INDIVIDUALES CONDENSADOS INTERMEDIOS 2.1 Bases de preparación

Laetitia Le Goff, Tracey J Lamb, Kathryn Lamza, Judith E. Allen.

Institute of Cell Animal And Population Biology, Ashworth Laboratories, University of Edinburgh, Edinburgh , United Kingdom

Previous studies have shown that inbred strains of mice differ in their susceptibility to L.

sigmodontis infection. BALB/c mice are fully susceptible to the infection and parasites

develop through to patency. In contrast, mice on the C57BL background are resistant to the infection and never produce a patent infection.

Th 2 lymphocyte responses under the control of IL-4 and IL-5 are frequently associated with immune responses to parasitic helminths but their actual role in protection remain controversial. We have shown previously that interleukin-5 is not involved in innate resistance to filarial infection. In this study, we have used the IL-4 deficient mice on C57BL/6 background to assess the effect of IL-4 on the development of L. sigmodontis. We analysed the parasite recovery rate and the microfilaraemia over the course of the development in wild type and IL-4 deficient mice. Two months after the infection, adult worms were found in 93% infected IL-4 deficient mice and the percentage of microfilaraemic mice was 33% comparable to that seen in fully susceptible BALB/c mice. We also compared the immune responses evoked during the course of chronic and patent infections. Significant differences in antibody isotypes were found between wild type and IL-4 deficient mice. However no differences were found in parasite recovery between infected wild type and B-cell deficient mice. Thus it does not appear that antibody is a critical component mediated resistance,

These results suggest that IL-4 but not antibody is an essential component in innate immune mechanisms that control filarial parasites following a primary infection.

Genes differentially expressed during development of Brugia malayi infective larvae

B-W Li, A. Hofer, M.L. Michalski, U.R. Rao, G.J. Weil, Infectious Diseases Division, Washington University School of Medicine, St. Louis, MO, USA

Two methods were used to identify genes that are differentially expressed by Brugia malayi shortly after infection by comparing L3 that were cultured in vitro under conditions that mimic the mammalian environment. The first method, 5= spliced leader differential display PCR (SL DD-PCR), used primer pairs comprised of SL1 and a random primer to amplify cDNA templates from L3 before or after culture. PCR products amplified from different templates were compared on sequencing gels. 3 of 11 candidate genes were confirmed to be differentially expressed in cultured L3 by RTPCR. None of the sequences had significant homology to sequences in GenBank. The second method involved electronic subtraction (ES) of EST cluster databases to identify clusters with 3 or more ESTs in the L3 post culture database but absent in the mosquito-derived (day 0) ESTs . 1 of 9 candidates identified by ES was confirmed as differentially expressed in the day 6 post culture L3 library. Additional studies are in progress to characterize the differentially expressed genes we have already identified and to identify other examples. We believe these studies will improve understanding of adaptations employed by filarial parasites to survive and develop in the mammalian host.

Glyoxalase in Onchocerca volvulus and Caenorhabditis elegans

Eva Liebau, Inga Wilde, Alexandra Sommer, Gary Moulder and Rolf-Dieter Walter

The glyoxalase system, consisting of two consecutive enzymatic reactions catalysed by glyoxalase I and II, is an important but frequently neglected component of the glutathione- dependent enzymology. The physiological substrates of glyoxalase I are glyoxal, methylglyoxal and other α-oxoaldehydes formed by the lipid peroxidation, glycation and degradation of glycolytic intermediates. The glyoxalase I from Onchocerca volvulus has been cloned, recombinantly expressed and kinetic as well as inhibitor constants were determined. By semi-quantitative PCR ELISA it was demonstrated that the O. volvulus glyoxalase I is expressed at elevated levels under oxidative stress conditions. The heterologous free-living nematode Caenorhabditis elegans was employed in the characterisation of nematode glyoxalase I to enable a level of analysis unapproachable in the parasitic system. Microinjection of a promoter-"green fluorescent protein (GFP)" reporter construct of C.

elegans glyoxalase I showed moderate GFP-expression in all larval stages and tissues; strong

GFP-signals were observed in the terminal bulbus of the pharynx. A genetic knockout of C.

elegans D2030.5, the first mitochondrial glyoxalase I to be described, was generated. When

incubating the worms with methylglyoxal and hydrogenperoxide, the mutant worms were more susceptible to oxidative stress than the wild type worms.

