84 results on '"Fernandez-Becerra C"'
Search Results
2. Advancing Key Gaps in the Knowledge of Plasmodium vivax Cryptic Infections Using Humanized Mouse Models and Organs-on-Chips
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Herraiz, IA, Caires, HR, Castillo-Fernandez, O, Sima, N, Mendez-Mora, L, Risueno, RM, Sattabongkot, J, Roobsoong, W, Hernandez-Machado, A, Fernandez-Becerra, C, Barrias, CC, and del Portillo, HA
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models ,humanized mouse ,Key gaps in the knowledge ,Plasmodium vivax ,organs-on-a-chip - Abstract
Plasmodium vivax is the most widely distributed human malaria parasite representing 36.3% of disease burden in the South-East Asia region and the most predominant species in the region of the Americas. Recent estimates indicate that 3.3 billion of people are under risk of infection with circa 7 million clinical cases reported each year. This burden is certainly underestimated as the vast majority of chronic infections are asymptomatic. For centuries, it has been widely accepted that the only source of cryptic parasites is the liver dormant stages known as hypnozoites. However, recent evidence indicates that niches outside the liver, in particular in the spleen and the bone marrow, can represent a major source of cryptic chronic erythrocytic infections. The origin of such chronic infections is highly controversial as many key knowledge gaps remain unanswered. Yet, as parasites in these niches seem to be sheltered from immune response and antimalarial drugs, research on this area should be reinforced if elimination of malaria is to be achieved. Due to ethical and technical considerations, working with the liver, bone marrow and spleen from natural infections is very difficult. Recent advances in the development of humanized mouse models and organs-on-a-chip models, offer novel technological frontiers to study human diseases, vaccine validation and drug discovery. Here, we review current data of these frontier technologies in malaria, highlighting major challenges ahead to study P. vivax cryptic niches, which perpetuate transmission and burden.
- Published
- 2022
3. Extracellular Vesicles in Trypanosoma cruzi Infection: Immunomodulatory Effects and Future Perspectives as Potential Control Tools against Chagas Disease
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Cortes-Serra, N, Gualdron-Lopez, M, Pinazo, MJ, Torrecilhas, AC, and Fernandez-Becerra, C
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Article Subject - Abstract
Chagas disease, caused by the protozoa parasite Trypanosoma cruzi, is a neglected tropical disease and a major public health problem affecting more than 6 million people worldwide. Many challenges remain in the quest to control Chagas disease: the diagnosis presents several limitations and the two available treatments cause several side effects, presenting limited efficacy during the chronic phase of the disease. In addition, there are no preventive vaccines or biomarkers of therapeutic response or disease outcome. Trypomastigote form and T. cruzi-infected cells release extracellular vesicles (EVs), which are involved in cell-to-cell communication and can modulate the host immune response. Importantly, EVs have been described as promising tools for the development of new therapeutic strategies, such as vaccines, and for the discovery of new biomarkers. Here, we review and discuss the role of EVs secreted during T. cruzi infection and their immunomodulatory properties. Finally, we briefly describe their potential for biomarker discovery and future perspectives as vaccine development tools for Chagas Disease.
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- 2022
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4. Cryptic Plasmodium chronic infections: was Maurizio Ascoli right?
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Monteiro, W, Brito-Sousa, JD, Elizalde-Torrent, A, Botto-Menezes, C, Melo, GC, Fernandez-Becerra, C, Lacerda, M, and del Portillo, HA
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Cryptic infection ,Plasmodium ,Parasite recurrence ,Ascoli's method ,Spleen - Abstract
Cryptic Plasmodium niches outside the liver possibly represent a major source of hypnozoite-unrelated recrudescences in malaria. Maurizio Ascoli, an Italian physician and scientist, suggested that infection was maintained as a result of the persistence of endoerythrocytic parasites in the circulatory bed of some internal organs, mainly the spleen. This would explain a proportion of the recurrences in patients, regardless of the Plasmodium species. Ascoli proposed a method that included the co-administration of adrenaline, in order to induce splenic contraction, and quinine to clear expelled forms in major vessels. Driven by controversy regarding safety and effectiveness, along with the introduction of new drugs, the Ascoli method was abandoned and mostly forgotten by the malaria research community. To date, however, the existence of cryptic parasites outside the liver is gaining supportive data. This work is a historical retrospective of cryptic malaria infections and the Ascoli method, highlighting key knowledge gaps regarding these possible parasite reservoirs.
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- 2020
5. Plasmodium vivax spleen -dependent genes encode antigens associated with cytoadhesion and clinical protection
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Fernandez-Becerra, C, Bernabeu, M, Castellanos, A, Correa, BR, Obadia, T, Ramirez, M, Rui, ED, Hentzschel, F, Lopez-Montanes, M, Ayllon-Hermida, A, Martin-Jaular, L, Elizalde-Torrent, A, Siba, P, Vencio, RZ, Arevalo-Herrera, M, Herrera, S, Alonso, PL, Mueller, I, and del Portillo, MHA
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cytoadherence ,global transcription ,spleen-dependent genes ,Plasmodium vivax - Abstract
Plasmodium vivax, the most widely distributed human malaria parasite, causes severe clinical syndromes despite low peripheral blood parasitemia. This conundrum is further complicated as cytoadherence in the microvasculature is still a matter of investigations. Previous reports in Plasmodium knowlesi, another parasite species shown to infect humans, demonstrated that variant genes involved in cytoadherence were dependent on the spleen for their expression. Hence, using a global transcriptional analysis of parasites obtained from spleen-intact and splenectomized monkeys, we identified 67 P. vivax genes whose expression was spleen dependent. To determine their role in cytoadherence, two Plasmodium falciparum transgenic lines expressing two variant proteins pertaining to VIR and Pv-FAM-D multigene families were used. Cytoadherence assays demonstrated specific binding to human spleen but not lung fibroblasts of the transgenic line expressing the VIR14 protein. To gain more insights, we expressed five P. vivax spleen-dependent genes as recombinant proteins, including members of three different multigene families (VIR, Pv-FAM-A, Pv-FAM-D), one membrane transporter (SECY), and one hypothetical protein (HYP1), and determined their immunogenicity and association with clinical protection in a prospective study of 383 children in Papua New Guinea. Results demonstrated that spleen-dependent antigens are immunogenic in natural infections and that antibodies to HYP1 are associated with clinical protection. These results suggest that the spleen plays a major role in expression of parasite proteins involved in cytoadherence and can reveal antigens associated with clinical protection, thus prompting a paradigm shift in P. vivax biology toward deeper studies of the spleen during infections.
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- 2020
6. Spleen-Dependent Immune Protection Elicited by CpG Adjuvanted Reticulocyte-Derived Exosomes from Malaria Infection Is Associated with Changes in T Cell Subsets' Distribution (vol 4, 131, 2016)
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Martin-Jaular, L, de Menezes-Neto, A, Monguio-Tortajada, M, Elizalde-Torrent, A, Diaz-Varela, M, Fernandez-Becerra, C, Borras, FE, Montoya, M, and del Portillo, HA
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PD-1 cells ,vaccine ,effector memory T-cells ,malaria ,spleen ,reticulocyte-derived exosomes - Published
- 2017
7. Functional analysis of Plasmodium vivax VIR proteins reveals different subcellular localizations and cytoadherence to the ICAM-1 endothelial receptor
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Bernabeu, M., Lopez, F. J., Ferrer, M., Martin-Jaular, L., Razaname, A., Corradin, G., Maier, A. G., del Portillo, H. A., and Fernandez-Becerra, C.
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- 2012
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8. A functional microengineered model of the human splenon-on-a-chip
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Rigat-Brugarolas, L. G., primary, Elizalde-Torrent, A., additional, Bernabeu, M., additional, De Niz, M., additional, Martin-Jaular, L., additional, Fernandez-Becerra, C., additional, Homs-Corbera, A., additional, Samitier, J., additional, and del Portillo, H. A., additional
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- 2014
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9. Functional analysis of Plasmodium vivax VIR proteins reveals different subcellular localizations and cytoadherence to the ICAM-1 endothelial receptor
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Bernabeu, M, Lopez, FJ, Ferrer, M, Martin-Jaular, L, Razaname, A, Corradin, G, Maier, Alex, del Portillo, HA, Fernandez-Becerra, C, Bernabeu, M, Lopez, FJ, Ferrer, M, Martin-Jaular, L, Razaname, A, Corradin, G, Maier, Alex, del Portillo, HA, and Fernandez-Becerra, C
- Abstract
The subcellular localization and function of variant subtelomeric multigene families in Plasmodium vivax remain vastly unknown. Among them, the vir superfamily is putatively involved in antigenic variation and in mediating adherence to endothelial receptors. In the absence of a continuous in vitro culture system for P. vivax, we have generated P. falciparum transgenic lines expressing VIR proteins to infer location and function. We chose three proteins pertaining to subfamilies A (VIR17), C (VIR14) and D (VIR10), with domains and secondary structures that predictably traffic these proteins to different subcellular compartments. Here, we showed that VIR17 remained inside the parasite and around merozoites, whereas VIR14 and VIR10 were exported to the membrane of infected red blood cells (iRBCs) in an apparent independent pathway of Maurer's clefts. Remarkably, VIR14 was exposed at the surface of iRBCs and mediated adherence to different endothelial receptors expressed in CHO cells under static conditions. Under physiological flow conditions, however, cytoadherence was only observed to ICAM-1, which was the only receptor whose adherence was specifically and significantly inhibited by antibodies against conserved motifs of VIR proteins. Immunofluorescence studies using these antibodies also showed different subcellular localizations of VIR proteins in P. vivax-infected reticulocytes from natural infections. These data suggest that VIR proteins are trafficked to different cellular compartments and functionally demonstrates that VIR proteins can specifically mediate cytoadherence to the ICAM-1 endothelial receptor.
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- 2011
10. Naturally-acquired humoral immune responses against the N- and C-termini of the Plasmodium vivax MSP1 protein in endemic regions of Brazil and Papua New Guinea using a multiplex assay
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Fernandez-Becerra, C, Sanz, S, Brucet, M, Stanisic, DI, Alves, FP, Camargo, EP, Alonso, PL, Mueller, I, del Portillo, HA, Fernandez-Becerra, C, Sanz, S, Brucet, M, Stanisic, DI, Alves, FP, Camargo, EP, Alonso, PL, Mueller, I, and del Portillo, HA
- Abstract
BACKGROUND: Progress 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. METHODS: Glutathione 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. RESULTS: The 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. CONCLUSIONS: This study validates the use of the multiplex assay to measure naturally-acquired IgG antibodies against the merozoite surface protein 1 of P. vivax.
