Detection of cytokine protein expression in mouse lung homogenates using suspension bead array

BACKGROUND: The objective for this present study was to determine whether or not suspension bead array is a feasible method to detect changes in cytokine protein expression in mouse lung tissue homogenates. Here, we report on suspension bead array as a feasible method for detection of lipopolysaccharide (LPS)-evoked changes in cytokine protein expression in mouse lung tissue homogenates. MATERIALS AND METHODS: Mice were treated (0.2 ml, intraperitoneal, i.p.) with phosphate buffered saline (PBS) or LPS (0.25 mg/ml) and sacrificed at either 2- or 24-hours post treatment. Formalin-fixed and paraffin-embedded lung sections were evaluated by light microscopy. Flash frozen lung tissues were homogenized for measurement of various cytokine protein expression levels using suspension bead array, antibody array and ELISA. Comparison between groups was performed using a Wilcoxon signed rank test. RESULTS: Pulmonary perivascular edema and an accumulation of mixed cell infiltrates within blood and lymphatic vessels, as well as in the adjacent interstitium, were present at both 2- and 24-hours following LPS treatment. A minimal increase in the number of alveolar macrophages was also observed in the 24-hour LPS-treated mice only. The suspension bead array assay revealed statistically significant increases in mouse lung tissue homogenate levels of interleukin-6 (IL-6) and granulocyte/macrophage colony-stimulating factor (GM-CSF) proteins and a decrease in IL-2 protein at 24-hours post LPS-treatment only. Similar cytokine protein expression patterns were observed using antibody array. Significantly increased IL-6 protein expression levels were also detected using ELISA, which correlated with the suspension bead array data. CONCLUSION: The present study shows that suspension bead array is a feasible method to detect changes in cytokine protein expression in mouse lung tissue homogenates

The Role of Hypoxia in 2-Butoxyethanol–Induced Hemangiosarcoma

To understand the molecular mechanisms underlying compound-induced hemangiosarcomas in mice, and therefore, their human relevance, a systems biology approach was undertaken using transcriptomics and Causal Network Modeling from mice treated with 2-butoxyethanol (2-BE). 2-BE is a hemolytic agent that induces hemangiosarcomas in mice. We hypothesized that the hemolysis induced by 2-BE would result in local tissue hypoxia, a well-documented trigger for endothelial cell proliferation leading to hemangiosarcoma. Gene expression data from bone marrow (BM), liver, and spleen of mice exposed to a single dose (4 h) or seven daily doses of 2-BE were used to develop a mechanistic model of hemangiosarcoma. The resulting mechanistic model confirms previous work proposing that 2-BE induces macrophage activation and inflammation in the liver. In addition, the model supports local tissue hypoxia in the liver and spleen, coupled with increased erythropoeitin signaling and erythropoiesis in the spleen and BM, and suppression of mechanisms that contribute to genomic stability, events that could be contributing factors to hemangiosarcoma formation. Finally, an immunohistochemistry method (Hypoxyprobe) demonstrated that tissue hypoxia was present in the spleen and BM. Together, the results of this study identify molecular mechanisms that initiate hemangiosarcoma, a key step in understanding safety concerns that can impact drug decision processes, and identified hypoxia as a possible contributing factor for 2-BE–induced hemangiosarcoma in mice

Early Pathogenesis of Transmucosal Feline Immunodeficiency Virus Infection

To identify the early target cells and tissues in transmucosal feline immunodeficiency virus (FIV) infection, cats were exposed to a clade C FIV isolate via the oral-nasal or vaginal mucosa and multiple tissues were examined by virus isolation coculture (VI), DNA PCR, catalyzed tyramide signal-amplified in situ hybridization (TSA-ISH), and immunohistochemistry between days 1 and 12 postinoculation (p.i.). FIV RNA was detected in tonsil and oral or vaginal mucosa as early as 1 day p.i. by TSA-ISH and in retropharyngeal, tracheobronchial, or external iliac lymph nodes and sometimes in spleen or blood mononuclear cells by day 2, indicating that regional and distant spread of virus-infected cells occurred rapidly after mucosal exposure. By day 8, viral RNA, DNA, and culturable virus were uniformly detected in regional and distant tissues, connoting systemic infection. TSA-ISH proved more sensitive than DNA PCR in detecting early FIV-infected cells. In mucosal tissues, the earliest demonstrable FIV-bearing cells were either within or subjacent to the mucosal epithelium or were in germinal centers of regional lymph nodes. The FIV(+) cells were of either of two morphological types, large stellate or small round. Those FIV RNA(+) cells which could be colabeled for a phenotype marker, were labeled for either dendritic-cell-associated protein p55 or T-lymphocyte receptor antigen CD3. These studies indicate that FIV crosses mucous membranes within hours after exposure and rapidly traffics via dendritic and T cells to systemic lymphoid tissues, a pathway similar to that thought to occur in the initial phase of infection by the human and simian immunodeficiency viruses

