A primate model for human cerebral malaria: Plasmodium coatneyi-infected rhesus monkeys.

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.     

A quantitative ultrastructural study of renal pathology in fatal Plasmodium falciparum malaria

OBJECTIVE: To use electron microscopy to examine the role of parasitized red blood cell (PRBC) sequestration in the pathogenesis of acute renal failure in severe falciparum malaria. METHODS: Ultrastructural pathological examination of renal tissues from Southeast Asian adults (n = 63) who died from severe falciparum malaria. Qualitative and quantitative determination of the major pathological features of disease, including PRBC and leukocyte sequestration. Clinico-pathological correlation with the pre-mortem clinical picture and peripheral parasite count. RESULTS: There was a high incidence of malaria-associated renal failure in this population (> 40%) and a correlation between this incidence, severe malarial anaemia and shock. Pathological features included PRBC sequestration in glomerular and tubulo-interstitial vessels, acute tubular damage and mild glomerular hypercellularity resulting from the accumulation of host monocytes within glomerular capillaries. No evidence for an immune complex mediated glomerulonephritis was found. There was a correlation between parasite sequestration in the kidney and pre-mortem renal failure, although overall levels of sequestration were relatively low. Levels of sequestration (Knob+ PRBC) were significantly higher in malaria-associated renal failure than in fatal cases without renal failure (P = 0.005). CONCLUSION: Malaria-associated renal failure is a common and serious complication of severe Plasmodium falciparum malaria in this population, associated with acute tubular injury rather than glomerulonephritis, and linked to localization of host monocytes in the kidney as well as sequestration of PRBCs.     

Apoptosis: a potential triggering mechanism of neurological manifestation in Plasmodium falciparum malaria

Plasmodium falciparum infection can lead to a life threatening disease and the pathogenetic mechanisms of severe manifestations are not fully understood. Here, we investigated the capacity of P. falciparum-parasitized red blood cells (PRBC) from 45 children with clinical malaria to induce endothelial cell (EC) apoptosis. In all subjects, PRBC that cytoadhered to ECs could be found albeit to a variable degree. By contrast, PRBC that induce EC apoptosis were found only in nine (20%) subjects. Interestingly, children with neurological manifestations were significantly more likely to harbour apoptogenic strains. There was no quantitative relationship between the capacity of these isolates to cytoadhere and apoptosis induction. We hypothesize that P. falciparum-encoded molecules could be responsible for apoptosis induction and therefore suggest new insights in the pathogenesis of P. falciparum malaria. Further investigations are currently in progress to determine whether these results can be confirmed and to identify putative parasite apoptogenic factors.     

A quantitative ultrastructural study of the liver and the spleen in fatal falciparum malaria

We performed a retrospective study of 25 patients who died of severe falciparum malaria in Thailand and Vietnam using electron microscopy. The aims of the study were: to determine if there was any significant association between parasitized red blood cells (PRBC) sequestered in liver and spleen and particular pre-mortem clinical complications, and to compare the degree of parasite load between the liver and spleen within the same patients. PRBC sequestrations in each organ were compared with the pre-mortem parasitemia, to calculate the sequestration index (S.I.). The S.I. showed that the degree of PRBC sequestration in the spleen was higher than the liver (S.I. median = 3.13, 0.87, respectively) (p < 0.05). The results of quantitative ultrastructural study showed a significantly high parasite load in the liver of patients with jaundice, hepatomegaly and liver enzyme elevation (p < 0.05). We found a significant correlation between PRBC sequestration in the liver and a high serum bilirubin level, a high aspartate aminotransferase (AST) level and an increase in the size of the liver (Spearman's correlation coefficient = 0.688, 0.572, 0.736, respectively). Furthermore, a higher parasite load was found in the liver of patients with acute renal failure (ARF) compared to patients without ARF (p < 0.05). These findings suggest that PRBC sequestration in the liver is quantitatively associated with pre-mortem hepatic dysfunction and renal impairment. There was no significant difference between splenomegaly and PRBC sequestration. The size of a palpable spleen was not correlated with parasite load in the spleen. When ultrastructural features were compared between the two reticuloendothelial organs, we found that the spleen had more PRBC and phagocytes than the liver. The spleen of non-cerebral malaria (NCM) patients had more phagocytes than cerebral malaria (CM) patients. This observation reveals that the spleen plays a major role in malaria parasite clearance, and is associated with host defence mechanisms against malaria.     