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- 2010
11. In vitro culture of Phytomonas sp. isolated from Euphorbia characias: metabolic studies by H NMR
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Sanchez Moreno, Manuel, Fernandez Becerra, C., Entrala, E., Opperdoes, F.R., Dollet, Michel, and Osuna, A.
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Culture in vitro ,Trypanosoma ,Identification ,Spectroscopie RMN ,Phytomonas ,Maladie des plantes ,Métabolisme ,Microscopie électronique ,H20 - Maladies des plantes - Abstract
L'article porte sur la description d'espèces de Phytomonas isolées à partir de Euphorbia characias et cultivées in vitro. Un des buts de cette étude était aussi de trouver le milieu de culture optimal pour ces Phytomonas. Par microscopie électronique, les particularités de l'ultrastructure des formes obtenues in vitro ont pu être caractérisées. Par des méthodes métaboliques qualitatives (spectroscopie par résonnance magnétique nucléaire) et quantitatives (tests enzymatiques), le processus métabolique et la production de métabolites sont analysés
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- 1995
12. Characterization of trypanosomatids isolated from plants in Southeastern Spain by lectin-agglutination and isoenzyme and DNA-kinetoplast analysis
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Fernandez Becerra, C., Luque, F., Fernandez Ramos, C., Osuna, A., Dollet, Michel, and Sanchez Moreno, Manuel
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Trypanosoma ,Identification ,ADN ,Analyse enzymatique ,Maladie des plantes ,Réaction d'agglutination ,Solanum lycopersicum ,Cocos nucifera ,H20 - Maladies des plantes ,Enzyme de restriction ,Amaranthus ,Isoenzyme ,Phytomonas ,méthode ,Trifolium - Abstract
Utilisation de tests d'agglutination avec des lectines, ainsi que des analyses d'ADN kinétoplastique et isoenzymatiques pour la caractérisation de trypanosomatidae isolés à partir de plantes dans le sud-est de l'Espagne (Lycopersicum esculentum, Anona cherimolia, Mangus sp., Trifolium, Amaranthus sp.)
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- 1995
13. Trypanosomatids protozoa isolated from several plants in Southeastern Spain
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Fernandez Becerra, C., Luque, F., Fernandez Ramos, C., Osuna, A., Dollet, Michel, and Sanchez Moreno, Manuel
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Trypanosoma ,Amaranthus ,Pouvoir pathogène ,Phytomonas ,Maladie des plantes ,Solanum lycopersicum ,Trifolium ,Plante de culture ,Microscopie ,H20 - Maladies des plantes - Abstract
Isolement de protozoaires flagellés à partir de plusieurs plantes d'intérêt économique susceptible de se comporter comme un réservoir pour le parasite. Pour examiner les racines, les tiges, feuilles et fruits on a utilisé un microscope en contraste de phase. On a constaté que chez Lycopersicum esculentum et Anona cherimolia seul les fruits étaient infectés alors que pour Mangus sp, Trifolium et Amaranthus sp les protozoaires ont été retrouvés uniquement dans les tiges. La virulence des protozoaires n'est pas modifiée lorsqu'ils sont cultivés in vitro bien que leur forme soit modifiée. Les résultats préliminaires sur l'étude expérimentale des relations possibles entre les symptômes du parasitisme et de la pathologie montrent une influence du parasite sur le développement normal de la plante (croissance, dépigmentation des fruits)
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- 1995
14. Functional analysis of Plasmodium vivax VIR proteins reveals different subcellular localizations and cytoadherence to the ICAM-1 endothelial receptor
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Bernabeu, M., primary, Lopez, F. J., additional, Ferrer, M., additional, Martin-Jaular, L., additional, Razaname, A., additional, Corradin, G., additional, Maier, A. G., additional, del Portillo, H. A., additional, and Fernandez-Becerra, C., additional
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- 2011
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15. Inhibition of Superoxide Dismutase from Ascaris suum by Benzimidazoles and Synthesized Pyrimidine and Glycine Derivatives
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Sanchez-Moreno, M., primary, Entrala, E., additional, Janssen, D., additional, Fernandez-Becerra, C., additional, Salas-Peregrin, J.M., additional, and Osuna, A., additional
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- 1996
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16. A new computational approach redefines the subtelomeric vir superfamily of Plasmodium vivax
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Lopez Francisco Javier, Bernabeu Maria, Fernandez-Becerra Carmen, and del Portillo Hernando A
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Malaria ,Plasmodium vivax ,vir genes ,VIR proteins ,Subtelomeric multigene families ,Sequence clustering ,Similarity networks ,Homology blocks ,Biotechnology ,TP248.13-248.65 ,Genetics ,QH426-470 - Abstract
Abstract Background Subtelomeric multigene families of malaria parasites encode virulent determinants. The published genome sequence of Plasmodium vivax revealed the largest subtelomeric multigene family of human malaria parasites, the vir super-family, presently composed of 346 vir genes subdivided into 12 different subfamilies based on sequence homologies detected by BLAST. Results A novel computational approach was used to redefine vir genes. First, a protein-weighted graph was built based on BLAST alignments. This graph was processed to ensure that edge weights are not exclusively based on the BLAST score between the two corresponding proteins, but strongly dependant on their graph neighbours and their associations. Then the Markov Clustering Algorithm was applied to the protein graph. Next, the Homology Block concept was used to further validate this clustering approach. Finally, proteome-wide analysis was carried out to predict new VIR members. Results showed that (i) three previous subfamilies cannot longer be classified as vir genes; (ii) most previously unclustered vir genes were clustered into vir subfamilies; (iii) 39 hypothetical proteins were predicted as VIR proteins; (iv) many of these findings are supported by a number of structural and functional evidences, sub-cellular localization studies, gene expression analysis and chromosome localization (v) this approach can be used to study other multigene families in malaria. Conclusions This methodology, resource and new classification of vir genes will contribute to a new structural framing of this multigene family and other multigene families of malaria parasites, facilitating the design of experiments to understand their role in pathology, which in turn may help furthering vaccine development.
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- 2013
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17. Plasmodium vivax malaria in Mali: a study from three different regions
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Bernabeu Maria, Gomez-Perez Gloria P, Sissoko Sibiri, Niambélé Mohamed B, Haibala Allassane Ag, Sanz Ariadna, Théra Mahamadou A, Fernandez-Becerra Carmen, Traoré Klénon, Alonso Pedro L, Bassat Quique, del Portillo Hernando A, and Doumbo Ogobara
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Mali ,Sub-Saharan Africa ,Plasmodium vivax ,Vivax malaria ,Nested-PCR ,DNA sequencing ,SSU RNA ,Giemsa-smears ,Arctic medicine. Tropical medicine ,RC955-962 ,Infectious and parasitic diseases ,RC109-216 - Abstract
Abstract Background Plasmodium vivax has traditionally been considered virtually absent from Western and Central Africa, due to the absence of the Duffy blood group in most of the population living in these areas. Recent reports, however, suggest the circulation of P. vivax in sub-Saharan Africa. Methods Giemsa/Field-stained smears from febrile patients recruited in five different cities (Goundam, Tombouctou, Gao, Bourem and Kidal) pertaining to three regions from Northern Mali were examined. Nested-PCR and DNA sequence analyses of selected samples were performed to fully confirm the presence of P. vivax infections. Results Results demonstrated the presence of P. vivax infections in close to 30% of the cases as detected by Giemsa/Field-stained smears and nested-PCR and DNA-sequence analyses of selected samples unequivocally confirmed the presence of P. vivax. Conclusions The diagnostics of this human malaria parasite should be taken into account in the context of malaria control and elimination efforts, not only in Mali, but also in sub-Saharan Africa.
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- 2012
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18. Plasmodium vivax: comparison of immunogenicity among proteins expressed in the cell-free systems of Escherichia coli and wheat germ by suspension array assays
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Tsuboi Takafumi, Lacerda Marcus VG, Sanz Sergi, Takeo Satoru, Fernandez-Becerra Carmen, Rui Edmilson, and del Portillo Hernando A
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Arctic medicine. Tropical medicine ,RC955-962 ,Infectious and parasitic diseases ,RC109-216 - Abstract
Abstract Background In vitro cell-free systems for protein expression with extracts from prokaryotic (Escherichia coli) or eukaryotic (wheat germ) cells coupled to solid matrices have offered a valid approach for antigen discovery in malaria research. However, no comparative analysis of both systems is presently available nor the usage of suspension array technologies, which offer nearly solution phase kinetics. Methods Five Plasmodium vivax antigens representing leading vaccine candidates were expressed in the E. coli and wheat germ cell-free systems at a 50 μl scale. Products were affinity purified in a single-step and coupled to luminex beads to measure antibody reactivity of human immune sera. Results Both systems readily produced detectable proteins; proteins produced in wheat germ, however, were mostly soluble and intact as opposed to proteins produced in E. coli, which remained mostly insoluble and highly degraded. Noticeably, wheat germ proteins were recognized in significantly higher numbers by sera of P. vivax patients than identical proteins produced in E. coli. Conclusions The wheat germ cell-free system offers the possibility of expressing soluble P. vivax proteins in a small-scale for antigen discovery and immuno-epidemiological studies using suspension array technology.
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- 2011
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19. Naturally-acquired humoral immune responses against the N- and C-termini of the Plasmodium vivax MSP1 protein in endemic regions of Brazil and Papua New Guinea using a multiplex assay
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Alonso Pedro L, Camargo Erney P, Alves Fabiana P, Stanisic Danielle I, Brucet Marina, Sanz Sergi, Fernandez-Becerra Carmen, Mueller Ivo, and del Portillo Hernando A
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Arctic medicine. Tropical medicine ,RC955-962 ,Infectious and parasitic diseases ,RC109-216 - Abstract
Abstract Background Progress 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. Methods Glutathione 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. Results The 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. Conclusions This study validates the use of the multiplex assay to measure naturally-acquired IgG antibodies against the merozoite surface protein 1 of P. vivax.
