Diagnostic Methods for Non-Falciparum Malaria

Malaria is a serious public health problem that affects mostly the poorest countries in the world, killing more than 400,000 people per year, mainly children under 5 years old. Among the control and prevention strategies, the differential diagnosis of the Plasmodium–infecting species is an important factor for selecting a treatment and, consequently, for preventing the spread of the disease. One of the main difficulties for the detection of a specific Plasmodium sp is that most of the existing methods for malaria diagnosis focus on detecting P. falciparum. Thus, in many cases, the diagnostic methods neglect the other non-falciparum species and underestimate their prevalence and severity. Traditional methods for diagnosing malaria may present low specificity or sensitivity to non-falciparum spp. Therefore, there is high demand for new alternative methods able to differentiate Plasmodium species in a faster, cheaper and easier manner to execute. This review details the classical procedures and new perspectives of diagnostic methods for malaria non-falciparum differential detection and the possibilities of their application in different circumstances.Fil: Gimenez, Alba Marina. Universidade de Sao Paulo; Brasil. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ferreira Marqués, Rodolfo. Universidade de Sao Paulo; BrasilFil: Regiart, Daniel Matias Gaston. Universidade de Sao Paulo; Brasil. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Química de San Luis. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia. Instituto de Química de San Luis; ArgentinaFil: Bargieri, Daniel Youssef. Universidade de Sao Paulo; Brasi

TLR5-dependent immunogenicity of a recombinant fusion protein containing an immunodominant epitope of malarial circumsporozoite protein and the FliC flagellin of Salmonella Typhimurium

Recently, we described the improved immunogenicity of new malaria vaccine candidates based on the expression of fusion proteins containing immunodominant epitopes of merozoites and Salmonella enterica serovar Typhimurium flagellin (FliC) protein as an innate immune agonist. Here, we tested whether a similar strategy, based on an immunodominant B-cell epitope from malaria sporozoites, could also generate immunogenic fusion polypeptides. A recombinant His6-tagged FliC protein containing the C-terminal repeat regions of the VK210 variant of Plasmodium vivax circumsporozoite (CS) protein was constructed. This recombinant protein was successfully expressed in Escherichia coli as soluble protein and was purified by affinity to Ni-agarose beads followed by ion exchange chromatography. A monoclonal antibody specific for the CS protein of P. vivax sporozoites (VK210) was able to recognise the purified protein. C57BL/6 mice subcutaneously immunised with the recombinant fusion protein in the absence of any conventional adjuvant developed protein-specific systemic antibody responses. However, in mice genetically deficient in expression of TLR5, this immune response was extremely low. These results extend our previous observations concerning the immunogenicity of these recombinant fusion proteins and provide evidence that the main mechanism responsible for this immune activation involves interactions with TLR5, which has not previously been demonstrated for any recombinant FliC fusion protein.FAPESPCNPq - INCTVCoordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES

Violacein-Induced Chaperone System Collapse Underlies Multistage Antiplasmodial Activity

Antimalarial drugs with novel modes of action and wide therapeutic potential are needed to pave the way for malaria eradication. Violacein is a natural compound known for its biological activity against cancer cells and several pathogens, including the malaria parasite, Plasmodium falciparum (Pf). Herein, using chemical genomic profiling (CGP), we found that violacein affects protein homeostasis. Mechanistically, violacein binds Pf chaperones, PfHsp90 and PfHsp70-1, compromising the latter's ATPase and chaperone activities. Additionally, violacein-treated parasites exhibited increased protein unfolding and proteasomal degradation. The uncoupling of the parasite stress response reflects the multistage growth inhibitory effect promoted by violacein. Despite evidence of proteotoxic stress, violacein did not inhibit global protein synthesis via UPR activation - a process that is highly dependent on chaperones, in agreement with the notion of a violacein-induced proteostasis collapse. Our data highlight the importance of a functioning chaperone-proteasome system for parasite development and differentiation. Thus, a violacein-like small molecule might provide a good scaffold for development of a novel probe for examining the molecular chaperone network and/or antiplasmodial drug design.publishersversionpublishe

Role of interferon-gamma during CpG oligodeoxynucleotide-adjuvanted immunization with recombinant proteins

Synthetic oligonucleotides (ODNs) containing immunostimulatory CpG motifs (CpG) are a new class of adjuvants suitable for the development of recombinant vaccines. Here we describe that endogenous interferon (IFN) was critical for the adjuvant activity of CpG ODN as genetically deficient mice developed significantly lower IgG antibody titers following immunization with recombinant proteins. in contrast, the absence of endogenous IL-12/IL-23 or IL-4 had little impact on the magnitude of the antibody response but instead caused a dramatic change in the pattern of IgG isotypes. the dependence on IFN-gamma was specific for CpG ODN and it was not observed with other adjuvants tested. IFN-gamma was produced by NK, dendritic cells, CD4(+) and CD8(+) T cells stimulated in vitro with CpG ODN. Adoptive transfer experiments confirmed that CD4(+) or CD8(+) T cells were in fact relevant sources of IFN-gamma in vivo. Following CpG ODN injection, splenic dendritic cells from IFN-gamma deficient mice did not up-regulate CD86 or CD40 expression, suggesting a role for these molecules. the importance of CD28 (CD86 ligand) was confirmed using CD28 deficient mice which presented severely impaired immune responses following CpG ODN-assisted immunization. (c) 2007 Elsevier B.V. All rights reserved.Universidade Federal de São Paulo, Escola Paulista Med, CINTERGEN, BR-04023062 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Immunol & Parasitol, BR-04023062 São Paulo, BrazilUniv São Paulo, Inst Biomed Sci, Dept Immunol, BR-05508900 São Paulo, BrazilUniv São Paulo, Fac Pharmaceut Sci, Dept Clin Anal Toxicol & Bromatol, BR-14040903 Ribeirao Preto, SP, BrazilUniv São Paulo, Fac Ciencias Farmaceut, Dept Anal Clin & Toxicol, BR-05508900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, CINTERGEN, BR-04023062 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Immunol & Parasitol, BR-04023062 São Paulo, BrazilWeb of Scienc