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An improved protocol for efficient engraftment in NOD/LTSZ-SCIDIL-2Rγ mice allows HIV replication and development of anti-HIV immune responses.

Singh M, Singh P, Gaudray G, Musumeci L, Thielen C, Vaira D, Vandergeeten C, Delacroix L, Van Gulck E, Vanham G, de Leval L, Rahmouni S, Moutschen M - PLoS ONE (2012)

Bottom Line: In the present study, we further ameliorated the busulfan myeloablation regimen with fresh CB-CD34+cell transplantation in 3-4 week old NSG mice.Optimal engraftment promoted early and increased CD3+T cell levels, with better lymphoid tissue development and prolonged human cell chimerism over 300 days.We also saw a gradual decline of the CD4+T cell count, widespread immune activation, up-regulation of inflammation marker and microbial translocation after HIV-1 infection.

View Article: PubMed Central - PubMed

Affiliation: Immunology and infectious diseases unit GIGA-I3, University of Liege, Liege, Belgium.

ABSTRACT
Cord blood hematopoietic progenitor cells (CB-HPCs) transplanted immunodeficient NOD/LtsZ-scidIL2Rγ() (NSG) and NOD/SCID/IL2Rγ() (NOG) mice need efficient human cell engraftment for long-term HIV-1 replication studies. Total body irradiation (TBI) is a classical myeloablation regimen used to improve engraftment levels of human cells in these humanized mice. Some recent reports suggest the use of busulfan as a myeloablation regimen to transplant HPCs in neonatal and adult NSG mice. In the present study, we further ameliorated the busulfan myeloablation regimen with fresh CB-CD34+cell transplantation in 3-4 week old NSG mice. In this CB-CD34+transplanted NSG mice engraftment efficiency of human CD45+cell is over 90% in peripheral blood. Optimal engraftment promoted early and increased CD3+T cell levels, with better lymphoid tissue development and prolonged human cell chimerism over 300 days. These humanized NSG mice have shown long-lasting viremia after HIV-1JRCSF and HIV-1Bal inoculation through intravenous and rectal routes. We also saw a gradual decline of the CD4+T cell count, widespread immune activation, up-regulation of inflammation marker and microbial translocation after HIV-1 infection. Humanized NSG mice reconstituted according to our new protocol produced, moderate cellular and humoral immune responses to HIV-1 postinfection. We believe that NSG mice reconstituted according to our easy to use protocol will provide a better in vivo model for HIV-1 replication and anti-HIV-1 therapy trials.

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Sustained high-level of HIV dissemination and infection progression.(A) Humanized mice were i.v. injected with HIV-1 JRCSF and HIV-1 Bal at 10,000 TCID. Mice were bled at 20, 40, 60, 80, 100 and 120 days to detect plasma viral load, curve represent the viral load at different time point. Error bars (B) Sections of lymph node (a) and spleen (b) of humanized HIV infected mice were stained using anti-p24 antibody. Brown color show p24 specific staining. Lymph node of HIV-1 infected human (c) is shown as positive control and engrafted noninfected mouse spleen (d) is shown as negative control. (C) DNA was extracted from peripheral blood of HIV-1JRCSF-infected mice 9 week after infection. Determination of HIV-1 DNA copy was performed by PCR assay for gag and pol gene. 8E5 cell DNA was amplified as positive control (PC) along with humanized mice DNA (hm1, hm2, and hm3) (D, E) Percentage of CD4+ cells of T-cells following HIV-1 infection, each circle represent one mouse infected with HIV-1 JRCSF and each square represent one mouse infected with HIV-1 Bal means of CD4% of CD3+ cells at time point of infection are shown in solid line.
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pone-0038491-g004: Sustained high-level of HIV dissemination and infection progression.(A) Humanized mice were i.v. injected with HIV-1 JRCSF and HIV-1 Bal at 10,000 TCID. Mice were bled at 20, 40, 60, 80, 100 and 120 days to detect plasma viral load, curve represent the viral load at different time point. Error bars (B) Sections of lymph node (a) and spleen (b) of humanized HIV infected mice were stained using anti-p24 antibody. Brown color show p24 specific staining. Lymph node of HIV-1 infected human (c) is shown as positive control and engrafted noninfected mouse spleen (d) is shown as negative control. (C) DNA was extracted from peripheral blood of HIV-1JRCSF-infected mice 9 week after infection. Determination of HIV-1 DNA copy was performed by PCR assay for gag and pol gene. 8E5 cell DNA was amplified as positive control (PC) along with humanized mice DNA (hm1, hm2, and hm3) (D, E) Percentage of CD4+ cells of T-cells following HIV-1 infection, each circle represent one mouse infected with HIV-1 JRCSF and each square represent one mouse infected with HIV-1 Bal means of CD4% of CD3+ cells at time point of infection are shown in solid line.