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- 2010
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20. Increased expression levels of the pvcrt-o and pvmdr1 genes in a patient with severe Plasmodium vivax malaria
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del Portillo Hernando A, Alonso Pedro L, González Ana, Pinazo Maria, Fernández-Becerra Carmen, and Gascón Joaquim
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Arctic medicine. Tropical medicine ,RC955-962 ,Infectious and parasitic diseases ,RC109-216 - Abstract
Abstract Background There are increasing reports of severe clinical cases exclusively associated with Plasmodium vivax infections. Notably, this severity has been recently suggested to be associated with chloroquine resistance. Patients Two different patients presented at the Hospital Clinic in Barcelona with P. vivax malaria episodes. One patient had severe symptoms and the other mild symptoms. Both patients traveled through the Brazilian Amazon (Manaus) in 2007. For both patients the current diagnosis of malaria was the first. Two other patients with mild symptoms presented to the "Centro de Pesquisa em Medicina Tropical", also in the Brazilian Amazon (Rondônia) in 2000. Methods To exclude the possibility that the patient's severe symptoms were due to Plasmodium falciparum, a nested PCR was performed. A magnetic method was used to purify P. vivax free of human leukocytes. Quantitative real-time PCR was performed to compare the transcript levels of two main transporters likely to be involved in chloroquine resistance in P. vivax, namely the P. vivax chloroquine resistance transporter, pvcrt-o, and the P. vivax multidrug resistance transporter, pvmdr 1. Results Results demonstrated that the severe clinical symptoms were exclusively due to P. vivax. The patient presented acute respiratory conditions requiring admission to the intensive care unit. The magnetic method showed highly purified infected-reticulocytes with mature stages. In addition, it was found that parasites obtained from the severe patient had up to 2.9-fold increase in pvmdr1 levels and up to 21.9-fold increase in pvcrt-o levels compared to expression levels of parasites from the other patients with mild symptoms. Conclusion This is the first clinical case of severe disease exclusively associated with vivax malaria in Spain. Moreover, these findings suggest that clinical severity could be associated with increased expression levels of parasite genes likely involved in chloroquine resistance. It is necessary to further explore the potential of pvmdr1 and particularly pvcrt-o expression levels as molecular markers of severe disease in P. vivax.
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- 2009
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21. Evaluation of the acquired immune responses to Plasmodium vivax VIR variant antigens in individuals living in malaria-endemic areas of Brazil
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Soares Irene S, Del Portillo Hernando A, Jimenez Maria Carolina S, Fernandez-Becerra Carmen, and Oliveira Tatiane R
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Arctic medicine. Tropical medicine ,RC955-962 ,Infectious and parasitic diseases ,RC109-216 - Abstract
Abstract Background The naturally-acquired immune response to Plasmodium vivax variant antigens (VIR) was evaluated in individuals exposed to malaria and living in different endemic areas for malaria in the north of Brazil. Methods Seven recombinant proteins representing four vir subfamilies (A, B, C, and E) obtained from a single patient from the Amazon Region were expressed in Escherichia coli as soluble glutathione S-transferase fusion proteins. The different recombinant proteins were compared by ELISA with regard to the recognition by IgM, IgG, and IgG subclass of antibodies from 200 individuals with patent infection. Results The frequency of individuals that presented antibodies anti-VIR (IgM plus IgG) during the infection was 49%. The frequencies of individuals that presented IgM or IgG antibodies anti-VIR were 29.6% or 26.0%, respectively. The prevalence of IgG antibodies against recombinant VIR proteins was significantly lower than the prevalence of antibodies against the recombinant proteins representing two surface antigens of merozoites of P. vivax: AMA-1 and MSP119 (57.0% and 90.5%, respectively). The cellular immune response to VIR antigens was evaluated by in vitro proliferative assays in mononuclear cells of the individuals recently exposed to P. vivax. No significant proliferative response to these antigens was observed when comparing malaria-exposed to non-exposed individuals. Conclusion This study provides evidence that there is a low frequency of individuals responding to each VIR antigens in endemic areas of Brazil. This fact may explain the host susceptibility to new episodes of the disease.
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- 2006
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22. Pilot survey of expressed sequence tags (ESTs) from the asexual blood stages of Plasmodium vivax in human patients
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Gruber Arthur, Durham Alan, Machado Ariane L, Madeira Alda MBN, Fernandez-Becerra Carmen, Merino Emilio F, Hall Neil, and del Portillo Hernando A
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Arctic medicine. Tropical medicine ,RC955-962 ,Infectious and parasitic diseases ,RC109-216 - Abstract
Abstract Background Plasmodium vivax is the most widely distributed human malaria, responsible for 70–80 million clinical cases each year and large socio-economical burdens for countries such as Brazil where it is the most prevalent species. Unfortunately, due to the impossibility of growing this parasite in continuous in vitro culture, research on P. vivax remains largely neglected. Methods A pilot survey of expressed sequence tags (ESTs) from the asexual blood stages of P. vivax was performed. To do so, 1,184 clones from a cDNA library constructed with parasites obtained from 10 different human patients in the Brazilian Amazon were sequenced. Sequences were automatedly processed to remove contaminants and low quality reads. A total of 806 sequences with an average length of 586 bp met such criteria and their clustering revealed 666 distinct events. The consensus sequence of each cluster and the unique sequences of the singlets were used in similarity searches against different databases that included P. vivax, Plasmodium falciparum, Plasmodium yoelii, Plasmodium knowlesi, Apicomplexa and the GenBank non-redundant database. An E-value of -30 was used to define a significant database match. ESTs were manually assigned a gene ontology (GO) terminology Results A total of 769 ESTs could be assigned a putative identity based upon sequence similarity to known proteins in GenBank. Moreover, 292 ESTs were annotated and a GO terminology was assigned to 164 of them. Conclusion These are the first ESTs reported for P. vivax and, as such, they represent a valuable resource to assist in the annotation of the P. vivax genome currently being sequenced. Moreover, since the GC-content of the P. vivax genome is strikingly different from that of P. falciparum, these ESTs will help in the validation of gene predictions for P. vivax and to create a gene index of this malaria parasite.
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- 2003
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23. Advancing research on parasitic infections: Standardized extracellular vesicle guideline.
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Fernandez-Becerra C, Xander P, Olivier M, and Torrecilhas AC
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- 2024
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24. Proteomics of circulating extracellular vesicles reveals diverse clinical presentations of COVID-19 but fails to identify viral peptides.
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Gualdrón-López M, Ayllon-Hermida A, Cortes-Serra N, Resa-Infante P, Bech-Serra JJ, Aparici-Herraiz I, Nicolau-Fernandez M, Erkizia I, Gutierrez-Chamorro L, Marfil S, Pradenas E, Ávila Nieto C, Cucurull B, Montaner-Tarbés S, Muelas M, Sotil R, Ballana E, Urrea V, Fraile L, Montoya M, Vergara J, Segales J, Carrillo J, Izquierdo-Useros N, Blanco J, Fernandez-Becerra C, de La Torre C, Pinazo MJ, Martinez-Picado J, and Del Portillo HA
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- Animals, Humans, Male, Antibodies, Neutralizing blood, Antibodies, Neutralizing immunology, Female, Peptides metabolism, Antibodies, Viral blood, Mesocricetus, Adult, Middle Aged, Cricetinae, Viral Proteins metabolism, Extracellular Vesicles metabolism, COVID-19 virology, COVID-19 immunology, Proteomics, SARS-CoV-2
- Abstract
Extracellular vesicles (EVs) released by virus-infected cells have the potential to encapsulate viral peptides, a characteristic that could facilitate vaccine development. Furthermore, plasma-derived EVs may elucidate pathological changes occurring in distal tissues during viral infections. We hypothesized that molecular characterization of EVs isolated from COVID-19 patients would reveal peptides suitable for vaccine development. Blood samples were collected from three cohorts: severe COVID-19 patients (G1), mild/asymptomatic cases (G2), and SARS-CoV-2-negative healthcare workers (G3). Samples were obtained at two time points: during the initial phase of the pandemic in early 2020 (m0) and eight months later (m8). Clinical data analysis revealed elevated inflammatory markers in G1. Notably, non-vaccinated individuals in G1 exhibited increased levels of neutralizing antibodies at m8, suggesting prolonged exposure to viral antigens. Proteomic profiling of EVs was performed using three distinct methods: immunocapture (targeting CD9), ganglioside-capture (utilizing Siglec-1) and size-exclusion chromatography (SEC). Contrary to our hypothesis, this analysis failed to identify viral peptides. These findings were subsequently validated through Western blot analysis targeting the RBD of the SARS-CoV-2 Spike protein's and comparative studies using samples from experimentally infected Syrian hamsters. Furthermore, analysis of the EV cargo revealed a diverse molecular profile, including components involved in the regulation of viral replication, systemic inflammation, antigen presentation, and stress responses. These findings underscore the potential significance of EVs in the pathogenesis and progression of COVID-19., Competing Interests: HP, MMo, and LF are shareholders of Innovex Therapeutics. SM-T was a former employee of Innovex Therapeutics. JM-P has received institutional grants and educational/consultancy fees from AbiVax; AstraZeneca; Gilead Sciences; Grifols; Janssen; Merck Sharp & Dohme; and ViiV Healthcare; all outside the submitted work. JC and JB are shareholders of Albajuna Therapeutics SL, NI-U reports institutional grants from Grifols, Dentaid, Hipra and Amassence. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Gualdrón-López, Ayllon-Hermida, Cortes-Serra, Resa-Infante, Bech-Serra, Aparici-Herraiz, Nicolau-Fernandez, Erkizia, Gutierrez-Chamorro, Marfil, Pradenas, Ávila Nieto, Cucurull, Montaner-Tarbés, Muelas, Sotil, Ballana, Urrea, Fraile, Montoya, Vergara, Segales, Carrillo, Izquierdo-Useros, Blanco, Fernandez-Becerra, de La Torre, Pinazo, Martinez-Picado and del Portillo.)
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- 2024
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25. Plasmodium vivax spleen-dependent protein 1 and its role in extracellular vesicles-mediated intrasplenic infections.
