Characterization of chromosomal instability in murine colitis-associated colorectal cancer

Background

Patients suffering from ulcerative colitis (UC) bear an increased risk for colorectal cancer. Due to the sparsity of colitis-associated cancer (CAC) and the long duration between UC initiation and overt carcinoma, elucidating mechanisms of inflammation-associated carcinogenesis in the gut is particularly challenging. Adequate murine models are thus highly desirable. For human CACs a high frequency of chromosomal instability (CIN) reflected by aneuploidy could be shown, exceeding that of sporadic carcinomas. The aim of this study was to analyze mouse models of CAC with regard to CIN. Additionally, protein expression of p53, beta-catenin and Ki67 was measured to further characterize murine tumor development in comparison to UC-associated carcinogenesis in men.

Methods

The AOM/DSS model (n = 23) and IL-10^−/− mice (n = 8) were applied to monitor malignancy development via endoscopy and to analyze premalignant and malignant stages of CACs. CIN was assessed using DNA-image cytometry. Protein expression of p53, beta-catenin and Ki67 was evaluated by immunohistochemistry. The degree of inflammation was analyzed by histology and paralleled to local interferon-γ release.

Results

CIN was detected in 81.25% of all murine CACs induced by AOM/DSS, while all carcinomas that arose in IL-10^−/− mice were chromosomally stable. Beta-catenin expression was strongly membranous in IL-10^−/− mice, while 87.50% of AOM/DSS-induced tumors showed cytoplasmatic and/or nuclear translocation of beta-catenin. p53 expression was high in both models and Ki67 staining revealed higher proliferation of IL-10^−/−-induced CACs.

Conclusions

AOM/DSS-colitis, but not IL-10^−/− mice, could provide a powerful murine model to mechanistically investigate CIN in colitis-associated carcinogenesis.     

Rapid detection of total and viable Legionella pneumophila in tap water by immunomagnetic separation,double fluorescent staining and flow cytometry

We developed a rapid detection method for Legionella pneumophila (Lp) by filtration, immunomagnetic separation, double fluorescent staining, and flow cytometry (IMS‐FCM method). The method requires 120 min and can discriminate ‘viable’ and ‘membrane‐damaged’ cells. The recovery is over 85% of spiked Lp SG 1 cells in 1 l of tap water and detection limits are around 50 and 15 cells per litre for total and viable Lp, respectively. The method was compared using water samples from house installations in a blind study with three environmental laboratories performing the ISO 11731 plating method. In 53% of the water samples from different taps and showers significantly higher concentrations of Lp were detected by flow cytometry. No correlation to the plate culture method was found. Since also ‘viable but not culturable’ (VNBC) cells are detected by our method, this result was expected. The IMS‐FCM method is limited by the specificity of the used antibodies; in the presented case they target Lp serogroups 1–12. This and the fact that no Lp‐containing amoebae are detected may explain why in 21% of all samples higher counts were observed using the plate culture method. Though the IMS‐FCM method is not yet fit to completely displace the established plating method (ISO 11731) for routine Lp monitoring, it has major advantages to plating and can quickly provide important insights into the ecology of this pathogen in water distribution systems.     

Cutting edge: IL-23 cross-regulates IL-12 production in T cell-dependent experimental colitis

Although IL-12 and IL-23 share the common p40 subunit, IL-23, rather than IL-12, seems to drive the pathogenesis of experimental autoimmune encephalomyelitis and arthritis, because IL-23/p19 knockout mice are protected from disease. In contrast, we describe in this study that newly created LacZ knockin mice deficient for IL-23 p19 were highly susceptible for the development of experimental T cell-mediated TNBS colitis and showed even more severe colitis than wild-type mice by endoscopic and histologic criteria. Subsequent studies revealed that dendritic cells from p19-deficient mice produce elevated levels of IL-12, and that IL-23 down-regulates IL-12 expression upon TLR ligation. Finally, in vivo blockade of IL-12 p40 in IL-23-deficient mice rescued mice from lethal colitis. Taken together, our data identify cross-regulation of IL-12 expression by IL-23 as novel key regulatory pathway during initiation of T cell dependent colitis.     

