IL-17Aは赤痢アメーバの腸管感染においてIFN-γ/IL-4比の減少と持続感染に寄与する

Amebiasis is an infectious disease caused by Entamoeba histolytica, an anaerobic protozoan parasite, and is a major public health problem worldwide, particularly in areas with inadequate sanitation and poor hygiene. Th1 responses, represented by interferon gamma (IFN-γ), play a protective role by clearing the amebae from the gut, whereas Th2 responses are responsible for chronic infection. Th17 responses preconditioned by vaccination or by modulating the intestinal microbiome protect mice from the settlement of E. histolytica. However, the role of interleukin-17A (IL-17A), which is upregulated during the natural course of intestinal amebiasis, has not been clarified. The aim of this study was to investigate the role of IL-17A during intestinal amebiasis in a mouse model. IL-17A knockout and wild-type CBA/J mice were challenged intracecally with 2 × 106 E. histolytica trophozoites, and their infection, pathology, and immune responses were monitored. Neither the initial settlement of E. histolytica nor the inflammation of the cecum was affected by the absence of IL-17A for week 1, but the infection rate and parasite burden declined in a late stage of infection, accompanied by an increased IFN-γ/IL-4 ratio. Therefore, IL-17A contributes to the persistence of E. histolytica and modulates the immune response, including the IFN-γ/IL-4 ratio, which may be responsible for the reduction of the parasite burden in the IL-17A knockout mice during the chronic phase of intestinal amebiasis.長崎大学学位論文 学位記番号:博(医歯薬)甲第1005号 学位授与年月日:平成29年12月6日Author: Sharmina Deloer, Risa Nakamura, Mihoko Kikuchi, Taeko Moriyasu, Yombo Dan Justin Kalenda, Eman Sayed Mohammed, Masachika Senba, Yoichiro Iwakura, Hiroki Yoshida, Shinjiro HamanoCitation: Parasitology International, 66(6), pp.817-823; 2017Nagasaki University (長崎大学)課程博

Prevalence of anopheline species and their Plasmodium infection status in epidemic-prone border areas of Bangladesh

<p>Abstract</p> <p>Background</p> <p>Information related to malaria vectors is very limited in Bangladesh. In the changing environment and various <it>Anopheles </it>species may be incriminated and play role in the transmission cycle. This study was designed with an intention to identify anopheline species and possible malaria vectors in the border belt areas, where the malaria is endemic in Bangladesh.</p> <p>Methods</p> <p><it>Anopheles </it>mosquitoes were collected from three border belt areas (Lengura, Deorgachh and Matiranga) during the peak malaria transmission season (May to August). Three different methods were used: human landing catches, resting collecting by mouth aspirator and CDC light traps. Enzyme-linked immunosorbent assay (ELISA) was done to detect <it>Plasmodium falciparum</it>, <it>Plasmodium vivax</it>-210 and <it>Plasmodium vivax</it>-247 circumsporozoite proteins (CSP) from the collected female species.</p> <p>Results</p> <p>A total of 634 female <it>Anopheles </it>mosquitoes belonging to 17 species were collected. <it>Anopheles vagus </it>(was the dominant species (18.6%) followed by <it>Anopheles nigerrimus </it>(14.5%) and <it>Anopheles philippinensis </it>(11.0%). Infection rate was found 2.6% within 622 mosquitoes tested with CSP-ELISA. Eight (1.3%) mosquitoes belonging to five species were positive for <it>P. falciparum</it>, seven (1.1%) mosquitoes belonging to five species were positive for <it>P. vivax </it>-210 and a single mosquito (0.2%) identified as <it>Anopheles maculatus </it>was positive for <it>P. vivax</it>-247. No mixed infection was found. Highest infection rate was found in <it>Anopheles karwari </it>(22.2%) followed by <it>An. maculatus </it>(14.3%) and <it>Anopheles barbirostris </it>(9.5%). Other positive species were <it>An. nigerrimus </it>(4.4%), <it>An. vagus </it>(4.3%), <it>Anopheles subpictus </it>(1.5%) and <it>An. philippinensis </it>(1.4%). <it>Anopheles vagus </it>and <it>An. philippinensis </it>were previously incriminated as malaria vector in Bangladesh. In contrast, <it>An. karwari</it>, <it>An. maculatus</it>, <it>An. barbirostris</it>, <it>An. nigerrimus </it>and <it>An. subpictus </it>had never previously been incriminated in Bangladesh.</p> <p>Conclusion</p> <p>Findings of this study suggested that in absence of major malaria vectors there is a possibility that other <it>Anopheles </it>species may have been playing role in malaria transmission in Bangladesh. Therefore, further studies are required with the positive mosquito species found in this study to investigate their possible role in malaria transmission in Bangladesh.</p