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Ayllon-Hermida A, Nicolau-Fernandez M, Larrinaga AM, Aparici-Herraiz I, Tintó-Font E, Llorà-Batlle O, Orban A, Yasnot MF, Graupera M, Esteller M, Popovici J, Cortés A, Del Portillo HA, and Fernandez-Becerra C
- Subjects
- Humans, Erythrocytes parasitology, Erythrocytes metabolism, Fibroblasts parasitology, Fibroblasts metabolism, Plasmodium falciparum genetics, Plasmodium falciparum metabolism, Plasmodium falciparum physiology, Cell Adhesion, Host-Parasite Interactions, Extracellular Vesicles metabolism, Plasmodium vivax genetics, Plasmodium vivax metabolism, Spleen metabolism, Spleen parasitology, Malaria, Vivax parasitology, Protozoan Proteins metabolism, Protozoan Proteins genetics
- Abstract
Recent studies indicate that human spleen contains over 95% of the total parasite biomass during chronic asymptomatic infections caused by Plasmodium vivax . Previous studies have demonstrated that extracellular vesicles (EVs) secreted from infected reticulocytes facilitate binding to human spleen fibroblasts (hSFs) and identified parasite genes whose expression was dependent on an intact spleen. Here, we characterize the P. vivax spleen-dependent hypothetical gene (PVX_114580). Using CRISPR/Cas9, PVX_114580 was integrated into P. falciparum 3D7 genome and expressed during asexual stages. Immunofluorescence analysis demonstrated that the protein, which we named P. vivax Spleen-Dependent Protein 1 (PvSDP1), was located at the surface of infected red blood cells in the transgenic line and this localization was later confirmed in natural infections. Plasma-derived EVs from P. vivax -infected individuals (PvEVs) significantly increased cytoadherence of 3D7_PvSDP1 transgenic line to hSFs and this binding was inhibited by anti-PvSDP1 antibodies. Single-cell RNAseq of PvEVs-treated hSFs revealed increased expression of adhesion-related genes. These findings demonstrate the importance of parasite spleen-dependent genes and EVs from natural infections in the formation of intrasplenic niches in P. vivax , a major challenge for malaria elimination., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ayllon-Hermida, Nicolau-Fernandez, Larrinaga, Aparici-Herraiz, Tintó-Font, Llorà-Batlle, Orban, Yasnot, Graupera, Esteller, Popovici, Cortés, del Portillo and Fernandez-Becerra.)
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- 2024
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26. Extracellular vesicles as biomarkers in parasitic disease diagnosis.
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Barnadas-Carceller B, Del Portillo HA, and Fernandez-Becerra C
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- Humans, Animals, Extracellular Vesicles metabolism, Biomarkers metabolism, Biomarkers blood, Parasitic Diseases diagnosis, Parasitic Diseases metabolism, Parasitic Diseases blood, Parasitic Diseases parasitology
- Abstract
Parasitic diseases constitute a major global health problem, affecting millions of people worldwide. Recent advances in the study of extracellular vesicles (EVs) have opened up new strategies for biomarker discovery in protozoan and helminth infections. Analyses of EVs in cultures and biological fluids have identified numerous potential biomarkers that could be useful for early and differential diagnosis, monitoring therapeutic responses, and the overall management and control of these diseases. Despite the potential of these biomarkers, several challenges must be addressed, including limited research, the need for standardized protocols, and the reproducibility of results across studies. In many parasitic infections, EVs have been obtained from various sample types, including plasma from human patients and mouse models, as well as cultures of the parasites at different stages. EVs were isolated by various methods and predominantly characterized through proteomic analysis or RNA sequencing to assess their cargo and identify potential biomarkers. These biomarker candidates were investigated and validated using different assays such as ELISA, Western Blot, and ROC curves. Overall, the use of EVs is considered a promising new diagnostic strategy for parasite infections, but further research with larger cohorts, standardized methods, and additional validation tests are essential for effective diagnosis and management of these diseases., (Copyright © 2024. Published by Elsevier Inc.)
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- 2024
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27. Guidelines for the purification and characterization of extracellular vesicles of parasites.
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Fernandez-Becerra C, Xander P, Alfandari D, Dong G, Aparici-Herraiz I, Rosenhek-Goldian I, Shokouhy M, Gualdron-Lopez M, Lozano N, Cortes-Serra N, Karam PA, Meneghetti P, Madeira RP, Porat Z, Soares RP, Costa AO, Rafati S, da Silva AC, Santarém N, Fernandez-Prada C, Ramirez MI, Bernal D, Marcilla A, Pereira-Chioccola VL, Alves LR, Portillo HD, Regev-Rudzki N, de Almeida IC, Schenkman S, Olivier M, and Torrecilhas AC
- Abstract
Parasites are responsible for the most neglected tropical diseases, affecting over a billion people worldwide (WHO, 2015) and accounting for billions of cases a year and responsible for several millions of deaths. Research on extracellular vesicles (EVs) has increased in recent years and demonstrated that EVs shed by pathogenic parasites interact with host cells playing an important role in the parasite's survival, such as facilitation of infection, immunomodulation, parasite adaptation to the host environment and the transfer of drug resistance factors. Thus, EVs released by parasites mediate parasite-parasite and parasite-host intercellular communication. In addition, they are being explored as biomarkers of asymptomatic infections and disease prognosis after drug treatment. However, most current protocols used for the isolation, size determination, quantification and characterization of molecular cargo of EVs lack greater rigor, standardization, and adequate quality controls to certify the enrichment or purity of the ensuing bioproducts. We are now initiating major guidelines based on the evolution of collective knowledge in recent years. The main points covered in this position paper are methods for the isolation and molecular characterization of EVs obtained from parasite-infected cell cultures, experimental animals, and patients. The guideline also includes a discussion of suggested protocols and functional assays in host cells., Competing Interests: No potential conflicts of interest were reported by the authors., (© 2023 The Authors. Journal of Extracellular Biology published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)
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- 2023
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28. Characterization and Proteomic Analysis of Plasma EVs Recovered from Healthy and Diseased Dogs with Canine Leishmaniosis.
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Esteves S, Lima C, Costa I, Osório H, Fernandez-Becerra C, Santarém N, and Cordeiro-da-Silva A
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- Dogs, Animals, Chromatography, Liquid, Proteomics, Tandem Mass Spectrometry, Biomarkers, Leishmaniasis, Visceral veterinary, Leishmania infantum, Leishmaniasis veterinary, Dog Diseases
- Abstract
Dogs are highly valued companions and work animals that are susceptible to many life-threatening conditions such as canine leishmaniosis (CanL). Plasma-derived extracellular vesicles (EVs), exploited extensively in biomarker discovery, constitute a mostly untapped resource in veterinary sciences. Thus, the definition of proteins associated with plasma EVs recovered from healthy and diseased dogs with a relevant pathogen would be important for biomarker development. For this, we recovered, using size-exclusion chromatography (SEC), EVs from 19 healthy and 20 CanL dogs' plasma and performed proteomic analysis by LC-MS/MS to define their core proteomic composition and search for CanL-associated alterations. EVs-specific markers were identified in all preparations and also non-EVs proteins. Some EVs markers such as CD82 were specific to the healthy animals, while others, such as the Integrin beta 3 were identified in most samples. The EVs-enriched preparations allowed the identification of 529 canine proteins that were identified in both groups, while 465 and 154 were only identified in healthy or CanL samples, respectively. A GO enrichment analysis revealed few CanL-specific terms. Leishmania spp. protein identifications were also found, although with only one unique peptide. Ultimately, CanL-associated proteins of interest were identified and a core proteome was revealed that will be available for intra- and inter-species comparisons.
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- 2023
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29. Mass Spectrometry Identification of Biomarkers in Extracellular Vesicles From Plasmodium vivax Liver Hypnozoite Infections.
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Gualdrón-López M, Díaz-Varela M, Zanghi G, Aparici-Herraiz I, Steel RWJ, Schäfer C, Cuscó P, Chuenchob V, Kangwangransan N, Billman ZP, Olsen TM, González JR, Roobsoong W, Sattabongkot J, Murphy SC, Mikolajczak SA, Borràs E, Sabidó E, Fernandez-Becerra C, Flannery EL, Kappe SHI, and Del Portillo HA
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- Humans, Mice, Animals, Plasmodium vivax, Proteomics, Proteome, Filamins, Liver, Biomarkers, Mass Spectrometry, Malaria, Vivax drug therapy, Malaria, Vivax parasitology, Parasites, Extracellular Vesicles
- Abstract
Latent liver stages termed hypnozoites cause relapsing Plasmodium vivax malaria infection and represent a major obstacle in the goal of malaria elimination. Hypnozoites are clinically undetectable, and presently, there are no biomarkers of this persistent parasite reservoir in the human liver. Here, we have identified parasite and human proteins associated with extracellular vesicles (EVs) secreted from in vivo infections exclusively containing hypnozoites. We used P. vivax-infected human liver-chimeric (huHEP) FRG KO mice treated with the schizonticidal experimental drug MMV048 as hypnozoite infection model. Immunofluorescence-based quantification of P. vivax liver forms showed that MMV048 removed schizonts from chimeric mice livers. Proteomic analysis of EVs derived from FRG huHEP mice showed that human EV cargo from infected FRG huHEP mice contain inflammation markers associated with active schizont replication and identified 66 P. vivax proteins. To identify hypnozoite-specific proteins associated with EVs, we mined the proteome data from MMV048-treated mice and performed an analysis involving intragroup and intergroup comparisons across all experimental conditions followed by a peptide compatibility analysis with predicted spectra to warrant robust identification. Only one protein fulfilled this stringent top-down selection, a putative filamin domain-containing protein. This study sets the stage to unveil biological features of human liver infections and identify biomarkers of hypnozoite infection associated with EVs., Competing Interests: Conflict of interest V. C., S. A. M., and E. L. F. are employed by and/or is a shareholder of Novartis Pharma AG. Other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
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- 2022
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30. Advancing Key Gaps in the Knowledge of Plasmodium vivax Cryptic Infections Using Humanized Mouse Models and Organs-on-Chips.
