Pathogenesis and host responses in human ...

Microbes and Infection 6 (2004) 113–128 www.elsevier.com/locate/micinf

Review

Pathogenesis and host responses in human onchocerciasis: impact of Onchocerca filariae and Wolbachia endobacteria N.W. Brattig * Tropical Medicine Section, Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Strasse 74, 20359 Hamburg, Germany Dedicated to Professor Dr. Dietrich W. Büttner on his 70th birthday

Abstract Onchocerca volvulus is a tissue-invasive parasitic nematode causing skin and eye pathology in human onchocerciasis. The filariae habour abundant intracellular Wolbachia bacteria, now recognised as obligatory symbionts, and therefore emerging as a novel target for chemotherapy. Recent research demonstrates that both the filariae and endobacteria contribute to the pathogenesis of onchocerciasis, and molecules have been identified that promote inflammatory or counter-inflammatory immune mechanisms, divert the host’s immune response or procure evasion of the parasite. © 2003 Elsevier SAS. All rights reserved. Keywords: Onchocerciasis; Skin and eye pathology; Wolbachia; Inflammation; Immunosuppression; Parasite evasion

1. Introduction Onchocerciasis is one of the 10 major tropical diseases. It is caused by the filaria Onchocerca volvulus, which affects the skin and eyes, sometimes culminating in blindness (river blindness). Approximately 18 million people are infected, and another 120 million people residing in endemic areas that are located near fast-flowing rivers in tropical Africa, Latin America, and the Yemen are at risk of infection [1]. The disease exerts a burden of a million disability-adjusted annually lost life years (DALY) and constitutes an obstacle to socioeconomic development. Over some 25 years, progress has been made in the control of onchocerciasis by the Onchocerciasis Control Programme (OCP) of the WHO in 11 West African countries and the subsequent African Programme for Onchocerciasis Control (APOC). The strategy of the OCP was the interruption of the Abbreviations: ALT, abundant larval transcript; AMM, alternatively activated macrophages; APOC, African Programme for Onchocerciasis Control; CHI, chitinase; CPI, cysteine protease inhibitor; CTLA-4, cytotoxic T-lymphocyte-associated protein 4; DEC, diethylcarbamazine; GPX, glutathione peroxidase; IFN-gamma, interferon gamma; MIF, macrophage migration inhibitory factor; OCP, Onchocerciasis Control Programme; OvAg, Onchocerca volvulus antigens; PAMP, pathogen-associated molecular patterns; PRX, peroxiredoxins; TGF-beta, transforming growth factor beta; TNF-alpha, tumour necrosis factor alpha; TLR, Toll-like receptor; Tr1, regulatory T cells; WSP, Wolbachia surface protein. * Tel.: +49-40-42818-530; fax: +49-40-42818-377. E-mail address: [email protected] (N.W. Brattig). © 2003 Elsevier SAS. All rights reserved. doi:10.1016/j.micinf.2003.11.003

transmission of infection by applying larvicides to the breeding sites of the mosquito vector, the blackfly Simulium spp. Since 1987, as an adjunct to the vector control, mass treatment was applied broadly in the OCP with the microfilaricidal macrocyclic lactone, ivermectin, a derivative of the antibiotic avermectin. The success of the control programme had reduced the risk of O. volvulus infection for 30 million people; however, a reinvasion of Simulium and a recrudescence of infection have occurred in areas where the transmission had been interrupted [2]. Using current tools, onchocerciasis appears to be ineradicable in Africa by both vector control and by chemotherapy, but the disease still causes a high burden of morbidity and disability and remains a public health problem [2]. Therefore, macrofilaricidal drugs and other intervention strategies must be envisaged. For the development of a vaccine, an increased understanding of the pathogenesis of onchocerciasis, of the evasion strategies of the parasite and of the immune mechanisms involved in parasite clearance are needed. A long-lasting vaccine could prevent infection or reduce inflammatory reactions and could lead to better control or the elimination of onchocerciasis [3]. 2. Biology of the filaria and its endobacteria 2.1. O. volvulus The genus Onchocerca principally causes a zoonosis in ungulates, while humans represent atypical and evolution-

114

N.W. Brattig / Microbes and Infection 6 (2004) 113–128

arily younger hosts, possibly since some 10 000 years. The human parasitosis seems, therefore, less balanced, often leading to more exaggerated reactions. Onchocerca filariae are long-lived parasites going through a five-stage life cycle. The long persistence of the parasite indicates highly adapted mechanisms of immune evasion (see Section 4.4). The small blackflies, Simulium spp., are the obligatory vectors transmitting the infective third larvae that moult in the human host and mature to adult filariae for a year, leading to the formation of subcutaneous nodules, onchocercomas, where the coiled females are sessile for up to 10–15 years (Fig. 1). In contrast to the sessile females (30–80 cm in length), the males, 10 times shorter, do not induce the formation of nodules but migrate in the subcutaneous tissue and may enter and leave the nodules to inseminate a number of resident females. The numbers of females in infected persons may range from 1 to 60 or more [4]. The microfilaria is the most abundant stage, and it is critical in the maintenance of the parasite’s life cycle, as well

as being the pathological agent (see Section 3). Fertilised females release cyclically 1000–3000 microfilariae per day for the lifetime of the female—up to 15 years—resulting in a total microfilarial load of 100–150 million in highly infected persons harbouring some dozen females [5]. Thus, the parasite has an enormous reproductive capacity. A sufficient number of microfilariae have to exceed the distance between the onchocercoma and the dermis in order to ensure their uptake by the vector. To reach this aim, an armada of microfilariae is produced in the nodules [5]. The life span of the microfilariae has been calculated to last 1–1.5 years; the daily turnover rate, which varies between 20 000 and 300 000 microfilariae, results in a balanced microfilarial population. Current analyses in the “River Blindness Genome Project” (website: http://www.math.smith.edu/~sawlab/ OnchoNet/RBGP.html), sponsored by the Edna McConell Clark Foundation from 1985 to 2000, reveal that the nuclear genome of Onchocerca is roughly 150 million base pairs in size and codes for a predicted 20 000 genes [6,7]. Many

Fig. 1. Life cycle of O. volvulus (Leuckart 1893) in the human host and the vector Simulium damnosum. From: D.D. Despommier, R.W. Gwadz, P.J. Hotez (Eds.), Parasitic Diseases, fourth ed.; with permission of Apple Trees Production, LCC.


115

Table 1 W. pipientis is an abundant intracellular bacterium in many filariae Feature Characteristics of Wolbachia

Interaction between Wolbachia and the filaria Interaction between Wolbachia and the human host

Characteristic • Alpha 2 proteobacteria; gram-negative; order Rickettsiales • Obligatory intracellular bacteria • Presence in all embryonic stages, in the hypodermis and oocyte • Transmission vertically via oocytes • Co-evolution and co-adaptation with filariae • Wolbachia: obligatory symbiont • Wolbachia: essential for embryogenesis, growth, fertility of the filaria • Wolbachia: novel target for treatment of filariae with antibiotics • Role of Wolbachia in pathogenesis of infection • Wolbachia: stimulator of innate and possibly adaptive immune responses (Th1-type) • Implication of Wolbachia in inflammatory reactions after microfilaricidal drugs

genes have been identified that appear to be stage specific or highly upregulated at specific stages of development and may become potential vaccine and drug target candidates (website: http://helios.bto.ed.ac.uk/mbx/fgh/OnchoNet/onchotable1.html; expressed sequence tag database: http://www. ncbi.nlm.nih.gov/dbEST/dbESTsummary.html) [8]. The O. volvulus Gene Index (OvGI) is documented at the website http://www.tigr.org/tdb/tgi/ovgi/. 2.2. Wolbachia pipientis Wolbachia bacteria inhabit a wide range of arthropods and most filarial nematodes [9]. The presence of endobacteria in the filaria was discovered by McLaren and Kozek some 20 years ago. Based on DNA sequence data, the bacteria of the filariae have been identified as belonging to the species W. pipientis, as demonstrated for arthropods before [10,11]. Wolbachia belongs to the alpha-2 proteobacteria in the order of Rickettsiales (Table 1). The divergence date between Wolbachia of filariae and arthropods is estimated at approximately 100 million years. It could be speculated that filarial nematodes acquired Wolbachia from arthropods. In contrast to the arthropods, in filariae, the phylogeny of Wolbachia is congruent with that of the host. Phylogenetic analysis of the genes coding for the small subunit ribosomal RNA (16S rDNA), for the cell-cycle protein FTSZ, and the Wolbachia surface protein (WSP) [12] revealed that the phylogeny of Wolbachia in filariae appears to be congruent with that of their hosts due to the long co-evolutionary history [9,11]. While in arthropods, Wolbachia have parasitic habits manipulating arthropods’ reproduction, the endobacteria live as symbionts in the filariae. The symbiont in filariae increases its survival by increasing the fitness of its host. The long co-evolution of the symbiont and host is expected to result in co-adaption and reciprocal dependence. The Wolbachia appears essential for embryogenesis and larval development of the filaria and is, therefore, designated as obligatory symbiotic [9]. As known from other mutualistic endobacteria, the filarial symbiotic Wolbachia has a genome size smaller by approximately 1 Mb than that of its parasitic relatives (1.6 Mb) in arthropods and free-living bacteria (4.7 Mb) [13]. The Wolbachia genome is likely to encode 2–3000 proteins. Adult filariae harbour the endobacteria abundantly in the hypodermis and in female reproductive organs, including

Reference [9–13]

[9,14–17]

[9,18–24]

oocytes, and in all embryonal stages. The Wolbachia are transovarially transmitted. All developmental stages and individual parasites are infected; only the spermatocytes are endobacteria free. As obligatory symbionts, the Wolbachia can be used as a target for therapy. Tetracycline treatment, known to be effective against Rickettsia-like bacteria, was used for treatment of various filarial infections, resulting in reduced growth, sterility and even killing of filariae [14–16]. In humans, treatment trials with onchocerciasis patients also resulted in an interruption of embryogenesis and sterility of worms, and tetracycline was recommended for the development of new treatment strategies for the control of filarial infections [2,17]. Already in 1977, the first observers of filarial endobacteria suggested a possible involvement of the endobacteria in the pathogenesis of filarial disease and the possibility that they might represent a target for therapy. Recent studies indeed provided evidence that these bacteria can contribute to the pathogenesis of lymphatic filariasis and onchocerciasis ([18– 22]; C. Bazzocchi et al. Vet. Parasitol. 117 (2003) 73–83). The innate and acquired immune systems of the human host were implicated in Wolbachia-induced pathogenesis (see Section 4.3). Furthermore, it was reported that antigens of Wolbachia from filarial parasites are recognised by the humoral immune system of infected hosts [23,24].