Spermidine synthesis in Caenorhabditis elegans

Kai Lüersen, Marie-Luise Eschbach, Eva Liebau and Rolf D. Walter Bernhard Nocht Institute for Tropical Medicine Hamburg, Germany

The polyamines spermidine and spermine are ubiquitous aliphatic polycations. They are essential for growth and differentiation. Intracellular polyamine concentrations are highly regulated by a complex system of biosynthesis, uptake, oxidation and acetylation. The gene encoding the biosynthetic enzyme spermidine synthase was cloned from Caenorhabditis

elegans. Recombinant expression of the spermidine synthase in Escherichia coli yielded an

enzymatically active protein with a homodimeric structure. The pattern of spermidine synthase gene expression in C. elegans was established by microinjection of green fluorescent protein (GFP) reporter gene constructs under the control of the spermidine synthase promotor region. In transgenic C. elegans abundant GFP expression was observed in intestinal cells of larvae and adult worms and during the embryonic development within the eggs. In addition, faint fluorescence was also detecetd in some cells of the nervous system. However, most of the 959 cells of the nematode did not show GFP expression. Unexpectedly, a GFP fusion protein containing the first 78 amino acids of the spermidine synthase was membrane and nuclear envelope associated. Omitting the N-terminal part of the spermidine synthase results in a cytoplasmatic localization of the reporter-protein. Interestingly, a specific insertion of 27 amino acids was identified within the N-terminus of all other known nematode spermidine synthases including Brugia and other parastic nematodes. Currently, we investigate whether this nematode specific insertion is responsible for the peculiar subcellular localization in

Protective immune responses to O. volvulus larval proteins

Sara Lustigman1, Angus MacDonald1 and David Abraham2 1

New York Blood Center, New York, NY 10021; 2Thomas Jefferson University, Philadelphia, PA 19107.

Both putatively immune (PI) and infected (INF) PI individuals have a Th2 response to larval (L-3) antigens that is distinct from the responses to female adult worm and microfilarial antigens. In a mouse model, immunization with irradiated-L3 induced protection against challenge infection that was also Th2-mediated. Furthermore, using gene knockout mice and depletion experiments it was determined that the mechanism of larval killing was antibody and granulocyte dependent. Therefore, in the present study the induction of cytophilic antibodies against L3 in an endemic population was studied. Antibody responses to F-OvAg, Ov-L3 and the recombinant antigen rOv-ALT-1were examined in INF in relation to age and microfilariae counts. The anti-F-OvAg IgE response was negatively correlated with age whereas the anti-L3 and the anti-rOv-ALT-1 responses were elevated in all ages. Anti-Ov-L3 and anti-rOv-ALT-1 specific IgG1 and IgG3 responses were elevated in all ages or increased with age, respectively. In addition, antibodies from the majority of PI and INF reacted with the surface of L3 and less with the surface of microfilariae. In the INF this reactivity, as well as the percentage of circulating eosinophils, was age dependent. Collectively, these data strongly implicate ADCC in limiting the survival of L3 in humans and mice.