Characterization of Entamoeba histolytica adenosine 5′-phosphosulfate (APS) kinase; validation as a target and provision of leads for the development of new drugs against amoebiasis

BACKGROUND: Amoebiasis, caused by Entamoeba histolytica infection, is a global public health problem. However, available drugs to treat amoebiasis are currently limited, and no effective vaccine exists. Therefore, development of new preventive measures against amoebiasis is urgently needed. METHODOLOGY/PRINCIPAL FINDINGS: Here, to develop new drugs against amoebiasis, we focused on E. histolytica adenosine 5\u27-phosphosulfate kinase (EhAPSK), an essential enzyme in Entamoeba sulfolipid metabolism. Fatty alcohol disulfates and cholesteryl sulfate, sulfolipids synthesized in Entamoeba, play important roles in trophozoite proliferation and cyst formation. These processes are closely associated with clinical manifestation and severe pathogenesis of amoebiasis and with disease transmission, respectively. We validated a combination approach of in silico molecular docking analysis and an in vitro enzyme activity assay for large scale screening. Docking simulation ranked the binding free energy between a homology modeling structure of EhAPSK and 400 compounds. The 400 compounds were also screened by a 96-well plate-based in vitro APSK activity assay. Among fifteen compounds identified as EhAPSK inhibitors by the in vitro system, six were ranked by the in silico analysis as having high affinity toward EhAPSK. Furthermore, 2-(3-fluorophenoxy)-N-[4-(2-pyridyl)thiazol-2-yl]-acetamide, 3-phenyl-N-[4-(2-pyridyl)thiazol-2-yl]-imidazole-4-carboxamide, and auranofin, which were identified as EhAPSK inhibitors by both in silico and in vitro analyses, halted not only Entamoeba trophozoite proliferation but also cyst formation. These three compounds also dose-dependently impaired the synthesis of sulfolipids in E. histolytica. CONCLUSIONS/SIGNIFICANCE: Hence, the combined approach of in silico and in vitro-based EhAPSK analyses identified compounds that can be evaluated for their effects on Entamoeba. This can provide leads for the development of new anti-amoebic and amoebiasis transmission-blocking drugs. This strategy can also be applied to identify specific APSK inhibitors, which will benefit research into sulfur metabolism and the ubiquitous pathway terminally synthesizing essential sulfur-containing biomolecules

<i>Schistosoma mansoni</i> infection suppresses the growth of <i>Plasmodium yoelii</i> parasites in the liver and reduces gametocyte infectivity to mosquitoes

<div><p>Malaria and schistosomiasis are major parasitic diseases causing morbidity and mortality in the tropics. Epidemiological surveys have revealed coinfection rates of up to 30% among children in Sub-Saharan Africa. To investigate the impact of coinfection of these two parasites on disease epidemiology and pathology, we carried out coinfection studies using <i>Plasmodium yoelii</i> and <i>Schistosoma mansoni</i> in mice. Malaria parasite growth in the liver following sporozoite inoculation is significantly inhibited in mice infected with <i>S</i>. <i>mansoni</i>, so that when low numbers of sporozoites are inoculated, there is a large reduction in the percentage of mice that go on to develop blood stage malaria. Furthermore, gametocyte infectivity is much reduced in mice with <i>S</i>. <i>mansoni</i> infections. These results have profound implications for understanding the interactions between <i>Plasmodium</i> and <i>Schistosoma</i> species, and have implications for the control of malaria in schistosome endemic areas.</p></div

Information for PCR primers.

<p>Information for PCR primers.</p

Malaria parasite liver burden in BALB/c, C57BL/6, and CBA/J mice.

