Drug transporter expression and activity in cryopreserved human hepatocytes isolated from chimeric TK-NOG mice with humanized livers.

Chimeric mice with humanized liver are thought to represent a sustainable source of isolated human hepatocytes for in vitro studying detoxification of drugs in humans. Because drug transporters are now recognized as key-actors of the hepatic detoxifying process, the present study was designed to characterize mRNA expression and activity of main hepatic drug transporters in cryopreserved human hepatocytes isolated from chimeric TK-NOG mice and termed HepaSH cells. Such cells after thawing were shown to exhibit a profile of hepatic solute carrier (SLC) and ATP-binding cassette (ABC) drug transporter mRNA levels well correlated to those found in cryopreserved primary human hepatocytes or human livers. HepaSH cells used either as suspensions or as 24 h-cultures additionally displayed notable activities of uptake SLCs, including organic anion transporting polypeptides (OATPs), organic anion transporter 2 (OAT2) or sodium-taurocholate co-transporting polypeptide (NTCP). SLC transporter mRNA expression, as well as SLC activities, nevertheless fell in HepaSH cells cultured for 120 h, which may reflect a partial dedifferentiation of these cells with time in culture in the conventional monolayer culture conditions used in the study. These data therefore support the use of cryopreserved HepaSH cells as either suspensions or short-term cultures for drug transport studies.

The Objective Stretch Marks Photonumeric Assessment Scale: A New and Complete Method to Assess Striae Distensae.

BACKGROUND: Striae distensae evaluation criteria have been recently described, but none is focused on objective striae assessment. With the purpose of better and objectively estimating the severity of striae distensae, the Objective Stretch Marks Assessment Scale has been developed by the authors' team. METHODS: Seven hundred White patients were included in the study and assessed. To assess the severity of striae distensae, abdomen, breasts, hips, gluteal area, back area, thighs, calves, and upper limbs photonumeric grading scales were developed. The Rasch model was used as part of the validation process. A score was attributed to each patient, based on the scales we developed. The interrater reliability and test-retest reliability were analyzed. RESULTS: Eight photonumeric scales for striae distensae treatment outcomes assessment were developed. All scales exceeded criteria for acceptability, reliability and validity. The interrater and intrarater reliabilities were good, with a substantial or virtually perfect interrater reliability for the total score (P = 0.16). CONCLUSIONS: The authors' results allowed them to validate the Objective Stretch Marks Assessment Scale as a reliable and reproducible tool to assess striae distensae treatment outcomes. This scale could be also considered as an important new metric that can be used in clinical research.

Liposome-Mediated Gene Transfer in Differentiated HepaRG™ Cells: Expression of Liver Specific Functions and Application to the Cytochrome P450 2D6 Expression.

The goal of this study was to establish a procedure for gene delivery mediated by cationic liposomes in quiescent differentiated HepaRG™ human hepatoma cells. We first identified several cationic lipids promoting efficient gene transfer with low toxicity in actively dividing HepG2, HuH7, BC2 and progenitor HepaRG™ human hepatoma cells. The lipophosphoramidate Syn1-based nanovector, which allowed the highest transfection efficiencies of progenitor HepaRG™ cells, was next used to transfect differentiated HepaRG™ cells. Lipofection of these cells using Syn1-based liposome was poorly efficient most likely because the differentiated HepaRG™ cells are highly quiescent. Thus, we engineered the differentiated HepaRG™ Mitogenic medium supplement (ADD1001) that triggered robust proliferation of differentiated cells. Importantly, we characterized the phenotypical changes occurring during proliferation of differentiated HepaRG™ cells and demonstrated that mitogenic stimulation induced a partial and transient decrease in the expression levels of some liver specific functions followed by a fast recovery of the full differentiation status upon removal of the mitogens. Taking advantage of the proliferation of HepaRG™ cells, we defined lipofection conditions using Syn1-based liposomes allowing transient expression of the cytochrome P450 2D6, a phase I enzyme poorly expressed in HepaRG cells, which opens new means for drug metabolism studies in HepaRG™ cells.

Striae Distensae: In Vitro Study and Assessment of Combined Treatment With Sodium Ascorbate and Platelet-Rich Plasma on Fibroblasts.