Reduced microbicidal and anti-tumour activities of human monocytes after ingestion of Plasmodium falciparum-infected red blood cells

Oxidatively stressed red blood cells (RBC) and Plasmodium falciparum-infected RBC (PRBC) are avidly phago-cytosed by human peripheral monocytes. Following the ingestion of PRBC the monocytes' ability to phagocytose PRBC and to generate aggressive oxidative compounds is severely impaired. In the present work the microbicidal and anti-tumour capacities of monocytes fed with diamide-treated RBC and PRBC harbouring mature (trophozoite) parasites have been investigated. The capacity of the latter, but not of the former, to phagocytose Escherichia coli and Staphylococcus aureus and to kill them, as well as ingested Candida albicans cells intracellularly, was found to be markedly impaired. Monocytes that have ingested PRBC had a significantly reduced cytostatic and cytolytic activities against a lymphoblastic tumour cell line. Monocytesfed with oxidatively stressed RBC had normal or sometimes even greater anti-tumour activities. Monocytes that have ingested PRBC showed a reduced capability to produce superoxide following stimulation with phorbol ester. Such impairment in monocyte functions may explain the reduced antibacterial and anti-tumour activities of monocytes in malaria patients, and could be consequential to their ability to resist bacterial infections and to provide means for the control of tumour development in those patients.     

Platelet-induced clumping of Plasmodium falciparum-infected erythrocytes from Malawian patients with cerebral malaria-possible modulation in vivo by thrombocytopenia

Platelets may play a role in the pathogenesis of human cerebral malaria (CM), and they have been shown to induce clumping of Plasmodium falciparum-parasitized red blood cells (PRBCs) in vitro. Both thrombocytopenia and platelet-induced PRBC clumping are associated with severe malaria and, especially, with CM. In the present study, we investigated the occurrence of the clumping phenomenon in patients with CM by isolating and coincubating their plasma and PRBCs ex vivo. Malawian children with CM all had low platelet counts, with the degree of thrombocytopenia directly proportional to the density of parasitemia. Plasma samples obtained from these patients subsequently induced weak PRBC clumping. When the assays were repeated, with the plasma platelet concentrations adjusted to within the physiological range considered to be normal, massive clumping occurred. The results of this study suggest that thrombocytopenia may, through reduction of platelet-mediated clumping of PRBCs, provide a protective mechanism for the host during CM.     

Identifying polymorphic regions of the p190 protein from different Plasmodium falciparum strains by using specific T cells

The p190 protein (also called MSA1 or MSP1) of the asexual blood stage forms of Plasmodium falciparum, a human malaria vaccine candidate, shows polymorphism between different isolates. Mice were immunized with p190–3, a recombinant protein which contains mostly conserved sequences derived from the p190 protein of the K1 parasite isolate. Proliferative T-cell responses of lymph node cells from immunized mice were assessed by stimulation in vitro with p190–3 or preparations of parasitized red blood cells (PRBC) containing the native protein. The p190–3-specific T cells from C57BL/6 mice consistently responded to some P. falciparum isolates, representing either the K1 or MAD20 serotype of p190, but not to other P. falciparum strains or to rodent malaria parasite-infected red blood cells. p190–3-specific T-cell responses from other mouse strains (BALB/c, C3H/He) did not distinguish between P. falciparum isolates. The polymorphic epitopes which were preferentially recognized by T cells from C57BL/6 mice were identified.     

Cytoadherence in human falciparum malaria as a cause of respiratory distress

The ultrastructure of three cases of fatal human falciparum malaria was studied in order to identify the cytoadherence of the endothelial cells in relation to parasitized red blood cells and septal interstitial changes which could be related to respiratory distress. Two cases showed marked endothelial oedema narrowing the capillary lumen with areas of adherence preferentially related to knobs, accompanied by septal interstitial oedema. One case showed no endothelial cells oedema, no knobs in parasitized red blood cells with no cytoadherence, no septal interstitial oedema and no respiratory distress. Cytoadherence seems to be the mechanism responsible for the septal pulmonary changes in severe falciparum malaria.     

Is there any artemisinin resistance in falciparum malaria?

We reported two cases of complicated falciparum malaria who had poor response to artesunate with delayed parasite clearance times. They were splenectomized patients who were treated with high doses of artemisinin derivatives. Our cases showed the importance of the spleen in the clearance of malaria parasites and had different clinical outcome, one fatal and one recovery. The host factors, the parasitemia count, the quality of antimalarial chemotherapy and blood level of the antimalarial drugs must be considered in relation to the causes of the delayed clearance of parasitemia.     

