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Rottenberg, P., M. Freret, S. Calbo, T. Lequerré, and O. Vittecoq. "Effet de l’alpha-énolase sur la différenciation des monocytes in vitro." Revue du Rhumatisme 83 (November 2016): A143. http://dx.doi.org/10.1016/s1169-8330(16)30298-8.
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Mandelbaum, Jacqueline. "Différenciation in vitro d’ovocytes fonctionnels à partir de souris mâles [1]." Médecine de la Reproduction 25, no. 2 (June 2023): 195–200. http://dx.doi.org/10.1684/mte.2023.0953.
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RICCOBONO, D., E. ROTA GRAZIOSI, S. FRANÇOIS, M. GAUTHIER, M. DROUET, and N. JULLIEN. "Régénération musculaire après une irradiation localisée à forte dose." Revue Médecine et Armées, Volume 50, Numéro 2 (June 6, 2024): 87–100. http://dx.doi.org/10.17184/eac.8641.
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Ces vingt dernières années, une augmentation des accidents d'irradiation localisée à forte dose a été constatée, nécessitant le développement de nouvelles contre-mesures médicales pour assurer le traitement difficile des conséquences des rayonnements. L’exposition de la peau, mais aussi de la musculature sous-jacente, entraîne le développement d’un syndrome cutané radio-induit. Actuellement, le traitement de référence consiste en une chirurgie complétée par thérapie cellulaire ; ce qui améliore la survie fonctionnelle et globale du patient, mais laisse subsister un défaut musculaire limitant la mobilité, la motricité et la qualité de vie. L’identification de nouvelles cibles thérapeutiques s’avère donc indispensable pour améliorer la régénération musculaire post-irradiation. Ainsi, ce projet vise à évaluer la modulation de la voie de signalisation Hedgehog comme potentielle stratégie médicale. Une étude in vitro a permis d'analyser la prolifération, la différenciation, la survie et les variations d'expression génique et protéique des cellules musculaires C2C12 irradiées et traitées avec deux conditionnements post-irradiation (prolifération ou différenciation), en présence d'un agoniste (Sonic Hedgehog) ou d'un antagoniste de la voie Hedgehog (la cyclopamine). Il a été mis en évidence que l'irradiation présente un impact négatif sur les mécanismes de prolifération et de différenciation des C2C12 qui peuvent être modulées par la voie Hedgehog. En effet, son activation permet de stimuler la survie et la prolifération cellulaire alors que son inhibition favorise la différenciation cellulaire. Ces résultats suggèrent qu'un traitement séquentiel des lésions radio-induites pourrait être envisagé, consistant 1- en l'activation de la voie Hedgehog pour permettre à un nombre suffisant de précurseurs myogéniques de survivre et de proliférer, suivi 2- d'une inhibition favorisant leur différenciation et donc la régénération musculaire. L'efficacité thérapeutique de ce traitement séquentiel in vivo reste à être évaluée, ce qui constitue la dernière étape de ce projet.
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Nakazawa, Shusuke, Takashi Maoka, Haruki Uemura, Yoshihiro Ito, and Hiroji Kanbara. "Malaria Parasites Giving Rise to Recrudescence In Vitro." Antimicrobial Agents and Chemotherapy 46, no. 4 (April 2002): 958–65. http://dx.doi.org/10.1128/aac.46.4.958-965.2002.
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ABSTRACT Recrudescences were simulated in vitro with drug treatment to examine how drug-sensitive parasites survive the treatment. Various numbers of cultured parasites were treated with lethal doses of pyrimethamine or mefloquine for various lengths of time. Recrudescences were observed in parasite populations with larger initial numbers of parasites when the treatment duration was prolonged. Equal numbers of parasitized erythrocytes were treated with various concentrations of pyrimethamine or mefloquine. There was no clear linear relationship between the incidence of recrudescence and the drug concentration. Parasites that had recrudesced were continuously allowed to recrudesce in the succeeding recrudescence experiments. Both the duration from the cessation of treatment to the time at which the recrudescent parasitemia level reached 1% and the growth rate of recrudescent parasites were equal among these recrudescences. The recrudescent parasites in these experiments were as sensitive to the drugs as the parasites tested before treatment were. These results suggest that a parasite culture may contain parasites in some phases that are not killed by drug for up to 10 days, which explains the recrudescences that occur even after treatment.
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Kocken, Clemens H. M., Hastings Ozwara, Annemarie van der Wel, Annette L. Beetsma, Jason M. Mwenda, and Alan W. Thomas. "Plasmodium knowlesi Provides a Rapid In Vitro and In Vivo Transfection System That Enables Double-Crossover Gene Knockout Studies." Infection and Immunity 70, no. 2 (February 2002): 655–60. http://dx.doi.org/10.1128/iai.70.2.655-660.2002.
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ABSTRACT Transfection technology for malaria parasites provides a valuable tool for analyzing gene function and correlating genotype with phenotype. Transfection models are even more valuable when appropriate animal models are available in addition to complete in vitro systems to be able to fully analyze parasite-host interactions. Here we describe the development of such a model by using the nonhuman primate malaria Plasmodium knowlesi. Blood-stage parasites were adapted to long-term in vitro culture. In vitro-adapted parasites could readapt to in vivo growth and regain wild-type characteristics after a single passage through an intact rhesus monkey. P. knowlesi parasites, either in vitro adapted or in vivo derived, were successfully transfected to generate circumsporozoite protein (CSP) knockout parasites by double-crossover mechanisms. In vitro-transfected and cloned CSP knockout parasites were derived in a time span of only 18 days. Microscopic evaluation of developing oocysts from mosquitoes that had fed on CSP knockout parasites confirmed the impairment of sporozoite formation observed in P. berghei CSP knockout parasites. The P. knowlesi model currently is the only malaria system that combines rapid and precise double-crossover genetic manipulation procedures with complete in vitro as well as in vivo possibilities. This allows for full analysis of P. knowlesi genotype-phenotype relationships and host-parasite interactions in a system closely related to humans.
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Bouthors, Charlie, Charles-Henri Flouzat-lachaniette, Béatrice Laurent, Jérôme Allain, Hélène Rouard, and Nicolas Jullien. "Stimulation des cellules stromales mésenchymateuses humaines vers une différenciation nucléopulpogénique in vitro." Revue de Chirurgie Orthopédique et Traumatologique 100, no. 7 (November 2014): S316. http://dx.doi.org/10.1016/j.rcot.2014.09.256.
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Sereno, D., A. Monte Alegre, R. Silvestre, B. Vergnes, and A. Ouaissi. "In Vitro Antileishmanial Activity of Nicotinamide." Antimicrobial Agents and Chemotherapy 49, no. 2 (February 2005): 808–12. http://dx.doi.org/10.1128/aac.49.2.808-812.2005.
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ABSTRACT Our study represents the first report demonstrating the antileishmanial activity of nicotinamide (NAm), a form of vitamin B3. A 5 mM concentration of NAm significantly inhibited the intracellular growth of Leishmania amastigotes and the NAD-dependent deacetylase activity carried by parasites overexpressing Leishmania major SIR2 (LmSIR2). However, the transgenic parasites were as susceptible as the wild-type parasites to NAm-induced cell growth arrest. Therefore, we conclude that NAm inhibits leishmanial growth and that overexpression of LmSIR2 does not overcome this inhibition. The mechanism of the inhibition is not defined but may include other in vivo targets. NAm may thus represent a new antileishmanial agent which could potentially be used in combination with other drugs during therapy.
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BARTLEY, P. M., S. WRIGHT, J. SALES, F. CHIANINI, D. BUXTON, and E. A. INNES. "Long-term passage of tachyzoites in tissue culture can attenuate virulence of Neospora caninum in vivo." Parasitology 133, no. 4 (June 9, 2006): 421–32. http://dx.doi.org/10.1017/s0031182006000539.
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To determine whether prolonged in vitro passage would result in attenuation of virulence in vivo, Neospora caninum tachyzoites were passaged for different lengths of time in vitro and compared for their ability to cause disease in mice. Groups of Balb/c mice were inoculated intraperitoneally with 5×106 or 1×107 of low-passage or high-passage N. caninum tachyzoites. The mice were monitored for changes in their demeanour and body weight, and were culled when severe clinical symptoms of murine neosporosis were observed. Mice inoculated with the high-passage parasites survived longer (P<0·05), and showed fewer clinical symptoms of murine neosporosis, compared to the mice receiving the low-passage parasites. The parasite was detected in the brains of inoculated mice using immunohistochemistry and ITS1 PCR. Tissue cysts containing parasites were seen in mice inoculated with both low-passage and high-passage parasites. When the in vitro growth rates of the parasites were compared, the high-passage parasites initially multiplied more rapidly (P<0·001) than the low-passage parasites, suggesting that the high-passage parasites had become more adapted to tissue culture. These results would suggest that it is possible to attenuate the virulence of N. caninum tachyzoites in mice through prolonged in vitro passage.
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Visvesvara, Govinda S., and Lynne S. Garcia. "Culture of Protozoan Parasites." Clinical Microbiology Reviews 15, no. 3 (July 2002): 327–28. http://dx.doi.org/10.1128/cmr.15.3.327-328.2002.
