No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Shear, H. L., Marino, M. W., Wanidworanun, C. J., Berman, W. & Nagel, R. L. Correlation of increased expression of intercellular adhesion molecule-1, but not high levels of tumor necrosis factor-, with lethality of Plasmodium yoelii 17XL, a rodent model of cerebral malaria. Am. J. Trop. Med. Hyg. 59, 852-858
Abstract
Previous studies demonstrated that Plasmodium yoelii 17XL, a lethal strain of rodent malaria, causes a syndrome in SW mice that resembles human cerebral malaria. The mouse brain pathology is characterized by cytoadherence of parasitized erythrocytes. Here, the possible mechanisms mediating cerebral malaria in this model were studied and the results were compared with a nonlethal strain of this parasite, P. yoelii 17XNL (nonlethal), which does not cause cerebral malaria. Immunostaining for intercellular adhesion molecule-1 (ICAM-1) revealed an increase in expression of this protein in the small venules and capillaries of the brains of infected mice that increased with time after infection. Staining was more pronounced during the lethal infection than the nonlethal infection. Some staining with monoclonal antibody to vascular cell adhesion molecule-1 was also observed, but it was quantitatively less than ICAM-1 staining and was limited to larger venules. During the lethal infection, levels of tumor necrosis factor-alpha (TNF-alpha) increased rapidly, peaking on day 4. In contrast, mice infected with nonlethal P. yoelii had a slower serum TNF-alpha response that peaked on day 10, prior to the maximum parasitemia. In addition, mice with a targeted disruption of the TNF-alpha gene (TNF-alpha-/- mice) were infected with the lethal and nonlethal strains of P. yoelii 17X. The TNF-alpha-/- mice infected with the nonlethal parasite had significantly higher levels of parasitemia than controls, whereas TNF-alpha-/- mice infected with the lethal strain had slightly higher levels of infected erythrocytes but were equally susceptible to death from this infection. Thus, TNF-alpha does not appear to be essential in mediating death. These results demonstrate that P. yoelii 17XL infection has features in common with human cerebral malaria and suggest that this model may be useful in testing strategies to alleviate this syndrome.
... Mice were injected intraperitoneally with P. yoelii 17XL parasitized blood, kindly provided by Dr. Christine Olver (Department of Pathology, Colorado State University, USA). This rodent malaria strain causes a syndrome that resembles human malaria, char acterized by fever, spleno-and hepatomegaly by day eight post-infection [14,15] . Parasitaemia was determined in a total count of 300 to 500 red blood cells (RBCs) on Wright-Giemsa-stained (Sigma Diagnostics, USA) thin blood smears. ...
... All the mice infected with P. yoelii 17XL parasites developed malaria-related-symptoms, which included the appearance of ruffled fur and shivering at peak parasitaemia by day eight post-infection (Figure 1). Examination of the viscera of dissected mice confirmed spleno-and hepatomegaly at peak parasitaemia, concordant with reported P. yoelii 17XL malaria infections [14,15]. None of the control or uninfected mice showed any of these signs. ...
... The cytoadherence of infected red blood cells (IRBCs) to the postcapillary venules is the major cause of IRBC sequestration and vessel blockage in the cerebral form of human malaria. In both human cerebral malaria caused by P. falciparum and the P. yoelii 17XL-infected rodent model of malaria, the sequestration of IRBCs in the brain vessels is secondary to the cytoadherence of IRBCs to the postcapillary venules [14,15]. This observation has resulted in the general suggestion that the P. yoelii 17XL mouse model resembles human P. falciparum infection more closely than the P. berghei ANKA mouse model, since it shows little accumulation of monocytes /macrophages in the brain microvessels [14,15]. ...
Abstract
Background
Malaria afflicts 300–500 million people causing over 1 million deaths globally per year. The immunopathogenesis of malaria is mediated partly by co mplex cellular and immunomodulator interactions involving co-regulators such as cytokines and adhesion molecules. However, the role of chemokines and their receptors in malaria immunopathology remains unclear. RANTES (Regulated on Activation Normal T-Cell Expressed and Secreted) is a chemokine involved in the generation of inflammatory infiltrates. Recent studies indicate that the degradation of cell-cell junctions, blood-brain barrier dysfunction, recruitment of leukocytes and Plasmodium -infected erythrocytes into and occlusion of microvessels relevant to malaria pathogenesis are associated with RANTES expression. Additionally, activated lymphocytes, platelets and endothelial cells release large quantities of RANTES, thus suggesting a unique role for RANTES in the generation and maintenance of the malaria-induced inflammatory response. The hypothesis of this study is that RANTES and its corresponding receptors (CCR1, CCR3 and CCR5) modulate malaria immunopathogenesis. A murine malaria model was utilized to evaluate the role of this chemokine and its receptors in malaria.
Methods
The alterations in immunomodulator gene expression in brains of Plasmodium yoelii 17XL-infected mice was analysed using cDNA microarray screening, followed by a temporal comparison of mRNA and protein expression of RANTES and its corresponding receptors by qRT-PCR and Western blot analysis, respectively. Plasma RANTES levels was determined by ELISA and ultrastructural studies of brain sections from infected and uninfected mice was conducted.
Results
RANTES (p < 0.002), CCR1 (p < 0.036), CCR3 (p < 0.033), and CCR5 (p < 0.026) mRNA were significantly upregulated at peak parasitaemia and remained high thereafter in the experimental mouse model. RANTES protein in the brain of infected mice was upregulated (p < 0.034) compared with controls. RANTES plasma levels were significantly upregulated; two to three fold in infected mice compared with controls (p < 0.026). Some d istal microvascular endothelium in infected cerebellum appeared degraded, but remained intact in controls.
Conclusion
The upregulation of RANTES, CCR1, CCR3, and CCR5 mRNA, and RANTES protein mediate inflammation and cellular degradation in the cerebellum during P. yoelii 17XL malaria.
... Mice were injected intraperitoneally with P. yoelii 17XL parasitized blood, kindly provided by Dr. Christine Olver (Department of Pathology, Colorado State University, USA). This rodent malaria strain causes a syndrome that resembles human malaria, char acterized by fever, spleno-and hepatomegaly by day eight post-infection [14,15] . Parasitaemia was determined in a total count of 300 to 500 red blood cells (RBCs) on Wright-Giemsa-stained (Sigma Diagnostics, USA) thin blood smears. ...
... All the mice infected with P. yoelii 17XL parasites developed malaria-related-symptoms, which included the appearance of ruffled fur and shivering at peak parasitaemia by day eight post-infection (Figure 1). Examination of the viscera of dissected mice confirmed spleno-and hepatomegaly at peak parasitaemia, concordant with reported P. yoelii 17XL malaria infections [14,15]. None of the control or uninfected mice showed any of these signs. ...
... The cytoadherence of infected red blood cells (IRBCs) to the postcapillary venules is the major cause of IRBC sequestration and vessel blockage in the cerebral form of human malaria. In both human cerebral malaria caused by P. falciparum and the P. yoelii 17XL-infected rodent model of malaria, the sequestration of IRBCs in the brain vessels is secondary to the cytoadherence of IRBCs to the postcapillary venules [14,15]. This observation has resulted in the general suggestion that the P. yoelii 17XL mouse model resembles human P. falciparum infection more closely than the P. berghei ANKA mouse model, since it shows little accumulation of monocytes /macrophages in the brain microvessels [14,15]. ...