Glycolysis in Plasmodium falciparum results in modulation of host enzyme activities

BACKGROUND & OBJECTIVES: Plasmodium falciparum, the causative agent of the most serious form of malaria, infects about 5-10% of the world human population per year. It is well established that the erythrocytic stages of the malaria parasite rely mainly on glycolysis for their energy supply. In the present study, the glucose utilisation of erythrocyte population with parasitaemia levels similar to that of malaria patients was measured. The results allowed us to assess the effect of the parasites on the glucose utilisation of the vast majority of uninfected erythrocytes. METHODS: Using [2-13C]glucose and nuclear magnetic resonance (NMR) technique, the glucose utilisation in normal red blood cell (RBC) and P. falciparum infected red blood cell (IRBC) populations was measured. The IRBC population consisted of > 96% RBC and < 4% of parasite infected red blood cells (PRBC). The glycolytic enzymes were assayed to assess the effect of infected red cells on the enzymatic activities of uninfected ones. RESULTS: The rate of glucose utilisation by IRBC was considerably higher than that of RBC. Upon addition of 25% v/v conditioned culture medium (CM) of IRBC, RBCs exhibited a significant decrease in glucose utilisation. The CM could directly inhibit the activities of RBC glycolytic enzymes-phosphofructokinase (PFK) and pyruvate kinase (PK), without interfering with the activity of the pentose phosphate pathway enzyme-glucose-6-phosphate dehydrogenase (G-6-PD). INTERPRETATION & CONCLUSION: The present study showed that the clinical level of P. falciparum infected RBCs (< 4% parasitaemia) significantly enhance the glycolytic flux as well as down-regulate the glucose utilisation rate in the majority of uninfected RBC population. The mechanism of inhibition seems to be direct inhibition of the regulatory glycolytic enzymes-PFK and PK.     

Decreased transfusion requirements in patients given stem cell allografts using a non-myeloablative conditioning regimen: a single institution experience

We report our experience of allogeneic peripheral blood stem cell transplantation using non-myeloablative conditioning regimens delivered and supported on an outpatient basis. A group of 44 patients underwent 47 allograft procedures using peripheral blood stem cells. Approximately one third of the individuals did not require red blood cells transfusions: the median of transfused red blood cells units was 1 (range 0-10). In addition one out of three did not require platelet transfusions either, the median of platelet transfusions being 1 (range 0-6). In fourteen allografts (30%) neither red blood cells nor platelet transfusions were used. An inverse correlation was found between the number of CD34 cells infused and the PRBC and PLT transfusion requirements, those patients receiving high numbers of CD34 cells needing fewer transfusions of both PRBC and platelets. The possibility of conducting allografts without transfusion of blood products in some patients may result in a decrease in both cost and the risks stemming from exposure to human blood derivatives.     

Microvascular sequestration of parasitized erythrocytes in human falciparum malaria: a pathological study

Thirty-nine falciparum malaria autopsy cases from the Hospital for Tropical Diseases, Mahidol University, Bangkok, Thailand were divided into two groups that had had either cerebral malaria (CM) or non-cerebral malaria (NCM). We then studied significant pathological differences between these groups in order to investigate the correlation between parasitized erythrocyte (PRBC) sequestration in small blood vessels in the brain, heart, lungs and small intestines. We found that the percentage of PRBC sequestration in the organs which we studied was higher in the CM patients than in the NCM patients. The difference of PRBC sequestration among the organs of two groups was significant (P less than 0.05). In the CM group, the average percentage of PRBC sequestration in the brain was significantly higher than in the heart, lungs and small intestines (P less than 0.05). No statistically significant difference was found between PRBC sequestration in the brains, hearts, lungs and small intestines in the NCM group (P greater than 0.05). Our study indicates that severity of malaria in the CM patients depends on PRBC sequestration, especially in the brain. A combination of functional disturbances of the other organs, in addition to the cerebral pathology, may augment the severity of the disease.     

Antibodies to variant surface antigens of Plasmodium falciparum-infected erythrocytes and adhesion inhibitory antibodies are associated with placental malaria and have overlapping and distinct targets

We measured antibodies to chondroitin sulfate A (CSA)-binding and placental Plasmodium falciparum-infected red blood cells (PRBCs) among pregnant women with or without placental malaria. Immunoglobulin G to PRBC surface antigens was rare in uninfected primigravidae (3.7%), more prevalent in infected primigravidae (70%; P<.001), and common in infected (77%) and uninfected (83%) multigravidae. Similar patterns were seen for agglutinating antibodies, and antibodies were similar among women with past or active placental infection. PRBC adhesion to CSA was inhibited 60% by serum from infected primigravidae but 24% by serum from uninfected primigravidae (P=.025), whereas infection did not alter adhesion inhibition by multigravidae (77% inhibition)[corrected]. There was substantial heterogeneity in antibody type and levels. Antibodies did not correlate with parasite density or pregnancy outcome. Comparisons between antibodies suggest that adhesion-inhibitory antibodies and those to PRBC variant antigens have distinct and overlapping epitopes, may be acquired independently, and have different roles in immunity.     

