Estudio conformacional de p��ptidos modificados de las prote��nas STARP, CelTOS, TRSP y SERA 5 de Plasmodium falciparum y su relaci��n con una respuesta inmune en malaria

El desarrollo de una vacuna contra malaria es un ��rea de exploraci��n activa pero con enormes retos debido especialmente a la complejidad del ciclo del par��sito. As��, es necesario bloquear las diferentes etapas de la invasi��n que tiene el Plasmodium falciparum y extraer de ellas la mayor informaci��n posible de la artiller��a que utiliza para su ataque. Para esto, p��ptidos de las prote��nas STARP, CelTOS y TRSP (del esporozoito) y SERA 5 (del merozoito) que tienen alta afinidad de uni��n a c��lulas HepG2 y a gl��bulos rojos respectivamente (conocidos como cHABPs), han sido modificados (conocidos como mHABPs), sintetizados y evaluados a nivel de respuesta inmune en monos Aotus as�� como estudiados en su conformaci��n estructural por RMN de 1H. Los resultados muestran que los p��ptidos nativos no son inmunog��nicos, pero pueden inducir altos t��tulos de anticuerpos cuando sus residuos cr��ticos o sus vecinos son reemplazados por otro con un volumen y masa similar, pero diferente polaridad. El estudio conformacional pone de manifiesto que las estructuras de los p��ptidos nativos son diferentes de sus p��ptidos modificados ya sea que muestren regiones estructuradas m��s cortas o m��s largas o que no presenten ninguna, en comparaci��n con sus an��logos modificados altamente inmunog��nicos. Las caracter��sticas estereoqu��micas particulares en las cadenas laterales de algunos residuos de amino��cidos de estos p��ptidos modificados as�� como los rasgos fisicoqu��micos parecen jugar un rol importante en la respuesta inmune apropiada cuando estos fueron inmunizados en grupos de monos Aotus confiriendo un avance al dise��o de una vacuna contra malaria totalmente eficaz.

Revisi��n sistem��tica de la literatura para el manejo de dolor central en esclerosis m��ltiple

El dolor es un s��ntoma muy com��n en pacientes con Esclerosis M��ltiple, pues del 42 al 65% de los enfermos lo presentan, y es calificado como el s��ntoma m��s severo entre el 8 y el 32%. Todos los s��ndromes dolorosos centrales se presentan por lesi��n o disfunci��n del sistema nervioso central, causando discapacidad severa y deterioro de la calidad de vida de los pacientes.

"Genetic Diversity and Selection in Three Plasmodium vivax Merozoite Surface Protein 7 (Pvmsp-7) Genes in a Colombian Population"

A completely effective vaccine for malaria (one of the major infectious diseases worldwide) is not yet available; different membrane proteins involved in parasite-host interactions have been proposed as candidates for designing it. It has been found that proteins encoded by the merozoite surface protein (msp)-7 multigene family are antibody targets in natural infection; the nucleotide diversity of three Pvmsp-7 genes was thus analyzed in a Colombian parasite population. By contrast with P. falciparum msp-7 loci and ancestral P. vivax msp-7 genes, specie-specific duplicates of the latter specie display high genetic variability, generated by single nucleotide polymorphisms, repeat regions, and recombination. At least three major allele types are present in Pvmsp-7C, Pvmsp-7H and Pvmsp-7I and positive selection seems to be operating on the central region of these msp-7 genes. Although this region has high genetic polymorphism, the C-terminus (Pfam domain ID: PF12948) is conserved and could be an important candidate when designing a subunit-based antimalarial vaccine.

Genetic diversity and selection in three plasmodium vivax merozoite surface protein 7 (pvmsp-7) genes in a colombian population

A completely effective vaccine for malaria (one of the major infectious diseases worldwide) is not yet available; different membrane proteins involved in parasite-host interactions have been proposed as candidates for designing it. It has been found that proteins encoded by the merozoite surface protein (msp)-7 multigene family are antibody targets in natural infection; the nucleotide diversity of three Pvmsp-7 genes was thus analyzed in a Colombian parasite population. By contrast with P. falciparum msp-7 loci and ancestral P. vivax msp-7 genes, specie-specific duplicates of the latter specie display high genetic variability, generated by single nucleotide polymorphisms, repeat regions, and recombination. At least three major allele types are present in Pvmsp-7C, Pvmsp-7H and Pvmsp-7I and positive selection seems to be operating on the central region of these msp-7 genes. Although this region has high genetic polymorphism, the C-terminus (Pfam domain ID: PF12948) is conserved and could be an important candidate when designing a subunit-based antimalarial vaccine.

