Human cerebral malaria. A quantitative ultrastructural analysis of parasitized erythrocyte sequestration.

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.     

Killing of human malaria parasites by macrophage secretory products.

The susceptibility of the human malaria parasite, Plasmodium falciparum, to killing in vitro by macrophage secretory products was investigated. The effect of O2 radicals and tumor necrosis factor on parasite viability was assessed both morphologically and by following the uptake of [3H]hypoxanthine. H2O2 produced by the interaction of glucose and glucose oxidase was found to reduce viability; this effect was reversed by the addition of exogenous catalase. Further studies indicated that the catalase level within the erythrocyte was not altered upon parasite invasion. O2 radicals produced during the xanthine-xanthine oxidase interaction also killed P. falciparum. The addition of various O2 radical scavengers (including catalase) did not reverse this effect; therefore, it was not possible to determine which of the O2 radicals were involved in the killing process. Samples from three different sources containing tumor necrosis factor, a nonspecific soluble mediator derived from Mycobacterium bovis BCG-activated macrophages treated with endotoxin, also killed the parasite. There was no evidence that tumor necrosis factor or the products of the xanthine-xanthine oxidase interaction caused damage to the erythrocyte membrane that could be implicated as an important aspect of the killing process. These findings all strongly suggest that such macrophage products play an important role in immunity to malaria.     

Comparative morphology of human and animal malaria parasites

Human and animal malaria parasites (Plasmodium falciparum, P. malariae, P. vivax, P. berghei, P. gallinaceum) were studied using special fixation and standardized methods, with special attention to their effects on host cells. Morphological alterations induced by the parasites in infected erythrocytes included knobs, invaginations, and caveola-vesicle complexes on the surface of the host cell and clefts, microvesicles, and small vesicles in the cytoplasm of the infected erythrocytes. ForP. malariae, the ultrastructural study revealed invaginations with associated microvesicles, but knobs did not occur on the surface of infected erythrocytes. The development of invaginations and microvesicles inP. malariae-infected erythrocytes corresponded to the morphological alterations induced byP. vivax. A new hypothesis concerning the origin of Schüffner's dots is discussed.Abbreviations bz budding zone - c cluster - cl cleft - cv caveolavesicle complex - e erythrocyte - g gametocyte - i mvagination - k knob - m merozoite - mv microvesicle - n nucleus - p pigment - pv parasitophorous vacuole - sm small vesicle - t trophozoite Supported by Deutsche Forschungsgemeinschaft (DFG)     

Sera from patients with falciparum malaria induce substance P gene expression in cultured human brain microvascular endothelial cells.

Substance P is a pluripotent neuropeptide capable of inducing neurogenic inflammation, immunoregulation, and vasodilatation. In an effort to contribute to the understanding of the pathophysiology of cerebral malaria, we have evaluated the effects of sera obtained from patients suffering from severe or mild malaria and from a healthy donor with no previous history of exposure to malaria on the expression of the substance P gene by cultured human brain microvascular endothelial cells (HBMEC) and human umbilical-vein endothelial cells. PCR, Southern blotting, hybridization with an internal probe, and densitometry demonstrated that treatment of HBMEC with sera from patients with severe malaria caused remarkably increased expression of the substance P gene. In HBMEC, substance P was not significantly influenced by serum from a healthy donor. Substance P was expressed at almost undetectable levels in untreated HBMEC. Treatment of cultured human umbilical-vein endothelial cells with the same sera produced no signal. The influence of different sera on the expression of substance P by HBMEC suggests that substance P expression may be involved in events leading to the development of severe malaria.     

Real-time quantitative PCR for analysis of genetically mixed infections of malaria parasites: technique validation and applications