Malaria afflicts 300-500 million people causing over 1 million deaths globally per year. The immunopathogenesis of malaria is mediated partly by co mplex cellular and immunomodulator interactions involving co-regulators such as cytokines and adhesion molecules. However, the role of chemokines and their receptors in malaria immunopathology remains unclear. RANTES (Regulated on Activation Normal T-Cell Expressed and Secreted) is a chemokine involved in the generation of inflammatory infiltrates. Recent studies indicate that the degradation of cell-cell junctions, blood-brain barrier dysfunction, recruitment of leukocytes and Plasmodium-infected erythrocytes into and occlusion of microvessels relevant to malaria pathogenesis are associated with RANTES expression. Additionally, activated lymphocytes, platelets and endothelial cells release large quantities of RANTES, thus suggesting a unique role for RANTES in the generation and maintenance of the malaria-induced inflammatory response. The hypothesis of this study is that RANTES and its corresponding receptors (CCR1, CCR3 and CCR5) modulate malaria immunopathogenesis. A murine malaria model was utilized to evaluate the role of this chemokine and its receptors in malaria.
The alterations in immunomodulator gene expression in brains of Plasmodium yoelii 17XL-infected mice was analysed using cDNA microarray screening, followed by a temporal comparison of mRNA and protein expression of RANTES and its corresponding receptors by qRT-PCR and Western blot analysis, respectively. Plasma RANTES levels was determined by ELISA and ultrastructural studies of brain sections from infected and uninfected mice was conducted.
RANTES (p < 0.002), CCR1 (p < 0.036), CCR3 (p < 0.033), and CCR5 (p < 0.026) mRNA were significantly upregulated at peak parasitaemia and remained high thereafter in the experimental mouse model. RANTES protein in the brain of infected mice was upregulated (p < 0.034) compared with controls. RANTES plasma levels were significantly upregulated; two to three fold in infected mice compared with controls (p < 0.026). Some distal microvascular endothelium in infected cerebellum appeared degraded, but remained intact in controls.
The upregulation of RANTES, CCR1, CCR3, and CCR5 mRNA, and RANTES protein mediate inflammation and cellular degradation in the cerebellum during P. yoelii 17XL malaria.
... Conversely, cognitive impairment identified by our analytic instruments was not restricted to the C57BL/6 background since it was also observed in SW mice. SW mice infected with lethal strain PyXL [21] developed early signs of cerebral dysfunction that was not detected after infection with a non-lethal PyNXL strain (Table 1). SW mice infected with PyNXL showed a significant reduction in the numbers of test events when training and testing sessions were compared and the pattern was not different from non-infected control animals (Figure 3C, D). ...
... Contrary to PbA, Py 17XL has been described to induce high parasitemia, massive anemia and kidney failure without CM (for review see Engwerda et al., [11]). On the other hand, other studies report that Py 17XL induces clear signs of CM and is a useful model of this condition in the laboratory setting [13,21,36]. We detected high parasitemia (32%) at day 7 after Py 17XL infection and these animals exhibited signs of cerebral dysfunction when submitted to the SIRPA protocol. ...
Neurological impairments are frequently detected in children surviving cerebral malaria (CM), the most severe neurological complication of infection with Plasmodium falciparum. The pathophysiology and therapy of long lasting cognitive deficits in malaria patients after treatment of the parasitic disease is a critical area of investigation. In the present study we used several models of experimental malaria with differential features to investigate persistent cognitive damage after rescue treatment. Infection of C57BL/6 and Swiss (SW) mice with Plasmodium berghei ANKA (PbA) or a lethal strain of Plasmodium yoelii XL (PyXL), respectively, resulted in documented CM and sustained persistent cognitive damage detected by a battery of behavioral tests after cure of the acute parasitic disease with chloroquine therapy. Strikingly, cognitive impairment was still present 30 days after the initial infection. In contrast, BALB/c mice infected with PbA, C57BL6 infected with Plasmodium chabaudi chabaudi and SW infected with non lethal Plasmodium yoelii NXL (PyNXL) did not develop signs of CM, were cured of the acute parasitic infection by chloroquine, and showed no persistent cognitive impairment. Reactive oxygen species have been reported to mediate neurological injury in CM. Increased production of malondialdehyde (MDA) and conjugated dienes was detected in the brains of PbA-infected C57BL/6 mice with CM, indicating high oxidative stress. Treatment of PbA-infected C57BL/6 mice with additive antioxidants together with chloroquine at the first signs of CM prevented the development of persistent cognitive damage. These studies provide new insights into the natural history of cognitive dysfunction after rescue therapy for CM that may have clinical relevance, and may also be relevant to cerebral sequelae of sepsis and other disorders.
... Interestingly, ICAM-1 upregulation coincided with a labeling pattern that outlined the endothelial junctions. In agreement with earlier reports636465, ICAM-1 was also significantly upregulated in postcapillary venules from PyXLinfected mice with hyperparasitemia (Figure 5B and 5E, Video S18) compared to uninfected control mice (Tukey's Test: T = 18.79, P,0.001;Figure 5C, Video S19). ...
Cerebral malaria claims the lives of over 600,000 African children every year. To better understand the pathogenesis of this devastating disease, we compared the cellular dynamics in the cortical microvasculature between two infection models, Plasmodium berghei ANKA (PbA) infected CBA/CaJ mice, which develop experimental cerebral malaria (ECM), and P. yoelii 17XL (PyXL) infected mice, which succumb to malarial hyperparasitemia without neurological impairment. Using a combination of intravital imaging and flow cytometry, we show that significantly more CD8+ T cells, neutrophils, and macrophages are recruited to postcapillary venules during ECM compared to hyperparasitemia. ECM correlated with ICAM-1 upregulation on macrophages, while vascular endothelia upregulated ICAM-1 during ECM and hyperparasitemia. The arrest of large numbers of leukocytes in postcapillary and larger venules caused microrheological alterations that significantly restricted the venous blood flow. Treatment with FTY720, which inhibits vascular leakage, neurological signs, and death from ECM, prevented the recruitment of a subpopulation of CD45hi CD8+ T cells, ICAM-1+ macrophages, and neutrophils to postcapillary venules. FTY720 had no effect on the ECM-associated expression of the pattern recognition receptor CD14 in postcapillary venules suggesting that endothelial activation is insufficient to cause vascular pathology. Expression of the endothelial tight junction proteins claudin-5, occludin, and ZO-1 in the cerebral cortex and cerebellum of PbA-infected mice with ECM was unaltered compared to FTY720-treated PbA-infected mice or PyXL-infected mice with hyperparasitemia. Thus, blood brain barrier opening does not involve endothelial injury and is likely reversible, consistent with the rapid recovery of many patients with CM. We conclude that the ECM-associated recruitment of large numbers of activated leukocytes, in particular CD8+ T cells and ICAM+ macrophages, causes a severe restriction in the venous blood efflux from the brain, which exacerbates the vasogenic edema and increases the intracranial pressure. Thus, death from ECM could potentially occur as a consequence of intracranial hypertension.
