Immunohistochemical Method to Identify Epstein Barr Virus in Tonsil Tissue - Essay Example

Your first and here The of your teacher here The of the here 17 November 2008 Immunohistochemical method to identify Epstein barr virus in tonsil tissue removed from a patient. One of the most widespread human virus, which expands worldwide during human lives is a virus of the herpes family, Epstein-Barr virus (EBV), also called Human herpesvirus 4. According to National Center of Infection disease, in the United States 95% of adults between 35 and 40 years of age have been infected and the most common age of infection is 21 years. Disease carriers of EBV could also be infants as soon as immunological connection with the mother has disappeared. Children usually appear asymptomatic, which in the most cases depend of development of the country, where no disease carrier are established in developed countries and adolescent carrier which cause mononucleosis 35% to 50% of the time. It has a complex managing of viral lifecycle not usual as many viruses, and Henle and Hausen has succeed leading, maintaining and manipulating in continual latency, discovering EBV through immortalized B cells after infection in which it persist as episome -circularized genome. The path of spreading through distinct gene expression categorized in two groups , lytic cycle try expressing viral protein and spreading through virions and latent cycle in which different set of viral protein are produced such as antigen, membrane protein encoded RNAs spreading try at least twenty micro RNAs in infected cell. Clinical symptoms of primary infection often manifested as mononucleosis and appear through fever, sore throat and swollen gland and rarely it could develop in hepatosplenomegaly, heart problem or attack on the central nervous system and lymphadenopathy. Because the infection is almost never fatal, it usually resolves during 1 or 2 months, persisting in rest of the person's life EBV remains dormant in few cells in the throat and blood. Usually the virus periodically reactivates without symptoms manly in seropositive individuals and it could spread through macrophages via placenta infecting the fetus, commonly found in saliva. EBV also could be dormant in some of the body's immune system, establishing important role in malignancies, such as Burkett's lymphoma and nasopharyngeal carcinoma. Transmission could occur contacting the saliva of an infected person, such as persons with infective mononucleosis, not usually through air or blood. Although healthy people can carry and spread the virus, known as primary reservoir for person-to-person transmission, so preventing the cause entirely is unachievable. During routine diagnosis, primary infection of EBV can be established through serologic testing or specific laboratory, funding elevated blood cell increased lymphocytes positive "mono spot" test or specific antibody high levels, although as indirect detection of EBV, could be sighs in blood and lymphoid tissue where leading through research we consider them as a research tool. So symptoms compatible to infection mononucleosis and positive Paul-Bunnell heterophile antibody test result is diagnostic positive, also it is relevant to maintain on considerably high levels of heterophile antibodies, which are seen during first month of illness. Patient with infective monuclesosis show large expansion of EB specific CD8 T cell in the blood and so while latent infection of B cell poll is quickly controlled, virus shedding from lyticaly infected cell in the oropharynx remains high for several month. During primary infection response is localize to the tonsil. In acute IM, EBV specific effector inacute IM, EBV-specific effectors were poorly represented among CD8+ T cells in tonsil compared with blood, coincident with absence of the CCR7 lymphoid homing marker on these highly activated cells. In patients who had recently recovered from IM, latent epitope reactivities were quicker than lytic reactivities both to acquire CCR7 and to accumulate in the tonsil, with some of these cells now expressing the CD103 integrin, which mediates retention at mucosal sites. By contrast, in long-term virus carriers in whom both lytic and latent infections had been controlled, there was 2- to 5-fold enrichment of lytic epitope reactivates and 10- to 20-foldenrichment of latent epitope reactivates in tonsil compared with blood; up to 20% of tonsillar CD8+ T cells were EBV specific, and many now expressed CD103. Therefore efficient control of EBV infection requires appropriate CD8+ T cell homing to oropharyngeal sites. We know that, relevant for the identification of EBV are the places where they exist manly or where they cause consistent modification such as malignancies, so locating could be during search through immunohistochemical method in tonsil tissue removed from infected patient. The virus can be determined through a specific protocol. During cutting, the tonsil tissue is soaked with ethanol 2. M NaCl. Using the gavage needle attached to the syringe, the neck is flushed with 10 ml of 0.15 M NaCl. Using the Curved scissors, the tonsil tissue is dissected. The tonsil tissue is transfered into the Erlenmeyer flask contanin 20 ml RMPI and a stirring bar. It is gently stirred for 10 min at room temperature. The supernatant is discarded and the process is repeated three times. The washing medium is replaced with 10 ml dispase/DNAase solution and it is gently stirred for 30 min at 37 C. The supernatant is then collected in a 50-ml conical tube containing 35 ml RMPI with 5%FCS and is kept on ice. The last two steps are repeated four times and then the tubes are centrifuged at 250g for 10 min at 4C. The example is resuspended and pooled in RPMI with 5% FCS and filter through a nylon cell strainer. Only sterile material and aseptic technique must be used so a proper research could be established with remarkable precision. Also researching EBV could be through viral cultures, immunologic assay try detecting HSV antigens in ELISA, tzanck smear, cytology examination try researching base of the skin looking for nonspecific giant cell and eosiophilic intranuclear inclusion, serologic tests, PCR, detecting HSV DNA in very sensitive method, and lumbar puncture, radiologic and brain biopsy. Tonsillectomy specimens are disaggregated to single-cell suspensions by testing apart the tissue and fine mincing. Mononuclear cells are isolated by purification over a Lymphoprep gradient (Nycomed) per the manufacturer's instructions. PBMCs are isolated in a similar manner from peripheral blood specimens taken at the time of tonsillectomy. In both cases, mononuclear cells are aliquoted, cryopreserved, and stored in liquid nitrogen. DNA was isolated from an aliquot of the tonsillar cells for HLA typing by sequence-specific oligonucleotide PCR analysis. Also it could be research made through flow cytometry anylasyse of the analyses of the sample, ELISpot analyses of a sample , virus genom load analyses, and statical analyses. References: 1. Hausen, Harald zur. Infections causing human cancer. Darmstadt, Germany: Wiley-VCH, 2006 2. Killeen, Anthony A. Molecular Pathology protocols. Totowa, New Jersey: Humana press, 2001. 3. Kinchington, Derek and Raymond F. Schinazi. Antiviral Methods and Protocols. Totowa, New Jersey: Humana press, 1999 Read More