Limits...
Molecular detection and quantification of Plasmodium falciparum-infected human hepatocytes in chimeric immune-deficient mice.

Foquet L, Hermsen CC, van Gemert GJ, Libbrecht L, Sauerwein R, Meuleman P, Leroux-Roels G - Malar. J. (2013)

Bottom Line: Adequate assessment of hepatic infections is generally compromised by the limited number of human hepatocytes infected by developing parasites.The sensitivity of the protocol, which comprises approximately 25% of the total chimeric liver, allows for the detection of a single infected hepatocyte in the analysed tissue.It will be a useful tool when studying the in vivo therapeutic and/or prophylactic qualities of novel compounds, small molecules or antibodies directed against the liver stage of P. falciparum infections.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Vaccinology, Ghent University and University Hospital, De Pintelaan 185, Ghent 9000, Belgium. geert.lerouxroels@ugent.be.

ABSTRACT

Background: Chimeric mice with humanized livers represent a promising tool for infections with Plasmodium falciparum to evaluate novel methods for prevention and treatment of pre-erythrocytic stages. Adequate assessment of hepatic infections is generally compromised by the limited number of human hepatocytes infected by developing parasites.

Methods: A qPCR-based method has been developed that sensitively and reliably detects P. falciparum liver stage infection of humanized mice and quantitatively expresses the results as the number of parasites per human hepatocyte.

Results: This assay allows for detection of liver stage parasites after challenging humanized mice with infected mosquito bites or after intravenous injection with sporozoites. The sensitivity of the protocol, which comprises approximately 25% of the total chimeric liver, allows for the detection of a single infected hepatocyte in the analysed tissue.

Conclusions: This method allows for the detection and quantification of P. falciparum parasites in chimeric mice repopulated with human hepatocytes. It will be a useful tool when studying the in vivo therapeutic and/or prophylactic qualities of novel compounds, small molecules or antibodies directed against the liver stage of P. falciparum infections.

Show MeSH

Related in: MedlinePlus

Histology and determination of polyploidy of a chimeric liver. (A) A representative section of a chimeric liver (haematoxylin and eosin staining) showing the morphological differences between pale human hepatocytes (upper zone), diseased mouse parenchyma (middle zone) and healthy mouse parenchyma originating from mouse hepatocytes that have eliminated both uPA transgene copies, known as red nodules (lower zone). Detailed view of chimeric liver sections, showing human hepatocytes (B) none of which is multinucleated and mouse hepatocytes (C), one of which is binucleated (arrow). Original magnification A: 200 x, B and C: 400 x.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4222492&req=5

Figure 4: Histology and determination of polyploidy of a chimeric liver. (A) A representative section of a chimeric liver (haematoxylin and eosin staining) showing the morphological differences between pale human hepatocytes (upper zone), diseased mouse parenchyma (middle zone) and healthy mouse parenchyma originating from mouse hepatocytes that have eliminated both uPA transgene copies, known as red nodules (lower zone). Detailed view of chimeric liver sections, showing human hepatocytes (B) none of which is multinucleated and mouse hepatocytes (C), one of which is binucleated (arrow). Original magnification A: 200 x, B and C: 400 x.

Mentions: Injection of P. falciparum sporozoites in a chimeric mouse will result in infection of an unknown and low number of infected human hepatocytes distributed in a large background of non-infected mouse and human cells (Figure 4). Consequently, an analysis of the complete liver by cell lysis and subsequent DNA extraction will result in a strong dilution of parasitic DNA and loss of sensitivity. Therefore, exactly 25 mg of liver tissue was sampled from 12 standardized sections (Figure 1) for DNA extraction in a volume of 100 μL. Five μL of each extract was used in duplicate to measure the number of parasites by qPCR and 1 μL to determine the fraction (percentage) of human hepatocytes in each fragment (Figure 2). This approach allowed for the analysis of several millions of human hepatocytes randomly distributed in each chimeric liver (Additional file 1: Table S1).


