Var transcription profiling of Plasmodium falciparum 3D7: assignment of cytoadherent phenotypes to dominant transcripts

Background\ud Cytoadherence of Plasmodium falciparum -infected red blood cells is mediated by var gene-encoded P. falciparum erythrocyte membrane protein-1 and host receptor preference depends in most cases on which of the 50–60 var genes per genome is expressed. Enrichment of phenotypically homogenous parasites by panning on receptor expressing cells is fundamental for the identification of the corresponding var transcript.\ud \ud \ud Methods\ud P. falciparum 3D7 parasites were panned on several transfected CHO-cell lines and their var transcripts analysed by i) reverse transcription/PCR/cloning/sequencing using a universal DBLα specific oligonucleotide pair and ii) by reverse transcription followed by quantitative PCR using 57 different oligonucleotide pairs.\ud \ud \ud Results\ud Each cytoadherence selected parasite line also adhered to untransfected CHO-745 cells and upregulation of the var gene PFD995/PFD1000c was consistently associated with cytoadherence to all but one CHO cell line. In addition, parasites panned on different CHO cell lines revealed candidate var genes which reproducibly associated to the respective cytoadherent phenotype. The transcription profile obtained by RT-PCR/cloning/sequencing differed significantly from that of RT-quantitative PCR.\ud \ud \ud Conclusion\ud Transfected CHO cell lines are of limited use for the creation of monophenotypic cytoadherent parasite lines. Nevertheless, 3D7 parasites can be reproducibly selected for the transcription of different determined var genes without genetic manipulation. Most importantly, var transcription analysis by RT-PCR/cloning/sequencing may lead to erroneous interpretation of var transcription profiles.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) [06/51873-0]CNP

Preliminary studies of biosynthesis of heme O and heme A in intraerythrocytic stages of P. falciparum

Intermediates of the isoprenoid metabolism can bind to heme groups. Heme is critical and its derivatives, heme O and heme A, are the main compounds of aerobic respiration. They are synthesized by farnesylation of heme groups by ?heme O synthase? (HOS or Cox10) and ?heme A synthase? (HAS or Cox15). The identification of heme O and heme A and characterization of enzymes involved in their synthesis are important for exploring the possible products biosynthesized by the parasite derived from the MEP pathway. Importantly, hemes A and O are essential molecules for numerous living organisms, since they are critical components of the mitochondrial electron transport chain, catalyzing the reduction of O2 to H2O. Additionally, the heme group is an important target of drugs such as artemisinin and quinolines.Fil: Simao Gurge, Raquel. Universidade de Sao Paulo; BrasilFil: Cricco, Julia Alejandra. Universidad Nacional de Rosario; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Wunderlich, Gerhard. Universidade de Sao Paulo; BrasilFil: Kimura, Emilia. Universidade de Sao Paulo; BrasilFil: Cirulli, Brenda Analía. Universidad Nacional de Rosario; ArgentinaFil: Katzin, Alejandro. Universidade de Sao Paulo; Brasi

Diversidade antigênica e evasão imune nos parasitos da malária

Clinical immunity against blood stage malaria is achieved only after multiple infections with the same species. A major reason for this is the extense diversity of antigens located at the parasites surface or even at the surface of the infected red blood cell. There are two sources for antigenic diversity: first, there is allelic polymorphism with the existence of different and genetically stable versions of antigen encoding genes, generated through mutations and recombination. The second source is antigenic variation, a mechanism by which different antigens are expressed successively without change of the underlying genotype (Ferreira and col., 2007). Herein, we discuss the mechanisms and origins of diversity and antigenic variationUma das principais razões do porquê a imunidade clínica contra a malária se desenvolve somente após diversas infecções pela mesma espécie de parasito se deve à extensa diversidade dos antígenos de superfície dos plasmódios e de hemácias infectadas. Existem duas origens para tal diversidade antigênica: uma é o polimorfismo alélico, com a existência de formas alternativas e estáveis de genes que codificam antígenos, gerados através de mutações e recombinações; outra é devido à variação antigênica, mecanismo pelo qual uma linhagem clonal de parasitos expressa sucessivamente formas alternativas de um antígeno sem alterações de genótipo (Ferreira e col., 2007). Aqui, os mecanismos e origens da diversidade e variação antigênica são discutido

Rápida mudança de transcritos var e de génotipos de Plasmodium falciparum em infecções assintomáticas naturalmente adquiridas

Os genes var de Plasmodium falciparum codificam as proteínas variantes da superfície do eritrócito infectado (PfEMP1). Neste estudo examinamos a mudança de transcritos destes genes var em duas infecções assintomáticas durante um curto prazo e estimamos simultaneamente o número de genomas circulantes nas mesmas amostras por análise de microssatélites. Nas duas infecções observamos uma rápida mudança de genótipos e transcritos de genes var. A mudança acelerada do repertório de transcritos possivelmente foi causada pela rápida eliminação de parasitas circulantes transcrevendo genes var a partir de genomas iguais ou diferentes, ou pela mudança acelerada da própria transcrição (switching) de genes var.The var genes of Plasmodium falciparum code for the antigenically variant erythrocyte membrane proteins 1 (PfEMP1), a major factor for cytoadherence and immune escape of the parasite. Herein, we analyzed the var gene transcript turnover in two ongoing, non-symptomatic infections at sequential time points during two weeks. The number of different circulating genomes was estimated by microsatellite analyses. In both infections, we observed a rapid turnover of plasmodial genotypes and var transcripts. The rapidly changing repertoire of var transcripts could have been caused either by swift elimination of circulating var-transcribing parasites stemming from different or identical genetic backgrounds, or by accelerated switching of var gene transcription itself