A technique that can distinguish and quantify genetically different malaria parasite clones in a mixed infection reliably and with speed and accuracy would be very useful for researchers. Many current methods of genotyping and quantification fall down on a number of aspects relating to their ease of use, sensitivity, cost, reproducibility and, not least, accuracy. Here we report the development and validation of a method that offers several advantages in terms of cost, speed and accuracy over conventional PCR or antibody-based methods. Using real-time quantitative PCR (RTQ-PCR) with allele-specific primers, we have accurately quantified the relative proportions of clones present in laboratory prepared ring-stage mixtures of two genetically distinct clones of the rodent malaria parasite Plasmodium chabaudi chabaudi. Accurate and reproducible measurement of the amount of genomic DNA representing each clone in a mixture was achieved over 100-fold range, corresponding to 0.074% parasitised erythrocytes at the lower end. To demonstrate the potential utility of this method, we include an example of the type of application it could be used for. In this case, we studied the growth rate dynamics of mixed-clone infections of P. chabaudi using an avirulent/virulent clone combination (AS (PYR) and AJ) or two clones with similar growth rate profiles (AQ and AJ). The modification of the technique described here should enable researchers to quickly extract accurate and reliable data from in-depth studies covering broad areas of interest, such as analyses of clone-specific responses to drugs, vaccines or other selection pressures in malaria or other parasite species that also contain highly polymorphic DNA sequences.     

Immunopathology of cerebral malaria: morphological evidence of parasite sequestration in murine brain microvasculature

A murine model that closely resembles human cerebral malaria is presented, in which characteristic features of parasite sequestration and inflammation in the brain are clearly demonstrable. "Young" (BALB/c x C57BL/6)F(1) mice infected with Plasmodium berghei (ANKA) developed typical neurological symptoms 7 to 8 days later and then died, although their parasitemias were below 20%. Older animals were less susceptible. Immunohistopathology and ultrastructure demonstrated that neurological symptoms were associated with sequestration of both parasitized erythrocytes and leukocytes and with clogging and rupture of vessels in both cerebral and cerebellar regions. Increases in tumor necrosis factor alpha and CD54 expression were also present. Similar phenomena were absent or substantially reduced in older infected but asymptomatic animals. These findings suggest that this murine model is suitable both for determining precise pathogenetic features of the cerebral form of the disease and for evaluating circumventive interventions.     

The sequestration hypothesis: an explanation for the sensitivity of malaria parasites to nitric oxide-mediated immune effector function in vivo

While the effect of reactive nitrogen intermediates (RNI) against macrophage-dwelling protozoa, such as Leishmania and Toxoplasma has become established, the possible antiparasitic function of nitric oxide (NO) and RNI against the intracellular blood stages of malaria and babesia has, until recently, been less well accepted. This was, at least in part, due to the long-standing notion that haemoglobin (Hb) universally scavenges NO and thus that erythrocytes act as a permanent sink for this molecule. It is now known that NO can be released as well as scavenged by Hb, and that the less oxygenated the blood the lower the affinity of Hb for NO. As a consequence, NO is preferentially released by venous erythrocytes. Based on the increased sensitivity of Plasmodium falciparum -infected erythrocytes to RNI in venous blood that was recently demonstrated, it is proposed that the noted greater susceptibility of mature intra-erythrocytic forms of malaria, late-stage schizonts, is coincidental with their peripheral blood withdrawal by sequestration to deep-tissue capillaries. This environment is non NO-scavenging in nature and one which would bring schizonts and macrophages into intimate proximity, providing diffusion distances sufficiently short for RNI to be effective. Given its short half-life, this hypothesis explains the potential for NO to be toxic for malaria parasites in vivo, and suggests that sequestration, a mechanism adopted by the parasite to supposedly avoid immune surveillance, may in fact have a partially counteractive effect.     

Identification of four species of human malaria parasites by fast PCR

Malaria is one of the most prevalent infectious diseases in the world. Accurate identification of four species of human malaria parasite is essential for appropriate treatment. Here, we developed a simple and rapid method of identifying Plasmodium species using a fast polymerase chain reaction (PCR) assay. Based on the previous literature, we amplified small subunit ribosomal RNA genes of four human malaria parasites. To establish a minimum detection limit, a blood sample with a known number of P. falciparum parasites (parasitemia: 3%) was diluted serially(from 0.03% to 0.000003%). We compared the detection limits between single (one-step) PCR and nested (two-step) PCR. Other clinical blood samples, which were infected with P. falciparum (parasitemia: 2.8%), P. vivax (parasitemia: 0.13%), P. ovale (parasitemia: 0.04%), respectively, were also tested by our PCR system. The PCR findings were compared to those of blood film Giemsa staining and rapid diagnostic tests (RDT). The sensitivity of our method is less than one parasite in 1 microl of blood(estimated parasitemia: 0.000003%) for both single PCR and nested PCR, though an increased number of cycles (40 cycles) was required for single PCR. Using clinical samples, it was proven that amplified products by single PCR could clearly distinguish between P. falciparum, P. vivax, and P. ovale. To detect P. vivax and P. ovale, the PCR system was more sensitive than RDT. The total required time for our method was within three to four hours from DNA extraction to PCR detection. Taken together, our method is easier and faster than the previously reported PCR-based malaria parasite identification systems, and is also useful for cases in which diagnosis by Giemsa staining and RDT is difficult.     

