Activities of fenbendazole in comparison with albendazole against Echinococcus multilocularis metacestodes in vitro and in a murine infection model.

The current chemotherapeutic treatment of alveolar echinococcosis (AE) in humans is based on albendazole and/or mebendazole. However, the costs of treatment, life-long consumption of drugs, parasitostatic rather than parasiticidal activity of chemotherapy, and high recurrence rates after treatment interruption warrant more efficient treatment options. Experimental treatment of mice infected with Echinococcus multilocularis metacestodes with fenbendazole revealed similar efficacy to albendazole. Inspection of parasite tissue from infected and benzimidazole-treated mice by transmission electron microscopy (TEM) demonstrated drug-induced alterations within the germinal layer of the parasites, and most notably an almost complete absence of microtriches. On the other hand, upon in vitro exposure of metacestodes to benzimidazoles, no phosphoglucose isomerase activity could be detected in medium supernatants during treatment with any of these drugs, indicating that in vitro treatment did not severely affect the viability of metacestode tissue. Corresponding TEM analysis also revealed a dramatic shortening/retraction of microtriches as a hallmark of benzimidazole action, and as a consequence separation of the acellular laminated layer from the cellular germinal layer. Since TEM did not reveal any microtubule-based structures within Echinococcus microtriches, this effect cannot be explained by the previously described mechanism of action of benzimidazoles targeting β-tubulin, thus benzimidazoles must interact with additional targets that have not been yet identified. In addition, these results indicate the potential usefulness of fenbendazole for the chemotherapy of AE.

The choice of anesthesia influences oxidative energy metabolism and tissue survival in critically ischemic murine skin

In surgical animal studies anesthesia is used regularly. Several reports in the literature demonstrate respiratory and cardiovascular side effects of anesthesiologic agents. The aim of this study was to compare two frequently used anesthesia cocktails (ketamine/xylazine [KX] versus medetomidine/climazolam/fentanyl [MCF]) in skin flap mouse models. Systemic blood values, local metabolic parameters, and surgical outcome should be analyzed in critical ischemic skin flap models. Systemic hypoxia was found in the animals undergoing KX anesthesia compared with normoxia in the MCF group (sO(2): 89.2% +/- 2.4% versus 98.5% +/- 1.2%, P < 0.01). Analysis of tissue metabolism revealed impaired anaerobic oxygen metabolism and increased cellular damage in critical ischemic flap tissue under KX anesthesia (lactate/pyruvate ratio: KX 349.86 +/- 282.38 versus MCF 64.53 +/- 18.63; P < 0.01 and glycerol: KX 333.50 +/- 83.91 micromol/L versus MCF 195.83 +/- 29.49 micromol/L; P < 0.01). After 6 d, different rates of flap tissue necrosis could be detected (MCF 57% +/- 6% versus KX 68% +/- 6%, P < 0.01). In summary we want to point out that the type of anesthesia, the animal model and the goal of the study have to be well correlated. Comparing the effects of KX and MCF anesthesia in mice on surgical outcome was a novel aspect of our study.

Quercetin aglycone is bioavailable in murine pancreas and pancreatic xenografts

Quercetin is a potential chemopreventive and chemotherapeutic agent for pancreatic and other cancers. This study examined the distribution of quercetin in plasma, lung, liver, pancreas, and pancreatic cancer xenografts in a murine in vivo model and the uptake of quercetin in pancreatic cancer MiaPaCa-2 cells in a cellular in vitro model. Mice were randomly allocated to control or 0.2 and 1% quercetin diet groups utilizing the AIN93G-based diet (n = 12 per group) for 6 weeks. In addition, 6 mice from each group were injected weekly with the chemotherapeutic drug gemcitabine (120 mg/kg mouse, ip). MiaPaCa cells were collected from culture medium after cells were exposed to 30 muM quercetin for 0.5, 1, 2, 4, 8, and 24 h. Levels of quercetin and 3-O'-methylquercetin in mouse tissues and MiaPaCa-2 cells were measured by high-pressure liquid chromatography following enzymatic hydrolysis and then extraction. The study showed that quercetin is accumulated in pancreatic cancer cells and is absorbed in the circulating system, tumors, and tissues of pancreas, liver, and lung in vivo. A higher proportion of total quercetin found in tumors and pancreas is aglycones. Gemcitabine cotreatment with quercetin reduced absorption of quercetin in the mouse circulatory system and liver. Results from the study provide important information on the interpretation of the chemotherapeutic efficacy of quercetin.

