OFERTA PÚBLICA DE ADQUISICIÓN OBLIGATORIA POR CAMBIO DE CONTROL (“OPA”)

Cathy Steel and Thomas B. Nutman. Laboratory of Parasitic Diseases, National Institutes of Health, Bethesda, MD 20892, USA

To identify T cell populations responsible for regulating immune responses in chronic filarial infections, the frequency of various T cell populations were measured (ex vivo) in both filarial [loiasis, lymphatic filariasis, onchocerciasis]-infected and -uninfected individuals. Flow cytometry was used for both cell surface expression of CD4, CD8, and CD25 and intracellular expression for IL-10 and CTLA-4 in patients with longstanding (endemic) and less longstanding (expatriate) patent filarial infection. Although the frequencies of IL-10- producing T cells were higher in uninfected than in filarial-infected individuals, the frequencies of IL-10 producing CD4+ cells were higher in those with chronic, longstanding infection compared with those having relatively recent infection, particularly in individuals with patent infections (Mf+). Following definitive cure of the filarial infection, the frequencies of CD4+IL10+ cells diminished significantly (p = 0.004).

Further analysis of patients with bancroftian filariasis demonstrated that the frequency of CTLA-4+ T cells was greater than in those uninfected individuals from the same endemic region. Moreover, higher CTLA4+ frequencies were seen in those with patent infections compared to those without. Interestingly, some of the highest upregulation of CTLA-4 occurred in CD4+ cells also positive for CD25 (a cell population purported to include suppressive cells). These data suggest there exists a network of cells with differing functions and phenotypes that together serve to regulate the immune response to filarial parasites.

Age patterns in prevalence of lymphatic filariasis infection

W.A. Stolk1

, K.D. Ramaiah2

, G.J. van Oortmarssen1

, J.D.F. Habbema1 1

Dept of Public Health, Erasmus University Rotterdam, The Netherlands 2

Vector Control Research Centre, Pondicherry, India

Age-patterns of prevalence or intensity in lymphatic filariasis reflect the underlying dynamics of transmission and infection. We reviewed patterns of Wuchereria bancrofti prevalence by age in Sub-Saharan Africa to determine the dominant pattern in this region and study the variation occurring between areas. Relevant studies were identified from Medline, complemented by reference tracing and a search through WHO’s library for unpublished reports. Different types of curves were fitted to the data. The database for Sub-Saharan Africa contained data from 21 studies, containing 37 datasets from 10 countries. The overall prevalence in these studies varied from 3% (Ghana) to 48% (Tanzania). A straight line increasing to a maximum level provided a good fit to the data. The age at which the maximum level was reached was on average 23.1 years. Results do not exhibit a relatioship between the prevalence level among adults or total prevalence and the age at which the maximum is reached in Africa. Results are discussed in relation to possibly underlying dynamics. The analyses are currently being repeated for India; preliminary results show a similar age-pattern.

High prevalence of Brugia timori infection in the highland of Alor island, Indonesia

Taniawati Supali, Herry Wibowo, Paul Rückert, Kerstin Fischer, Is S. Ismid, Purnomo, Yenny Djuardi, and Peter Fischer.

Department of Parasitology, Faculty of Medicine, University of Indonesia, Jakarta; German Agency for Technical Co-operation GTZ/SISKES, Kupang, Indonesia; Bernhard Nocht

Institute for Tropical Medicine, Hamburg, Germany; U.S. Naval Medical Research Unit No. 2, Jakarta, Indonesia.

During a filariasis field survey Brugia timori and Wuchereria bancrofti infections were identified on Alor, East Nusa Tenggara Timor, Indonesia. On this island elephantiasis was reported by villagers as a major health problem. In the coastal areas W. bancrofti was detected, whereas in four rice-farming villages B. timori was identified. No mixed infections with both species were found. In the highland village Mainang (880 m altitude) 586 individuals were examined for B. timori infection and 157 (27%) microfilaria (mf) carriers were detected. The prevalence of microfilaraemics standardized by sex and age was 25% and the geometric mean mf density of microfilaraemics was 138 mf/ml. Among teenagers and adults, male individuals tended to have a higher mf prevalence than females. Microfilaria prevalence increased with the age and a maximum was observed in the fifth decade of life. In infected individuals the mf density increased rapidly and high levels were already noticed in the age group of 11-20 years. The highest mf density was found in a 27-year-old woman with 6028 mf/ml. B. timori on Alor was nocturnally periodic, but in patients with high parasite loads, a small number of mf was also detected in the day blood. The disease rate was high and many persons reported a history of acute filarial attacks. Seventy-seven (13%) individuals showed lymphoedema of the leg, sometimes leading to severe elephantiasis. No hydrocele or genital lymphoedema were observed. To evaluate new control strategies for B. timori, infected persons were treated by DEC or a combination of DEC and albendazole. Thirty adults were divided in 2 groups and treated in the hospital either by DEC alone (6 mg/kg, 100 mg on day 1 and the rest given on day 3), or by a single dose combination of DEC (6 mg/kg) and albendazole (400 mg). Although adverse reaction were observed in both treatment groups, no serious or long-lasting side effects were noted. A strong reduction of mf density was observed in the first treatment group on day 3 after the total dose was given and in the second group 12 h after treatment. The rest of the infected individuals were treated in the community with a combination of DEC/albendazole. Further studies have to show the long- term effects of these treatment regimens. This study showed that B. timori infection is not restricted to the lowlands of the lesser Sunda archipelago. In this region B. timori is still of public health relevance and self-sustaining control programs are required.

