open access

Vol 47, No 3 (2009)
ORIGINAL PAPERS
Published online: 2010-02-19
Submitted: 2011-12-19
Get Citation

An optimization of protocol for mixed chimerism induction in mice model.

M Baśkiewicz-Masiuk, K Grymuła, E Pius, M Hałasa, V Dziedziejko, Ch Schmidt, M Walczak, B Machaliński
DOI: 10.2478/v10042-009-0086-z
·
Folia Histochem Cytobiol 2009;47(3):395-400.

open access

Vol 47, No 3 (2009)
ORIGINAL PAPERS
Published online: 2010-02-19
Submitted: 2011-12-19

Abstract

Studies on mixed chimerism are currently focused primarily on obtaining less toxic conditioning protocols. With these issues in mind, we have undertaken the attempt to optimize the procedure of mixed chimerism induction in mice. In order to reduce toxicity, we used decreasing doses of total body irradiation (TBI) together with combination of blocking antibodies. We also tried to eliminate immunosuppression (cyclophosphamide - CP) treatment after bone marrow transplantation. B6.SJL-PtprcaPep3b mice were injected with 20-30 x 106 bone marrow cells from Balb C mice. Mice were treated with TBI (3 - 1.5 - 0 Gy) on "-1" day of the experiment and blocking antibodies against CD40L ("0", and "4" days) and additionally anti-CD8 ("-2" day) and/or anti-NK1.1 ("-3" day). Mice in certain groups also received CP (175 mg/kg) on "2" day. Presence of mixed chimerism was assessed in peripheral blood cells by flow cytometry on the 1st, 2nd, 3rd, 4th, 6th and 8th weeks of the experiment by detecting of CD45.1 (characteristic for B6.SJL-PtprcaPep3b strain) and CD45.2 (characteristic for Balb C strain) antigens expression. We also analyzed the percentage of peripheral blood CD8 T-cells (CD3e/CD8a) and NK cells (Ly-49D/NK1.1). We found that reduction of TBI dose and elimination of CP decrease the rate of mixed chimerism formation. The highest percentage of donor cells was obtained in the group of animals treated with 3 Gy of TBI, CP and combination of anti-CD40L, anti-CD8, and anti-NK1.1 antibodies. The 3 Gy TBI was necessary to induce stable mixed chimerism, but it could be obtained without the CP use. The percentage of CD3e/CD8a and Ly-49D/NK1.1 cells was significantly lower in the groups of mice treated by corresponding antibodies. Moreover, we observed the lowest number of peripheral blood Ly-49D/NK1.1 cells in the group of animals with highest mixed chimerism. Our experiments in mice model can help in better understanding of mixed chimerism phenomenon and in selecting the method of mixed chimerism induction with lowest possible toxicity. This also might improve the protocols of stable mixed chimerism induction in humans, and in the future, the effectiveness of vascularized organ transplantation.

Abstract

Studies on mixed chimerism are currently focused primarily on obtaining less toxic conditioning protocols. With these issues in mind, we have undertaken the attempt to optimize the procedure of mixed chimerism induction in mice. In order to reduce toxicity, we used decreasing doses of total body irradiation (TBI) together with combination of blocking antibodies. We also tried to eliminate immunosuppression (cyclophosphamide - CP) treatment after bone marrow transplantation. B6.SJL-PtprcaPep3b mice were injected with 20-30 x 106 bone marrow cells from Balb C mice. Mice were treated with TBI (3 - 1.5 - 0 Gy) on "-1" day of the experiment and blocking antibodies against CD40L ("0", and "4" days) and additionally anti-CD8 ("-2" day) and/or anti-NK1.1 ("-3" day). Mice in certain groups also received CP (175 mg/kg) on "2" day. Presence of mixed chimerism was assessed in peripheral blood cells by flow cytometry on the 1st, 2nd, 3rd, 4th, 6th and 8th weeks of the experiment by detecting of CD45.1 (characteristic for B6.SJL-PtprcaPep3b strain) and CD45.2 (characteristic for Balb C strain) antigens expression. We also analyzed the percentage of peripheral blood CD8 T-cells (CD3e/CD8a) and NK cells (Ly-49D/NK1.1). We found that reduction of TBI dose and elimination of CP decrease the rate of mixed chimerism formation. The highest percentage of donor cells was obtained in the group of animals treated with 3 Gy of TBI, CP and combination of anti-CD40L, anti-CD8, and anti-NK1.1 antibodies. The 3 Gy TBI was necessary to induce stable mixed chimerism, but it could be obtained without the CP use. The percentage of CD3e/CD8a and Ly-49D/NK1.1 cells was significantly lower in the groups of mice treated by corresponding antibodies. Moreover, we observed the lowest number of peripheral blood Ly-49D/NK1.1 cells in the group of animals with highest mixed chimerism. Our experiments in mice model can help in better understanding of mixed chimerism phenomenon and in selecting the method of mixed chimerism induction with lowest possible toxicity. This also might improve the protocols of stable mixed chimerism induction in humans, and in the future, the effectiveness of vascularized organ transplantation.
Get Citation
About this article
Title

An optimization of protocol for mixed chimerism induction in mice model.

Journal

Folia Histochemica et Cytobiologica

Issue

Vol 47, No 3 (2009)

Pages

395-400

Published online

2010-02-19

DOI

10.2478/v10042-009-0086-z

Bibliographic record

Folia Histochem Cytobiol 2009;47(3):395-400.

Authors

M Baśkiewicz-Masiuk
K Grymuła
E Pius
M Hałasa
V Dziedziejko
Ch Schmidt
M Walczak
B Machaliński

Important: This website uses cookies. More >>

The cookies allow us to identify your computer and find out details about your last visit. They remembering whether you've visited the site before, so that you remain logged in - or to help us work out how many new website visitors we get each month. Most internet browsers accept cookies automatically, but you can change the settings of your browser to erase cookies or prevent automatic acceptance if you prefer.

By "Via Medica sp. z o.o." sp.k., ul. Świętokrzyska 73, 80–180 Gdańsk

tel.:+48 58 320 94 94, faks:+48 58 320 94 60, e-mail:  viamedica@viamedica.pl