Disruption of BSEP Function in HepaRG Cells Alters Bile Acid Disposition and Is a Susceptive Factor to Drug-Induced Cholestatic Injury.

In the present study, we characterized in vitro biosynthesis and disposition of bile acids (BAs) as well as hepatic transporter expression followed by ABCB11 (BSEP) gene knockout in HepaRG cells (HepaRG-KO cells). BSEP KO in HepaRG cells led to time-dependent BA accumulation, resulting in reduced biosynthesis of BAs and altered BA disposition. In HepaRG-KO cells, the expression of NTCP, OATP1B1, OATP2B1, BCRP, P-gp, and MRP2 were reduced, whereas MRP3 and OCT1 were up-regulated. As a result, BSEP KO altered the disposition of BAs and subsequently underwent adaptive regulations of BA synthesis and homeostasis to enable healthy growth of the cells. Although BSEP inhibitors caused no or slight increase of BAs in HepaRG wild type cells (HepaRG-WT cells), excessive intracellular accumulation of BAs was observed in HepaRG-KO cells exposed to bosentan and troglitazone, but not dipyridamole. LDH release in the medium was remarkably increased in HepaRG-KO cultures exposed to troglitazone (50 µM), suggesting drug-induced cellular injury. The results revealed that functional impairment of BSEP predisposes the cells to altered BA disposition and is a susceptive factor to drug-induced cholestatic injury. In total, BSEP inhibition might trigger the processes but is not a sole determinant of cholestatic cellular injury. As intracellular BA accumulation is determined by BSEP function and the subsequent adaptive gene regulation, assessment of intracellular BA accumulation in HepaRG-KO cells could be a useful approach to evaluate drug-induced liver injury (DILI) potentials of drugs that could disrupt other BA homeostasis pathways beyond BSEP inhibition.

Correlation Analysis of Potential Breast Cancer Resistance Protein Probes in Different Monolayer Systems.

Breast cancer resistance protein (BCRP) is a point of interest in drug-drug interaction safety testing. Therefore, a consensus probe that can be applied as victim in multiple experimental settings is of great benefit. Identification of candidates has been driven by the amount and quality of available clinical data, and as a result, drugs such as sulfasalazine and rosuvastatin have been suggested. In this article, the in vitro performance of 5 possible alternatives was evaluated: atorvastatin, chlorothiazide, dantrolene, topotecan, and teriflunomide, and benchmarked against sulfasalazine and rosuvastatin in reference in vitro assays for BCRP drug-drug interaction testing. Based on the results, teriflunomide is proposed as an alternate in vitro BCRP probe.

Murine cytomegalovirus displays selective infection of cells within hours after systemic administration.

A distinctive feature of the cytomegaloviruses is their wide tissue tropism, demonstrated by the infection of many organs and cell types in an active infection. However, in experimental models of systemic infection, the earliest stages of infection are not well characterized, and it is unclear whether only certain cells are initially infected. Using a recombinant murine cytomegalovirus (MCMV) expressing green fluorescent protein (GFP), we tracked viral infection after systemic administration via intraperitoneal injection and showed that specific cells are infected within the first hours. We provide evidence that MCMV traffics as free virus from the peritoneal cavity into the mediastinal lymphatics, providing access to the bloodstream. We demonstrate that MCMV productively infected CD169(+) subcapsular sinus macrophages in the mediastinal lymph nodes, ER-TR7(+) CD29(+) reticular fibroblasts in the spleen and hepatocytes. Infection in the spleen followed a distinctive pattern, beginning in the marginal zone at 6 h and spreading into the red pulp by 17 h. By 48 h after infection, there was widespread infection in the spleen and liver with degeneration of infected cells. In addition, infected dendritic cells appeared in the white pulp of the spleen at 48 h post-infection. On the other hand, cowpox virus showed a different pattern of infectivity in the spleen and liver. Thus, early MCMV infection produces a distinct pattern of infection of selective cells.

Chronic lymphocytosis of functionally immature natural killer cells.

BACKGROUND: The development of natural killer (NK) cells in the bone marrow is not well characterized. We recently described a mouse (referred to as an NK cell-deficient [NKD] mouse) with a selective deficiency in NK cells caused by the insertion of a transgene construct into the genetic locus for the basic leucine zipper transcription factor ATF-2. NK cells in this mouse were both phenotypically and functionally immature and accumulated in the bone marrow at a stage at which constitutive NK cell proliferation occurs in wild-type mice. OBJECTIVE: We hypothesized that excess IL-15 could potentially overcome this developmental block, allowing normal emigration of mature NK cells from the bone marrow to the periphery. METHODS: Double-transgenic mice were generated by crossing the NKD mice with transgenic mice overexpressing IL-15. RESULTS: The double-transgenic mice had a dramatic accumulation of phenotypically immature NK cells in the bone marrow and subsequently in the blood, liver, and spleen. NK cells from these double-transgenic mice manifested functional deficits similar to those observed in NK cells from NKD mice, as assessed by decreased cytokine production and cytotoxicity. CONCLUSION: Rather than bypass the observed developmental defect in NKD mice, excess IL-15 drove a massive accumulation of phenotypically and functionally immature NK cells in the bone marrow and periphery. CLINICAL IMPLICATIONS: We propose that these double-transgenic mice will serve as a murine model of chronic NK cell lymphocytosis in human patients.

Arrested natural killer cell development associated with transgene insertion into the Atf2 locus.

Natural killer (NK) cell development in the bone marrow is not fully understood. Following lineage commitment, these cells appear to advance through a series of developmental stages that are beginning to be characterized. We previously reported a selective deficiency of NK cells in a C57BL/6 mouse with a transgenic construct consisting of the cDNA for the Ly49A major histocompatibility complex (MHC) class 1-specific inhibitory receptor driven by the granzyme A gene. This mouse has few NK cells in peripheral tissues with relative preservation of other immune cells, including T and B cells. Herein we demonstrate that these mice have an accumulation of NK cells with an immature phenotype in the bone marrow, consistent with a block at a previously proposed stage in normal NK-cell development. The phenotype is associated with transgenic insertion into Atf2, the gene for the basic leucine zipper (bZIP) transcription factor family member ATF-2. Although analysis of Atf2-null NK cells shows no defect, the transgenic mice express abnormal truncated Atf2 transcripts that may mediate a repressor effect because ATF2 can heterodimerize with other bZIP molecules. The defect is cell intrinsic, suggesting that certain bZIP molecules play significant roles in NK-cell development.