New Jersey Medical School



Aspergillus fumigatus (Af): Exposure to the opportunistic fungal pathogen Af leads to a pulmonary infection. To recapitulate the natural route of infection, mice will be challenged with Af via the intra-tracheal route using a non-invasive procedure. Prior to infection mice will be anesthesized by isoflurane inhalation to effect until changes in breathing pattern are observed (approx. 2-3 min. in the isoflurane chamber). Mice will receive different doses of live fungal conidia suspended in 50 ?l of PBS. In previous studies we have shown that wild type mice can survive Af infection without the development of detectable illnessPEVuZE5vdGU+PENpdGU+PEF1dGhvcj5SaXZlcmE8L0F1dGhvcj48WWVhcj4yMDA5PC9ZZWFyPjxS

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ADDIN EN.CITE.DATA (15, 17, 24, 25). In experiments where the use of genetically deficient mice might be needed the animals will be monitored daily for signs of disease progression (ruffled fur, listless, dehydrated). When necessary animals will be euthanized by CO2 asphyxiation. For all experimental procedures mice will be subjected to the minimal discomfort possible. Nippostrongylus brasiliensis (Nb): The intestinal nematode parasite Nb, is a widely used model for examining the development and function of the in vivo type 2 immune response. In the murine model, Nb host exposure begins with subcutaneous inoculation of third stage larvae (L3) under the skin, the site of natural infection. L3 migrate in the circulation to the lungs as early as 11 h after invasion of subcutaneous tissues. L3 molts to fourth stage larvae (L4) and remain in the lungs ≤50 h post-infection before migrating through the trachea to the intestine. Worm expulsion, mediated in the intestine, usually occurs by days 9-12 after primary inoculation. This infection and migration pattern is similar to that of several human intestinal roundworm infections, including Ancylostoma duodenale (Old World hookworm) and Necator americanus (New World hookworm), and recent studies suggest that the immune response to these hookworms has many similar properties to type 2 responses identified in experimental mouse models including the immune response to Nb ADDIN EN.CITE <EndNote><Cite><Author>Moon</Author><Year>2015</Year><RecNum>25</RecNum><DisplayText>(2)</DisplayText><record><rec-number>25</rec-number><foreign-keys><key app="EN" db-id="zzvr2adearz202ewpsz5w2wh2vxxdt0waar0">25</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Moon, C.</author><author>Baldridge, M. T.</author><author>Wallace, M. A.</author><author>Burnham, C. A.</author><author>Virgin, H. W.</author><author>Stappenbeck, T. S.</author></authors></contributors><auth-address>Department of Pathology and Immunology, Washington University School of Medicine, St Louis, Missouri 63110, USA.</auth-address><titles><title>Vertically transmitted faecal IgA levels determine extra-chromosomal phenotypic variation</title><secondary-title>Nature</secondary-title><alt-title>Nature</alt-title></titles><periodical><full-title>Nature</full-title></periodical><alt-periodical><full-title>Nature</full-title></alt-periodical><pages>90-3</pages><volume>521</volume><number>7550</number><edition>2015/02/18</edition><dates><year>2015</year><pub-dates><date>May 7</date></pub-dates></dates><isbn>1476-4687 (Electronic)&#xD;0028-0836 (Linking)</isbn><accession-num>25686606</accession-num><work-type>Research Support, N.I.H., Extramural&#xD;Research Support, Non-U.S. Gov&apos;t</work-type><urls><related-urls><url>;(2). To model the natural route of infection Infectious (100-500) L3 Nb will be injected subcutaneously to establish infection. For the studies in this PPG lung tissue will be collected at specific time points after inoculation. Influenza A Virus: Studies with influenza A virus will be done using the mouse-adapted (H1N1) PR8 strain that has been used in many similar studies. Animals will be anesthetized with avertin, and inoculated intranasally with 200 pfu in 50 μl PBS. Virus stocks are prepared in embryonated chicken eggs, and virus concentration is measured by focus forming assay done on MDCK cells, with foci visualized by immunostaining. Stocks are maintained at -80°C, at concentrations on the order of 106 pfu/ml. All virus stocks are plaque-purified, and tested for LPS contamination before use.Streptococcus pneumonia (Sp): Type 3 Sp (ATCC 6303 clinical isolate with capsular serotype 3) will be used. This serotype is chosen because it is virulent in mice and man. Bacteria will be grown in Todd-Hewitt broth with yeast extract at 37°C until log phase. The concentration of bacteria in broth is determined by measuring the absorbance at 600 nm and then plotting the OD on a standard curve against known colony counts. The bacterial culture is then diluted to a concentration of 4 x 10 4 cfu/ml, and 25 μl aliquots delivered intranasally will be administered to anesthetized animals. Secondary bacterial challenge of influenza-infected mice will be done in the window of maximal susceptibility, from day 5 to day 7 post-influenza infection. Bacterial load is determined by plating serial 10-fold dilutions of culture or lung homogenates onto blood agar plates incubated overnight at 37°C in 5% CO2.Toxoplasma gondii (Tg): For studies with T. gondii, mice are infected intraperitoneally with either a uracil auxotrophic carbamoyl phosphate synthase (CPS) mutant strain (2 million parasites per mouse) or a TdTomato-expressing Prugiaud strain (500 parasites per mouse) of T. gondii. All strains are maintained and propagated in confluent human foreskin fibroblast cultures. Upon 60–80% lysis of the HFF monolayer, T. gondii tachyzoites are released by passing through 25G needle twice and spun down by centrifugation at 2,000 × g for 10 min. For the CPS strain, the pellet was resuspended in PBS and gamma (γ) irradiated at 15,000 rads before priming of mouse. For lung studies the TdTomato-expressing Prugiaud strain is used, this is a virulent strain and infects the lung starting at day 7 after i.p administration. All T. gondii strains were routinely monitored with MycoSensor PCR assay Kit (Agilent Technologies) and were maintained free of Mycoplasma. ................
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