Keith A. Charlton

Analysis of the Diversity of a Sheep Antibody Repertoire as Revealed from a Bacteriophage Display Library

We have applied bacteriophage display technology to construct and analyze the diversity of an IgG library of >1 × 108 clones from an adult sheep immunized against the hapten atrazine. We have identified eight new VH gene families (VH2–VH9) and five new Vκ gene families (VκV–VκIX). The heavy and κ light chain variable region gene loci were found to be far more diverse than previously thought.

AR42J-B-13 cell: An expandable progenitor to generate an unlimited supply of functional hepatocytes

Hepatocytes are the preparation of choice for Toxicological research in vitro. However, despite the fact that hepatocytes proliferate in vivo during liver regeneration, they are resistant to proliferation in vitro, do not tolerate sub-culture and tend to enter a de-differentiation program that results in a loss of hepatic function. These limitations have resulted in the search for expandable rodent and human cells capable of being directed to differentiate into functional hepatocytes. Research with stem cells suggests that it may be possible to provide the research community with hepatocytes in vitro although to date, significant challenges remain, notably generating a sufficiently pure population of hepatocytes with a quantitative functionality comparable with hepatocytes. This paper reviews work with the AR42J-B-13 (B-13) cell line. The B-13 cell was cloned from the rodent AR42J pancreatic cell line, express genes associated with pancreatic acinar cells and readily proliferates in simple culture media. When exposed to glucocorticoid, 75-85% of the cells trans-differentiate into hepatocyte-like (B-13/H) cells functioning at a level quantitatively similar to freshly isolated rat hepatocytes (with the remaining cells retaining the B-13 phenotype). Trans-differentiation of pancreatic acinar cells also appears to occur in vivo in rats treated with glucocorticoid; in mice with elevated circulating glucocorticoid and in humans treated for long periods with glucocorticoid. The B-13 response to glucocorticoid therefore appears to be related to a real pathophysiological response of a pancreatic cell to glucocorticoid. An understanding of how this process occurs and if it can be generated or engineered in human cells would result in a cell line with the ability to generate an unlimited supply of functional human hepatocytes in a cost effective manner.

Serine/threonine protein kinase SGK1 in glucocorticoid-dependent transdifferentiation of pancreatic acinar cells to hepatocytes

Elevated glucocorticoid levels result in the transdifferentiation of pancreatic acinar cells into hepatocytes through a process that requires a transient repression of WNT signalling upstream of the induction of C/EBP-β. However, the mechanism by which glucocorticoid interacts with WNT signalling is unknown. A screen of microarray data showed that the serine/threonine protein kinase SGK1 (serum- and glucocorticoid-regulated kinase 1) was markedly induced in the model B-13 pancreatic rat acinar cell line after glucocorticoid treatment (which converts them into hepatocyte-like ‘B-13/H’ cells) and this was confirmed at the level of mRNA (notably an alternatively transcribed SGK1C form) and protein. Knockdown of SGK1 using an siRNA designed to target all variant transcripts inhibited glucocorticoid-dependent transdifferentiation, whereas overexpression of the human C isoform (and also the human SGK1F isoform, for which no orthologue in the rat has been identified) alone – but not the wild-type A form – inhibited distal WNT signalling Tcf/Lef transcription factor activity, and converted B-13 cells into B-13/H cells. These effects were lost when the kinase functions of SGK1C and SGK1F were mutated. Inhibition of SGK1 kinase activity also inhibited glucocorticoid-dependent transdifferentiation. Expression of SGK1C and SGK1F resulted in the appearance of phosphorylated β-catenin, and recombinant SGK1 was shown to directly phosphorylate purified β-catenin in vitro in an ATP-dependent reaction. These data therefore demonstrate a crucial role for SGK1 induction in B-13 cell transdifferentiation to B-13/H hepatocytes and suggest that direct phosphorylation of β-catenin by SGK1C represents the mechanism of crosstalk between glucocorticoid and WNT signalling pathways.

High-Sensitivity Monoclonal Antibodies Specific for Homoserine Lactones Protect Mice from Lethal Pseudomonas aeruginosa Infections

ABSTRACT A number of bacteria, including pathogens like Pseudomonas aeruginosa, utilize homoserine lactones (HSLs) as quorum sensing (QS) signaling compounds and engage in cell-to-cell communication to coordinate their behavior. Blocking this bacterial communication may be an attractive strategy for infection control as QS takes a central role in P. aeruginosa biology. In this study, immunomodulation of HSL molecules by monoclonal antibodies (MAbs) was used as a novel approach to prevent P. aeruginosa infections and as tools to detect HSLs in bodily fluids as a possible first clue to an undiagnosed Gram-negative infection. Using sheep immunization and recombinant antibody technology, a panel of sheep-mouse chimeric MAbs were generated which recognized HSL compounds with high sensitivity (nanomolar range) and cross-reactivity. These MAbs retained their nanomolar sensitivity in complex matrices and were able to recognize HSLs in P. aeruginosa cultures grown in the presence of urine. In a nematode slow-killing assay, HSL MAbs significantly increased the survival of worms fed on the antibiotic-resistant strain PA058. The therapeutic benefit of these MAbs was further studied using a mouse model of Pseudomonas infection in which groups of mice treated with HSL-2 and HSL-4 MAbs survived, 7 days after pathogen challenge, in significantly greater numbers (83 and 67%, respectively) compared with the control groups. This body of work has provided early proof-of-concept data to demonstrate the potential of HSL-specific, monoclonal antibodies as theranostic clinical leads suitable for the diagnosis, prevention, and treatment of life-threatening bacterial infections.

