Roz Jenkins

Phenotypic and functional analyses show stem cell-derived hepatocyte-like cells better mimic fetal rather than adult hepatocytes

Graphical abstract

Identification of Ca2+-dependent binding partners for the neuronal calcium sensor protein neurocalcin δ: interaction with actin, clathrin and tubulin

The neuronal calcium sensors are a family of EF-hand-containing Ca(2+)-binding proteins expressed predominantly in retinal photoreceptors and neurons. One of the family members is neurocalcin delta, the function of which is unknown. As an approach to elucidating the protein interactions made by neurocalcin delta, we have identified brain cytosolic proteins that bind to neurocalcin delta in a Ca(2+)-dependent manner. We used immobilized recombinant myristoylated neurocalcin delta combined with protein identification using MS. We demonstrate a specific interaction with clathrin heavy chain, alpha- and beta-tubulin, and actin. These interactions were dependent upon myristoylation of neurocalcin delta indicating that the N-terminal myristoyl group may be important for protein-protein interactions in addition to membrane association. Direct binding of neurocalcin delta to clathrin, tubulin and actin was confirmed using an overlay assay. These interactions were also demonstrated for endogenous neurocalcin delta by co-immunoprecipitation from rat brain cytosol. When expressed in HeLa cells, neurocalcin delta was cytosolic at resting Ca(2+) levels but translocated to membranes, including a perinuclear compartment (trans-Golgi network) where it co-localized with clathrin, following Ca(2+) elevation. These data suggest the possibility that neurocalcin delta functions in the control of clathrin-coated vesicle traffic.

Proteome‐wide analyses of human hepatocytes during differentiation and dedifferentiation

Failure to predict hepatotoxic drugs in preclinical testing makes it imperative to develop better liver models with a stable phenotype in culture. Stem cell‐derived models offer promise, with differentiated hepatocyte‐like cells currently considered to be “fetal‐like” in their maturity. However, this judgment is based on limited biomarkers or transcripts and lacks the required proteomic datasets that directly compare fetal and adult hepatocytes. Here, we quantitatively compare the proteomes of human fetal liver, adult hepatocytes, and the HepG2 cell line. In addition, we investigate the proteome changes in human fetal and adult hepatocytes when cultured in a new air‐liquid interface format compared to conventional submerged extracellular matrix sandwich culture. From albumin and urea secretion, and luciferase‐based cytochrome P450 activity, adult hepatocytes were viable in either culture model over 2 weeks. The function of fetal cells was better maintained in the air‐liquid interface system. Strikingly, the proteome was qualitatively similar across all samples but hierarchical clustering showed that each sample type had a distinct quantitative profile. HepG2 cells more closely resembled fetal than adult hepatocytes. Furthermore, clustering showed that primary adult hepatocytes cultured at the air‐liquid interface retained a proteome that more closely mimicked their fresh counterparts than conventional culture, which acquired myofibroblast features. Principal component analysis extended these findings and identified a simple set of proteins, including cytochrome P450 2A6, glutathione S transferase P, and alcohol dehydrogenases as specialized indicators of hepatocyte differentiation. Conclusion: Our quantitative datasets are the first that directly compare multiple human liver cells, define a model for enhanced maintenance of the hepatocyte proteome in culture, and provide a new protein “toolkit” for determining human hepatocyte maturity in cultured cells. (Hepatology 2013;58:799–809)

β-Lactam antibiotics form distinct haptenic structures on albumin and activate drug-specific T-lymphocyte responses in multiallergic patients with cystic fibrosis.

