more significant than originally thought in people with prediabetes. Rather than being in a normal state before diabetes occurs, it appears that subjects are already in an early diabetic condition resembling more closely T2D.

Human Placenta-Derived ECM Supports Tri-Lineage Differentiation of Human Induced Pluripotent Stem Cells.

Human pluripotent stem cells (hPSCs) hold great promise for future applications in drug discovery and cell therapies. hPSC culture protocols require specific substrates and medium supplements to support cell expansion and lineage specific differentiation. The animal origin of these substrates is a severe limitation when considering the translation of hPSC derivatives to the clinic and in vitro disease modeling. The present study evaluates the use of a human placenta-derived extracellular matrix (ECM) hydrogel, HuGentraⓇ, to support tri-lineage differentiation of human induced pluripotent stem cells (hiPSCs). Lineage-specific embryoid bodies (EBs) were plated onto three separate matrices, and differentiation efficiency was evaluated based on morphology, protein, and gene expression. HuGentra was found to support the differentiation of hiPSCs to all three germ layers: ectodermal, mesodermal, and endodermal lineages. hiPSCs differentiated into neurons, cardiomyocytes, and hepatocytes on HuGentra had similar morphology, protein, and gene expression compared to differentiation on Matrigel or other cell preferred matrices. HuGentra can be considered as a suitable human substrate for hiPSC differentiation.

Scaffold-free bioprinted osteogenic and chondrogenic systems to model osteochondral physiology.

Three-dimensional bioprinted culture platforms mimic the native microenvironment of tissues more accurately than two-dimensional cell cultures or animal models. Scaffold-free bioprinting eliminates many complications associated with traditional scaffold-dependent printing as well as provides better cell-to-cell interactions and long-term functionality. In this study, constructs were produced from bone marrow derived mesenchymal stem cells (BM-MSCs) using a scaffold-free bioprinter. These constructs were cultured in either osteogenic, chondrogenic, a 50:50 mixture of osteogenic and chondrogenic ('osteo-chondro'), or BM-MSC growth medium. Osteogenic and chondrogenic differentiation capacity was determined over an 8-week culture period using histological and immunohistochemical staining and RT-qPCR (Phase I). After 6 weeks in culture, individual osteogenic and chondrogenic differentiated constructs were adhered to create a bone-cartilage interaction model. Adhered differentiated constructs were cultured for an additional 8 weeks in either chondrogenic or osteo-chondro medium to evaluate sustainability of lineage specification and transdifferentiation potential (Phase II). Constructs cultured in their respective osteogenic and/or chondrogenic medium differentiated directly into bone (model of intramembranous ossification) or cartilage. Positive histological and immunohistochemical staining for bone or cartilage identification was shown after 4 and 8 weeks in culture. Expression of osteogenesis and chondrogenesis associated genes increased between weeks 2 and 6. Adhered individual osteogenic and chondrogenic differentiated constructs sustained their differentiated phenotype when cultured in chondrogenic medium. However, adhered individual chondrogenic differentiated constructs cultured in osteo-chondro medium were converted to bone (model of metaplastic transformation). These bioprinted models of bone-cartilage interaction, intramembranous ossification, and metaplastic transformation of cartilage into bone offer a useful and promising approach for bone and cartilage tissue engineering research. Specifically, these models can be potentially used as functional tissue systems for studying osteochondral defect repair, drug discovery and response, and many other potential applications.

12-Lipoxygenase Inhibitor Improves Functions of Cytokine-Treated Human Islets and Type 2 Diabetic Islets.

Context: The 12-lipoxygenase (12-LO) pathway produces proinflammatory metabolites, and its activation is implicated in islet inflammation associated with type 1 and type 2 diabetes (T2D). Objectives: We aimed to test the efficacy of ML355, a highly selective, small molecule inhibitor of 12-LO, for the preservation of islet function. Design: Human islets from nondiabetic donors were incubated with a mixture of tumor necrosis factor α , interluekin-1ß, and interferon-γ to model islet inflammation. Cytokine-treated islets and human islets from T2D donors were incubated in the presence and absence of ML355. Setting: In vitro study. Participants: Human islets from organ donors aged >20 years of both sexes and any race were used. T2D status was defined from either medical history or most recent hemoglobin A1c value >6.5%. Intervention: Glucose stimulation. Main Outcome Measures: Static and dynamic insulin secretion and oxygen consumption rate (OCR). Results: ML355 prevented the reduction of insulin secretion and OCR in cytokine-treated human islets and improved both parameters in human islets from T2D donors. Conclusions: ML355 was efficacious in improving human islet function after cytokine treatment and in T2D islets in vitro. The study suggests that the blockade of the 12-LO pathway may serve as a target for both form of diabetes and provides the basis for further study of this small molecule inhibitor in vivo.

