The study of the chemical constituents of the roots of Newbouldia laevis (Bignoniaceae) has resulted in the isolation and characterization of a naphthoquinone-anthraquinone coupled pigment named newbouldiaquinone A (1) together with 14 known compounds: apigenin, chrysoeriol, newbouldiaquinone, lapachol, 2-methylanthraquinone, 2-acetylfuro-1,4-naphthoquinone, 2,3-dimethoxy-1,4-benzoquinone, oleanolic acid, canthic acid, 2-(4-hydroxyphenyl)ethyl triacontanoate, newbouldiamide, 5,7-dihydroxydehydroiso-alpha-lapachone, beta-sitosterol, and beta-sitosterol glucopyranoside. The structure elucidation of the isolated compounds was established based on spectroscopic studies, notably of the 2D NMR spectra. The antimalarial activity of compound (1) against Plasmodium falciparum in vitro shows moderate chemo suppression of parasitic growth. Its antimicrobial activity against a wide range of microorganisms was 13- and 24-fold more active against Candida gabrata and Enterobacter aerogens than the reference antibiotics nystatin and gentamycin.

The increasing drug resistance of malaria parasites against chemotherapeutics enforces new strategies in finding new drugs. Here, we describe a new class of compounds the piperidone 3-carboxylates which show an antiplasmodial effect in vitro and in vivo. This effect might be caused by inhibition of eukaryotic initiation factor (eIF-5A).

In order to chronically infect their hosts, filarial nematodes have generated a range of strategies to evade and down-modulate the host's immune system. The recent concept of suppression of immune responses by regulatory T cells has in part benefited from examinations in human and murine filariasis. Its further development in basic immunology animal models has in turn helped to better understand down-regulatory immune mechanisms in filariasis. Thus, filarial nematodes orchestrate down-regulation by inducing regulatory T cells and alternatively activated macrophages, which are able to suppress both Th1 and Th2 responses. Regulatory T cells can also induce the secretion of IgG4 from B cells as another arm of modulation. Dendritic cells are down-regulated upon first encounter with infective L3 larvae. Failure to respond to down-regulatory induction is based on genetic traits in hosts and leads to reduced parasite loads, albeit at the expense of pathology and disease. Since down-regulation in chronically and heavily infected hosts extends to third-party antigens, it is essential to analyse the impact of filarial infection for vaccination, allergy and important coinfections such as malaria, in order to foresee and avert potentially disastrous consequences of filariasis control programmes.

ABSTRACTThere has been a prevailing perception that Th1 and Th2 immune responses induce antagonistic immune effector mechanisms during an infection. We investigated the role of the Th1 cytokine gamma interferon (IFN-γ) and the Th2 cytokine interleukin-5 (IL-5) in murine filariasis infections with the rodent filarial nematodeLitomosoides sigmodontiswith regard to immune responses to the parasite. Earlier data showed an important role for IL-5 and IFN-γ in effective immune responses to filarial infection. Therefore, in this study it was asked whether IL-5 and IFN-γ act synergistically or antagonistically. Indeed, IL-5 as well as IFN-γ knockout (KO) mice show a higher worm load than the wild-type controls. IFN-γ/IL-5 double-KO mice had a significantly higher worm load than any of the single-KO mice, suggesting a synergism between IFN-γ and IL-5 in controlling worm infection. Neutrophils are known to play an important role for the containment and encapsulation process of the worms. In infected IFN-γ KO, IL-5 KO, and IFN-γ/IL-5 double-KO mice, neutrophils were significantly reduced in chemotactic activity levels compared to controls. In addition, the level of phagocytosis activity of neutrophils from IFN-γ/IL-5 double-KO mice was further decreased in comparison to that of the single-KO mice. Levels of tumor necrosis factor alpha, which is an important factor for neutrophil activation, were found to be reduced in macrophages from KO mice. In conclusion, these results argue for immune effector mechanisms in murine filarial infection that are dependent on both IFN-γ and IL-5. Synergistic effects of the two cytokines may be mediated, at least in part, by neutrophils for the control of adult worms.