3. Pathological manifestations in onchocerciasis 3.1. The majority of the filariae are enclosed in onchocercomas Over a period of years, adult female worms become enveloped by host tissue, forming a characteristic subcutaneous nodule (onchocercoma). The formation of the nodule appears as a response of the host to an organism continuously producing foreign proteins, causing perivascular infiltration of leukocytes and tissue cells, including fibroblasts and histiocytes, thereby forming a granuloma. The principal cells of a granuloma are macrophages that mature into epitheloid cells and fuse to multinucleate so-called foreign body giant cells. The infiltrate surrounding the encased female worm(s) consists not only of macrophages but also of eosinophilic and neutro-

116


philic granulocytes, mast cells, T and B lymphocytes (plasma cells) and NK cells. T cells, like mast cells, are involved as regulators of the cellular infiltrate. The density and composition of the inflammatory infiltrate depend on the viability and productivity of the parasite and the immune state of the host. In contrast to neutrophils, macrophages and giant cells, the mast cells were never found adherent to the cuticle of the filaria. While macrophages are most intensely associated with the adult stage of filariae, an infiltration of eosinophils into nodules is dependent on microfilariae production. Vital microfilariae appear to be hardly attacked by host cells, but degenerating microfilariae are often encircled, and inflammatory cells adhere and degranulate, and fragments of the larvae can be taken up [4]. Older nodules often contain necrotic material with degenerating or calcified remnants of filariae. The parasite itself also may contribute to the development of the nodule. Recently, one abundant onchocercal-expressed sequence tag was identified to encode an angiogenic protein [25]. This activation-associated secreted protein may promote vascularisation during nodule formation. The nutritional supply and survival of the parasite and of the onchocercoma depend on angiogenesis. Furthermore, the fatty-acid binding protein Ov-FAR-1 is shown to induce collagen synthesis and has been suggested to be involved in nodule formation [26] (see Section 4.4). The size of the nodules varies between 2 mm and 6 cm and more, depending on the differing immunological states of the individual human hosts and the numbers of worms. The nodules are usually palpable at the pelvic girdle and also on the ribs and the skull. They harbour, on the average, from 2–3 up to 10–50 females and 1–2 up to 5–10 males. In the rare, hyperreactive form of onchocerciasis, only a few, very large onchocercomas, with few adult parasites are observed [27] (see Section 4.1). The aggregation pattern of female clustering and formation of nodule conglomerates characterises onchocerciasis in humans and distinguishes this disorder from the evolutionarily older bovine parasitosis, showing usually solitary females in the nodules [28]. 3.2. The skin is the principal organ affected The skin is the principal site of infection, containing the mass of microfilariae, which represent the causative stage of cutaneous pathology. The prevalence and expression of reactive skin lesions in onchocerciasis depend on the intensity of infection but also on inherited factors [29]. Thus, the absence of external symptoms or mild skin alteration can be observed in infections with low, but also with high, parasite loads. Over years of chronic infection, extended fibrosis and keratosis can develop, along with pigmentary changes and lymphatic dilatation as a result of cumulative effects. In a systematic study, Murdoch et al. [30] classified cutaneous changes into acute papular, chronic papular and lichenified onchodermatitis, atrophy and depigmentation, and graded them (Fig. 2). Acute papular onchodermatitis (Fig. 2A) consists of small, scattered pruritic papules, which

Fig. 2. Skin lesions showing the spectrum of onchocerciasis [30]. (A) Acute papular dermatitis on the ventral torso of a 36-year-old woman in Burkina Faso with generalised onchocerciasis 24 h after treatment with DEC. (B) Chronic dermatitis with postpapular lesions (arrow) and scars on the foot of a Liberian woman with generalised onchocerciasis. (C) Severe chronic dermatitis with dark black hyperpigmentation (sowda) [4], lichenification, and enlarged femoral lymph nodes (arrows) on a 32-year-old male Liberian with hyperreactive onchocerciasis. The patient shows the typical asymmetry of the skin lesions (localised form). (D) Severe atrophic skin on the shoulder of a 13-year-old boy from Burkina Faso.

can progress to vesicles, pustules, erythema and oedema. Histologically, intradermal microabscesses around microfilariae are seen. Chronic onchodermatitis (Fig. 2B) shows larger lesions of varying size, with papules and postinflammatory hyperpigmentation. Lichenified onchodermatitis is frequently observed in patients with the chronic hyperreactive form (see Section 4.1). It is characterised by pruritic, hyperkeratotic, often confluent, plaques, ulceration as well as enlarged draining lymph nodes and is associated with low parasite loads. The affected skin is hyperpigmented, leading to the term “aswad” (the Arabic word for dark black coloured) that was introduced as “sowda(h)” (Fig. 2C). In chronic onchocerciasis, the skin is often characterised by atrophy (Fig. 2D), which is especially prominent at the site of enlarged lymph nodes (“hanging groin”) and which shows wrinkling of the skin and loss of elasticity. A degradation of the dermal extracellular matrix and elastic fibres can be caused by parasite or host proteases [31]. Finally, onchocercal depigmentation is often described as “leopard skin” showing patches of complete pigment loss. While atrophy can already affect young patients [32,33], depigmentation is more often observed in older, chronically infected persons.


These skin lesions are associated with a spectrum of T helper 2 (Th2)-type responses (see Section 4.2) ranging from low reactivities, in patients with high microfilarial loads to strong reactivities, in cases of lichenification (sowda) [32,33]. Following microfilaricidal treatment e.g. by the piperazine derivative diethylcarbamazine (DEC), acute inflammatory adverse reactions occur with varying intensity, depending on the microfilarial load. This so-called Mazzotti reaction is characterised by an acute papular onchodermatitis, lymphadenitis, pruritus, rash as well as fever, and rarely, hypotension that may exacerbate to shock in the case of very high microfilarial loads. Microfilariae are mobilised, found in body fluids and destroyed in lymph nodes and dermal microabcesses or microgranulomas [34]. During the first 24 h after drug exposure, dermal eosinophils and mast cells degranulate, eosinophil microabcesses and microgranulomas appear, degranulated eosinophil toxic proteins like major basic protein and eosinophil-derived neurotoxin are deposited around microfilariae, and degenerated microfilariae are observed [35]. The peripheral blood shows a transient eosinopenia, which is most pronounced after 8 h, followed by transient neutrophilia, with the highest numbers after 24 h. During the first 2 days, helminth-associated circulating eosinophil degranulation products [36] increase; subsequently, during the second to fourth day, elevated plasma levels of the eosinophil regulator, interleukin (IL)-5, occur that are followed by newly produced eosinophils and transient eosinophilia 5–10 days after provocation [37]. 3.3. Local lymphadenitis is associated with onchodermatitis The regional lymph nodes are often enlarged and involved in O. volvulus infection. They show follicular hyperplasia, infiltration of eosinophils and neutrophils and high numbers of plasma cells. With chronic infection, fibrosis and atrophy of the lymph node develop. The majority of microfilariae are often surrounded by eosinophils and macrophages. Direct contact between eosinophils and microfilariae, degranulation of eosinophils and free eosinophil granules around the degenerating microfilariae are observed. Increased numbers of IgE-binding mast cells and IgE- and IgG-producing plasma cells are observed [38]. 3.4. Visual impairment and blindness represent the severest pathology in onchocerciasis Infection with O. volvulus can lead to severe visual impairment and to blindness. In sub-Saharan Africa, 500 000 persons were estimated to suffer from visual impairment, and blindness occurred in more than 250 000 people, rendering river blindness the second most frequent cause of infectious blindness in that area [1,39]. In patients with a high microfilarial load and in the rare cases with onchocercoma in the upper part of the body, including the head, wandering microfilariae can invade the

