RESUMO
Primaquine is the mainstream antimalarial drug to prevent Plasmodium vivax relapses. However, this drug can induce hemolysis in patients with glucose-6-phosphate dehydrogenase deficiency. Nanostructure formulations of primaquine loaded with D-galactose were used as a strategy to target the drug to the liver and decrease the hemolytic risks. Nanoemulsion (NE-Pq) and nanochitosan (NQ-Pq) formulations of primaquine diphosphate containing D-galactose were prepared and characterized by their physicochemistry properties. Pharmacokinetic and biodistribution studies were conducted using Swiss Webster mice. A single dose of 10 mg/kg of each nanoformulation or free primaquine solution was administered by gavage to the animals, which were killed at 0.5, 1, 2, 4, 8, and 24 hours. Blood samples and tissues were collected, processed, and analyzed by high-performance liquid chromatography. The nanoformulation showed sizes around 200 nm (NE-Pq) and 400 nm (NQ-Pq) and physicochemical stability for over 30 days. Free primaquine solution achieved higher primaquine Cmax in the liver than NE-Pq or NQ-Pq at 0.5 hours. However, the half-life and mean residence time (MRT) of primaquine in the liver were three times higher with the NQ-Pq formulation than with free primaquine, and the volume distribution was four times higher. Conversely, primaquine's half-life, MRT, and volume distribution in the plasma were lower for NQ-Pq than for free primaquine. NE-Pq, on the other hand, accumulated more in the lungs but not in the liver. Galactose-coated primaquine nanochitosan formulation showed increased drug targeting to the liver compared to free primaquine and may represent a promising strategy for a more efficient and safer radical cure for vivax malaria.
Assuntos
Antimaláricos , Quitosana , Galactose , Fígado , Primaquina , Primaquina/farmacocinética , Primaquina/química , Animais , Camundongos , Fígado/metabolismo , Fígado/efeitos dos fármacos , Galactose/química , Quitosana/química , Antimaláricos/farmacocinética , Nanopartículas/química , Distribuição Tecidual , Nanoestruturas/química , MasculinoRESUMO
Aotus and Saimiri are non-human primate models recommended by the World Health Organization for experimental studies in malaria, especially for vaccine pre-clinical trials. However, research using these primates is hindered by the lack of specific reagents to evaluate immune responses to infection or vaccination. As a step toward developing molecular tools for cytokine expression studies in these species, primer pairs for 18 cytokine gene fragments were designed based on human DNA sequences and used to amplify the corresponding genes in Aotus infulatus and Saimiri sciureus genomic DNA samples. IFNγ, TNFα, LTA, IL2, IL3, IL4, IL5, IL6, IL10, IL12, IL13, CSF2 and TGFß2 gene fragments were amplified and sequenced. Primer pairs for IL8, IL17, IL18, IL27 and MIF failed to generate amplification products. When compared to the available corresponding human and non-human primate sequences, most--except IL3 and IL4--showed identity degrees above 90%. Small variations in sequence can help to explain the failure to amplify certain genes or the amplification only at lower annealing temperatures as compared to human DNA samples for several primer pairs. The sequences made available provide the basis for designing molecular tools such as primers for real time PCR specific for A. infulatus and/or S. sciureus. The nucleotide sequences reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned accession numbers DQ985386 to DQ985389, DQ989356 to DQ989369, FJ89020 to FJ89024, and FJ89029.
Assuntos
Citocinas/genética , Modelos Animais de Doenças , Malária/genética , Análise de Sequência de DNA , Animais , Aotidae , Sequência de Bases , Primers do DNA , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , SaimiriRESUMO
Cassava is one of the most important tropical food crops for more than 600 million people worldwide. Transgenic technologies can be useful for increasing its nutritional value and its resistance to viral diseases and insect pests. However, tissue-specific promoters that guarantee correct expression of transgenes would be necessary. We used inverse polymerase chain reaction to isolate a promoter sequence of the Mec1 gene coding for Pt2L4, a glutamic acid-rich protein differentially expressed in cassava storage roots. In silico analysis revealed putative cis-acting regulatory elements within this promoter sequence, including root-specific elements that may be required for its expression in vascular tissues. Transient expression experiments showed that the Mec1 promoter is functional, since this sequence was able to drive GUS expression in bean embryonic axes. Results from our computational analysis can serve as a guide for functional experiments to identify regions with tissue-specific Mec1 promoter activity. The DNA sequence that we identified is a new promoter that could be a candidate for genetic engineering of cassava roots.
Assuntos
Genes de Plantas , Manihot/genética , Proteínas de Plantas/genética , Raízes de Plantas/metabolismo , Regiões Promotoras Genéticas , Sequência de Bases , Sítios de Ligação , DNA Complementar/metabolismo , DNA de Plantas/metabolismo , Ácido Glutâmico/análise , Manihot/metabolismo , Dados de Sequência Molecular , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
The immunogenicity and protective efficacy of various antigen-adjuvant formulations derived either from the merozoite-surface protein-3 (MSP-3) or the glutamate-rich protein (GLURP) of Plasmodium falciparum were evaluated in Saimiri sciureus monkeys. These proteins were selected for immunogenicity studies based primarily on their capacity of inducing an antibody-dependent cellular inhibition effect on parasite growth. Some of the S. sciureus monkeys immunized with MSP-3(212-380)-AS02 or GLURP(27-500)-alum were able to fully or partially control parasitaemia upon an experimental P. falciparum [Falciparum Uganda Palo Alto (FUP-SP) strain] blood-stage infection, and this protection was related to the prechallenge antibody titres induced. The data are indicative that MSP-3 and GLURP can induce protective immunity against an experimental P. falciparum infection using adjuvants that are acceptable for human use and this should trigger further studies with those new antigens.