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Aparici Herraiz I, Caires HR, Castillo-Fernández Ó, Sima N, Méndez-Mora L, Risueño RM, Sattabongkot J, Roobsoong W, Hernández-Machado A, Fernandez-Becerra C, Barrias CC, and Del Portillo HA
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- Animals, Bone Marrow parasitology, Disease Models, Animal, Humans, Mice, Plasmodium vivax, Antimalarials, Malaria drug therapy, Malaria, Vivax prevention & control
- Abstract
Plasmodium vivax is the most widely distributed human malaria parasite representing 36.3% of disease burden in the South-East Asia region and the most predominant species in the region of the Americas. Recent estimates indicate that 3.3 billion of people are under risk of infection with circa 7 million clinical cases reported each year. This burden is certainly underestimated as the vast majority of chronic infections are asymptomatic. For centuries, it has been widely accepted that the only source of cryptic parasites is the liver dormant stages known as hypnozoites. However, recent evidence indicates that niches outside the liver, in particular in the spleen and the bone marrow, can represent a major source of cryptic chronic erythrocytic infections. The origin of such chronic infections is highly controversial as many key knowledge gaps remain unanswered. Yet, as parasites in these niches seem to be sheltered from immune response and antimalarial drugs, research on this area should be reinforced if elimination of malaria is to be achieved. Due to ethical and technical considerations, working with the liver, bone marrow and spleen from natural infections is very difficult. Recent advances in the development of humanized mouse models and organs-on-a-chip models, offer novel technological frontiers to study human diseases, vaccine validation and drug discovery. Here, we review current data of these frontier technologies in malaria, highlighting major challenges ahead to study P. vivax cryptic niches, which perpetuate transmission and burden., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Aparici Herraiz, Caires, Castillo-Fernández, Sima, Méndez-Mora, Risueño, Sattabongkot, Roobsoong, Hernández-Machado, Fernandez-Becerra, Barrias and del Portillo.)
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- 2022
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31. Isolation and molecular characterization of circulating extracellular vesicles from blood of chronic Chagas disease patients.
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Madeira RP, Meneghetti P, de Barros LA, de Cassia Buck P, Mady C, Ianni BM, Fernandez-Becerra C, and Torrecilhas AC
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- Anticoagulants, Biomarkers metabolism, Humans, Chagas Disease, Extracellular Vesicles metabolism, Trypanosoma cruzi metabolism
- Abstract
Extracellular vesicles (EVs) are lipid bilayer envelopes that encase several types of molecules. Their contents mostly reflect their cell origin and possible targets at other locations in the organism and can be modified in pathological conditions to interfere with intercellular communication, thus promoting disease establishment and development. These characteristics, in addition to their presence in virtually all body fluids, make such vesicles ideal for biomarker discovery in human diseases. Here, we describe the effect of different anticoagulants and the combination of two purification methods for isolation and characterization of circulating EVs from blood of chronic Chagas disease (CCD) patients. We illustrated this procedure by studying a population of patients with Chagas disease at the indeterminate chronic stage, in which the Trypanosoma cruzi is very scarce in circulation. EVs were harvested from blood collected without or with different anticoagulants. Protein and nanoparticle tracking analysis was used to measure EVs size and concentration. The EVs were purified by ultracentrifugation, followed by size-exclusion chromatography and characterized by chemiluminescent enzyme-linked immunosorbent assay and dot blot using antibodies that recognized parasite-derived EVs, such as hyperimmune sera, polyclonal and monoclonal antibodies against trans-sialidase and mucins. In parallel, antibodies against classical human EV markers CD9, CD63, CD81, and CD82, were also analyzed. The results showed that anticoagulants did not interfere with the analyzed parameters and circulating EVs from CCD patients contain T. cruzi antigens and classical human exosomal markers. Overall, our protocol is adequate for the isolation of the total circulating EVs and can serve as an important basis for further studies on biomarker discovery in Chagas' disease., (© 2022 International Federation for Cell Biology.)
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- 2022
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32. Cryptic erythrocytic infections in Plasmodium vivax, another challenge to its elimination.
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Fernandez-Becerra C, Aparici-Herraiz I, and Del Portillo HA
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- Animals, Antimalarials therapeutic use, Exosomes parasitology, Humans, Malaria, Vivax drug therapy, Malaria, Vivax epidemiology, Plasmodium vivax, Reticulocytes parasitology, Reticulocytes ultrastructure, Bone Marrow parasitology, Erythrocytes parasitology, Malaria, Vivax blood, Malaria, Vivax prevention & control, Spleen parasitology
- Abstract
Human malaria caused by Plasmodium vivax infection (vivax malaria) is a major global health issue. It is the most geographically widespread form of the disease, accounting for 7 million annual clinical cases, the majority of cases in America and Asia and an estimation of over 2.5 billion people living under risk of infection. The general perception towards vivax malaria has shifted recently, following a series of reports, from being viewed as a benign infection to the recognition of its potential for more severe manifestations including fatal cases. However, the underlying pathogenic mechanisms of vivax malaria remain largely unresolved. Asymptomatic carriers of malaria parasites are a major challenge for malaria elimination. In the case of P. vivax, it has been widely accepted that the only source of cryptic parasites is hypnozoite dormant stages. Here, we will review new evidence indicating that cryptic erythrocytic niches outside the liver, in particular in the spleen and bone marrow, can represent a major source of asymptomatic infections. The origin of such parasites is being controversial and many key gaps in the knowledge of such infections remain unanswered. Yet, as parasites in these niches seem to be sheltered from immune response and antimalarial drugs, research on this area should be reinforced if elimination of malaria is to be achieved. Last, we will glimpse into the role of reticulocyte-derived exosomes, extracellular vesicles of endocytic origin, as intercellular communicators likely involved in the formation of such cryptic erythrocytic infections., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)
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- 2022
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33. Morphological and Transcriptional Changes in Human Bone Marrow During Natural Plasmodium vivax Malaria Infections.
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Brito MAM, Baro B, Raiol TC, Ayllon-Hermida A, Safe IP, Deroost K, Figueiredo EFG, Costa AG, Armengol MDP, Sumoy L, Almeida ACG, Hounkpe BW, De Paula EV, Fernandez-Becerra C, Monteiro WM, Del Portillo HA, and Lacerda MVG
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- Animals, Bone Marrow, Erythropoiesis, Humans, Plasmodium vivax genetics, Anemia, Malaria, Vivax parasitology
- Abstract
Background: The presence of Plasmodium vivax malaria parasites in the human bone marrow (BM) is still controversial. However, recent data from a clinical case and experimental infections in splenectomized nonhuman primates unequivocally demonstrated the presence of parasites in this tissue., Methods: In the current study, we analyzed BM aspirates of 7 patients during the acute attack and 42 days after drug treatment. RNA extracted from CD71+ cell suspensions was used for sequencing and transcriptomic analysis., Results: We demonstrated the presence of parasites in all patients during acute infections. To provide further insights, we purified CD71+ BM cells and demonstrated dyserythropoiesis and inefficient erythropoiesis in all patients. In addition, RNA sequencing from 3 patients showed that genes related to erythroid maturation were down-regulated during acute infections, whereas immune response genes were up-regulated., Conclusions: This study thus shows that during P. vivax infections, parasites are always present in the BM and that such infections induced dyserythropoiesis and ineffective erythropoiesis. Moreover, infections induce transcriptional changes associated with such altered erythropoietic response, thus highlighting the importance of this hidden niche during natural infections., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America.)
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- 2022
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34. Antigen Discovery in Circulating Extracellular Vesicles From Plasmodium vivax Patients.
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Aparici-Herraiz I, Gualdrón-López M, Castro-Cavadía CJ, Carmona-Fonseca J, Yasnot MF, Fernandez-Becerra C, and Del Portillo HA
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- Antibodies, Protozoan, Antigens, Protozoan, Erythrocytes parasitology, Humans, Plasmodium vivax, Protozoan Proteins metabolism, Reticulocytes metabolism, Reticulocytes parasitology, Extracellular Vesicles metabolism, Malaria, Vivax parasitology
- Abstract
Plasmodium vivax is the most widely distributed human malaria parasite with 7 million annual clinical cases and 2.5 billion people living under risk of infection. There is an urgent need to discover new antigens for vaccination as only two vaccine candidates are currently in clinical trials. Extracellular vesicles (EVs) are small membrane-bound vesicles involved in intercellular communication and initially described in reticulocytes, the host cell of P. vivax , as a selective disposal mechanism of the transferrin receptor (CD71) in the maturation of reticulocytes to erythrocytes. We have recently reported the proteomics identification of P. vivax proteins associated to circulating EVs in P. vivax patients using size exclusion chromatography followed by mass spectrometry (MS). Parasite proteins were detected in only two out of ten patients. To increase the MS signal, we have implemented the direct immuno-affinity capture (DIC) technique to enrich in EVs derived from CD71-expressing cells. Remarkably, we identified parasite proteins in all patients totaling 48 proteins and including several previously identified P. vivax vaccine candidate antigens (MSP1, MSP3, MSP7, MSP9, Serine-repeat antigen 1, and HSP70) as well as membrane, cytosolic and exported proteins. Notably, a member of the Plasmodium helical interspersed sub-telomeric (PHIST-c) family and a member of the Plasmodium exported proteins, were detected in five out of six analyzed patients. Humoral immune response analysis using sera from vivax patients confirmed the antigenicity of the PHIST-c protein. Collectively, we showed that enrichment of EVs by CD71-DIC from plasma of patients, allows a robust identification of P. vivax immunogenic proteins. This study represents a significant advance in identifying new antigens for vaccination against this human malaria parasite., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Aparici-Herraiz, Gualdrón-López, Castro-Cavadía, Carmona-Fonseca, Yasnot, Fernandez-Becerra and del Portillo.)
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- 2022
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35. Antibody Profile Comparison against MSP1 Antigens of Multiple Plasmodium Species in Human Serum Samples from Two Different Brazilian Populations Using a Multiplex Serological Assay.
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Monteiro EF, Fernandez-Becerra C, Curado I, Wunderlich G, Hiyane MI, and Kirchgatter K
- Abstract
Plasmodium malariae has a wide geographic distribution, but mainly at very low parasitemias and in co-infections, leading to an underestimated prevalence of this species. Studies for the detection of antibodies against Plasmodium recombinant proteins are increasingly used to map geographical distributions, seroprevalence and transmission intensities of malaria infection. However, no seroepidemiological survey using recombinant P. malariae proteins has been conducted in Brazil. This work evaluated the antibody response in serum samples of individuals from endemic regions of Brazil (the Amazon region and Atlantic Forest) against five recombinant proteins of P. malariae merozoite surface protein 1 (MSP1), and the MSP1 C-terminal portions of P. vivax and P. falciparum , in a multiplex assay. The positivity was 69.5% of samples recognizing at least one MSP1 recombinant protein. The mean of the Reactivity Index for the C-terminal portion of the P. falciparum was significantly higher compared to the other recombinant proteins, followed by the C-terminal of P. vivax and the N-terminal of P. malariae . Among the recombinant P. malariae proteins, the N-terminal of P. malariae showed the highest Reactivity Index alone. This study validates the use of the multiplex assay to measure naturally acquired IgG antibodies against Plasmodium MSP1 proteins and demonstrate that these proteins are important tools for seroepidemiological surveys and could be used in malaria surveillance.
