High-Throughput Assay for the Identification of Compounds Regulating Osteogenic Differentiation of Human Mesenchymal Stromal Cells

Human mesenchymal stromal cells are regarded as the golden standard for cell-based therapies. They present multilineage differentiation potential and trophic and immunosuppressive abilities, making them the best candidate for clinical applications. Several molecules have been described to increase bone formation and were mainly discovered by candidate approaches towards known signaling pathways controlling osteogenesis. However, their bone forming potential is still limited, making the search for novel molecules a necessity. High-throughput screening (HTS) not only allows the screening of a large number of diverse chemical compounds, but also allows the discovery of unexpected signaling pathways and molecular mechanisms for a certain application, even without the prior knowledge of the full molecular pathway. Typically HTS is performed in cell lines, however, in this manuscript we have performed a phenotypical screen on more clinically relevant human mesenchymal stromal cells, as a proof of principle that HTS can be performed in those cells and can be used to find small molecules that impact stem cell fate. From a library of pharmacologically active small molecules, we were able to identify novel compounds with increased osteogenic activity. These compounds allowed achieving levels of bone-specific alkaline phosphatase higher than any other combination previously known. By combining biochemical techniques, we were able to demonstrate that a medium to high-throughput phenotypic assay can be performed in academic research laboratories allowing the discovery of novel molecules able to enhance stem cell differentiation

An inter-laboratory comparison of standard membrane-feeding assays for evaluation of malaria transmission-blocking vaccines.

BACKGROUND: An effective malaria transmission-blocking vaccine may play an important role in malaria elimination efforts, and a robust biological assay is essential for its development. The standard membrane-feeding assay (SMFA) for Plasmodium falciparum infection of mosquitoes is considered a "gold standard" assay to measure transmission-blocking activity of test antibodies, and has been utilized widely in both non-clinical and clinical studies. While several studies have discussed the inherent variability of SMFA within a study group, there has been no assessment of inter-laboratory variation. Therefore, there is currently no assurance that SMFA results are comparable between different studies. METHODS: Mouse anti-Pfs25 monoclonal antibody (mAb, 4B7 mAb), rat anti-Pfs48/45 mAb (85RF45.1 mAb) and a human polyclonal antibody (pAb) collected from a malaria-exposed adult were tested at the same concentrations (6-94 μg/mL for 4B7, 1.2-31.3 μg/mL for 85RF45.1 and 23-630 μg/mL for human pAb) in two laboratories following their own standardized SMFA protocols. The mAbs and pAb, previously shown to have strong inhibition activities in the SMFA, were tested at three or four concentrations in two or three independent assays in each laboratory, and percent inhibition in mean oocyst intensity relative to a control in the same feed was determined in each feeding experiment. RESULTS: Both monoclonal and polyclonal antibodies dose-dependently reduced oocyst intensity in all experiments performed at the two test sites. In both laboratories, the inter-assay variability in percent inhibition in oocyst intensity decreased at higher levels of inhibition, regardless of which antibody was tested. At antibody concentrations that led to a >80 % reduction in oocyst numbers, the inter-laboratory variations were in the same range compared with the inter-assay variation observed within a single laboratory, and the differences in best estimates from multiple feeds between the two laboratories were <5 percentage points. CONCLUSIONS: This study confirms previous reports that the precision of the SMFA increases with increasing percent inhibition. Moreover, the variation between the two laboratories is not greater than the variation observed within a laboratory. The findings of this study provide guidance for comparison of SMFA data from different laboratories

Salinomycin and Other Ionophores as a New Class of Antimalarial Drugs with Transmission-Blocking Activity

The drug target profile proposed by the Medicines for Malaria Venture for a malaria elimination/eradication policy focuses on molecules active on both asexual and sexual stages of Plasmodium, thus with both curative and transmission-blocking activities. The aim of the present work was to investigate whether the class of monovalent ionophores, which includes drugs used in veterinary medicine and that were recently proposed as human anticancer agents, meets these requirements. The activity of salinomycin, monensin, and nigericin on Plasmodium falciparum asexual and sexual erythrocytic stages and on the development of the Plasmodium berghei and P. falciparum mosquito stages is reported here. Gametocytogenesis of the P. falciparum strain 3D7 was induced in vitro, and gametocytes at stage II and III or stage IV and V of development were treated for different lengths of time with the ionophores and their viability measured with the parasite lactate dehydrogenase (pLDH) assay. The monovalent ionophores efficiently killed both asexual parasites and gametocytes with a nanomolar 50% inhibitory concentration (IC50). Salinomycin showed a fast speed of kill compared to that of standard drugs, and the potency was higher on stage IV and V than on stage II and III gametocytes. The ionophores inhibited ookinete development and subsequent oocyst formation in the mosquito midgut, confirming their transmission-blocking activity. Potential toxicity due to hemolysis was excluded, since only infected and not normal erythrocytes were damaged by ionophores. Our data strongly support the downstream exploration of monovalent ionophores for repositioning as new antimalarial and transmission-blocking leads

Generation of a genetically modified chimeric plasmodium falciparum parasite expressing plasmodium vivax circumsporozoite protein for malaria vaccine development

