Formal proposal

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Formal proposal

  1. 1. University of Puerto Rico at Cayey<br />RISE Summer Bridge Program<br />Tuesday, June 12, 2011<br />Tittle:<br />Increase antibody production as treatment against development of Lissencephaly caused by CMV in pregnant Mus musculus<br />Abstract<br />The main purpose of this Project is to develop a treatment with antibodies against the development of Lissencephaly caused by Citomegalovirus (for now on, CMV). The research model used is Mus musculus. Knowing that the presence of CMV activates the immune system in order to combat the antigen is why it is reasoned that the augment of specialized antibodies IgG and IgM may reduce the presence of CVM, and consecuently, the risks of the fetus of developing Lissencephaly. The antibody presence will be directly induced by injecting IgG or IgM; also, it will be indirectly induced injecting Interleukins (for now on, IL) 6 or 28. The injections to CMV infected pregnant Mus musculus will be given in different lapses of time. After fifteen days (four to five days before birth), Chronic Villus Sampling test will be done for a Direct Immunoflorescence Assay, will be extracted blood for the Antigenemia Assay, and a MRI in order to compare results and determine whether or not the experimental treatment is effective to the hypothesis.<br />Introduction<br />Researches as Malformations of cortical development in children with congenital cytomegalovirus infection - A study of nine children with proven congenital cytomegalovirus infection affirm the development of Lissencephaly in the fetus as direct consequence of early infection. Also states: “Congenital cytomegalovirus (CMV) infection is the most common vertically transmitted disease with the rate of the infection ranging from 0.2 to 2.4% in newborn infants”. A chemical remedy had already been experimented in Oral Hexadecyloxypropyl-Cidofovir Therapy in Pregnant Guinea Pigs Improves Outcome in the Congenital Model of Cytomegalovirus Infection as an alternative orally supply analog of the existing drug, Cidofovir, against CMV. Otherwise, the the use of direct supply of antibodies as a method against the virus, wasn’t found while research. A journal named Seroprevalence of cytomegalovirus infection in pregnant women and associated role in obstetric complications: a preliminary study notes that from 125 pregnant women tested, IgG antibody was found 84% of the times, but IgM only on 7.2%. It means that they weren’t always presences, signifying lack of antibody levels. Therefore, that can be reasoned by the augment of antibodies IgG and IgM may reduce the presence of CVM, representing a minor risk to the fetus in developing Lissencephaly. <br />Materials and methods<br />The chosen organism to be worked with is Mus musculus, because its resemblance to human, able to be infected with CMV, more ethical to work with than humans, small sized, highly fertile, and principally because is grades as ideal for ‘in vivo’ hybridomas to produce continuous supply of antibody. The total of female pregnant Mus musculus will be 39: 9 for each experimental group (IgG, IgM, IL 9, IL 28), plus 3 for the control group. All 39 will be injected with CMV Pp38 (UL80a), Cytomegalovirus Antigen, Recombinant according to Selective Optical Control of Synaptic Transmission in the Subcortical Visual Pathway by Activation of Viral Vector-Expressed Halorhodopsin. Next, the corresponding substances of each group will be injected: group 1, Mouse IgG control (Whole Molecule), Purified; group 2, IgM Antibody, mAb, Mouse; group 3, IL-4 Antibody (2G6A8), mAb, Mouse; group 4, Mouse IL-28A/B (IFN-lambda 2/3) Biotin MAb (Clone 244707); group 5, none (it will be the control group). Each group will be divided in sub-groups A, B, and C (each having three mice), which will represent different timings of supply. To the sub-group A the substance will be supplied every 48 hours; sub-group B, every 3 days; sub-group C, every week. Each will be for a complete of period fifteen days, because the sampling can be made days before the offspring born. After fifteen days of gestation, the CVS (Chronic Villous Sampling) in order to analyze the placenta tissue. For the analysis of the placental tissue will be made an Immunofluorescent test with Light Diagnostics CMV Direct Immunofluorescence Assay (DFA) Kit. To quantify results, will be used a Image Cytometri. Also will be used all four steps (isolation