BALB/c interleukin-4 (IL-4(-/-)) or IL-4 receptor-alpha (IL-4ralpha(-/-)) knockout (KO) mice were used to assess the roles of the IL-4 and IL-13 pathways during infections with the blood or liver stages of plasmodium in murine malaria. Intraperitoneal infection with the blood-stage erythrocytes of Plasmodium berghei (ANKA) resulted in 100% mortality within 24 days in BALB/c mice, as well as in the mutant mouse strains. However, when infected intravenously with the sporozoite liver stage, 60 to 80% of IL-4(-/-) and IL-4ralpha(-/-) mice survived, whereas all BALB/c mice succumbed with high parasitemia. Compared to infected BALB/c controls, the surviving KO mice showed increased NK cell numbers and expression of inducible nitric oxide synthase (iNOS) in the liver and were able to eliminate parasites early during infection. In vivo blockade of NO resulted in 100% mortality of sporozoite-infected KO mice. In vivo depletion of NK cells also resulted in 80 to 100% mortality, with a significant reduction in gamma interferon (IFN-gamma) production in the liver. These results suggest that IFN-gamma-producing NK cells are critical in host resistance against the sporozoite liver stage by inducing NO production, an effective killing effector molecule against Plasmodium. The absence of IL-4-mediated functions increases the protective innate immune mechanism identified above, which results in immunity against P. berghei infection in these mice, with no major role for IL-13.

Natural killer cell-associated direct cytotoxicity and cytokine production are crucial mechanisms for early innate host resistance against viruses, bacteria, or protozoa. The engagement of inhibitory NK cell receptors can influence host responses to viruses. However, these receptors have not been investigated to date in parasitic infections, and little is known about the role of NK cells in the defense against helminths. Therefore, we have correlated the frequencies of cells expressing the pan-NK marker DX5 and subsets bearing inhibitory Ly-49 receptors with worm survival and cytokine production during infection with Litomosoides sigmodontis in BALB/c mice (H2(d)), the only fully permissive model of filariasis. A marked influx of DX5(+)/CD3(-) NK cells and DX5(+)/CD3(+) T cells into the pleural cavity, where the parasites were located, was observed. The frequency of pleural NK cells expressing the H2(d)-reactive inhibitory receptors Ly-49A, Ly-49C, or Ly-49G2 declined most strongly compared with spleen and blood. In the peripheral blood, longitudinal analysis revealed an early and stable reduction of Ly-49C(+) and Ly-49G2(+) NK cells, a subsequent significant increase of the entire NK cell and DX5(+)/CD3(+) T cell populations, and a reduction in the Ly-49A(+) subset. The in vivo depletion of NK cells strongly enhanced the worm load and influenced IL-4 and IL-5 plasma levels. These data demonstrate a new role for NK cells in the host defense against filariae and, for the first time, alterations of Ly-49 receptor-expressing NK cell subsets in a parasitic infection.

Trypanosoma cruzi, the causative agent of Chagas' disease, is known to be susceptible to nitric oxide (NO)-dependent killing by gamma interferon-activated macrophages. Mice deficient for inducible nitric oxide synthase (iNOS) are highly susceptible to T. cruzi, and inhibition of iNOS from the beginning of infection was reported to lead to an increase in trypomastigotes in the blood and to high mortality. In the present study, we investigated whether NO production is essential for the control of T. cruzi in all phases of the infection. BALB/c mice were treated at different time intervals after T. cruzi infection with an iNOS inhibitor, aminoguanidine or L-N6-(1-iminoethyl)-lysine (L-NIL). Treatment initiated with the beginning of the infection resulted in 100% mortality by day 16 postinfection (p.i.). If treatment was started later during the acute phase at the peak of parasitemia (day 20 p.i.), all the mice survived. Parasitemia was cleared and tissue amastigotes became undetectable in these mice even in the presence of the iNOS inhibitor L-NIL. Inhibition of iNOS in the chronic phase of the infection, i.e., from day 60 to day 120 p.i., with L-NIL did not result in a reappearance of parasitemia. These data suggest that while NO is essential for T. cruzi control in the early phase of acute infection, it is dispensable in the late acute and chronic phase, revealing a fundamental difference in control mechanisms compared to those in infections by other members of the order Kinetoplastida, e.g., Leishmania major.