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SUMMARY The in vitro culture of protozoan parasites involves highly complex procedures, which are subject to many variables. These parasites have very complex life cycles and, depending on the life cycle stage, may require different culture parameters. However, in vitro cultivation is important for many reasons, some of which include: diagnosis, antigen and antibody production, assessment of parasite immune modulating capabilities, drug screening, improvements in chemotherapy, differentiation of clinical isolates, determination of strain differences, vaccine production, development of attenuated strains, and the continued supply of viable organisms for studying host-parasite interactions.
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Bazin, Hervé. "L’histoire des vaccinations. 2e partie : des vaccins pastoriens aux vaccins modernes." Bulletin de la Société Française d'Histoire de la Médecine et des Sciences Vétérinaires 13, no. 1 (2013): 45–63. https://doi.org/10.3406/bhsv.2013.1146.
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Cette présentation concerne la mise en route des vaccins à virulence atténuée par Pasteur et son équipe, donnant l’espoir d’obtenir un vaccin pour chaque maladie infectieuse. De nombreuses techniques ont été mises en oeuvre : vaccins chimiques, sérothérapie, séro-vaccination, vaccins tués (microbes) ou inactivés (virus), auto-vaccins, anatoxines, irradiation de parasites… pour les vaccins de première génération. La naissance de la biologie moléculaire et du génie génétique en microbiologie et en immunologie a conduit à une explosion de vaccins OGM dont les premiers exemples sont le vaccin hépatite B (production d’un ou plusieurs antigènes d’un agent pathogène dans un système d’expression : cellules eucaryotes, levures…), le vaccin vaccine-rage (expression d’antigènes dans un vecteur…) mais aussi, des vaccins à propriétés nouvelles d’emploi comme le premier vaccin à marqueur, appelé DIVA pour « Différenciation des animaux infectés de ceux vaccinés ».
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Yarlett, Nigel, Mary Morada, Deborah A. Schaefer, Kevin Ackman, Elizabeth Carranza, Rodrigo de Paula Baptista, Michael W. Riggs, and Jessica Kissinger. "Genomic and virulence analysis of in vitro cultured Cryptosporidium parvum." PLOS Pathogens 20, no. 2 (February 28, 2024): e1011992. http://dx.doi.org/10.1371/journal.ppat.1011992.
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Recent advances in the in vitro cultivation of Cryptosporidium parvum using hollow fiber bioreactor technology (HFB) have permitted continuous growth of parasites that complete all life cycle stages. The method provides access to all stages of the parasite and provides a method for non-animal production of oocysts for use in clinical trials. Here we examined the effect of long-term (>20 months) in vitro culture on virulence-factors, genome conservation, and in vivo pathogenicity of the host by in vitro cultured parasites. We find low-level sequence variation that is consistent with that observed in calf-passaged parasites. Further using a calf model infection, oocysts obtained from the HFB caused diarrhea of the same volume, duration and oocyst shedding intensity as in vivo passaged parasites.
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Nsobya, Samuel L., Moses Kiggundu, Sarah Nanyunja, Moses Joloba, Bryan Greenhouse, and Philip J. Rosenthal. "In Vitro Sensitivities of Plasmodium falciparum to Different Antimalarial Drugs in Uganda." Antimicrobial Agents and Chemotherapy 54, no. 3 (January 11, 2010): 1200–1206. http://dx.doi.org/10.1128/aac.01412-09.
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ABSTRACT The control of malaria is challenged by resistance of Plasmodium falciparum to multiple drugs. New combination regimens are now advocated for the treatment of uncomplicated falciparum malaria, but the extent of resistance to newer agents is incompletely understood. We measured the in vitro sensitivity of P. falciparum parasites cultured from children enrolled in a drug efficacy trial in Kampala, Uganda, from 2006 to 2008. Sensitivities were measured by comparing levels of histidine-rich protein-2 in parasites incubated with different concentrations of drugs with those in untreated controls. The cultured parasites exhibited a wide range of sensitivities to chloroquine (CQ); monodesethylamodiaquine (MDAQ), the major active metabolite of amodiaquine; and quinine (QN). Mean 50% inhibitory concentration (IC50) results were above standard cutoffs for resistance for CQ and MDAQ. Parasites were generally sensitive to dihydroartemisinin (DHA), lumefantrine (LM), and piperaquine (PQ). For CQ, MDAQ, and QN but not the other drugs, activities against individual strains were highly correlated. We also assessed known resistance-mediating polymorphisms in two putative transporters, pfcrt and pfmdr1. When parasites that were least and most sensitive to each drug were compared, the pfmdr1 86Y mutation was significantly more common in parasites that were most resistant to CQ and MDAQ, and the pfmdr1 D1246Y mutation was significantly more common in parasites that were most resistant to MDAQ and QN. In summary, we demonstrated in parasites from Kampala a range of sensitivities to older drugs; correlation of sensitivities to CQ, MDAQ, and QN; and good activity against nearly all strains for DHA, LM, and PQ.
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Luce, Eléanor, Antonietta Messina, Amandine Caillaud, Karim Si-Tayeb, Bertrand Cariou, Etienne Bur, Anne Dubart-Kupperschmitt, and Jean-Charles Duclos-Vallée. "Les organoïdes hépatiques." médecine/sciences 37, no. 10 (October 2021): 902–9. http://dx.doi.org/10.1051/medsci/2021119.
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L’étude et la compréhension de l’organogenèse du foie ont permis le développement de protocoles de différenciation des cellules souches pluripotentes afin de pallier le manque de cellules primaires, offrant ainsi une source quasi illimitée de cellules hépatiques. La différenciation de ces cellules dans des systèmes de culture conventionnels en deux dimensions (2D) ayant cependant montré ses limites, des organoïdes hépatiques ont été dérivés de cellules souches pluripotentes humaines et représentent désormais une alternative prometteuse. Ces structures 3D, complexes et organisées, intégrant un ou plusieurs types cellulaires, permettent de reproduire in vitro une ou plusieurs fonctions de l’organe, et ouvrent ainsi la voie à de nombreuses applications, comme l’étude du développement du foie, la production en masse de cellules hépatiques fonctionnelles pour la transplantation ou le développement de foies bioartificiels, sans oublier la modélisation de pathologies hépatiques permettant le criblage à haut débit de médicaments ou des études de toxicité. Des enjeux économiques et éthiques doivent également être pris en considération avant une utilisation de ces organoïdes pour des applications thérapeutiques.
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Cudjoe, Elizabeth, Dickson Donu, Ruth E. Okonu, Jones A. Amponsah, and Linda E. Amoah. "The In Vitro Antiplasmodial Activities of Aqueous Extracts of Selected Ghanaian Herbal Plants." Journal of Parasitology Research 2020 (May 20, 2020): 1–8. http://dx.doi.org/10.1155/2020/5041919.
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Background. The asexual and sexual stages (gametocytes) of Plasmodium falciparum parasites are known to respond differently to antimalarial drugs. Herbal products with extended treatment regimens and inadequate dosing information are widely used to treat malaria in Ghana. This study set out to determine the in vitro activity of selected herbal extracts on the development of asexual and sexual stage malaria parasites. Methods. The 72-hour SYBR Green 1-based in vitro drug assay was used to determine the asexual parasite growth inhibitory effects exhibited by aqueous extracts of Alchornea cordifolia, Polyalthia longifolia, Moringa oleifera, and Mangifera indica on the NF54, CamWT_C580Y, and IPC 4912 strains of Plasmodium falciparum. The effects of exposure of asexual and early-stage NF54 gametocytes to varying concentrations of the aqueous herbal extracts were assessed by microscopy after 7 days of continuous culturing in the presence of the herbal extract. Qualitative and quantitative phytochemical screening were also performed on the herbal extracts. Results. In the SYBR Green 1 assay, aqueous extracts of Alchornea cordifolia exhibited moderate (IC50 of 5.8, 17.4, and 15.8 μg/ml) and Mangifera indica exhibited low (IC50 of 65.4, 96.7, and 81.7 μg/ml) activities against the NF54, Cam WT_C580Y, and IPC 4912 parasites, respectively, whilst Polyalthia longifolia and Moringa oleifera were inactive. Long-term treatment of NF54 parasites with 1 mg/ml of Polyalthia longifolia produced the highest densities of gametocytes and the least (56%) inhibition of asexual parasites on Day 7. Long-term treatment of NF54 parasites with 10 μg/ml Alchornea cordifolia resulted in complete parasite (asexual and gametocyte) clearance on Day 7. Conclusions. Alchornea cordifolia exhibited a ‘moderate’ activity against the three parasites tested in the 72-hour SYBR Green 1 assay and also effectively cleared both asexual parasites and gametocytes. Long-term treatment of malaria parasites with herbal extracts mimics a treatment regimen and should be used to determine the antimalarial properties of herbal extracts.
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Sanchez, C., M. Deberg, P. Msika, C. Baudoin, and Y. Henrotin. "Étude de conditions in vitro favorables à la différenciation hypertrophique des chondrocytes articulaires arthrosiques." Revue du Rhumatisme 74, no. 10-11 (November 2007): 1085. http://dx.doi.org/10.1016/j.rhum.2007.10.121.