Differential human cytokine and antibody responses to adult and larval stages of

Onchocerca volvulus consistent with a state of concomitant immunity

Angus J. MacDonald, Prasad S.D. Turaga, Carolyn Harmon-Brown & Sara Lustigman Molecular Parasitology, New York Blood Center, 310 E. 67th Street, New York, NY 10021, USA

The possibility of concomitant immunity and its potential mechanisms in human Onchocerca

volvulus (Ov) infection was examined by analyzing cytokine and antibody responses to

infective larval (L3) adult female (F-OvAg) and skin microfilarial (Smf) antigens in infected individuals in a hyperendemic region of Cameroon as a function of age. Peripheral blood mononuclear cell (PBMC) IL-5 responses to both F-OvAg and Smf declined significantly with age (equivalent to years of exposure to Ov in this cohort). In marked contrast, IL-5 secretion to L3 antigens remained elevated with increasing age. IFN-γ responses to L3 and F- OvAg were low or suppressed and unrelated to age, except Smf which stimulated this cytokine in older subjects. IL-10 was uniformly elevated, regardless of age, in response to L3 and F-OvAg, except to Smf where levels declined with age. 49 – 60% of all subjects’ PBMC had GM-CSF responses to all four Ov antigens with no effect of age. Analysis of stage- specific, IgG3 and IgE levels revealed a striking, age-dependent dissociation between antibody responses to the larval antigens (L3 and the recombinant L3 –specific protein Ov- ALT-1) which were significantly increased or maintained with age, and those towards F- OvAg which declined with duration of exposure to Ov. IgG1 to L3 and F-OvAg were elevated regardless of age and IgG4 to both these stages increased significantly with age, although not to Ov-ALT-1 which may have unique L3-specific epitopes. By immunofluorescent staining of whole larvae, total anti-L3 immunoglobulin also increased with age of the serum donor. Separate and distinct cytokine and antibody responses exist to adult and infective larval stages of Ov which are a function of the duration of exposure to infection and are consistent with the acquisition of a state of concomitant immunity in infected individuals.

Filarial genes in immune evasion and host modulation

R M Maizels (1), N Gomez-Escobar (1), W Gregory (1), J Murray (1), A L Scott (2), P Taylor (3), M D Walkinshaw (3) and X Zang (1).

(1) ICAPB, and (3) ICMB, University of Edinburgh, UK; (2) Johns Hopkins University, Baltimore, MD

Many viruses encode cytokine-like molecules and receptor mimics, which mediate microbial immune evasion strategies. We have now identified a set of candidate immune evasion products from filarial nematode parasites. Two sets of cytokine homologues from Brugia malayi are related to the Transforming Growth Factor-beta (TGF-beta) and the Macrophage Migration Inhibitory Factor (MIF) gene families. The Brugia TGF-beta (Bm-TGH-1 and -2) and MIF (Bm-MIF-1 and -2) homologues are members of ancient gene families conserved across metazoan organisms, bearing 30% amino acid identity between filarial and human proteins.While Bm-TGH-1 is more closely related to tissue differentiation-inducing molecules, TGH-2 is expressed by arrested microfilariae as well as adult Brugia, is secreted in vitro, and binds to host TGF-beta receptors. The two Brugia MIF molecules display cytokine- like activity (eg macrophage kinesis) and mimic the enzyme activity (dopachrome tautomerase) described for human MIF. We have also determined the crystal structure of Bm-MIF-2, which despite being only 29% identical to human MIF, presents a highly similar 3-dimensional structure including trimerisation around an intriguing central channel. Other immune evasion genes include protease inhibitors which target cysteine proteases required for antigen processing by human B cells. Finally, a group of highly-expressed genes uniquely found in invasive nematode larvae have been discovered, which are candidate proteins both for novel immune evasion functions and for new, stage-specific vaccination.

The antifilarial activity of moxidectin against patent infections of Brugia pahangi in dogs

McCall JW, Dzimianski MT, Supakorndej P, and Jun JJ.

Department of Medical Microbiology and Parasitology, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602, USA.