Tumor necrosis factor sensitizes primary murine hepatocytes to Fas/CD95-induced apoptosis in a Bim- and Bid-dependent manner

Fas/CD95 is a critical mediator of cell death in many chronic and acute liver diseases and induces apoptosis in primary hepatocytes in vitro. In contrast, the proinflammatory cytokine tumor necrosis factor α (TNFα) fails to provoke cell death in isolated hepatocytes but has been implicated in hepatocyte apoptosis during liver diseases associated with chronic inflammation. Here we report that TNFα sensitizes primary murine hepatocytes cultured on collagen to Fas ligand (FasL)-induced apoptosis. This synergism is time-dependent and is specifically mediated by TNFα. Fas itself is essential for the sensitization, but neither Fas up-regulation nor endogenous FasL is responsible for this effect. Although FasL is shown to induce Bid-independent apoptosis in hepatocytes cultured on collagen, the sensitizing effect of TNFα is clearly dependent on Bid. Moreover, both c-Jun N-terminal kinase activation and Bim, another B cell lymphoma 2 homology domain 3 (BH3)-only protein, are crucial mediators of TNFα-induced apoptosis sensitization. Bim and Bid activate the mitochondrial amplification loop and induce cytochrome c release, a hallmark of type II apoptosis. The mechanism of TNFα-induced sensitization is supported by a mathematical model that correctly reproduces the biological findings. Finally, our results are physiologically relevant because TNFα also induces sensitivity to agonistic anti-Fas-induced liver damage. CONCLUSION: Our data suggest that TNFα can cooperate with FasL to induce hepatocyte apoptosis by activating the BH3-only proteins Bim and Bid.

Morphology and Hemodynamics during Vascular Regeneration in Critically Ischemic Murine Skin Studied by Intravital Microscopy Techniques

With the understanding of angiogenesis and arteriogenesis, new theories about the orchestration of these processes have emerged. The aim of this study was to develop an in vivo model that enables visualization of vascular regenerating mechanisms by intravital microscopy techniques in collateral arteriolar flap vascularity.

Paracrine effects of mesenchymal stem cells enhance vascular regeneration in ischemic murine skin

New theories on the regeneration of ischemic vasculature have emerged indicating a pivotal role of adult stem cells. The aim of this study was to investigate homing and hemodynamic effects of circulating bone marrow-derived mesenchymal stem cells (MSCs) in a critically ischemic murine skin flap model. Bone marrow-derived mesenchymal stem cells (Lin(-)CD105(+)) were harvested from GFP(+)-donor mice and transferred to wildtype C57BL/6 mice. Animals receiving GFP(+)-fibroblasts served as a control group. Laser scanning confocal microscopy and intravital fluorescence microscopy were used for morphological analysis, monitoring and quantitative assessment of the stem cell homing and microhemodynamics over two weeks. Immunohistochemical staining was performed for GFP, eNOS, iNOS, VEGF. Tissue viability was analyzed by TUNEL-assay. We were able to visualize perivascular homing of MSCs in vivo. After 4 days, MSCs aligned along the vascular wall without undergoing endothelial or smooth muscle cell differentiation during the observation period. The gradual increase in arterial vascular resistance observed in the control group was abolished after MSC administration (P<0.01). At capillary level, a strong angiogenic response was found from day 7 onwards. Functional capillary density was raised in the MSC group to 197% compared to 132% in the control group (P<0.01). Paracrine expression of VEGF and iNOS, but not eNOS could be shown in the MSC group but not in the controls. In conclusion, we demonstrated that circulating bone marrow-derived MSCs home to perivascular sites in critically ischemic tissue, exhibits paracrine function and augment microhemodynamics. These effects were mediated through arteriogenesis and angiogenesis, which contributed to vascular regeneration.

