Limits...
Humanized HLA-DR4.RagKO.IL2RγcKO.NOD (DRAG) mice sustain the complex vertebrate life cycle of Plasmodium falciparum malaria.

Wijayalath W, Majji S, Villasante EF, Brumeanu TD, Richie TL, Casares S - Malar. J. (2014)

Bottom Line: Malaria is a deadly infectious disease affecting millions of people in tropical and sub-tropical countries.Among the five species of Plasmodium parasites that infect humans, Plasmodium falciparum accounts for the highest morbidity and mortality associated with malaria.Four week-old DRAG mice were infused with HLA-matched human haematopoietic stem cells (HSC) and examined for reconstitution of human liver cells and erythrocytes.

View Article: PubMed Central - PubMed

Affiliation: US Military Malaria Vaccine Program, Naval Medical Research Center/Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910, USA. sofia.a.casares.civ@mail.mil.

ABSTRACT

Background: Malaria is a deadly infectious disease affecting millions of people in tropical and sub-tropical countries. Among the five species of Plasmodium parasites that infect humans, Plasmodium falciparum accounts for the highest morbidity and mortality associated with malaria. Since humans are the only natural hosts for P. falciparum, the lack of convenient animal models has hindered the understanding of disease pathogenesis and prompted the need of testing anti-malarial drugs and vaccines directly in human trials. Humanized mice hosting human cells represent new pre-clinical models for infectious diseases that affect only humans. In this study, the ability of human-immune-system humanized HLA-DR4.RagKO.IL2RγcKO.NOD (DRAG) mice to sustain infection with P. falciparum was explored.

Methods: Four week-old DRAG mice were infused with HLA-matched human haematopoietic stem cells (HSC) and examined for reconstitution of human liver cells and erythrocytes. Upon challenge with infectious P. falciparum sporozoites (NF54 strain) humanized DRAG mice were examined for liver stage infection, blood stage infection, and transmission to Anopheles stephensi mosquitoes.

Results: Humanized DRAG mice reconstituted human hepatocytes, Kupffer cells, liver endothelial cells, and erythrocytes. Upon intravenous challenge with P. falciparum sporozoites, DRAG mice sustained liver to blood stage infection (average 3-5 parasites/microlitre blood) and allowed transmission to An. stephensi mosquitoes. Infected DRAG mice elicited antibody and cellular responses to the blood stage parasites and self-cured the infection by day 45 post-challenge.

Conclusions: DRAG mice represent the first human-immune-system humanized mouse model that sustains the complex vertebrate life cycle of P. falciparum without the need of exogenous injection of human hepatocytes/erythrocytes or P. falciparum parasite adaptation. The ability of DRAG mice to elicit specific human immune responses to P. falciparum parasites may help deciphering immune correlates of protection and to identify protective malaria antigens.

Show MeSH

Related in: MedlinePlus

DRAG mice develop human hepatocytes. A) Plasma levels of human transferrin in DRAG mice (n = 14) at 14 weeks post-infusion of HSC. No human transferrin was detected in serum from control (non-HSC infused) DRAG mice (n = 7). Data represent mean ± SD of mice analysed individually (left panel). Kinetics of human transferrin in plasma of two DRAG mice analysed individually (right panel). B) Staining of liver sections with anti-human transferrin. Arrow shows the presence of human hepatocytes. C) Identification of human hepatocytes in livers of HSC-infused DRAG mice using human anti-Hep Par-1 (red) and counterstained with DAPI (DNA, blue). D) Frequency of human hepatocytes in livers of DRAG mice as measured by FACS using anti-human albumin. Left panel shows binding of anti-human albumin to primary human hepatocytes (black histogram) but not to mouse hepatocytes (white histogram). Middle and right panels show anti-human albumin staining in livers of control (non-HSC infused) and in HSC-infused DRAG mice, respectively. Data represent mean and interval frequencies from three mice analysed individually.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4197321&req=5