... A larger set of differentially expressed genes, including cytokine genes (for interleukin-1, interleukin-6, gamma interferon , and TNF-), chemokine genes, interferon-induced genes, and genes critical for adaptive immune responses were similarly induced by both lethal and nonlethal parasites as the parasite density rose to 5% (Fig. 2B). Many transcriptional changes precede the action of inflammatory cytokines such as TNF-and gamma interferon and have been shown to vary with parasite density and return to baseline upon resolution of infection (16, 19, 25). The changes in expression in genes associated with the defense response in murine malaria were similarly observed in an early infection in nonhuman primates infected with the nonlethal parasite Plasmodium cynomolgi (35). ...
High-density oligonucleotide microarrays are widely used to study gene expression in cells exposed to a variety of pathogens.
This study addressed the global genome-wide transcriptional activation of genes in hosts infected in vivo, which result in
radically different clinical outcomes. We present an analysis of the gene expression profiles that identified a set of host
biomarkers which distinguish between lethal and nonlethal blood stage Plasmodium yoelii malaria infections. Multiple biological replicates sampled during the course of infection were used to establish statistically
valid sets of differentially expressed genes. These genes that correlated with the intensity of infection were used to identify
pathways of cellular processes related to metabolic perturbations, erythropoiesis, and B-cell immune responses and other innate
and cellular immune responses. The transcriptional apparatus that controls gene expression in erythropoiesis was also differentially
expressed and regulated the expression of target genes involved in the host's response to malaria anemia. The biological systems
approach provides unprecedented opportunities to explore the pathophysiology of host-pathogen interactions in experimental
malaria infection and to decipher functionally complex networks of gene and protein interactions.
Co-infection of malaria and intestinal parasites is widespread in sub-Saharan Africa and causes severe disease especially among the poorest populations. It has been shown that an intestinal parasite (helminth), mixed intestinal helminth or Plasmodium parasite infection in a human induces a wide range of cytokine responses, including anti-inflammatory, pro-inflammatory as well as regulatory cytokines. Although immunological interactions have been suggested to occur during a concurrent infection of helminths and Plasmodium parasites, different conclusions have been drawn on the influence this co-infection has on cytokine production. This review briefly discusses patterns of selected cytokine (IL-6, IL-8, IL-10, TNF-α and INF-γ) responses associated with infections caused by Plasmodium, intestinal parasites as well as a Plasmodium-helminth co-infection.
Coevolution of humans and malaria parasites has generated an intricate balance between the immune system of the host and virulence factors of the parasite, equilibrating maximal parasite transmission with limited host damage. Focusing on the blood stage of the disease, we discuss how the balance between anti-parasite immunity versus immunomodulatory and evasion mechanisms of the parasite may result in parasite clearance or chronic infection without major symptoms, whereas imbalances characterized by excessive parasite growth, exaggerated immune reactions or a combination of both cause severe pathology and death, which is detrimental for both parasite and host. A thorough understanding of the immunological balance of malaria and its relation to other physiological balances in the body is of crucial importance for developing effective interventions to reduce malaria-related morbidity and to diminish fatal outcomes due to severe complications. Therefore, we discuss in this review the detailed mechanisms of anti-malarial immunity, parasite virulence factors including immune evasion mechanisms and pathogenesis. Furthermore, we propose a comprehensive classification of malaria complications according to the different types of imbalances.
There is an urgent need to develop and test novel compounds against malaria infection. Carrageenans, sulphated polysaccharides derived from seaweeds, have been previously shown to inhibit Plasmodium falciparum in vitro. However, they are inflammatory and alter the permeability of the blood-brain barrier, raising concerns that their use as a treatment for malaria could lead to cerebral malaria (CM), a severe complication of the disease. In this work, the authors look into the effects of the administration of lamda-carrageenan to the development and severity of CM in BALB/c mice, a relatively non-susceptible model, during infection with the ANKA strain of Plasmodium berghei.
Five-week-old female BALB/c mice were infected with P. berghei intraperitoneally. One group was treated with lamda-carrageenan (PbCGN) following the 4-day suppressive test protocol, whereas the other group was not treated (PbN). Another group of healthy BALB/c mice was similarly given lamda-carrageenan (CGN) for comparison. The following parameters were assessed: parasitaemia, clinical signs of CM, and mortality. Brain and other vital organs were collected and examined for gross and histopathological lesions. Evans blue dye assays were employed to assess blood-brain barrier integrity.
Plasmodium berghei ANKA-infected BALB/c mice treated with lamda-carrageenan died earlier than those that received no treatment. Histopathological examination revealed that intracerebral haemorrhages related to CM were present in both groups of infected BALB/c mice, but were more numerous in those treated with lamda-carrageenan than in mock-treated animals. Inflammatory lesions were also observed only in the lamda-carrageenan-treated mice. These observations are consistent with the clinical signs associated with CM, such as head tilt, convulsions, and coma, which were observed only in this group, and may account for the earlier death of the mice.
The results of this study indicate that the administration of lamda-carrageenan exacerbates the severe brain lesions and clinical signs associated with CM in BALB/c mice infected with P. berghei ANKA.
In the present study, we found that 129S1 mice are resistant to the infection with Plasmodium yoelii 17XL, which is highly virulent and causes lethal infection in various strains of mice. In contrast, IFN-γ receptor-deficient (IFN-γR(-/-)) mice on the 129S1 background were much more susceptible than 129S1 mice with intraperitoneal infection with 1 × 10(5) parasitized erythrocytes. The mortality in 129S1 and IFN-γR(-/-) mice was 11.6 and 79.4 %, respectively. Following inoculation of the parasites, both 129S1 and IFN-γR(-/-) mice showed a progressive increase in parasitemia. Growth rate of malaria parasites at the early stages of infection in the IFN-γR(-/-) mice was faster than that in 129S1 mice, and this difference in growth rate might cause the earlier death of IFN-γR(-/-) host from day 8 of infection than that of 129S1. In surviving mice of both strains, however, malaria parasites in their bloodstream began to decrease in number right after a peak of parasitemia and were not detectable by a microscopic examination during the observation period. Next, we investigated the cytokine and antibody production in 129S1 and IFN-γR(-/-) mice during infection. An analysis of cytokines showed that serum IFN-γ and IL-4 levels elevated significantly from day 1 and day 4 of infection, respectively, in both 129S1 and IFN-γR(-/-) mice when compared with the levels from the uninfected controls. Following the infection, significantly higher levels of malaria-specific IgG1 and IgG2a antibodies in the infected 129S1 mice were detected from day 15, and these elevations were coincident with the decrease of parasitemia. On the other hand, the levels of malaria-specific antibodies in IFN-γR(-/-) mice had a tendency to elevate on day 21 but did not reach statistical significance. The present data indicate that IFN-γR plays an essential role in mediating the early immune mechanisms induced by the infection of erythrocytic stages of P. yoelii 17XL parasite, leading to host survival.
Pyrimethamine is an antimalarial drug that has also been used successfully to treat autoimmune diseases such as lymphoproliferative syndrome. In this work, the effect of pyrimethamine (PYR) on the production of free radicals in malaria-infected mice was studied to better understand the drug's immunomodulatory properties. BALB/c and CBA/Ca mice were infected with Plasmodium yoelii 17XL. Seven days after infection, mice were treated with PYR or vehicle and sacrificed 24h later. Treatment with PYR increased superoxide dismutase and glutathione peroxidase activities in erythrocytes and the liver, augmented the levels of nitric oxide in the serum, and upregulated mRNA levels of superoxide dismutase, glutathione peroxidase, catalase, and iNOS in the spleen. In addition, PYR increased lipoperoxidation and protein carbonylation in infected mice. Our results indicate that P. yoelii 17XL reduces oxidative stress in infected cells, while PYR induces it, which is associated with increased parasite elimination. Thus, it is possible that oxidative stress generated by pyrimethamine is also involved in its immunomodulatory mechanism of action.
