Towards an In Vitro Model of Plasmodium Hypnozoites Suitable for Drug Discovery

Contains fulltext : 96475.pdf (publisher's version ) (Open Access)BACKGROUND: Amongst the Plasmodium species in humans, only P. vivax and P. ovale produce latent hepatic stages called hypnozoites, which are responsible for malaria episodes long after a mosquito bite. Relapses contribute to increased morbidity, and complicate malaria elimination programs. A single drug effective against hypnozoites, primaquine, is available, but its deployment is curtailed by its haemolytic potential in glucose-6-phosphate dehydrogenase deficient persons. Novel compounds are thus urgently needed to replace primaquine. Discovery of compounds active against hypnozoites is restricted to the in vivo P. cynomolgi-rhesus monkey model. Slow growing hepatic parasites reminiscent of hypnozoites had been noted in cultured P. vivax-infected hepatoma cells, but similar forms are also observed in vitro by other species including P. falciparum that do not produce hypnozoites. METHODOLOGY: P. falciparum or P. cynomolgi sporozoites were used to infect human or Macaca fascicularis primary hepatocytes, respectively. The susceptibility of the slow and normally growing hepatic forms obtained in vitro to three antimalarial drugs, one active against hepatic forms including hypnozoites and two only against the growing forms, was measured. RESULTS: The non-dividing slow growing P. cynomolgi hepatic forms, observed in vitro in primary hepatocytes from the natural host Macaca fascicularis, can be distinguished from similar forms seen in P. falciparum-infected human primary hepatocytes by the differential action of selected anti-malarial drugs. Whereas atovaquone and pyrimethamine are active on all the dividing hepatic forms observed, the P. cynomolgi slow growing forms are highly resistant to treatment by these drugs, but remain susceptible to primaquine. CONCLUSION: Resistance of the non-dividing P. cynomolgi forms to atovaquone and pyrimethamine, which do not prevent relapses, strongly suggests that these slow growing forms are hypnozoites. This represents a first step towards the development of a practical medium-throughput in vitro screening assay for novel hypnozoiticidal drugs

Gene Disruption of Plasmodium falciparum p52 Results in Attenuation of Malaria Liver Stage Development in Cultured Primary Human Hepatocytes

Difficulties with inducing sterile and long lasting protective immunity against malaria with subunit vaccines has renewed interest in vaccinations with attenuated Plasmodium parasites. Immunizations with sporozoites that are attenuated by radiation (RAS) can induce strong protective immunity both in humans and rodent models of malaria. Recently, in rodent parasites it has been shown that through the deletion of a single gene, sporozoites can also become attenuated in liver stage development and, importantly, immunization with these sporozoites results in immune responses identical to RAS. The promise of vaccination using these genetically attenuated sporozoites (GAS) depends on translating the results in rodent malaria models to human malaria. In this study, we perform the first essential step in this transition by disrupting, p52, in P. falciparum an ortholog of the rodent parasite gene, p36p, which we had previously shown can confer long lasting protective immunity in mice. These P. falciparum P52 deficient sporozoites demonstrate gliding motility, cell traversal and an invasion rate into primary human hepatocytes in vitro that is comparable to wild type sporozoites. However, inside the host hepatocyte development is arrested very soon after invasion. This study reveals, for the first time, that disrupting the equivalent gene in both P. falciparum and rodent malaria Plasmodium species generates parasites that become similarly arrested during liver stage development and these results pave the way for further development of GAS for human use

