Formal proposal
-
Upload
celizbetscolon -
Category
Documents
-
view
432 -
download
0
description
Transcript of Formal proposal
University of Puerto Rico at Cayey
RISE Summer Bridge Program
Tuesday, June 12, 2011
Tittle:
Increase antibody production as treatment against development of Lissencephaly caused by CMV in pregnant Mus musculus
Abstract
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.
Introduction
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.
Materials and methods
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.1 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 PMN2 by dextran separation with
Dextran Leukocyte Separation Kit and preparation of slides, fixation, immunostaining, and 1 Use mice from 12-13 days and 8-9 weeks. Anestethiaze mice with ketamine (60 mg/kg,ip) and xylazine (10 mg/kg, ip) Inject mice with
unilateral pressure injections of 1.0–1.5 µl of CMV vector solution into the body using a glass micropipette with a tip diameter of 20–30
µm.Place pipette for an 30 s and then slowly withdrawn.
reading and quantification) of Antigenemia Assay3 . 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-404, because of
the good evidence of the clearer signal and the higher sensitivity with immunofluorescence
staining it provides. For the Immunostaining will be used MAbs5 against the lower matrix protein
pp656, 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.
Expected results and discussion
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
2 PMN (Polymorphonuclear leukocytes)= CMV DNA can be detected in different fractions of leukocytes like this one during active infection3 PCR for CMV DNA. “Due to the high sensitivity of these assays, CMV is also detectable in a substantial number of patients with asymptomatic infection who never progress to disease.” Quantitation of Cytomegalovirus: Methodologic Aspects and Clinical Applications4 A solution made of a simple Aldehyde and a detergent.5 Acronym for Monoclonal Antibodies.6 Specific protein prodeuced by CMV.
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.
References:
1. 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. 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. 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. 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. 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