β-Lactam antibiotics provide the cornerstone of treatment for respiratory exacerbations in patients with cystic fibrosis. Unfortunately, approximately 20% of patients develop multiple nonimmediate allergic reactions that restrict therapeutic options. The purpose of this study was to explore the chemical and immunological basis of multiple β-lactam allergy through the analysis of human serum albumin (HSA) covalent binding profiles and T-cell responses against 3 commonly prescribed drugs; piperacillin, meropenem, and aztreonam. The chemical structures of the drug haptens were defined by mass spectrometry. Peripheral blood mononuclear cells (PBMC) were isolated from 4 patients with multiple allergic reactions and cultured with piperacillin, meropenem, and aztreonam. PBMC responses were characterized using the lymphocyte transformation test and IFN-γ /IL-13 ELIspot. T-cell clones were generated from drug-stimulated T-cell lines and characterized in terms of phenotype, function, and cross-reactivity. Piperacillin, meropenem, and aztreonam formed complex and structurally distinct haptenic structures with lysine residues on HSA. Each drug modified Lys190 and at least 6 additional lysine residues in a time- and concentration-dependent manner. PBMC proliferative responses and cytokine release were detected with cells from the allergic patients, but not tolerant controls, following exposure to the drugs. 122 CD4+, CD8+, or CD4+CD8+ T-cell clones isolated from the allergic patients were found to proliferate and release cytokines following stimulation with piperacillin, meropenem, or aztreonam. Cross-reactivity with the different drugs was not observed. In conclusion, our data show that piperacillin-, meropenem-, and aztreonam-specific T-cell responses are readily detectable in allergic patients with cystic fibrosis, which indicates that multiple β-lactam allergies are instigated through priming of naïve T-cells against the different drug antigens. Characterization of complex haptenic structures on distinct HSA lysine residues provides a chemical basis for the drug-specific T-cell response.

Characterization of Drug-Specific Signaling Between Primary Human Hepatocytes and Immune Cells

It is now apparent that antigen-specific T-cells are activated in certain patients with drug-induced liver injury (DILI). Since cross-talk between hepatocytes and immune cells is likely to be critical in determining the outcome of drug exposure, the aim of this study was to profile the signals released by drug-treated hepatocytes and to characterize the impact of these molecules on dendritic cells. Human hepatocytes were exposed to 3 drugs (flucloxacillin, amoxicillin, and isoniazid) associated with DILI potentially mediated by the adaptive immune system as drug-specific T-cells have been isolated from DILI patients, and the metabolite nitroso-sulfamethoxazole (SMX-NO). Hepatocyte toxicity, cytokine release and activation of oxidative stress pathways were measured. Supernatants were transferred to monocyte-derived dendritic cells and cell phenotype and function were assessed. High-mobility group box 1 protein (HMGB1) and lactate dehydrogenase release as well as adenosine triphosphate depletion occurred in a drug-, time-, and concentration-dependent manner with SMX-NO and flucloxacillin, whereas isoniazid and amoxicillin were nontoxic. Furthermore, drug-induced activation of nuclear factor (erythroid-derived 2)-like 2 marker genes was observed when hepatocytes were exposed to test drugs. The disulfide isoform of HMGB1 stimulated dendritic cell cytokine release and enhanced the priming of naive T-cells. Incubation of dendritic cells with supernatant from drug-treated hepatocytes resulted in 2 distinct cytokine profiles. SMX-NO/flucloxacillin stimulated secretion of TNF-α, IL-6, IL-1α, and IL-1-β. Isoniazid which did not induce significant hepatocyte toxicity, compared with SMX-NO and flucloxacillin, stimulated the release of a panel of cytokines including the above and IFN-γ, IL-12, IL-17A, IP-10, and IL-10. Collectively, our study identifies drug-specific signaling pathways between hepatocytes and immune cells that could influence whether drug exposure will result in an immune response and tissue injury.

Comparison of Hepatic 2D Sandwich Cultures and 3D Spheroids for Long-term Toxicity Applications: A Multicenter Study