The Plasmodium berghei-infected mouse model is a well-recognized model for human cerebral malaria. Mice infected withP. berghei exhibit (i) metabolic acidosis (pH < 7.3) associated with elevated plasma lactate concentrations, (ii) significant (P < 0.05) vascular leakage in their lungs, hearts, kidneys, and brains, (ii) significantly (P < 0.05) higher cell and serum glutamate concentrations, and (iv) significantly (P < 0.05) lower mean arterial blood pressures. Because these complications are similar to those of septic shock, the simplest
interpretation of these findings is that the mice develop shock brought on by the P. berghei infection. To determine whether the immune system and specifically CD8+ T cells mediate the key features of shock during P. berghei malaria, we depleted CD8+T cells by monoclonal antibody (mAb) treatment and assessed the complications of malarial shock. P. berghei-infected mice depleted of CD8+ T cells by mAb treatment had significantly reduced vascular leakage in their hearts, brains, lungs, and kidneys compared
with infected controls treated with rat immunoglobulin G. CD8-depleted mice were significantly (P < 0.05) protected from lactic acidosis, glutamate buildup, and diminished HCO3
−levels. Although the blood pressure decreased in anti-CD8 mAb-treated mice infected with P. berghei, the cardiac output, as assessed by echocardiography, was similar to that of uninfected control mice. Collectively, our results
indicate that (i) pathogenesis similar to septic shock occurs during experimental P. bergheimalaria, (ii) respiratory distress with lactic acidosis occurs duringP. berghei malaria, and (iii) most components of circulatory shock are ameliorated by depletion of CD8+ T cells.
In this study, we report that infection with Plasmodium yoelii 17XL, a lethal strain of rodent malaria, does not result in death in the DBA/2 strain of mice. In contrast to BALB/c mice, DBA/2 mice developed significantly less parasitemia and never manifested symptoms of cerebral malaria (CM) on infection with this parasite. Moreover, the histological changes evident in the brain of susceptible BALB/c were absent in DBA/2 mice. Interestingly, the resistant DBA/2 mice when treated with recombinant interleukin (IL)-2, were found to develop CM symptoms and the infection became fatal by 6 to 8 days after infection. This condition was associated with an augmented interferon-gamma and nitric oxide production. Unexpectedly, IL-10 levels were also elevated in IL-2-treated DBA/2 mice during late stage of infection (at day 6 of infection) whereas the inverse relationship between IL-10 and interferon-gamma or nitric oxide was maintained in the early stage of infection (at day 3 after infection). The level of tumor necrosis factor-alpha production was moderately increased in the late phase of infection in these mice. Histology of brain from IL-2-treated mice demonstrated the presence of parasitized erythrocytes and infiltration of lymphocytes in cerebral vessels, and also displayed some signs of endothelial degeneration. Confocal microscopical studies demonstrated preferential accumulation of gammadelta T cells in the cerebral vessels of IL-2-treated and -infected mice but not in mice treated with IL-2 alone. The cells recruited in the brain were activated because they demonstrated expression of CD25 (IL-2R) and CD54 (intercellular adhesion molecule 1) molecules. Administration of anti-gammadelta mAb prevented development of CM in IL-2-treated mice until day 18 after infection whereas mice treated with control antibody showed CM symptoms by day 6 after infection. The information concerning creating pathological sequelae and death in an otherwise resistant mouse strain provides an interesting focus for the burden of pathological attributes on death in an infectious disease.
Vascular endothelial integrity, assessed by Evans blue dye extrusion and radiolabeled monoclonal antibody leakage, was markedly
compromised in the brain, lung, kidney, and heart duringPlasmodium berghei infection, a well-recognized model for human cerebral malaria. The results for vascular permeability from both methods were
significantly (P < 0.001) related.
Malaria is caused by the protozoan Plasmodium, transmitted to humans by Anopheles mosquitoes. The most dangerous of the plasmodia infecting humans is Plasmodium falciparum. Most of the clinical signs of this disease are caused by the parasite at stages in which it multiplies asexually in red blood cells. P. falciparum infection is most severe in children. However, only a small proportion of infected children develop severe complications; in nonimmune individuals these can cause severe and life-threatening disease. The reasons for these differences are not fully understood, but it is likely that host genetic, immune, and social and geographic factors play a role. Malaria is the world's most prevalent parasitic disease and against which effective control measures are urgently needed. Many attempts have been made by using several Plasmodium antigens both in model systems and in humans to develop a malaria vaccine. However, the results, although encouraging, are far from satisfactory (36, 73). One of the difficulties hindering the design of a successful vaccine against Plasmodium is our current incomplete knowledge of protective immunity and how it can be induced. Moreover, the pathogenesis of two of the most severe complications of P. falciparum malaria, cerebral malaria (CM) and severe malarial anemia (SA), both appear to involve dysregulation of the immune system (56). Therefore, a greater appreciation of the mechanisms of protective immunity on the one hand and of immunopathology on the other would provide crucial clues as to how manipulation of the immune system may best be achieved in order to reach the goal of better vaccines.
The purpose of this review is to summarize recent work on the role of cytokines in antiparasitic immune responses and immunopathology caused by blood-stage parasites. Although, other points in the biological cycle of Plasmodium offer attractive sites for prophylactic intervention, all clinical symptoms are provoked by this parasitic stage.
Plasmodium berghei-infected mice, a well-recognized model of experimental cerebral malaria (ECM), exhibit many of the hallmarks of a systemic
inflammatory response, with organ damage in brain, lung, and kidneys. Identification of the molecules mediating pathogenesis
of the inflammatory response, such as leukocyte adhesion, may lead to new therapies. Indeed, mice lacking the cell adhesion
molecule P-selectin were significantly (P = 0.005) protected from death due to P. berghei malaria compared with C57BL/6 controls despite similar parasitemia (P = 0.6) being found in both groups of mice. P-selectin levels assessed by the quantitative dual radiolabeled monoclonal antibody
technique increased significantly (P < 0.05) in several organs in C57BL/6 mice infected with P. berghei, supporting the concept of a systemic inflammatory response mediating malarial pathogenesis. Intravital microscopic analysis
of the brain microvasculature demonstrated significant (P < 0.001) leukocyte rolling and adhesion in brain venules of P. berghei-infected mice compared with those found in uninfected controls. The maximum leukocyte adhesion occurred on day 6 of P. berghei infection, when the mice become moribund and exhibit marked vascular leakage into the brain, lung, and heart. However, P-selectin
levels were significantly (P < 0.005) increased in brain, lung, and kidneys during P. berghei malaria in ECM-resistant BALB/c mice compared with those found in uninfected BALB/c controls, indicating that increased P-selectin
alone is not sufficient to mediate malarial pathogenesis. Leukocyte adhesion to brain microvessels of P-selectin-deficient
mice with P. berghei malaria was similar to that observed in control mice. Collectively, these results indicate that P-selectin is important for
the development of malarial pathogenesis but is not required for leukocyte adhesion in brain.