Interleukin-12 (IL-12)/STAT4 Axis Is an Important Element for ß-Cell Dysfunction Induced by Inflammatory Cytokines.

Pathology driving ß-cell loss in diabetes is poorly defined. Chronic subclinical inflammation is associated with ß-cell dysfunction. Acute in vitro exposure of islets and ß-cells to an inflammatory cytokine cocktail (IL-1ß/TNF-α/IFN-γ) results in loss of cell function and viability. The contribution of each cytokine alone or in combination has been evaluated in homogeneous mouse ß-cell lines and primary mouse islets. Cytokine cooperation is required for ß-cell apoptosis with the most potent combinations including IL-1ß. Single cytokine exposure did not induce ß-cell apoptosis. Expression of endogenous interleukin-12 in ß-cells correlated with inflammatory cytokine combinations that induced ß-cell apoptosis. Uncoupling of the IL-12 axis by a block of IL-12 production, inhibition of IL-12 receptor/ligand interaction or disruption of IL-12 receptor signaling conferred protection to ß-cells from apoptosis induced by inflammatory cytokine stimulation. Signaling through STAT4 is indicated since disruption of IL-12 concomitantly reduced inflammatory cytokine stimulation of endogenous IFN-γ expression. Primary mouse islets isolated from mice deficient in STAT4 show resistance to inflammatory-cytokine-induced cell death when compared to islets isolated from wild type mice. Collectively, the data identify IL-12 as an important mediator of inflammation induced ß-cell apoptosis. Modulation of IL-12/STAT4 signaling may be a valuable therapeutic strategy to preserve islet/ß-cell viability in established diabetes.

Inhibition of NADPH oxidase-1 preserves beta cell function.

AIMS/HYPOTHESIS: Upregulation of the reactive oxygen species (ROS)-producing enzyme NADPH oxidase (NOX)-1 in islets and beta cells follows acute exposure to inflammatory cytokines and is concomitant with beta cell dysfunction. NOX-1 is a candidate mediator of inflammation-induced beta cell dysfunction. This study aimed to determine whether selective inhibition of NADPH oxidase-1 presents a new strategy to preserve beta cell function. METHODS: Induced beta cell dysfunction was studied in primary human donor islets, isolated mouse islets and murine beta cell lines. Islets and beta cells were stimulated with inflammatory cytokines (TNF-α, IL-1ß, IFN-γ). NOX-1 activity was blocked by the selective inhibitor ML171. RESULTS: Cytokine induction of intracellular ROS was reduced 80% with 1 µmol/l ML171 in murine beta cell lines (p < 0.01). Cytokine-induced apoptosis, measured by caspase-3 activation or quantified fluorescence microscopy, was prevented in islets and beta cell lines up to 100% with ML171 in a concentration-dependent manner (p < 0.05). Functionally, glucose-stimulated insulin secretion was abolished by cytokine exposure but preserved by ML171 in isolated mouse islets and murine beta cell lines. A feed-forward regulation of NOX-1 in islets and beta cell lines was disrupted by ML171. CONCLUSIONS/INTERPRETATION: Stimulation of NOX-1 activity is a major component of inflammatory cytokine-induced beta cell dysfunction. Significant protection of beta cells is conferred with selective inhibition of NOX-1. Suppression of NOX-1 activity may present a new therapeutic strategy to preserve and protect beta cell function in diabetes.

Integration of pro-inflammatory cytokines, 12-lipoxygenase and NOX-1 in pancreatic islet beta cell dysfunction.

