Project description:A 7-month inhalation study in C57BL/6 mice was conducted to evaluate long-term respiratory toxicity of e-vapor aerosols compared to cigarette smoke and to assess the impact of smoking cessation or switching to an e-vapor product after 3 months of exposure to 3R4F cigarette smoke (CS). In this study, we performed a chronic inhalation (4 h/day, 5 d/week, up to 7 months) study in C57BL/6 mice using a commercial (MarkTen®) e-vapor product and a combustible reference cigarette (3R4F) using a Switching and Cessation study design. A commercial e-vapor product (MarkTen® device [version 2.6.8]; “Test Red”) was supplied by Altria Client Services LLC (Richmond, VA, USA). The Test Red formulation was composed of aerosol formers (propylene glycol [PG] and vegetable glycerol [VG]), ~4% nicotine by weight, and flavors (non-menthol). The 3R4F commercial reference cigarettes were purchased from the University of Kentucky (Lexington, KY). HEPA filtered air at the testing facility (Battelle, West Jefferson, OH) was used as Sham Control. General procedures for animal care and housing met the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) recommendations and requirements stated in the “Guide for Care and Use of Laboratory Animals” [National Research Council (NRC)] and approved by the Institutional Animal Care and Use Committee (IACUC). Female C57BL/6 mice were received from Charles River Kingston (Stone Ridge, NY). Test atmosphere was generated from smoking machines and delivered to the mice through a nose-only exposure system. The modified Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA) Reference Method 81 regimen (55/30/5: a 55 ± 0.3 mL puff volume, a puff every 30 seconds, a 5-second puff duration) was used to generate e-vapor aerosol for 130 puffs/cartridge. Mainstream smoke from 3R4F cigarette was generated using a modified Health Canada Intense regimen (55/30/2: a 55 ± 0.3 mL puff volume, a puff every 30 seconds, a 2-second puff duration, and a near-square puff profile) for 8 puffs/cigarette. Female C57BL/6 mice (~10 weeks old) were randomly assigned based on body weight to one of five exposure groups: Sham Control, 3R4F CS, Test Red, Switching, and Cessation. Mice were exposed to 3R4F CS (550 µg/L TPM) or e-vapor aerosols (Test Red; 1100 µg/L TPM) via nose-only inhalation up to 4 h/day, 5 d/week for up to 7 months. After the first 3 months of exposure, groups of 3R4F CS mice were subjected to exposures of: (1) Test Red aerosol (“Switching”) or (2) filtered air (“Cessation”), while a group of mice continued to be exposed to 3R4F CS. Here, the protein expression data for lung tissue assessed by iTRAQ®-based quantitative proteomics is reported.

Project description:The objective of this study was to compare the biological and toxicological response of apolipoprotein E-deficient (Apoe-/-) mice to 3R4F mainstream smoke exposure for 2 months in whole-body exposure chambers (WBEC) and nose-only exposure chambers (NOEC). Female ApoE-/- mice were randomized into four groups: two Sham groups, exposed to filtered air, and two 3R4F groups, exposed to CS from the 3R4F reference cigarette (550 Âµg TPM/L). Half the number of mice in the Sham- and CS-exposed groups were exposed in WBECs and the other half in NOECs. The exposure phase lasted 9 weeks and included 1 week of adaption, during which exposure in both chamber types was escalated in dose and duration to a maximum of 4 h per day. The TPM concentration and exposure duration in WBECs were matched to those in NOECs on the basis of the regimen that the mice tolerated, as determined by in-life findings on acute signs of nicotine toxicity. Fresh air breaks were introduced during the exposure period to maintain carboxyhemoglobin (COHb) concentrations at acceptable levels. More frequent and longer fresh air breaks were required for exposure in the WBEC than in the NOEC because of the greater internal volumeâ€”and, consequently, the longer durationâ€”required to clear smoke from the WBEC than from the NOEC. For animals in NOECs, a 30-min fresh air break was introduced after 2 and 3 h of exposure. For animals in WBECs, a 30-min fresh air break was introduced after 1 and 2 h of exposure and a 60-min fresh air break after the third hour of exposure. The general condition and health of the mice following exposure were monitored throughout the study. Full necropsy was performed 16-20 h after the last exposure without prior fasting, in accordance with previously described methods (Vanscheeuwijck et al., 2002). Differences between WBEC and NOEC expsoure were analyzed with regard to aerosol uptake, disease endpoints (adaptive changes in nasal epithelia, changes in lung function and inflammatory parameters, plasma cholesterol/triglyceride levels in lipoprotein fractions, and atherosclerosis plaque development), and systems biology endpoints (changes in the lung proteome and liver, nasal epithelial, and heart transcriptomes). All procedures involving animals were performed in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International and licensed by the Agri-Food & Veterinary Authority of Singapore, with approval from an Institutional Animal Care and Use Committee and in compliance with the National Advisory Committee for Laboratory Animal Research Guidelines on the Care and Use of Animals for Scientific Purposes (NACLAR, 2004). Here, the protein expression data for lung tissue assessed by iTRAQ®-based quantitative proteomics will be described.

