The liver is a critical tissue for maintaining glucose, fatty acid, and cholesterol homeostasis. Primary hepatocytes represent the gold standard for studying the mechanisms controlling hepatic glucose, lipid, and cholesterol metabolism in vitro. However, access to primary hepatocytes can be limiting, and therefore, other immortalized hepatocyte models are commonly used. Here, we describe substrate metabolism of cultured AML12, IHH, and PH5CH8 cells, hepatocellular carcinoma-derived HepG2s, and primary mouse hepatocytes (PMH) to identify which of these cell lines most accurately phenocopy PMH basal and insulin-stimulated metabolism. Insulin-stimulated glucose metabolism in PH5CH8 cells, and to a lesser extent AML12 cells, responded most similarly to PMH. Notably, glucose incorporation in HepG2 cells were 14-fold greater than PMH. The differences in glucose metabolic activity were not explained by differential protein expression of key regulators of these pathways, for example glycogen synthase and glycogen content. In contrast, fatty acid metabolism in IHH cells was the closest to PMHs, yet insulin-responsive fatty acid metabolism in AML12 and HepG2 cells was most similar to PMH. Finally, incorporation of acetate into intracellular-free cholesterol was comparable for all cells to PMH; however, insulin-stimulated glucose conversion into lipids and the incorporation of acetate into intracellular cholesterol esters were strikingly different between PMHs and all tested cell lines. In general, AML12 cells most closely phenocopied PMH in vitro energy metabolism. However, the cell line most representative of PMHs differed depending on the mode of metabolism being investigated, and so careful consideration is needed in model selection.

The acute phase protein serum amyloid A (SAA), a marker of inflammation, induces expression of pro-inflammatory and pro-thrombotic mediators including ICAM-1, VCAM-1, IL-6, IL-8, MCP-1 and tissue factor (TF) in both monocytes/macrophages and endothelial cells, and induces endothelial dysfunction—a precursor to atherosclerosis. In this study, we determined the effect of pharmacological inhibition of known SAA receptors on pro-inflammatory and pro-thrombotic activities of SAA in human carotid artery endothelial cells (HCtAEC). HCtAEC were pre-treated with inhibitors of formyl peptide receptor-like-1 (FPRL-1), WRW4; receptor for advanced glycation-endproducts (RAGE), (endogenous secretory RAGE; esRAGE) and toll-like receptors-2/4 (TLR2/4) (OxPapC), before stimulation by added SAA. Inhibitor activity was also compared to high-density lipoprotein (HDL), a known inhibitor of SAA-induced effects on endothelial cells. SAA significantly increased gene expression of TF, NFκB and TNF and protein levels of TF and VEGF in HCtAEC. These effects were inhibited to variable extents by WRW4, esRAGE and OxPapC either alone or in combination, suggesting involvement of endothelial cell SAA receptors in pro-atherogenic gene expression. In contrast, HDL consistently showed the greatest inhibitory action, and often abrogated SAA-mediated responses. Increasing HDL levels relative to circulating free SAA may prevent SAA-mediated endothelial dysfunction and ameliorate atherogenesis.

Activation of TLR2 or TLR4 by endogenous ligands such as high mobility group box 1 (HMGB1) may mediate inflammation causing diabetic kidney injury. We determined whether blockade of HMGB1 signaling by: (1) supra-physiological production of endogenous secretory Receptor for Advanced Glycation End-products (esRAGE), a receptor for HMGB1; (2) administration of HMGB1 A Box, a specific competitive antagonist, would inhibit development of streptozotocin induced diabetic nephropathy (DN). Wild-type diabetic mice developed albuminuria, glomerular injuries, interstitial fibrosis and renal inflammation. Using an adeno-associated virus vector, systemic over-expression of esRAGE afforded significant protection from all parameters. No protection was achieved by a control vector which expressed human serum albumin. Administration of A Box was similarly protective against development of DN. To determine the mechanism(s) of protection, we found that whilst deficiency of TLR2, TLR4 or RAGE afforded partial protection from development of DN, over-expression of esRAGE provided additional protection in TLR2−/−, modest protection against podocyte damage only in TLR4−/− and no protection in RAGE−/− diabetic mice, suggesting the protection provided by esRAGE was primarily through interruption of RAGE and TLR4 pathways. We conclude that strategies to block the interaction between HMGB1 and its receptors may be effective in preventing the development of DN.

Adeno-associated viral vector-mediated (AAV-mediated) expression of allogeneic major histocompatibility complex class I (MHC class I) in recipient liver induces donor-specific tolerance in mouse skin transplant models in which a class I allele (H-2Kb or H-2Kd) is mismatched between donor and recipient. Tolerance can be induced in mice primed by prior rejection of a donor-strain skin graft, as well as in naive recipients. Allogeneic MHC class I may be recognized by recipient T cells as an intact molecule (direct recognition) or may be processed and presented as an allogeneic peptide in the context of self-MHC (indirect recognition). The relative contributions of direct and indirect allorecognition to tolerance induction in this setting are unknown. Using hepatocyte-specific AAV vectors encoding WT allogeneic MHC class I molecules, or class I molecules containing a point mutation (D227K) that impedes direct recognition of intact allogeneic MHC class I by CD8+ T cells without hampering the presentation of processed peptides derived from allogeneic MHC class I, we show here that tolerance induction depends upon recognition of intact MHC class I. Indirect recognition alone yielded a modest prolongation of subsequent skin graft survival, attributable to the generation of CD4+ Tregs, but it was not sufficient to induce tolerance.