As part of the ongoing World Health Organization program to develop filaricides, the filaricidal activity of the macrocyclic lactone moxidectin is being investigated. In the present study, 90 microfilaremic beagles without lymphedema were selected from a group of 120 dogs infected 162 days earlier with 200 infective larvae (100 in each hind paw) of B. pahangi. These 90 dogs were blocked within sex by pretreatment microfilarial counts and randomly allocated to 15 groups of 6 dogs each (3 female and 3 male dogs in each group). Two groups served as nontreated controls (1 group for 6-month necropsy; 1 group for 12-month necropsy) while the remaining 13 groups were treated orally with moxidectin. Moxidectin was given at either 250 mcg/kg or 1000 mcg/kg for 1, 3, 6, or 12 doses. Dogs receiving more than one dose were treated monthly (28 day intervals). Dogs were bled for microfilarial counts on Day 0 prior to the first dose of moxidectin and at Days 1, 3, 7, 14, 28, and months 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 after the first dose. Six months after the first dose, dogs in 6 groups (1 control, 250 mcg/kg x 3 or 6 doses, and 1000 mcg/kg x 1, 3, or 6 doses) were necropsied to evaluate the effect on adult worms. The remaining 54 dogs in 9 groups (1 control, 250 mcg/kg x 1, 3, 6 or 12 doses, and 1000 mcg/kg x 1, 3, 6, or 12 doses) were necropsied at 12 months. After the first dose of moxidectin, the microfilarial counts for all of the treated dogs gradually declined from pretreatment levels during the first two weeks after treatment. By one month after the first dose, the microfilarial counts for dogs in all of the treated groups

were significantly less (p<.05) than the counts for both groups of controls. By 3 months, the microfilarial counts were suppressed 90% or more from pretreatment levels. The microfilarial counts remain suppressed to the end of the study at 12 months, with only 3 dogs not cleared of microfilariae. At the first necropsy 6 months after the first dose, dogs treated with moxidectin at 250 mcg/kg for 3 and 6 doses had 77% and 92% fewer worms, respectively, than controls, and dogs treated with moxidectin at 1000 mcg/kg for 1, 3, and 6 doses had 30%, 87% and 89% fewer worms, respectively, than controls. . At the second necropsy 12 months after the first dose, the dogs given one or more doses of moxidectin at either 250 mcg/kg or 1000 mcg/kg had at least 92% fewer worms than the controls, with only 6 of the 48 treated dogs having live adult worms at necropsy. The worm recoveries from all of the moxidectin treatments were significantly less (p<.05) than the recoveries for the controls, but there was no significant difference (p>.05) among the worm recoveries for the different moxidectin treatments. Dogs receiving a single dose of moxidectin at 1000 mcg/kg had significantly less (p<.05) live worms when necropsied 12 months after the start of treatment than when necropsied at 6 months. No difference in worm recoveries between the two necropsy times were seen with the other treatments. A follow-up study comparing a single oral dose of moxidectin (250 mcg/kg) with a single oral dose of ivermectin (250 mcg/kg) in dogs with patent infections of B. pahangi is underway. Dogs are being bled to monitor microfilarial counts at the same schedule as in the above trial. All dogs will be necropsied 12 months after treatment to assess the effects of these two drugs on the adult worms.

The -tubulin gene of human hookworm

James S. McCarthy1, Wendy Chung1, Richard M. Hopkins2 and John Hawdon3

University of Western Australia Dept of Medicine Fremantle Hospital, 2Division of Leukemia and Cancer Research, TVW Telethon Institute for Child Health Research, Subiaco Western Australia, and 3Dept. Microbiology, and Tropical Medicine, The George Washington University, Washington DC

Albendazole is a key component of the strategy to eliminate filarial parasites. Its broad- spectrum anthelmintic activity in addition offers the potential to reduce the significant morbidity due to intestinal nematode infections, including ascariasis, trichuriasis, hookworm and strongyloidiasis. In animal husbandry, widespread use of benzimidazole anthelmintics has led to the development of drug resistance. Such resistance is strongly associated with a phe-tyr transition at the 200 position in the parasite β-tubulin gene. With the growing use of albendazole, there is a need to develop tools to monitor for the development of resistance. Towards this objective we have cloned and sequenced the β-tubulin genes of human hookworm. Free-living larvae were retrieved from infected human faeces by Harada-Mori culture. Parasite RNA and DNA was then extracted by standard means and subject to RT- PCR and PCR. Using primers designed from the sequence of related nematodes, the full length β-tubulin cDNA of Ancylostoma duodenale has been cloned. It encodes a peptide of 448 amino acids in length. As predicted from knowledge that no benzimidazole therapy had been undertaken in the community from where the isolates had been collected, the sequence encoded a tyrosine residue at the critical 200 amino acid residue. We are in the process of optimising a PCR-based assay that will enable genotyping of field isolates for significant polymorphism in this important gene.