Evaluation of a Red Cell Leukofilter Performance and Effect of Buffy Coat Removal on Filtration Efficiency and Post Filtration Storage

Prestorage leukoreduction of red cells is effective in reducing the incidence of HLA alloimmunization and improving the quality of stored packed red blood cells (PRBC). This study was conducted to evaluate the effectiveness of Imugard III-RC 4P in removing the leukocyte from packed red cells and the storage effects thereafter. The effects of buffy coat removal on the efficiency of leukofiltration, storage parameters of leukofiltered packed red blood cells and feasibility of prestorage leukofiltration were also assessed. Sixteen units each of buffy coat-depleted (LP) and nondepleted (NLP) PRBC were taken. Every unit was divided into two equal halves, one leukofiltered and other, non-leukofiltered. Cell counts, volume, hematocrit and hemoglobin were measured before and after filtration. Levels of K⁺, lactate dehydrogenase (LDH) and hemolysis were assessed in all the units weekly, post leukofiltration. Post leukofiltration, red cell and volume loss was within the specified limit in all the units. Residual leukocytes were significantly lesser in LP- PRBC compared to NLPPRBC. K⁺, LDH and hemolysis were significantly elevated in NLP- PRBC. Leukofiltered PRBC showed lesser elevation of K⁺, LDH and hemolysis towards the end of the storage period as compared to their unfiltered counterparts. Leukofilter is capable of performing ~4 log reduction. Buffy coat removal prior to filtration improves the efficiency of leukofilter and aids in improving the storage of red cells in terms of hemolysis.     

IgE deposition in brain microvessels and on parasitized erythrocytes from cerebral malaria patients

Postmortem brain tissues of 21 cerebral malaria cases were obtained in Myanmar and Vietnam. The tissues were examined by light microscopy and by an immunohistochemical method. Brain microvessels (capillaries and venules) were examined for the presence of immunoglobulins IgE and IgG, Plasmodium falciparum antigen, and parasitized erythrocytes (PRBC). Deposition of IgE, IgG, and P. falciparum antigen was observed in the microvessels from all specimens examined. Sequestered PRBC in the microvessels were positive for IgG in all 21 cases and for IgE in six cases. In the latter cases, the percentage of microvessels with sequestered PRBC was > 50%, with the frequency of IgE-positive cells ranging from 42% to 52%. In contrast, in five cases that were only weakly positive for IgE, the percentage of microvessels with sequestered PRBC was remarkably low (< 1%). These data indicate that the degree of deposition of IgE in microvessels and on PRBC from cerebral malaria patients correlated with that of PRBC sequestration. As IgE-containing immune complexes are known to induce local overproduction of tumor necrosis factor-alpha (TNF-alpha), a major pathogenic factor in cerebral malaria, IgE may contribute to the pathogenesis of this severe disease.     

Two-step process of cytoskeletal structural damage during long-term storage of packed red blood cells

BackgroundStorage of packed red blood cells (PRBC) for 42 days causes morphological, structural, and functional changes in the red cells. To assess the quality of stored PRBC, it is important to evaluate the main components of the product. The aim of this study was to evaluate the kinetics of the structural transformations in the cytoskeleton of red cells during long-term storage (up to 42 days).Materials and methodsBags of PRBC were stored with CPD/SAGM solution at +4 °C. Cytoskeletal parameters were measured on days 3, 12, 19, 21, 24, 28, 35, and 42 of storage to determine their changes. Atomic force microscopy was used to obtain images and analyse the parameters of the cytoskeletal network. As the storage time increased, a general PRBC test was performed. Membrane fixatives were not used at any stage of the preparation of the specimens for cytoskeletal imaging.ResultsWhen PRBC were stored for 42 days, the main changes to the cytoskeletal mesh included rupture of filaments, merger of small pores into larger ones, a decrease of the number of pores, thickening of filaments, and an increase of membrane stiffness. A process of irreversible changes to the cytoskeleton started on days 19–21. A kinetic model of changes in the parameters of the cytoskeletal mesh with time of PRBC storage was created.DiscussionTwo stages of impairment in cytoskeletal elements were found: rupture of filaments and clustering of protein components. The typical time of development and specifics of these stages are discussed. The consequences of the altered configuration of the cytoskeleton are also discussed. Destruction of the red cell cytoskeleton can have a negative effect on the efficacy of blood transfusion and increase the risk of post-transfusion complications. Our findings can be used in clinical medicine to evaluate the quality of PRBC for blood transfusion as well as for studies of the molecular organisation of red cells undergoing various types of physical and chemical treatment.     

