S7100 Sigma-AldrichApopTag® Peroxidase In Situ Apoptosis Detection Kit

Hepatitis B virus (HBV) is a major etiological factor of hepatocellular carcinoma (HCC). However, the precise pathogenetic mechanisms linking HBV infection and HCC remain uncertain. It has been reported that decreased antioxidant enzyme activities are associated with severe liver injury and hepatocarcinogenesis in mouse models. It is unclear if HBV can interfere with the activities of antioxidant enzymes. We established a HBV transgenic mouse line, which spontaneously developed HCC at 2 years of age. We studied the activities of the antioxidant enzymes in the liver of the HBV transgenic mice. Our results showed that the antioxidant enzymes glutathione peroxidase and superoxide dismutase 2 were down-regulated in HBV transgenic mice and correlated with JNK activation. HBV enhanced the Fas-mediated activation of caspase 6, caspase 8 and JNK without enhancing the activation of caspase 3 and hepatocellular apoptosis. As a proper redox balance is important for maintaining cellular homeostasis, these effects of HBV on the host antioxidant system and Fas-signaling may play an important role in HBV-induced hepatocarcinogenesis.

Neurotrophin-BDNF can be effectively encapsulated in nanoporous poly(L-glutamic acid) particles prepared via mesoporous silica templating. The loaded BDNF can be released in a sustained manner with retained biological activity. Animal experiments demonstrate the released BDNF can efficiently rescue the auditory neurons (as indicated by the arrows) in the cochlea of guinea pigs with sensorineural hearing loss.

Our previous study found that the artery interposed to vein did not develop atherosclerosis but rather underwent atrophic remodeling in hyperlipidemic rabbits, suggesting that local hemodynamic load was another important determinant for the development of atherosclerosis. This study focused on the cellular and molecular changes in autologous artery grafts derived from rabbits fed with high lipid diet for 1, 2, 4, 8, and 12 weeks. Thickness, area of vessel wall, and lumen area were measured and analyzed on the grafted common carotid artery (GCCA) interposed to vein and on the right common carotid artery. Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling. Both elastin and collagen of GCCA were identified by the method of double stains of elastin and collagen. Reverse transcription polymerase chain reaction was used to observe matrix metalloproteinases (MMPs) mRNA expression changes in the examined arteries. The lumen area increased gradually in control common carotid artery and remained unchanged in GCCA 3 months later, since the surgery and the start of high lipid diet, while significantly increased apoptosis was evidenced from inner to outer part of GCCA. Collagen content decreased gradually and elastic fibers remained unchanged in GCCA. At 1 week after operation, the mRNA expression of MMP(2) and MMP(9) increased significantly and returned to baseline thereafter. The artery interposed to a vein underwent atrophy, characterized by increased apoptosis in the vessel wall from intima to adventitia, possibly due to low shear stress circumference and reduced vessel collagen resulting from postsurgical upregulated MMP(2) and MMP(9) expression.

Intestinal mucositis is a serious complication of cancer chemotherapy and radiotherapy; it frequently compromises treatment and dramatically reduces the quality of life of patients. Different approaches to limit the damage to the intestine during anti-cancer therapy have been largely ineffective due to insufficient knowledge of the mechanism of mucositis development. This study aimed to define the role of TLR-2 and TLR-9 in the modulation of small intestinal damage in a model of doxorubicin-induced mucositis. Doxorubicin-induced intestinal damage was verified by a histological score (HS), analysis of leukocyte influx into the lamina propria, and determination of the number of apoptotic cells. Additionally, the activation status of glycogen synthase kinase 3? (GSK-3?) was assessed. Wild-type (WT) mice injected with doxorubicin demonstrated severe intestinal damage (HS 8.0 ± 0.81), reduction of villus length to 43.9% ± 13.7% of original length, and increased influx of leukocytes as compared to vehicle-injected mice (HS 1.33 ± 1.15). The protective effect of TLR-2 or TLR-9 deficiency was associated with a significant decrease of the HS as compared to WT mice. In the ileum, a minor reduction of villus length and a decreased number of infiltrating leukocytes and TUNEL-positive cells was observed. We demonstrate that the TLR-9 antagonist ODN2088 reduces doxorubicin-induced intestinal damage. Furthermore, we show that GSK-3? activity is inhibited in the absence of TLR-2. The protective capacity of GSK-3? suppression was observed in WT mice by inhibiting it with the specific inhibitor SB216763. Overall, our findings demonstrate that the TLR-2/GSK-3? and TLR-9 signalling pathways play a central role in the development of intestinal mucositis and we suggest a new therapeutic strategy for limiting doxorubicin-induced intestinal inflammation. Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Subacute toxicity of 14 nm nanoparticulate silver (Ag-NP) stabilised with polyvinylpyrrolidone and ionic silver in the form of silver acetate (Ag-acetate) was investigated in four-week-old Wistar rats. Animals received orally by gavage the following: vehicle control (10 ♀, 6 ♂); Ag-NP at doses: 2.25 (8 ♀), 4.5 (8 ♀) or 9 mg/kg bw/day (10 ♀, 6 ♂); or Ag-acetate 9 mg silver/kg bw/day (8 ♀) for 28 days. Clinical, haematolological and biochemical parameters, organ weights, macro- and microscopic pathological changes were investigated. Caecal bacterial phyla and their silver resistance genes were quantified. For the Ag-NP groups, no toxicological effects were recorded. For Ag-acetate, lower body weight gain (day 4-7, 11-14, 14-16, P < 0.05; overall, day 1-28, P < 0.01), increased plasma alkaline phosphatase (P < 0.05), decreased plasma urea (P < 0.05) and lower absolute (P < 0.01) and relative (P < 0.05) thymus weight were recorded. In conclusion, these findings indicate toxicity of 9 mg/kg bw/day ionic silver but not of an equimolar Ag-NP dose. This is in accordance with previously reported data showing that oral Ag-acetate, in comparison with an equimolar dose of Ag-NP, resulted in higher silver plasma and organ concentrations.