Fig1: DRAG mice develop human hepatocytes. A) Plasma levels of human transferrin in DRAG mice (n = 14) at 14 weeks post-infusion of HSC. No human transferrin was detected in serum from control (non-HSC infused) DRAG mice (n = 7). Data represent mean ± SD of mice analysed individually (left panel). Kinetics of human transferrin in plasma of two DRAG mice analysed individually (right panel). B) Staining of liver sections with anti-human transferrin. Arrow shows the presence of human hepatocytes. C) Identification of human hepatocytes in livers of HSC-infused DRAG mice using human anti-Hep Par-1 (red) and counterstained with DAPI (DNA, blue). D) Frequency of human hepatocytes in livers of DRAG mice as measured by FACS using anti-human albumin. Left panel shows binding of anti-human albumin to primary human hepatocytes (black histogram) but not to mouse hepatocytes (white histogram). Middle and right panels show anti-human albumin staining in livers of control (non-HSC infused) and in HSC-infused DRAG mice, respectively. Data represent mean and interval frequencies from three mice analysed individually.

Mentions: The ability of human HSC (CD34+) to differentiate into non-haematopoietic cells such as hepatocytes, cardiomyocytes, and endothelial cells has become evident in human and animal studies [27]. Thus it was investigated whether DRAG mice develop human hepatocytes by measuring plasma levels of human transferrin, a protein secreted by human hepatocytes [28]. At 14 weeks post-infusion of human HSCs the levels of human transferrin averaged 2–3 ng/ml (Figure 1A, left panel), which is about 5x105 fold lower than the levels of transferrin in human plasma (1 mg/ml) [28]. No human transferrin was detected in plasma of naïve (non-HSC infused) DRAG mice. Longitudinal analysis indicated that DRAG mice reconstituted human transferrin at eight weeks post-infusion of HSCs and the levels remained constant for up to 22 weeks (Figure 1A, right panel). Histological examination using anti-human transferrin (Figure 1B) and anti-human Hep Par-1 (a marker specific for human hepatocyte mitochondria) [29] (Figure 1C) revealed the presence of scattered human hepatocytes within the livers. To estimate the frequency of human hepatocytes, livers of HSC-infused DRAG mice were analysed by FACS using anti-human albumin. As illustrated in Figure 1D, approximately 0.023% of the hepatocytes in mouse livers were of human origin. Thus the results indicated that DRAG mice infused with HSC develop human hepatocytes.Figure 1


Humanized HLA-DR4.RagKO.IL2RγcKO.NOD (DRAG) mice sustain the complex vertebrate life cycle of Plasmodium falciparum malaria.

Wijayalath W, Majji S, Villasante EF, Brumeanu TD, Richie TL, Casares S - Malar. J. (2014)

DRAG mice develop human hepatocytes. A) Plasma levels of human transferrin in DRAG mice (n = 14) at 14 weeks post-infusion of HSC. No human transferrin was detected in serum from control (non-HSC infused) DRAG mice (n = 7). Data represent mean ± SD of mice analysed individually (left panel). Kinetics of human transferrin in plasma of two DRAG mice analysed individually (right panel). B) Staining of liver sections with anti-human transferrin. Arrow shows the presence of human hepatocytes. C) Identification of human hepatocytes in livers of HSC-infused DRAG mice using human anti-Hep Par-1 (red) and counterstained with DAPI (DNA, blue). D) Frequency of human hepatocytes in livers of DRAG mice as measured by FACS using anti-human albumin. Left panel shows binding of anti-human albumin to primary human hepatocytes (black histogram) but not to mouse hepatocytes (white histogram). Middle and right panels show anti-human albumin staining in livers of control (non-HSC infused) and in HSC-infused DRAG mice, respectively. Data represent mean and interval frequencies from three mice analysed individually.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4197321&req=5