Wolbachia endosymbionts

Marc Taylor, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool UK, ([email protected])

Studies aimed at characterizing inflammatory mediators of filarial nematodes have led to the discovery of LPS-like molecules derived from endosymbiotic Wolbachia bacteria. I will summarize current information on the characterization of LPS-like molecules and other bacterial and filarial inflammatory mediators including hsp60 and DNA, and discuss mechanisms by which Wolbachia initiates acute inflammatory responses associated with death of parasites, leading to acute filarial lymphangitis and adverse reactions to anti-filarial chemotherapy. I will discuss the potential role of Wolbachia in the development of chronic pathology, due to the repeated exposure to acute inflammatory responses leading to damage to infected lymphatics and desensitization of the innate immune system. These events will result in an increased susceptibility to opportunistic infections, which cause acute dermatolymphangitis associated with lymphodema and elephantiasis. The recognition of the contribution of endosymbiotic bacteria to filarial disease could be exploited for clinical

intervention by the targeting of bacteria with antibiotics in an attempt to reduce the development of filarial pathology.

Evaluation of drugs against Onchocerca parasites in the laboratory, including Wolbachia as a potential target

Simon Townson

Northwick Park Institute for Medical Research, Watford Rd, Harrow, HA1 3UJ, UK.

The discovery and development strategy for new drugs to treat human filarial diseases involves the evaluation of compounds from a variety of sources; these include active compounds from pharmaceutical high throughput anthelmintic screens, more specialized molecular target screens, hypothesis driven compound selection, and the evaluation and re- evaluation of existing products, near-products and combinations. The standard in vitro assay involves screening compounds against male Onchocerca gutturosa in a 5 day trial. Confirmed active compounds are then tested in vivo against Onchocerca microfilariae in mice and against transplanted Brugia pahangi macrofilariae in jirds. Promising compounds from these screens are further evaluated in preliminary toxicity studies before selection for testing against infective larvae induced infections of Brugia pahangi in dogs, followed by consideration as candidates for clinical trials. However, it has been clear for some time that the standard screening protocols may not be suitable for the evaluation of all types of compound, in which case a modified or more investigative approach may be required. Recent studies have demonstrated that, following extended exposure to drug, certain antibiotics have antifilarial properties, thought to be mediated by the destruction of the endosymbiont bacterium

Wolbachia. We have used a modified long-term (40 day) O. gutturosa in vitro assay to

examine a range of compounds from different classes of antibiotics. This has been useful in identifying 5 antibiotics (rifampicin, minocycline, doxycycline, clofazamine and ethambutol) with significant anti-Onchocerca activity. Ongoing studies indicate that minocycline is the most active drug when tested against Onchocerca microfilariae in mice.

Vaccination against onchocerciasis: field trial of recombinant antigens to protect against Onchocerca ochengi in cattle

Trees, Alexander

Immunogenic properties and domains of Wolbachia Surface Protein (WSP) from Brugia

malayi

George Tzertzinis, Jeremy Foster

New England Biolabs, Beverly, MA, USA 01915

Wolbachia bacterial endosymbionts have been identified in most filarial nematode species

with rare exceptions. Several lines of evidence suggest that, as in the case of arthropod

Wolbachia, these endosymbionts play an important role in the development of the nematode

host, and are perhaps involved in the pathology of filarial disease, making them a potential target for the control of filariasis (1,2).

Wolbachia Surface Protein (WSP) is a major surface antigen of insect Wolbachia and a close

homologue exists in nematode Wolbachia. Anti-WSP antibodies have been detected by western blots in sera of cats infected with D. immitis (3) and ELISA tests have shown that anti-WSP antibody response parallels anti–filarial IgG1 response and onset of lymphoedema in B. malayi-infected monkeys (4).We have expressed and purified recombinant B. malayi WSP in E. coli and have developed a polyclonal antibody that identifies WSP in crude extracts from B. malayi males, females, and microfilaria.

We tested sera from jirds experimentally infected with B. malayi for the presence of anti-WSP antibodies using different versions of recombinant WSP protein. All infected individuals tested produce antibodies directed against a C-terminal portion of WSP. These results suggest that the C-terminus of WSP is preferrentially exposed to the immune system of the infected animals and support the notion that endosymbiont protein antigens are immunogenic in filarial infection.

