ヘパドナウイルスのエンハンサー領域の保存された配列に結合する核タンパク質の精製とその性質

取得学位 : 博士（医学）, 学位授与番号 : 医博甲第979号, 学位授与年月日：平成3年3月25日,学位授与年：199

Resistance of a Rodent Malaria Parasite to a Thymidylate Synthase Inhibitor Induces an Apoptotic Parasite Death and Imposes a Huge Cost of Fitness

BACKGROUND: The greatest impediment to effective malaria control is drug resistance in Plasmodium falciparum, and thus understanding how resistance impacts on the parasite's fitness and pathogenicity may aid in malaria control strategy. METHODOLOGY/PRINCIPAL FINDINGS: To generate resistance, P. berghei NK65 was subjected to 5-fluoroorotate (FOA, an inhibitor of thymidylate synthase, TS) pressure in mice. After 15 generations of drug pressure, the 2% DT (the delay time for proliferation of parasites to 2% parasitaemia, relative to untreated wild-type controls) reduced from 8 days to 4, equalling the controls. Drug sensitivity studies confirmed that FOA-resistance was stable. During serial passaging in the absence of drug, resistant parasite maintained low growth rates (parasitaemia, 15.5%±2.9, 7 dpi) relative to the wild-type (45.6%±8.4), translating into resistance cost of fitness of 66.0%. The resistant parasite showed an apoptosis-like death, as confirmed by light and transmission electron microscopy and corroborated by oligonucleosomal DNA fragmentation. CONCLUSIONS/SIGNIFICANCE: The resistant parasite was less fit than the wild-type, which implies that in the absence of drug pressure in the field, the wild-type alleles may expand and allow drugs withdrawn due to resistance to be reintroduced. FOA resistance led to depleted dTTP pools, causing thymineless parasite death via apoptosis. This supports the tenet that unicellular eukaryotes, like metazoans, also undergo apoptosis. This is the first report where resistance to a chemical stimulus and not the stimulus itself is shown to induce apoptosis in a unicellular parasite. This finding is relevant in cancer therapy, since thymineless cell death induced by resistance to TS-inhibitors can further be optimized via inhibition of pyrimidine salvage enzymes, thus providing a synergistic impact. We conclude that since apoptosis is a process that can be pharmacologically modulated, the parasite's apoptotic machinery may be exploited as a novel drug target in malaria and other protozoan diseases of medical importance

Induction of Protective Immunity to Listeria monocytogenes with Dendritic Cells Retrovirally Transduced with a Cytotoxic T Lymphocyte Epitope Minigene

In the present study, we developed a cytotoxic T lymphocyte (CTL) epitope minigene-transduced dendritic cell (DC)-based vaccine against Listeria monocytogenes. Murine bone marrow-derived DCs were retrovirally transduced with a minigene for listeriolysin O (LLO) 91-99, a dominant CTL epitope of L. monocytogenes, and were injected into BALB/c mice intravenously. We found that the DC vaccine was capable of generating peptide-specific CD8(+) T cells exhibiting LLO 91-99-specific cytotoxic activity and gamma interferon production, leading to induction of protective immunity to the bacterium. Furthermore, we demonstrated that the retrovirally transduced DC vaccine was more effective than a CTL epitope peptide-pulsed DC vaccine and a minigene DNA vaccine for eliciting antilisterial immunity. These results provide an alternative strategy in which retrovirally transduced DCs are used to design vaccines against intracellular pathogens

Resistance induces internucleosomal DNA fragmentation in FOA-resistant parasite grown in mice without drug.

<p>Polyacrylamide gel electrophoretic fraction of DNA was done in two independent experiments, A and B. In A, lanes 1 represent DNA extracted from the wild-type parasite that shows no cleavage, while lanes 2 represent cleaved DNA of FOA-resistant parasite, with the main band at ≈200 bp. Similar results are represented in B, where the wild-type shows no cleaved DNA (lanes 1), while FOA-resistant parasite shows cleaved DNA and a clear band at ≈200 bp (lanes 2). Lane M is the molecular size marker (pSG5/Hinf I and φX174/HincII for A, and pSG5/Hinf I for B).</p

Parasitaemia patterns of mice infected with FOA-resistant parasite and orally treated with FOA.

<p>The mice were treated twice daily for 3 days with FOA (40 mg/kg cumulative dose) at passages 2, 5, 10 and 12. Note that the patterns mirror each other, confirming that the acquired FOA-resistance is stable. The discontinuous curve indicates that following FOA administration to mice infected with the wild-type parasite, no parasites could be observed under the microscope until day 14 p.i when recrudescent parasites were observed.</p

Electron micrographs of FOA-resistant and wild-type parasites grown in mice in the absence of drug.

<p>In (B), the trophozoite of the wild-type parasite shows normal ultrastructural morphology with various compartments and organelles well defined by membranes that have retained their integrity. In (A), late schizont of FOA-resistant parasite is seen with intact plasmalemma housing non-viable segmenters/merozoites that appear as electron dense bodies probably due to compaction of nuclear chromatin and condensation of cytoplasm. (C) is an immuno-gold electron micrograph adapted from Bhowmick et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021251#pone.0021251-Bhowmick1" target="_blank">[114]</a> showing a similar ‘syncytial’ cell from <i>P. falciparum</i> with viable merozoites. Note the distinct nucleus of segmenters. Abbreviations: CN, condensed segmenter; E, erythrocyte; FV, food vacuole; IPV, intraparasitic vacuole; M, mitochodria; N, nucleus; NM, nuclear membrane; PM, plasma membrane; PV, parasitophorous vacuole; PVM, parasitophorous vacuolar membrane; VL, vacuolization.</p