117

conjunctiva, the cornea and posterior regions of the eye. Increasing numbers of degenerating microfilariae in the eye lead to the release of multiple somatic antigens that can induce inflammatory responses through a breakdown of the so-called immune privilege, normally preventing inflammation in the eye. The Onchocerca-induced ocular pathology can involve all parts of the eye, resulting in conjunctivitis, uveitis, iridocyclitis and chorioretinitis. The host’s reaction to the infiltrated microfilariae initially presents as snowflake corneal opacities or punctate keratitis that can develop into corneal scarring and a sclerosing keratitis [39]. Clinical manifestations similar to onchocercal keratitis can be induced experimentally by injection of O. volvulus antigens (OvAg) into the corneal stroma [40,41]. Inflammatory disturbances of the retinal pigment epithelium can result in chorioretinitis and optic atrophy. An invasion of the posterior segment of the eye can also cause a progressive loss of vision up to blindness [39]. 4. Determinants of pathogenesis and host responses 4.1. Onchocerciasis presents a spectrum of disease manifestations and immune responsiveness The pathology of chronic onchocerciasis is principally considered to be a consequence of long-standing host inflammatory responses [1,39,42]. In addition, pathogenic molecules produced by the filariae, such as proteases, may also be involved [8,31,40]. For characterisation of the pathogenic mechanisms, histological studies and examination of circulating humoral and cellular components, in vitro lymphocyte responses, as well as granulocyte reactivities have been both examined and related to disease manifestations [42,43]. As stimulants, somatic extracts of adult or larval stages of O. volvulus, comprising endobacterial Wolbachia molecules, excretory–secretory antigens from cultured filarial stages, as well as a variety of recombinant antigens [44] of Onchocerca and recently, of Wolbachia, have been investigated. In these studies, patients with varying parasitological, pathological and immunological states were included. Furthermore, various animal models that appear to represent some manifestations similar to those of onchocerciasis have been studied [45]. Prominent antibody responses with characteristically high IgG4 and IgE levels reactive with OvAg are documented [42,43,46,47]. While O. volvulus-specific IgG1 and IgG3 are regarded as possibly protective, being operative in antibodydependent cellular cytotoxicity reactions against filarial larvae, IgG4 may act as a blocking antibody [48]. O. volvulusspecific IgE may (i) be protective when bound to larval stages and eosinophil FcE-receptors, which results in cell degranulation and (ii) be pro-inflammatory through mast cell activation and degranulation. In contrast, polyclonal IgE is presumed to block allergic reactions of mast cells by saturating IgE receptors and preventing the bridging of O. volvulusspecific IgE, thereby avoiding mast cell degranulation. One major allergenic molecule of filariae appears to be gamma-

118


Table 2 Onchocerciasis patients present a spectrum of parasitological, clinical and immununological states Feature Microfilarial load per mg skin Onchocercoma

Generalised, including intermediate onchocerciasis 1–>500

Hyperreactive form (sowda)

Putative immune persons

Reference

50 adult worms 1–>50 nodules

Few adult worms Few large nodules, strong inflammatory reaction

None

[1,4,27,28,42,43,51]

Severe dermatitis, lichenification, hyperpigmentation Severe lymphadenitis Strong Th2 response

None

[1,30,32,33,42]

No specific reaction observed Weak responses: more pronounced Th2 than Th1 Pronounced proliferative response to OvAg Low levels of OvAg-reactive IgG1, IgE

[1,30,32,38,42] [32,33,57,59–63,79]

NE d

[27,33,36,50,53]

Skin lesions

Moderate, partially inflammatory reaction Subclinical or mild dermatitis

Local lymph nodes T cell responses

Enlarged, often fibrotic Th2, Th3/Tr1 responses a

B cell responses

Effector responses

Weak proliferative response to OvAg b Elevated levels of OvAg-reactive IgG1, IgG4, IgE c Eosinophilia, mastocytosis, serum eosinophil cationic proteins increased Antimicrofilarial response in vivo only periodically or after anthelminthic treatment

Pronounced proliferative response to OvAg High levels of OvAg-reactive IgG1, IgG3, IgG4, IgE Strong eosinophilia, mastocytosis, serum eosinophil cationic proteins strongly increased Antimicrofilarial response in vivo without treatment

[42,46,47,51,55,59,89]

NE

a

Th, T helper cells. OvAg, O. volvulus antigens. c Ig, immunoglobulin. d NE, not examined. b

glutamyl transpeptidase, inducing IgE responses possibly associated with pathology but also protection [49]. Analyses of T cell responses of O. volvulus-infected persons include antigen-induced lymphocyte proliferation and cytokine release, observing characteristically elevated Th2 responses but also Th1 and Th3 reactivities [42,43]. Onchocerciasis represents a disease comprising a spectrum of manifestations and host responsiveness. The polar forms are described in Table 2. The majority of O. volvulusinfected persons present the common, so-called generalised form of onchocerciasis presenting varying, mostly mild or moderate skin dermatitis. The patients with high microfilarial loads show no obvious capacity to effectively reduce the numbers of microfilariae and adult filariae. In contrast, those with lower microfilarial densities intermittantly present onchodermatitis with signs of antimicrofilarial defence reactivities, expressing an intermediate state [4,50]. While with the polar, generalised form, the patients are considered to be hyporeactive, patients with the rare, hyperreactive form (sowda) exhibit extensive reactivity and skin pathology. This variation in manifestations in patients with onchocerciasis that includes intermediate forms has been reported [51,52]. In addition, a small number of long-time O. volvulusexposed residents of endemic areas do not show any parasitological and clinical signs of onchocerciasis, and therefore, are designated putatively immune individuals or “endemic normals”.

Sowda patients show a severe chronic papular dermatitis with hyperpigmentation, lichenification and lymphadenopathy (see Section 3.2), often presented asymmetrically on one limb, designated a “localised” or “regional” form of onchocerciasis (Fig. 2C). In comparison with the generalised form of onchocerciasis, in sowda patients, the numbers of onchocercomas, adult filariae and microfilariae are very low, and histological studies revealed massive perivascular infiltration in the large onchocercomas, the regional lymph nodes and the affected skin sites [27,38] (Fig. 3B). The few live or disintegrating microfilariae are found encircled predominantly by eosinophils and macrophages, and more rarely, neutrophils, forming multiple microgranulomas. Eosinophils and eosinophil constituents are found attached to the microfilarial cuticle, and fragments of microfilariae are engulfed by giant cells. Similarly, sowda patients exhibit greatly enlarged lymph nodes with extensive follicular hyperplasia and activation of germinal centres, strongly increased numbers of plasma cells producing IgE and IgG class antibodies, and high numbers of eosinophils surrounding degenerated microfilariae [38]. These patients with a hyperreactive form obviously have the capacity to kill microfilariae, as observed in patients with a generalised form after treatment with filaricidal drugs (DEC) and those with a state of intermittent dermatitis (see Section 3.2). The obligatory low numbers of macro- and microfilariae observed in sowda patients, exposed to similar transmission rates of the parasites as the patients


Fig. 3. Comparison of onchocercomas of the hyperreactive (sowda) and generalised form of onchocerciasis [4,27]. (A) Scheme of nodules from both forms, containing pairs of adult worms and depicting a broad subcapsular infiltrate in the sowda nodule (left). (B) Photographs of cross-sections of nodules from patients with the hyperreactive (top) and generalised (bottom) form. The nodule of the generalised form shows several worm pairs in two compartments with a small perivascular inflammatory infiltrate, while the nodule of the hyperreactive form shows a massive inflammatory infiltration around a single central worm pair (scale in cm).

119

with a generalised form, clearly indicates the ability to also eliminate invading infective larvae. The larvicidal potential of human as well as mouse eosinophils has been observed in vitro (Fig. 4) [53,54]. Eosinophils can damage filarial larvae and host cells by release of cytotoxic cationic proteins as well as newly formed mediators like oxygen radicals. Immunological studies revealed intense T helper 2-type reactivities in sowda patients. In vivo, high to excessive serum levels of total IgE and high antifilarial IgG antibodies are associated with pronounced eosinophilia and mastocytosis [27,33,50,51,55]. Furthermore, increased numbers of peripheral T cells and an increased delayed type of hypersensitivity were reported [42,56]. In vitro studies revealed strong Th2type cytokine production, and chemotactic responsiveness of peripheral eosinophils are observed in sowda patients [32,50,57]. The high levels of O. volvulus antigen-reactive IgG1 and IgG3 antibodies and of total IgE represent proinflammatory antibodies that probably refer to the microfilaricidal potency of the sowda patients [51,55]. Furthermore, in hyperreactive onchocerciasis, autoantibodies that cross-react with neutrophil bactericidal defensins, calreticulin or collagen are frequently observed [58]. From these observations, autoimmunity mediated by antigenic mimicry has been implicated in immunopathological mechanisms in onchoceriasis. While Th2 responses are consistently found in patients with sowda and less pronounced in the majority of patients with generalised onchocerciasis—with regard to in vivo effectors like IgE, eosinophils and mast cells, as well as in vitro production of regulatory cytokines like IL-4, IL-13 and IL-5—inconsistent findings of Th1 responses have been reported [42,43]. Most reports described weak proliferative responsiveness to onchocercal antigens of circulating lymphocytes from patients with the generalised form of onchocerciasis and low production or absence of interferon

Fig. 4. Comparison of eosinophil reactions to microfilariae (A, B) and infective larvae of O. volvulus [53] (C, D). (A, B) Adaptive immune responses: adherence reaction of eosinophilic granulocytes to microfilariae requires the presence of antibodies. Exposure time: (A) 1 h; (B) 24 h. (C, D) Innate immune responses: eosinophil adherence to the infective larva occurs in the absence of O. volvulus-specific antibodies. Exposure time: (C) 15 min; (D) 18 h. Bars: 10 µm (A), 50 µm (B), 30 µm (C), 35 µm (D).

