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De Novo Generated Human Red Blood Cells in Humanized Mice Support Plasmodium falciparum Infection.

Amaladoss A, Chen Q, Liu M, Dummler SK, Dao M, Suresh S, Chen J, Preiser PR - PLoS ONE (2015)

Bottom Line: The poor reconstitution is mainly the result of a deficiency of appropriate human cytokines that are necessary for the development and maintenance of these cell lineages.In vivo adaptation of K1 strain further improves the infection efficiency and parasites can be detected by microscopy for 3 cycles.The parasitemia ranges between 0.13 and 0.25% at the first cycle of infection, falls between 0.08 and 0.15% at the second cycle, and drops to barely detectable levels at the third cycle of infection.

View Article: PubMed Central - PubMed

Affiliation: Infectious Diseases Interdisciplinary Research Group, Singapore-Massachusetts Institute of Technology Alliance for Research and Technology, Singapore, 138602, Singapore.

ABSTRACT
Immunodeficient mouse-human chimeras provide a powerful approach to study host specific pathogens like Plasmodium (P.) falciparum that causes human malaria. Existing mouse models of P. falciparum infection require repeated injections of human red blood cells (RBCs). In addition, clodronate lipsomes and anti-neutrophil antibodies are injected to suppress the clearance of human RBCs by the residual immune system of the immunodeficient mice. Engraftment of NOD-scid Il2rg-/- mice with human hematopoietic stem cells leads to reconstitution of human immune cells. Although human B cell reconstitution is robust and T cell reconstitution is reasonable in the recipient mice, human RBC reconstitution is generally poor or undetectable. The poor reconstitution is mainly the result of a deficiency of appropriate human cytokines that are necessary for the development and maintenance of these cell lineages. Delivery of plasmid DNA encoding human erythropoietin and interleukin-3 into humanized mice by hydrodynamic tail-vein injection resulted in significantly enhanced reconstitution of erythrocytes. With this improved humanized mouse, here we show that P. falciparum infects de novo generated human RBCs, develops into schizonts and causes successive reinvasion. We also show that different parasite strains exhibit variation in their ability to infect these humanized mice. Parasites could be detected by nested PCR in the blood samples of humanized mice infected with P. falciparum K1 and HB3 strains for 3 cycles, whereas in other strains such as 3D7, DD2, 7G8, FCR3 and W2mef parasites could only be detected for 1 cycle. In vivo adaptation of K1 strain further improves the infection efficiency and parasites can be detected by microscopy for 3 cycles. The parasitemia ranges between 0.13 and 0.25% at the first cycle of infection, falls between 0.08 and 0.15% at the second cycle, and drops to barely detectable levels at the third cycle of infection. Compared to existing mouse models, our model generates human RBCs de novo and does not require the treatment of mice with immunomodulators.

No MeSH data available.


Related in: MedlinePlus

Infection of P. falciparum (3D7) in de novo generated human RBCs.RBCs from humanized mice and purified schizonts were stained with Red CMTPX and Green CMFDA dyes, respectively. After the overnight culture, the parasites were stained with DAPI and imaged under fluorescent microscope (Panel A). Panel B and C represent the images taken under Green (contaminant RBC from culture) and Red (humanized mice RBC) channels. A ring stage parasite can be seen in red stained RBC (Panel C) indicating the infection of de novo generated human RBC in humanized mice. Panel D and E represent the bright field and composite images respectively.
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pone.0129825.g001: Infection of P. falciparum (3D7) in de novo generated human RBCs.RBCs from humanized mice and purified schizonts were stained with Red CMTPX and Green CMFDA dyes, respectively. After the overnight culture, the parasites were stained with DAPI and imaged under fluorescent microscope (Panel A). Panel B and C represent the images taken under Green (contaminant RBC from culture) and Red (humanized mice RBC) channels. A ring stage parasite can be seen in red stained RBC (Panel C) indicating the infection of de novo generated human RBC in humanized mice. Panel D and E represent the bright field and composite images respectively.

Mentions: In order to confirm that the infected RBCs are in fact from humanized mouse origin and not from contaminant RBCs carried along with purified schizonts, we performed a double staining experiment. RBCs from humanized mice and in vitro parasite culture (source of schizonts) were stained with CellTracker Red CMTPX and Green CMFDA (Life Technologies) fluorescent dyes, respectively. These dyes freely pass through cell membranes into the cells, where they transform into a cell-impermeable, fluorescent product which are retained for several days. After overnight culture of the purified schizonts with blood from humanized mice, the parasites were stained with DAPI and imaged under fluorescent microscope. The parasites (blue) were observed in RBCs stained with red confirming that the parasites invaded only the de novo generated human RBC from mice (Fig 1).


