Table 1.
Comparison of the clinical data, histopathological changes and clinical complications in severe malaria cases with and without ARDS.
Fig 1.
Histopathological and ultrastructural appearance in normal and severe malarial lungs.
A: normal lung represented by a very thin alveolar septum and clearly defined alveolar sac in contrast to the non-PE lung (B) which was characterized by thickened alveolar septum congested with blood components (e.g. PRBCs and WBCs). Apart from the thickened membrane, fluid (C*) and the hyaline membrane (D*) were deposited in the PE and ARDS lungs, respectively. Fine morphology of hyaline membrane (E*), macrophage (F; arrow)-ladened hemozoin pigment (F*) and PRBCs (G; arrow) with endothelial cell damage (G-inset) were frequently observed in the ARDS lung patients. A positive correlation between some histopathological changes and ARDS severity was calculated using Spearman test (H-I).
Fig 2.
Immunohistochemical staining of EPCR in the normal (A), non-PE (B), PE (C) and ARDS (D) lungs. The EPCR expression in small blood vessels are shown in the inset of each micrograph. The H-score expression is demonstrated by bar graphs for PE (E) or ARDS (F) lungs, to compare those expression Friedman test was performed (**; p-value < 0.001, ***; p-value <0.0001). Negative correlation between the level of EPCR and some histopathological changes was observed using Spearman test (G-I).
Fig 3.
Immunohistochemical staining for thrombomodulin in normal (A), non-PE (B), PE (C) and ARDS (D) lungs. The level of thrombomodulin expression in the small blood vessels are shown in the inset of each micrograph, The H-score expression is demonstrated by bar graphs for PE (E) or ARDS (F) lungs, to compare those expressions Friedman test was performed (*; p-value < 0.05).
Fig 4.
Immunohistochemical staining for ICAM-1 thrombomodulin in normal (A), non-PE (B), PE (C) and ARDS (D) lungs. The H-score expression is demonstrated by bar graphs for PE (E) or ARDS (F) lungs, to compare those expression Friedman test was performed (***; p-value <0.0001).
Fig 5.
Immunohistochemical staining for TNF-α in normal (A), non-PE (B), PE (C) and ARDS (D) lungs. The H-score expression is demonstrated by bar graphs for PE (E) or ARDS (F) lungs, to compare those expression Friedman test was performed (***; p-value <0.0001).
Fig 6.
Masson’s trichrome staining for intravascular fibrin deposition in normal (A), non-PE (B), PE (C) and ARDS (D) lungs. The fibrin staining is exhibited in the yellowish material found in the small blood vessels, whereas red blood cells and collagen fibrils were stained red and blue, respectively. The H-score expression is demonstrated by bar graphs for PE (E) or ARDS (F) lungs, to compare those expression Friedman test was performed (***; p-value <0.0001).
Fig 7.
Scanning electron micrographs of A549 cells.
A549 cell morphology was composed of many cell types: 1) the mature form with (A) and without granules (B) were the largest cells with an irregular and granulate surface; 2) the progenitor form consisted of small and rounded cells with a smooth and irregular membrane (C) and 3) apoptotic cells (D) are the shrunken cells with numerous vacuolated membranes. Cells stained with trypan blue cells were shown in the inset of each micrograph.
Fig 8.
Percentage of apoptotic cells induced by cytokines.
The bar graph exhibited the difference in the number of apoptotic cells in each group using Friedman test (*; p-value<0.05, **; p-value <0.001, ***; p-value<0.001) (A) which was morphologically characterised by apoptotic (B) and normal cells (C).
Fig 9.
Apoptosis indicated by EB/AO staining and real-time RT-PCR.
S-hemozoin-induced pneumocytic apoptosis was characterized by morphological changes and staining patterns that included normal cells (A), early apoptotic cells with nuclear fragmentation or condense chromatin (B), membrane blebbing (C), cytoplasmic vacuolization (D), late apoptotic cells with condensed chromatin (E) and necrotic cells (F). The bar graph demonstrates that early apoptosis was predominately found following hemozoin treatment between 1–24 h with an increasing trend (G) in accordance with the mRNA expression of CARD-9 (H).
Fig 10.
Transmission electron micrograph of A459 cells exposed to s-hemozoin for 24 h.
A semi-thin section of A549 cells was characterized by toluidine blue staining, including normal cells with or without hemozoin, that adhered to the membrane and apoptotic cells (*) with hemozoin, which slipped out of the membrane (A). A number of hemozoin pigments (arrow) were ingested by intact (B-C) and apoptotic (E-F) A549 cells, were located in multivesicular (G) or lamellar (H-I) bodies characterized by dark electron dense material (*).
Fig 11.
Size comparison of synthetic and P. falciparum hemozoins.
Ultrastructural micrographs of the s-hemozoin in the A549 cell (A) and Pf-hemozoin in the mononuclear cell (B). The dot graph exhibited the difference in length (C) and width (D) of hemozoin crystal using Mann-Whitney test.