The filarial parasite, Mansonella perstans (Mp), is endemic in central and West Africa with a geographic distribution that overlaps those of Wuchereria bancrofti (Wb), Loa loa, and Onchocerca volvulus. The clinical and immunologic contributions of Mp infection are unknown, in part, because of the lack of response of Mp to standard antifilarial therapies. The recent discovery of bacterial endosymbionts (Wolbachia) in most filarial species, including Mp (Keiser et al. 2008 Mol Biochem Parasitol), provides new therapeutic options for reducing microfilaremia. In an open label, randomized trial, subjects with Mp microfilaremia with and without concomitant Wb infection were recruited from 4 villages in Mali and received doxycycline (200 mg daily for 6 weeks;n=106) or no treatment (n=110). At 6 months, Wb coinfected subjects were randomized further to receive a single dose of albendazole (400 mg) and ivermectin (150 g/kg) or no treatment. Subjects were monitored daily during treatment for adverse events. The effects of drug treatment on Mp and Wb microfilarial levels were assessed at 6, 12 and 36 months following the initiation of doxycycline therapy. Doxycycline was safe and effective at reducing Mp microfilaremia at all time points with complete clearance in 67/69 (97%) doxycycline-treated subjects at 12 months as compared to 10/63 untreated subjects. Clearance persisted in 48/74 (75%) at 36 months consistent with an effect of doxycycline on the adult worms. These results are consistent with previous findings that Mp harbors the intracellular endosymbiont, Wolbachia, and provide the first example of effective therapy for Mp infection (Coulibaly et al 2009 in press). Filariasis and malaria infection are co-endemic in many regions of the world, including subsaharan Africa. We have previously shown that pre-existent filarial infection does not influence susceptibility to malaria infection, but may lower the threshold parasitemia at which clinical symptoms occur. To determine whether the observed decrease in clinical threshold is accompanied by alterations in the cytokine response to acute malaria infection, blood samples were collected from 16 filaria-positive (FP) and 20 filaria-negative (FN) children and young adults (1-20 years of age) who presented with clinical malaria, defined as signs and symptoms consistent with malaria infection in the presence of malaria parasitemia. Plasma levels of IFN-g, IL-1a, IL-1b, IL-1Ra, IL-6, IL-8, IL-10, IL-12p70, IP-10 and TNF-a were measured by multiplex assay (Luminex). Interestingly, neither the level of malaria parasitemia, nor the presence of fever, was correlated with increased levels of pro-inflammatory cytokines. Plasma levels of IP-10 and IL1Ra, two cytokines that are strongly associated with clinical severity in malaria, were decreased in the FP subjects (GM 2130 and 82 pg/ml, respectively) during acute clinical malaria as compared to the FN subjects (GM 5308 and 489 pg/ml;p = 0.001 and 0.003, respectively). All other cytokine levels measured were comparable between the two groups. These findings are consistent with prior findings demonstrating a decrease in the in vitro production of IP-10 by PBMC from FP individuals in response to malaria antigen and provide further in vivo evidence that pre-existent filarial infection can modulate the immune response to incoming malaria parasites. To examine the chronic effects of pre-existent filarial infection on the immune response to malaria, filaria antigen- specific and malaria antigen-specific cytokine responses were examined in 20 FN and 19 FP individuals prior to the malaria transmission season. Of note, the Fil+ group mounted a markedly diminished IL-12p70, IFN- and IP10 response following MalAg stimulation compared to the Fil- group but a significantly higher IL-10 response. Whereas, anti-TGF- had little effect, anti-IL10 antibodies induced a significant reversal of the MalAg specific downregulation of IL-12p70, IFN- , and IP10. Taken together these data demonstrate that filarial infections clearly modulate the Pf-specific IL-12p70-IFN- axis known to be pivotal for resistance to malarial parasites and do so in an IL10-dependent manner (Metenou et al 2009 J Immunol). To determine whether this Pf-specific Type 1 modulation extends to the level of CD4+ T cell frequencies of effector or regulatory cells, the frequencies of cytokine-producing cells were examined in the same population. Filarial infection was associated with a higher frequency of total CD4+ cells producing IL-4, IL-10 and IL-17 in unstimulated cultures, however, in response to malaria stimulation, Fil+ individuals had lower frequencies of IFN-γ-, IL-17- and TNF-α- producing total CD4+ cells, but higher frequencies of IL-10-producing total CD4+. The Fil+ group also had a greater frequency of CD4+CD25+Foxp3+CD4 as well as T cells expressing CTLA-4 and/or IL10 in both unstimulated and MalAg-driven cells compared to the Fil- group. More interestingly, however, IL-10 was produced primarly by CD4+CD25- T cells and not the Tregs. Together these data demonstrate that filarial infection induces a regulatory environment dominated by CD4+CD25- IL10 producing T cells that modulates the Pf-specific Th1 pathways known to play a key role in resistance to malaria infection. To investigate the relative contribution of nTreg and/or IL-10 (Tr1) to the modulation of malaria-specific cytokine producing T cells, ongoing study is underway to assess the effect of nTreg depletion on the frequency of cytokine producing cells. Albendazole and ivermectin are currently used in combination for annual mass treatment of Wuchereria bancrofti (Wb) infection in Africa. Although the drugs have been donated, the cost of such programs is very high and has proven to be a major impediment to the success of programs in countries with limited financial resources. To determine the effect of increased dose and frequency of albendazole/ivermectin (A/I) treatment on microfilarial clearance, 42 Wb microfilaremic residents of an endemic area in Mali, were randomized to receive standard annual A/I therapy (400 mg/150 mcg/kg;n=22) or biannual increased dose A/I therapy (800 mg/400 mcg/kg;n=20). There was a significant decrease in mf counts both groups with complete clearance of detectable mf at 12 months in all 19 subjects in the biannual group as compared to 10/21 in the annual group (p<0.001, Fishers exact test). This difference between the two groups was sustained at 18 months with no detectable mf in the subjects receiving biannual treatment as compared to 6/21 in the annual treatment group (p<0.001). There were no significant differences in measures of adult worm burden (circulating antigen levels or worm nests) between the two groups. These findings suggest that increasing the dose and frequency of treatment enhances the efficacy of A/I therapy at suppressing microfilarial levels, although longer therapy may be necessary to demonstrate a significant effect on adult worms. Analysis of the 24 month data is underway.
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