De Novo Generated Human Red Blood Cells in Humanized Mice Support Plasmodium falciparum Infection.

Amaladoss A, Chen Q, Liu M, Dummler SK, Dao M, Suresh S, Chen J, Preiser PR - PLoS ONE (2015)

Infection of P. falciparum (3D7) in de novo generated human RBCs.RBCs from humanized mice and purified schizonts were stained with Red CMTPX and Green CMFDA dyes, respectively. After the overnight culture, the parasites were stained with DAPI and imaged under fluorescent microscope (Panel A). Panel B and C represent the images taken under Green (contaminant RBC from culture) and Red (humanized mice RBC) channels. A ring stage parasite can be seen in red stained RBC (Panel C) indicating the infection of de novo generated human RBC in humanized mice. Panel D and E represent the bright field and composite images respectively.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4476714&req=5

pone.0129825.g001: Infection of P. falciparum (3D7) in de novo generated human RBCs.RBCs from humanized mice and purified schizonts were stained with Red CMTPX and Green CMFDA dyes, respectively. After the overnight culture, the parasites were stained with DAPI and imaged under fluorescent microscope (Panel A). Panel B and C represent the images taken under Green (contaminant RBC from culture) and Red (humanized mice RBC) channels. A ring stage parasite can be seen in red stained RBC (Panel C) indicating the infection of de novo generated human RBC in humanized mice. Panel D and E represent the bright field and composite images respectively.
Mentions: In order to confirm that the infected RBCs are in fact from humanized mouse origin and not from contaminant RBCs carried along with purified schizonts, we performed a double staining experiment. RBCs from humanized mice and in vitro parasite culture (source of schizonts) were stained with CellTracker Red CMTPX and Green CMFDA (Life Technologies) fluorescent dyes, respectively. These dyes freely pass through cell membranes into the cells, where they transform into a cell-impermeable, fluorescent product which are retained for several days. After overnight culture of the purified schizonts with blood from humanized mice, the parasites were stained with DAPI and imaged under fluorescent microscope. The parasites (blue) were observed in RBCs stained with red confirming that the parasites invaded only the de novo generated human RBC from mice (Fig 1).

Bottom Line: The poor reconstitution is mainly the result of a deficiency of appropriate human cytokines that are necessary for the development and maintenance of these cell lineages.In vivo adaptation of K1 strain further improves the infection efficiency and parasites can be detected by microscopy for 3 cycles.The parasitemia ranges between 0.13 and 0.25% at the first cycle of infection, falls between 0.08 and 0.15% at the second cycle, and drops to barely detectable levels at the third cycle of infection.

View Article: PubMed Central - PubMed

Affiliation: Infectious Diseases Interdisciplinary Research Group, Singapore-Massachusetts Institute of Technology Alliance for Research and Technology, Singapore, 138602, Singapore.

ABSTRACT
Immunodeficient mouse-human chimeras provide a powerful approach to study host specific pathogens like Plasmodium (P.) falciparum that causes human malaria. Existing mouse models of P. falciparum infection require repeated injections of human red blood cells (RBCs). In addition, clodronate lipsomes and anti-neutrophil antibodies are injected to suppress the clearance of human RBCs by the residual immune system of the immunodeficient mice. Engraftment of NOD-scid Il2rg-/- mice with human hematopoietic stem cells leads to reconstitution of human immune cells. Although human B cell reconstitution is robust and T cell reconstitution is reasonable in the recipient mice, human RBC reconstitution is generally poor or undetectable. The poor reconstitution is mainly the result of a deficiency of appropriate human cytokines that are necessary for the development and maintenance of these cell lineages. Delivery of plasmid DNA encoding human erythropoietin and interleukin-3 into humanized mice by hydrodynamic tail-vein injection resulted in significantly enhanced reconstitution of erythrocytes. With this improved humanized mouse, here we show that P. falciparum infects de novo generated human RBCs, develops into schizonts and causes successive reinvasion. We also show that different parasite strains exhibit variation in their ability to infect these humanized mice. Parasites could be detected by nested PCR in the blood samples of humanized mice infected with P. falciparum K1 and HB3 strains for 3 cycles, whereas in other strains such as 3D7, DD2, 7G8, FCR3 and W2mef parasites could only be detected for 1 cycle. In vivo adaptation of K1 strain further improves the infection efficiency and parasites can be detected by microscopy for 3 cycles. The parasitemia ranges between 0.13 and 0.25% at the first cycle of infection, falls between 0.08 and 0.15% at the second cycle, and drops to barely detectable levels at the third cycle of infection. Compared to existing mouse models, our model generates human RBCs de novo and does not require the treatment of mice with immunomodulators.

No MeSH data available.


Related in: MedlinePlus