Abstract Primary human hepatocytes (PHHs) are commonly used for in vitro studies of drug-induced liver injury. However, when cultured as 2D monolayers, PHH lose crucial hepatic functions within hours. This dedifferentiation can be ameliorated when PHHs are cultured in sandwich configuration (2Dsw), particularly when cultures are regularly re-overlaid with extracellular matrix, or as 3D spheroids. In this study, the 6 participating laboratories evaluated the robustness of these 2 model systems made from cryopreserved PHH from the same donors considering both inter-donor and inter-laboratory variability and compared their suitability for use in repeated-dose toxicity studies using 5 different hepatotoxins with different toxicity mechanisms. We found that expression levels of proteins involved in drug absorption, distribution, metabolism, and excretion, as well as catalytic activities of 5 different CYPs, were significantly higher in 3D spheroid cultures, potentially affecting the exposure of the cells to drugs and their metabolites. Furthermore, global proteomic analyses revealed that PHH in 3D spheroid configuration were temporally stable whereas proteomes from the same donors in 2Dsw cultures showed substantial alterations in protein expression patterns over the 14 days in culture. Overall, spheroid cultures were more sensitive to the hepatotoxic compounds investigated, particularly upon long-term exposures, across testing sites with little inter-laboratory or inter-donor variability. The data presented here suggest that repeated-dosing regimens improve the predictivity of in vitro toxicity assays, and that PHH spheroids provide a sensitive and robust system for long-term mechanistic studies of drug-induced hepatotoxicity, whereas the 2Dsw system has a more dedifferentiated phenotype and lower sensitivity to detect hepatotoxicity.

Detection of drug‐responsive B lymphocytes and antidrug IgG in patients with β‐lactam hypersensitivity

Delayed‐type β‐lactam hypersensitivity develops in subset of patients. The cellular immunological processes that underlie the drug‐specific response have been described; however, little is known about involvement of the humoral immune system. Thus, the aim of this study was to utilize piperacillin hypersensitivity as an exemplar to (i) develop cell culture methods for the detection of drug‐specific B‐cell responses, (ii) characterize drug‐specific IgG subtypes and (iii) assess reactivity of IgG antibodies against proteins modified to different levels with piperacillin haptens.

Characterization of Flucloxacillin-specific CD8+ T-cells in a mouse model

Drug-induced liver injury (DILI) involving the adaptive immune system is a serious complication in both patient treatment and drug development. Flucloxacillin, a -lactam antibiotic effective against -lactamase-producing bacteria, is a common cause of immunological DILI through cholestasis. We have recently isolated CD8+ flucloxacillin-specific T-cells from patients with DILI, which indicates that T-cells participate in the pathogenesis of the disease. There are currently no animal models of DILI where the adaptive immune system has shown to damage liver, and therefore, it is difficult to explore the mechanistic basis of the tissue injury. Thus, the aim of this study was to use C57/Bl6 CD4 deficient mice with a mutation in the gene encoding for MHC class II molecules to explore the immunogenicity of flucloxacillin and whether flucloxacillin-responsive CD8+ T-cells damage hepatocytes. In initial experiments sensitization was achieved through epicutaneous application (1g/ml; in 70% DMSO; 50L). Proliferation, IFN- and granzyme-B secretion from draining lymph node cells was measured ex vivo following treatment with flucloxacillin. Flucloxacillin-specific CD8+ T-cell mediated killing of hepatocytes was measured using the ApoTox-Glo kit (Promega, UK). In separate experiments, flucloxacillin-specific T-cells were forced to migrate to the mesenteric lymph nodes using retinoic acid, prior to administration of oral flucloxacillin for 10 days, followed by analysis of plasma biomarkers of liver injury. CD8+ T-cells from draining lymph nodes of flucloxacillin-treated mice proliferated in a concentration-dependent manner following ex vivo secondary stimulation. The proliferative response was associated with IFN- and granzyme-B secretion. In contrast, proliferative responses and cytokine secretion was not detected with cells from vehicle control mice. Flucloxacillin-specific hepatocyte toxicity and apoptosis was observed when CD8+ T-cells were cultured with dendritic cells and flucloxacillin for 24h, washed and transferred to the hepatocyte cultures. Flucloxacillin sensitization followed by 10 day oral exposure resulted in marked gall bladder swelling and mild elevations in ALT. Other plasma biomarkers of liver damage were negative. In conclusion, these data show successful sensitization of mice against flucloxacillin. Thus, the C57/Bl6 CD4 deficient mouse represents a promising model to study the role of the adaptive immune system in DILI.

