Novel rational drug design strategies with potential to revolutionize malaria chemotherapy.

Efforts to develop an effective malaria vaccine are yet to be successful and thus chemotherapy remains the mainstay of malaria control strategy. Plasmodium falciparum, the parasite that causes about 90% of all global malaria cases is increasingly becoming resistant to most antimalarial drugs in clinical use. This dire situation is aggravated by reports from Southeast Asia, of the parasite becoming resistant to the "magic bullet" artemisinins, the last line of defense in malaria chemotherapy. Drug development is a laborious and time consuming process, and thus antimalarial drug discovery approaches currently being deployed largely include optimization of therapy with available drugs--including combination therapy and developing analogues of the existing drugs. However, the latter strategy may be hampered by crossresistance, since agents that are closely related chemically may share similar mechanisms of action and/or targets. This may render new drugs ineffective even before they are brought to clinical use. Evaluation of drug-resistance reversers (chemosensitizers) against quinoline-based drugs such as chloroquine and mefloquine is another approach that is being explored. Recently, evaluation of new chemotherapeutic targets is gaining new impetus as knowledge of malaria parasite biology expands. Also, single but hybrid molecules with dual functionality and/or targets have been developed through rational drug design approach, termed as "covalent bitherapy". Since desperate times call for radical measures, this review aims to explore novel rational drug-design strategies potentially capable of revolutionizing malaria therapy. We thus explore malaria apoptosis machinery as a novel drug target, and also discuss the potential of hybrid molecules as well as prodrugs and double prodrugs in malaria chemotherapy.

Cytokine and antibody production during the course of resolution in Plasmodium yoelii 17XL-infected BALB/c mice treated with febrifugine and isofebrifugine mixture from leaves of Hydrangea macrophylla var. Otaksa.

Cytokine and antibody production was investigated during the course of resolution of primary infection in Plasmodium yoelii 17XL-infected BALB/c mice treated with a mixture of febrifugine and isofebrifugine. The infected mice in an untreated control group showed a progressively increasing parasitemia, leading to mouse death. In contrast, infected mice given the mixture orally showed low parasitemia levels during administration. Following a transient increase in parasitemia in the bloodstream of the treated mice, no parasites could be detected by microscopic examination. Analysis of cytokines in plasma showed that the plasma IFN-gamma levels elevated significantly within the first week of infection in both groups. Furthermore, on day 20 the plasma IFN-gamma and IL-4 levels elevated significantly in the treated mice and the production of both cytokines was sustained until at least day 40. The production of both cytokines in the treated mice was coincident with a decrease in parasitemia. The production of parasite-specific antibodies in the course of P. yoelii 17XL infection was also monitored. In the drug-treated mice, the titers of parasite-specific IgG1, IgG2a, IgG2b and IgG3 elevated significantly from day 20; and the production of parasite-specific antibodies was coincident with a decrease in parasite numbers in the bloodstream.

Hymenolepis pseudodiminuta Tenora et al. 1994 from Apodemus speciosus and H. diminuta: a comparison of experimental infections in rats.

The successful maintenance of Hymenolepis pseudodiminuta, isolated from Apodemus speciosus, is described for the first time. In the laboratory, the flour beetle, Tribolium confusum, and F344 rats could serve as intermediate and definitive hosts, respectively. In single worm infections with H. pseudodiminuta, which were carried in two groups of rats, adult worms were recovered from eight and seven out of ten rats, respectively, while Hymenolepis diminuta was found in all of ten rats 6 weeks after inoculation. The worm weight of H. pseudodiminuta in rats was significantly lower than that of H. diminuta. The egg output of H. pseudodiminuta occurred significantly earlier than that of H. diminuta. The number of eggs in the faeces of H. diminuta-infected rats was approximately twofold higher than the number in the faeces of H. pseudodiminuta-infected rats throughout the course of the infection. Mucosal mast cells in rats infected with H. pseudodiminuta were significantly more common than in rats infected with H. diminuta. No detectable IgE antibodies were found in the uninfected and H. diminuta-infected rat groups; however total IgE was detected in H. pseudodiminuta-infected rats but the concentrations were variable between individuals.

