Month: May 2017

Exposure to amphibole asbestos has been associated with production of autoantibodies in mice and humans, and increases the risk of systemic autoimmune disease. the spleen and lungs. The results show that amphibole, but not chrysotile, asbestos increases the frequency of ANA/ENA in mice. Amphibole and chrysotile both increased multiple serum cytokines, but only amphibole increased IL-17. Both fibers decreased IgG1, without significant changes in other immunoglobulin isotypes. Although there were no gross changes in overall percentages of T- and B-cells in the spleen or lung, there was a significant increase in the normally rare populations of suppressor B-cells (CD19+, CD5+, CD1d+) in both the spleen and lungs of chrysotile-exposed mice. Overall, the results suggest that, while there may be an inflammatory response to both forms of asbestos, there is an autoimmune response in only the amphibole-exposed, but not the chrysotile-exposed mice. These data have crucial implications in terms of screening and health outcomes of asbestos-exposed populations. access to standard rodent chow and filtered water. Only female mice between 6C8-weeks-of-age were used. Initially, 12 mice were to be instilled in each group, but mice were lost due to death during the 8 months, or excess skin barbering that required euthanasia. Thus, the study ended up examining a total of seven saline, 10 BIBR 953 chrysotile, and 11 6-Mix uncovered mice. Intratracheal instillations Exposing mice to 6-Mix, chrysotile, or saline was carried out by injection of the suspension directly into the BIBR 953 trachea. The fiber suspension was created by collecting 1.0 mg dry fiber in a 1.5 ml microcentrifuge tube and adding 1.0 ml sterile PBS, followed by sonication. Mice were injected in the peritoneum with a mixture of 50 l Xylazine, 40 l Ketamine, Rabbit Polyclonal to TRIM16. and 40 l Torbugesic answer (all from MWI Veterinary Supply, Boise, ID) for sedation and then their necks were wiped down with ethanol wipes and shaved. An incision uncovered the trachea and 30 l of the 1 mg/ml fiber suspension (or saline) was injected down the trachea. The incision was closed using an adhesive and cleaned using Betadyne. All mice were allowed to recover from the procedure in a warmed cage. All mice received two instillations 3C4 weeks apart (total exposure = 60 g of either BIBR 953 6-Mix or chrysotile) and were monitored for 8 months thereafter. Because is it very difficult to extrapolate an appropriate bolus dose that would represent accumulated human exposure over time, an exposure regimen was used that induced fibrosis or autoantibodies as in previous studies with 6-Mix in these mice (Pfau et al., 2008; Smartt et al., 2010). While a single exposure regimen has induced autoantibodies in these mice, the second exposure enhances the reproducibility and magnitude of the autoantibody response (unpublished data). At the end of the 8 month period, mice were euthanized by CO2 asphyxiation (according to IACUC guidelines) and tissues including blood, spleen, and lungs were harvested. Anti-nuclear antibody (ANA) assay Serum samples from cardiac punctures were analyzed for antinuclear antibodies (ANA). At necropsy, blood was drawn from your heart using a 1.0 ml syringe and collected into microtainer tubes that contained BIBR 953 a serum separator (Becton-Dickinson, San Jose, CA). Once the serum was collected, it was stored at 4 C. The ANA test (ImmunoConcepts, Sacramento, CA), a semi-quantitative analysis that uses indirect immunofluorescence to detect any auto-reactive antibodies in the serum, was altered for the use of mouse serum. Each serum sample was diluted 1:40 (for screening) by adding 5 l serum to 195 l PBS. Diluted sample (20 l) was then added to dedicated wells on a substrate slide and incubated at room heat (RT) for BIBR 953 30 min. The slides were then washed with PBS, being careful not to cross-contaminate any samples. The slide was then submerged in PBS for 10 min and then dipped in deionized water 5-times. The slide was then cautiously dried by blotting in between the wells. Fluorescent antibody reagent was prepared by adding 2 l of goat anti-mouse IgG anti-body conjugated to AlexaFluor 488 (Invitrogen, Eugene, OR) to 1 1 ml PBS. Antibody answer (20 l) was then added to each well and incubated in the dark at RT for 30 min. The slide was washed as layed out before and 4C5 drops of Fluorsave (Calbiochem, La Jolla, CA) was added along the midline of the slide. A coverslip was then placed on the slide (at angle) and any air bubbles pushed out of the wells. The slides were then viewed using a FITC (488 nm) filter on a Leica DMRB fluorescent microscope in the Advanced Imaging Core Facility at ISU. RELISA ENA multiparameter antibody screening test Nuclear antibodies.