BackgroundProgress towards the development of a malaria vaccine against Plasmodium vivax, the most widely distributed human malaria parasite, will require a better understanding of the immune responses that confer clinical protection to patients in regions where malaria is endemic.MethodsGlutathione S-transferase (GST) and GST-fusion proteins representing the N- terminus of the merozoite surface protein 1 of P. vivax, PvMSP1-N, and the C-terminus, PvMSP1-C, were covalently coupled to BioPlex carboxylated beads. Recombinant proteins and coupled beads were used, respectively, in ELISA and Bioplex assays using immune sera of P. vivax patients from Brazil and PNG to determine IgG and subclass responses. Concordances between the two methods in the seropositivity responses were evaluated using the Kappa statistic and the Spearman's rank correlation.ResultsThe results using this methodology were compared with the classical microtitre enzyme-linked immnosorbent assay (ELISA), showing that the assay was sensitive, reproducible and had good concordance with ELISA; yet, further research into different statistical analyses seems desirable before claiming conclusive results exclusively based on multiplex assays. As expected, results demonstrated that PvMSP1 was immunogenic in natural infections of patients from different endemic regions of Brazil and Papua New Guinea (PNG), and that age correlated only with antibodies against the C-terminus part of the molecule. Furthermore, the IgG subclass profiles were different in these endemic regions having IgG3 predominantly recognizing PvMSP1 in Brazil and IgG1 predominantly recognizing PvMSP1 in PNG.ConclusionsThis study validates the use of the multiplex assay to measure naturally-acquired IgG antibodies against the merozoite surface protein 1 of P. vivax.
TREX1 is the most abundant mammalian 3 3 5 DNA exonuclease. It has been described to form part of the SET complex and is responsible for the Aicardi-Goutières syndrome in humans. Here we show that the exonuclease activity is correlated to the binding preferences toward certain DNA sequences. In particular, we have found three motifs that are selected, GAG, ACA, and CTGC. To elucidate how the discrimination occurs, we determined the crystal structures of two murine TREX1 complexes, with a nucleotide product of the exonuclease reaction, and with a single-stranded DNA substrate. Using confocal microscopy, we observed TREX1 both in nuclear and cytoplasmic subcellular compartments. Remarkably, the presence of TREX1 in the nucleus requires the loss of a C-terminal segment, which we named leucine-rich repeat 3. Furthermore, we detected the presence of a conserved proline-rich region on the surface of TREX1. This observation points to interactions with proline-binding domains. The potential interacting motif "PPPVPRPP" does not contain aromatic residues and thus resembles other sequences that select SH3 and/or Group 2 WW domains. By means of nuclear magnetic resonance titration experiments, we show that, indeed, a polyproline peptide derived from the murine TREX1 sequence interacted with the WW2 domain of the elongation transcription factor CA150. Coimmunoprecipitation studies confirmed this interaction with the full-length TREX1 protein, thereby suggesting that TREX1 participates in more functional complexes than previously thought.
The genes of the transporter associated with antigen processing (Tap)-1, and the low molecular weight peptide (Lmp)-2, are crucial for class I major histocompatibility complex function and share a common bidirectional promoter. In murine bone marrow-derived macrophages, interferon gamma (IFN-g) induced Tap-1 and upregulated Lmp-2, which is constitutively expressed at low levels. The IFN-g-induction was independent of early gene synthesis. The mRNA induced by IFN-g was very stable. In macrophages from STAT1 knockout mice, IFN-g did not induce the expression of Tap-1 or Lmp-2. Several areas in the promoter can be controlled by IFN-g, such as proximal and distal GAS boxes in the direction of the Tap-1 gene, NFgB and IRF-1 boxes. By making deletions of the promoter, we found that only the proximal GAS and IRF-1 boxes are required for IFN-g induction of Tap-1 and Lmp-2. Experiments using nuclear extracts from macrophages treated for 30 min with IFN-g and gel shift analysis indicated that STAT1 binds to the GAS box. The nuclear extracts from macrophages treated for at least 2 h with IFN-g bound to the IRF-1 box. These results indicate that both STAT1 and IRF-1 are required for the IFN-g induction of Tap-1 and Lmp-2 genes.
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