Natural killer-cell deficiency alters placental development in rats.

Natural killer (NK) cells are the most prevalent leukocyte population in the uterus during early pregnancy. Natural killer cells contribute to uterine vascular (spiral artery) remodeling in preparation for the increased demand on these vessels later in pregnancy. A second wave of spiral artery modification is directed by invasive trophoblast cells. The significance of the initial wave of NK-cell-mediated vascular remodeling in species exhibiting deep trophoblast invasion such as humans and rats is not known. The purpose of this study was to generate a genetic model of NK-cell deficiency in rats, and determine the consequences of NK-cell deficiency on spiral artery remodeling and reproductive outcomes. To accomplish this task, we utilized zinc finger nuclease-mediated genome editing of the rat interleukin-15 (Il15) gene. Il15 encodes a cytokine required for NK-cell lineage development. Using this strategy, a founder rat was generated containing a frameshift deletion in Il15. Uteri of females harboring a homozygous mutation at the Il15 locus contained no detectable NK cells. NK-cell deficiency did not impact fetal growth or viability. However, NK-cell deficiency caused major structural changes to the placenta, including expansion of the junctional zone and robust, early-onset activation of invasive trophoblast-guided spiral artery remodeling. In summary, we successfully generated an NK-cell-deficient rat and showed, using this model, that NK cells dampen the extent of trophoblast invasion and delay trophoblast-directed spiral artery remodeling. This study furthers our understanding of the role of NK cells on uterine vascular remodeling, trophoblast invasion, and placental development.

A focused microarray for screening rat embryonic stem cell lines.

Here, we describe a focused microarray for screening rat embryonic stem cells (ESCs) and provide validation data that this array can distinguish undifferentiated rat ESCs from rat trophoblast stem (TS) cells, rat extraembryonic endoderm cells, mouse embryonic fibroblast feeder cells, and differentiated rat ESCs. Using this tool, genuine rat ESC lines, which have been expanded in a conventional rat ESC medium containing two inhibitors (2i), for example, glycogen synthase kinase 3 (GSK3) and mitogen-activated protein kinase (MEK) inhibitors, and leukemia inhibitory factor, and genuine rat ESCs, which have been expanded in rat ESC medium containing four inhibitors (4i), for example, GSK3, MEK, Alk5, and Rho-associated kinase inhibitors were compared; as were genuine rat ESCs from 4 different strains of rats. Expression of Cdx2, a gene associated with trophoblast determination, was observed in genuine, undifferentiated rat ESCs from 4 strains and from both 2i and 4i ESC derivation medium. This finding is in contrast to undifferentiated mouse ESCs that do not express Cdx2. The rat ESC focused microarray described in this report has utility for rapid screening of rat ESCs. This tool will enable optimization of culture conditions in the future.

Administration of PPARß/δ agonist reduces copper-induced liver damage in mice: possible implications in clinical practice.

In this study we investigated if peroxisome proliferator-activated receptor ß/δ activation protects from copper-induced acute liver damage. Mice treated with copper had significant body weight loss, serum alanine aminotransferase increase, modest changes in liver histology, increase of tumor necrosis factor α and macrophage inflammatory protein 2 mRNA and 8-hydroxy-2'-deoxyguanosine. Mice treated with copper and peroxisome proliferator-activated receptor ß/δ agonist GW0742 had significantly less body weight loss, less serum alanine aminotransferase increase, less tumor necrosis factor α, macrophage inflammatory protein-2 and 8-hydroxy-2'-deoxyguanosine upregulation than copper treated mice. The opposite effect was observed in mice treated with copper and peroxisome proliferator-activated receptor ß/δ antagonist GSK0660. In vitro, copper induced reactive oxygen species, which was lower in cells treated with GW0742 or transfected with peroxisome proliferator-activated receptor ß/δ expression vector; together, transfection and GW0742 had an additive reactive oxygen species-reducing effect. Copper also upregulated Fas ligand and Caspase 3/7 activity, effects that were significantly lower in cells also treated with GW0742. In conclusion, peroxisome proliferator-activated receptor ß/δ activation reduced copper-induced reactive oxygen species, pro-inflammatory and acute phase reaction cytokines in mice liver. Peroxisome proliferator-activated receptor ß/δ agonists could become useful in the management of copper-induced liver damage.

Therapeutic targeting of toll-like receptors in gastrointestinal inflammation.