Intestinal granuloma formation in normal and SCID BALB/c mice infected with Angiostrongylus costaricensis.

We studied the histomorphology of granuloma formation and cytokine production in Angiostrongylus costaricensis-infected immunocompetent and immunodeficient BALB/c mice. Histological examination of the infected intestine showed well developed granulomas in BALB/c mice. In contrast, in SCID mice, clusters of eggs and larvae and a progression of infiltration of inflammatory cells were also observed, but little fibroblastic activity was seen. Analysis of plasma of the infected mice demonstrated a dramatic contrast in the cytokine profile between normal and SCID mice. Normal mice infected with A. costaricensis showed highly elevated plasma IFN-gamma levels at week 3 of infection, but plasma IL-4 remained close to the background levels obtained from non-infected mice. In contrast, the amount of those cytokines in the plasma of SCID animals was little affected by this parasitic infection. These results suggest that Th1-mediated immunity is required for granuloma formation in response to A. costaricensis infection in mice.

A potent antimalarial activity of Hydrangea macrophylla var. Otaksa leaf extract against Plasmodium yoelii 17XL in mice.

The antimalarial activity of the hot-water extract of Hydrangea macrophylla var. Otaksa leaves was evaluated against Plasmodium yoelii 17XL in mice. Non-treated control mice died from 6 to 7 days after infection, but mice treated with the leaf extract survived during the experiment. Mice given the extract orally showed low parasitemia levels during administration. Following a transient recrudescence of malaria parasites in the bloodstream of treated mice, no parasites could be detected by a microscopic examination. Furthermore, the 30% MeOH aq. eluate and 50% MeOH aq. eluate from dried leaves of H. macrophylla var. Otaksa showed an antimalarial activity in vivo. Sulfamonomethoxine was orally given to infected mice to compare with the antimalarial activity of the hot-water extract of leaves. Sulfamonomethoxine given orally reduced parasitemia, but no complete cure of mice was observed.

A brief survey of free-living amebae in Thailand and Hamamatsu District, Japan.

The aim of this study was to determine the presence of free-living amebae in aquatic habitats of human environments in Thailand and Hamamatsu district, Japan. Genus identification was based on the morphology of cyst and trophozoite forms and a flagellation test for genus Naegleria. The pathogenic potential was tested in mice by nasal instillation for genus Naegleria and Acanthameba. In 14 provinces of Thailand, amebae were isolated in 43 from 95 water samples and 67 from 120 soil swabs. Amebae of 49 isolates from waters were identified as Acanthameba (36.7%), Naegleria (28.6%), Hartmannella (20.4%), Vahlkampfia (12.2%) and Vannella (2%). Soil samples have significantly higher levels of Acanthameba and Hartmannella (p<0.05) but lower for Naegleria (p<0.05) and 7 unidentified amebae were found. In Hamamatsu district, Japan, 62 amebae of the same genera were isolated from 47 of 95 water samples. There were significantly higher levels of Acanthameba (22.6%) (p<0.05) but lower for Naegleria (4.8%) (p<0.05) than those of Thailand which each of them caused death in mice. Three unidentified amebae were isolated. This finding serves as additional evidence for the presence of free-living amebae under natural and the difference in distribution between tropic and subtropic areas.

Effects of anthelmintics on the development of eggs of Angiostrongylus costaricensis in vitro.

Effects of the anthelmintics, pyrantel and levamisole, on egg development of Angiostrongylus costaricensis were studied in vitro. After 7 days, about 80% of eggs developed to first-stage larvae in Ham's F-12 medium with 10% foetal calf serum under 5% CO2. Significant inhibition of development was caused by pyrantel (10(-9) - 10(-8) g ml(-1)) and levamisole (10(-9) - 10(-8) g ml(-1)) (Mann-Whitney U-test; ), and none of the eggs developed to first-stage larvae in higher concentrations of these anthelmintics (10(-7) g ml(-1)). Furthermore, incubation with these drugs at 10(-8) g ml(-1) for at least 3 h or at 10(-4) g ml(-1) for 1 h caused irreversible effects on egg development.

Chromosomal mapping of the host resistance locus to rodent malaria (Plasmodium yoelii) infection in mice.