120


gamma (IFN-gamma) and IL-2 in the cell cultures, indicating a suppressed immune state in the majority of the infected persons [42,59]. In contrast, higher Th1 responses have repeatedly been reported in putatively immune individuals exposed to filarial infection [42,60]. More recently, Th2-type immune responses have been shown in this group of exposed persons [57,61–63]. In experimental mouse models of filariasis, infective larvae affect the innate immune system and elicit Th2-dependent protective immunity [45,64]. For a long time, intrinsic human factors have been presumed to influence factors in the pathogenesis of onchocerciasis. Thus, in West Africa, the sowda form is more frequently observed in young women than in men, which may indicate the influence of sex-related factors. Distinct HLA class II alleles are found associated with either the generalised or sowda form of onchocerciasis and in putatively immune individuals, indicating that the HLA-D variants may influence the host response to O. volvulus [65]. Furthermore, a distinct gene variant of IL-13, the regulator of IgE production and of Th2 responses in the skin, is associated with the sowda form [66]. Thus, further studies will possibly enable the discovery of still unknown inherited factors including mediators of affecting O. volvulus infection and pathogenesis. Although similar attraction of the vector (anthropophily) by onchocerciasis patients and putatively immune individuals could be observed [67], other influences on the expression of the pathogenesis and the host–parasite relationship have to be envisaged, such as variation and polymorphism of the filarial parasite, and duration and intensity of transmission [68]. Allelic variation of protein-coding genes may modify biological activity or provide diversity in the face of specific host immune responses. Polymorphisms are found in some filarial surface antigens like the antioxidant enzyme glutathione peroxidase (GPX), the cysteine protease inhibitor (cystatin, CPI), chitinase proteins (CHI) and the abundant larval transcript (ALT) [68] (see Section 4.4). Antigens of microfilariae may display more diversity than those of adult filariae, since this stage is under stronger selective pressure. Interestingly, proteins of sheathed microfilariae of other filarial species revealed a high level of polymorphism [68]. For Onchocerca, only limited diversity has been observed so far. 4.2. The balance between pro-inflammation and antiinflammation Evolution appears to have selected mechanisms of interaction between parasite and host that limit defence and inflammatory reactions, which would decrease the parasite’s survival but also increase the host’s pathology. The result is that vital microfilariae appear to wander unharmed through the subcutaneous tissue, and the vital adult worms reside for about 10 years encapsulated in the onchocercoma. This persistence supports the view that the parasite modulates the host response to prevent immune-mediated damage. Al-

though in many microfilaria carriers, significant antiparasitic host responses are not evident and pro-inflammatory mechanisms appear well contained [42], the potential for proinflammatory host responses exists and can be demonstrated in vivo in the form of increased numbers of tissue-residing effector cells. While the balance inclines to limitation of inflammatory reactions towards vital migrating and secreting microfilariae, the containment of the inflammation can be periodically overcome e.g. when larger numbers of filariae degenerate, resulting in the release of a multitude of breakdown metabolites. Invasion of infective larvae of O. volvulus into the skin introduces foreign (non-self) antigens, recognised primarily by the host’s innate, and subsequently, its acquired immune system. Recent research on the early interaction between microbial pathogens and the human host revealed that initial recognition (sensing) of an agent is mediated by pathogenassociated molecular patterns (PAMP) that specifically ligate to pathogen recognition receptors, e.g. Toll-like receptors (TLR) expressed on cells of the innate immune system, predominantly on macrophages but also on mast cells and granulocytes [69]. TLR signalling and regulation of cytokine gene expression link early innate responses and the ensuing specific immune responses. Lately, they have been shown to stimulate not only pro-inflammatory but also antiinflammatory mechanisms (see Section 4.3) [70]. PAMPs have been identified on bacteria, fungi, protozoa and recently, as phospholipids in the tegument of the trematode Schistosoma [71]. The existence of PAMPs of filaria is unknown; however, similarly to Schistosoma, the filarial surface also contains distinct lipid and carbohydrate compounds like phosphorylcholine (PC)containing glycoprotein ES-62, reported to affect macrophages, thereby polarising the immune responses [72]. Since the early in vivo responses of innate immune cells to infective larvae in the human skin cannot be studied, only in vitro experiments revealed that the O. volvulus infective third-stage larvae, but not the fourth-stage larvae or onchocercoma-released microfilariae, are attacked by cells of the innate immune system in the absence of antibodies (Fig. 4) [53]. There are numerous studies indicating strong immune responses, predominantly in the early phase of Onchocerca infection. Early after infection, patients showed elevated proliferation of peripheral lymphocytes in response to filarial antigens and higher cytokine responses in comparison with long-term infection ([73], Brattig et al., unpublished). Similarly, after experimental infection of chimpanzees with O. volvulus or of cattle with the natural parasite O. ochengi, the cellular immune responses were most pronounced in the early and prepatent phase of infection, and subsequently, with patency, decreased while the antibody levels persisted [74,75]. Of interest, most of the patients with the hyperreactive sowda form of onchocerciasis are young. Persisting transmission and chronic infection result in an accumulation of parasites, leading to downregulation of parasite-specific cellular immune responses [43,63,75,76] (Fig. 5; Table 3). The mechanisms of immunosuppression are


Fig. 5. Relation between microfilarial load and in vitro cellular immune responsiveness. Negative correlation (P < 0.0001) of microfilaria counts (0–460 microfilariae per mg skin) with proliferative (A) and IL-5 (B) responses of O. volvulus antigen-exposed peripheral blood cells from 551 O. volvulus-exposed persons [63].

still poorly understood. The antiinflammatory cytokine IL-10 has been implicated as one major mediator of hyporesponsiveness in filariasis, as known in other chronic infections [77,78]. IL-10 can be demonstrated in culture supernatants of O. volvulus antigen-exposed mononuclear cells as well as T cell clones [57,59,79], and cell responsiveness increased by neutralisation of IL-10 [73]. Mononuclear cells from miTable 3 Filarial and endobacterial molecules activate pro-inflammatory and antiinflammatory mechanisms Response

Regulatory cells

Mechanisms operative in immunopathogenesis of onchocerciasis Pro-inflammatory Antiinflammatory • Th3 • Th2, Th1a • Macrophage • Alternatively activated • Mast cell macrophage

Regulatory molecules

• IL-10, TGF-betac • IL-5, IL-4, IL-13b • IFN-gammad • IL-4 • TNF-alphae, IL-8, IL-12

Effector cells

• B cell • Eosinophil, neutrophil • Macrophage, mast cell

• B cell • Alternatively activated macrophage

Effector molecules

• IgG1, IgG3, IgEf • MBP, EDN, ECP • Peroxidases (EPO, MPO) • Defensins • Oxygen, nitrogen radicals • Proteases

• IgG4, polyclonal IgE

a

Th, T helper cells. IL, interleukin. c TGF, transforming growth factor. d IFN, interferon. e TNF, tumour necrosis factor. f Ig, immunoglobulin. b

121

crofilaria carriers also released IL-10 spontaneously [59,77]. The major sources of IL-10 are regulatory T cells (Tr1; Th3) and macrophages [77,79]. Antigen-specific Tr1 cells appear to act as mediators of peripheral tolerance [79]. IL-10 has been shown to inhibit the maturation and activation of antigen-presenting cells and to suppress their proinflammatory functions and migration and downregulate T cell function, but IL-10 promotes the growth and differentiation of B cells [78]. Immunosuppression has been reported to relate to duration and intensity of filarial infection [76], and products of the frequent microfilariae and of the large female filariae [80] may be operative. Of importance will be the identification of involved filarial or endobacterial molecules constantly exposed to the human immune system. Interestingly, after transient reduction in microfilarial burden by microfilaricidal therapy with ivermectin, the cellular hyporesponsiveness was temporarily reversed [81]. In accordance with these observations, in utero exposure to O. volvulus microfilariae and their antigens has been shown to exert long-term effects on the subsequent newborn cellular immune responses, which may render the child more susceptible to O. volvulus infection postnatally [82]. In utero contact with microfilarial antigens also caused a strong upregulation of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) [83]. In addition to IL-10 and transforming growth factor beta (TGF-beta), Tr1 cells express CTLA-4, representing a counter-receptor in co-stimulation between T cells and antigen-presenting cells. CTLA-4 binds with much higher affinity to the adhesion molecule CD80 on antigen-presenting cells than its homologous T cell receptor CD28, and delivers an inhibitory signal that generates IL-10. Microfilaria carriers demonstrated higher frequencies of CTLA-4-positive T cells than microfilaria-negative persons, and blocking experiments indicated an influence on the Th1/Th2 balance [83]. Thus, activation of CTLA-4-positive T cells may contribute to the diminished T cell reactivity in onchocerciasis. It is well known that immune responses to filariae and other helminths share key features with the allergic responses, such as the predominance of Th2-type mechanisms (see Sections 3.2 and 4.1). Studies have indicated that little allergic disease is found in areas where helminth infections are endemic. In onchocerciasis, patients with a generalised form, with high parasite burdens often are characterised by mild allergic skin reactions associated with strong downregulatory mechanisms, while in the rare sowda group, severe pathology and the highest allergy-type inflammation is associated with a low worm load, indicating that sowda patients fail to control allergic inflammation [42,43]. The allergic potential in general is much higher in filarial infections than that observed in non-helminth allergic persons who obviously miss the downregulation. Recent epidemiological and clinical studies in helminth-endemic areas in Africa have suggested an inverse association between helminth infection and allergy [84]. Chronic experimental helminth infection