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Fressinaud, C., and J. Eyer. "Les peptides des neurofilaments liant la tubuline accroissent la différenciation des oligodendrocytes in vitro." Revue Neurologique 169 (April 2013): A104—A105. http://dx.doi.org/10.1016/j.neurol.2013.01.250.
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Arrowood, Michael J. "In Vitro Cultivation of Cryptosporidium Species." Clinical Microbiology Reviews 15, no. 3 (July 2002): 390–400. http://dx.doi.org/10.1128/cmr.15.3.390-400.2002.
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SUMMARY The in vitro cultivation of protozoan parasites of the genus Cryptosporidium has advanced significantly in recent years. These obligate, intracellular parasites colonize the epithelium of the digestive and respiratory tracts, are often difficult to obtain in significant numbers, produce durable oocysts that defy conventional chemical disinfection methods, and are persistently infectious when stored at refrigerated temperatures (4 to 8°C). While continuous culture and efficient life cycle completion (oocyst production) have not yet been achieved in vitro, routine methods for parasite preparation and cell culture infection and assays for parasite life cycle development have been established. Parasite yields may be limited, but in vitro growth is sufficient to support a variety of research studies, including assessing potential drug therapies, evaluating oocyst disinfection methods, and characterizing life cycle stage development and differentiation.
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Genetu Bayih, Abebe, Anjan Debnath, Edward Mitre, Christopher D. Huston, Benoît Laleu, Didier Leroy, Benjamin Blasco, et al. "Susceptibility Testing of Medically Important Parasites." Clinical Microbiology Reviews 30, no. 3 (April 26, 2017): 647–69. http://dx.doi.org/10.1128/cmr.00111-16.
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SUMMARY In the last 2 decades, renewed attention to neglected tropical diseases (NTDs) has spurred the development of antiparasitic agents, especially in light of emerging drug resistance. The need for new drugs has required in vitro screening methods using parasite culture. Furthermore, clinical laboratories sought to correlate in vitro susceptibility methods with treatment outcomes, most notably with malaria. Parasites with their various life cycles present greater complexity than bacteria, for which standardized susceptibility methods exist. This review catalogs the state-of-the-art methodologies used to evaluate the effects of drugs on key human parasites from the point of view of drug discovery as well as the need for laboratory methods that correlate with clinical outcomes.
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Fernandez-Baca, Martha V., Cristian Hoban, Rodrigo A. Ore, Pedro Ortiz, Young-Jun Choi, César Murga-Moreno, Makedonka Mitreva, and Miguel M. Cabada. "The Differences in the Susceptibility Patterns to Triclabendazole Sulfoxide in Field Isolates of Fasciola hepatica Are Associated with Geographic, Seasonal, and Morphometric Variations." Pathogens 11, no. 6 (May 28, 2022): 625. http://dx.doi.org/10.3390/pathogens11060625.
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Triclabendazole (TCBZ) resistance is an emerging problem in fascioliasis that is not well understood. Studies including small numbers of parasites fail to capture the complexity of susceptibility variations between and within Fasciolahepatica populations. As the first step to studying the complex resistant phenotype–genotype associations, we characterized a large sample of adult F. hepatica with diverging TCBZ susceptibility. We collected parasites from naturally infected livestock slaughtered in the Cusco and Cajamarca regions of Peru. These parasites were exposed to TCBZ sulfoxide (TCBZ.SO) in vitro to determine their susceptibility. We used a motility score to determine the parasite’s viability. We titrated drug concentrations and times to detect 20% non-viable (susceptible conditions) or 80% non-viable (resistant conditions) parasites. We exposed 3348 fully motile parasites to susceptible (n = 1565) or resistant (n = 1783) conditions. Three hundred and forty-one (21.8%) were classified as susceptible and 462 (25.9%) were classified as resistant. More resistant parasites were found in Cusco than in Cajamarca (p < 0.001). Resistant parasites varied by slaughterhouse (p < 0.001), month of the year (p = 0.008), fluke length (p = 0.016), and year of collection (p < 0.001). The in vitro susceptibility to TCBZ.SO in wildtype F. hepatica was associated with geography, season, and morphometry.
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Rosenberg, Alex, Madeline R. Luth, Elizabeth A. Winzeler, Michael Behnke, and L. David Sibley. "Evolution of resistance in vitro reveals mechanisms of artemisinin activity inToxoplasma gondii." Proceedings of the National Academy of Sciences 116, no. 52 (December 5, 2019): 26881–91. http://dx.doi.org/10.1073/pnas.1914732116.
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Artemisinins are effective against a variety of parasites and provide the first line of treatment for malaria. Laboratory studies have identified several mechanisms for artemisinin resistance inPlasmodium falciparum, including mutations in Kelch13 that are associated with delayed clearance in some clinical isolates, although other mechanisms are likely involved. To explore other potential mechanisms of resistance in parasites, we took advantage of the genetic tractability ofToxoplasma gondii, a related parasite that shows moderate sensitivity to artemisinin. Resistant populations ofT. gondiiwere selected by culture in increasing concentrations and whole-genome sequencing identified several nonconservative point mutations that emerged in the population and were fixed over time. Genome editing using CRISPR/Cas9 was used to introduce point mutations conferring amino acid changes in a serine protease homologous to DegP and a serine/threonine protein kinase of unknown function. Single and double mutations conferred a competitive advantage over wild-type parasites in the presence of drug, despite not changing EC50values. Additionally, the evolved resistant lines showed dramatic amplification of the mitochondria genome, including genes encoding cytochromeband cytochromecoxidase I. Prior studies in yeast and mammalian tumor cells implicate the mitochondrion as a target of artemisinins, and treatment of wild-type parasites with high concentrations of drug decreased mitochondrial membrane potential, a phenotype that was stably altered in the resistant parasites. These findings extend the repertoire of mutations associated with artemisinin resistance and suggest that the mitochondrion may be an important target of inhibition of resistance inT. gondii.
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Ozwara, Hastings, Jan A. M. Langermans, Clemens H. M. Kocken, Annemarie van der Wel, Peter H. van der Meide, Richard A. W. Vervenne, Jason M. Mwenda, and Alan W. Thomas. "Transfected Plasmodium knowlesi Produces Bioactive Host Gamma Interferon: a New Perspective for Modulating Immune Responses to Malaria Parasites." Infection and Immunity 71, no. 8 (August 2003): 4375–81. http://dx.doi.org/10.1128/iai.71.8.4375-4381.2003.
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ABSTRACT Transgenic pathogenic microorganisms expressing host cytokines such as gamma interferon (IFN-γ) have been shown to manipulate host-pathogen interaction, leading to immunomodulation and enhanced protection. Expression of host cytokines in malaria parasites offers the opportunity to investigate the potential of an immunomodulatory approach by generating immunopotentiated parasites. Using the primate malaria parasite Plasmodium knowlesi, we explored the conditions for expressing host cytokines in malaria parasites. P. knowlesi parasites transfected with DNA constructs for expressing rhesus monkey (Macaca mulatta) IFN-γ under the control of the heterologous P. berghei apical membrane antigen 1 promoter, produced bioactive IFN-γ in a developmentally regulated manner. IFN-γ expression had no marked effect on in vitro parasite development. Bioactivity of the parasite-produced IFN-γ was shown through inhibition of virus cytopathic effect and confirmed by using M. mulatta peripheral blood cells in vitro. These data indicate for the first time that it is feasible to generate malaria parasites expressing bioactive host immunomodulatory cytokines. Furthermore, cytokine-expressing malaria parasites offer the opportunity to analyze cytokine-mediated modulation of malaria during the blood and liver stages of the infection.
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Vincendeau, P., and M. Daëron. "Trypanosoma musculi co-express several receptors binding rodent IgM, IgE, and IgG subclasses." Journal of Immunology 142, no. 5 (March 1, 1989): 1702–9. http://dx.doi.org/10.4049/jimmunol.142.5.1702.
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Abstract The present work demonstrates the expression of receptors for the Fc portion of rodent Ig by the murine parasite Trypanosoma musculi. By using a rosette assay adapted to the parasite morphology and by flow cytometry analysis, three distinct receptors were identified. A receptor binding rabbit or rat polyclonal IgG and mouse monoclonal IgG1, IgG2a, and IgG2b was found on parasites purified from the blood and the peritoneal cavity of infected mice and on parasites maintained in culture conditions. This IgG receptor was degraded by pepsin. A separate receptor, binding only mouse monoclonal IgG3 was observed on cultured parasites. A receptor binding rabbit, rat, and mouse IgM was found on cultured and peritoneal parasites, but not on blood parasites. This receptor did not bind IgG or IgA but it bound mouse and rat IgE as well as IgM. It was degraded by trypsin. IgG and IgM/IgE receptors were co-expressed on single parasites. They were not of host origin but synthesized by trypanosomes as shown by reexpression in vitro after proteolytic degradation. Their expression was variable with the development of trypanosomes both in vitro and in vivo.
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Manley, K. M., and J. A. Embil. "In vitro effect of ivermectin on Pseudoterranova decipiens survival." Journal of Helminthology 63, no. 1 (March 1989): 72–74. http://dx.doi.org/10.1017/s0022149x00008750.