1. Hoerauf et al. (2000). Lancet 355:1242-3. 2.Taylor et al. (2000) Parasitol Today. 16:179-80. 3. Bazzocchi et al. (2000) Proc. R. Soc. Lond. B 267: 2511-2516. 4. Punkosdy et al. (2001) J. Infect. Dis. 184(3): 385-389.

An active retrotransposon resides embedded in the MboI repeat that is found solely on the Y chromsome of Brugia malayi.

Anthony Underwood, Molecular Biology and Immunology Division, Liverpool School of Tropical Medicine, University of Liverpool, U.K.

The gridded B.malayi BAC library constructed as part of the Filarial Genome Project was differentially hybridised with 32P-labelled genomic DNA from L4 females and adult males. Those BAC clones that hybridised only to male DNA were confirmed to be unique to the male genome by PCR and therefore were postulated to be from the Y chromosome. An overview of the contents of these clones was achieved by sequencing subclones generated using the Genome Priming System from NEB. This revealed that many of the Y clones consisted mostly of the previously described MboI repeat. Hybridisation with the MboI repeat sequence demonstrated that this was only found on the Y BACs. Apart from the repeat many of the Y BACs contained sequences with homology to reverse transcriptase and integrase. Probes derived from these sequences hybridised to multiple restriction enzyme digest products that differed in size between strains of B.malayi. Reverse-transcriptase PCR analysis revealed these sequences to be transcribed in adults. We propose that these sequences are part of one or more active retrotransposons that have accumulated on the Y chromosome of

B.malayi.

Methods for transient transfection of B. malayi

Tarig B. Higazi1, Limin Shu1, Jane C. Burns2 and Thomas R. Unnasch1

1Division of Geographic Medicine, University of Alabama at Birmingham and 2Department of Pediatrics, University of California at San Diego

To develop a method for the introduction DNA into filarial parasites, several methods that have proven successful in other organisms were evaluated for their ability to transform B. malayi. Experiments utilized a variety of B. malayi promoter constructs driving the expression of both luciferase and green florescent protein (GFP) reporter genes. Similar to what has been noted in Ascaris suum, biolostic bombardment was found to transiently transform B. malayi embryos and adult parasites. Luciferase activity was detectable in embryos bombarded with gold particles coated with a construct consisting of a luciferase reporter gene under the control of the spliced leader promoter. Similar levels of luciferase activity to those obtained from the biolositically transformed embryos were seen in adult

parasites microinjected with the same construct. In similar experiments employing the SL promoter driving a GFP reporter, expression of the reporter was detectable in the intrauterine embryos of the microinjected adult parasites, and in the hypodermis of biolistically trasnfected adult female parasites. A similar pattern of GFP expression to that seen in the SL promoter construct transfected parasites was noted in parasites transfected with constructs consisting of the upstream domain derived from an aminoacyl tRNA synthetase gene of B. malayi. Finally, cultured B. malayi embryos were shown to be susceptible to transduction by pantropic retroviral vectors, suggesting that these vectors might be used as a means to develop stable transfected lines of B. malayi. The ability to transfect B. malayi embryos may provide a foundation for studies of the regulation of gene expression and function in these organisms.

Filarial Genome Projects: current status and prospects for the future

Steve Williams

Co-endemicity of Onchocerca volvulus and Loa loa in forested villages of south-west province of Cameroon : implications on ivermectin mass distribution programmes S. WANJI 1,3; M. ESUM2; N. TENDONGFOR 1,3; P. ENYONG2

University of Buea, Faculty of Science, Department of Life Sciences. P. O. Box, 63, Buea, Cameroon

Tropical Medicine Research Station, P.O. Box 55, Kumba, Cameroon

Research Foundation in Tropical Diseases and Environment, P.O. Box 474, Buea, Cameroon

Encephalopathy has ben reported in Cameroon in individuals heavily infected with Loa loa microfilaria who were treated with ivermectin against onchocerciasis.

Prior to the initiation of the community directed treatment of onchocerciasis with ivermectin (CDTI) in the South West Province of Cameroon, an epidemiological survey of loiasis was conducted to evaluate the intensity of the disease. Thick blood films were made from 1228 blood samples collected during the day. Rapid epidemiological assessment of onchocerciasis (REA) was conducted among 614 individuals in the upper Bayang area. 16.0% of the population examined were carriers of Loa loa microfilaria. The males (20.1%) were more infected than females (12.4%). The community mean microfilaria of the different villages were low (<1100mf/ml). However, one person was found harbouring 174000mf/ml of blood and 1% of the study population (12) had microfilaraemia > 8100mf/ml. Results of the rapid epidemiological assessment (REA) of onchocerciasis show that 31.3% of the population investigated in the Upper Bayang area are infected with this disease. These results show that loiasis and onchocerciasis are co-endemic in the area. The implications on these findings on the policy of mectizan distribution will be discussed.

SINGLE DOSE VERSUS MULTIDOSE DIETHYLCARBAMAZINE WITH