122


was reported to bias the IgE response to non-helminth allergens. The ability of helminth infections to protect against allergic disease is linked to the generation of immunoregulatory cytokines. Thus, high levels of IL-10 correlated with reduced skin test reactivity to non-helminth allergens, and IL-10 has been shown to downregulate mast cell activation. Thus, Tr cells and IL-10 have been anticipated as regulative in allergic processes [85]. Cells of the macrophage lineage in addition to Tr1 cells can function as producers of immunoregulative IL-10, TGFbeta and other regulators [77,86,87]. Recently, such macrophages had been named alternatively activated macrophages (AAM) and were distinguished from classically activated macrophages [88]. It had been hypothesised that (i) secreted products of vital filariae may activate Th2 responses and AAM, while degenerating parasites deliberate metabolites and activate classically activated macrophages, and that (ii) a critical balance between both divergent pathways of macrophages could be an important factor in the pathogenesis of filariasis [86]. Recently, the in vivo gene expression phenotype of IL-4-activated AAM was analysed [87]. The expression profile revealed the upregulation of arginase and an eosinophil attractant promoting Th2-mediated responses. The activation of AAM could be achieved by filarial molecules, including a homologue of the human mediator macrophage migration inhibitory factor (MIF) (see Section 4.4). 4.3. Onchocerca- and Wolbachia-related responses The high turnover rates of microfilariae [5] cause a persistent release of somatic constituents consisting of filarial and endobacterial products. The exposure of these antigens to the host defence system can induce immune responses to filarial as well as endobacterial molecules. While Th2-like immune responses principally characterise helminth infections, Th1type responses are attributed generally to microbial pathogens. OvAg have widely been used in immunological analyses in the form of somatic total extracts of various adult and larval stages [42,57,59,62,63,89]. More recently, with the research program supported by the Edna McConell Clark Foundation, more than 5000 genes of O. volvulus were identified, and approximately 50 recombinant proteins were cloned and examined immunologically in vitro and in vivo [3,7,44]. Predominantly Th2 immune responses were elicited by OvAg in patients with onchocerciasis or in experimental rodent models [42,45,62,63], and rarely, filarial antigens have been reported as Th1 stimulus [90]. Furthermore, the activation of Tr1/Th3 cells has recently been demonstrated [79] (see Section 4.2), and filarial molecules have been implicated in the induction of counter-inflammatory responses (see Section 4.4). While the dominant Th2 responsiveness to filarial antigen is well documented, recent and increasing evidence strengthens the notion that innate immune responses, neutrophil and Th1-type responses—observed in filarial infections prefer-

Fig. 6. Association between the presence of intracellular Wolbachia bacteria in the filaria and the host’s neutrophilic granulocytes in the vicinity of the O. volvulus female [20]. (A) Demonstration of Wolbachia in the hypodermis of an untreated female worm using polyclonal antibodies against endobacterial heat-shock protein 60 and staining by immunohistology (APAAP). (B) Double-staining of heat-shock protein-stained (PAP) endobacteria in the hypodermis and defensin-stained (APAAP) neutrophils adjacent to the filaria. (C) Absence of neutrophil accumulation around the worm depleted of Wolbachia by doxycycline treatment of the patients [17]. Only a few elastase-positive neutrophils (arrow head) are shown by immunostaining (Bars = 100 µm).

entially after microfilaricidal treatment—may depend on stimuli deriving from the Wolbachia bacteria. (I) Histological and in vitro studies revealed that the accumulation of neutrophils around Onchocerca worms in the nodules and the in vitro chemotaxis towards O. volvulus somatic extract are related to the presence of Wolbachia (Fig. 6) [20]. It is reported that defensin, one major neutrophil constituent, is prominently bound to the surface of adult females [58], which may be the result of Wolbachia-induced cell activation. Neutrophils have been reported as effectors against O. volvulus larval stages [91]. Neutrophils are also thought to be involved in the generation of a cyst at the anterior end of females, which appear to aid the uptake of host nutrients and to facilitate, mating with males. Thus, neutrophils recruited by endobacterial chemoattractants may contribute to the survival of the parasites. In tetracyclinetreated patients, no accumulations of neutrophils in the vicin-


123

Table 4 Wolbachia- and filaria-related responses are involved in the pathogenesis and after exposure to antifilaricidal drug DEC Origin Filaria-related responses

Wolbachia-related responses

Type of immune response Adaptive (Th2-type) and innate immune responses In vivo • Th2 response: peripheral and tissue eosinophilia, mastocytosis, increased serum levels of OvAg-reactive IgG4, IgE, total IgE and eosinophil cationic proteins • Th2 response stimulated after antifilaricidal treatment (DEC) – early eosinophil degranulation, transient eosinopenia – increased serum levels of IL-5, RANTES, followed by eosinophilia In vitro • Predominant Th2 responses to OvAg: IL-4, IL-13, IL-5 • Eosinophil chemotaxis, adherence to larvae (Mf, L3, not L4) degranulation, microfilaria killing by eosinophils, neutrophils and macrophages Stimulation of innate immune and Th1-like responses In vivo • Early systemic inflammatory responses 6–24 h after antifilaricidal treatment (DEC) – high peripheral levels of TNF-alpha, IL-6 antibacterial acute phase reactants • focal neutrophil accumulation • Release of Wolbachia (DNA) • Control of embryogenesis • IgG1 antibodies reactive with Wolbachia molecules (WSP, AAT, heat-shock protein) In vitro • Release of TNF-alpha, IL-1–beta, IL-8, nitric oxide by host cells • Neutrophil chemotaxis dependent on Wolbachia • Inflammatory response in the cornea

Reference [1,4,27,34–37,42,46,47,51,55]

[1,42,50,53,91]

[20–24,40,41,92–94]

[18–20]

For abbreviations see Table 2: Th, T helper cells; OvAg, O. volvulus antigens; Ig, immunoglobulin.

ity of filariae are observed, and extracts of O. volvulus from these patients show reduced chemotactic activity. (II) In the early phase of the Mazzotti reaction (see 3.2.), observed after microfilaricidal treatment by DEC in onchocerciasis, and similary in lymphatic filariasis, a burst of circulating inflammatory mediators (e.g. TNF-a and IL-6) and a transient neutrophilia coincide with pathological manifestations (Table 4) [42,92,93]. Large amounts of endobacterial molecules are supposed to be released in consequence of the drug-induced breakdown of microfilariae, and the levels of pro-inflammatory cytokines and antibacterial neutrophilic peptides correlated well with the level of detectable endobacterial DNA [21,93]. In the case of high microfilarial burden, the clinical manifestations may resemble in some aspects a sepsis-type reaction attributed to bacterial products. In this drug-induced reaction to microfilarial products delayed Th2-type responses occurred, in second wave (see 3.2.). The impact of filarial molecules in the induction of the Mazzotti reaction can be deduced from the observation of skin inflammation associated with recruitment of eosinophils, but not of neutrophils, after DEC administration to onchocerciasis patients whose endobacteria had been depleted by preceding antibiotic treatment (D.W. Büttner, unpublished), contesting the view that the inflammation may depend on endobacterial stimulants. (III) Similarly, neutrophil as well as eosinophil granulocyte responses are also involved in the inflammation of eyes affected by microfilariae (see Section 3.4), and produced by microfilarial breakdown products. In a murine model of ocular onchocerciasis, the intrastromal injection of OvAg results in a biphasic recruitment of inflammatory cells, with an early

and transient influx of neutrophils peaking 12–24 h after injection, followed by a long-time mobilisation of eosinophils 3–14 days after exposure [41]. Activation of resident corneal cells by breakdown products of the microfilariae leads to secretion of proinflammatory cytokines, which stimulate synthesis of chemokines by keratocytes and induce the expression of adhesion molecules on vascular endothelial cells followed by early recruitment of neutrophils. The neutrophil infiltration increases stromal thickness and corneal opacification characteristic of Onchocerca keratitis [40,41]. By comparing the reactions induced by Wolbachiacontaining extract from untreated O. volvulus with extract prepared from worms obtained from tetracycline-treated patients, the endobacteria were implicated in the inflammatory responses in the experimental keratitis model. In contrast, the recruitment of eosinophils appears not to be dependent on endobacteria [22,41]. In vivo and in vitro experiments using mouse strains with mutation in the TLR4 gene indicated that distinct molecules of Wolbachia mediate an activation of macrophages through the engagement of TLR4, leading to the release of inflammatory cytokines, radicals and neutrophil activators [18,22,94]. TLR4 is one of the best characterized and most important TLRs that mediates signals for a broad spectrum of ligands [69] in addition to lipopolysaccharides, including lipoteichoic acid, fatty acids, oligosaccharides, heat shock proteins, viral proteins, fibronectin, and terpenoids. So far, lipopolysaccharide-like molecules have been considered candidate Wolbachia stimulants of the human innate immune system, although the biosynthetic genes have not yet been identified. Until now, only a few Wolbachia molecules have

124


Table 5 Filarial molecules putatively involved in parasite evasion Group Antioxidative enzymes

Protease inhibitors

Fatty-acid-binding molecules

Cytokine homologues, cytokine receptor homologues Miscellaneous

Molecule Glutathione peroxidase (GPX-1) Superoxide dismutase (SOD-2) Thioredoxin peroxidase (TPX-1,2) Glutathione S-transferase (GST-1) Cysteine protease inhibitor, cystatin (CPI-2) Serine protease inhibitor, serpin (SPN-2) Aspartyl protease inhibitor (API-1; Ov33) Fatty-acid-binding protein (FAR-1; Ov20)

Polyprotein gp15/400 (NPA) MIF homologue (MIF) TGF-beta homologue (TGH) TGF-beta receptor (TRK) Activation-associated secreted protein (ASP-1), PC-containing glycoprotein (ES-62)

been characterised, including a heat shock protein 60, the cell cycle protein FTSZ, a surface protein WSP, an aspartate aminotransferase and a putative periplasmic HtrA-type serine protease [9,11,12,24,95]. A recent study indicated that the WSP expresses a TLR-2- and TLR-4-dependent inflammatory potential on macrophages and activates neutrophils (Brattig et al., unpublished; C. Bazzocchi et al., Vet. Parasitol. 117 (2003) 73–83). After demonstration of both TLRs in addition to macrophages also on mast cells and neutrophils (see Section 4.2), an involvement of these cells of the innate immune system in response to Wolbachia molecules must also be considered. Interestingly, the engagement of TLR-2 and TLR-4 on mast cells has lately been reported to cause the release of Th2 cytokines [96]. In contrast to high doses of TLR-4 ligands, known to stimulate Th1-type reactions, low doses can promote allergen-type Th2 responses. These observations appear to indicate a link between Wolbachia- and filaria-related host responses and emphasise the significance of the dose of stimuli. 4.4. Parasite molecules maintaining its survival The O. volvulus filariae predominantly reside and migrate in the human skin and subcutaneous tissue that represents a milieu of potent defence mechanisms against invading agents. The multitude of reactive cells includes macrophages, Langerhans’ cells, lymphocytes, granulocytes, mast cells, keratinocytes, and endothelial cells. Since filarial parasites enjoy life spans of years in this immunocompetent environment, they must have acquired mechanisms to resist the host defence system. Helminths as multicellular macroparasites have evolved survival strategies different from those of microparasites like protozoa, bacteria or viruses. During the long-standing co-evolutionary interaction between filariae and the human defence system, helminths have accomplished genes encoding products that divert or block the host defence mechanisms.