Transmission intensity and the immunoepidemiology and control of bancroftian filariasis

E. Michael1, P.E. Simonsen2, M.N. Malecela-Lazaro3, W.G. Jaoko4, E.M. Pedersen2, D. Mukoko5, R.T. Rwegoshora3 & D.W. Meyrowitsch2

1

Imperial College School of Medicine, London, U.K.; 2Danish Bilharziasis Laboratory, Denmark; 3

National Institute for Medical Research, Tanzania;

4

College of Health Sciences, University of Nairobi, Kenya; 5Division of Vector Borne Diseases, Kenya

The role of transmission or exposure intensity on community patterns of infection, morbidity and parasite control remains an unresolved question in human lymphatic filariasis epidemiology. This is despite field observations and theoretical work that highlight the central role that transmission intensity may play in infection, disease, immune reponses and the dynamics of control of helminth infections. Here, we apply a combined mathematical and statistical approach to parallel data collected on vector infection intensity (as measured by the Annual Transmission Potential (ATP)), microfilaraemia and chronic disease (hydrocele and lymphoedema) prevalences from communities in East Africa in order to investigate the importance and mechanistic interactions between these variables in underlying filariasis epidemiology and control. The models are based on decribing adult worm population dynamics, worm initiated chronic disease and two major forms of acquired immunity (larval versus adult-worm generated) explicitly linked to ATP in the community, and discrimination between different suggested mechanisms were done by comparison of the goodness of fit of competing models to the observed data. The results indicate a profound effect of transmission intensity on patent infection, chronic disease and the generation and impact of immunity on these variables in East African filariasis. These findings have important implications for the new mass chemotherapy-based global initiative to achieve control of this parasitic disease, as the models indicate that the duration of such community-based treatment could increase with transmission intensity owning to transmission-generated acquired immunity in areas of high transmission. This may argue for the need for a geographically targeted strategy in the control of lymphatic filariasis.

Active Sites in Brugia malayi and C. elegans Cystatins

Janice Murray, William F. Gregory and Rick Maizels, ICAPB, University of Edinburgh

Cystatins form part of a large family of molecules called cysteine protease inhibitors. Along with stefins and kininogens they are found in various tissues in mammals, plants and parasitic helminths. Each group differs in structure and function. Stefins and cystatins are smaller proteins while kinnogens are much more complicated plasma glycoproteins. Stefins lack carbohydrate and disulphide bonds whilst cystatins are disuphide bonded, secreted molecules. Due to their function as enzyme inhibitors, their expression throughout the lifecycle and their localisation these molecules pose interesting questions as to their relationship with the parasite and the host.

We have characterised a set of cystatins from Brugia malayi and C.elegans. One of the

B.malayi cystatins, Bm-CPI-2, contains two separate active sites. The corresponding sites which

are found in mammalian cystatin C are thought to be exposed on opposite faces of the molecule. The first site blocks papain and cathepsin B activity, and is also found in the other nematode

cystatins. The second has activity against asparaginyl endopeptidases (AEP), and enzyme known to be involved in MHC class II antigen processing. Using site-directed mutagenesis we have altered the AEP-inhibiting site to test whether this explains the recently discovered ability of Bm-CPI-2 to inhibit class II antigen processing. Interestingly, the C. elegans cystatin sequence around the AEP-inhibiting site differs from the consensus for activity. We have cloned two homologues found in C.elegans and intend to examine the functionality of this site.