INTRODUCTION: Striae distensae (SD) appear clinically as parallel striae, lying perpendicular to the tension lines of the skin. SD evolve into two clinical phases, an initial inflammatory phase in which they are called "striae rubrae" (SR) and a chronic phase in which they are called striae albae (SA). Fibroblasts seem to play a key role in the pathogenesis of stretch marks. This study was aimed at describing and analyzing stretch marks-derived fibroblasts (SMF), the differences between SR- and SA-derived fibroblasts (SRF, SAF), testing two treatments in vitro (sodium ascorbate and PrP) on SAF. MATERIAL AND METHODS: To characterize the SMF, the expression of alpha smooth muscle actin (alpha SMA) was investigated. Type I collagen expression was measured in SAF, before and after adding different PrP concentrations and sodium ascorbate in the culture medium. Results were processed through statistical analysis models using the Student's t-test. RESULTS: A significant increase in alpha SMA (P <0.001) was observed in SRF. SAF treated with PrP and sodium ascorbate showed a resumption of their metabolic activity by an increase in collagen type I production and cell proliferation. After 24 h of incubation with PrP 1% and PrP 5% + sodium ascorbate, cell viability was increased by 140% and 151% and by 156 and 178% after 48 h, respectively, compared to the control. CONCLUSION: Our study shows that a biologically mediated improvement in SMF metabolic activity is possible. Our promising results require further trials to be able to confirm the reproducibility of this combined treatment, particularly in vivo. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable.

Recruitment of CXCR3+ T cells into injured tissues in adult IgA vasculitis patients correlates with disease activity.

OBJECTIVES: Adult immunoglobulin A vasculitis (IgAV) is an immune complex small vessel vasculitis. So far, the involvement of T cells in this pathology has been poorly studied. The aim of this study was to analyze T-cell homeostasis as well as cytokine and chemokine concentrations in the blood and tissues of IgAV patients. METHODS: T cells, cytokine and chemokine concentrations were analyzed in peripheral blood using flow cytometry and multiplex assays. T-cell infiltrates in the kidney and the skin were characterized by immunohistochemistry. This study prospectively included 44 adult patients with biopsy-proven IgAV and 24 age- and sex-matched healthy controls. RESULTS: We observed reduced proportions of circulating CXCR5-and CXCR3-expressing memory CD4 T cells at diagnosis but normal values at remission. The plasma levels of Th1-related cytokines (IL-12, IL-27 and IFNγ) and of the TFH-related cytokine, IL-21, were paradoxically not reduced in patients. We observed increased plasma concentrations of the CXCR5 ligand, CXCL13, and of the CXCR3 ligands, CXCL10/11, suggesting a potential relocation of the corresponding T cells into inflamed tissues. We then confirmed the recruitment of CXCR3-expressing T cells into the skin and kidneys. In the skin, T-cell infiltrates mainly co-localized with damaged dermal small vessels. Finally, patients with the largest kidney T-cell infiltrates were also those with the highest proteinuria. CONCLUSION: Altogether, our results strongly suggest that, in IgAV patients, CXCL10/11 orchestrate the recruitment of CXCR3-expressing T cells in injured tissues, contributing to tissue damage and disease activity.

Idiopathic nephrotic syndrome: the EBV hypothesis.

Steroid sensitive nephrotic syndrome is marked by a massive proteinuria and loss of podocytes foot processes. The mechanism of the disease remains debated but recent publications suggest a primary role of Epstein-Barr Virus (EBV). EBV replication in the peripheral blood is found in 50% of patients during the first flare of the disease. The genetic locus of steroid sensitive nephrotic syndrome was also identified as influencing antibodies directed against EBNA1. EBV is able to establish, latent benign infection in memory B cells that display phenotypes similar to antigen-selected memory B cells. Consistently, memory B cells reconstitution after rituximab infusion is a predictor of the relapse of proteinuria. We suggest that a specific anti-EBNA1 antibody internalized in the podocytes via the neonatal Fc receptor might cross-react with a major protein present in the same cell trafficking compartment. The diversion of this major podocyte protein in the urinary space and the subsequent depletion is supposed to result in podocyte damages with loss of foot processes and massive proteinuria. Immunosuppression of B cells and subsequent clearance of anti-EBNA1 antibodies would lead to a restoration of the normal level of the protein allowing recovery of proteinuria and of normal podocyte morphology.

Fatal effects of a neonatal high-protein diet in low-birth-weight piglets used as a model of intrauterine growth restriction.