The disease outcome in malaria caused by the protozoan parasite Plasmodium is influenced by host genetic factors. To identify host genes conferring resistance to infection with the malaria parasite, we undertook chromosomal mapping using a whole-genome scanning approach in cross-bred mice. NC/Jic mice all died with high parasitemia within 8 days of infection with 1 x 10(5) parasitized erythrocytes. In contrast, 129/SvJ mice all completely excluded malaria parasites from the circulation and remained alive 21 days after infection. We performed linkage analysis in backcross [(NC/Jic x 129/SvJ)xNC/Jic] mice. The Pymr ( Plasmodium yoelii malaria resistance) locus was mapped to the telomeric portion of mouse Chromosome (Chr) 9. This locus controls host survival and parasitemia after infection. The Char1 locus ( P. chabaudi resistance locus 1), controlling host survival and peak parasitemia in P. chabaudi infection, was previously mapped to the same region. This host resistance locus mapping to Chr 9 may represent a ubiquitous locus controlling susceptibility to rodent malaria. Elucidation of the function of this gene will provide valuable insights into the mechanism of host defense against malaria parasite infection.

Immunoglobulin E and mast cell responses are related to worm biomass but not expulsion of Hymenolepis diminuta during low dose infection in rats.

It is well known that the destrobilation and later expulsion are characteristics of multiple Hymenolepis diminuta infections in rats. This process is suggested to be mediated by a variety of host cellular responses. It has also been suggested that immunoglobulin (Ig) E may have a beneficial role for some cestodes including H. diminuta. We examined the intestinal mast cell and serum IgE responses to a 10-H. diminuta infection in three different rat strains. Tapeworm infection induced no increased mast cell and IgE responses in F344 rats in which neither worm biomass nor worm burden decreased during 6 weeks of observation. The number of mast cells and amounts of serum rat mast cell protease (RMCP) II and IgE markedly increased from 3 weeks postinfection (p.i.) in BN rats. The worm biomass in BN rats was significantly lower than that in F344 rats, but worm burden was not different from that in F344 rats at 3 or 6 weeks p.i. In DA rats, the number of mast cells and levels of serum RMCP II and IgE increased at 6 weeks but not at 3 weeks p.i. Although numbers of mast cells and serum RMCP II and IgE levels were lower in DA rats than in BN rats, smaller and fewer worms were recovered in DA rats than in F344 and BN rats at from 3 and 6 weeks p.i. Worms were recovered from all of F344 and BN rats, while only 40% of DA rats harboured worms at 6 weeks p.i. These results suggested that the worm biomass was related to mast cell and IgE responses, but these responses were not required for worm expulsion during low dose H. diminuta infection in rats.

Genetic analysis of mortality in murine angiostrongyliasis costaricensis using SMXA recombinant inbred mouse strains.

The SMXA recombinant inbred mouse strain set was produced by systematic inbreeding from the F2 generation of a cross between two progenitor inbred strains, A/J and SM/J, which differed markedly with respect to the patterns of infection with Angiostrongylus costaricensis. We have applied this set to genetic analysis of mouse susceptibility to this nematode infection. The mortality was variable among substrains of the SMXA RI strains, indicating the involvement of multiple genes. Linkage analysis showed several chromosomal regions closely linked to mortality; chromosome 6 (D6Rik86, 87; P<==0.001), 10 (D10Rik66-D10Mit12; P=0.0058), 13 (D13Rik79, 80; P=0.0096) and 17 (D17Mit28-D17Rik76; P=0.0088).

Differential responses of SM/J and A/J mice to experimental Angiostrongylus costaricensis infection.

Angiostrongylus costaricensis matures in mice, but shows variation in mouse mortality and worm burden among inbred strains. Differences in response to infection may be controlled genetically. The patterns of infection with A. costaricensis in SM/J and A/J mouse strains differed markedly in terms of level of haematocrit and the magnitude of splenomegaly.

Effects of PF1022A on adult Angiostrongylus cantonensis in the pulmonary arteries and larvae migrating into the central nervous system of rats.