Antibiotics-induced oxidative stress

This review comes from a themed issue on Oxidative Toxicology; Edited by Yvonne Will, Moorthy Bhagavatula, Martin van den Berg and Jose ManautouInternational audienceAround one hundred drugs of our modern pharmacopeia are efficacious and useable as antibiotics (ATBs) in medicine; they are used to kill or block growth of bacteria. Bactericidal ATBs can induce a common oxidative damage pathway, leading to the production of reactive oxygen species and cell death. ATBs can also damage various mammalian cell types and tissues but mechanisms of action remain relatively unclear. Both bactericidal and bacteriostatic ATBs can target mitochondria but only the former usually induce mitochondrial dysfunction and oxidative stress at clinically relevant doses. Human liver is a major target of ATBs of which toxicity is mostly idiosyncratic. Interestingly, β-lactam penicillinase-resistant ATBs, which are known to cause mostly immune reactions in patients, induce an early endoplasmic reticulum stress in in vitro human hepatocytes at low concentrations; this stress is inhibited by activation of the HSP27 protein which acts as a protective response associated with occurrence of cholestatic features. In this review, we analyze the importance of oxidative and endoplasmic reticulum stress in cellular damage induced by ATBs, especially in hepatocytes and highlight specific cellular protection mechanisms associated with penicillinase-resistant ATB treatments

HepaRG Cells as a Model for Hepatotoxicity Studies

International audienceThe human HepaRG cell line has been established from a hepatocholangiocarcinoma and first described in 2002; it possesses bipotent properties allowing commitment into two cell lineages hepatocytes expressing various functions of mature hepatocytes and primitive biliary cells. HepaRG cells can retrodifferentiate and transdifferentiate. Mature HepaRG hepatocytes express most phase 1 (including the major cytochromes P450 3A4, 2B6, and 1A2) and phase 2 enzymes, as well as the main transporters involved in drug and bile acid metabolism and excretion, and exhibit appropriate responsiveness to prototypical inducers and inhibitors. They can remain functionally stable in a nonproliferative state for several weeks; meanwhile, they progress toward aging. HepaRG cells are widely used to investigate different types of chemical-induced hepatotoxicity, including cell death, cholestasis, steatosis, and phospholipidosis, as well as genotoxicity, after acute and/or repeated treatment with reference xenobiotics. Overall, they represent a reliable source of metabolically competent human liver cells, provide consistent responses to xenobiotics and, therefore, can be used as a surrogate to primary human hepatocytes for investigating drug metabolism parameters and both acute and chronic effects of xenobiotics in human liver. © 2018 John Wiley and Sons, Inc

Endoplasmic reticulum stress precedes oxidative stress in antibiotic-induced cholestasis and cytotoxicity in human hepatocytes

International audienceEndoplasmic reticulum (ER) stress has been associated with various drug–induced liver lesions but its participation in drug-induced cholestasis remains unclear. We first aimed at analyzing liver damage caused by various hepatotoxic antibiotics, including three penicillinase-resistant antibiotics (PRAs), i.e. flucloxacillin, cloxacillin and nafcillin, as well as trovafloxacin, levofloxacin and erythromycin, using human differentiated HepaRG cells and primary hepatocytes. All these antibiotics caused early cholestatic effects typified by bile canaliculi dilatation and reduced bile acid efflux within 2 h and dose-dependent enhanced caspase-3 activity within 24 h. PRAs induced the highest cholestatic effects at non cytotoxic concentrations. Then, molecular events involved in these lesions were analyzed. Early accumulation of misfolded proteins revealed by thioflavin-T fluorescence and associated with phosphorylation of the unfolded protein response sensors, eIF2α and/or IRE1α, was evidenced with all tested hepatotoxic antibiotics. Inhibition of ER stress markedly restored bile acid efflux and prevented bile canaliculi dilatation. Downstream of ER stress, ROS were also generated with high antibiotic concentrations. The protective HSP27-PI3K-AKT signaling pathway was activated only in PRA-treated cells and its inhibition increased ROS production and aggravated caspase-3 activity. Overall, our results demonstrate that (i) various antibiotics reported to cause cholestasis and hepatocellular injury in the clinic can also induce such effects in in vitro human hepatocytes; (ii) PRAs cause the strongest cholestatic effects in the absence of cytotoxicity; (iii) cholestatic features occur early through ER stress; (iv) cytotoxic lesions are observed later through ER stress-mediated ROS generation; and (v) activation of the HSP27-PI3K-AKT pathway protects from cytotoxic damage induced by PRAs only. © 2017 Elsevier Inc