Toll-like receptors (TLRs) are sensors of microbial products that initiate host defense responses in multicellular organisms. They are mainly linked to innate immunity and bridging to adaptive immunity, signaling through different TLRs responsible for a wide range of biological responses. The intracellular signaling pathways through Toll/interleukin-1 receptor (IL-1R) domains result in recruitment of the cytoplasmic adaptor molecules, with subsequent activation of a signaling cascade leading to nuclear factor-kappa B (NF-kappaB). TLR-signaling induces host inflammatory response and the inflammation becomes more severe in the absence of several extra and intra cellular negative regulators of TLR-signaling. In the intestine, TLR-dependent activation of NF-kappaB plays a vital role in maintaining epithelial homeostasis as well as regulating infections and inflammation, while dysregulation of TLR-signaling is associated with the pathogenesis of inflammatory bowel diseases (IBD). Recent findings regarding innate immunity-mediated regulation of intestinal pathophysiology prove that development of new drugs targeting TLRs including antagonists of TLR-signaling and agonists of their negative regulators has a potential impact on therapeutic strategies for intestinal inflammatory diseases.

Kinetics of indocyanine green removal from blood can be used to predict the size of the area removed by radiofrequency ablation of hepatic nodules.

BACKGROUND AND AIM: The size of radiofrequency ablation (RFA) in the liver can be negatively influenced by the surrounding blood flow. The indocyanine green (ICG) test can be used to evaluate the effective blood flow in the liver, and distance from the hilus can affect local blood flow. The aim of this study was to assess whether the ICG test or distance from the hilus could be used to predict the size of the ablated area in liver by RFA treatment of hepatocellular carcinoma (HCC) nodules. METHODS: The RFA measurements of 44 HCC nodules in 39 patients were retrospectively studied. Cases were included if they met the following criteria: (i) no catheter treatment before RFA; (ii) no movement of the RFA device; (iii) strict ablation time; and (iv) only one ablation. In all patients, ICG-R15 testing was done immediately before RFA and the initial therapeutic efficacy was evaluated by dynamic computed tomography scanning 2-5 days after RFA. The correlation between the maximum size of the RFA area and the ICG test results or the distance of the target area from the hilus (site of first portal vein divergence) were analyzed statistically. RESULTS: The ICG-R15 result was significantly correlated with the maximum diameter of the ablated area both in 2 cm-electrode tip length (R2 = 0.35, P = 0.0012), and in 3 cm-tip length (R2 = 0.26, P = 0.0377). Multiple-regression analysis showed that the electrode tip length (P = 0.0010) and ICG-R15 (P = 0.0046) were independent factors that could predict the maximum diameter of the RFA area. CONCLUSION: The results of ICG testing can be used to predict the size of the area that will be ablated at a target liver site before RFA treatment.

Epithelial toll-like receptor 5 is constitutively localized in the mouse cecum and exhibits distinctive down-regulation during experimental colitis.

We recently demonstrated that the pattern recognition receptors (PRRs) toll-like receptor 2 (TLR2), TLR4, and CD14 are expressed in mouse colonic epithelium in a compartmentalized manner. Here we report the localization of TLR5, the receptor for bacterial flagellin, and its distinctive down-regulation during experimental colitis. Guts from normal BALB/c mice and those with dextran sodium sulfate (DSS)-induced colitis were compared. Each gut was divided into seven segments (stomach, small intestine [three parts], and colon [three parts]), and epithelial cells and crypt units were collected by scraping and EDTA treatment, respectively. Northern blotting showed that TLR5 mRNA was preferentially expressed in the epithelium of the proximal colon in normal mice. Laser capture microdissection coupled to reverse transcriptase PCR confirmed this localization. TLR5 protein expression reflected mRNA expression, as evidenced by Western blotting. In mice with acute colitis, inflammation occurred mainly in the distal colon. Interestingly, while TLR2, TLR4, and CD14 were up-regulated in the inflamed colon, TLR5 was down-regulated at both the mRNA and protein levels. Decreased TLR5 expression was more evident during chronic colitis. Additional in vitro studies using a mouse cell line, Colon-26, showed that gamma interferon (IFN-gamma) time- and dose-dependently down-regulates TLR5. In conclusion, epithelial cells, mainly in the proximal colon, constitutively express TLR5. TLR5 expression is down-regulated in vivo during acute and chronic DSS-induced colitis, in contrast to the expression of TLR2, TLR4, and CD14. The mechanism governing TLR5 regulation may therefore differ from that controlling other PRRs. Finally, IFN-gamma may be involved in down-regulating TLR5 expression.