We examined the effects of PF1022A, newly developing in Japan, on adult Angiostrongylus cantonensis in the pulmonary arteries of rats. Following five and ten successive oral doses at 10 mg/kg per day, the first-stage larvae in rat faeces disappeared completely at 2 weeks after treatment. The treatment completely killed the female worms, but not the male worms. However, numbers of male worms were also decreased after the administration of either five successive oral doses at 10 mg/kg per day for four courses or five successive intraperitoneal doses at 0.5 mg/kg per day. Next, we examined the effects of PF1022A on larval A. cantonensis migrating into the central nervous system (CNS) of rats. Following five successive oral doses at 5 or 10 mg/kg per day and five successive intraperitoneal doses at 0.5 mg/kg per day, lesser killing effects were observed on male as well as female worms. On the basis of these results it is apparent that PF1022A will become a promising anthelmintic available as treatment for tissue-dwelling as well as intestinal nematodes.

Worm burden and mucosal mast cell response in DA and F344/N rat strains infected with Hymenolepis diminuta.

After oral administration of 1 or 5 cysticercoids of Hymenolepis diminuta, 5-week-old DA male rats showed significant mastocytosis. In F344/N rats, however, neither mastocytosis nor worm loss occurred during a 6 week infection. With regard to mucosal mast cell response to infection with H. diminuta, DA rats can be looked on as high responders and F344/N rats as low responders.

[Effect of the anthelminthic levamisole on the first-stage larva of Angiostrongylus cantonensis in infected rats]. / Die Wirkung des Anthelminthikums Levamisol auf erste Angiostrongylus cantonensis-Larven in infizierten Ratten.

Effects of levamisole, orally administered at 1, 3, 5 or 7 successive daily doses of 30 mg/kg, on the number of first-stage larvae of Angiostrongylus cantonensis released in the faeces of treated rats were examined. First-stage larval counts (larvae per gram of faeces per female worm recovered, LPG/female) were conspicuously reduced 1 day after administration in all treated groups. In the group treated with a single dose, the larval counts increased 2 days after administration, but no larvae could be detected in the other treated groups. Histological observations of lung tissues of the host showed the degenerative changes in the reproductive system of the worms. These results suggest that levamisole affects the larval output of A. cantonensis through a direct paralyzing action and an indirect mode of action including inhibition in energy metabolism.

Effects of sensitization on efficacy of mebendazole in mice infected with adult Angiostrongylus costaricensis.

The relation between immunopotentiation and efficacy of mebendazole in sensitized mice infected with adult Angiostrongylus costaricensis was investigated. When compared with the non-treated control, the sensitized control showed better results in almost all parameters with a few mice being positive for the first-stage larvae in feces and eggs in the intestinal section. The results suggest development of protective immunity in the sensitized mice though it was not completely effective for inhibiting infection, worm growth and their functions. The immunity seemed to be developed by producing specific antibodies against the larvae by sensitization. Among 4 infected groups, the sensitized-treated group had the best results in all parameters especially in worm recovery and worm body length which referred that drug action was enhanced by the sensitization.

Studies on chemotherapy of parasitic helminths: efficacy of artemether on Japanese strain of Schistosoma japonicum in mice.

Effects of artemether were examined on Schistosoma japonicum in mice. When the drug was given at a daily dosage of 200 mg/kg for 4 successive days from 46 days post-infection, a significant reduction in worm recovery was observed. A significant reduction in size of worms from the medicated mice was also seen compared with that from non-medicated controls.

Mucosal mast cell response to Hymenolepis diminuta infection in different rat strains.

Five-week-old DA male rats infected with 10 Hymenolepis diminuta cysticercoids showed significant mastocytosis 6 weeks post-infection and low persistence of worms. In F344/N rats, however, no mastocytosis and no worm loss occurred during a 6 week infection. Mucosal mast cells appear to be associated with the expulsion of H. diminuta from DA rats.

A case of ileus caused by a spiruroid nematode.

A 34-year-old male living in Aichi Prefecture, Japan, complained of lower abdominal pain. Ileus was suspected based on his clinical history and symptoms, and a laparotomy was performed. Four sections of a nematode were found in a large eosinophilic granuloma in the intestinal wall, and were identified as the larva of a spiruroid nematode. This is the third reported case of a spiruroid nematode infection found in the ileum.