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- 2021
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36. Hidden Biomass of Intact Malaria Parasites in the Human Spleen.
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Kho S, Qotrunnada L, Leonardo L, Andries B, Wardani PAI, Fricot A, Henry B, Hardy D, Margyaningsih NI, Apriyanti D, Puspitasari AM, Prayoga P, Trianty L, Kenangalem E, Chretien F, Safeukui I, Del Portillo HA, Fernandez-Becerra C, Meibalan E, Marti M, Price RN, Woodberry T, Ndour PA, Russell BM, Yeo TW, Minigo G, Noviyanti R, Poespoprodjo JR, Siregar NC, Buffet PA, and Anstey NM
- Subjects
- Adolescent, Adult, Animals, Asymptomatic Infections, Biomass, Child, Female, Humans, Malaria, Falciparum parasitology, Malaria, Vivax parasitology, Male, Splenectomy, Young Adult, Erythrocytes parasitology, Plasmodium falciparum isolation & purification, Plasmodium vivax isolation & purification, Spleen parasitology
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- 2021
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37. Evaluation of splenic accumulation and colocalization of immature reticulocytes and Plasmodium vivax in asymptomatic malaria: A prospective human splenectomy study.
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Kho S, Qotrunnada L, Leonardo L, Andries B, Wardani PAI, Fricot A, Henry B, Hardy D, Margyaningsih NI, Apriyanti D, Puspitasari AM, Prayoga P, Trianty L, Kenangalem E, Chretien F, Brousse V, Safeukui I, Del Portillo HA, Fernandez-Becerra C, Meibalan E, Marti M, Price RN, Woodberry T, Ndour PA, Russell BM, Yeo TW, Minigo G, Noviyanti R, Poespoprodjo JR, Siregar NC, Buffet PA, and Anstey NM
- Subjects
- Adolescent, Adult, Asymptomatic Infections, Female, Humans, Indonesia, Malaria, Vivax parasitology, Malaria, Vivax physiopathology, Male, Middle Aged, New Guinea, Prospective Studies, Young Adult, Plasmodium vivax physiology, Reticulocytes metabolism, Spleen metabolism, Spleen parasitology, Splenectomy statistics & numerical data
- Abstract
Background: A very large biomass of intact asexual-stage malaria parasites accumulates in the spleen of asymptomatic human individuals infected with Plasmodium vivax. The mechanisms underlying this intense tropism are not clear. We hypothesised that immature reticulocytes, in which P. vivax develops, may display high densities in the spleen, thereby providing a niche for parasite survival., Methods and Findings: We examined spleen tissue in 22 mostly untreated individuals naturally exposed to P. vivax and Plasmodium falciparum undergoing splenectomy for any clinical indication in malaria-endemic Papua, Indonesia (2015 to 2017). Infection, parasite and immature reticulocyte density, and splenic distribution were analysed by optical microscopy, flow cytometry, and molecular assays. Nine non-endemic control spleens from individuals undergoing spleno-pancreatectomy in France (2017 to 2020) were also examined for reticulocyte densities. There were no exclusion criteria or sample size considerations in both patient cohorts for this demanding approach. In Indonesia, 95.5% (21/22) of splenectomy patients had asymptomatic splenic Plasmodium infection (7 P. vivax, 13 P. falciparum, and 1 mixed infection). Significant splenic accumulation of immature CD71 intermediate- and high-expressing reticulocytes was seen, with concentrations 11 times greater than in peripheral blood. Accordingly, in France, reticulocyte concentrations in the splenic effluent were higher than in peripheral blood. Greater rigidity of reticulocytes in splenic than in peripheral blood, and their higher densities in splenic cords both suggest a mechanical retention process. Asexual-stage P. vivax-infected erythrocytes of all developmental stages accumulated in the spleen, with non-phagocytosed parasite densities 3,590 times (IQR: 2,600 to 4,130) higher than in circulating blood, and median total splenic parasite loads 81 (IQR: 14 to 205) times greater, accounting for 98.7% (IQR: 95.1% to 98.9%) of the estimated total-body P. vivax biomass. More reticulocytes were in contact with sinus lumen endothelial cells in P. vivax- than in P. falciparum-infected spleens. Histological analyses revealed 96% of P. vivax rings/trophozoites and 46% of schizonts colocalised with 92% of immature reticulocytes in the cords and sinus lumens of the red pulp. Larger splenic cohort studies and similar investigations in untreated symptomatic malaria are warranted., Conclusions: Immature CD71+ reticulocytes and splenic P. vivax-infected erythrocytes of all asexual stages accumulate in the same splenic compartments, suggesting the existence of a cryptic endosplenic lifecycle in chronic P. vivax infection. Findings provide insight into P. vivax-specific adaptions that have evolved to maximise survival and replication in the spleen., Competing Interests: The authors have declared that no competing interests exist.
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- 2021
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38. Multiparameter Flow Cytometry Analysis of the Human Spleen Applied to Studies of Plasma-Derived EVs From Plasmodium vivax Patients.
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Gualdrón-López M, Díaz-Varela M, Toda H, Aparici-Herraiz I, Pedró-Cos L, Lauzurica R, Lacerda MVG, Fernández-Sanmartín MA, Fernandez-Becerra C, and Del Portillo HA
- Subjects
- Flow Cytometry, Humans, Plasmodium vivax, Spleen, Extracellular Vesicles, Malaria, Vivax
- Abstract
The spleen is a secondary lymphoid organ with multiple functions including the removal of senescent red blood cells and the coordination of immune responses against blood-borne pathogens, such as malaria parasites. Despite the major role of the spleen, the study of its function in humans is limited by ethical implications to access human tissues. Here, we employed multiparameter flow cytometry combined with cell purification techniques to determine human spleen cell populations from transplantation donors. Spleen immuno-phenotyping showed that CD45
+ cells included B (30%), CD4+ T (16%), CD8+ T (10%), NK (6%) and NKT (2%) lymphocytes. Myeloid cells comprised neutrophils (16%), monocytes (2%) and DCs (0.3%). Erythrocytes represented 70%, reticulocytes 0.7% and hematopoietic stem cells 0.02%. Extracellular vesicles (EVs) are membrane-bound nanoparticles involved in intercellular communication and secreted by almost all cell types. EVs play several roles in malaria that range from modulation of immune responses to vascular alterations. To investigate interactions of plasma-derived EVs from Plasmodium vivax infected patients (PvEVs) with human spleen cells, we used size-exclusion chromatography (SEC) to separate EVs from the bulk of soluble plasma proteins and stained isolated EVs with fluorescent lipophilic dyes. The integrated cellular analysis of the human spleen and the methodology employed here allowed in vitro interaction studies of human spleen cells and EVs that showed an increased proportion of T cells (CD4+ 3 fold and CD8+ 4 fold), monocytes (1.51 fold), B cells (2.3 fold) and erythrocytes (3 fold) interacting with PvEVs as compared to plasma-derived EVs from healthy volunteers (hEVs). Future functional studies of these interactions can contribute to unveil pathophysiological processes involving the spleen in vivax malaria., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gualdrón-López, Díaz-Varela, Toda, Aparici-Herraiz, Pedró-Cos, Lauzurica, Lacerda, Fernández-Sanmartín, Fernandez-Becerra and del Portillo.)- Published
- 2021
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39. Cryptic Plasmodium chronic infections: was Maurizio Ascoli right?
- Author
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Monteiro W, Brito-Sousa JD, Elizalde-Torrent A, Bôtto-Menezes C, Melo GC, Fernandez-Becerra C, Lacerda M, and Del Portillo HA
- Subjects
- Chronic Disease prevention & control, History, 20th Century, Antimalarials administration & dosage, Asymptomatic Infections, Epinephrine administration & dosage, Malaria prevention & control, Quinine administration & dosage, Spleen drug effects
- Abstract
Cryptic Plasmodium niches outside the liver possibly represent a major source of hypnozoite-unrelated recrudescences in malaria. Maurizio Ascoli, an Italian physician and scientist, suggested that infection was maintained as a result of the persistence of endoerythrocytic parasites in the circulatory bed of some internal organs, mainly the spleen. This would explain a proportion of the recurrences in patients, regardless of the Plasmodium species. Ascoli proposed a method that included the co-administration of adrenaline, in order to induce splenic contraction, and quinine to clear expelled forms in major vessels. Driven by controversy regarding safety and effectiveness, along with the introduction of new drugs, the Ascoli method was abandoned and mostly forgotten by the malaria research community. To date, however, the existence of cryptic parasites outside the liver is gaining supportive data. This work is a historical retrospective of cryptic malaria infections and the Ascoli method, highlighting key knowledge gaps regarding these possible parasite reservoirs.
- Published
- 2020
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40. Extracellular vesicles derived from Plasmodium-infected and non-infected red blood cells as targeted drug delivery vehicles.
- Author
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Borgheti-Cardoso LN, Kooijmans SAA, Chamorro LG, Biosca A, Lantero E, Ramírez M, Avalos-Padilla Y, Crespo I, Fernández I, Fernandez-Becerra C, Del Portillo HA, and Fernàndez-Busquets X
- Subjects
- Drug Delivery Systems, Erythrocytes, Humans, Liposomes, Plasmodium falciparum, Extracellular Vesicles, Plasmodium
- Abstract
Among several factors behind drug resistance evolution in malaria is the challenge of administering overall doses that are not toxic for the patient but that, locally, are sufficiently high to rapidly kill the parasites. Thus, a crucial antimalarial strategy is the development of drug delivery systems capable of targeting antimalarial compounds to Plasmodium with high specificity. In the present study, extracellular vesicles (EVs) have been evaluated as a drug delivery system for the treatment of malaria. EVs derived from naive red blood cells (RBCs) and from Plasmodium falciparum-infected RBCs (pRBCs) were isolated by ultrafiltration followed by size exclusion chromatography. Lipidomic characterization showed that there were no significant qualitative differences between the lipidomic profiles of pRBC-derived EVs (pRBC-EVs) and RBC-derived EVs (RBC-EVs). Both EVs were taken up by RBCs and pRBCs, although pRBC-EVs were more efficiently internalized than RBC-EVs, which suggested their potential use as drug delivery vehicles for these cells. When loaded into pRBC-EVs, the antimalarial drugs atovaquone and tafenoquine inhibited in vitro P. falciparum growth more efficiently than their free drug counterparts, indicating that pRBC-EVs can potentially increase the efficacy of several small hydrophobic drugs used for the treatment of malaria., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)
- Published
- 2020
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41. Plasma-Derived Extracellular Vesicles as Potential Biomarkers in Heart Transplant Patient with Chronic Chagas Disease.