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ABSTRACTThird larval stages (L3) removed from fish fillets, fourth larval stages (L4) raised in in vitro culture, and adults of Pseudoterranova decipiens, collected from grey seal (Halichoerus grypus) stomachs, were exposed to the broad spectrum anthelmintic, ivermectin. L3 and L4 parasites were exposed, in vitro, to 500, 100, 50, 20, 5 and 1 μg/ml concentrations of the drug, in culture media. Adult P. decipiens were exposed in vitro to a concentration of 500 μg/ml ivermectin, only. Controls consisted of parasites placed in culture media alone or culture media plus drug vehicle. These three developmental stages of P. decipiens were all found to be susceptible to the effects of ivermectin.
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Mutoro, Christine N., Johnson K. Kinyua, Joseph K. Ng'ang'a, Daniel W. Kariuki, Johnstone M. Ingonga, and Christopher O. Anjili. "In vitro study of the efficacy of Solanum nigrum against Leishmania major." F1000Research 7 (August 22, 2018): 1329. http://dx.doi.org/10.12688/f1000research.15826.1.
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Leishmania parasites (Kinetoplastida: Trypanosomatidae) are obligate intracellular parasites of macrophages that causes visceral and cutaneous leishmaniases. Currently, there is inadequate therapeutic interventions to manage this endemic tropical disease, transmitted mainly by phlebotomine sandflies hence there is need to develop affordable and effective therapeutic measures. This study determined the in vitro efficacy of Solanum nigrum methanolic and aqueous plant extracts on Leishmania major parasites. Cytotoxic effects of the extracts were determined using vero cells and reported as percentage viability of the cells. The promastigote parasites of Leishmania major were cultured and grown for 3 days in different concentrations of extracts to determine the MIC and IC50 values. The in vitro antileishmanial efficacy was done on macrophages infected with L. major amastigote parasites and then treated with extracts in varying concentrations. The study revealed that all the test extracts had lower toxicity than control drugs, pentostam (IC50= 0.0 92 mg/ml) and amphotericin B (IC50=0.049 mg/ml). The extracts tended to show a dose dependent cytotoxic effect which corresponded to high vero cells viability as their concentration increased. Methanolic extract of S. nigrum from Kisii seemed to be more efficacious in vitro since it knocked out the promastigotes at a lower MIC level (0.5 mg/ml) when compared to all other extracts whose effective MIC level was ≥ 1 mg/ml. High concentrations of the test extracts and control drugs resulted to low infectivity and multiplication of L. major amastigotes. Findings from this study demonstrate that S. nigrum extracts have potential antileishmanial activities however; further investigation needs to be done on pure compound isolation, in vivo assays and clinical trials so as to use the promising compounds as effective antileishmanial agents.
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BUCKLING, A., L. C. RANFORD-CARTWRIGHT, A. MILES, and A. F. READ. "Chloroquine increases Plasmodium falciparum gametocytogenesis in vitro." Parasitology 118, no. 4 (April 1999): 339–46. http://dx.doi.org/10.1017/s0031182099003960.
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Malaria parasites are capable of modulating the diversion of resources from asexual growth to the production of stages infective to mosquitoes (gametocytes). Increased rates of gametocytogenesis appear to be a general response to stress, both naturally encountered and novel. We have previously reported earlier and greater gametocytogenesis in response to subcurative antimalarial chemotherapy in the rodent malaria, Plasmodium chabaudi, in vivo. Using an immunofluorescent assay to detect parasites that had invaded red blood cell monolayers, we demonstrate a 5-fold increase in gametocytogenesis in the human malaria, P. falciparum, in vitro, in response to treatment with the antimalarial drug chloroquine. In all clones used, gametocytogenesis increased with increasing inhibition of asexual growth by chloroquine. Furthermore, there were clone differences in the relationship between stress and gametocyte production, implying the response was genetically variable. This was not, however, associated with chloroquine resistance. The epidemiological significance of these results is discussed.
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Ye, Qing, Hui-Fen Dong, Christoph G. Grevelding, and Min Hu. "In vitro cultivation of Schistosoma japonicum-parasites and cells." Biotechnology Advances 31, no. 8 (December 2013): 1722–37. http://dx.doi.org/10.1016/j.biotechadv.2013.09.003.
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Ramharter, M., H. Noedl, H. Winkler, W. Graninger, W. H. Wernsdorfer, P. G. Kremsner, and S. Winkler. "In Vitro Activity and Interaction of Clindamycin Combined with Dihydroartemisinin against Plasmodium falciparum." Antimicrobial Agents and Chemotherapy 47, no. 11 (November 2003): 3494–99. http://dx.doi.org/10.1128/aac.47.11.3494-3499.2003.
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ABSTRACT Combination regimens are considered a valuable tool for the fight against drug-resistant falciparum malaria. This study was conducted to evaluate the antimalarial potential of clindamycin in combination with dihydroartemisinin in continuously cultured and in freshly isolated Plasmodium falciparum parasites, measuring the inhibition of Plasmodium falciparum histidine-rich protein II synthesis. Interaction analysis revealed a synergistic or additive mode of interaction at various concentration ratios in all continuously cultured parasites at the 50% effective concentration (EC50) level. Antagonism was not found for any of the culture-adapted parasites. In fresh P. falciparum isolates, a fixed clindamycin-dihydroartemisinin combination exhibited additive activity at the EC50 and EC90 levels. The drug mixture showed no significant activity correlation to other commonly used antimalarials. The clindamycin-dihydroartemisinin combination appears to be a promising candidate for clinical investigation.
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FRAME, M. J., J. C. MOTTRAM, and G. H. COOMBS. "Analysis of the roles of cysteine proteinases of Leishmania mexicana in the host–parasite interaction." Parasitology 121, no. 4 (October 2000): 367–77. http://dx.doi.org/10.1017/s0031182099006435.
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Promastigotes of Leishmania mexicana mutants lacking the multicopy CPB cysteine proteinase genes (ΔCPB) are markedly less able than wild-type parasites to infect macrophages in vitro. ΔCPB promastigotes invade macrophages in large numbers but are unable to survive in the majority of the cells. In contrast, ΔCPB amastigotes invade and survive within macrophages in vitro. This extreme in vitro stage-specific difference was not mimicked in vivo; both promastigotes and amastigotes of ΔCPB produced lesions in BALB/c mice, but in each case the lesions grew considerably more slowly than those caused by wild-type parasites and only small lesions resulted. Inhibition of CPB in situ using cell-permeant peptidyldiazomethylketones had no measurable effect on parasite growth or differentiation axenically in vitro. In contrast, N-benzoyloxycarbonyl-phe-ala-diazomethylketone reduced the infectivity of wild-type parasites to macrophages by 80%. Time-course experiments demonstrated that application of the inhibitor caused effects not seen with ΔCPB, suggesting that CPB may not be the prime target of this inhibitor. The data show that the CPB genes of L. mexicana encode enzymes that have important roles in intracellular survival of the parasite and more generally in its interaction with its mammalian host.
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Walz, Annabelle, Maëlle Duffey, Ghaith Aljayyoussi, Sibylle Sax, Didier Leroy, Dominique Besson, Jeremy N. Burrows, et al. "The Parasite Reduction Ratio (PRR) Assay Version 2: Standardized Assessment of Plasmodium falciparum Viability after Antimalarial Treatment In Vitro." Pharmaceuticals 16, no. 2 (January 23, 2023): 163. http://dx.doi.org/10.3390/ph16020163.
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With artemisinin-resistant Plasmodium falciparum parasites emerging in Africa, the need for new antimalarial chemotypes is persistently high. The ideal pharmacodynamic parameters of a candidate drug are a rapid onset of action and a fast rate of parasite killing or clearance. To determine these parameters, it is essential to discriminate viable from nonviable parasites, which is complicated by the fact that viable parasites can be metabolically inactive, whilst dying parasites can still be metabolically active and morphologically unaffected. Standard growth inhibition assays, read out via microscopy or [3H] hypoxanthine incorporation, cannot reliably discriminate between viable and nonviable parasites. Conversely, the in vitro parasite reduction ratio (PRR) assay is able to measure viable parasites with high sensitivity. It provides valuable pharmacodynamic parameters, such as PRR, 99.9% parasite clearance time (PCT99.9%) and lag phase. Here we report the development of the PRR assay version 2 (V2), which comes with a shorter assay duration, optimized quality controls and an objective, automated analysis pipeline that systematically estimates PRR, PCT99.9% and lag time and returns meaningful secondary parameters such as the maximal killing rate of a drug (Emax) at the assayed concentration. These parameters can be fed directly into pharmacokinetic/pharmacodynamic models, hence aiding and standardizing lead selection, optimization, and dose prediction.
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Ursing, Johan, Rasmus Johns, Berit Aydin-Schmidt, Carla Calçada, Poul-Erik Kofoed, Najia Karim Ghanchi, Maria Isabel Veiga, and Lars Rombo. "Chloroquine-susceptible and -resistant Plasmodium falciparum strains survive high chloroquine concentrations by becoming dormant but are eliminated by prolonged exposure." Journal of Antimicrobial Chemotherapy 77, no. 4 (February 8, 2022): 1005–11. http://dx.doi.org/10.1093/jac/dkac008.