Mentions: Humanized mice were inoculated i.v. with two different HIV-1 isolates. We usedCCR5-tropic (R5-tropic) JRCSF and HIV-1Bal isolates inoculated at 10,000 TCID50 doses for infection. Mice were bled at 20, 40, 60, 80, 100 and 120 days postinfection and plasma viral loads were determined by cobas AmpliPrep/cobas Taq Man Version 2.0 HIV-1 assay. Productive infection was observed after inoculation with both isolates. For HIV-1Bal, viral load reached 4.2×105±2.2×105 copies/ml on day 40 and remained over 1.9×105±5.8×104 copies/ml at the three following time points. Viral loads were lower for HIV-1JRCSF reaching 1.6×105±3.5×104 copies/ml at day 60 and 1.85×105±3.2×104 copies/ml 100 days after infection. Viral loads were still detected at day 120 for both isolates (Fig. 4A). In a preliminary experiment, mice infected by the rectal route (BAL isolate at 2500 TCID50) and showed high levels of plasma viral load 0.93×105±2.2×104 copies/ml (n = 3) 35 days after inoculation.


An improved protocol for efficient engraftment in NOD/LTSZ-SCIDIL-2Rγ mice allows HIV replication and development of anti-HIV immune responses.

Singh M, Singh P, Gaudray G, Musumeci L, Thielen C, Vaira D, Vandergeeten C, Delacroix L, Van Gulck E, Vanham G, de Leval L, Rahmouni S, Moutschen M - PLoS ONE (2012)

Sustained high-level of HIV dissemination and infection progression.(A) Humanized mice were i.v. injected with HIV-1 JRCSF and HIV-1 Bal at 10,000 TCID. Mice were bled at 20, 40, 60, 80, 100 and 120 days to detect plasma viral load, curve represent the viral load at different time point. Error bars (B) Sections of lymph node (a) and spleen (b) of humanized HIV infected mice were stained using anti-p24 antibody. Brown color show p24 specific staining. Lymph node of HIV-1 infected human (c) is shown as positive control and engrafted noninfected mouse spleen (d) is shown as negative control. (C) DNA was extracted from peripheral blood of HIV-1JRCSF-infected mice 9 week after infection. Determination of HIV-1 DNA copy was performed by PCR assay for gag and pol gene. 8E5 cell DNA was amplified as positive control (PC) along with humanized mice DNA (hm1, hm2, and hm3) (D, E) Percentage of CD4+ cells of T-cells following HIV-1 infection, each circle represent one mouse infected with HIV-1 JRCSF and each square represent one mouse infected with HIV-1 Bal means of CD4% of CD3+ cells at time point of infection are shown in solid line.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3366932&req=5