of the PMN by dextran separation with Dextran Leukocyte Separation Kit and preparation of slides, fixation, immunostaining, and reading and quantification) of Antigenemia Assay . According to the recommendation of Quantitation of Cytomegalovirus: Methodologic Aspects and Clinical Applications slides will be the same day of the assay for optimal results, which are considered by some investigators from 150,000 to 200,000 cells. For the Fixation will be used Formaldehyde-Noidet P-40, because of the good evidence of the clearer signal and the higher sensitivity with immunofluorescence staining it provides. For the Immunostaining will be used MAbs against the lower matrix protein pp65, because it is required a shorter processing time and a higher sensitivity with immunofluorescence staining than with others. Finally it will be read and quantificated using the method of numbering the positive cells per slide. It will be determined using Image Cytometri. In order to organize and analyze the obtained data, some graphics and tables will be done. They help the direct comparing of the experimental group results with the control group results and determine whether or not the method was efficient. Is recommended to repeat tests two more times. After the mice are born, a MRI will be made in order to search for Lysencephaly characteristics like absence of gyres, fissures or grooves.<br />Expected results and discussion<br />After the methodology is correctly done and without any other complication, is expected to have results in Immunofluorescent assay and Antigenemia Assay that support the hypothesis. Is projected that the results for Direct Immunofluorescence will include descriptions that will conclude in a brighter image for control group than for experimental groups. That means that will be more tagged antibodies supplied for that test in the control group (which will have an increased number of CMV) attached to CMV than to the tissues of control groups. The Antigenia Assay results will demonstrate less positive results in the experimental groups and more in control groups –Tables 4 and 5 of the Quantitation of Cytomegalovirus: Methodologic Aspects and Clinical Applications show quantitave results of other experiments made with the Antigenemia Assay and shows clear positive and negative numbers. Even though doesn’t show any experiment done with placenta, it is commonly used for detection of CMV in different patients settings. – If those tests show a lower level of the CMV, is expected that the MRI test will show normal cephalic mice. If something goes wrong and it is found what didn’t function well, it will be improved and re-done, but with the necessary improvements. After being successful, the augment of antibodies IgG and IgM will be used as a treatment to reduce the presence of CMV and reduce the risks of fetus of developing Lissencephaly.<br />References:<br /><ul><li>Bosnjak VM, Daković I, et al. Malformations of cortical development in children with congenital cytomegalovirus infection - A study of nine children with proven congenital cytomegalovirus infection. January, 2011. http://www.ncbi.nlm.nih.gov/pubmed/21648339
  2. 2. Ravo FJ, Bernstein DI, et al. Oral Hexadecyloxypropyl-Cidofovir Therapy in Pregnant Guinea Pigs Improves Outcome in the Congenital Model of Cytomegalovirus Infection. November 15, 2011. http://www.ncbi.nlm.nih.gov/pubmed?term=Oral%20Hexadecyloxypropyl-Cidofovir%20Therapy%20in%20Pregnant%20Guinea%20Pigs%20Improves%20Outcome%20in%20the%20Congenital%20Model%20of%20Cytomegalovirus%20Infection%20
  3. 3. Araswathy TS, Az-Ulhusna A, et al. Seroprevalence of cytomegalovirus infection in pregnant women and associated role in obstetric complications: a preliminary study. March, 2011. http://www.ncbi.nlm.nih.gov/pubmed/21710852
  4. 4. Kaneda K, Kasahara H, et al. Selective Optical Control of Synaptic Transmission in the Subcortical Visual Pathway by Activation of Viral Vector-Expressed Halorhodopsin. April 5, 2011. http://www.ncbi.nlm.nih.gov/pubmed?term=Selective%20Optical%20Control%20of%20Synaptic%20Transmission%20in%20the%20Subcortical%20Visual%20Pathway%20by%20Activation%20of%20Viral%20Vector-Expressed%20Halorhodopsin
  5. 5. Boeckh, M. and Boivin, G. Quantitation of Cytomegalovirus: Methodologic Aspects and Clinical Applications. Clinical Microbiology Reviews, July 1998, p. 533-554, Vol. 11, No. 3. http://cmr.asm.org/cgi/content/full/11/3/533

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