Elevated cellular reactive species, which can be produced by diabetic serum conditions such as elevated inflammatory cytokines, lipotoxicity or glucotoxicity contribute to islet beta cell dysfunction and cell death. Cellular pathways that result in beta cell oxidative stress are poorly resolved. In this study, stimulation of human donor islets, primary mouse islets or homogeneous beta cell lines with a cocktail of inflammatory cytokines (TNFα, IL-1ß, and INFγ) significantly induced NADPH oxidase-1 (NOX-1) gene expression (p<0.05). This pro-inflammatory cytokine cocktail concomitantly induced loss of islet glucose stimulated insulin response (p<0.05), elevated expression of MCP-1 (p<0.01), increased cellular reactive oxygen species (ROS) and induced cell death. Inhibitors of NADPH oxidase, apocynin and diphenyleneiodonium, and a dual selective NOX1/4 inhibitor, blocked ROS generation (p<0.01) and induction of MCP-1 (p<0.05) by pro-inflammatory cytokines in beta cells. It has previously been reported that pro-inflammatory cytokine stimulation induces 12-lipoxygenase (12-LO) expression in human islets. 12-Hydroxyeicosatetraenoic acid (12-HETE), a product of 12-LO activity, stimulated NOX-1 expression in human islets (p<0.05). A novel selective inhibitor of 12-LO blocked induction of NOX-1, production of ROS and pro-caspase 3 cleavage by pro-inflammatory cytokines in INS-1 beta cells (p<0.01). Inhibition was not seen with a structurally related but inactive analog. Importantly, islets from human type 2 diabetic donors have an elevated expression of NOX-1 (p<0.05). This study describes an integrated pathway in beta cells that links beta cell dysfunction induced by pro-inflammatory cytokines with 12-lipoxygenase and NADPH oxidase (NOX-1) activation. Inhibitors of this pathway may provide a new therapeutic strategy to preserve beta cell mass in diabetes.

Targeted expression of islet neogenesis associated protein to beta cells enhances glucose tolerance and confers resistance to streptozotocin-induced hyperglycemia.

Islet neogenesis associated protein (INGAP) stimulates experimental pancreatic islet growth, as evidenced by elevated markers of beta cell mass, in rodents, dogs and primates. Previous analyses of mice that have a transgenic expression of INGAP targeted to the exocrine pancreas have indicated additional biological activity attributed to INGAP. In this study we report on mice with a targeted expression of INGAP to the islet beta cell. The beta cell transgenic mice (IP-INGAP) showed enhanced normalization of blood glucose during IPGTT. Further, IP-INGAP mice had a significant delay in development of hyperglycemia following a diabetogenic dose of streptozotocin. INGAP conferred beta cell protection and enhanced islet function. Analysis of oxidative stress genes in IP-INGAP mice revealed a decrease in islet expression of the NADPH oxidase, NOX1, in both basal state and in response to pro-inflammatory cytokine stimulation. These data are consistent with a pleiotropic role for INGAP and reveal new pathways to target in the discovery of improved diabetic therapies.

Listeria monocytogenes delivery of HPV-16 major capsid protein L1 induces systemic and mucosal cell-mediated CD4+ and CD8+ T-cell responses after oral immunization.

Neutralizing antibodies are thought to be required at mucosal surfaces to prevent human papillomavirus (HPV) transmission. However, the potential for cell-mediated immunity in mediating protection against HPV infection has not been well explored. We generated recombinant Listeria monocytogenes (Lm) constructs that secrete listeriolysin O (LLO) fused with overlapping N-terminal (LLO-L1(1-258)) or C-terminal (LLO-L1(238-474)) fragments of HPV type 16 major capsid protein L1 (HPV-16-L1). Oral immunization of mice with either construct induced IFN-gamma-producing CD8+ and CD4+ T cells in the spleen and in the Peyer's patches with the C-terminal construct. Oral immunization with both constructs resulted in diminished viral titers in the cervix and uterus of mice after intravaginal challenge with vaccinia virus expressing HPV-16-L1.

Monkeypox virus and insights into its immunomodulatory proteins.