Acute trichinellosis increases susceptibility to Giardia lamblia infection in the mouse model

The intestinal protozoan parasite Giardia lamblia causes diarrhoea in humans and animals. In the present study, we used the C57BL/6 inbred mouse model to assess the impact of a nematode (Trichinella spiralis) infection on the course of a G. lamblia (clone GS/M-83-H7) infection. Acute trichinellosis coincided with transient intestinal inflammation and generated an intestinal environment that strongly promoted growth of G. lamblia trophozoites although the local anti-Giardia immunoglobulin (Ig) A production was not affected. This increased G. lamblia infection intensity correlated with intestinal mast cell infiltration, mast cell degranulation, and total IgE production. Furthermore, a G. lamblia single-infection investigated in parallel also resulted in intestinal mast cell accumulation but severe infiltration was triggered in the absence of IgE. Recently, intestinal mast cells emerging during a G. lamblia infection were reported to be involved in those immunological mechanisms that control intestinal proliferation of the parasite in mice. This anti-giardial activity was assumed to be related to the capacity of mast cells to produce IL-6. However, this previous assumption was questioned by our present immunohistological findings indicating that murine intestinal mast cells, activated during a G. lamblia infection were IL-6-negative. In the present co-infection experiments, mast cells induced during acute trichinellosis were not able to control a concurrent G. lamblia infection. This observation makes it feasible that the T. spiralis infection created an immunological and physiological environment that superimposed the anti-giardial effect of mast cells and thus favoured intestinal growth of G. lamblia trophozoites in double-infected mice. Furthermore, our findings raise the possibility that intestinal inflammation e.g. as a consequence of a 'pre-existing' nematode infection is a factor which contributes to increased susceptibility of a host to a G. lamblia infection. The phenomenon of a 'pre-existing' nematode infection prior to a G. lamblia infection is a frequent constellation in endemic areas of giardiasis and may therefore have a direct impact on the epidemiological situation of the disease.

The impact of different nanoparticle surface chemistry and size on uptake and toxicity in a murine macrophage cell line

This study investigated the uptake, kinetics and cellular distribution of different surface coated quantum dots (QDs) before relating this to their toxicity. J774.A1 cells were treated with organic, COOH and NH2 (PEG) surface coated QDs (40 nM). Model 20 nm and 200 nm COOH-modified coated polystyrene beads (PBs) were also examined (50 microg ml(-1)). The potential for uptake of QDs was examined by both fixed and live cell confocal microscopy as well as by flow cytometry over 2 h. Both the COOH 20 nm and 200 nm PBs were clearly and rapidly taken up by the J774.A1 cells, with uptake of 20 nm PBs being relatively quicker and more extensive. Similarly, COOH QDs were clearly taken up by the macrophages. Uptake of NH2 (PEG) QDs was not detectable by live cell imaging however, was observed following 3D reconstruction of fixed cells, as well as by flow cytometry. Cells treated with organic QDs, monitored by live cell imaging, showed only a small amount of uptake in a relatively small number of cells. This uptake was insufficient to be detected by flow cytometry. Imaging of fixed cells was not possible due to a loss in cell integrity related to cytotoxicity. A significant reduction (p<0.05) in the fluorescent intensity in a cell-free environment was found with organic QDs, NH2 (PEG) QDs, 20 nm and 200 nm PBs at pH 4.0 (indicative of an endosome) after 2 h, suggesting reduced stability. No evidence of exocytosis was found over 2 h. These findings confirm that surface coating has a significant influence on the mode of NP interaction with cells, as well as the subsequent consequences of that interaction.

Wnt activation of immortalized brain endothelial cells as a tool for generating a standardized model of the blood brain barrier in vitro

Reproducing the characteristics and the functional responses of the blood-brain barrier (BBB) in vitro represents an important task for the research community, and would be a critical biotechnological breakthrough. Pharmaceutical and biotechnology industries provide strong demand for inexpensive and easy-to-handle in vitro BBB models to screen novel drug candidates. Recently, it was shown that canonical Wnt signaling is responsible for the induction of the BBB properties in the neonatal brain microvasculature in vivo. In the present study, following on from earlier observations, we have developed a novel model of the BBB in vitro that may be suitable for large scale screening assays. This model is based on immortalized endothelial cell lines derived from murine and human brain, with no need for co-culture with astrocytes. To maintain the BBB endothelial cell properties, the cell lines are cultured in the presence of Wnt3a or drugs that stabilize β-catenin, or they are infected with a transcriptionally active form of β-catenin. Upon these treatments, the cell lines maintain expression of BBB-specific markers, which results in elevated transendothelial electrical resistance and reduced cell permeability. Importantly, these properties are retained for several passages in culture, and they can be reproduced and maintained in different laboratories over time. We conclude that the brain-derived endothelial cell lines that we have investigated gain their specialized characteristics upon activation of the canonical Wnt pathway. This model may be thus suitable to test the BBB permeability to chemicals or large molecular weight proteins, transmigration of inflammatory cells, treatments with cytokines, and genetic manipulation.