Reduced microcirculatory flow in severe falciparum malaria: pathophysiology and electron-microscopic pathology

The pathophysiology of severe falciparum malaria is complex, but evidence is mounting that its central feature is the old concept of a mechanical microcirculatory obstruction. Autopsy studies, but also in vivo observations of the microcirculation, demonstrate variable obstruction of the microcirculation in severe malaria. The principal cause of this is cytoadherence to the vascular endothelium of erythrocytes containing the mature forms of the parasite, leading to sequestration and obstruction of small vessels. Besides, parasitized red cells become rigid, compromising their flow through capillaries whose lumen has been reduced by sequestered erythrocytes. Adhesive forces between infected red cells (auto-agglutination), between infected and uninfected red cells (rosetting) and between uninfected erythrocytes (aggregation) could further slow down microcirculatory flow. A more recent finding is that uninfected erythrocytes also become rigid in severe malaria. Reduction in the overall red cell deformability has a strong predictive value for a fatal outcome. Rigidity may be caused by oxidative damage to the red blood cell membrane by malaria pigment released at the moment of schizont rupture. Anti-oxidants, such as N-acetylcysteine can reverse this effect and are promising as adjunctive treatment in severe malaria.     

Microwave warming of resuscitation fluids

Hypothermia is a common complication in fluid resuscitation of the hypovolemic patient. Warm intravenous (IV) fluids have been shown to be a valuable adjunct in volume replacement to prevent this complication. A rapid method of warming IV fluids is the microwave oven. Heating time for liter bags of crystalloid to 39 C was determined to be two minutes at high power, 600 W. Fresh frozen plasma was thawed with five 30-second exposures to microwave radiation. Microwave warming of packed red blood cells (PRBC), 4 C to 37 C, resulted in a 17-fold increase in plasma hemoglobin over that of water bath controls, (P greater than .01). Heating on a warm cycle to room temperature, 21 C, caused an average 26% increase in plasma hemoglobin. Therefore, we do not advocate microwave warming of PRBC because of the possible danger of local overheating, which causes hemolysis. We warm PRBC secondarily by diluting with microwave-warmed, calcium-free crystalloid.     

Pathology of falciparum malaria in Vietnam.

Autopsy samples from the brains of 20 patients who died of falciparum malaria were examined by light microscopy and by an immunohistologic method. Particular attention was paid to a comparison of the pathologic features of the white matter and the cortex. In the high-sequestration (greater than 50%) group (n = 8), the mean +/- SD percentage of cerebral microvessels that showed parasitized red blood cell (PRBC) sequestration was 71.2 +/- 8.1% in the cortex and 84.0 +/- 6.7% in the white matter. The difference in the PRBC sequestration rate between cortex and white matter was statistically significant (P less than 0.01). Perivascular and ring hemorrhages were seen more frequently in the white matter than in the cortex. Deposition of IgG and Plasmodium falciparum antigen in the cerebral microvessels was more highly significant in the white matter than in the cortex (P less than 0.01). Our study demonstrated that the localized concentration of PRBC sequestration in the brain correlated with the marked immunohistologic differences in the microvessels of cortex and white matter.     

Plasmodium falciparum malaria in splenectomized patients: two case reports in French Guiana and a literature review

Some of the immunologic mechanisms involved in malaria physiopathology remain unclear. In animals, the spleen seems to play a key role in protecting the host against malaria. However, little is known about the effect of spleen dysfunction on human malaria. We report two severe cases of Plasmodium falciparum infection with unusual clinical and parasitologic features in two splenectomized men living in French Guiana. The peripheral blood of these cases showed hyperparasitemia, with a high proportion of mature parasites and leukocytes with malaria pigment. Despite appropriate treatment and adequate absorption, hyperparasitemia persisted. Parasite clearance was delayed and one patient died. Only the patient who died had the merozoite surface protein 1 allele B-K1 and the varD gene genotype, which is considered to be a probable parasite virulence factor. These uncommon cases differ from most of those described in the literature, illustrating the complexity of the mechanisms underlying the protective function of the spleen in human malaria.