Transforming growth factor-alpha directly augments histidine decarboxylase and vesicular monoamine transporter 2 production in rat enterochromaffin-like cells.

For the production and vesicle storage of histamine, Enterochromaffin-like (ECL) cells express histidine decarboxylase (HDC) and vesicular monoamine transporter 2 (VMAT2). Although HDC and VMAT2 show dynamic changes during gastric ulcer healing, the control system of their expression has not been fully investigated. In the present study, we investigated the effect of transforming growth factor-alpha (TGF-alpha) and proinflammatory cytokines on HDC and VMAT2 expression in rat ECL cells. Time course changes in the expression of TGF-alpha during the healing of acetic acid-induced ulcers were studied. EGF receptor (EGFR) expression was also examined in ECL cells, whereas the direct effects of TGF-alpha and proinflammatory cytokines on HDC and VMAT2 expression in ECL cells were investigated using in vivo and in vitro models. During the process of ulcer healing, expression of TGF-alpha mRNA was markedly augmented. Furthermore, EGFR was identified in isolated ECL cells. TGF-alpha stimulated HDC and VMAT2 mRNA expression and protein production and also increased histamine release from ECL cells. Selective EGFR tyrosine kinase inhibitor tyrphostin AG1478 almost completely inhibited HDC and VMAT2 gene expression induced by TGF-alpha in vivo and in vitro. During gastric mucosal injury, TGF-alpha was found to stimulate ECL cell functions by increasing HDC and VMAT2 expression.

Strategic compartmentalization of Toll-like receptor 4 in the mouse gut.

Pattern recognition receptors (PRRs), which include the Toll-like receptors (TLRs), are involved in the innate immune response to infection. TLR4 is a model for the TLR family and is the main LPS receptor. We wanted to determine the expression of TLR4 and compare it with that of TLR2 and CD14 along the gastrointestinal mucosa of normal and colitic BALB/c mice. Colitis was induced with 2.5% dextran sodium sulfate (DSS). Mucosa from seven segments of the digestive tract (stomach, small intestine in three parts, and colon in three parts) was isolated by two different methods. Mucosal TLR4, CD14, TLR2, MyD88, and IL-1beta mRNA were semiquantified by Northern blotting. TLR4 protein was determined by Western blotting. TLR4/MD-2 complex and CD14 were evaluated by immunohistochemistry. PRR genes were constitutively expressed and were especially stronger in colon. TLR4 and CD14 mRNA were increased in the distal colon, but TLR2 mRNA was expressed more strongly in the proximal colon, and MyD88 had a uniform expression throughout the gut. Accordingly, TLR4 and CD14 protein levels were higher in the distal colon. TLR4/MD-2 and CD14 were localized at crypt bottom epithelial cells. TLR4/MD2, but not CD14, was found in mucosal mononuclear cells. Finally, DSS-induced inflammation was localized in the distal colon. All genes studied were up-regulated during DSS-induced inflammation, but the normal colon-stressed gut distribution was preserved. Our findings demonstrate that TLR4, CD14, and TLR2 are expressed in a compartmentalized manner in the mouse gut and provide novel information about the in vivo localization of PRRs.

Hepatocyte growth factor expression in dextran sodium sulfate-induced colitis in rats.

Hepatocyte growth factor (HGF), a potent inducer of cell migration with morphogenic and mitogenic actions was reported to have key roles in the repair of various tissues. In order to evaluate the role of HGF in the repair process of inflammatory bowel disease, we have investigated the HGF expression in a dextran sodium sulfate (DSS) colitis model. We randomly assigned rats to a colitis group or to a placebo group; the former received a 7-day course of 5% DSS (mw 5 kDa) in drinking water. DSS-induced severe colitis in rats manifested with weight loss, diarrhea, and intestinal bleeding. Animals were killed from day 1 through 7 and on days 9 and 14 after the end of DSS administration. After DSS was withdrawn, disease activity subsided gradually and HGF expression was significantly enhanced along with the augmented expression of IL-1beta, TNF-alpha, and cyclooxygenase-2, accompanied by an increased number of proliferating epithelial cells in colon. These findings suggest that proinflammatory cytokines and cyclooxygenase-2 may have an important role in the mucosal repair in inflammatory bowel disease through increased production of HGF.