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Abstract Background Plasmodium falciparum strains that are resistant to standard-dose chloroquine can be treated by higher chloroquine concentrations maintained for a longer time in vivo. Objectives To determine the relative importance of chloroquine concentrations versus exposure time for elimination of chloroquine-susceptible and -resistant P. falciparum in vitro. Methods Chloroquine-susceptible (3D7) and -resistant (FCR3) strains were exposed in vitro to 1, 2, 4, 8, 16 or 32 times their respective 90% inhibitory chloroquine concentrations for 3, 5, 7 or 14 days and then followed until recrudescence, or not, by 42 days after the end of exposure. Results Exposure to chloroquine appeared to eliminate susceptible and resistant parasites, leaving small pyknotic apparently dead parasites. Chloroquine-susceptible and -resistant parasites recrudesced after 3 and 5 days of chloroquine exposure. Recrudescence occurred in one out of four 7 day exposure series but not after 14 days exposure. The median time to recrudescence was 13 to 28 days with a range of 8 to 41 days after the end of exposure. Time to recrudescence after the end of exposure increased with duration of exposure for susceptible and resistant strains (P < 0.001). Time to recrudescence did not correlate with concentrations greater than 1× IC90. Conclusions Chloroquine-susceptible and -resistant P. falciparum probably become dormant. Elimination of dormant parasites is primarily dependent upon the duration of chloroquine exposure. Exposure to effective drug concentrations for 7 days eliminates most parasites in vitro. The results support in vivo data indicating that elimination of chloroquine-resistant P. falciparum correlates with Day 7 chloroquine concentrations.
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Kelly, Ben L., Daniel B. Stetson, and Richard M. Locksley. "Leishmania major LACK Antigen Is Required for Efficient Vertebrate Parasitization." Journal of Experimental Medicine 198, no. 11 (December 1, 2003): 1689–98. http://dx.doi.org/10.1084/jem.20031162.
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The Leishmania major LACK antigen is a key target of the immune response in susceptible BALB/c mice and remains a viable vaccine candidate for human leishmaniasis. We describe the genomic organization of the four lack genes in the L. major diploid genome together with results of selected lack gene targeting. Parasites containing a single lack gene in either the upstream or downstream locus grew comparably to wild-type promastigotes in vitro, but failed to parasitize BALB/c mice efficiently, even in a T cell–deficient environment. The replication of single copy lack mutants as amastigotes was attenuated in macrophages in vitro, and parasites failed to increase in numbers in immunodeficient mice, despite their persistence over months. Complementation with an additional lack copy was sufficient to induce robust lesion development, which also occurred using parasites with two lack genes. Conversely, attempts to generate lack-null parasites failed, suggesting that LACK is required for parasite viability. These data suggest that LACK is critical for effective mammalian parasitization and thus represents a potential drug target for leishmaniasis.
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Horta, M. F., F. J. Ramalho-Pinto, and M. Fatima. "Role of human decay-accelerating factor in the evasion of Schistosoma mansoni from the complement-mediated killing in vitro." Journal of Experimental Medicine 174, no. 6 (December 1, 1991): 1399–406. http://dx.doi.org/10.1084/jem.174.6.1399.
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Decay-accelerating factor (DAF) is a 70-kD membrane glycoprotein that prevents complement (C)-mediated hemolysis by blocking the assembly or accelerating the decay of C3 convertase. Purified DAF is known to incorporate into the membrane of DAF-deficient cells, inhibiting lysis. Since Schistosoma mansoni is a blood-dwelling parasite, we investigated whether DAF can be transferred from human erythrocytes to the worm and protect it against C-mediated killing in vitro. We have found that schistosomula (schla) incubated with normal human erythrocytes (N-HuE), but not with DAF-deficient erythrocytes, become resistant to C damage in vitro. Protected parasites acquire a 70-kD surface protein which can be immunoprecipitated by anti-DAF antibodies. The acquired resistance is abrogated by treatment of N-HuE-incubated parasites with anti-DAF antibody. These results indicate that, in vitro, N-HuE DAF can be transferred to schla, and suggest its participation in preventing their C-mediated killing. This could represent an important strategy of parasites to evade the host's immune response in vivo.
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Teuscher, Franka, Nanhua Chen, Dennis E. Kyle, Michelle L. Gatton, and Qin Cheng. "Phenotypic Changes in Artemisinin-Resistant Plasmodium falciparum LinesIn Vitro: Evidence for Decreased Sensitivity to Dormancy and Growth Inhibition." Antimicrobial Agents and Chemotherapy 56, no. 1 (October 10, 2011): 428–31. http://dx.doi.org/10.1128/aac.05456-11.
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ABSTRACTThe appearance ofPlasmodium falciparumparasites with decreasedin vivosensitivity but no measurablein vitroresistance to artemisinin has raised the urgent need to characterize the artemisinin resistance phenotype. Changes in the temporary growth arrest (dormancy) profile of parasites may be one aspect of this phenotype. In this study, we investigated the link between dormancy and resistance, using artelinic acid (AL)-resistant parasites. Our results demonstrate that the AL resistance phenotype has (i) decreased sensitivity of mature-stage parasites, (ii) decreased sensitivity of the ring stage to the induction of dormancy, and (iii) a faster recovery from dormancy.
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de Morais-Teixeira, Eliane, Ana Rabello, and Marta Marques Gontijo Aguiar. "In vitro activity and in vivo efficacy of fexinidazole against New World Leishmania species." Journal of Antimicrobial Chemotherapy 74, no. 8 (May 2, 2019): 2318–25. http://dx.doi.org/10.1093/jac/dkz172.
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Abstract Objectives To evaluate the in vitro activity and in vivo efficacy of fexinidazole against the main species that cause visceral and cutaneous New World leishmaniasis. Methods The inhibitory concentrations of fexinidazole against Leishmania (Leishmania) infantum chagasi, Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis in amastigotes were determined by in vitro activity assays. For the in vivo evaluation, animals were infected with L. (L.) infantum chagasi, L. (L.) amazonensis, L. (V.) braziliensis or Leishmania (Viannia) guyanensis and divided into groups: (i) control; and (ii) treated with oral fexinidazole, from 50 to 300 mg/kg/day. For cutaneous leishmaniasis, the size of the lesion was determined weekly after the beginning of the treatment. Upon completion, parasites were recovered from the spleen and liver, or skin lesion and spleen, and evaluated by a limiting dilution assay. Results All Leishmania isolates were susceptible to fexinidazole in the in vitro assays. The viable parasites in the liver and spleen were reduced with 100 and 300 mg/kg/day, respectively, for L. (L.) infantum chagasi. For the species causing cutaneous leishmaniasis, the viable parasites in lesions and the size of the lesions were reduced, starting from 200 mg/kg/day. The viable parasites in the spleen were also reduced with 200 and 300 mg/kg/day for L. (V.) braziliensis and L. (L.) amazonensis. Conclusions Considering the defined parameters, fexinidazole showed in vitro and in vivo activity against all tested species. This drug may represent an alternative treatment for the New World species.
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Rizk, Mohamed Abdo, Shimaa Abd El-Salam El-Sayed, and Ikuo Igarashi. "Effects of Methanolic Extract from Turmeric (Curcuma longa) against the In Vitro Multiplication of Several Babesia Species and Theileria equi." Parasitologia 1, no. 4 (September 24, 2021): 188–96. http://dx.doi.org/10.3390/parasitologia1040020.
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Anti-piroplasm drugs currently on the market have proven toxicity to the host and parasite resistance. Plants are possible sources of novel drugs. Subsequently, a novel strategy should be used to find new anti-piroplasm agents that are both effective and safe. In the present study, we have evaluated the effect of turmeric (Curcuma longa) methanolic extract on the in vitro growth of Babesia (B.) bovis, B. divergens, B. caballi, and Theileria (T.) equi. The in vitro inhibitory effectiveness of turmeric was assessed using a fluorescence test. The enhancement in the in vitro inhibitory efficacy of turmeric when administrated in combination with diminazene aceturate (DA) was investigated using in vitro cultures of different piroplasm parasites. Turmeric reduced the in vitro growth of B. bovis, B. divergens, T. equi, and B. caballi with IC50 values of 0.830 ± 0.078, 0.375 ± 0.055, 1.405 ± 0.575, and 0.720 ± 0.090 mg/mL, respectively. An amount of 1 mg/mL turmeric for B. bovis, 0.5 mg/mL turmeric for B. divergens, 1 mg/mL turmeric for T. equi, and 0.5 mg/mL turmeric for B. caballi exhibited 73.43%, 80.065%, 73.47%, and 47.375% inhibitions in the growth of the parasites, respectively. When turmeric was combined with DA, its in vitro inhibitory impact on bovine Babesia and equine Babesia/Theileria parasites was amplified. These findings show that a methanolic extract of turmeric could be a promising medicinal plant for the treatment of babesiosis, especially when administered in conjunction with DA.
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Nzila, Alexis, Eddy Mberu, Pat Bray, Gilbert Kokwaro, Peter Winstanley, Kevin Marsh, and Steve Ward. "Chemosensitization of Plasmodium falciparum by Probenecid In Vitro." Antimicrobial Agents and Chemotherapy 47, no. 7 (July 2003): 2108–12. http://dx.doi.org/10.1128/aac.47.7.2108-2112.2003.