Bone marrow-derived mesenchymal stromal cells improve vascular regeneration and reduce leukocyte-endothelium activation in critical ischemic murine skin in a dose-dependent manner

BACKGROUND AIMS Stem cells participate in vascular regeneration following critical ischemia. However, their angiogenic and remodeling properties, as well as their role in ischemia-related endothelial leukocyte activation, need to be further elucidated. Herein, we investigated the effect of bone marrow-derived mesenchymal stromal cells (BM-MSCs) in a critically ischemic murine skin flap model. METHODS Groups received either 1 × 10(5), 5 × 10(5), or 1 × 10(6) BM-MSCs or cell-free conditioned medium (CM). Controls received sodium chloride. Intravital fluorescence microscopy was performed for morphological and quantitative assessment of micro-hemodynamic parameters over 12 days. RESULTS Tortuosity and diameter of conduit-arterioles were pronounced in the MSC groups (P < 0.01), whereas vasodilation was shifted to the end arteriolar level in the CM group (P < 0.01). These effects were accompanied by angiopoietin-2 expression. Functional capillary density and red blood cell velocity were enhanced in all treatment groups (P < 0.01). Although a significant reduction of rolling and sticking leukocytes was observed in the MSC groups with a reduction of diameter in postcapillary venules (P < 0.01), animals receiving CM exhibited a leukocyte-endothelium interaction similar to controls. This correlated with leukocyte common antigen expression in tissue sections (P < 0.01) and p38 mitogen-activated protein kinase expression from tissue samples. Cytokine analysis from BM-MSC culture medium revealed a 50% reduction of pro-inflammatory cytokines (interleukin [IL]-1β, IL-6, IL-12, tumor necrosis factor-α, interferon-γ) and chemokines (keratinocyte chemoattractant, granulocyte colony-stimulating factor) under hypoxic conditions. DISCUSSION We demonstrated positive effects of BM-MSCs on vascular regeneration and modulation of endothelial leukocyte adhesion in critical ischemic skin. The improvements after MSC application were dose-dependent and superior to the use of CM alone.

Bilateral uterine vessel ligation as a model of intrauterine growth restriction in mice

BACKGROUND Intrauterine growth restriction (IUGR) occurs in up to 10% of pregnancies and is considered as a major risk to develop various diseases in adulthood, such as cardiovascular diseases, insulin resistance, hypertension or end stage kidney disease. Several IUGR models have been developed in order to understand the biological processes linked to fetal growth retardation, most of them being rat or mouse models and nutritional models. In order to reproduce altered placental flow, surgical models have also been developed, and among them bilateral uterine ligation has been frequently used. Nevertheless, this model has never been developed in the mouse, although murine tools display multiple advantages for biological research. The aim of this work was therefore to develop a mouse model of bilateral uterine ligation as a surgical model of IUGR. RESULTS In this report, we describe the set up and experimental data obtained from three different protocols (P1, P2, P3) of bilateral uterine vessel ligation in the mouse. Ligation was either performed at the cervical end of each uterine horn (P1) or at the central part of each uterine horn (P2 and P3). Time of surgery was E16 (P1), E17 (P2) or E16.5 (P3). Mortality, maternal weight and abortion parameters were recorded, as well as placentas weights, fetal resorption, viability, fetal weight and size. Results showed that P1 in test animals led to IUGR but was also accompanied with high mortality rate of mothers (50%), low viability of fetuses (8%) and high resorption rate (25%). P2 and P3 improved most of these parameters (decreased mortality and improved pregnancy outcomes; improved fetal viability to 90% and 27%, respectively) nevertheless P2 was not associated to IUGR contrary to P3. Thus P3 experimental conditions enable IUGR with better pregnancy and fetuses outcomes parameters that allow its use in experimental studies. CONCLUSIONS Our results show that bilateral uterine artery ligation according to the protocol we have developed and validated can be used as a surgical mouse model of IUGR.