Abstract Schistosomiasis is a major global health problem caused by blood-dwelling parasitic worms and is currently treated by the mass administration of the drug praziquantel. Appropriate drug treatment strategies are informed by diagnostics that establish the prevalence and intensity of infection, which, in regions of low transmission should be highly sensitive. To identify sensitive new serological markers of Schistosoma mansoni infections, we have compiled a recombinant protein library of 115 parasite cell surface and secreted proteins expressed in mammalian cells. The vast majority of them were shown to be immunoreactive and to contain heat-labile conformational epitopes when tested against pooled human sera from endemic regions. After probing the library against a time series of sera samples from experimental infections in mice, we identified several markers of infection, the majority of which belong to the saposin-domain-containing and cathepsin families of proteins. These new markers will be a valuable tool to detect ongoing and previous S. mansoni infections, including in regions of low transmission. We envisage that this new recombinant protein resource will be used in a wide range of cellular and molecular assays to further our understanding of Schistosoma biology.
No vaccine has yet proven effective against the blood-stages of Plasmodium falciparum, which cause the symptoms and severe manifestations of malaria. We recently found that PfRH5, a P. falciparum-specific protein expressed in merozoites, is efficiently targeted by broadly-neutralizing, vaccine-induced antibodies. Here we show that antibodies against PfRH5 efficiently inhibit the in vitro growth of short-term-adapted parasite isolates from Cambodia, and that the EC(50) values of antigen-specific antibodies against PfRH5 are lower than those against PfAMA1. Since antibody responses elicited by multiple antigens are speculated to improve the efficacy of blood-stage vaccines, we conducted detailed assessments of parasite growth inhibition by antibodies against PfRH5 in combination with antibodies against seven other merozoite antigens. We found that antibodies against PfRH5 act synergistically with antibodies against certain other merozoite antigens, most notably with antibodies against other erythrocyte-binding antigens such as PfRH4, to inhibit the growth of a homologous P. falciparum clone. A combination of antibodies against PfRH4 and basigin, the erythrocyte receptor for PfRH5, also potently inhibited parasite growth. This methodology provides the first quantitative evidence that polyclonal vaccine-induced antibodies can act synergistically against P. falciparum antigens and should help to guide the rational development of future multi-antigen vaccines.
Significance Malaria is caused by a parasite that is deposited in the skin through the bite of an infected mosquito. From the skin, parasites navigate through host tissues where they must locate and invade liver cells. We know that a parasite surface protein called TRAP is important for this process, making it a leading vaccine candidate. TRAP is thought to work by specifically binding a defined host cell surface protein, but its identity has remained a long-standing mystery. Our research has identified an integrin—a class of host cell surface proteins—as a TRAP receptor. This finding provides an important piece of the puzzle relating to TRAP function and may help improve the development of an effective malaria vaccine.
Background. Plasmodium falciparum reticulocyte-binding protein homologue 5 (PfRH5) is a blood-stage parasite protein essential for host erythrocyte invasion. PfRH5-specific antibodies raised in animals inhibit parasite growth in vitro, but the relevance of naturally acquired PfRH5-specific antibodies in humans is unclear.
Abstract Background Monoclonal antibodies with high affinity and selectivity that work on wholemount fixed tissues are valuable reagents to the cell and developmental biologist, and yet isolating them remains a long and unpredictable process. Here we report a rapid and scalable method to select and express recombinant mouse monoclonal antibodies that are essentially equivalent to those secreted by parental IgG-isotype hybridomas. Results Increased throughput was achieved by immunizing mice with pools of antigens and cloning - from small numbers of hybridoma cells - the functionally rearranged light and heavy chains into a single expression plasmid. By immunizing with the ectodomains of zebrafish cell surface receptor proteins expressed in mammalian cells and screening for formalin-resistant epitopes, we selected antibodies that gave expected staining patterns on wholemount fixed zebrafish embryos. Conclusions This method can be used to quickly select several high quality monoclonal antibodies from a single immunized mouse and facilitates their distribution using plasmids.
Introduction: Heterozygous mutations in JAGGED1 (JAG1), encoding a ligand for Notch receptors, have been identified in 70 % of patients with Alagille syndrome (AGS), using PCR-SSCP (single strand conformation polymorphism). These mutations are located in the extracellular and transmembrane domains of the protein and 70 % of them lead to a premature termination codon (PTC). However, only few studies of the modified mRNAs have been performed. Methods: and aims: To improve the molecular diagnosis of AGS, we have performed RT-PCR analyses and sequencing from mRNAs of lymphoblastoid cell lines derived from 11 patients with no mutations identified by PCR-SSCP. In addition, we have studied the mutant transcript levels in 21 cell lines from AGS patients with already identified mutations, and from the tissues of a 23-week-old fetus with AGS. Results: 1/ in 3 of the patients without previously identified mutations, JAG1 mutations were found by RT-PCR analysis, 2/ in the 8 other patients, no mutation was found in the cDNA of JAG1, supporting the hypothesis of genetic heterogeneity, 3/ mutant transcripts were recovered from lymphoblastoid cells of all patients with missense mutations (5) or in-phase deletions (2), and from all but one of the 16 with PTC, 4/ mutant transcripts were present in tissues of the 23 week-old fetus, albeit in different relative amounts; this could originate from tissue specificity of nonsense mediated mRNA decay. Conclusion: We found that some JAGGED1 mutations not detected by PCR-SSCP were identified by RT-PCR analysis on lymphoblastoid cells with no treatment against the nonsense mediated mRNA decay, but that some AGS patients have no mutation in the cDNA of JAGGED1 supporting the hypothesis of genetic heterogeneity. Mutant transcripts were recovered whatever the mutations, even with PTC, suggesting that both haploinsufficiency and a dominant negative effect could be involved in molecular mechanisms of AGS.
Des modifications de certaines proteines de la voie de signalisation de notch, impliquee dans le developpement, provoquent des pathologies humaines. Parmi celles-ci, le syndrome d'alagille (mim118450), associant 5 elements majeurs affectant le foie, le cur, les vertebres, l'il et le visage, est du a des mutations du gene jagged1, codant un ligand des recepteurs notch. Une grande variabilite phenotypique et de nombreux elements associes affectant d'autres organes sont observes. La recherche de mutations a ete effectuee pour 150 patients, permettant l'identification de 92 mutations de jagged1 dont 84 differentes. L'analyse de ces mutations montre une absence de correlation genotype-phenotype. Toutes les mutations sont localisees dans la partie du gene correspondant au domaine extracellulaire de la proteine et sont surtout des mutations non-sens. Cependant, des mutations faux-sens, jusqu'alors peu decrites, ont ete identifiees. L'etude de la transmission demontre le caractere sporadique de la maladie (70% de mutations de novo). Par ailleurs, l'expression du gene jagged1 au cours de l'embryogenese humaine normale a ete etudiee par hybridation in situ. Jagged1 est tres fortement exprime dans le systeme cardiovasculaire, suggerant que certains elements du syndrome (en particulier hepatiques et vertebraux) sont dus a des atteintes ischemiques. Hormis le systeme nerveux, une correlation a ete montree entre les territoires d'expression de jagged1 et les elements majeurs et mineurs du syndrome. Ces resultats et l'observation frequente d'une atteinte renale chez les patients conduisent a redefinir le syndrome en considerant l'atteinte renale comme le sixieme element majeur et en prenant en compte les elements associes dans le diagnostic. Differentes hypotheses concernant les mecanismes physiopathologiques responsables du syndrome sont abordees dans la discussion.
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