- Author
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Cortes-Serra N, Mendes MT, Mazagatos C, Segui-Barber J, Ellis CC, Ballart C, Garcia-Alvarez A, Gállego M, Gascon J, Almeida IC, Pinazo MJ, and Fernandez-Becerra C
- Subjects
- Biomarkers, Humans, Chagas Disease diagnosis, Extracellular Vesicles, Heart Transplantation adverse effects, Trypanosoma cruzi
- Abstract
Chagas disease is emerging in countries to which it is not endemic. Biomarkers for earlier therapeutic response assessment in patients with chronic Chagas disease are needed. We profiled plasma-derived extracellular vesicles from a heart transplant patient with chronic Chagas disease and showed the potential of this approach for discovering such biomarkers.
- Published
- 2020
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42. State-of-the-art in host-derived biomarkers of Chagas disease prognosis and early evaluation of anti-Trypanosoma cruzi treatment response.
- Author
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Cortes-Serra N, Losada-Galvan I, Pinazo MJ, Fernandez-Becerra C, Gascon J, and Alonso-Padilla J
- Subjects
- Biomarkers blood, Chagas Disease blood, Chagas Disease epidemiology, Chagas Disease parasitology, Chronic Disease drug therapy, Gastrointestinal Tract parasitology, Gastrointestinal Tract pathology, Heart parasitology, Heart physiopathology, Humans, Prognosis, Treatment Outcome, Trypanosoma cruzi pathogenicity, Chagas Disease drug therapy, Host-Parasite Interactions genetics, Nifurtimox therapeutic use, Nitroimidazoles therapeutic use
- Abstract
Chagas disease is caused by infection with the parasite Trypanosoma cruzi, which might lead to a chronic disease state and drive to irreversible damage to the heart and/or digestive tract tissues. Endemic in 21 countries in the Americas, it is the neglected disease with a highest burden in the region. Current estimates point at ~6 million people infected, of which ~30% will progress onto the symptomatic tissue disruptive stage. There is no vaccine but there are two anti-parasitic drugs available: benznidazole and nifurtimox. However, their efficacy is variable at the chronic symptomatic stage and both have frequent adverse effects. Since there are no prognosis markers, drugs should be administered to all T. cruzi-infected individuals in the indeterminate and early symptomatic stages. Nowadays, there are no tests-of-cure either, which greatly undermines patients follow-up and the search of safer and more efficacious drugs. Therefore, the identification and validation of biomarkers of disease progression and/or treatment response on which to develop tests of prognosis and/or cure is a major research priority. Both parasite- and host-derived markers have been investigated. In the present manuscript we present an updated outlook of the latter., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)
- Published
- 2020
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43. Naturally Acquired Humoral Immunity against Malaria Parasites in Non-Human Primates from the Brazilian Amazon, Cerrado and Atlantic Forest.
- Author
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Monteiro EF, Fernandez-Becerra C, Araujo MDS, Messias MR, Ozaki LS, Duarte AMRC, Bueno MG, Catao-Dias JL, Chagas CRF, Mathias BDS, Santos MGD, Santos SV, Holcman MM, Jr JCS, and Kirchgatter K
- Abstract
Non-human primates (NHPs) have been shown to be infected by parasites of the genus Plasmodium , the etiological agent of malaria in humans, creating potential risks of zoonotic transmission. Plasmodium brasilianum , a parasite species similar to P. malariae of humans, have been described in NHPs from Central and South America, including Brazil. The merozoite surface protein 1 (MSP1), besides being a malaria vaccine candidate, is highly immunogenic. Due to such properties, we tested this protein for the diagnosis of parasite infection. We used recombinant proteins of P. malariae MSP1, as well as of P. falciparum and P. vivax , for the detection of antibodies anti-MSP1 of these parasite species, in the sera of NHPs collected in different regions of Brazil. About 40% of the NHP sera were confirmed as reactive to the proteins of one or more parasite species. A relatively higher number of reactive sera was found in animals from the Atlantic Forest than those from the Amazon region, possibly reflecting the former more intense parasite circulation among NHPs due to their proximity to humans at a higher populational density. The presence of Plasmodium positive NHPs in the surveyed areas, being therefore potential parasite reservoirs, needs to be considered in any malaria surveillance program., Competing Interests: The authors declare no conflict of interest.
- Published
- 2020
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44. Plasmodium vivax spleen-dependent genes encode antigens associated with cytoadhesion and clinical protection.
- Author
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Fernandez-Becerra C, Bernabeu M, Castellanos A, Correa BR, Obadia T, Ramirez M, Rui E, Hentzschel F, López-Montañés M, Ayllon-Hermida A, Martin-Jaular L, Elizalde-Torrent A, Siba P, Vêncio RZ, Arevalo-Herrera M, Herrera S, Alonso PL, Mueller I, and Del Portillo HA
- Subjects
- Animals, Antibodies, Viral blood, Antibodies, Viral immunology, Antigens, Protozoan genetics, Aotidae, CHO Cells, Cell Adhesion genetics, Cell Adhesion immunology, Child, Cricetulus, Disease Models, Animal, Fibroblasts, Gene Expression Profiling, Host-Pathogen Interactions genetics, Humans, Malaria, Vivax blood, Malaria, Vivax parasitology, Multigene Family, Papua New Guinea, Plasmodium vivax genetics, Spleen cytology, Spleen parasitology, Splenectomy, Tissue Array Analysis, Antigens, Protozoan immunology, Genes, Protozoan, Malaria, Vivax immunology, Plasmodium vivax immunology, Spleen metabolism
- Abstract
Plasmodium vivax , the most widely distributed human malaria parasite, causes severe clinical syndromes despite low peripheral blood parasitemia. This conundrum is further complicated as cytoadherence in the microvasculature is still a matter of investigations. Previous reports in Plasmodium knowlesi , another parasite species shown to infect humans, demonstrated that variant genes involved in cytoadherence were dependent on the spleen for their expression. Hence, using a global transcriptional analysis of parasites obtained from spleen-intact and splenectomized monkeys, we identified 67 P. vivax genes whose expression was spleen dependent. To determine their role in cytoadherence, two Plasmodium falciparum transgenic lines expressing two variant proteins pertaining to VIR and Pv-FAM-D multigene families were used. Cytoadherence assays demonstrated specific binding to human spleen but not lung fibroblasts of the transgenic line expressing the VIR14 protein. To gain more insights, we expressed five P. vivax spleen-dependent genes as recombinant proteins, including members of three different multigene families (VIR, Pv-FAM-A, Pv-FAM-D), one membrane transporter (SECY), and one hypothetical protein (HYP1), and determined their immunogenicity and association with clinical protection in a prospective study of 383 children in Papua New Guinea. Results demonstrated that spleen-dependent antigens are immunogenic in natural infections and that antibodies to HYP1 are associated with clinical protection. These results suggest that the spleen plays a major role in expression of parasite proteins involved in cytoadherence and can reveal antigens associated with clinical protection, thus prompting a paradigm shift in P. vivax biology toward deeper studies of the spleen during infections., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)
- Published
- 2020
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45. Plasma-derived extracellular vesicles from Plasmodium vivax patients signal spleen fibroblasts via NF-kB facilitating parasite cytoadherence.
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Toda H, Diaz-Varela M, Segui-Barber J, Roobsoong W, Baro B, Garcia-Silva S, Galiano A, Gualdrón-López M, Almeida ACG, Brito MAM, de Melo GC, Aparici-Herraiz I, Castro-Cavadía C, Monteiro WM, Borràs E, Sabidó E, Almeida IC, Chojnacki J, Martinez-Picado J, Calvo M, Armengol P, Carmona-Fonseca J, Yasnot MF, Lauzurica R, Marcilla A, Peinado H, Galinski MR, Lacerda MVG, Sattabongkot J, Fernandez-Becerra C, and Del Portillo HA
- Subjects
- Animals, Cell Adhesion, Cell-Derived Microparticles, Disease Models, Animal, Extracellular Vesicles parasitology, Fibroblasts pathology, Host-Parasite Interactions physiology, Humans, Intercellular Adhesion Molecule-1 metabolism, Malaria, Vivax parasitology, Male, Mice, Mice, Inbred C57BL, Microvessels parasitology, Proteomics, Reticulocytes parasitology, Spleen pathology, Extracellular Vesicles metabolism, Fibroblasts metabolism, NF-kappa B metabolism, Plasma, Plasmodium vivax physiology, Reticulocytes metabolism, Spleen metabolism
- Abstract
Plasmodium vivax is the most widely distributed human malaria parasite. Previous studies have shown that circulating microparticles during P. vivax acute attacks are indirectly associated with severity. Extracellular vesicles (EVs) are therefore major components of circulating plasma holding insights into pathological processes. Here, we demonstrate that plasma-derived EVs from Plasmodium vivax patients (PvEVs) are preferentially uptaken by human spleen fibroblasts (hSFs) as compared to the uptake of EVs from healthy individuals. Moreover, this uptake induces specific upregulation of ICAM-1 associated with the translocation of NF-kB to the nucleus. After this uptake, P. vivax-infected reticulocytes obtained from patients show specific adhesion properties to hSFs, reversed by inhibiting NF-kB translocation to the nucleus. Together, these data provide physiological EV-based insights into the mechanisms of human malaria pathology and support the existence of P. vivax-adherent parasite subpopulations in the microvasculature of the human spleen.
- Published
- 2020
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46. Characterization of Plasmodium vivax Proteins in Plasma-Derived Exosomes From Malaria-Infected Liver-Chimeric Humanized Mice.