Characteristics Antioxidative enzymes Protection from host reactive oxidants Surface-association, secretory

Reference [97–100]

Surface-association in larvae, adult filariae Immunosuppression, polymorph Inhibition of host serine proteases (controvers) Sequestering, transport of fatty acids (retinol, arachidonic acid, possibly ivermectin) Surface protein, allergen (IgE response) Secretion Counter-inflammation suggested Downregulation Angiogenesis in mice, allergen Immunosuppression T, B cell responses

[97,98,101,102]

[26,97,103]

[87,98,106,107]

[25,105]

A number of molecules of the parasite have been characterised that neutralise or affect host reactants against the parasite. The parasite, furthermore, appears to modulate immune responses in order to evade mechanisms leading to its elimination (Table 5). Thus, vital adult filariae and microfilariae seem to provoke little or restricted host responses, while molecules released by degenerating and dead microfilariae and adult worms appear to evoke inflammatory reactions. Immune evasion genes include genes encoding surface and secreted proteins, antioxidative enzymes, protease inhibitors, fatty acid-binding protein, and homologues of mammalian regulatory cytokines [97,98]. Secreted antioxidant enzymes of O. volvulus, as in other filariae, cope with reactive oxygen species like hydrogen peroxide, superoxide anion and hydroxyl radical to defend the parasite from radicals strongly generated by eosinophils and less pronounced by neutrophils and macrophages. The oxidative reactants damage proteins, lipids and DNA at the parasite surface and intracellularly. The involved enzymes include peroxiredoxins (PRX), GPXs, superoxide dismutases (SOD), and glutathione S-transferase (GST), several of which have been found surface-associated and secreted as GPX, SOD, PRX, GST in filariae [99]. A second major group of neutralising, often exported, parasite molecules are protease inhibitors identified for cysteine proteases (cystatins, CPI), serine proteases (serpins, SPN) and aspartyl proteases (API) [97,98]. These inhibitors may regulate parasite proteases as well as neutralising those of the host. In addition, cystatins (CPI) have been demonstrated to be potent immunomodulators interfering with central immune reactions such as antigen presentation, effector functions like chemotaxis, phagocytosis and respiratory burst as well as the polarisation of Th responses by promoting IL-10 production [101,102]. A further abundant and secreted filarial product is the fatty acid-binding protein (FAR-1; Ov20) that may function in scavenge, uptake, transport and metabolism of fatty acids,


sterols, and retinol [26,103]. The parasite requires retinol for its growth, differentiation, and embryogenesis, and as antioxidant. Retinol deficiency observed in onchocerciasis has been implicated in visual impairment and dermal pathology. A recent controversial hypothesis suggested that onchocerciasis-associated morbidity may be due in part to a FAR-related accumulation of retinol, creating a retinoid toxicity [104]. Furthermore, the filarial PC-containing glycoprotein ES-62 has been characterised as an immunomodulatory molecule [105]. ES-62 inhibits T and B cell activation, induces the maturation of dendritic cell type 2 with the capacity to induce Th2 responses. Of special interest is the recent identification of filarial gene products homologous to human regulatory cytokines that may interfere with the host cytokine network [97]. While viruses have been shown to capture genes encoding immunomodulatory cytokines like IL-10 or its receptors from their hosts, helminths appear to evolve ancestral genes in parallel with their hosts and target the host’s cytokine network by mimicry and modulation to increase their success. Thus, filariae express homologues of human MIF and TGF-beta [106,107]. Filarial MIF showed striking conservation of structure and inflammatory function to the human homologue. However, MIF showed a dosedependent pro-inflammatory and also a counterinflammatory effect. A continuous secretion of the mediator homologue was assumed to induce a counter-inflammatory state [98]. Filarial MIF was reported to generate AMMs in mice shown to downregulate T cell proliferation and to promote Th2 differentiation, including the secretion of eosinophil chemotactic factor [87] (see Section 4.2). Thus, filarial molecules themselves (MIF, TGF-beta) may function as immunomodulators and preferentially mitigate host inflammatory responses, or they express molecules (CPI, PC) that exert suppressive mechanisms by the generation of AMMs or regulatory T cells [79,87].

125

especially after massive degeneration of the filariae by filaricidal treatment. Inflammation appears to result from innate immune system-activating endobacterial molecules as well as from Th2-activating filarial molecules. The chronic exposure of Onchocerca-associated filarial and endobacterial molecules to the host evokes antiinflammatory mechanisms, including Tr1 cells and presumably, AMMs, thereby controlling the inflammatory potential. Filarial and possibly endobacterial molecules are thought to sustain counterinflammation. With the advent of genome research (River Blindness Genome Project), molecular insights led to the identification of over 5000 genes, including numerous immune evasion genes encoding various antioxidants, enzyme inhibitors, lytic enzymes, cytokine homologues and immune modulators. Secretory and somatic filarial molecules can block and divert the host immune mechanisms. Future immunological, molecular and genome work will expand our understanding of the biology of O. volvulus and molecular pathogenesis, which can provide tools for identification of target molecules for possible intervention by chemotherapy and vaccination.

Acknowledgements I acknowledge the important work of colleagues in the research field who may have been left out of this report. I wish to thank Frank Geisinger and Wilfried Groenwoldt for their technical contribution. I am grateful to Dietrich W. Büttner for providing photographs of patients with onchocerciasis and of onchocercomas, for helpful discussion and comments on the manuscript. This work was supported in parts by grants from the Deutsche Forschungsgemeinschaft, from the Ministry of Education, Science, Research and Technology, and the Alexander von Humboldt Foundation, Bonn, Germany.

5. Concluding remarks References The limited pathology provoked by one of the largest and longest-surviving parasites of human tissue is thought to result from long-time co-habitation, co-evolution and coadaptation of the filaria with its intracellular Rickettsiarelated Wolbachia, on the one hand, and over a shorter period, with its mammalian hosts, on the other hand. Thus, the migrating stages of the parasite, predominantly the microfilariae, appear to actively evade effective immune mechanisms in the skin. Recent evidence revealed that, as an obligatory symbiont, Wolbachia contributes to the embryogenesis and larval development of the filariae, thereby emerging as a novel target for chemotherapy. Further research efforts will lead to the identification of essential metabolites sustaining the mutualistic relationship between the co-habitants. Endobacterial molecules also have been suggested to contribute to the pathogenesis of onchocerciasis,

[1]

WHO, Expert Committee on Onchocerciasis Control, WHO Tech. Rep. Ser. 852 (1995) 1–103.

[2]

Y. Dadzie, M. Neira, D. Hopkins, Final report of the Conference on the Eradicability of Onchocerciasis, Filaria J. 2 (2003) 2–20.

[3]

J.A. Cook, C. Steel, E.A. Ottesen, Towards a vaccine for onchocerciasis, Trends Parasitol. 17 (2001) 555–558.

[4]

D.W. Büttner, P. Racz, Macro- and microfilariae in nodules from onchocerciasis patients in the Yemen Arab Republic, Tropenmed. Parasitol. 34 (1983) 113–121.

[5]

B.O. Duke, The population dynamics of Onchocerca volvulus in the human host, Trop. Med. Parasitol. 44 (1993) 61–68.

[6]

T.R. Unnasch, S.A. Williams, The genomes of Onchocerca volvulus, Int. J. Parasitol. 30 (2000) 543–552.

[7]

S.A. Williams, S.J. Laney, M. Lizotte-Waniewski, L.A. Bierwert, T.R. Unnasch, The River Blindness Genome Projekt, Trends Parasitol. 18 (2002) 86–90.