Cigarette smoke elicits leukocyte adhesion to endothelium in hamsters: Inhibition by CuZn-SOD

Although cigarette smoking has been identified as a major risk factor for cardiovascular diseases, the underlying pathomechanism is largely unknown. Using a dorsal skinfold chamber model in Syrian golden hamsters for intravital microscopy on striated muscle microcirculation, we investigated whether cigarette smoke (CS) affects the adhesion of circulating leukocytes to the endothelium, a constant feature of early atherogenesis and a hallmark of ischemia-reperfusion injury. Awake hamsters were exposed for 5 min to the mainstream smoke of one cigarette (2R 1 research cigarette), inducing nicotine, cotinine, and carboxyhemoglobin plasma levels comparable to levels found in human smokers. In control animals (n = 7), CS exposure elicited the rolling and subsequent adhesion of fluorescently stained leukocytes to the endothelium of arterioles and postcapillary venules. Leukocyte/endothelium interaction was preceded by an early rise in xanthine oxidase activity and intravascular hemolysis. Leukocyte adhesion and xanthine oxidase (Xo) activation were significantly attenuated in hamsters pretreated with superoxide dismutase (5 mg/kg, 10 min prior to CS, n= 7), suggesting a key role of superoxide in this event. These in vitro results suggest a novel pathomechanism of CS-induced cardiovascular pathology.     

Impact of vitamin E supplement in standard laboratory animal diet on microvascular manifestation of ischemia/reperfusion injury

Aimed at improving animal fertility and health, diets for farm and laboratory animals have over the last few years been supplemented with increasing amounts of the antioxidant vitamin E. We now demonstrate by intravital microscopy that feeding hamsters with a vitamin E-supplemented “standard” rodent diet (60 ppm vitamin E) significantly reduces the microvascular manifestations of ischemia/reperfusion injury when compared to animals fed a nonsupplemented diet. Postischemic leukocyte adhesion to venular endothelium was reduced from 770 ± 204 cells/mm² at 24 h after reperfusion in control animals on the nonsupplemented diet to 403 ± 105 cells/mm² in animals on the “standard” rodent diet (means ± SD, N = 7 animals per group, p < 0.01). Animals on the nonsupplemented diet showed a dramatic loss of capillary perfusion density until 7 days after reperfusion (to 21 ± 13% of preischemic baseline values), whereas this loss was significantly attenuated (to 71 ± 12% of preischemic values, p < 0.01) in animals on the “standard” rodent diet. No difference in the extent of reperfusion injury was seen between animals on the “standard” rodent diet and animals on diets with substantially higher vitamin E supplements (300 ppm–30.000 ppm). Besides underscoring the benefit of vitamin E in reducing the extent of ischemia/reperfusion injury, this study raises the concern that vitamin E supplements in “standard” laboratory animal diets may have a far-reaching impact on biomedical research by jeopardizing established animal models of disease.     

Midostaurin upregulates eNOS gene expression and preserves eNOS function in the microcirculation of the mouse

Nitric oxide (NO) derived from endothelial NO synthase (eNOS) is a powerful vasodilator and possesses vasoprotective effects. Therefore, augmentation of eNOS expression and -activity by pharmacological means could provide protection against cardiovascular disease. However, this concept has been questioned recently, because in several disease models, eNOS upregulation was associated with a dysfunctional enzyme (referred to as eNOS uncoupling). In contrast, the present study demonstrates that an eNOS gene expression-enhancing compound with additional protein kinase C (PKC) inhibitory properties can upregulate eNOS while preserving its enzymatic function. Apolipoprotein E-knockout mice were treated for 7 days with midostaurin (4'-N-benzoyl staurosporine, compound CGP 41251, 50-125 mg/kg/day), a PKC inhibitor previously shown to increase eNOS expression and NO production in cultured human endothelial cells. Midostaurin treatment enhanced eNOS mRNA expression (RNase protection assay) in mouse aorta, kidney, and heart in a dose-dependent fashion. In the dorsal skinfold microcirculation, midostaurin produced an arteriolar vasorelaxation (intravital microscopy), which could be prevented by the NOS inhibitor L-NAME, indicating that the upregulated eNOS remained functional. In organ chamber experiments, the aorta from midostaurin-treated mice showed an enhanced NO-mediated relaxation in response to acetylcholine. Accordingly, serum levels of nitrite/nitrate (NO-Analyzer) were increased, and the production of reactive oxygen species in the aorta (L-012 chemiluminescence) was reduced by midostaurin. Thus, in mice in vivo, midostaurin treatment results in enhanced expression of eNOS with preserved enzyme function and enhanced production of bioactive NO. Given the beneficial effects of endothelial-derived NO, vasoprotective and anti-atherosclerotic effects are likely to ensue.