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ABSTRACT Resistance to drugs can result from changes in drug transport, and this resistance can sometimes be overcome by a second drug that modifies the transport mechanisms of the cell. This strategy has been exploited to partly reverse resistance to chloroquine in Plasmodium falciparum. Studies with human tumor cells have shown that probenecid can reverse resistance to the antifolate methotrexate, but the potential for reversal of antifolate resistance has not been studied in P. falciparum. In the present study we tested the ability of probenecid to reverse antifolate resistance in P. falciparum in vitro. Probenecid, at concentrations that had no effect on parasite viability alone (50 μM), was shown to increase the sensitivity of a highly resistant parasite isolate to the antifolates pyrimethamine, sulfadoxine, chlorcycloguanil, and dapsone by seven-, five-, three-, and threefold, respectively. The equivalent effects against an antifolate-sensitive isolate were activity enhancements of approximately 3-, 6-, 1.2-, and 19-fold, respectively. Probenecid decreased the level of uptake of radiolabeled folic acid, suggesting a transport-based mechanism linked to folate salvage. When probenecid was tested with chloroquine, it chemosensitized the resistant isolate to chloroquine (i.e., enhanced the activity of chloroquine). This enhancement of activity was associated with increased levels of chloroquine accumulation. In conclusion, we have shown that probenecid can chemosensitize malaria parasites to antifolate compounds via a mechanism linked to reduced folate uptake. Notably, this effect is observed in both folate-sensitive and -resistant parasites. In contrast to the activities of antifolate compounds, the effect of probenecid on chloroquine sensitivity was selective for chloroquine-resistant parasites (patent P407595GB [W. P. Thompson & Co., Liverpool, United Kingdom] has been filed to protect this intellectual property).
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Mairet-Khedim, Mélissa, Flore Nardella, Nimol Khim, Saorin Kim, Nimol Kloeung, Sopheakvatey Ke, Chhayleang Kauy, et al. "In vitro activity of ferroquine against artemisinin-based combination therapy (ACT)-resistant Plasmodium falciparum isolates from Cambodia." Journal of Antimicrobial Chemotherapy 74, no. 11 (September 13, 2019): 3240–44. http://dx.doi.org/10.1093/jac/dkz340.
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Abstract Background Cambodia is the epicentre of resistance emergence for virtually all antimalarial drugs. Selection and spread of parasites resistant to artemisinin-based combination therapy (ACT) is a major threat for malaria elimination, hence the need to renew the pool of effective treatments. Objectives To determine whether ACT resistance haplotypes could have an effect on ferroquine in vitro antimalarial activity. Methods In vitro susceptibility to ferroquine was measured for 80 isolates from Cambodia characterized for their molecular resistance profile to artemisinin, piperaquine and mefloquine. Results Among the 80 isolates tested, the overall median (IQR) IC50 of ferroquine was 10.9 nM (8.7–18.3). The ferroquine median (IQR) IC50 was 8.9 nM (8.1–11.8) for Pfk13 WT parasites and was 12.9 nM (9.5–20.0) for Pfk13 C580Y parasites with no amplification of Pfpm2 and Pfmdr1 genes. The median (IQR) IC50 of ferroquine for Pfk13 C580Y parasites with amplification of the Pfpm2 gene was 17.2 nM (14.5–20.5) versus 9.1 nM (7.9–10.7) for Pfk13 C580Y parasites with amplification of the Pfmdr1 gene. Conclusions Ferroquine exerts promising efficacy against ACT-resistant isolates. Whereas Pfpm2 amplification was associated with the highest parasite tolerance to ferroquine, the susceptibility range observed was in accordance with those measured in ACT resistance-free areas. This enables consideration of ferroquine as a relevant therapeutic option against ACT-resistant malaria.
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Castelli, Germano, Eugenia Oliveri, Viviana Valenza, Susanna Giardina, Flavia Facciponte, Francesco La Russa, Fabrizio Vitale, and Federica Bruno. "Cultivation of Protozoa Parasites In Vitro: Growth Potential in Conventional Culture Media versus RPMI-PY Medium." Veterinary Sciences 10, no. 4 (March 27, 2023): 252. http://dx.doi.org/10.3390/vetsci10040252.
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The in vitro cultivation of Leishmania and Trypanosoma parasites plays an important role in the diagnosis and treatment of parasitic diseases. Although Evans’s modified Tobie and Novy–MacNeal–Nicolle media, for Leishmania spp. and Trypanosoma cruzi, respectively, are the two commonly used media for both isolation and maintenance of strains in vitro, their preparation is expensive and laborious and requires fresh rabbit blood from housed animals. The purpose of this study was to evaluate the in vitro growth of both parasites with an alternative monophasic, blood-free, easy, and affordable medium called RPMI-PY, which was previously demonstrated suitable for the in vitro growth of Leishmania infantum. The potential growth of different Leishmania species and Trypanosoma cruzi was evaluated in traditional culture media versus RPMI-PY medium, and we recorded the protozoa parasites’ morphology via orange acridine–ethidium bromide staining. The results of our study show that RPMI-PY medium can be used for Trypanosoma cruzi, Leishmania amazonensis, Leishmania major, and Leishmania tropica species since in all the species except Leishmania braziliensis, the exponential growth of the parasite was observed, in many cases higher than conventional media. The staining confirmed not only their growth during the 72 h investigation but also the optimal morphology and viability of the protozoa in the RPMI-PY medium.
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Vigan-Womas, Inès, Micheline Guillotte, Cécile Le Scanf, Sébastien Igonet, Stéphane Petres, Alexandre Juillerat, Cyril Badaut, et al. "An In Vivo and In Vitro Model of Plasmodium falciparum Rosetting and Autoagglutination Mediated by varO, a Group A var Gene Encoding a Frequent Serotype." Infection and Immunity 76, no. 12 (September 22, 2008): 5565–80. http://dx.doi.org/10.1128/iai.00901-08.
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ABSTRACTIn theSaimiri sciureusmonkey, erythrocytes infected with the varO antigenic variant of thePlasmodium falciparumPalo Alto 89F5 clone bind uninfected red blood cells (rosetting), form autoagglutinates, and have a high multiplication rate, three phenotypic characteristics that are associated with severe malaria in human patients. We report here that varO parasites express avargene having the characteristics of group Avargenes, and we show that the varO Duffy binding-like 1α1(DBL1α1) domain is implicated in the rosetting of bothS. sciureusand human erythrocytes. The soluble varO N-terminal sequence (NTS)-DBL1α1recombinant domain, produced in a baculovirus-insect cell system, induced high titers of antibodies that reacted with varO-infected red blood cells and disrupted varO rosettes. varO parasites were culture adapted in vitro using human erythrocytes. They formed rosettes and autoagglutinates, and they had the same surface serotype and expressed the samevarOgene as the monkey-propagated parasites. To develop an in vitro model with highly homogeneous varO parasites, rosette purification was combined with positive selection by panning with a varO NTS-DBL1α1-specific mouse monoclonal antibody. The single-variant, clonal parasites were used to analyze seroprevalence for varO at the village level in a setting where malaria is holoendemic (Dielmo, Senegal). We found 93.6% (95% confidence interval, 89.7 to 96.4%) seroprevalence for varO surface-reacting antibodies and 86.7% (95% confidence interval, 82.8 to 91.6%) seroprevalence for the recombinant NTS-DBL1α1domain, and virtually all permanent residents had seroconverted by the age of 5 years. These data imply that the varO model is a relevant in vivo and in vitro model for rosetting and autoagglutination that can be used for rational development of vaccine candidates and therapeutic strategies aimed at preventing malaria pathology.
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Tavares-Dias, Marcos. "Current knowledge on use of essential oils as alternative treatment against fish parasites." Aquatic Living Resources 31 (2018): 13. http://dx.doi.org/10.1051/alr/2018001.
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This review article focuses on current knowledge about in vitro and in vivo experimentation relating to use of essential oils (EOs) to combat fish parasites. In addition, we discuss the existing methodologies used in studies to determine the antiparasitic activity of EOs, along with their toxicity and major compounds. The methodological approaches used to describe the anthelmintic properties of EOs were demonstrated. The consistency of their activity and thus their potential use for fish ectoparasites (in vitro and in vivo) and endoparasites (in vitro) control was reviewed. There is a clear need to find EOs and active agents of EOs to treatment in vivo against endoparasites of fish. Thus, progress may be achieved through considering the beneficial aspects of EOs when their complementarity and potentiality are exploited. EOs are therefore viable alternative sources of therapeutic products against fish parasites. On the other hand, use of chemotherapeutics has been increasingly questioned, such that constant environmental and consumer concerns regarding them now exist. The synergistic functions of EOs, in comparison with the action of one or two major compounds of these oils, seems unquestionable. It is possible that their activity is modulated by several molecules of the major compounds. Lastly, EOs are bioactive products that are viable sources of therapy against fish parasites. Although more than 3000 EOs are known, less than 0.4% of them have been tested on fish parasites. Thus, it has become clear that more studies testing these therapeutic alternatives are required, in order to evaluate the antiparasitic potential of other EOs for controlling fish parasites and to maximize their benefits to hosts.