A quantitative analysis of the crossed septohippocampal projection in the rat brain

Summary The projection from the medial septal-diagonal band complex (MSDB) to the hippocampal formation is generally considered to be primarily an uncrossed projection. The present study examined the number of MSDB neurons which project to the contralateral hippocampus, the position of these cells, their identity as either cholinergic or GABAergic, and the question of bilateral collateral projections. The fluorescent dyes Fluoro-Gold or diamidino yellow were injected into 5 equally-spaced sites along the septotemporal extent of the right hippocampus of adult female Sprague-Dawley rats. In some animals True Blue (5%) was also injected in a similar fashion into the left hippocampus. Five days later the brains were processed for fluorescence microscopic examination. The number of retrogradely labeled cells on each side was counted in every fifth section (200 m interval) through the MSDB and the percentage of contralaterally projecting neurons was calculated. Averaging across all sections and all brains, it was found that approximately 15% of the medial septal neurons and 17% of the diagonal band neurons project to the contralateral hippocampus. Most of the labeled neurons, both ipsilateral and contralateral, were present in the caudal half of the MSDB. Within the medial septum contralaterally placed cells were distributed uniformly throughout the mediolateral extent and some of these stained immunocytochemically for choline acetyltransferase or glutamic acid decarboxylase. In brains in which diamidino yellow was injected into the right hippocampus and True Blue into the left numerous neurons were observed to contain both dyes, thus indicating projections to both hippocampi.Abbreviations DY diamidino yellow - FG Fluoro-Gold - MSDB medial septum-diagonal band complex - TB True Blue     

Synthetic microvascular networks for quantitative analysis of particle adhesion

We have developed a methodology to study particle adhesion in the microvascular environment using microfluidic, image-derived microvascular networks on a chip accompanied by Computational Fluid Dynamics (CFD) analysis of fluid flow and particle adhesion. Microfluidic networks, obtained from digitization of in vivo microvascular topology were prototyped using soft-lithography techniques to obtain semicircular cross sectional microvascular networks in polydimethylsiloxane (PDMS). Dye perfusion studies indicated the presence of well-perfused as well as stagnant regions in a given network. Furthermore, microparticle adhesion to antibody coated networks was found to be spatially non-uniform as well. These findings were broadly corroborated in the CFD analyses. Detailed information on shear rates and particle fluxes in the entire network, obtained from the CFD models, were used to show global adhesion trends to be qualitatively consistent with current knowledge obtained using flow chambers. However, in comparison with a flow chamber, this method represents and incorporates elements of size and complex morphology of the microvasculature. Particle adhesion was found to be significantly localized near the bifurcations in comparison with the straight sections over the entire network, an effect not observable with flow chambers. In addition, the microvascular network chips are resource effective by providing data on particle adhesion over physiologically relevant shear range from even a single experiment. The microfluidic microvascular networks developed in this study can be readily used to gain fundamental insights into the processes leading to particle adhesion in the microvasculature.     

Methodology and application of flow cytometry for investigation of human malaria parasites

Historically, examinations of the inhibition of malaria parasite growth/invasion, whether using drugs or antibodies, have relied on the use of microscopy or radioactive hypoxanthine uptake. These are considered gold standards for measuring the effectiveness of antimalarial treatments, however, these methods have well known shortcomings. With the advent of flow cytometry coupled with the use of fluorescent DNA stains allowed for increased speed, reproducibility, and qualitative estimates of the effectiveness of antibodies and drugs to limit malaria parasite growth which addresses the challenges of traditional techniques. Because materials and machines available to research facilities are so varied, different methods have been developed to investigate malaria parasites by flow cytometry. This review is intended to serve as a reference guide for advanced users and importantly, as a primer for new users, to support expanded use and improvements to malaria flow cytometry, particularly in endemic countries.     