Copyright © 2020 Miyazaki, Marin-Mogollon, Imai, Mendes, van der Laak, Sturm, Geurten, Miyazaki, Chevalley-Maurel, Ramesar, Kolli, Kroeze, van Schuijlenburg, Salman, Wilder, Reyes-Sandoval, Dechering, Prudencio, Janse, Khan and ̂ Franke-Fayard. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Chimeric rodent malaria parasites with the endogenous circumsporozoite protein (csp) gene replaced with csp from the human parasites Plasmodium falciparum (Pf) and P. vivax (Pv) are used in preclinical evaluation of CSP vaccines. Chimeric rodent parasites expressing PfCSP have also been assessed as whole sporozoite (WSP) vaccines. Comparable chimeric P. falciparum parasites expressing CSP of P. vivax could be used both for clinical evaluation of vaccines targeting PvCSP in controlled human P. falciparum infections and in WSP vaccines targeting P. vivax and P. falciparum. We generated chimeric P. falciparum parasites expressing both PfCSP and PvCSP. These Pf-PvCSP parasites produced sporozoite comparable to wild type P. falciparum parasites and expressed PfCSP and PvCSP on the sporozoite surface. Pf-PvCSP sporozoites infected human hepatocytes and induced antibodies to the repeats of both PfCSP and PvCSP after immunization of mice. These results support the use of Pf-PvCSP sporozoites in studies optimizing vaccines targeting PvCSP.CM-M was, in part, supported by Colciencias Ph.D. fellowship (Call 568 from 2012 Resolution 01218 Bogotá, Colombia). TI was, in part, supported by Uehara Memorial Foundation grant. Work performed at IMM was supported by Fundação para a Ciência e Tecnologia (FCT-Portugal)’s grants PTDC/BBB-BMD/2695/2014 and PTDC-SAU-INF-29550-2017. AR-S is supported by the MRC-DPFS grant MR/N019008/1.info:eu-repo/semantics/publishedVersio

Functional antibodies against Plasmodium falciparum sporozoites are associated with a longer time to qPCR-detected infection among schoolchildren in Burkina Faso.

Background: Individuals living in malaria-endemic regions develop immunity against severe malaria, but it is unclear whether immunity against pre-erythrocytic stages that blocks initiation of blood-stage infection after parasite inoculation develops following continuous natural exposure. Methods: We cleared schoolchildren living in an area (health district of Saponé, Burkina Faso) with highly endemic seasonal malaria of possible sub-patent infections and examined them weekly for incident infections by nested PCR. Plasma samples collected at enrolment were used to quantify antibodies to the pre-eryhrocytic-stage antigens circumsporozoite protein (CSP) and Liver stage antigen 1 (LSA-1). In vitro sporozoite gliding inhibition and hepatocyte invasion inhibition by naturally acquired antibodies were assessed using Plasmodium falciparum NF54 sporozoites. Associations between antibody responses, functional pre-erythrocytic immunity phenotypes and time to infection detected by 18S quantitative PCR were studied. Results: A total of 51 children were monitored. Anti-CSP antibody titres showed a positive association with sporozoite gliding motility inhibition (P<0.0001, Spearman's ρ=0.76). In vitro hepatocyte invasion was inhibited by naturally acquired antibodies (median inhibition, 19.4% [IQR 15.2-40.9%]), and there were positive correlations between invasion inhibition and gliding inhibition (P=0.005, Spearman's ρ=0.67) and between invasion inhibition and CSP-specific antibodies (P=0.002, Spearman's ρ=0.76). Survival analysis indicated longer time to infection in individuals displaying higher-than-median sporozoite gliding inhibition activity (P=0.01), although this association became non-significant after adjustment for blood-stage immunity (P = 0.06). Conclusions: In summary, functional antibodies against the pre-erythrocytic stages of malaria infection are acquired in children who are repeatedly exposed to Plasmodium parasites. This immune response does not prevent them from becoming infected during a malaria transmission season, but might delay the appearance of blood stage parasitaemia. Our approach could not fully separate the effects of pre-erythrocytic-specific and blood-stage-specific antibody-mediated immune responses in vivo; epidemiological studies powered and designed to address this important question should become a research priority

Creation and preclinical evaluation of genetically attenuated malaria parasites arresting growth late in the liver.

Whole-sporozoite (WSp) malaria vaccines induce protective immune responses in animal malaria models and in humans. A recent clinical trial with a WSp vaccine comprising genetically attenuated parasites (GAP) which arrest growth early in the liver (PfSPZ-GA1), showed that GAPs can be safely administered to humans and immunogenicity is comparable to radiation-attenuated PfSPZ Vaccine. GAPs that arrest late in the liver stage (LA-GAP) have potential for increased potency as shown in rodent malaria models. Here we describe the generation of four putative P. falciparum LA-GAPs, generated by CRISPR/Cas9-mediated gene deletion. One out of four gene-deletion mutants produced sporozoites in sufficient numbers for further preclinical evaluation. This mutant, PfΔmei2, lacking the mei2-like RNA gene, showed late liver growth arrest in human liver-chimeric mice with human erythrocytes, absence of unwanted genetic alterations and sensitivity to antimalarial drugs. These features of PfΔmei2 make it a promising vaccine candidate, supporting further clinical evaluation. PfΔmei2 (GA2) has passed regulatory approval for safety and efficacy testing in humans based on the findings reported in this study