Annotation and characterization of the Plasmodium vivax rhoptry neck protein 4 (PvRON4)

Background: The tight junction (TJ) is one of the most important structures established during merozoite invasion of host cells and a large amount of proteins stored in Toxoplasma and Plasmodium parasites��� apical organelles are involved in forming the TJ. Plasmodium falciparum and Toxoplasma gondii apical membrane antigen 1 (AMA-1) and rhoptry neck proteins (RONs) are the two main TJ components. It has been shown that RON4 plays an essential role during merozoite and sporozoite invasion to target cells. This study has focused on characterizing a novel Plasmodium vivax rhoptry protein, RON4, which is homologous to PfRON4 and PkRON4. Methods: The ron4 gene was re-annotated in the P. vivax genome using various bioinformatics tools and taking PfRON4 and PkRON4 amino acid sequences as templates. Gene synteny, as well as identity and similarity values between open reading frames (ORFs) belonging to the three species were assessed. The gene transcription of pvron4, and the expression and localization of the encoded protein were also determined in the VCG-1 strain by molecular and immunological studies. Nucleotide and amino acid sequences obtained for pvron4 in VCG-1 were compared to those from strains coming from different geographical areas. Results: PvRON4 is a 733 amino acid long protein, which is encoded by three exons, having similar transcription and translation patterns to those reported for its homologue, PfRON4. Sequencing PvRON4 from the VCG-1 strain and comparing it to P. vivax strains from different geographical locations has shown two conserved regions separated by a low complexity variable region, possibly acting as a ���smokescreen���. PvRON4 contains a predicted signal sequence, a coiled-coil ��-helical motif, two tandem repeats and six conserved cysteines towards the carboxyterminus and is a soluble protein lacking predicted transmembranal domains or a GPI anchor. Indirect immunofluorescence assays have shown that PvRON4 is expressed at the apical end of schizonts and co-localizes at the rhoptry neck with PvRON2.

Identification, characterization and antigenicity of the Plasmodium vivax rhoptry neck protein 1 (PvRON1)

Background: Plasmodium vivax malaria remains a major health problem in tropical and sub-tropical regions worldwide. Several rhoptry proteins which are important for interaction with and/or invasion of red blood cells, such as PfRONs, Pf92, Pf38, Pf12 and Pf34, have been described during the last few years and are being considered as potential anti-malarial vaccine candidates. This study describes the identification and characterization of the P. vivax rhoptry neck protein 1 (PvRON1) and examine its antigenicity in natural P. vivax infections. Methods: The PvRON1 encoding gene, which is homologous to that encoding the P. falciparum apical sushi protein (ASP) according to the plasmoDB database, was selected as our study target. The pvron1 gene transcription was evaluated by RT-PCR using RNA obtained from the P. vivax VCG-1 strain. Two peptides derived from the deduced P. vivax Sal-I PvRON1 sequence were synthesized and inoculated in rabbits for obtaining anti-PvRON1 antibodies which were used to confirm the protein expression in VCG-1 strain schizonts along with its association with detergent-resistant microdomains (DRMs) by Western blot, and its localization by immunofluorescence assays. The antigenicity of the PvRON1 protein was assessed using human sera from individuals previously exposed to P. vivax malaria by ELISA. Results: In the P. vivax VCG-1 strain, RON1 is a 764 amino acid-long protein. In silico analysis has revealed that PvRON1 shares essential characteristics with different antigens involved in invasion, such as the presence of a secretory signal, a GPI-anchor sequence and a putative sushi domain. The PvRON1 protein is expressed in parasite's schizont stage, localized in rhoptry necks and it is associated with DRMs. Recombinant protein recognition by human sera indicates that this antigen can trigger an immune response during a natural infection with P. vivax. Conclusions: This study shows the identification and characterization of the P. vivax rhoptry neck protein 1 in the VCG-1 strain. Taking into account that PvRON1 shares several important characteristics with other Plasmodium antigens that play a functional role during RBC invasion and, as shown here, it is antigenic, it could be considered as a good vaccine candidate. Further studies aimed at assessing its immunogenicity and protection-inducing ability in the Aotus monkey model are thus recommended.

