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