Characterization of anti-drug antibodies and drug-responsive B-lymphocytes in piperacillin hypersensitive patients with cystic fibrosis

Cystic fibrosis is the most common autosomal recessive condition in Caucasians and recurrent infections lead to a plethora of complications. Repeated courses of high dose intravenous β-lactam antibiotics, such as piperacillin, are employed for the treatment of respiratory exacerbations. Unfortunately, delayed-type hypersensitivity reactions develop in between 26-50% of treated patients. We have recently described the cellular immunological processes that underlie drug-specific response in hypersensitive patients; however, the involvement of the humoral immune system has not been studied. The aim of this study was to quantify piperacillin-specific antibodies in plasma of hypersensitive and tolerant patients and investigate whether B-cells can be stimulated to secrete antibodies in vitro following drug stimulation. Drug-specific antibodies were quantified by ELISA using piperacillin-modified BSA as an antigen. Adducts generated using different drug-protein ratios were used to measure the degree of conjugation that elicits an antibody response. BSA was modified with different β-lactam antibiotics to define structural specificity. Specificity for the piperacillin BSA adduct was confirmed by hapten inhibition. Mass spectrometry was used to characterize the Lys residues modified with piperacillin. B-cells isolated from PBMC were cultured with piperacillin for 5 days and IgG secretion and B-cell activation was measured using ELISpot and flow cytometry (CD19, CD27), respectively. A significantly higher level of anti-piperacillin IgG antibody was detected in plasma of hypersensitive patients when hypersensitive and tolerant patients were compared. Hapten inhibition ELISA confirmed specificity for the piperacillin antigenic determinant. Antibody binding was detected with adducts generated at piperacillin:BSA ratios between 1:1 and 100:1. In contrast, antibody binding was not detectable with penicillin G, amoxicillin or aztreonam BSA conjugates. IgG antibody secretion was also detected from purified B-cells from hypersensitive patients cultured with soluble piperacillin. Drug treatment was associated with increased expression of the B-cell activation marker CD27. These data begin to describe the drug-specific humoral immune response in piperacillin hypersensitive patients with cystic fibrosis. Further work is needed to define the role different antibody subtypes play in the disease pathogenesis.

Antigen exposure required for T cell activation

Background A high frequency of hypersensitivity reactions to -lactam antibiotics are observed in patients with cystic fibrosis. -lactam antibiotics form protein conjugates in vitro and in vivo, and a core group of lysine residues of human serum albumin have been shown to be penicilloylated by mass spectrometric methods. Furthermore, protein/ peptide conjugates have been shown to stimulate T cells isolated from patients with -lactam hypersensitivity. However, the threshold level of protein conjugation required to trigger immune responses has not been studied. Method Thus, we focused on piperacillin, a commonly used drug in patients with cystic fibrosis, to (a) quantify the piperacillin antigens formed in patients using mass spectrometry and a synthetic piperacillin-modified albumin peptide (539ATK(Pip)EQLK545) as a standard and (b) determine the quantity of the piperacillin protein adducts formed at the time of T cell activation. Plasma was collected prior to commencing treatment and after a 14 day treatment course from 10 patients and the level of piperacillin-modified albumin in patient plasma was measured. Piperacillin-specific CD4+ T-cell clones were generated from hypersensitive patients by serial dilution and cultured with soluble drug or antigen presenting cells pulsed with piperacillin for 1-48h for the analysis of drug-specific proliferative responses. At each time-point piperacillin albumin binding was quantified. Results Piperacillin-modified lysine was detected in human serum albumin in all 10 patients; the level of piperacillinmodified lysine 541 was found to range from 2.6 to 6.5%. Antigen presenting cells pulsed with piperacillin for 1 and 4h did not stimulate a strong T-cell proliferative response and this coincided with low levels of albumin modification. In contrast, antigen presenting cells pulsed with piperacillin for 24 or 48h activated all of the clones. Quantitative analysis of incubation medium revealed that approximately 3% of lysine 541 was modified after 24h. Conclusion In conclusion, these data quantify for the first time, the level of piperacillin albumin binding in drug exposed patients and in vitro at drug antigen concentrations that activate piperacillin-specific T-cells.