126 [8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

N.W. Brattig / Microbes and Infection 6 (2004) 113–128 M. Lizotte-Waniewski, W. Tawe, D.B. Guiliano, W. Lu, J. Liu, S.A. Williams, S. Lustigman, Identification of potential vaccine and drug target candidates by expressed sequence tag analysis and immunoscreening of Onchocerca volvulus larval cDNA libraries, Infect. Immun. 68 (2000) 3491–3501. C. Bandi, A.J. Trees, N.W. Brattig, Wolbachia in filarial nematodes: evolutionary aspects and implications for the pathogenesis and treatment of filarial diseases, Vet. Parasitol. 98 (2001) 215–238. M. Sirioni, C. Bandi, L. Sacchi, B. Di Sacco, G. Damiani, C. Genchi, Molecular evidence for a close relative of the arthropod endosymbiont Wolbachia in a filarial worm, Mol. Biochem. Parasitol. 74 (1995) 223–227. C. Bandi, T.J. Anderson, C. Genchi, M.L. Blaxter, Phylogeny of Wolbachia in filarial nematodes, Proc. R. Soc. Lond. B. Biol. Sci. 265 (1998) 2407–2413. C. Bazzocchi, W. Jamnongluk, S.L. O’Neill, T.J. Anderson, C. Genchi, C. Bandi, WSP gene sequences from the Wolbachia of filarial nematodes, Curr. Microbiol. 41 (2000) 96–100. L.V. Sun, J.M. Foster, G. Tzertzinis, M. Ono, C. Bandi, B.E. Slatko, S.L. O’Neill, Determination of Wolbachia genome size by pulsedfield gel electrophoresis, J. Bacteriol. 183 (2001) 2219–2225. S.C. Bosshardt, J.W. McCall, S.U. Coleman, K.L. Jones, T.A. Petit, T.R. Klei, Prophylactic activity of tetracycline against Brugia pahangi infection in jirds (Meriones unguiculatus), J. Parasitol. 79 (1993) 775–777. C. Bandi, J.W. McCall, C. Genchi, S. Corona, L. Venco, L. Sacchi, Effects of tetracycline on the filarial worms Brugia pahangi and Dirofilaria immitis and their bacterial endosymbionts Wolbachia, Int. J. Parasitol. 29 (1999) 357–364. N.G. Langworthy, A. Renz, U. Mackenstedt, K. Henkle-Dührsen, M.B. de Bronsvoort, V.N. Tanya, M.J. Donnelly, A.J. Trees, Macrofilaricidal activity of tetracycline against the filarial nematode Onchocerca ochengi: elimination of Wolbachia precedes worm death and suggests a dependent relationship, Proc. R. Soc. Lond. B. Biol. Sci. 267 (2000) 1063–1069. A. Hoerauf, L. Volkmann, C. Hamelmann, O. Adjei, I.B. Autenrieth, B. Fleischer, D.W. Büttner, Endosymbiotic bacteria in worms as targets for a novel chemotherapy in filariasis, Lancet 355 (2000) 1242–1243. M.J. Taylor, H.F. Cross, K. Bilo, Inflammatory responses induced by the filarial nematode Brugia malayi are mediated by lipopolysaccharide-like activity from endosymbiotic Wolbachia bacteria, J. Exp. Med. 191 (2000) 1429–1436. N.W. Brattig, U. Rathjens, M. Ernst, F. Geisinger, A. Renz, F.W. Tischendorf, Lipopolysaccharide-like molecules derived from Wolbachia endobacteria of the filaria Onchocerca volvulus are candidate mediators in the sequence of inflammatory and antiinflammatory responses of human monocytes, Microbes Infect. 2 (2000) 1147– 1157. N.W. Brattig, D.W. Büttner, A. Hoerauf, Neutrophil accumulation around Onchocerca worms and chemotaxis of neutrophils are dependent on Wolbachia endobacteria, Microbes Infect. 3 (2001) 439–446. P.B. Keiser, S.M. Reynolds, K. Awadzi, E.A. Ottesen, M.J. Taylor, T.B. Nutman, Bacterial endosymbionts of Onchocerca volvulus in the pathogenesis of posttreatment reactions, J. Infect. Dis. 185 (2002) 805–811. A. Saint André, N.M. Blackwell, L.R. Hall, A. Hoerauf, N.W. Brattig, L. Volkmann, M.J. Taylor, L. Ford, A.G. Hise, J.H. Lass, E. Diaconu, E. Pearlman, The role of endosymbiotic Wolbachia bacteria in the pathogenesis of river blindness, Science 295 (2002) 1892–1895. C. Bazzocchi, F. Ceciliani, J.W. McCall, I. Ricci, C. Genchi, C. Bandi, Antigenic role of the endosymbionts of filarial nematodes: IgG response against the Wolbachia surface protein in cats infected with Dirofilaria immitis, Proc. R. Soc. Lond. B. Biol. Sci. 267 (2000) 2511–2516.

[24] P. Fischer, I. Bonow, D.W. Büttner, I.H. Kamal, E. Liebau, An aspartate aminotransferase of Wolbachia endobacteria from Onchocerca volvulus is recognized by IgG1 antibodies from residents of endemic areas, Parasitol. Res. 90 (2003) 38–47. [25] W. Tawe, E. Pearlman, T.R. Unnasch, S. Lustigman, Angiogenic activity of Onchocerca volvulus recombinant proteins similar to vespid venom antigen 5, Mol. Biochem. Parasitol. 109 (2000) 91–99. [26] J.E. Bradley, N. Nirmalan, S.L. Klager, H. Faulkner, M.W. Kennedy, River blindness: a role for parasite retinoid-binding proteins in the generation of pathology? Trends Parasitol. 17 (2001) 471–475. [27] S. Korten, G. Wildenburg, K. Darge, D.W. Büttner, Mast cells in onchocercomas from patients with hyperreactive onchocerciasis (sowda), Acta Trop. 70 (1998) 217–231. [28] H.P. Duerr, K. Dietz, D.W. Büttner, H. Schulz-Key, A stochastic model for the aggregation of Onchocerca volvulus in nodules, Parasitology 123 (2001) 193–201. [29] M.E. Murdoch, A. Payton, A. Abiose, W. Thomson, V.K. Panicker, P.A. Dyer, B.R. Jones, R.M. Maizels, W.E. Ollier, HLA-DQ alleles associate with cutaneous features of onchocerciasis. The Kaduna– London–Manchester Collaboration for Research on Onchocerciasis, Hum. Immunol. 55 (1997) 46–52. [30] M.E. Murdoch, R.J. Hay, C.D. Mackenzie, J.F. Williams, H.W. Ghalib, S. Cousens, A. Abiose, B.R. Jones, A clinical classification and grading system of the cutaneous changes in onchocerciasis, Br. J. Dermatol. 129 (1993) 260–269. [31] A. Haffner, A.Z. Guilavogui, F.W. Tischendorf, N.W. Brattig, Onchocerca volvulus: microfilariae secrete elastinolytic and males nonelastinolytic matrix-degrading serine and metalloproteases, Exp. Parasitol. 90 (1998) 26–33. [32] M.M. Ali, O.Z. Baraka, S.I. AbdelRahman, S.M. Sulaiman, J.F. Williams, M.M. Homeida, C.D. Mackenzie, Immune responses directed against microfilariae correlate with severity of clinical onchodermatitis and treatment history, J. Infect. Dis. 187 (2003) 714–717. [33] C. Timmann, R. Abraha, C. Hamelmann, D.W. Büttner, B. Lepping, Y. Marfo, N. Brattig, R.D. Horstmann, Cutaneous pathology in onchocerciasis associated with pronounced systemic Th2 responses to Onchocerca volvulus, Br. J. Dermatol. 149 (2003) 782–787. [34] H. Francis, K. Awadzi, E.A. Ottesen, The Mazzotti reaction following treatment of onchocerciasis with diethylcarbamazine: clinical severity as a function of infection intensity, Am. J. Trop. Med. Hyg. 34 (1985) 529–536. [35] S.J. Ackerman, G.M. Kephart, H. Francis, K. Awadzi, G.J. Gleich, E.A. Ottesen, Eosinophil degranulation. An immunologic determinant in the pathogenesis of the Mazzotti reaction in human onchocerciasis, J. Immunol. 144 (1990) 3961–3969. [36] F.W. Tischendorf, N.W. Brattig, D.W. Büttner, A. Pieper, M. Lintzel, Serum levels of eosinophil cationic protein, eosinophil-derived neurotoxin and myeloperoxidase in infections with filariae and schistosomes, Acta Trop. 62 (1996) 171–182. [37] A.P. Limaye, J.S. Abrams, J.E. Silver, K. Awadzi, H.F. Francis, E.A. Ottesen, T.B. Nutman, Interleukin-5 and the posttreatment eosinophilia in patients with onchocerciasis, J. Clin. Invest. 88 (1991) 1418–1421. [38] P. Racz, K. Tenner-Racz, B. Luther, D.W. Büttner, E.J. Albiez, Immunopathologic aspects in human onchocercal lymphadenitis, Bull. Soc. Pathol. Exot. Filiales 76 (1983) 676–680. [39] WHO, Expert Committee on Onchocerciasis. Third report, WHO Tech. Rep. Ser. 752 (1987) 1–167. [40] M.Y. Gallin, D. Murray, J.H. Lass, H.E. Grossniklaus, B.M. Greene, Experimental interstitial keratitis induced by Onchocerca volvulus antigens, Arch. Ophthalmol. 106 (1988) 1447–1452. [41] E. Pearlman, L.R. Hall, Immune mechanisms in Onchocerca volvulus-mediated corneal disease (river blindness), Parasite Immunol. 22 (2000) 625–631. [42] E.A. Ottesen, Immune responsiveness and the pathogenesis of human onchocerciasis, J. Infect. Dis. 171 (1995) 659–671.