BACKGROUND: Although full-term infants suffering intrauterine growth restriction (IUGR) are routinely fed high-protein (HP) formulas to ensure catch-up growth, the effects of HP intake are poorly understood. An IUGR piglet model provides an opportunity to investigate these effects. METHODS AND RESULTS: Twelve IUGR piglets were artificially fed HP formulas (50% more protein in comparison to sow milk) from the 2nd day of life (d2) until d28. Unexpectedly, all HP piglets developed poor growth, severe hypotonia and polypnea between d10 and d16. One third died spontaneously. This syndrome was investigated to understand its pathophysiology and to adopt a strategy to restore health. Blood and urine biochemistry and amino acid concentrations were investigated in 10 HP piglets and 8 piglets that were fed a normal-protein (NP) formula. In comparison to NP piglets, HP piglets showed significant hypokalemia (2.7 +/- 0.6 vs. 3.6 +/- 0.6 mmol/l; p < 0.01), hypophosphatemia (1.5 +/- 0.2 vs. 3.0 +/- 0.3 mmol/l; p > 0.01), hypercalcemia (3.0 +/- 0.3 vs. 2.5 +/- 0.2 mmol/l; p < 0.01), hyperammonemia (365 +/- 4 vs. 242 +/- 15 micromol/l; p < 0.05), elevated blood urea (6.5 +/- 0.4 vs. 1.3 +/- 0.4 mmol/l; p < 0.01) and elevated taurine concentrations (50.2 +/- 8.5 vs. 17.7 +/- 2.7 micromol/l; p < 0.01). CONCLUSIONS: These altered parameters indicated inadequate potassium and phosphorus dietary supplies in HP piglets. When the HP formula was supplemented with monocalcium phosphate and monopotassium phosphate (HP-sup), serum biochemistry was normalized in piglets fed this formula (n = 8). This experimental strategy restored growth in IUGR piglets fed HP-sup, without a toxic effect. The current findings suggest that use of an HP formula without a proportional increase in its phosphorus and potassium content induces pathology similar to the refeeding syndrome in IUGR piglets.

Maternal stress during late gestation has moderate but long-lasting effects on the immune system of the piglets.

Events acting prenatally on developing foetuses are important determinants for disorders later in life. Prenatal stress (PNS) is one of these events. The purpose of this study was to determine the consequences of a repeated social stress applied during late gestation of the pregnant gilt on the immune system and hypothalamo-pituitary-adrenal (HPA) axis activity of the piglets from birth to two months of age. Pregnant gilts were submitted to repeated social stress which was induced by housing unfamiliar gilts in pairs modified twice a week during 4 weeks between days 77 and 105 of gestation (S group, n=18). Control gilts were housed in stable pairs during the same period (C group, n=18). Blood cortisol, haptoglobin and IgG levels, immune cell counts, mitogen-induced whole-blood proliferation and TNF-alpha production were evaluated in piglets at 4 days of age (D4), before and after weaning (D26 and 28) and before and after relocation to a new building (D60 and 62). We found that PNS did not affect growth rate of the progeny. It decreased the relative weight of adrenal glands on D4 (P<0.05) but plasma cortisol levels were similar in both groups at all ages. IgG levels in colostrum and in the serum of piglets were not affected. PNS decreased the total numbers of white blood cells, lymphocytes and granulocytes from D26 to D60 (P<0.05), the CD4(+)/CD8(+) T cell ratio on D4 (P<0.05), and LPS induced-TNF-alpha production on D60 (P<0.05). PNS increased the ConA-induced lymphocyte proliferation on D4 and D60 and the PWM-induced proliferation on D60 (P<0.05). Our results demonstrate that a repeated social stress applied to pregnant sows during late gestation can induce long-lasting effects on several parameters of the immune function of the offspring. These effects are not due to modifications of the HPA axis activity and may impair the abilities of the piglets to efficiently react against infections during the suckling period and around weaning.

Classical swine fever virus induces activation of plasmacytoid and conventional dendritic cells in tonsil, blood, and spleen of infected pigs.

Classical swine fever virus (CSFV) compromises the host immune system, causing indirect leucopoenia and disruption of in vitro T cell stimulation capacity. In order to explore the potential role of dendritic cells (DC) in such phenomena, the activation of conventional DC (cDC) and plasmacytoid DC (pDC) in blood and secondary lymphoid organs of infected pigs was investigated in the early time course post-inoculation (pi), together with viral components dissemination and cytokine production in serum. Whereas CD11R1+CD172a+ cDC frequencies were markedly reduced in blood and spleen, analysis of CD4+CD172a+ pDC numbers revealed a rapid turn-over of this DC subset in tissues pi. Both subsets matured and were activated after infection, as demonstrated by down-regulation of CD1a, up-regulation of the co-stimulation molecule CD80/86 and expression of cytokines. cDC essentially expressed tumor necrosis factor alpha (TNF-alpha) and interleukin (IL)-10, whereas pDC produced alpha interferon (IFN-alpha) and IL-12. IFN-alpha and TNF-alpha productions revealed an enhancement of innate anti-viral immune responses. Detection of antigen activated B lymphocytes in tonsil T-cell areas at 72 h pi, subsequently to the transient translocation of the viral E2 protein within germinal centres at 48 h pi, indicates the initiation of humoral response. This response was also evidenced by an important IL-10 production in serum one week pi. IL-12 expression in organs, as well as transient detection of IL-18 and IFN-gamma in serum, reflected the initiation of cellular immune responses. However, the uncommonly high levels of TNF-alpha and IFN-alpha produced by DC and measured in serum early post-infection, together with IL-10 expression in spleen, could play a role in the disruption of immune system cells, either inducing apoptosis or impairing DC functionalities themselves.