<p>(A) Copy number of <i>Plasmodium yoelii</i> 18s RNA gene per 1×10⁶ mouse G3PDH gene measured at 42 h post sporozoite inoculation. Female BALB/c, B6, and CBA/J mice (N = 5) infected with 50 <i>Schistosoma mansoni</i>-cercaria 10 weeks previously were challenged with 1,500 SPZ of <i>P</i>. <i>yoelii</i> along with <i>S</i>. <i>mansoni-</i>non-infected controls. BALB/c: ***P<0.001, t = 6.283, df = 8; B6: *P<0.05, t = 2.511, df = 8; CBA: **P<0.01, t = 4.220, df = 7. (B) Copy number of <i>Plasmodium berghei</i> 18s RNA gene per 1×10⁶ mouse G3PDH gene measured at 42 h post sporozoite inoculation. Female BALB/c, B6, and CBA/J mice (N = 5) infected with 50 <i>S</i>. <i>mansoni</i>-cercaria 10 weeks previously were challenged with 1,500 SPZ of <i>P</i>. <i>berghei</i> along with <i>S</i>. <i>mansoni-</i>non-infected controls. BALB/c: *P<0.05, t = 2.306, df = 8; B6: ***P<0.001, t = 5.336, df = 8; CBA: *P<0.05, t = 2.846, df = 7. All data were statistically examined using Student’s two-tailed t-test.</p

Gametocyte infectivity.

<p>(A) Parasitemia. Female BALB/c mice were each inoculated with 1 x 10⁶ <i>Plasmodium yoelii</i> parasitized erythrocytes intravenously with (n = 4 mice) or without (n = 5) pre-existing <i>Schistosoma mansoni</i> infection. Parasitaemia was determined by microscopic examination on days 3, 4, and 5 post-inoculation; day 3: Student’s two-tailed t-test; **P<0.01, t = -4.906, df = 7; Day5: *P<0.05, t = -2.922, df = 5 (<b>B</b>) Gametocyte density. Gametocyte density was determined on days 3 and 4 post-inoculation of 1 x 10⁶ <i>P</i>. <i>yoelii</i>-parasitized erythrocytes intravenously. Day 3: Student’s two-tailed t-test; **P<0.01, t = 3.813, df = 5; Day 4: **P<0.01, t = 3.608, df = 5. Error bars show the geometric mean with 95% confidence intervals. (C) Percentage of mosquitoes with one or more oocysts present on the midgut eight days post-feeding on infected mice. A minimum of eight mosquitoes were allowed to feed on each individual mouse in the group per day **P = 0.0003, (2-way ANOVA, F = 22.23, DFn = 1, DFd = 14). Error bars mar the standard error of the mean per mouse group. (D) Oocyst numbers per mosquito. The numbers of oocysts present on mosquito midguts were determined eight days post-mosquito feeding; day 3: Student’s two-tailed t-test, **P<0.01, t = 3.077, df = 25. Error bars show the geometric mean with 95% confidence intervals. Data is representative of three independent experiments.</p

Liver immunopathology in <i>Schistosoma mansoni</i>-cercariae infection and intraportal infusion of frozen <i>S</i>. <i>mansoni</i>-eggs.

<p>(A) Malaria parasite liver burden with/without intraportal infusion of frozen <i>S</i>. <i>mansoni</i>-eggs. Female B6 mice were intraportally inoculated 3,000/10,000 frozen <i>S</i>. <i>mansoni</i>-eggs and challenged with 1,500 sporozoites of <i>Plasmodium yoelii</i> along with controls inoculated with PBS (control: N = 3; 3,000 frozen eggs: N = 5, ***P<0.001, t = 1.943, df = 6; 10,000 frozen eggs: N = 3, *P<0.05, Student’s two-tailed t-test, t = 4.072, df = 4). (B) Macroscopic and microscopic images of liver pathology. The black lines indicate 250 micro meters. (C) The number of granulomas in the liver. Female BALB/c mice and B6 mice were inoculated with 50 <i>S</i>. <i>mansoni</i>-cercariae subcutaneously (naive: N = 5; 50 cercariae: N = 3) or inoculated with 3,000 frozen <i>S</i>. <i>mansoni</i>-eggs along with controls inoculated with PBS intraportaly (PBS: N = 5; 3,000 frozen eggs: N = 5). The numbers of granuloma were counted in 20 microscopic fields at 100 x magnification. *P<0.05, ***P<0.001. All data were statistically examined using Student’s two-tailed t-test. (D) The numbers of immune cells induced by intraportal infusion of 3,000 frozen <i>S</i>. <i>mansoni</i>-eggs. Female B6 mice (N = 4/group) were intraportaly inoculated 3,000 <i>S</i>. <i>mansoni</i>-eggs. (E) The numbers of immune cells induced by 50 <i>S</i>. <i>mansoni</i>-cercaria infection. Female BALB/c mice (naive: N = 9, 8 weeks: N = 9, 10 weeks N = 6) were infected with 50 <i>S</i>. <i>mansoni</i>-cercariae. *P<0.05, **P<0.01, ***P<0.001. All data were statistically examined using Student’s two-tailed t-test.</p