SUMMARY: Monkeypox is a disease that is endemic in Central and Western Africa. However, in 2003, there was an outbreak in the United States, representing the first documented monkeypox cases in the Western hemisphere. Although monkeypox virus is less fatal and not as transmissible as variola virus, the causative agent of smallpox, there is concern that monkeypox virus could become a more efficient human pathogen. The reason for this may lie in the virus' genetic makeup, ecological changes, changes in host behavior, and the fact that with the eradication of variola virus, routine smallpox vaccination is no longer carried out. In this review, we focus on the viral proteins that are predicted to modulate the host immune response and compare the genome of monkeypox virus with the genomes of variola virus and the vaccinia virus, the orthopoxvirus that represented the smallpox vaccine. There are differences found in several of these immune-modulating genes including genes that express proteins that affect cytokines such as interleukin-1, tumor necrosis factor, and interferon. There are also differences in genes that code for virulence factors and host range proteins. Genetic differences likely also explain the differences in virulence between two strains of monkeypox virus found in two different regions of Africa. In the current setting of limited smallpox vaccination and little orthopoxvirus immunity in parts of the world, monkeypox could become a more efficient human pathogen under the right circumstances.

The identification and characterization of a monoclonal antibody to the vaccinia virus E3 protein.

Monoclonal antibodies with reactivity to vaccinia virus specific proteins are useful reagents to study the proteins as well as to help understand aspects of the poxvirus life cycle. Using a vaccinia virus proteomics microarray, we found a hybridoma (MAb 3015B2) from a vaccinia virus vaccinated mouse that reacted with the product of the E3L gene. The specificity to the E3 protein was confirmed by Western blotting and immunofluorescence of cells infected with either wild-type vaccinia virus or a mutant virus with the E3L gene deleted. Antibody reactivity with E3 was also seen in cells transfected with a plasmid expressing the E3 protein. A panel of mutated vaccinia viruses with truncations in the E3L gene revealed that while MAb 3015B2 reacted with E3 lacking the C-terminal 7 amino acids, it lost reactivity with a mutant E3 lacking the C-terminal 26 amino acids. This indicates that the antigenic site recognized by 3015B2 is on the C-terminus, somewhere between amino acids 164 through 183. The antibody also recognizes the E3 protein encoded by other orthopoxviruses. This antibody will be useful for further investigations of the E3 protein as well as a useful reagent to indicate vaccinia virus early protein expression.

Characterization of the sequence and architectural constraints of the regulatory and core regions of the human interleukin-2 promoter.

The cytokine interleukin-2 (IL-2) is produced by T cells when they recognize a foreign antigen. Transcription of the IL-2 gene is tightly controlled by the combined actions of multiple transcriptional activators. However, the contribution of sequences in the IL-2 core promoter and the architecture of the IL-2 regulatory region to setting levels of IL-2 transcription are not understood. We have probed these properties of the human IL-2 promoter to understand how the regulatory and core promoter regions cooperate in response to T cell stimulation, thereby setting high levels of inducible transcription. We found that the IL-2 core promoter contains a TATA box that is critical for inducible expression. Moreover, the spacing and orientation between the IL-2 regulatory and core promoter regions is important for setting the level of transcription. The regulatory region of the IL-2 promoter is capable of mediating high levels of expression even when the helical phasing between transcription factor binding sites is perturbed. Although long considered an enhancer, our studies indicate that the regulatory region in the IL-2 promoter is better considered as a proximal regulatory element, since it lacks multiple properties associated with enhancer elements.

The sequence at specific positions in the early transcribed region sets the rate of transcript synthesis by RNA polymerase II in vitro.

To further understand the mechanism of promoter escape by RNA polymerase II, we have systematically investigated the effect of core promoter sequence on the rate of transcript synthesis in vitro. Chimeric and mutant promoters were made by swapping sequences between the human interleukin-2 promoter and the adenovirus major late promoter, which exhibit different rates of transcript synthesis. Kinetic studies at these promoters revealed that sequences downstream of the start sites set the rate of transcript synthesis. Specifically, the sequences at +2 and +7/+8 are critical for determining the rate; when either +2 is a C (nontemplate strand) or +7/+8 is a TT (nontemplate strand), transcript synthesis is slow. At +7/+8, the thermodynamic stability of the RNA:DNA hybrid controls the overall rate of transcript synthesis. Our data support a model in which the rate-limiting step during transcript synthesis by RNA polymerase II in vitro occurs at the point in the reaction at which early ternary complexes transform into elongation complexes.