Acanthamoeba interactions with human brain microvascular endothelial cells

Acanthamoeba are opportunistic protozoan parasites that can cause fatal granulomatous amoebic encephalitis, however, the pathogenic mechanisms associated with this disease remain unclear. One of the primary factors in Acanthamoeba encephalitis is the haematogenous spread, followed by invasion of the blood-brain barrier resulting in the transmigration of Acanthamoeba into the central nervous system. In this study, we have used human brain microvascular endothelial cells, which constitute the blood-brain barrier and studied their interactions with Acanthamoeba. Using in vitro cultures, we showed that Acanthamoeba isolates belonging to genotypes T3, T4 and T11, exhibited increased cytotoxicity on human brain microvascular endothelial cells as well as exhibited higher binding and were considered potential pathogens. In contrast, Acanthamoeba isolates belonging to genotypes T2 and T7 exhibited minimal cytotoxicity and significantly less binding to human brain microvascular endothelial cells (P< 0.01). Furthermore, exogenous alpha-mannose inhibited binding but increased cytotoxicity of human brain microvascular endothelial cells. This is the first demonstration of Acanthamoeba interactions with primary human brain microvascular endothelial cells.     

Peptide-based subunit vaccines against pre-erythrocytic stages of malaria parasites.

Malaria currently ranks among the most prevalent infections in tropical and sub-tropical areas throughout the world with relatively high morbidity and mortality particularly in young children. The widespread occurrence and the increased incidence of malaria in many countries, caused by drug-resistant parasites (Plasmodium falciparum and P. vivax) and insecticide-resistant vectors (Anopheles mosquitoes), indicate the need to develop new methods of controlling this disease. Experimental vaccination with radiation-attenuated sporozoites can protect animals and humans against the disease, demonstrating the feasibility of developing an effective malaria vaccine. However, vaccines based on radiation-attenuated sporozoites are not feasible for large scale application due to lack of in vitro culture system. Therefore, the development of peptide-based subunit vaccines has been undertaken as an alternative approach. Synthetic peptides containing defined B- and T-cell epitopes of different antigens expressed in sporozoites and/or liver stages have been used as subunit vaccines in experimental animal models. They have been shown to be highly immunogenic and capable of inducing protective immunity mediated by antibodies, as well as CD4+ and CD8+ T-cells.     

Phospholipases during membrane dynamics in malaria parasites

Plasmodium parasites, the causative agents of malaria, display a well-regulated lipid metabolism required to ensure their survival in the human host as well as in the mosquito vector. The fine-tuning of lipid metabolic pathways is particularly important for the parasites during the rapid erythrocytic infection cycles, and thus enzymes involved in lipid metabolic processes represent prime targets for malaria chemotherapeutics. While plasmodial enzymes involved in lipid synthesis and acquisition have been studied in the past, to date not much is known about the roles of phospholipases for proliferation and transmission of the malaria parasite. These phospholipid-hydrolyzing esterases are crucial for membrane dynamics during host cell infection and egress by the parasite as well as for replication and cell signaling, and thus they are considered important virulence factors. In this review, we provide a comprehensive bioinformatic analysis of plasmodial phospholipases identified to date. We further summarize previous findings on the lipid metabolism of Plasmodium, highlight the roles of phospholipases during parasite life-cycle progression, and discuss the plasmodial phospholipases as potential targets for malaria therapy.     