The determination of the sequence selectivity of DNA-damaging agents is very important in elucidating the mechanism of action of anti-tumour drugs. The development of automated capillary DNA sequencers with fluorescent labelling has enabled a more precise method for DNA sequence specificity analysis. In this work we utilized the ABI 3730 capillary sequencer with laser-induced fluorescence to examine the sequence selectivity of cisplatin with purified DNA sequences. The use of this automated machine enabled a higher degree of precision of both position and intensity of cisplatin-DNA adducts than previously possible with manual and automated slab gel procedures. A problem with artefact bands was overcome by ethanol precipitation. It was found that cisplatin strongly formed adducts with telomeric DNA sequences.

The blood kinetics and tissue distribution of a conjugate of daunomycin and a monoclonal antibody (791T/36) have been examined in mice, including nude mice with human tumour xenografts reactive with the antibody. For this the antibody moiety of the conjugate was labelled with 125I and the drug moiety assayed by radioimmunoassay. After radioiodination, the preparation had an immunoreactive fraction in isotopic binding tests with 791T cells of 74%. Both drug and antibody moieties were precipitable with anti-mouse Ig anti-serum. Following i.v. injection, blood clearance of the two components of the conjugate was essentially identical, and with the serum-borne conjugate both radiolabel and drug were co-precipitable. In mice with 791T xenografts, the tumour showed localisation of both drug and antibody moieties and at the time of analysis (3 days) tumour levels of drug were over 100 times those seen with free drug. In parallel studies with mice with antigen negative xenografts, there was no preferential localisation of antibody or drug moieties of the conjugate. These studies have shown in vivo stability of this conjugate, and site-specific targetting of an anti-tumour anthracycline.

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While direct allorecognition underpins both solid organ allograft rejection and tolerance induction, the specific molecular targets of most directly-alloreactive CD8 + T cells have not been defined. In this study, we used a combination of genetically-engineered MHC class I (MHC I) constructs, mice with a hepatocyte-specific mutation in the class I antigen-presentation pathway and immunopeptidomic analysis to provide definitive evidence for the contribution of the peptide cargo of allogeneic MHC I molecules to transplant tolerance induction. We established a systematic approach for the discovery of directly-recognised pMHC epitopes, and identified 17 strongly immunogenic H-2K b -associated peptides recognised by CD8 + T cells from B10.BR (H-2 k ) mice, 13 of which were also recognised by BALB/c (H-2 d ) mice. As few as five different tetramers used together were able to identify a high proportion of alloreactive T cells within a polyclonal population, suggesting that there are immunodominant allogeneic MHC-peptide complexes that can account for a large component of the alloresponse. 5 Results Single chain trimer constructs exclude presentation of endogenous peptides.In preceding studies(1, 3), we have used AAV vectors encoding the donor MHC I HC. Within transduced hepatocytes, allogeneic HC associates with native b2m and the resulting heterodimers are loaded with a repertoire of endogenous peptides (Figure 1A). To express allogeneic MHC I at high levels on recipient hepatocytes while excluding the presentation of naturally processed peptides, we engineered SCT constructs, each encoding the HC of H-2K b , b2m and a single, defined H-2K b -restricted peptide [SIINFEKL (SIIN) or KIITYRNL (KIIT), Figure 1A], and packaged them in hepatocyte-specific AAV2/8 vectors.Sequences are shown in Supplementary Figure 1. Transgene expression in hepatocytes was close to maximal by d7 following intravenous (iv) inoculation, and persisted through to at least d100, no significant increases in serum aspartate aminotransferase (AST) or alanine aminotransferase (ALT) levels were observed, and minimal cellular infiltration was detected by histology (Supplementary Figure 2). SCT molecules were expressed on transduced hepatocytes at equivalent levels to the heterotrimer formed by transgenic H-2K b HC with native b2m and peptide (Figure 1B). To demonstrate exclusion of naturally processed peptides, we co-transduced B10.BR (H-2 k ) hepatocytes with AAV vectors encoding full-length chicken ovalbumin (OVA) and either HC-K b or SCT-K b -KIIT, and stained them with a monoclonal antibody, 25D-1.16, which is specific for the OVA peptide SIINFEKL complexed with K b . K b -SIINFEKL was only detected at the surface of cells co-transduced with HC-K b and not those expressing SCT-K b -KIIT (Figure 1C-D). We extended this analysis to the broader endogenous peptide repertoire of K b -transduced hepatocytes using immunoaffinity purification with the H-2K b -specific antibody K9-178, followed by reverse phase high performance liquid chromatography (RP-HPLC...