Malaria is a major cause of disease and death in tropical countries. A safe and effective vaccine is essential to achieve significant and sustained reductions in malaria-related morbidity and mortality. Driven by this need, research on the immunology of malaria has tended to focus on adaptive immunity. The potential for innate immune mechanisms to provide rapid protection against malaria has been largely neglected. On the basis of data from animal models, and clinical and epidemiological studies, this review considers the potential for innate immune mechanisms directed against Plasmodium parasites both to contribute to protection from malaria and to modulate adaptive immune responses.
A major factor in the pathogenesis of human cerebral malaria is blockage of cerebral microvessels by the sequestration of parasitized human red blood cells (PRBC). In vitro studies indicate that sequestration of PRBC in the microvessels is mediated by the attachment of knobs on PRBC to receptors on the endothelial cell surface such as CD36, thrombospondin (TSP), and intercellular adhesion molecule-1 (ICAM-1). However, it is difficult to test this theory in vivo because fresh human brain tissues from cerebral malarial autopsy cases are not easy to obtain. Although several animal models for human cerebral malaria have been proposed, none have shown pathologic findings that are similar to those seen in humans. In order to develop an animal model for human cerebral malaria, we studied brains of rhesus monkeys infected with the primate malaria parasite, Plasmodium coatneyi. Our study demonstrated PRBC sequestration and cytoadherence of knobs on PRBC to endothelial cells in the cerebral microvessels of these monkeys. Cerebral microvessels with sequestered PRBC were shown by immunohistochemical analysis to possess CD36, TSP, and ICAM-1. These proteins were not evident in the cerebral microvessels of uninfected control monkeys. Thus, our study indicates, for the first time, that rhesus monkeys infected with P. coatneyi can be used as a primate model to study human cerebral malaria. By using this animal model, we may be able to evaluate strategies for the development of vaccines to prevent human cerebral malaria.
The clinical complications associated with severe and cerebral malaria occur as a result of the intravascular mechanical obstruction of erythrocytes infected with the asexual stages of the parasite, Plasmodium falciparum. We now report that a primary P. falciparum-infected erythrocyte (parasitized red blood cell [PRBC]) isolate from a patient with severe complicated malaria binds to cytokine-induced human vascular endothelial cells, and that this adhesion is in part mediated by endothelial leukocyte adhesion molecule 1 (ELAM-1) and vascular cell adhesion molecule 1 (VCAM-1). PRBC binding to tumor necrosis factor alpha (TNF-alpha)-activated human vascular endothelial cells is partially inhibited by antibodies to ELAM-1 and ICAM-1 and the inhibitory effects of these antibodies is additive. PRBCs selected in vitro by sequential panning on purified adhesion molecules bind concurrently to recombinant soluble ELAM-1 and VCAM-1, and to two previously identified endothelial cell receptors for PRBCs, ICAM-1, and CD36. Post-mortem brain tissue from patients who died from cerebral malaria expressed multiple cell adhesion molecules including ELAM-1 and VCAM-1 on cerebral microvascular endothelium not expressed in brains of individuals who died from other causes. These results ascribe novel pathological functions for both ELAM-1 and VCAM-1 and may help delineate alternative adhesion pathways PRBCs use to modify malaria pathology.
Paroxysms are sharp episodes of high fever accompanied by chills and rigors that occur periodically, once in every 48 hr in Plasmodium vivax infections. We have measured the changing levels of serum tumor necrosis factor (TNF) during paroxysms in non-immune patients infected with P. vivax malaria. The changes in TNF levels closely paralleled the rise and fall in temperature during the paroxysms but tended to precede them by 30-60 min. These observations suggest that the rise and fall in temperature during P. vivax paroxysm may be directly related to the periodic changes in TNF levels induced during these infections. The peak TNF levels reached during P. vivax infections were much higher than even those which have been recorded during severe and fatal P. falciparum infections in which TNF has been postulated to contribute to the severe manifestations of this disease.
To determine whether isolates of Plasmodium falciparum have intrinsically different cytoadherent properties and whether these differences contribute to the clinical severity of human falciparum malaria, we studied the cytoadherence to C32 melanoma cells in vitro of 59 parasite isolates from patients with naturally acquired infections in Thailand. Parasitized erythrocytes adhere to these melanoma cells principally via the glycoprotein CD36, which is also expressed on most vascular endothelium. In vitro cytoadherence was significantly greater for isolates from patients with biochemical evidence of severe malaria. The cytoadherent properties of P. falciparum parasites may thus be a virulence factor in human falciparum malaria. However, there was no correlation between the degree of in vitro cytoadherence and cerebral symptoms, which suggests that other receptors and/or host factors may be important in the adherence of malaria parasites to cerebral vascular endothelium. The cytokines tumor necrosis factor, interleukin-1, and gamma interferon, which have been implicated in the pathogenesis of cerebral malaria and are known to promote intercellular adhesion in other systems, did not enhance the cytoadherence of P. falciparum isolates to C32 melanoma cells.
To understand the molecular mechanisms that lead to sequestration of red blood cells infected with mature stages of Plasmodium falciparum and to examine the relevance of earlier studies on adherence properties of laboratory-derived P falciparum parasites to the natural parasite population, we analyzed Gambian and Tanzanian isolates for in vitro cytoadherence and antibody-mediated microagglutination. Eighteen cryopreserved isolates of ring-stage parasites were cultured for 20 to 30 hours in vitro, in the patients original erythrocytes, to the trophozoite and schizont stage. All parasites were positive in the microagglutination assay with at least one of four African hyperimmune sera. In a rosetting assay, only 2 of the 18 isolates were strongly positive (35% and 41% of parasitized erythrocytes with more than two uninfected cells bound). Thirteen isolates showed either intermediate (5% to 18%) or low (less than 5%) rosetting while three isolates did not form rosettes. Infected cell-binding of the different isolates to immobilized CD36 or thrombospondin, or C32 melanoma cells correlated with the percentage of mature parasites in the blood samples (r = .932 for CD36, r = .946 for thrombospondin, and r = .881 for C32 melanoma cells). There was a high correlation between binding to CD36 and thrombospondin (r = .982). The extent of infected cell rosetting with uninfected cells in these blood samples was not correlated with these other receptor properties. We also observed coexpression of rosetting and cytoadherence receptors on the same parasitized erythrocytes.
The sequestration of parasitized erythrocytes in the microvasculature of vital organs is central to the pathogenesis of severe Plasmodium falciparum malaria. This process is mediated by specific interactions between parasite adherence ligands and host receptors on vascular endothelium such as intercellular adhesion molecule-1 (ICAM-1) and CD36. Using immunohistochemistry we have examined the distribution of putative sequestration receptors in different organs from fatal cases of P. falciparum malaria and noninfected controls. Receptor expression and parasite sequestration in the brain were quantified and correlated. Fatal malaria was associated with widespread induction of endothelial activation markers, with significantly higher levels of ICAM-1 and E-selectin expression on vessels in the brain. In contrast, cerebral endothelial CD36 and thrombospondin staining were sparse, with no evidence for increased expression in malaria. There was highly significant co-localization of sequestration with the expression of ICAM-1, CD36, and E-selectin in cerebral vessels but no cellular inflammatory response. These results suggest that these receptors have a role in sequestration in vivo and indicate that systemic endothelial activation is a feature of fatal malaria.