Adenosine A1 receptors in the human brain: a quantitative autoradiographic study

The distribution of adenosine A1 receptors in the human brain was studied by autoradiography in post mortem brain tissues from 26 subjects without reported neurological disease. N6-[3H]Cyclohexyl-adenosine was used as the ligand. For comparison, adjacent sections of some regions were examined histochemically for 5'-nucleotidase activity. The receptor sites were heterogeneously distributed throughout the CNS. The highest receptor densities were found in the stratum oriens, pyramidale and radiatum of the hippocampus. High densities were also found in the cerebral cortex and the striatum. In the thalamus there was a heterogeneous distribution of binding sites with a high density in structures such as the medial and anterior nucleus. Intermediate receptor densities were found in the accumbens, the olfactory tubercle and most parts of the amygdala among others. The hypothalamus had low receptor densities. In the brainstem and the spinal cord very low receptor concentrations were found. However, in some structures such as the substantia nigra, the colliculus superior and the substantia gelatinosa of the spinal cord a low level of binding could be measured. The cerebellar cortex showed low densities of receptors. Structures showing high levels of 5'-nucleotidase activity were the hippocampus, the striatum and parts of the cerebral cortex among other regions. In general there was a poor correlation between the localization of A1 receptors and the 5'-nucleotidase activity. Some regions, however, showed a similar distribution of these two markers. In general, the distribution of adenosine A1 receptors found in the human brain is comparable to that found in previous autoradiographic studies in the rat brain. However, some regional differences were observed in, for example, the cerebral cortex, the striatum and the cerebellar cortex. These differences may prove to be functionally relevant.     

Differential reactivity of brain microvascular endothelial cells to TNF reflects the genetic susceptibility to cerebral malaria

Upon infection with Plasmodium berghei ANKA (PbA), various inbred strains of mice exhibit different susceptibility to the development of cerebral malaria (CM). Tumor necrosis factor-α (TNF) and interferon-γ (IFN-γ) have been shown to be crucial mediators in the pathogenesis of this neurovascular complication. Brain microvascular endothelial cells (MVEC) represent an important target of both cytokines. In the present study, we show that brain MVEC purified from CM-susceptible (CM-S) CBA / J mice and CM-resistant (CM-R) BALB / c mice exhibit a different sensitivity to TNF. CBA / J brain MVEC displayed a higher capacity to produce IL-6 and to up-regulate intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in response to TNF than BALB / c brain MVEC. In contrast, no difference was found in the induction of E-selectin after TNF challenge. CM-S brain MVEC were also significantly more sensitive to TNF-induced lysis. This differential reactivity to TNF was further substantiated by comparing TNF receptor expression on CM-S and CM-R brain MVEC. Although the constitutive expression of TNF receptors was comparable on cells from the two origins, TNF induced an up-regulation of both p55 and p75 TNF receptors in CM-S, but not in CM-R brain MVEC. A similar regulation was found at the level of TNF receptor mRNA, but not for receptor shedding. Although a protein kinase C inhibitor blocked the response to TNF in both the brain MVEC, an inhibitor of protein kinase A selectively abolished the response to TNF in CM-R, but not CM-S brain MVEC, suggesting a differential protein kinase involvement in TNF-induced activation of CM-S and CM-R brain MVEC. These results indicate that brain MVEC purified from CM-S and CM-R mice exhibit distinctive sensitivity to TNF. This difference may be partly due to a differential regulation of TNF receptors and via distinct protein kinase pathways.     

Prognostic utility of quantitative image analysis of microvascular density in prostate cancer

The walls of angiogenic blood vessel capillaries are composed of two principal cell types, blood vessel endothelial cells (BEC) and pericytes (PC), whereas the walls of lymphatic capillaries are composed of lymphatic endothelial cells (LEC). In this investigation we describe a practical application of NIH ImageJ software for quantitative image analysis for pericytes and endothelial cells in prostate cancer. We used a tissue microarray that contained 49 tissue cores (normal prostate tissue or prostatic carcinomas with Gleason scores of 6 through 10). These prostate cancer samples represented AJCC prognostic stages II, III, and IV. Slides were immunostained with anti‐PDGFR‐β antibody for identification of PC, and quantified as microvascular pericyte density (MVPD); they were also immunostained with anti‐CD34 antibody for identification of LEC and BEC simultaneously, and quantified as microvascular endothelial density (MVED). CD31 and D2‐40 immunostains were used to quantify BEC and lymphatic endothelial cells, respectively. Our results showed higher MVPD and MVED in prostate cancers with higher Gleason scores and higher stages, suggesting the prognostic utility of vascular image analysis in prostate pathology. This investigation demonstrates the feasibility, versatility, and ease of use of ImageJ software and pericyte‐specific and endothelial‐specific immunohistochemistry for quantitative image analysis in prostate pathology.