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Improved methods for haemozoin quantification in tissues yield organ-and parasite-specific information in malaria-infected mice.

Deroost K, Lays N, Noppen S, Martens E, Opdenakker G, Van den Steen PE - Malar. J. (2012)

Bottom Line: Furthermore, total Hz contents correlated with peripheral parasitaemia and were significantly higher in mice with a lethal P. berghei ANKA or P. berghei NK65-infection than in mice with a self-resolving P. chabaudi AS-infection, despite similar peripheral parasitaemia levels.An organ-specific Hz deposition pattern was found and was independent of the parasite strain used.Highest Hz levels were identified in mice infected with lethal parasite strains suggesting that Hz accumulation in tissues is associated with malaria-related mortality.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Immunobiology, Rega Institute, University of Leuven, Leuven, Belgium.

ABSTRACT

Background: Despite intensive research, malaria remains a major health concern for non-immune residents and travelers in malaria-endemic regions. Efficient adjunctive therapies against life-threatening complications such as severe malarial anaemia, encephalopathy, placental malaria or respiratory problems are still lacking. Therefore, new insights into the pathogenesis of severe malaria are imperative. Haemozoin (Hz) or malaria pigment is produced during intra-erythrocytic parasite replication, released in the circulation after schizont rupture and accumulates inside multiple organs. Many in vitro and ex vivo immunomodulating effects are described for Hz but in vivo data are limited. This study aimed to improve methods for Hz quantification in tissues and to investigate the accumulation of Hz in different organs from mice infected with Plasmodium parasites with a varying degree of virulence.

Methods: An improved method for extraction of Hz from tissues was elaborated and coupled to an optimized, quantitative, microtiter plate-based luminescence assay with a high sensitivity. In addition, a technique for measuring Hz by semi-quantitative densitometry, applicable on transmitted light images, was developed. The methods were applied to measure Hz in various organs of C57BL/6 J mice infected with Plasmodium berghei ANKA, P. berghei NK65 or Plasmodium chabaudi AS. The used statistical methods were the Mann-Whitney U test and Pearsons correlation analysis.

Results: Most Hz was detected in livers and spleens, lower levels in lungs and kidneys, whereas sub-nanomolar amounts were observed in brains and hearts from infected mice, irrespectively of the parasite strain used. Furthermore, total Hz contents correlated with peripheral parasitaemia and were significantly higher in mice with a lethal P. berghei ANKA or P. berghei NK65-infection than in mice with a self-resolving P. chabaudi AS-infection, despite similar peripheral parasitaemia levels.

Conclusions: The developed techniques were useful to quantify Hz in different organs with a high reproducibility and sensitivity. An organ-specific Hz deposition pattern was found and was independent of the parasite strain used. Highest Hz levels were identified in mice infected with lethal parasite strains suggesting that Hz accumulation in tissues is associated with malaria-related mortality.

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PcAS-infected mice contain less total Hz thanPlasmodium berghei-infected mice. The total amount of Hz (nmol haematin)/mouse was estimated from the sum of the Hz content in the individual organs. Panel A shows the average amount of Hz found in C57BL/6 J mice infected with PbANKA, PbNK65 or PcAS on different time points post-infection, with the contribution of each organ to the total amount of Hz ± SEM. Asterisks on top of data sets indicate the statistical significances compared with the uninfected control group. Horizontal lines with asterisks on top indicate statistical differences between infected groups. * p < 0.05, ** p < 0.01 and *** p < 0.001. In panel B, the total amount of Hz (nmol haematin) was correlated with the level of parasitaemia (%) for each parasite strain separately and the individual regression lines are shown. The regression line of PbANKA was prolonged under the form of a hatched line to better distinguish it from the PbNK65 line. The equation of the regression lines and the R2-values, together with Spearman r-values and p-values are shown in panel C
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Figure 4: PcAS-infected mice contain less total Hz thanPlasmodium berghei-infected mice. The total amount of Hz (nmol haematin)/mouse was estimated from the sum of the Hz content in the individual organs. Panel A shows the average amount of Hz found in C57BL/6 J mice infected with PbANKA, PbNK65 or PcAS on different time points post-infection, with the contribution of each organ to the total amount of Hz ± SEM. Asterisks on top of data sets indicate the statistical significances compared with the uninfected control group. Horizontal lines with asterisks on top indicate statistical differences between infected groups. * p < 0.05, ** p < 0.01 and *** p < 0.001. In panel B, the total amount of Hz (nmol haematin) was correlated with the level of parasitaemia (%) for each parasite strain separately and the individual regression lines are shown. The regression line of PbANKA was prolonged under the form of a hatched line to better distinguish it from the PbNK65 line. The equation of the regression lines and the R2-values, together with Spearman r-values and p-values are shown in panel C

Mentions: As brains and hearts contained only subnanomolar amounts of Hz, individual total Hz levels were calculated by making the sum of organ-specific Hz levels from liver, spleen, lungs and kidneys. Mice infected with parasites of the P. berghei strains contained significantly more Hz than mice infected with PcAS-parasites (Figure 4A). Moreover, the total amounts of Hz correlated with the peripheral parasitaemia levels for all parasite strains (Figure 4B and C). PbANKA and PbNK65-parasites had a similar Hz-production pattern, i.e. comparable amounts of Hz at similar peripheral parasitaemia levels. In contrast, PcAS-infected mice seemed to have significantly less Hz in relation with their parasitaemia compared with PbANKA or PbNK65 as indicated by the lower slope on the regression curve. The difference between the slopes of the regression lines was significantly different between PbNK65 and PcAS-infected mice (p < 0.0001).