Plasmodium vivax lineages: geographical distribution, tandem repeat polymorphism, and phylogenetic relationship

Background: Multi-drug resistance and severe/ complicated cases are the emerging phenotypes of vivax malaria, which may deteriorate current anti-malarial control measures. The emergence of these phenotypes could be associated with either of the two Plasmodium vivax lineages. The two lineages had been categorized as Old World and New World, based on geographical sub-division and genetic and phenotypical markers. This study revisited the lineage hypothesis of P. vivax by typing the distribution of lineages among global isolates and evaluated their genetic relatedness using a panel of new mini-satellite markers. Methods: 18S SSU rRNA S-type gene was amplified from 420 Plasmodium vivax field isolates collected from different geographical regions of India, Thailand and Colombia as well as four strains each of P. vivax originating from Nicaragua, Panama, Thailand (Pak Chang), and Vietnam (ONG). A mini-satellite marker panel was then developed to understand the population genetic parameters and tested on a sample subset of both lineages. Results: 18S SSU rRNA S-type gene typing revealed the distribution of both lineages (Old World and New World) in all geographical regions. However, distribution of Plasmodium vivax lineages was highly variable in every geographical region. The lack of geographical sub-division between lineages suggests that both lineages are globally distributed. Ten mini-satellites were scanned from the P. vivax genome sequence; these tandem repeats were located in eight of the chromosomes. Mini-satellites revealed substantial allelic diversity (7-21, AE = 14.6 +/- 2.0) and heterozygosity (He = 0.697-0.924, AE = 0.857 +/- 0.033) per locus. Mini-satellite comparison between the two lineages revealed high but similar pattern of genetic diversity, allele frequency, and high degree of allele sharing. A Neighbour-Joining phylogenetic tree derived from genetic distance data obtained from ten mini-satellites also placed both lineages together in every cluster. Conclusions: The global lineage distribution, lack of genetic distance, similar pattern of genetic diversity, and allele sharing strongly suggested that both lineages are a single species and thus new emerging phenotypes associated with vivax malaria could not be clearly classified as belonging to a particular lineage on basis of their geographical origin.

Estudio de las modificaciones conformacionales y su influencia en la actividad inmunol��gica de p��ptidos derivados de las familias de las prote��nas spect-1 y -2, siap-1 y -2 de plasmodium falciparum

SPECT-1 y -2 y SIAP-1 y -2 son prote��nas pertenecientes al esporozo��to de Plasmodium falciparum causante de la malaria m��s agresiva en los humanos. Estas prote��nas est��n involucradas en el paso del par��sito a trav��s de las c��lulas del hospedero humano y en la invasi��n del hepatocito, haci��ndolas blancos atractivos para ser estudiadas. P��ptidos conservados de alta capacidad de uni��n (cHABPs) a c��lulas HeLa y HepG2 derivados de estas mol��culas son no inmunog��nicos porque son incapaces de generar una respuesta inmunitaria, pero son claves para el par��sito porque cumplen una funci��n importante durante la infecci��n del hospedero humano. En este trabajo se encontr�� que algunos cHABPs pertenecientes a las prote��nas SPECT-1 y -2, est��n posiblemente involucrados con la uni��n y formaci��n de poros sobre la membrana de las c��lulas hospederas, ayudando al esporozo��to a abrirse paso trav��s de las c��lulas del hospedero. Por otro lado, con el fin de cambiar el comportamiento inmunol��gico de cHABPs derivados de SPECT-1 y -2 y SIAP-1 y -2, se obtuvieron nuevos p��ptidos mediante el reemplazo de amino��cidos cr��ticos por otros residuos cuya masa molecular sea similar, pero diferente en su polaridad. En este trabajo se reporta que dichas modificaciones promovieron cambios en la estructura secundaria (determinada por DC o 1H-RMN) de los p��ptidos modificados (mHABPs) cuando se compar�� con la estructura de los cHABPs nativos; adicionalmente, estos mHABPs invirtieron su comportamiento inmunol��gico convirti��ndose en p��ptidos inmunog��nicos inductores de anticuerpos. Lo que permite establecer la existencia de una relaci��n entre la estructura que adoptan estos mHABPs con su actividad inmunol��gica. Adem��s, algunos de los mHABPs estudiados aqu��, pueden ser candidatos a ser incluidos en la vacuna contra la malaria qu��micamente sintetizada multi-epitope y multi-estadio que se est�� desarrollando en la Fundaci��n Instituto de Inmunolog��a de Colombia (FIDIC).