Pancreas transplantation is the definite treatment for type 1 diabetes that enables the achievement of long-term normoglycemia and insulin independence. However Post-Transplantation Pancreatitis (PTP) due to ischemia reperfusion (IR) injury and preservation is a major complication in pancreas transplantation. Owning the potential anti-inflammatory effect of Cisplatin (Cis) in liver IR injury, we have examined if Cis could attenuate PTP using a murine model. We found that Cis is able to prevent inflammatory response in PTP. Pretreatment of Cis in recipient mice reduce the impairments of the grafts and hyperamylasimea in the recipients. We documented that the protective mechanism of Cis in PTP involves improvement of microcirculation, reduction of the mononuclear cellular infiltration and apoptosis, suppression of inflammatory cytokine-cascade and inhibition of translocation of high-motility group box protein-1 (HMGB-1) from nucleus to cytoplasm. In short, our study demonstrated that pretreatment of Cis in recipients may reduce the onset of PTP in pancreas transplantation.

The effect of exogenously administered manganese (Mn) on developmental neurogenesis in the hippocampal dentate gyrus was examined in male mice after maternal exposure to MnCl(2) (0, 32, 160, or 800 ppm as Mn in diet) from gestational day 10 to day 21 after delivery on weaning. Immunohistochemistry was performed to monitor neurogenesis and interneuron subpopulations on postnatal days (PNDs) 21 and 77 (adult stage). Reelin-synthesizing γ-aminobutyric acid (GABA)ergic interneurons increased in the hilus with ≥ 160 ppm on weaning to sustain to PND 77 at 800 ppm. Apoptosis in the neuroblast-producing subgranular zone increased with 800 ppm and TUC4-expressing immature granule cells decreased with 800 ppm on weaning, whereas at the adult stage, immature granule cells increased. On PND 21, transcript levels increased with Reln and its receptor gene Lrp8 and decreased with Dpysl3 coding TUC4 in the dentate gyrus, confirming immunohistochemical results. Double immunohistochemistry revealed a sustained increase of reelin-expressing and NeuN-lacking or weakly positive immature interneurons and NeuN-expressing mature neurons in the hilus through to the adult stage as examined at 800 ppm. Brain Mn concentrations increased at both PNDs 21 and 77 in all MnCl(2)-exposed groups. These results suggest that Mn targets immature granule cells causing apoptosis and neuronal mismigration. Sustained increases in immature reelin-synthesizing GABAergic interneurons may represent continued aberration in neurogenesis and following migration to cause an excessive response for overproduction of immature granule cells through to the adult stage. Sustained high concentration of Mn in the brain may be responsible for these changes.

This investigation illustrates an important property of eukaryote-type serine/threonine phosphatase (SP-STP) of group A Streptococcus (GAS) in causing programmed cell death of human pharyngeal cells. The secretory nature of SP-STP, its elevated expression in the intracellular GAS, and the ability of wild-type GAS but not the GAS mutant devoid of SP-STP to cause apoptosis of the host cell both in vitro and in vivo suggest that GAS deploys SP-STP as an important virulence determinant to exploit host cell machinery for its own advantage during infection. The exogenously added SP-STP is able to enter the cytoplasm and subsequently traverses into the nucleus in a temporal fashion to cause apoptosis of the pharyngeal cells. The programmed cell death induced by SP-STP, which requires active transcription and de novo protein synthesis, is also caspase-dependent. Furthermore, the entry of SP-STP into the cytoplasm is dependent on its secondary structure as the catalytically inactive SP-STP with an altered structure is unable to internalize and cause apoptosis. The ectopically expressed wild-type SP-STP was found to be in the nucleus and conferred apoptosis of Detroit 562 pharyngeal cells. However, the catalytically inactive SP-STP was unable to cause apoptosis even when intracellularly expressed. The ability of SP-STP to activate pro-apoptotic signaling cascades both in the cytoplasm and in the nucleus resulted in mitochondrial dysfunctioning and perturbation in the phosphorylation status of histones in the nucleus. SP-STP thus not only functions as a virulence regulator but also as an important factor responsible for host-related pathogenesis.

Sepsis-induced lymphocyte and dendritic cell apoptosis contributes to immunosuppression, which results in an inability to eradicate the primary infection as well as a propensity to acquire new, secondary infections. Another cellular process, autophagy, is also activated in immune cells and plays a protective role. In the present study, we demonstrate that interferon regulatory factor 1 (IRF-1) regulates both immune cell apoptosis and autophagy in a murine endotoxemia model. Interferon regulatory factor 1 is activated at an early phase through a Toll-like receptor 4-dependent, myeloid differentiation primary response gene 88-independent manner in splenocytes. Furthermore, IRF-1 knockout (KO) mice are protected from a lethal endotoxemia model. This protection is associated with decreased apoptosis and increased autophagy in splenocytes. Interferon regulatory factor 1 KO mice experience decreased apoptotic cell loss, especially in CD4⁺ T lymphocytes and myeloid antigen-presenting cells. Meanwhile, IRF-1 KO mice demonstrate increased autophagy and improved mitochondrial integrity. This increased autophagy in KO mice is attributable, at least in part, to deactivation of mammalian target of rapamycin/P70S6 signaling--a main negative regulator of autophagy. Therefore, we propose a novel role for IRF-1 in regulating both apoptosis and autophagy in splenocytes in the setting of endotoxemia with IRF-1 promoting apoptosis and inhibiting autophagy.