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Cai, Xiaomin, Keith M. Woods, Steve J. Upton, and Guan Zhu. "Application of Quantitative Real-Time Reverse Transcription-PCR in Assessing Drug Efficacy against the Intracellular Pathogen Cryptosporidium parvum In Vitro." Antimicrobial Agents and Chemotherapy 49, no. 11 (November 2005): 4437–42. http://dx.doi.org/10.1128/aac.49.11.4437-4442.2005.
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ABSTRACT We report here on a quantitative real-time reverse transcription-PCR (qRT-PCR) assay for assessing drug efficacy against the intracellular pathogen Cryptosporidium parvum. The qRT-PCR assay detects 18S rRNA transcripts from both parasites, that is, the cycle threshold for 18S rRNA from parasites (CT [P18S]) and host cells (CT [H18S]), and evaluates the relative expression between parasite and host rRNA levels (i.e., ΔCT = CT [P18S] − CT [H18S]) to minimize experimental and operational errors. The choice of qRT-PCR over quantitative PCR (qPCR) in this study is based on the observations that (i) the relationship between the logarithm of infected parasites (log[P]) and the normalized relative level of rRNA (ΔΔCT ) is linear, with a fourfold dynamic range, by qRT-PCR but sigmoidal (nonlinear) by qPCR; and (ii) the level of RNA represents that of live parasites better than that of DNA, because the decay of RNA (99% in ∼3 h) in dead parasites is faster than that of DNA (99% in ∼24 to 48 h) under in vitro conditions. The reliability of the qRT-PCR method was validated by testing the efficacies of nitazoxanide and paromomycin on the development of two strains of C. parvum (IOWA and KSU-1) in HCT-8 cells in vitro. Both compounds displayed dose-dependent inhibitions. The observed MIC50 values for nitazoxanide and paromomycin were 0.30 to 0.45 μg/ml and 89.7 to 119.0 μg/ml, respectively, comparable to the values reported previously. Using the qRT-PCR assay, we have also observed that pyrazole could inhibit C. parvum development in vitro (MIC50 = 15.8 mM), suggesting that the recently discovered Cryptosporidium alcohol dehydrogenases may be explored as new drug targets.
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Muangnoicharoen, Sant, David J. Johnson, Sornchai Looareesuwan, Srivicha Krudsood, and Stephen A. Ward. "Role of Known Molecular Markers of Resistance in the Antimalarial Potency of Piperaquine and Dihydroartemisinin In Vitro." Antimicrobial Agents and Chemotherapy 53, no. 4 (February 2, 2009): 1362–66. http://dx.doi.org/10.1128/aac.01656-08.
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ABSTRACT Using a range of laboratory-adapted and genetically modified Plasmodium falciparum parasite isolates, we investigated the interaction between dihydroartemisinin and piperaquine (PIP), the individual components of an artemisinin combination therapy currently under development, in addition to the role of known drug resistance genes in parasite susceptibility in vitro. All but one parasite line investigated displayed an interaction of dihydroartemisinin and PIP that was antagonistic, although the degree of antagonism was isolate dependent. In terms of resistance markers, the pfcrt haplotypes CVIET and SVMNT were positively associated with reduced sensitivity to PIP, with parasites carrying the South American CQR (SVMNT) allele being generally less sensitive than CVIET parasites. Parasites carrying the CQS (CVMNK) allele displayed a further increase in PIP sensitivity compared with CVIET and SVMNT parasites. Our data indicate that PIP sensitivity was not affected by pfmdr1 sequence status, despite positive correlations between the structurally related compound amodiaquine and pfmdr1 mutations in other studies. In contrast, neither the pfcrt nor pfmdr1 sequence status had any significant impact on susceptibility to dihydroartemisinin.
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Nuha S. Al-Bayatii, Fatima Sh.Al-Naserii, and JaladetM.S.Jubrael. "Epidermal Growth factor in human urine as promotor for the growth of Leishmania sp. In vitro." Tikrit Journal of Pure Science 20, no. 2 (February 9, 2023): 35–39. http://dx.doi.org/10.25130/tjps.v20i2.1155.
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Leishmania parasites are the causal agents of leishmaniasis, a group of protozoan diseases transmitted to mammals, including human beings, by phlebotomine sandflies. In culture media (at 25-28̊C ,pH =7.2-7.4),leishmania parasites develop as motile promastigotes similar to those found in the sand fly midgut . Traditionally media available do not meet the requirement for the bulk cultivation of Leishmania parasites ,it requires fetal calf serum (FCS),that is very expensive and not easily available in the market. A number of studies have shown that the addition of 5-10 % normal human urine stimulates growth, leading to more rapid multiplication and a higher concentration of parasites .Urine from patients with bladder cancer were used in this study to determine the effect of Epidermal growth factor ( which is increased in level in this type of urine),and our study showed that proliferation indexes were significantly increased in the culture media supplemented with human urine from patients with bladder cancer, we undertook a detailed study of such an effect in old world Leishmania isolates causing cutaneous Leishmaniasis. We also found that urine with high percentage of EGF. Could be used as an alternative of fetal calf serum.
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Schalkwijk, Joost, Erik L. Allman, Patrick A. M. Jansen, Laura E. de Vries, Julie M. J. Verhoef, Suzanne Jackowski, Peter N. M. Botman, et al. "Antimalarial pantothenamide metabolites target acetyl–coenzyme A biosynthesis in Plasmodium falciparum." Science Translational Medicine 11, no. 510 (September 18, 2019): eaas9917. http://dx.doi.org/10.1126/scitranslmed.aas9917.
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Malaria eradication is critically dependent on new therapeutics that target resistant Plasmodium parasites and block transmission of the disease. Here, we report that pantothenamide bioisosteres were active against blood-stage Plasmodium falciparum parasites and also blocked transmission of sexual stages to the mosquito vector. These compounds were resistant to degradation by serum pantetheinases, showed favorable pharmacokinetic properties, and cleared parasites in a humanized mouse model of P. falciparum infection. Metabolomics revealed that coenzyme A biosynthetic enzymes converted pantothenamides into coenzyme A analogs that interfered with parasite acetyl–coenzyme A anabolism. Resistant parasites generated in vitro showed mutations in acetyl–coenzyme A synthetase and acyl–coenzyme A synthetase 11. Introduction and reversion of these mutations in P. falciparum using CRISPR-Cas9 gene editing confirmed the roles of these enzymes in the sensitivity of the malaria parasites to pantothenamides. These pantothenamide compounds with a new mode of action may have potential as drugs against malaria parasites.
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Coetzer, Theresa L., and Dewaldt Engelbrecht. "To Each His Own: Fever Induces Different Suicide Mechanisms in Early and Late Stage Plasmodium falciparum Malaria Parasites in vitro." Blood 122, no. 21 (November 15, 2013): 3427. http://dx.doi.org/10.1182/blood.v122.21.3427.3427.
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Abstract Malaria is characterised by cyclical febrile episodes that result from the rupture of mature schizont-infected erythrocytes releasing merozoites. In patients infected with Plasmodium falciparum, fever may reach peak temperatures as high as 41°C, last for 2-6 hours and recur every 48 hours, in accordance with the parasite life cycle. Febrile episodes typically have a deleterious effect on parasites, and probably benefit the host by aiding parasite clearance; however, a reduction in parasitemia may also be an advantage for the parasite by limiting the burden of infection on the host and prolonging infection to ensure development and transmission of slow-maturing gametocytes. Although programmed cell death (PCD) has been identified in the mosquito stages of the parasite, data regarding the erythrocytic stages are limited and often conflicting and thus the occurrence of PCD and the phenotype remain controversial. This study aimed to characterise the cell death phenotype of P. falciparum in response to in vitro heat stress similar to fever periods experienced during malaria paroxysms. Synchronised early and late stage P. falciparum 3D7 cultures were exposed to 41°C for 2 hours or maintained as controls at 37°C. Parasitemia was monitored with thiazole orange flow cytometry. Numerous biochemical markers of PCD were assessed by flow cytometric assays immediately after heat stress and 24 hours later. DNA fragmentation was measured with the Terminal deoxynucleotidyl transferase-mediated Nick End Labelling (TUNEL) assay; mitochondrial membrane polarisation was measured with 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)]; and phosphatidylserine (PS) externalisation of infected erythrocytes was quantified by Annexin V binding. Morphological studies of Giemsa-stained thin smears and real-time microscopy were also utilised to characterise the parasite response to heat stress. Exposure to 41°C decreased the parasitemia of both early and late stage P. falciparum in vitro. Ring stage parasites were more affected than expected, although some ring stage parasites developed into trophozoites similar to control parasites maintained at 37°C. Biochemical markers showed evidence of DNA fragmentation and mitochondrial depolarisation suggesting that ring stage parasites exhibited an apoptosis-like phenotype of PCD, although infected erythrocytes showed no significant increase in PS externalisation. Late stage control parasites maintained at 37°C replicated within 24 hours to form newly-invaded rings with increased parasitemia. In contrast, heat stress caused a reduction in the number of late stage parasites and the remaining parasites failed to invade new erythrocytes. Although infected erythrocytes exhibited increased PS externalisation, the parasites exhibited very little DNA fragmentation and no mitochondrial dysregulation. Giemsa-stained thin smears showed some late stage parasites with cytoplasmic vacuolisation, which was suggestive of an autophagy-like form of PCD. Real-time microscopy revealed the movement of many late stage parasites inside host erythrocytes after heat stress, indicating that these parasites were alive, consistent with a recovery in parasitemia after two cycles of continued culture at 37°C. Data regarding the possible induction of PCD by heat stress in P. falciparum are scarce and conflicting. Our results showed biochemical and morphological markers of PCD that varied with intra-erythrocytic parasite development and suggested that P. falciparum exhibited both apoptosis- and autophagy-like phenotypes of PCD after exposure to febrile temperatures. However, these cell death markers may also represent different facets of a PCD pathway in P. falciparum that is distinct from that in metazoans. The identification and exploitation of an intrinsic cell death pathway unique to P. falciparum may provide novel targets for eliminating the parasite in malaria patients. Disclosures: No relevant conflicts of interest to declare.