- Author
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Gualdrón-López M, Flannery EL, Kangwanrangsan N, Chuenchob V, Fernandez-Orth D, Segui-Barber J, Royo F, Falcón-Pérez JM, Fernandez-Becerra C, Lacerda MVG, Kappe SHI, Sattabongkot J, Gonzalez JR, Mikolajczak SA, and Del Portillo HA
- Abstract
Exosomes are extracellular vesicles of endocytic origin containing molecular signatures implying the cell of origin; thus, they offer a unique opportunity to discover biomarkers of disease. Plasmodium vivax , responsible for more than half of all malaria cases outside Africa, is a major obstacle in the goal of malaria elimination due to the presence of dormant liver stages (hypnozoites), which after the initial infection may reactivate to cause disease. Hypnozoite infection is asymptomatic and there are currently no diagnostic tools to detect their presence. The human liver-chimeric (FRG huHep) mouse is a robust P. vivax infection model for exo-erythrocytic development of liver stages, including hypnozoites. We studied the proteome of plasma-derived exosomes isolated from P. vivax infected FRG huHep mice with the objective of identifying liver-stage expressed parasite proteins indicative of infection. Proteomic analysis of these exosomes showed the presence of 290 and 234 proteins from mouse and human origin, respectively, including canonical exosomal markers. Human proteins include proteins previously detected in liver-derived exosomes, highlighting the potential of this chimeric mouse model to study plasma exosomes derived unequivocally from human hepatocytes. Noticeably, we identified 17 parasite proteins including enzymes, surface proteins, components of the endocytic pathway and translation machinery, as well as uncharacterized proteins. Western blot analysis validated the presence of human arginase-I and an uncharacterized P. vivax protein in plasma-derived exosomes. This study represents a proof-of-principle that plasma-derived exosomes from P. vivax infected FRG-huHep mice contain human hepatocyte and P. vivax proteins with the potential to unveil biological features of liver infection and identify biomarkers of hypnozoite infection.
- Published
- 2018
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47. Production of recombinant PvDBPII, receptor binding domain of Plasmodium vivax Duffy binding protein, and evaluation of immunogenicity to identify an adjuvant formulation for vaccine development.
- Author
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Bhardwaj R, Shakri AR, Hans D, Gupta P, Fernandez-Becerra C, Del Portillo HA, Pandey G, and Chitnis CE
- Subjects
- Animals, Humans, Mice, Mice, Inbred BALB C, Plasmodium vivax immunology, Protein Domains, Recombinant Proteins biosynthesis, Recombinant Proteins immunology, Recombinant Proteins isolation & purification, Antigens, Protozoan biosynthesis, Antigens, Protozoan genetics, Antigens, Protozoan immunology, Antigens, Protozoan isolation & purification, Immunogenicity, Vaccine, Malaria Vaccines biosynthesis, Malaria Vaccines genetics, Malaria Vaccines immunology, Malaria Vaccines isolation & purification, Plasmodium vivax genetics, Protozoan Proteins biosynthesis, Protozoan Proteins genetics, Protozoan Proteins immunology, Protozoan Proteins isolation & purification, Receptors, Cell Surface biosynthesis, Receptors, Cell Surface genetics, Receptors, Cell Surface immunology, Receptors, Cell Surface isolation & purification
- Abstract
Plasmodium vivax is dependent on interaction with the Duffy antigen receptor for chemokines (DARC) for invasion of human erythrocytes. The P. vivax Duffy binding protein (PvDBP) mediates interaction of P. vivax merozoites with DARC. The DARC receptor-binding domain lies in a conserved N-terminal cysteine-rich region of PvDBP referred to as region II (PvDBPII). PvDBPII is an attractive vaccine candidate since antibodies raised against PvDBPII block erythrocyte invasion by P. vivax. Here, we describe methods to produce recombinant PvDBPII in its correctly folded conformation. A synthetic gene optimized for expression of PvDBPII in Escherichia coli and fed batch fermentation process based on exponential feeding strategy was used to achieve high levels of expression of recombinant PvDBPII. Recombinant PvDBPII was isolated from inclusion bodies, refolded by rapid dilution and purified by ion exchange chromatography. Purified recombinant PvDBPII was characterized for identity, purity and functional activity using standardized release assays. Recombinant PvDBPII formulated with various human compatible adjuvants including glycosylpyranosyl lipid A-stable emulsion (GLA-SE) and alhydrogel was used for immunogenicity studies in small animals to downselect a suitable formulation for clinical development. Sera collected from immunized animals were tested for recognition of PvDBPII and inhibition of PvDBPII-DARC binding. GLA-SE formulations of PvDBPII yielded higher ELISA and binding inhibition titres compared to PvDBPII formulated with alhydrogel. These data support further development of a recombinant vaccine for P. vivax based on PvDBPII formulated with GLA-SE., (Copyright © 2017 Elsevier Inc. All rights reserved.)
- Published
- 2017
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48. Size-exclusion chromatography as a stand-alone methodology identifies novel markers in mass spectrometry analyses of plasma-derived vesicles from healthy individuals.
- Author
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de Menezes-Neto A, Sáez MJ, Lozano-Ramos I, Segui-Barber J, Martin-Jaular L, Ullate JM, Fernandez-Becerra C, Borrás FE, and Del Portillo HA
- Abstract
Plasma-derived vesicles hold a promising potential for use in biomedical applications. Two major challenges, however, hinder their implementation into translational tools: (a) the incomplete characterization of the protein composition of plasma-derived vesicles, in the size range of exosomes, as mass spectrometric analysis of plasma sub-components is recognizably troublesome and (b) the limited reach of vesicle-based studies in settings where the infrastructural demand of ultracentrifugation, the most widely used isolation/purification methodology, is not available. In this study, we have addressed both challenges by carrying-out mass spectrometry (MS) analyses of plasma-derived vesicles, in the size range of exosomes, from healthy donors obtained by 2 alternative methodologies: size-exclusion chromatography (SEC) on sepharose columns and Exo-Spin™. No exosome markers, as opposed to the most abundant plasma proteins, were detected by Exo-Spin™. In contrast, exosomal markers were present in the early fractions of SEC where the most abundant plasma proteins have been largely excluded. Noticeably, after a cross-comparative analysis of all published studies using MS to characterize plasma-derived exosomes from healthy individuals, we also observed a paucity of "classical exosome markers." Independent of the isolation method, however, we consistently identified 2 proteins, CD5 antigen-like (CD5L) and galectin-3-binding protein (LGALS3BP), whose presence was validated by a bead-exosome FACS assay. Altogether, our results support the use of SEC as a stand-alone methodology to obtain preparations of extracellular vesicles, in the size range of exosomes, from plasma and suggest the use of CD5L and LGALS3BP as more suitable markers of plasma-derived vesicles in MS.
- Published
- 2015
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49. Extracellular vesicles in parasitic diseases.
- Author
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Marcilla A, Martin-Jaular L, Trelis M, de Menezes-Neto A, Osuna A, Bernal D, Fernandez-Becerra C, Almeida IC, and Del Portillo HA
- Abstract
Parasitic diseases affect billions of people and are considered a major public health issue. Close to 400 species are estimated to parasitize humans, of which around 90 are responsible for great clinical burden and mortality rates. Unfortunately, they are largely neglected as they are mainly endemic to poor regions. Of relevance to this review, there is accumulating evidence of the release of extracellular vesicles (EVs) in parasitic diseases, acting both in parasite-parasite inter-communication as well as in parasite-host interactions. EVs participate in the dissemination of the pathogen and play a role in the regulation of the host immune systems. Production of EVs from parasites or parasitized cells has been described for a number of parasitic infections. In this review, we provide the most relevant findings of the involvement of EVs in intercellular communication, modulation of immune responses, involvement in pathology, and their potential as new diagnostic tools and therapeutic agents in some of the major human parasitic pathogens.
- Published
- 2014
- Full Text
- View/download PDF
50. Expression levels of pvcrt-o and pvmdr-1 are associated with chloroquine resistance and severe Plasmodium vivax malaria in patients of the Brazilian Amazon.
- Author
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Melo GC, Monteiro WM, Siqueira AM, Silva SR, Magalhães BM, Alencar AC, Kuehn A, del Portillo HA, Fernandez-Becerra C, and Lacerda MV
- Subjects
- Adolescent, Adult, Antimalarials therapeutic use, Brazil, Child, Chloroquine therapeutic use, Drug Resistance, Female, Gene Expression, Gene Frequency, Humans, Malaria, Vivax parasitology, Male, Membrane Transport Proteins genetics, Middle Aged, Molecular Sequence Data, Multidrug Resistance-Associated Proteins genetics, Polymorphism, Genetic, Protozoan Proteins genetics, Young Adult, Antimalarials pharmacology, Chloroquine pharmacology, Malaria, Vivax drug therapy, Membrane Transport Proteins metabolism, Multidrug Resistance-Associated Proteins metabolism, Plasmodium vivax metabolism, Protozoan Proteins metabolism
- Abstract
Molecular markers associated with the increase of chloroquine resistance and disease severity in Plasmodium vivax are needed. The objective of this study was to evaluate the expression levels of pvcrt-o and pvmdr-1 genes in a group of patients presenting CQRPv and patients who developed severe complications triggered exclusively by P. vivax infection. Two different sets of patients were included to this comprehensive study performed in the Brazilian Amazon: 1) patients with clinically characterized chloroquine-resistant P. vivax compared with patients with susceptible parasites from in vivo studies and 2) patients with severe vivax malaria compared with patients without severity. Quantitative real-time PCR was performed to compare the transcript levels of two main transporters genes, P. vivax chloroquine resistance transporter (pvcrt-o) and the P. vivax multidrug resistance transporter (pvmdr-1). Twelve chloroquine resistant cases and other 15 isolates from susceptible cases were included in the first set of patients. For the second set, seven patients with P. vivax-attributed severe and 10 mild manifestations were included. Parasites from patients with chloroquine resistance presented up to 6.1 (95% CI: 3.8-14.3) and 2.4 (95% CI: 0.53-9.1) fold increase in pvcrt-o and pvmdr-1 expression levels, respectively, compared to the susceptible group. Parasites from the severe vivax group had a 2.9 (95% CI: 1.1-8.3) and 4.9 (95% CI: 2.3-18.8) fold increase in pvcrt-o and pvmdr-1 expression levels as compared to the control group with mild disease. These findings suggest that chloroquine resistance and clinical severity in P. vivax infections are strongly associated with increased expression levels of the pvcrt-o and pvmdr-1 genes likely involved in chloroquine resistance.
- Published
- 2014
- Full Text
- View/download PDF
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