pone-0038491-g004: Sustained high-level of HIV dissemination and infection progression.(A) Humanized mice were i.v. injected with HIV-1 JRCSF and HIV-1 Bal at 10,000 TCID. Mice were bled at 20, 40, 60, 80, 100 and 120 days to detect plasma viral load, curve represent the viral load at different time point. Error bars (B) Sections of lymph node (a) and spleen (b) of humanized HIV infected mice were stained using anti-p24 antibody. Brown color show p24 specific staining. Lymph node of HIV-1 infected human (c) is shown as positive control and engrafted noninfected mouse spleen (d) is shown as negative control. (C) DNA was extracted from peripheral blood of HIV-1JRCSF-infected mice 9 week after infection. Determination of HIV-1 DNA copy was performed by PCR assay for gag and pol gene. 8E5 cell DNA was amplified as positive control (PC) along with humanized mice DNA (hm1, hm2, and hm3) (D, E) Percentage of CD4+ cells of T-cells following HIV-1 infection, each circle represent one mouse infected with HIV-1 JRCSF and each square represent one mouse infected with HIV-1 Bal means of CD4% of CD3+ cells at time point of infection are shown in solid line.
Mentions: Humanized mice were inoculated i.v. with two different HIV-1 isolates. We usedCCR5-tropic (R5-tropic) JRCSF and HIV-1Bal isolates inoculated at 10,000 TCID50 doses for infection. Mice were bled at 20, 40, 60, 80, 100 and 120 days postinfection and plasma viral loads were determined by cobas AmpliPrep/cobas Taq Man Version 2.0 HIV-1 assay. Productive infection was observed after inoculation with both isolates. For HIV-1Bal, viral load reached 4.2×105±2.2×105 copies/ml on day 40 and remained over 1.9×105±5.8×104 copies/ml at the three following time points. Viral loads were lower for HIV-1JRCSF reaching 1.6×105±3.5×104 copies/ml at day 60 and 1.85×105±3.2×104 copies/ml 100 days after infection. Viral loads were still detected at day 120 for both isolates (Fig. 4A). In a preliminary experiment, mice infected by the rectal route (BAL isolate at 2500 TCID50) and showed high levels of plasma viral load 0.93×105±2.2×104 copies/ml (n = 3) 35 days after inoculation.

Bottom Line: In the present study, we further ameliorated the busulfan myeloablation regimen with fresh CB-CD34+cell transplantation in 3-4 week old NSG mice.Optimal engraftment promoted early and increased CD3+T cell levels, with better lymphoid tissue development and prolonged human cell chimerism over 300 days.We also saw a gradual decline of the CD4+T cell count, widespread immune activation, up-regulation of inflammation marker and microbial translocation after HIV-1 infection.

View Article: PubMed Central - PubMed

Affiliation: Immunology and infectious diseases unit GIGA-I3, University of Liege, Liege, Belgium.

ABSTRACT
Cord blood hematopoietic progenitor cells (CB-HPCs) transplanted immunodeficient NOD/LtsZ-scidIL2Rγ() (NSG) and NOD/SCID/IL2Rγ() (NOG) mice need efficient human cell engraftment for long-term HIV-1 replication studies. Total body irradiation (TBI) is a classical myeloablation regimen used to improve engraftment levels of human cells in these humanized mice. Some recent reports suggest the use of busulfan as a myeloablation regimen to transplant HPCs in neonatal and adult NSG mice. In the present study, we further ameliorated the busulfan myeloablation regimen with fresh CB-CD34+cell transplantation in 3-4 week old NSG mice. In this CB-CD34+transplanted NSG mice engraftment efficiency of human CD45+cell is over 90% in peripheral blood. Optimal engraftment promoted early and increased CD3+T cell levels, with better lymphoid tissue development and prolonged human cell chimerism over 300 days. These humanized NSG mice have shown long-lasting viremia after HIV-1JRCSF and HIV-1Bal inoculation through intravenous and rectal routes. We also saw a gradual decline of the CD4+T cell count, widespread immune activation, up-regulation of inflammation marker and microbial translocation after HIV-1 infection. Humanized NSG mice reconstituted according to our new protocol produced, moderate cellular and humoral immune responses to HIV-1 postinfection. We believe that NSG mice reconstituted according to our easy to use protocol will provide a better in vivo model for HIV-1 replication and anti-HIV-1 therapy trials.

Show MeSH
Related in: MedlinePlus