Natural antibody response to Plasmodium falciparum merozoite antigens MSP5, MSP9 and EBA175 is associated to clinical protection in the Brazilian Amazon

BACKGROUND: Antibodies have an essential role in the acquired immune response against blood stage P. falciparum infection. Although several antigens have been identified as important antibody targets, it is still elusive which antigens have to be recognized for clinical protection. Herein, we analyzed antibodies from plasmas from symptomatic or asymptomatic individuals living in the same geographic area in the Western Amazon, measuring their recognition of multiple merozoite antigens. METHODS: Specific fragments of genes encoding merozoite proteins AMA1 and members of MSP and EBL families from circulating P. falciparum field isolates present in asymptomatic and symptomatic patients were amplified by PCR. After cloning and expression of different versions of the antigens as recombinant GST-fusion peptides, we tested the reactivity of patients’ plasmas by ELISA and the presence of IgG subclasses in the most reactive plasmas. RESULTS: 11 out of 24 recombinant antigens were recognized by plasmas from either symptomatic or asymptomatic infections. Antibodies to MSP9 (X(2)(DF=1) = 9.26/p = 0.0047) and MSP5 (X(2)(DF=1) = 8.29/p = 0.0069) were more prevalent in asymptomatic individuals whereas the opposite was observed for MSP1 block 2-MAD20 (X(2)(DF=1) = 6.41/p = 0.0206, Fisher’s exact test). Plasmas from asymptomatic individuals reacted more intensely against MSP4 (U = 210.5, p < 0.03), MSP5 (U = 212, p < 0.004), MSP9 (U = 189.5, p < 0.002) and EBA175 (U = 197, p < 0.014, Mann-Whitney’s U test). IgG1 and IgG3 were predominant for all antigens, but some patients also presented with IgG2 and IgG4. The recognition of MSP5 (OR = 0.112, IC(95%) = 0.021-0.585) and MSP9 (OR = 0.125, IC(95%) = 0.030-0.529, cross tab analysis) predicted 8.9 and 8 times less chances, respectively, to present symptoms. Higher antibody levels against MSP5 and EBA175 were associated by odds ratios of 9.4 (IC(95%) = 1.29-69.25) and 5.7 (IC(95%) = 1.12-29.62, logistic regression), respectively, with an asymptomatic status. CONCLUSIONS: Merozoite antigens were targets of cytophilic antibodies and antibodies against MSP5, MSP9 and EBA175 were independently associated with decreased symptoms

A large aperture reflective wave-plate for high-intensity short-pulse laser experiments

We report on a reflective wave-plate system utilizing phase-shifting mirrors (PSM) for a continuous variation of elliptical polarization without changing the beam position and direction. The scalability of multilayer optics to large apertures and the suitability for high-intensity broad-bandwidth laser beams make reflective wave-plates an ideal tool for experiments on relativistic laser-plasma interaction. Our measurements confirm the preservation of the pulse duration and spectrum when a 30-fs Ti:Sapphire laser beam passes the system

Nucleosomes in serum of patients with early cerebral stroke

Background: Nucleosomes are cell death products that are elevated in serum of patients with diseases that are associated with massive cell destruction. We investigated the kinetics of circulating nucleosomes after cerebral stroke and their correlation with the clinical status. Methods: In total, we analyzed nucleosomes by ELISA in sera of 63 patients with early stroke daily during the first week after onset. For correlation with the clinical pathology, patients were grouped into those with medium to slight functional impairment (Barthel Index BI >= 50) and those with severe functional impairment (BI = 50 showed a continuous increase in nucleosomes until day 5 (median: 523 arbitrary units, AU) followed by a slow decline. In contrast, patients with BI = 50 (497 AU; p = 0.031). Concerning the infarction volume, nucleosomes showed significant correlations for the concentrations on day 3 (r = 0.43; p = 0.001) and for the area under the curve (r = 0.34; p = 0.016). Conclusion: Even if nucleosomes are nonspecific cell death markers, their release into serum after cerebral stroke correlates with the gross functional status as well as with the infarction volume and can be considered as biochemical correlative to the severity of stroke. Copyright (c) 2006 S. Karger AG, Basel

Violacein Extracted from Chromobacterium violaceum Inhibits Plasmodium Growth in Vitro and in Vivo

Violacein is a violet pigment extracted from the gram-negative bacterium Chromobacterium violaceum. It presents bactericidal, tumoricidal, trypanocidal, and antileishmanial activities. We show that micromolar concentrations efficiently killed chloroquine-sensitive and -resistant Plasmodium falciparum strains in vitro; inhibited parasitemia in vivo, even after parasite establishment; and protected Plasmodium chabaudi chabaudi-infected mice from a lethal challenge.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Univ Estadual Campinas, UNICAMP, Dept Parasitol, Inst Biol, BR-13083970 Campinas, SP, BrazilUniv Estadual Campinas, UNICAMP, Dept Microbiol & Imunol, Inst Biol, BR-13083970 Campinas, SP, BrazilUniversidade Federal de São Paulo, UNIFESP, Dept Bioquim, BR-04044020 São Paulo, BrazilCEPEM, IPEPATRO, BR-78900970 Porto Velho, RO, BrazilUniv São Paulo, Dept Parasitol, ICB2, São Paulo, BrazilUniv Estadual Campinas, Dept Fisiol & Biofis, Inst Biol, BR-13083970 Campinas, SP, BrazilUniv Estadual Campinas, Lab Quim Biol, Inst Quim, BR-13083970 Campinas, SP, BrazilUniversidade Federal de São Paulo, UNIFESP, Dept Bioquim, BR-04044020 São Paulo, BrazilFAPESP: 2004/00638-6CNPq: 470587/2006-7Web of Scienc