Tumor necrosis factor (TNF) is thought to playa key role in the pathogenesis of cerebral malaria. A double-blind, placebo-controlled
trial of an anti- TNF monoclonal antibody (B-C7) comprised 610 Gambian children with cerebral malaria, with mortality and
residual neurologic sequelae as primary study end points. Sixty (19.9%) of 302 children who received B-C7 died compared with
64 (20.8%) of 308 children who received placebo (adjusted odds ratio [OR], 0.90; 95% confidence interval [CI], 0.57–1.42).
Residual neurologic sequelae were detected in 15 (6.8%) of 221 survivors from the B-C7 group and in 5 (2.2%) of 225 survivors
of the placebo group (adjusted OR, 3.35; 95% CI, 1.08–10.4). The monoclonal antibody used in this study did not improve survival
in cerebral malaria and was associated with a significant increase in neurologic sequelae. A possible explanation of the latter
observation is that the antibody acts to retain TNF within the circulation and thereby prolongs its effects on vascular endothelium.
Although tumor necrosis factor (TNF) initially came to prominence because of its anti-tumor activity, most attention is now focused on its proinflammatory actions. TNF appears to play a critical role in both early and late events involved in inflammation, from localizing the noxious agent and amplifying the cellular and mediator responses at the local site and systemically, to editing (e.g., apoptosis) injured cells or effete immune cells and repairing inflammatory damage. We have generated mice deficient in TNF (TNF-/- mice) and have begun to examine the multiple functions attributed to TNF. TNF-/- mice develop normally and have no gross structural or morphological abnormalities. As predicted, they are highly susceptible to challenge with an infectious agent (Candida albicans), are resistant to the lethality of minute doses of lipopolysaccharide (LPS) following D-galactosamine treatment, have a deficiency in granuloma development, and do not form germinal centers after immunization. Phagocytic activity of macrophages appears relatively normal, as do T cell functions, as measured by proliferation, cytokine release, and cytotoxicity. B cell response to thymus-independent antigens is normal, but the Ig response to thymus-dependent antigen is reduced. Surprisingly, cytokine production induced by LPS appears essentially intact, with the exception of reduced colony-stimulating factor activity. Other unexpected findings coming from our initial analysis are as follows. (i) TNF has low toxicity in TNF-/- mice. (ii) TNF-/- mice show an anomalous late response to heat-killed Corynebacterium parvum. In contrast to the prompt response (granuloma formation, hepatosplenomegaly) and subsequent resolution phase in C. parvum-injected TNF+/+ mice, similarly treated TNF-/- mice show little or no initial response, but then develop a vigorous, disorganized inflammatory response leading to death. These results suggest that TNF has an essential homeostatic role in limiting the extent and duration of an inflammatory process-i.e., an anti-inflammatory function. (iii) In contrast to the expectation that TNF+/+ mice and TNF+/- mice would have identical phenotypes, TNF+/- mice showed increased susceptibility to high-dose LPS lethality, increased susceptibility to Candida challenge, and delayed resolution of the C. parvum-induced inflammatory process, indicating a strong gene dose requirement for different actions of TNF.
The cytoadherence of infected red blood cells (IRBCs) to the vascular endothelium is the major cause of IRBC sequestration and vessel blockage in the cerebral form of human malaria. Among the rodent models of malaria, Plasmodium yoelii 17XL-infected mice show many similarities with the human cerebral malaria caused by P. falciparum. In both, the sequestration of IRBCs in the brain vessels is secondary to the cytoadherence of IRBCs to the vascular endothelium. Similar to P. falciparum infection in the human but in contrast to P. berghei ANKA infection in mice, P. yoelii 17XL results in little, if any, accumulation of monocytes in the brain. In vivo microcirculatory studies reported here were designed to further understand the hemodynamic aspects and mechanisms underlying cytoadherence of IRBCs in the P. yoelii model using the easily accessible cremaster muscle vasculature. The results show significant decreases in arteriovenous red blood cell velocities (Vrbc) and wall shear rates in the microcirculation of P. yoelii-infected mice, with a maximal decrease occurring in small-diameter postcapillary venules, the main sites of cytoadherence. This reflects contributions from IRBC cytoadherence as well as from increased rigidity of parasitized red blood cells. No cytoadherence is observed in arterioles of the infected mice despite decreased wall shear rates, indicating that endothelial receptors for cytoadherence are restricted to venules. Infusion of a monoclonal antibody (MAb) against the intercellular adhesion molecule-1 (ICAM-1) resulted in significant increases in both arteriolar and venular Vrbc and wall shear rates, accompanied by detachment of adhered IRBCs at some venular sites. The peripheral blood smears taken after the MAb infusion showed a distinct increase in the percentage of schizonts, again indicating detachment and/or prevention of cytoadherence. An MAb against the vascular cell adhesion molecule-1 (VCAM-1) as well as an irrelevant control antibody had no effect on these parameters. These results provide the first in vivo microcirculatory evidence indicating involvement of ICAM-1, but not of VCAM-1, in the sequestration of IRBCs in a rodent model of cerebral malaria.
We observed considerable diversity in the cytoadherence of Plasmodium falciparum isolates from Malawi to melanoma cells, U937 cells and human peripheral monocytes. Each isolate exhibited a unique cytoadherence profile for the three human cell types. These isolates generally adhered well to U937 cells and fresh monocytes, moderately to melanoma cells and poorly to TE 671, MIA-Pa-Ca, WI 38, PLC/PRF/5 and HeLa cells. An antimalarial immunoglobulin pool inhibited binding to melanoma cells by 50% or more and to U937 cells by 25% or less. There was no correlation between in vitro cytoadherence to the three cells and clinical disease. These results suggest that malarial adherence ligands exposed on the surface of infected erythrocytes vary from one isolate to another.
Infection of the squirrel monkey, Saimiri sciureus, with several strains of Plasmodium falciparum leads in a proportion of animals to neurological symptoms with a fatal outcome. This first simian model for human cerebral malaria was studied with three strains of parasites, the uncloned Palo Alto(FUP-1) strain, the Palo AltoPLF3 clone MHB11, and the recently monkey-adapted P. falciparum strain IPC/RAY. Cerebral malaria could develop during primo infection of monkeys, whether the animals had been splenectomized or not. It did not occur in all animals and the appearance of neurological symptoms could not be predicted, as it was not related to the degree of parasitemia or duration of parasite infections.