The B-13 hepatocyte progenitor cell resists pluripotency induction and differentiation to non-hepatocyte cells

The rat pancreatic “B-13” acinar cell is a stable progenitor cell line that differentiates into hepatocyte-like cells (B-13/H cells) in 2D un-coated plastic culture with simple culture media in response to glucocorticoid exposure. Examination of cytochrome P450 indicated that the expression of a range of genes were similar to freshly isolated hepatocytes and that these gene products were functional on the basis of spectrophotometrically-detectable reduced carbon-monoxide haemoprotein and metabolism of several drugs. Since normal hepatocytes readily de-differentiate under similar conditions, we hypothesized that B-13 cells have undergone a variety of alterations that stabilise a progenitor phenotype and restrict differentiation to hepatocytes only (which if capitulated in human cells, could generate a readily accessible supply of functional human hepatocytes in vitro). To examine this hypothesis, the B-13 karyotype; pluripotency-inducing transcription factor expression and forced over-expression of these factors in B-13 cells were examined. B-13 cells were also injected into NOD/SCID mice and engraftment and differentiation assessed by RT-PCR, Western blotting, immunohistochemistry and fluorescent in situ hybridization (FISH). B-13 cells expressed four pluripotency-inducing transcription factors c-Myc, Klf4, Oct4 and Sox2 with only c-Myc expression maintained after glucocorticoid treatment. Over-expression of the pluripotency-inducing transcription factors blocked B-13/H formation in response to glucocorticoid. Injection of B-13 cells into NOD/SCID mice resulted in their engraftment to the pancreas and liver, with restricted differentiation to hepatocytes in the liver. The cells did not engraft to any other tissues examined. The ability of B-13 cells to specifically generate functional hepatocytes in vitro in response to glucocorticoid is therefore associated with genetic rearrangements that may facilitate expression of genes associated with plasticity (without leading to pluripotency), which are repressed by glucocorticoid treatment.

Adult human exocrine pancreas differentiation to hepatocytes – potential source of a human hepatocyte progenitor for use in toxicology research

Reduction in the use of animals in toxicology is an important goal despite the continued need to assess drug and chemical safety in man. However, a limitation to in vitro screening for drug and chemical toxicity is the lack of available human hepatocytes and the difficulties associated with generating fully functional hepatocytes from stem cells. Previously, we have shown that a rat pancreatic acinar cell line is capable of trans-differentiating into fully functional hepatocyte-like cells in response to glucocorticoid via a serine/threonine protein kinase mechanism alone. Here we demonstrated that differentiation only occurs with glucocorticoids, not other steroids. We also investigated the potential of human pancreatic cells to undergo the same process. Analysis of adult human pancreata at the level of mRNA, protein and by immunohistochemical staining demonstrated that long term systemic exposure to glucocorticoid therapy resulted in differentiation of exocrine tissue to hepatocyte-like tissue. Glucocorticoid treatment of human pancreatic acinar cells in culture also resulted in trans-differentiation to hepatocyte-like cells. Both in vivo and in vitro, trans-differentiation of pancreas cells to hepatocytes was associated with an induction of SGK1 variant transcripts that have been previously shown to drive B-13 differentiation to hepatocytes. Adult exocrine human pancreas therefore responds in a similar qualitative fashion to that previously observed in rodents exposed to elevated glucocorticoid – that of a differentiation into hepatocyte-like cells. Understanding the enhanced response of B-13 cells to glucocorticoid and engineering this response in a replicating human acinar cell could generate an unlimited supply of functional human hepatocytes in vitro that could be useful in a variety of applications, including screening drugs and chemicals for hepatic metabolism and toxicity.

Synthesis and characterisation of immunogens for the production of antibodies against small hydrophobic molecules with biosignature properties

In the present study, five different classes of small hydrophobic molecular targets, atypical for antibody generation, were structurally modified in order to introduce suitable reactive functionalities and/or spacers which allow covalent coupling to a carrier protein resulting in a stable carrier-hapten complex. These targets were chosen to serve as markers of extant and/or extinct life in the context of the development of the Life Marker Chip (LMC), an antibody-based instrument, which is being developed by a UK-led international consortium for flight to Mars on board the joint ESA/NASA Mars exploration ExoMars mission. The hapten-protein conjugates were designed to be used as immunogens for antibody generation and immunoassay reagents in subsequent stages of the LMC development. The extent of protein modification due to covalent attachment of hapten was determined by two independent methods, i.e. trinitrobenzenesulfonic acid (TNBSA) titrations of remaining protein reactive groups and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of the resultant hapten-protein conjugates. In a further quality validation step, the conjugates were presented to an animals immune system and polyclonal antibody titres with moderate specificity were obtained. These results suggest that conjugates synthesized as described herein can successfully be used in the generation of antibodies targeting small hydrophobic molecules.