Improved methods for haemozoin quantification in tissues yield organ-and parasite-specific information in malaria-infected mice.

Deroost K, Lays N, Noppen S, Martens E, Opdenakker G, Van den Steen PE - Malar. J. (2012)

PcAS-infected mice contain less total Hz thanPlasmodium berghei-infected mice. The total amount of Hz (nmol haematin)/mouse was estimated from the sum of the Hz content in the individual organs. Panel A shows the average amount of Hz found in C57BL/6 J mice infected with PbANKA, PbNK65 or PcAS on different time points post-infection, with the contribution of each organ to the total amount of Hz ± SEM. Asterisks on top of data sets indicate the statistical significances compared with the uninfected control group. Horizontal lines with asterisks on top indicate statistical differences between infected groups. * p < 0.05, ** p < 0.01 and *** p < 0.001. In panel B, the total amount of Hz (nmol haematin) was correlated with the level of parasitaemia (%) for each parasite strain separately and the individual regression lines are shown. The regression line of PbANKA was prolonged under the form of a hatched line to better distinguish it from the PbNK65 line. The equation of the regression lines and the R2-values, together with Spearman r-values and p-values are shown in panel C
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3473299&req=5

Figure 4: PcAS-infected mice contain less total Hz thanPlasmodium berghei-infected mice. The total amount of Hz (nmol haematin)/mouse was estimated from the sum of the Hz content in the individual organs. Panel A shows the average amount of Hz found in C57BL/6 J mice infected with PbANKA, PbNK65 or PcAS on different time points post-infection, with the contribution of each organ to the total amount of Hz ± SEM. Asterisks on top of data sets indicate the statistical significances compared with the uninfected control group. Horizontal lines with asterisks on top indicate statistical differences between infected groups. * p < 0.05, ** p < 0.01 and *** p < 0.001. In panel B, the total amount of Hz (nmol haematin) was correlated with the level of parasitaemia (%) for each parasite strain separately and the individual regression lines are shown. The regression line of PbANKA was prolonged under the form of a hatched line to better distinguish it from the PbNK65 line. The equation of the regression lines and the R2-values, together with Spearman r-values and p-values are shown in panel C
Mentions: As brains and hearts contained only subnanomolar amounts of Hz, individual total Hz levels were calculated by making the sum of organ-specific Hz levels from liver, spleen, lungs and kidneys. Mice infected with parasites of the P. berghei strains contained significantly more Hz than mice infected with PcAS-parasites (Figure 4A). Moreover, the total amounts of Hz correlated with the peripheral parasitaemia levels for all parasite strains (Figure 4B and C). PbANKA and PbNK65-parasites had a similar Hz-production pattern, i.e. comparable amounts of Hz at similar peripheral parasitaemia levels. In contrast, PcAS-infected mice seemed to have significantly less Hz in relation with their parasitaemia compared with PbANKA or PbNK65 as indicated by the lower slope on the regression curve. The difference between the slopes of the regression lines was significantly different between PbNK65 and PcAS-infected mice (p < 0.0001).

Bottom Line: Furthermore, total Hz contents correlated with peripheral parasitaemia and were significantly higher in mice with a lethal P. berghei ANKA or P. berghei NK65-infection than in mice with a self-resolving P. chabaudi AS-infection, despite similar peripheral parasitaemia levels.An organ-specific Hz deposition pattern was found and was independent of the parasite strain used.Highest Hz levels were identified in mice infected with lethal parasite strains suggesting that Hz accumulation in tissues is associated with malaria-related mortality.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Immunobiology, Rega Institute, University of Leuven, Leuven, Belgium.

ABSTRACT

Background: Despite intensive research, malaria remains a major health concern for non-immune residents and travelers in malaria-endemic regions. Efficient adjunctive therapies against life-threatening complications such as severe malarial anaemia, encephalopathy, placental malaria or respiratory problems are still lacking. Therefore, new insights into the pathogenesis of severe malaria are imperative. Haemozoin (Hz) or malaria pigment is produced during intra-erythrocytic parasite replication, released in the circulation after schizont rupture and accumulates inside multiple organs. Many in vitro and ex vivo immunomodulating effects are described for Hz but in vivo data are limited. This study aimed to improve methods for Hz quantification in tissues and to investigate the accumulation of Hz in different organs from mice infected with Plasmodium parasites with a varying degree of virulence.

Methods: An improved method for extraction of Hz from tissues was elaborated and coupled to an optimized, quantitative, microtiter plate-based luminescence assay with a high sensitivity. In addition, a technique for measuring Hz by semi-quantitative densitometry, applicable on transmitted light images, was developed. The methods were applied to measure Hz in various organs of C57BL/6 J mice infected with Plasmodium berghei ANKA, P. berghei NK65 or Plasmodium chabaudi AS. The used statistical methods were the Mann-Whitney U test and Pearsons correlation analysis.

Results: Most Hz was detected in livers and spleens, lower levels in lungs and kidneys, whereas sub-nanomolar amounts were observed in brains and hearts from infected mice, irrespectively of the parasite strain used. Furthermore, total Hz contents correlated with peripheral parasitaemia and were significantly higher in mice with a lethal P. berghei ANKA or P. berghei NK65-infection than in mice with a self-resolving P. chabaudi AS-infection, despite similar peripheral parasitaemia levels.

Conclusions: The developed techniques were useful to quantify Hz in different organs with a high reproducibility and sensitivity. An organ-specific Hz deposition pattern was found and was independent of the parasite strain used. Highest Hz levels were identified in mice infected with lethal parasite strains suggesting that Hz accumulation in tissues is associated with malaria-related mortality.

Show MeSH
Related in: MedlinePlus