Endothelial cells have long been viewed as a passive lining of blood vessels endowed essentially with negative properties such as that of being nonreactive to blood components. It is now evident that upon exposure to environmental signals, cytokines in particular, vascular cells undergo profound changes in gene expression and function that allow these cells to participate actively in inflammatory reactions, immunity, and thrombosis. Different mediators (e.g., interleukin-1 [IL-1] and interferon-gamma) activate relatively distinct sets of functions. These functional programs expressed in activated endothelial cells include the production by the same cells of cytokines (e.g., IL-1, IL-6, chemotactic cytokines, and colony-stimulating factors), which regulate hematopoiesis, the differentiation and proliferation of T and B lymphocytes, and the extravasation of leukocytes. The identification of cytokine circuits through which vascular cells participate to thrombotic, inflammatory, and immune reactions provides novel targets for therapeutic intervention.
rIL-1 beta treatment of cultured human endothelial cells (HEC) promotes polymorphonuclear leukocyte (PMN) adhesion and transmigration. Using in vitro quantitative monolayer adhesion and videomicroscopic transmigration assays, we have examined the contributions of endothelial-leukocyte adhesion molecule-1 (ELAM-1), intercellular adhesion molecule-1 (ICAM-1), and the leukocyte adhesion complex, CD11/CD18, to these processes. Maximal enhancement of PMN adhesion and transmigration were observed after 4 h of rIL-1 beta treatment, when surface expression of ELAM-1 had peaked and ICAM-1 was modestly increased. Blocking mAb directed to either ELAM-1 or ICAM-1 inhibited greater than 90% of the up-regulated PMN transmigration. Blocking mAb directed to either CD11a/CD18 (LFA-1, a ICAM-1 counter-receptor), CD11b/CD18 (Mo-1), or CD18 (common beta 2-integrin) also blocked greater than 90% of PMN transmigration. At later time points (24 or 48 h), ELAM-1 surface expression was markedly decreased, whereas ICAM-1 expression was increased over the 4-h level; PMN adhesion remained elevated (approximately 50 to 60% of 4 h level), but transmigration returned to levels seen with unactivated HEC. These data indicate that PMN interaction with at least two distinct HEC adhesion molecules is necessary for transendothelial migration and suggests that PMN adhesion and transmigration, although interrelated, are mechanistically distinct processes.
To investigate the role of tumor necrosis factor in Plasmodium falciparum infections, we measured serum concentrations of this cytokine in 65 Malawian children with severe falciparum malaria. Of these children (mean age, 5.3 years), 55 were unconscious and 10 had hypoglycemia at presentation. Although there was considerable overlap, the mean (+/- SEM) initial serum concentration of tumor necrosis factor was significantly higher in the 10 patients who died (709 +/- 312 pg per milliliter) than in the 55 who survived (184 +/- 32 pg per milliliter; P less than 0.02). The mortality rate increased with the concentration of tumor necrosis factor: at a level of less than 100 pg per milliliter, 1 of 24 patients died; at 100 to 500 pg per milliliter, 6 of 34 patients; and at more than 500 pg per milliliter, 3 of 7 patients. High concentrations of tumor necrosis factor were also associated with hypoglycemia (P less than 0.02), hyperparasitemia (P less than 0.002), age under three years (P less than 0.03), and severity of illness as measured by a prognostic index (P less than 0.0005). The highest serum concentrations of tumor necrosis factor were found in patients who died shortly after admission. The concentrations in cerebrospinal fluid were within the normal range in all patients. In serum samples obtained from 38 convalescent patients, the concentration of tumor necrosis factor declined to a mean of 16 +/- 3 pg per milliliter. We conclude that the level of tumor necrosis factor is frequently increased in patients with severe falciparum malaria, particularly in those with cerebral malaria or hypoglycemia. To determine whether it is important in the pathogenesis of the signs and symptoms of the disease requires further study.
We studied the relationship between presenting features and outcome in 131 Malawian children admitted with cerebral malaria (P. falciparum malaria and unrousable coma). A method was devised for the measurement of depth of coma in children too young to speak. Twenty patients (15 per cent) died and 12 (9 per cent) recovered with residual neurological sequelae. Presenting clinical signs significantly associated with adverse outcome (death or sequelae) were profound coma, signs of decerebration, absence of corneal reflexes, convulsions at the time of admission and age under three years. Laboratory findings of prognostic significance were hypoglycaemia, leucocytosis, hyperparasitaemia, elevated plasma concentrations of alanine and 5'-nucleotidase, and elevated plasma or cerebrospinal fluid lactate. A prognostic index based on eight of these risk factors that can readily be ascertained at the bedside or in a ward sideroom, was more accurately predictive of outcome than any single feature. Such an index may be valuable as a measure of severity of illness for establishing the comparability of study groups, and for evaluating the role of other factors in the pathogenesis of cerebral malaria.
Tumor necrosis factor, or cachectin (TNF-alpha), a protein with a wide range of biological activities, is produced mainly by macrophages and may be important in inflammatory processes. The role of TNF-alpha in the pathogenesis of cerebral malaria was investigated in a murine model. Most CBA mice infected with Plasmodium berghei anka die between days 6 and 14 with acute neurological manifestations unrelated to the level of parasitemia, whereas mice of some other strains have malaria of the same severity that ends in death after 3 to 4 weeks without neurological manifestations. The activity of serum TNF-alpha was considerably increased in CBA/Ca mice with cerebral malaria but not in Plasmodium berghei-infected mice that did not develop this complication. One injection of rabbit antibody to TNF-alpha on day 4 or 7 fully protected infected mice from cerebral malaria without modifying the parasitemia, whereas immunoglobulins from normal rabbit had no effect. In mice with cerebral malaria, the cerebral vessels showed focal accumulations of packed macrophages often containing infected erythrocytes; this lesion was not seen in mice treated with antibody to TNF-alpha or in untreated mice without cerebral malaria. These findings indicate that TNF-alpha has an important role in the pathogenesis of cerebral malaria in this murine model and suggest that local accumulation and activation of macrophages may lead to the predominance of lesions in the central nervous system.
We have studied the pathophysiology of the vascular obstruction induced by Plasmodium falciparum-parasitized erythrocytes with the use of an ex vivo microcirculatory preparation perfused with red cells infected with knobless and knobby clones of the FCR-3 strain. We find that parasitized erythrocyte membrane knobs are indispensable for the generation of the circulatory obstruction. Uninfected erythrocytes incubated in culture and erythrocytes infected with early or late forms of the knobless clones or the early forms of the knobby clone all failed to obstruct the microcirculation, although exhibiting various effects on bulk viscosity and peripheral resistance during flow. In contrast, late forms of the knobby clone produced significantly higher peripheral resistance during flow and significant obstruction as detected by changes in time of pressure flow recovery as well as by direct videorecorded microscopic observation. Optical and electron microscopy showed that the adherence of parasitized cells to the endothelium was limited to the venules and involved the knobs in junctions. In addition, we were able to follow the sequence of events during obstruction: initial red-cell adherence to the venular endothelium (sometimes only transitory) followed by progressive recruitment at the venule surface, finally leading to total obstruction that involved parasitized and nonparasitized erythrocytes. Sometimes, retrograde aggregation would extend the obstruction to the capillaries or even precapillary arterioles. These results show that knobs are necessary and sufficient to produce vascular obstruction and that other factors (spleen, immunological, etc.) can only have a modulating role. These results also exclude the possibility that the exclusive adherence to venules is the consequence of "plasma factors" found in the malaric patients.
For investigation of the pathogenesis of cerebral malaria, immediate postmortem samples from brain and other tissues of patients dying with Plasmodium falciparum malaria, with (CM) or without (NCM) cerebral malaria, were processed for electron microscopy. Counts of parasitized erythrocytes (PRBCs) in cerebral and other vessels showed that the proportion of PRBCs was higher in CM than in NCM, and also that the proportion of PRBCs was higher in the brain than in other organs examined in both CM and NCM. Cerebral vessels from CM patients were more tightly packed with RBCs than those from NCM patients, but there was no significant difference in the amount or degree of endothelial damage or numbers of vessels with endothelial pseudopodia. Fibrillar (fibrin) deposits were present in a small proportion of vessels, but no thrombosis was present. There was neither acute nor chronic inflammation, and leukocytes were absent within or outside cerebral vessels. There was no immune complex deposition in cerebral vessels. Parasites in cerebral vessels were mainly trophozoites or schizonts. Occasional RBC remnants following parasite release were seen. Some parasites were degenerate, resembling crisis forms. PRBCs adhered to endothelium via surface knobs. It is concluded that there is no evidence for an inflammatory or immune pathogenesis for human cerebral malaria and that the clinical effects probably relate to anoxia and the metabolic activities of the parasites.