Project description:Differential gene expression of Dictyostelium discoideum after infection with Legionella pneumophila was investigated using DNA microarrays. A detailed analysis of the 24 h time point post infection was performed in comparison to three controls, uninfected cells and co-incubation with Legionella hackeliae and L. pneumophila DeltadotA. One hundred and thirty-one differentially expressed D. discoideum genes were identified as common to all three experiments and are thought to be involved in the pathogenic response. Functional annotation of the differentially regulated genes revealed that apart from triggering a stress response Legionella apparently not only interferes with intracellular vesicle fusion and destination but also profoundly influences and exploits the metabolism of its host. The results provide the basis for a better understanding of the complex host-pathogen interactions and for further studies on the Dictyostelium response to Legionella infection. The bacterial strains used in this study were L. pneumophila Philadelphia I JR32, L. pneumophila Philadelphia I JR32 LELA 3118 (dotA3118:Tn903 DLL LacZ) and L. hackeliae (ATCC 35250). The Legionella strains were grown on buffered charcoal yeast extract agar (BCYE) at 37M-BM-0C with 5% CO2 atmosphere for 3 days. The D. discoideum wild-type strain AX2 was grown at 23M-BM-0C in 75 cm2 cell-culture flasks with 10 ml HL5 medium. For infection, Dictyostelium cells were harvested, resuspended in a 1:1 solution of HL5 medium and Soerensen buffer. Fifteen millilitres of a 1M-CM-^W10e6 cells/ml suspension were seeded into a 75 square-cm cell culture flask and the amoebae were inoculated with 10e7 bacteria/ml. Three different pairs of infection were compared: 1. AX2 infected with L. pneumophila JR32 versus uninfected cells; 19 microarrays of seven independent infections; 2. AX2 infected with L. pneumophila JR32 versus AX2 infected with L. pneumophila JR32 delta DotA; 4 microarrays of two independent infections; 3. AX2 infected with L. pneumophila JR32 versus AX2 infected with L. hackeliae; 4 microarrays of two independent infections. 24h post infection the RNA was isolated from 1.5M-CM-^W10e7 Dictyostelium cells and microarray analysis was performed as described (Farbrother et al., 2006).

Project description:Differential gene expression of Dictyostelium discoideum after infection with Legionella pneumophila in comparison to uninfected cells was investigated using DNA microarrays. Investigation of a 48 h time course of infection revealed several clusters of co-regulated genes, an enrichment of preferentially up- or downregulated genes in distinct functional categories and also showed that most of the transcriptional changes occurred 24 h after infection. Functional annotation of the differentially regulated genes revealed that apart from triggering a stress response Legionella apparently not only interferes with intracellular vesicle fusion and destination but also profoundly influences and exploits the metabolism of its host. For some of the identified genes, e.g. rtoA involvement in the host response has been demonstrated in a recent study, for others such a role appears plausible. The results provide the basis for a better understanding of the complex host-pathogen interactions and for further studies on the Dictyostelium response to Legionella infection. The bacterial strain used in this study was L. Pneumophila Philadelphia I JR32. The strain was grown on buffered charcoal yeast extract agar (BCYE) at 37M-BM-0C with 5% CO2 atmosphere for 3 days. The D. discoideum wild-type strain AX2 was grown at 23M-BM-0C in 75 cm2 cell-culture flasks with 10 ml HL5 medium. For infection, Dictyostelium cells were harvested, resuspended in a 1:1 solution of HL5 medium and Soerensen buffer. Fifteen millilitres of a 1M-CM-^W10e6 cells/ml suspension were seeded into a 75 cme2 cell culture flask and the amoebae were inoculated with 10e7 bacteria/ml. After different time intervals of incubation (1, 3, 6, 24, and 48 h) the RNA was isolated from 1.5M-CM-^W10e7 Dictyostelium cells. Usually two or three parallel cultures for the experiment and the control were inoculated per infection. The percentage of infected cells was determined by in situ hybridization with Legionella-specific 16S rRNA probes. The average from three independent determinations was 34, 42, 42, 57 and 82% of infected Dictyostelium cells after 1, 3, 6, 24 and 48 h, respectively.

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Recombinant Mycobacterium smegmatis LC-MSMS

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