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Barragan, Antonio, and L. David Sibley. "Transepithelial Migration of Toxoplasma gondii Is Linked to Parasite Motility and Virulence." Journal of Experimental Medicine 195, no. 12 (June 17, 2002): 1625–33. http://dx.doi.org/10.1084/jem.20020258.
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After oral ingestion, Toxoplasma gondii crosses the intestinal epithelium, disseminates into the deep tissues, and traverses biological barriers such as the placenta and the blood-brain barrier to reach sites where it causes severe pathology. To examine the cellular basis of these processes, migration of T. gondii was studied in vitro using polarized host cell monolayers and extracellular matrix. Transmigration required active parasite motility and the highly virulent type I strains consistently exhibited a superior migratory capacity than the nonvirulent type II and type III strains. Type I strain parasites also demonstrated a greater capacity for transmigration across mouse intestine ex vivo, and directly penetrated into the lamina propria and vascular endothelium. A subpopulation of virulent type I parasites exhibited a long distance migration (LDM) phenotype in vitro, that was not expressed by nonvirulent type II and type III strains. Cloning of parasites expressing the LDM phenotype resulted in substantial increase of migratory capacity in vitro and in vivo. The potential to up-regulate migratory capacity in T. gondii likely plays an important role in establishing new infections and in dissemination upon reactivation of chronic infections.
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Cowell, Annie, and Elizabeth Winzeler. "Exploration of the Plasmodium falciparum Resistome and Druggable Genome Reveals New Mechanisms of Drug Resistance and Antimalarial Targets." Microbiology Insights 11 (January 2018): 117863611880852. http://dx.doi.org/10.1177/1178636118808529.
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Plasmodium parasites, the causative agent of malaria infections, rapidly evolve drug resistance and escape detection by the human immune response via the incredible mutability of its genome. Understanding the genetic mechanisms by which Plasmodium parasites develop antimalarial resistance is essential to understanding why most drugs fail in the clinic and designing the next generation of therapies. A systematic genomic analysis of 262 Plasmodium falciparum clones with stable in vitro resistance to 37 diverse compounds with potent antimalarial activity was undertaken with the main goal of identifying new drug targets. Despite several challenges inherent to this method of in vitro drug resistance generation followed by whole genome sequencing, the study was able to identify a likely drug target or resistance gene for every compound for which resistant parasites could be generated. Known and novel P falciparum resistance mediators were discovered along with several new promising antimalarial drug targets. Surprisingly, gene amplification events contributed to one-third of the drug resistance acquisition events. The study can serve as a model for drug discovery and resistance analyses in other similar microbial pathogens amenable to in vitro culture.
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Sutrave, Smita, and Martin Heinrich Richter. "The Truman Show for Human Helminthic Parasites: A Review of Recent Advances in In Vitro Cultivation Platforms." Microorganisms 11, no. 7 (June 29, 2023): 1708. http://dx.doi.org/10.3390/microorganisms11071708.
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Throughout history, parasites and parasitic diseases have been humankind’s constant companions, as evidenced by the findings of tapeworm eggs in ancient, mummified remains. Helminths are responsible for causing severe, long-term, and debilitating infectious diseases worldwide, especially affecting economically challenged nations due to prevailing deficits in access to sanitation, proper hygiene practices, and healthcare infrastructure. Socio-ecological drivers, such as poverty, migration, and climate change, continue to contribute to parasites and their disease vectors being spread beyond known endemic zones. The study of parasitic diseases has had a fair amount of success leading to the development of new chemotherapeutic agents and the implementation of parasite eradication programs. However, further progress in this direction has been hampered by the challenges of culturing some of these parasites in in vitro systems for efficient availability, basic life cycle, infection studies, and effectiveness of novel treatment strategies. The complexity of the existing models varies widely, depending on the parasite and its life cycle, ranging from basic culture methods to advanced 3D systems. This review aims to highlight the research conducted so far in culturing and maintaining parasites in an in vitro setting, thereby contributing to a better understanding of pathogenicity and generating new insights into their lifecycles in the hopes of leading to effective treatments and prevention strategies. This work is the first comprehensive outline of existing in vitro models for highly transmissible helminth diseases causing severe morbidity and mortality in humans globally.
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Gysin, J., B. Pouvelle, N. Fievet, A. Scherf, and C. Lépolard. "Ex Vivo Desequestration of Plasmodium falciparum-Infected Erythrocytes from Human Placenta by Chondroitin Sulfate A." Infection and Immunity 67, no. 12 (December 1, 1999): 6596–602. http://dx.doi.org/10.1128/iai.67.12.6596-6602.1999.
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ABSTRACT We performed ex vivo experiments with Plasmodium falciparum-infected human placentas from primi- and multigravida women from Cameroon. All women, independent of their gravida status, had anti-chondroitin sulfate A (CSA) adhesion antibodies which cross-reacted with heterologous strains, such as FCR3 and Palo-Alto(FUP)1, which were selected for CSA binding. These antibodies, directed against the surface of infected erythrocytes obtained by flushing with CSA (IRBCCSA), were restricted to the immunoglobulin G3 isotypes. Massive desequestration of parasites was achieved with soluble CSA but not with anti-ICAM-1 and anti-CD36 monoclonal antibodies. All of the CSA-flushed parasites were analyzed immediately by using in vitro assays of binding to Saimiribrain endothelial cells (SBEC) expressing various adhesion receptors. Parasites derived from all six placentas displayed the CSA adhesion phenotype. However, only partial inhibition of adhesion was observed in the presence of soluble CSA or when Sc1D SBEC were treated with chondroitinase ABC. These results suggest that an additional adhesive molecule of IRBCCSA which binds to an unidentified receptor is present in the placenta. This new phenotype was lost once the parasites adapted to in vitro culture. We observed additional differences in the CSA adhesion phenotype between placental parasites and in vitro-cultured parasites panned on endothelial cells carrying CSA. The minimum size of fractionated CSA required for a significant inhibition of placental IRBCCSA adhesion to Sc1D cells was 1 to 2 kDa, which contrasts with the 4-kDa size necessary to reach equivalent levels of inhibition with panned IRBCCSA of this phenotype. All placental IRBCCSA cytoadhered to Sc17 SBEC, which express only the CSA receptor. Panning of IRBCCSA on these cells resulted in a significant quantitative increase of IRBC cytoadhering to the CSA of Sc1D cells but did not change their capacity for adhesion to CSA on normal placenta cryosections. Our results indicate that the CSA binding phenotype is heterogeneous and that several distinct genes may encode P. falciparum-CSA ligands with distinct binding properties.
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FRAGA, CAROLINA MIGUEL, TATIANE LUIZA DA COSTA, ANA MARIA DE CASTRO, OLIVIA REYNOSO-DUCOING, JAVIER AMBROSIO, ALICIA HERNÁNDEZ-CAMPOS, RAFAEL CASTILLO, and MARINA CLARE VINAUD. "Alternative energy production pathways in Taenia crassiceps cysticerci in vitro exposed to a benzimidazole derivative (RCB20)." Parasitology 143, no. 4 (December 28, 2015): 488–93. http://dx.doi.org/10.1017/s0031182015001729.
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SUMMARYBiochemical studies of benzimidazole derivatives are important to determine their mode of action and activity against parasites. The lack of antihelminthic alternatives to treat parasitic infections and albendazole resistance cases make the search for new antiparasitary drugs of utmost importance. The 6-chloro-5-(1-naphthyloxy)-2-(trifluoromethyl)-1H-benzimidazole (RCB20) is a benzimidazole derivative with promising effect. This study evaluated the effect of different concentrations of RCB20 in the alternative energetic pathway of in vitro Taenia crassiceps cysticerci. The parasites were in vitro exposed to 6·5 and 13 µm of RCB20 and albendazole sulfoxide (ABZSO). The quantification of acetate, acetoacetate, β-hydroxybutyrate, fumarate and propionate was performed by high-performance liquid chromatography. The quantification of urea, creatinine and total proteins was performed by spectrophotometry. The increase in β-hydroxybutyrate reflects the enhancement of the fatty acid oxidation in the treated groups. Volatile fatty acids secretion, acetate and propionate, was increased in the treated groups. The secretion mechanisms of the treated parasites were impaired due to organic acids increased concentrations in the cysticerci. It is possible to conclude that the metabolic effect on alternative energetic pathways is slightly increased in the parasites treated with RCB20 than the ones treated with ABZSO.