Molecular detection and quantification of Plasmodium falciparum-infected human hepatocytes in chimeric immune-deficient mice.

Foquet L, Hermsen CC, van Gemert GJ, Libbrecht L, Sauerwein R, Meuleman P, Leroux-Roels G - Malar. J. (2013)

Histology and determination of polyploidy of a chimeric liver. (A) A representative section of a chimeric liver (haematoxylin and eosin staining) showing the morphological differences between pale human hepatocytes (upper zone), diseased mouse parenchyma (middle zone) and healthy mouse parenchyma originating from mouse hepatocytes that have eliminated both uPA transgene copies, known as red nodules (lower zone). Detailed view of chimeric liver sections, showing human hepatocytes (B) none of which is multinucleated and mouse hepatocytes (C), one of which is binucleated (arrow). Original magnification A: 200 x, B and C: 400 x.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222492&req=5

Figure 4: Histology and determination of polyploidy of a chimeric liver. (A) A representative section of a chimeric liver (haematoxylin and eosin staining) showing the morphological differences between pale human hepatocytes (upper zone), diseased mouse parenchyma (middle zone) and healthy mouse parenchyma originating from mouse hepatocytes that have eliminated both uPA transgene copies, known as red nodules (lower zone). Detailed view of chimeric liver sections, showing human hepatocytes (B) none of which is multinucleated and mouse hepatocytes (C), one of which is binucleated (arrow). Original magnification A: 200 x, B and C: 400 x.
Mentions: Injection of P. falciparum sporozoites in a chimeric mouse will result in infection of an unknown and low number of infected human hepatocytes distributed in a large background of non-infected mouse and human cells (Figure 4). Consequently, an analysis of the complete liver by cell lysis and subsequent DNA extraction will result in a strong dilution of parasitic DNA and loss of sensitivity. Therefore, exactly 25 mg of liver tissue was sampled from 12 standardized sections (Figure 1) for DNA extraction in a volume of 100 μL. Five μL of each extract was used in duplicate to measure the number of parasites by qPCR and 1 μL to determine the fraction (percentage) of human hepatocytes in each fragment (Figure 2). This approach allowed for the analysis of several millions of human hepatocytes randomly distributed in each chimeric liver (Additional file 1: Table S1).

Bottom Line: Adequate assessment of hepatic infections is generally compromised by the limited number of human hepatocytes infected by developing parasites.The sensitivity of the protocol, which comprises approximately 25% of the total chimeric liver, allows for the detection of a single infected hepatocyte in the analysed tissue.It will be a useful tool when studying the in vivo therapeutic and/or prophylactic qualities of novel compounds, small molecules or antibodies directed against the liver stage of P. falciparum infections.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Vaccinology, Ghent University and University Hospital, De Pintelaan 185, Ghent 9000, Belgium. geert.lerouxroels@ugent.be.

ABSTRACT

Background: Chimeric mice with humanized livers represent a promising tool for infections with Plasmodium falciparum to evaluate novel methods for prevention and treatment of pre-erythrocytic stages. Adequate assessment of hepatic infections is generally compromised by the limited number of human hepatocytes infected by developing parasites.

Methods: A qPCR-based method has been developed that sensitively and reliably detects P. falciparum liver stage infection of humanized mice and quantitatively expresses the results as the number of parasites per human hepatocyte.

Results: This assay allows for detection of liver stage parasites after challenging humanized mice with infected mosquito bites or after intravenous injection with sporozoites. The sensitivity of the protocol, which comprises approximately 25% of the total chimeric liver, allows for the detection of a single infected hepatocyte in the analysed tissue.

Conclusions: This method allows for the detection and quantification of P. falciparum parasites in chimeric mice repopulated with human hepatocytes. It will be a useful tool when studying the in vivo therapeutic and/or prophylactic qualities of novel compounds, small molecules or antibodies directed against the liver stage of P. falciparum infections.

Show MeSH
Related in: MedlinePlus