Assuntos
Anticorpos/sangue , Antígenos de Protozoários/farmacologia , Vacinas Antimaláricas/farmacologia , Malária Falciparum/prevenção & controle , Plasmodium falciparum/imunologia , Proteínas de Protozoários/farmacologia , Animais , Anticorpos/imunologia , Antígenos de Protozoários/administração & dosagem , Antígenos de Protozoários/imunologia , Imunofluorescência , Memória Imunológica/efeitos dos fármacos , Memória Imunológica/imunologia , Vacinas Antimaláricas/administração & dosagem , Vacinas Antimaláricas/imunologia , Peptídeos/administração & dosagem , Peptídeos/imunologia , Peptídeos/farmacologia , Proteínas de Protozoários/administração & dosagem , Proteínas de Protozoários/imunologia , SaimiriRESUMO
More than 30 years after the first report of successful vaccination against malaria using radiation-attenuated sporozoites, an effective malaria vaccine is not yet available. However, field and experimental data indicate that it can be developed. An astonishing amount of data has accumulated concerning parasite biology, host-parasite interactions, immunity and escape mechanisms, targets and modulators of immune responses. Nevertheless, so far this knowledge has not been enough to make us understand how to properly manipulate the whole system to build an effective vaccine. In this article, we describe candidate antigens, mechanisms, targets and trials performed with potential malaria vaccines and discuss the approaches, in vivo and in vitro models, constraints and how technologies such as DNA vaccination, genomics/proteomics and reverse immunogenetics are providing exciting results and opening new doors to make malaria vaccine a reality.
Assuntos
Vacinas Antimaláricas , Adulto , Animais , Anopheles/parasitologia , Antígenos de Protozoários/imunologia , Cebidae , Criança , Desenho de Fármacos , Eritrócitos/parasitologia , Feminino , Previsões , Interações Hospedeiro-Parasita , Humanos , Insetos Vetores/parasitologia , Estágios do Ciclo de Vida , Fígado/parasitologia , Malária/epidemiologia , Malária/imunologia , Malária/prevenção & controle , Vacinas Antimaláricas/imunologia , Masculino , Camundongos , Plasmodium/genética , Plasmodium/imunologia , Plasmodium/fisiologia , Vacinas de DNA , Vacinas SintéticasRESUMO
The protein population of cassava root layers was characterized by SDS-PAGE and bidimensional polyacrylamide gel electrophoresis. SDS-Page revealed the presence of a protein population in the molecular weight range between 94 and 20 kDa. The expression pattern of these proteins was well-defined within the different layers. Partial protein sequence analyses and preliminary results on the layer-specific expression pattern obtained with Northern analyses are presented.
Assuntos
Manihot/genética , Proteínas de Plantas/genética , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , DNA de Plantas/química , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Amplificação de Genes , Manihot/química , Dados de Sequência Molecular , Peso Molecular , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Raízes de Plantas/química , Raízes de Plantas/genética , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase , Prolaminas , RNA de Plantas/análise , Análise de Sequência de DNARESUMO
Plasmodium berghei ANKA infection in CBA/J mice leads to the development of cerebral malaria (CM) that kills 80-90% of the animals in 6-9 days. This model has been used to study the pathogenesis of CM, which is a major cause of morbidity and mortality in Plasmodium falciparum-infected individuals. The role of cytokines in the induction of CM in the murine model has been well documented, but most studies have been restricted to the peak of neurological manifestations. Here we used a sequential approach to compare mice that developed CM with those that developed no cerebral pathology. Animals were examined for systemic histopathological changes and plasma Tumor Necrosis Factor-alpha (TNF) levels. The objectives were (a) to further determine the importance of factors commonly associated with murine CM-such as elevated levels of TNF and the presence of hemorrhage and vascular plugging-by comparing mice at different stages of infection and/or with different outcomes following infection and (b) to examine the importance of systemic changes-course of parasitemia and histopathological alterations in brain, liver, and lungs-in the development of CM. The data suggest that (a) the clinical manifestation of CM appears to be associated with a wave of merozoite release on days 6-7, (b) murine CM does not present reliable histopathological indicators, (c) there is no topographic association between the occurrence of intravascular plugging and the hemorrhagic foci, (d) monocyte-monocyte and monocyte-endothelial cell adherence were the most expressive histopathological events and were not restricted to brain vessels, (e) blood levels of TNF are not indicative of the local tissue reaction, (f) adhesiveness of monocyte/endothelial cells fluctuate during infection and is dissociated from the lymphocyte homing to the liver, and (g) pulmonary megakaryocytosis (megakaryopoiesis?) is a late event in the lungs.
Assuntos
Malária Cerebral/imunologia , Plasmodium berghei , Fator de Necrose Tumoral alfa/análise , Animais , Encéfalo/patologia , Modelos Animais de Doenças , Feminino , Fígado/patologia , Pulmão/patologia , Malária Cerebral/patologia , Camundongos , Camundongos Endogâmicos CBARESUMO
Aotus is one of the WHO-recommended primate models for studies in malaria, and several species can be infected with Plasmodium falciparum or P. vivax. Here we describe the successful infection of the species A. infulatus from eastern Amazon with blood stages of P. falciparum. Both intact and splenectomized animals were susceptible to infection; the intact ones were able to keep parasitemias at lower levels for several days, but developed complications such as severe anemia; splenectomized monkeys developed higher parasitemias but no major complications. We conclude that A. infulatus is susceptible to P. falciparum infection and may represent an alternative model for studies in malaria.