ABSTRACT To establish the role of B cells and antibodies in destroying filariae, mice lacking mature B cells and therefore unable to produce antibodies were used. Litomosoides sigmodontis offers a good opportunity for this study because it is the only filarial species that completes its life cycle in mice. Its development was compared in B-cell-deficient mice (BALB/c μMT mice) and wild-type BALB/c mice in two different in vivo situations, vaccination with irradiated larvae and primary infection. In all cases, mice were challenged with subcutaneous inoculation of 40 infective larvae. Vaccine-induced protection was suppressed in B-cell-deficient mice. In these mice, eosinophils infiltrated the subcutaneous tissue normally during immunization; however, their morphological state did not change following challenge inoculation, whereas in wild-type mice the percentage of degranulated eosinophils was markedly increased. From this, it may be deduced that the eosinophil–antibody–B-cell complex is the effector mechanism of protection in vaccinated mice and that its action is fast and takes place in the subcutaneous tissue. In primary infection, the filarial survival and growth was not modified by the absence of B cells. However, no female worm had uterine microfilariae, nor did any mice develop a patent infection. In these mice, concentrations of type 1 (gamma interferon) and type 2 (interleukin-4 [IL-4], IL-5 and IL-10) cytokines in serum were lower and pleural neutrophils were more numerous. The effects of the μMT mutation therefore differ from those in B1-cell-deficient mice described on the same BALB/c background, which reveal a higher filarial recovery rate and microfilaremia. This outlines B2-cell-dependent mechanisms as favorable to the late maturation of L. sigmodontis.

We investigated the role of IFN-gamma in host defense during murine filariasis. Using the fully permissive infection of BALB/c mice with the rodent filaria Litomosoides sigmodontis, we show that interferon (IFN)-gamma is essential for encapsulation of adult filarial worms in inflammatory nodules and for normal worm clearance. IFN-gamma knockout (KO) mice had only one third of the nodules of wild-type mice but displayed a more than twofold increase in worm burden and increased microfilaremia. Neutrophil granulocytes, but not macrophages or eosinophils, appear to directly control worm load and nodule formation. Neutrophils, which we showed earlier to be essential for the encapsulation process in the thoracic cavity, where the worms reside, were diminished at this location in IFN-gamma KO compared to wild-type mice; they also displayed strongly reduced chemotactic and phagocytic activity compared to neutrophils of controls. This argues for a distinct defect in neutrophil activation accounting for the low formation of inflammatory nodules. Tumor necrosis factor-alpha, a major neutrophil-activating cytokine expressed by macrophages in the thoracic cavity around the worms, was highly induced in wild-type but absent in KO mice. Diminished activation of neutrophils seems to be a general hallmark of IFN-gamma KO mice, since neutrophils from uninfected KO mice also showed a reduction in chemotactic and phagocytic activity when induced by casein. In conclusion, these data are the first to define an IFN-gamma-dependent immune effector mechanism in murine filarial infection, i.e. neutrophil-mediated control of the adult worm load.

ABSTRACTLitomosoides sigmodontisis the only filaria which develops from infective larvae into microfilaria-producing adults in immunocompetent laboratory mice. In this study we report that interleukin-4 knockout (IL-4 KO) mice have an up to 100-fold-higher and a significantly prolonged microfilaremia compared to wild-type BALB/c mice, as well as 20 times more microfilariae in the thoracic cavity, the site of infection. While worm development and adult worm persistence were equivalent in IL-4 KO and wild-type mice, the fertility and length of adult female worms in IL-4 KO mice was clearly enhanced. The high susceptibility to microfilariae in IL-4 KO mice required the presence of adult worms in a full infection cycle since microfilariae loads did not differ much between IL-4 KO and wild-type mice when purified microfilariae were injected into mice. In addition, we found that eosinophilia was diminished and immunoglobulin E (IgE) was absent in IL-4 KO mice. IgE, however, does not seem to be the essential factor for microfilarial containment since microfilaremia was not elevated in B-cell KO mice. In conclusion, IL-4 is shown for the first time to be essential for the control of microfilarial loads but not of adult worm loads in a fully permissive murine filarial infection. IL-4 dependent effector pathways seem to operate on adult worms rather than directly on microfilariae.