2006 merck/merial - School of Veterinary Medicine - Louisiana State ...

Enhancing the Quality and Reliability of Diagnostic ImmunocytochemistryElisabeth Peters*, Victor E. ValliCollege of Veterinary Medicine, University of Illinois at Urbana-ChampaignDiagnostic immunocytochemistry is frequently conducted on routine submissions from schools of veterinarymedicine as well as local practitioners. Cytology is practical because it offers valuable management information for manytypes of lesions at very reasonable cost, but it requires skilled diagnostic interpretation. A recurring problem with this serviceis that freshly made blood and cytological smears with heavy cellular spreads tend to detach from the slides in the stainingprocess. For our study, tissues were collected from animals that were electively euthanized. The tissues were selected fromsites where inflammatory, hyperplastic, and neoplastic lesions commonly occur such as the lymph nodes, spleen, bonemarrow, skin, thyroid, liver, pancreas, and adrenal gland. Dispersed cells were collected into a transport media and cytologicpreparations of these tissues were used in the study along with specimens submitted to the veterinary teaching hospital. Twomethods of fixation were used prior to staining. These include drying the slide for a day followed by immersion in acetonefor 5 minutes, or immediate fixation in 10% formalin for 30 seconds. Formalin fixation gives better cell preservation andadhesion, but prevents the diagnostic antibodies from penetrating the cell membranes and requires an antigen retrievalprocess. This retrieval is achieved by boiling the slide preparations for 5 minutes in a pressurized chamber containing citratebuffer at pH 6. We have established base protocols for CD3, CD20, CD79, cytokeratin, and vimentin. Other antibodies inadvanced development include CD4, CD8á, CD8â, CD172, FIP, FeLV, thyroglobulin, myeloperoxidase, chromogranin, andsynaptophysin. The results of this study will permit those using immunocytochemistry to access a wider array of reagentsand expand their capabilities for making a specific diagnosis.Quantifying the effect of CD30 signaling via NFkB in the neoplastically transformed cells on Marek’sDisease Virus Meq transcriptionMelissa Pflueger*, Dusan Kunec, Dr. Shane BurgessCollege of Veterinary Medicine, Mississippi State University, Mississippi State, MSMarek’s Disease herpesvirus (MDV) is a naturally occurring oncogenic virus causing T-cell lymphomas in poultryas well as a unique natural animal model for CD30 (“Hodgkin’s disease antigen”) over-expressing human lymphomas. CD30is thought to contribute to the neoplastic transformation of lymphocytes via its signaling pathway which includes the NFkBtranscription factor family. The MDV putative “oncogene” Meq promotes expression of the CD30 antigen. We hypothesizethat CD30 signaling via NFkB in turns promotes Meq transcription setting up a positive feedback loop. To test our hypothesiswe will examine whether chicken NFkB can promote transcription from the Meq promoter in vitro. Our aims here were tofirst construct recombinant plasmids expressing NFkB isoforms p50, p65, p100 and p105 and a plasmid in which the Meqpromoter is cloned 5’ of the GFP gene. These will be used in co-transfection experiments to identify whether or not the NFkBisoforms are active at the Meq promoter.Characterization of Carboxylesterases Derived from Lean and Obese/Atherosclerotic Rat LiverLloyd V. Reitz*, Tim M. Streit, Abdolsamad Borazjani, Alison C. Elder, Matthew K. RossCollege of Veterinary Medicine, Mississippi State University, Mississippi State, MS. Department ofEnvironmental Medicine, University of Rochester, Rochester, NY.Carboxylesterases (CEs) are promiscuous members of a xenobiotic-detoxifying enzyme family that hydrolyzescompounds with ester bonds. CEs are primarily found in liver and major isoforms in rat are termed Hydrolase A andHydrolase B. In this study we examined the levels of hepatic CE activities and protein (Hydrolase A) in lean (+/?) andobese/atherosclerotic (cp/cp) rats during basal conditions and following exposure to concentrated ultrafine particulates(CUPs). For biochemical study CEs were extracted from rat liver in the microsomal fraction (RLM). Hydrolytic reactionscatalyzed by CEs utilized the following esterified substrates: para-nitrophenol valerate, 4-methylumbelliferyl acetate, andselect pyrethroid insecticides. The levels of CE activities in the lean and cp/cp RLMs were not significantly different underbasal conditions (control). However, following exposure to CUPs, hydrolytic activities in cp/cp RLM were significantlyreduced compared to the lean RLM. Western blots of these same microsomal proteins demonstrated that the cp/cp RLMs hadsignificantly reduced levels of Hydrolase A protein compared to lean RLMs. When hydrolytic activities of lean RLMs fromcontrol and CUP groups were compared, evidence of robust CE induction was observed. In contrast, no significantdifferences in hydrolytic activities were observed between the cp/cp RLMs from the exposed and control groups. Thus, theseresults indicate an induction response is occurring in the lean animals exposed to the CUPs, while this response is blunted inthe cp/cp animals. In conclusion, the CE xenobiotic-detoxifying activities of the liver appear to be linked to health statuswith obesity being a prime indicator of decreased activity following xenobiotic challenge. [Trainee stipend supported byNIH-NCRR RR070710].106