A sudden enhancement in virulence of a mild Plasmodium berghei yoelii 17 x strain resulted in fulminating and fatal infections in CF1 and A/J mice. The virulent strain has maintained its characteristics
after ten cyclical transmissions through Anopheles stephensi. The visible expression of virulence of the mutated strain is its ability to cross the blood-brain barrier and cause intravascular
sequestration of injected erythrocytes and blockage of brain capillaries. We, therfore, believe that the virulent line of
Plasmodium berghei yoelii 17 x could serve as a useful laboratory model for the study of "cerebral malaria."
In an open, randomized, controlled therapeutic trial, 56 children with cerebral malaria (CM) were randomly assigned to receive
standard Quinine regimen with or without pentoxifylline (10 mg/kg/day by continuous intravenous infusion). Pentoxifylline
exerted an inhibitory effect on the synthesis of tumor necrosis factor (TNF), a possible mediator of CM. The 26 children who
received pentoxifylline had significantly shorter comas than controls (median, 6 vs. 46 h; P < .001). Pentoxifylline recipients showed a trend toward a lower mortality, with a borderline significant difference (P = .055). The better outcome in the pentoxifylline group was associated with a decline in TNF serum levels on the third day
of treatment in a few subjects that was not seen in controls. While alternative or concurrent mechanisms of action may be
of some relevance, larger double-blind trials are needed to determine whether pentoxifylline has a therapeutic role in CM.
Tumour-necrosis factor-alpha (TNF-alpha) is believed to have an important role in the pathogenesis of severe infectious disease and fatal cerebral malaria is associated with high circulating levels of this cytokine. In a large case-control study in Gambian children we find that homozygotes for the TNF2 allele, a variant of the TNF-alpha gene promoter region, have a relative risk of 7 for death or severe neurological sequelae due to cerebral malaria. Although the TNF2 allele is in linkage disequilibrium with several neighbouring HLA alleles, we show that this disease association is independent of HLA class I and class II variation. These data suggest that regulatory polymorphisms of cytokine genes can affect the outcome of severe infection. The maintenance of the TNF2 allele at a gene frequency of 0.16 in The Gambia implies that the increased risk of cerebral malaria in homozygotes is counterbalanced by some biological advantage.
To understand the microcirculatory events during cerebral malaria, we have studied the lethal strain of rodent Plasmodia, Plasmodium yoelii 17XL, originally described by Yoeli and Hargreaves in 1974. The virulence of P. yoelii 17XL is caused by intravascular sequestration of infected red blood cells (IRBCs), especially in the brain vessels and capillaries. This mouse model resembles human P. falciparum infection more closely than P. berghei ANKA infection since it shows little, if any, inflammation of the brain. In vivo microcirculatory studies on cytoadherence of IRBCs were performed using the cremaster muscle preparation, which is an easily accessible vasculature for intravital observations. Ex vivo assay of cytoadherence was carried out in the artificially perfused mesocecum preparation of the rat. The results in either preparation demonstrated cytoadherence of IRBCs that was restricted to postcapillary venules. Furthermore, the in vivo measurements showed the prevalence of cytoadherence in small-diameter (< 40 microns) venules in accordance with the local wall shear rates. The parasitized animals demonstrated significantly reduced red blood cell velocities and wall shear rates in the small-diameter postcapillary venules of the cremaster. The relationship between cytoadherence and venular wall shear rates was also reflected in the inverse correlation between the number of adhered cells and the venular diameter in the ex vivo mesocecum preparation. In the ex vivo preparation, cytoadherence of IRBCs was accompanied by a higher peripheral resistance. Transmission electron microscopy of the cremaster muscle and brain tissues showed a tight association of IRBCs with the endothelium of small venules. These observations demonstrate that cytoadherence of P. yoelii 17XL-infected mouse red blood cells is very similar to that of P. falciparum-infected cells. Thus, this model should allow a detailed analysis of the molecular mechanisms involved in the generation of cerebral malaria by cytoadherence of the infected red blood cells to the vascular endothelium.
The pathogenicity of Plasmodium falciparum is due largely to the parasite's unique ability to adhere to capillary and postcapillary venular endothelium during the second-half of the 48-hour life cycle. The resulting sequestration of infected erythrocytes (IRBC) in deep vascular beds leads to tissue hypoxia, metabolic disturbances, and organ dysfunction which characterize severe falciparum malaria. Several endothelial receptors of cytoadherence have been identified, but their clinical relevance remains controversial. In the present report, the receptor specificity of 60 clinical P falciparum isolates was determined using transfectants each expressing one of CD36, intercellular adhesion molecule-1 (ICAM-1), E-selectin, and vascular cell adhesion molecule-1 (VCAM-1). All isolates tested adhered to CD36 and ICAM-1, but the adherence to CD36 was at least 10-fold higher. Seven isolates adhered to E-selectin whereas none of 19 isolates adhered to VCAM-1. From a population standpoint, about 30% of IRBC in each isolate adhered to CD36, and 2% to 3% adhered to ICAM-1. The percentage adherent to E-selectin and VCAM-1 was negligible. IRBC selected on CD36 adhered almost exclusively to CD36 whereas 80% to 90% of IRBC selected on ICAM-1 could also adhere to CD36. Selected IRBC did not adhere to E-selectin or VCAM-1. These findings indicate that cytoadherence to multiple endothelial receptors is a rare occurrence with natural P falciparum isolates, but do not exclude a role for the adhesion molecules in promoting other IRBC-endothelial interactions such as rolling under flow conditions. Receptor specificity in vivo may be dictated by the ligand-receptor combination which provides the best survival potential for the parasite.
The central pathologic process in severe Plasmodium falciparum malaria is the cytoadherence of parasitized erythrocytes to capillary and postcapillary venular endothelium, with resultant tissue hypoxia, metabolic disturbances, and multiorgan dysfunction. The molecular basis of this process has been studied extensively using static adhesion assays. In the present study, we determined whether infected red blood cells (IRBC) from clinical parasite isolates would roll and adhere on CD36, ICAM-1, E-selectin, P-selectin, and VCAM-1 using a laminar flow system that allowed for the direct visualization of IRBC-substratum interactions. The results indicate that IRBC could tether and roll on CD36, ICAM-1, P-selectin, and VCAM-1 in a shear-dependent fashion, but significant adhesion was restricted to CD36. There was no interaction with E-selectin. When both CD36 and ICAM-1 were expressed on the same cellular substratum such as C32 melanoma cells, adhesion was significantly greater than when CD36 was present alone. The adhesive interactions were different from those between leukocytes and the same adhesion molecules. Furthermore, IRBC rolling on P-selectin and VCAM-1 was not inhibitable by Abs that entirely prevented leukocyte-receptor interactions. These findings suggest that cytoadherence under physiologic conditions may be a multistep process similar to that involved in the recruitment of a number of different cell types. Further elucidation of the molecular basis of these novel interactions is crucial for the development of therapeutic interventions aimed at inhibiting or reversing the process.