Fig1: DRAG mice develop human hepatocytes. A) Plasma levels of human transferrin in DRAG mice (n = 14) at 14 weeks post-infusion of HSC. No human transferrin was detected in serum from control (non-HSC infused) DRAG mice (n = 7). Data represent mean ± SD of mice analysed individually (left panel). Kinetics of human transferrin in plasma of two DRAG mice analysed individually (right panel). B) Staining of liver sections with anti-human transferrin. Arrow shows the presence of human hepatocytes. C) Identification of human hepatocytes in livers of HSC-infused DRAG mice using human anti-Hep Par-1 (red) and counterstained with DAPI (DNA, blue). D) Frequency of human hepatocytes in livers of DRAG mice as measured by FACS using anti-human albumin. Left panel shows binding of anti-human albumin to primary human hepatocytes (black histogram) but not to mouse hepatocytes (white histogram). Middle and right panels show anti-human albumin staining in livers of control (non-HSC infused) and in HSC-infused DRAG mice, respectively. Data represent mean and interval frequencies from three mice analysed individually.
Mentions: The ability of human HSC (CD34+) to differentiate into non-haematopoietic cells such as hepatocytes, cardiomyocytes, and endothelial cells has become evident in human and animal studies [27]. Thus it was investigated whether DRAG mice develop human hepatocytes by measuring plasma levels of human transferrin, a protein secreted by human hepatocytes [28]. At 14 weeks post-infusion of human HSCs the levels of human transferrin averaged 2–3 ng/ml (Figure 1A, left panel), which is about 5x105 fold lower than the levels of transferrin in human plasma (1 mg/ml) [28]. No human transferrin was detected in plasma of naïve (non-HSC infused) DRAG mice. Longitudinal analysis indicated that DRAG mice reconstituted human transferrin at eight weeks post-infusion of HSCs and the levels remained constant for up to 22 weeks (Figure 1A, right panel). Histological examination using anti-human transferrin (Figure 1B) and anti-human Hep Par-1 (a marker specific for human hepatocyte mitochondria) [29] (Figure 1C) revealed the presence of scattered human hepatocytes within the livers. To estimate the frequency of human hepatocytes, livers of HSC-infused DRAG mice were analysed by FACS using anti-human albumin. As illustrated in Figure 1D, approximately 0.023% of the hepatocytes in mouse livers were of human origin. Thus the results indicated that DRAG mice infused with HSC develop human hepatocytes.Figure 1

Bottom Line: Malaria is a deadly infectious disease affecting millions of people in tropical and sub-tropical countries.Among the five species of Plasmodium parasites that infect humans, Plasmodium falciparum accounts for the highest morbidity and mortality associated with malaria.Four week-old DRAG mice were infused with HLA-matched human haematopoietic stem cells (HSC) and examined for reconstitution of human liver cells and erythrocytes.

View Article: PubMed Central - PubMed

Affiliation: US Military Malaria Vaccine Program, Naval Medical Research Center/Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910, USA. sofia.a.casares.civ@mail.mil.

ABSTRACT

Background: Malaria is a deadly infectious disease affecting millions of people in tropical and sub-tropical countries. Among the five species of Plasmodium parasites that infect humans, Plasmodium falciparum accounts for the highest morbidity and mortality associated with malaria. Since humans are the only natural hosts for P. falciparum, the lack of convenient animal models has hindered the understanding of disease pathogenesis and prompted the need of testing anti-malarial drugs and vaccines directly in human trials. Humanized mice hosting human cells represent new pre-clinical models for infectious diseases that affect only humans. In this study, the ability of human-immune-system humanized HLA-DR4.RagKO.IL2RγcKO.NOD (DRAG) mice to sustain infection with P. falciparum was explored.

Methods: Four week-old DRAG mice were infused with HLA-matched human haematopoietic stem cells (HSC) and examined for reconstitution of human liver cells and erythrocytes. Upon challenge with infectious P. falciparum sporozoites (NF54 strain) humanized DRAG mice were examined for liver stage infection, blood stage infection, and transmission to Anopheles stephensi mosquitoes.

Results: Humanized DRAG mice reconstituted human hepatocytes, Kupffer cells, liver endothelial cells, and erythrocytes. Upon intravenous challenge with P. falciparum sporozoites, DRAG mice sustained liver to blood stage infection (average 3-5 parasites/microlitre blood) and allowed transmission to An. stephensi mosquitoes. Infected DRAG mice elicited antibody and cellular responses to the blood stage parasites and self-cured the infection by day 45 post-challenge.

Conclusions: DRAG mice represent the first human-immune-system humanized mouse model that sustains the complex vertebrate life cycle of P. falciparum without the need of exogenous injection of human hepatocytes/erythrocytes or P. falciparum parasite adaptation. The ability of DRAG mice to elicit specific human immune responses to P. falciparum parasites may help deciphering immune correlates of protection and to identify protective malaria antigens.

Show MeSH
Related in: MedlinePlus