Étude des pratiques enseignantes déclarées concernant le programme de sciences citoyennes Vigie-Nature École

International audienceThis research focuses on teaching practices related to the French citizen science program, Vigie-Nature École (VNE), initiated by the National Museum of Natural History, which studies ordinary biodiversity through standardized protocols. Our study aims to characterize the self-reported practices in order to understand how teachers appropriate this program, at the interface between a biological fieldwork and its dimension of citizen science. An a priori analysis of VNE allows us to define its epistemological stance and to identify its didactical and educational potentialities. A questionnaire collected 254 responses from primary and secondary teachers. The results indicate that the participants assign VNE many objectives, such as raising students' awareness of biodiversity outdoor, working on the scientific inquiry, changing the image of the Nature of Science, engaging in interdisciplinary projects and contributing to scientific research. While the latter objective is strongly emphasized, there is a significant discrepancy in the actual contribution to research. Only half of the participants send data to researchers. This contrast with the intentions of the Museum researchers reflects a misuse by some teachers. A categorization of practices shows contrasting profiles, revealing very different ways of implementing VNE.Cette recherche porte sur les pratiques enseignantes relatives au programme français de sciences citoyennes, Vigie-Nature École (VNE), initié par le Muséum national d’Histoire naturelle étudiant la biodiversité ordinaire par des protocoles standardisés. Notre étude vise à caractériser les pratiques déclarées afin de comprendre comment les enseignants s'approprient ce dispositif, à l’interface entre une étude de terrain et sa dimension de sciences citoyennes. Une analyse a priori de VNE permet de cerner son positionnement épistémologique et de dégager ses potentialités didactiques et éducatives. Un questionnaire a recueilli 254 réponses de professeurs des écoles et de collège-lycée. Les résultats indiquent que les participants assignent à VNE de nombreux objectifs, tels que sensibiliser les élèves à la biodiversité par le terrain, travailler la démarche scientifique, modifier l’image de la nature de la science, engager des projets interdisciplinaires et contribuer à la recherche scientifique. Si ce dernier objectif est fortement mis en avant, un écart important est constaté quant à la contribution effective à la recherche. Seule la moitié des participants envoie les données aux chercheurs. Ce décalage par rapport aux intentions des concepteurs de VNE traduit un détournement d’usage par certains enseignants. Une catégorisation des pratiques permet de dégager des profils contrastés, révélant des façons très différentes de mettre en œuvre VNE

Induction of endogenous retroelements as a potential mechanism for mouse-specific drug-induced carcinogenicity

<div><p>A number of chemical compounds have been shown to induce liver tumors in mice but not in other species. While several mechanisms for this species-specific tumorigenicity have been proposed, no definitive mechanism has been established. We examined the effects of the nongenotoxic rodent hepatic carcinogen, WY-14,643, in male mice from a high liver tumor susceptible strain (C3H/HeJ), and from a low tumor susceptible strain (C57BL/6). WY-14,643, a PPARα activator induced widespread increases in the expression of some endogenous retroelements, namely members of LTR and LINE elements in both strains. The expression of a number of known retroviral defense genes was also elevated. We also demonstrated that basal immune-mediated viral defense was elevated in C57BL/6 mice (the resistant strain) and that WY-14,643 further activated those immuno-defense processes. We propose that the previously reported >100X activity of retroelements in mice drives mouse-specific tumorigenicity. We also propose that C57BL/6’s competent immune to retroviral activation allows it to remove cells before the activation of these elements can result in significant chromosomal insertions and mutation. Finally, we showed that WY-14,643 treatment induced gene signatures of DNA recombination in the sensitive C3H/HeJ strain.</p></div

Volcano plot of LINE and LTR elements.

<p>Panels A and B show fold-change versus -log10 of p-value of LINE elements which show significant change in the respective strains. Panels C and D show fold-change versus -log10 of p-value for the LTR elements. Panels A and C show elements significantly changed in the C3H/HeJ strain while panels B and C show elements from the C57BL/6 strain. Panels were clipped at plus/minus 50X fold-change for clarity resulting in 15, 14, 4, & 5 outliers not being shown on panels A, B, C, & D respectively. Unclipped figures are available in the supplementary files (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176768#pone.0176768.s001" target="_blank">S1 Excel File</a>).</p