N.W. Brattig / Microbes and Infection 6 (2004) 113–128 [43] A. Hoerauf, N.W. Brattig, Resistance and susceptibility in human onchocerciasis—beyond Th1 vs. Th2, Trends Parasitol. 18 (2002) 25–31. [44] S. Lustigman, E.R. James, W. Tawe, D. Abraham, Towards a recombinant antigen vaccine against Onchocerca volvulus, Trends Parasitol. 18 (2002) 135–141. [45] D. Abraham, R. Lucius, A.J. Trees, Immunity to Onchocerca spp. in animal hosts, Trends Parasitol. 18 (2002) 164–171. [46] T.H. Dafa’alla, H.W. Ghalib, A. Abdelmageed, J.F. Williams, The profile of IgG and IgG subclasses of onchocerciasis patients, Clin. Exp. Immunol. 88 (1992) 258–263. [47] O. Garraud, C. Nkenfou, J.E. Bradley, F.B. Perler, T.B. Nutman, Identification of recombinant filarial proteins capable of inducing polyclonal and antigen-specific IgE and IgG4 antibodies, J. Immunol. 155 (1995) 1316–1325. [48] R. Hussain, R.W. Poindexter, E.A. Ottesen, Control of allergic reactivity in human filariasis. Predominant localization of blocking antibody to the IgG4 subclass, J. Immunol. 148 (1992) 2731–2737. [49] E. Lobos, R. Zahn, N. Weiss, T.B. Nutman, A major allergen of lymphatic filarial nematodes is a parasite homolog of the gammaglutamyl transpeptidase, Mol. Med. 2 (1996) 712–724. [50] M.T. Rubio de Krömer, C.E. Medina-De la Garza, N.W. Brattig, Differences in eosinophil and neutrophil chemotactic responses in sowda and generalized form of onchocerciasis, Acta Trop. 60 (1995) 21–33. [51] D.W. Büttner, G. von Laer, E. Mannweiler, M. Büttner, Clinical, parasitological and serological studies on onchocerciasis in theYemen Arab Republic, Tropenmed. Parasitol. 33 (1982) 201–212. [52] C.D. Mackenzie, J.F. Williams, B.M. Sisley, M.W. Steward, J. O’Day, Variations in host responses and the pathogenesis of human onchocerciasis, Rev. Infect. Dis. 7 (1985) 802–808. [53] N.W. Brattig, F.W. Tischendorf, G. Strote, C.E. Medina-de la Garza, Eosinophil–larval-interaction in onchocerciasis: heterogeneity of in vitro adherence of eosinophils to infective third and fourth stage larvae and microfilariae of Onchocerca volvulus, Parasite Immunol. 13 (1991) 13–22. [54] S.G. Folkard, P.J. Hogarth, M.J. Taylor, A.E. Bianco, Eosinophils are the major effector cells of immunity to microfilariae in a mouse model of onchocerciasis, Parasitology 112 (1996) 323–329. [55] N.W. Brattig, I. Krawietz, A.Z. Abakar, K.D. Erttmann, T.F. Kruppa, A. Massougbodji, Strong IgG isotypic antibody response in sowdah type onchocerciasis, J. Infect. Dis. 170 (1994) 955–961. [56] G.D. Burchard, N.W. Brattig, T.F. Kruppa, R.D. Horstmann, Delayedtype hypersensitivity reactions to Onchocerca volvulus antigens in exposed and non-exposed African individuals, Trans. R. Soc. Trop. Med. Hyg. 93 (1999) 103–105. [57] N.W. Brattig, C. Nietz, S. Hounkpatin, R. Lucius, F. Seeber, U. Pichlmeier, T. Pogonka, Differences in cytokine responses to Onchocerca volvulus extract and recombinant Ov33 and OvL3-1 proteins in exposed subjects with various parasitologic and clinical states, J. Infect. Dis. 176 (1997) 838–842. [58] M.Y. Gallin, A.B. Jacobi, D.W. Büttner, O. Schonberger, T. Marti, K.D. Erttmann, Human autoantibody to defensin: disease association with hyperreactive onchocerciasis (sowda), J. Exp. Med. 182 (1995) 41–47. [59] P.T. Soboslay, S.M. Geiger, N. Weiss, M. Banla, C.G. Lüder, C.M. Dreweck, E. Batchassi, B.A. Boatin, A. Stadler, H. Schulz-Key, The diverse expression of immunity in humans at distinct states of Onchocerca volvulus infection, Immunology 90 (1997) 592–599. [60] L.H. Elson, H.M. Calvopina, Y.W. Paredes, N.E. Araujo, J.E. Bradley, R.H. Guderian, T.B. Nutman, Immunity to onchocerciasis: putative immune persons produce a Th1-like response to Onchocerca volvulus, J. Infect. Dis. 171 (1995) 652–658. [61] C. Steel, T.B. Nutman, Regulation of IL-5 in onchocerciasis. A critical role for IL-2, J. Immunol. 150 (1993) 5511–5518.

127

[62] P.S. Turaga, T.J. Tierney, K.E. Bennett, M.C. McCarthy, S.C. Simonek, P.A. Enyong, D.W. Moukatte, S. Lustigman, Immunity to onchocerciasis: cells from putatively immune individuals produce enhanced levels of interleukin-5, gamma interferon, and granulocytemacrophage colony-stimulating factor in response to Onchocerca volvulus larval and male worm antigens, Infect. Immun. 68 (2000) 1905–1911. [63] N.W. Brattig, B. Lepping, C. Timmann, D.W. Büttner, Y. Marfo, C. Hamelmann, R.D. Horstmann, Onchocerca volvulus-exposed persons fail to produce interferon-gamma in response to O. volvulus antigen but mount proliferative responses with interleukin-5 and IL-13 production that decrease with increasing microfilarial density, J. Infect. Dis. 185 (2002) 1148–1154. [64] L. Volkmann, O. Bain, M. Saeftel, S. Specht, K. Fischer, F. Brombacher, K.I. Matthaei, A. Hoerauf, Murine filariasis: interleukin 4 and interleukin 5 lead to containment of different worm developmental stages, Med. Microbiol. Immunol. (Berlin) 192 (2003) 23–31. [65] C.G. Meyer, M. Gallin, K.D. Erttmann, N. Brattig, L. Schnittger, A. Gelhaus, E. Tannich, A.B. Begovich, H.A. Erlich, R.D. Horstmann, HLA-D alleles associated with generalized disease, localized disease, and putative immunity in Onchocerca volvulus infection, Proc. Natl. Acad. Sci. USA 91 (1994) 7515–7519. [66] A. Hoerauf, S. Kruse, N.W. Brattig, A. Heinzmann, B. MuellerMyhsok, K.A. Deichmann, The variant Arg110Gln of human IL-13 is associated with an immunologically hyper-reactive form of onchocerciasis (sowda), Microbes Infect. 4 (2002) 37–42. [67] T.F. Kruppa, G.D. Burchard, Similar blackfly attraction by onchocerciasis patients and individuals putatively immune to Onchocerca volvulus, Trans. R. Soc. Trop. Med. Hyg. 93 (1999) 365–367. [68] R.M. Maizels, A. Kurniawan-Atmadja, Variation and polymorphism in helminth parasites, Parasitology 125 (Suppl) (2002) S25–S37. [69] K. Takeda, T. Kaisho, S. Akira, Toll-like receptors, Annu. Rev. Immunol. 21 (2003) 335–376. [70] K. Ozato, H. Tsujimura, T. Tamura, Toll-like receptor signaling and regulation of cytokine gene expression in the immune system, Biotech. Suppl. 70 (2002) 66–68. [71] D. van der Kleij, E. Latz, J.F. Brouwers, Y.C. Kruize, M. Schmitz, E.A. Kurt-Jones, T. Espevik, E.C. de Jong, M.L. Kapsenberg, D.T. Golenbock, A.G. Tielens, M. Yazdanbakhsh, A novel host– parasite lipid cross-talk. Schistosomal lyso-phosphatidylserine activates toll-like receptor 2 and affects immune polarization, J. Biol. Chem. 277 (2002) 48122–48129. [72] M. Whelan, M.M. Harnett, K.M. Houston, V. Patel, W. Harnett, K.P. Rigley, A filarial nematode-secreted product signals dendritic cells to acquire a phenotype that drives development of Th2 cells, J. Immunol. 164 (2000) 6453–6460. [73] P.J. Cooper, T. Mancero, M. Espinel, C. Sandoval, R. Lovato, R.H. Guderian, T.B. Nutman, Early human infection with Onchocerca volvulus is associated with an enhanced parasite-specific cellular immune response, J. Infect. Dis. 183 (2001) 1662–1668. [74] P.T. Soboslay, C.M. Dreweck, H.R. Taylor, B. Brotman, P. Wenk, B.M. Greene, Experimental onchocerciasis in chimpanzees. Cellmediated immune responses, and production and effects of IL-1 and IL-2 with Onchocerca volvulus infection, J. Immunol. 147 (1991) 346–353. [75] S.P. Graham, A.J. Trees, R.A. Collins, D.M. Moore, F.M. Guy, M.J. Taylor, A.E. Bianco, Down-regulated lymphoproliferation coincides with parasite maturation and with the collapse of both gamma interferon and interleukin-4 responses in a bovine model of onchocerciasis, Infect. Immun. 69 (2001) 4313–4319. [76] C.L. King, Transmission intensity and human immune responses to lymphatic filariasis, Parasite Immunol. 23 (2001) 363–371. [77] S. Mahanty, S.N. Mollis, M. Ravichandran, J.S. Abrams, V. Kumaraswami, K. Jayaraman, E.A. Ottesen, T.B. Nutman, High levels of spontaneous and parasite antigen-driven interleukin-10 production are associated with antigen-specific hyporesponsiveness in human lymphatic filariasis, J. Infect. Dis. 173 (1996) 769–773.

128


[78] K.W. Moore, R. de Waal Malefyt, R.L. Coffman, A. O’Garra, Interleukin-10 and the interleukin-10 receptor, Annu. Rev. Immunol. 19 (2001) 683–765. [79] J. Satoguina, M. Mempel, J. Larbi, M. Badusche, C. Loliger, O. Adjei, G. Gachelin, B. Fleischer, A. Hoerauf, Antigen-specific T regulatory-1 cells are associated with immunosuppression in a chronic helminth infection (onchocerciasis), Microbes Infect. 4 (2002) 1291–1300. [80] W.H. Hoffmann, A.W. Pfaff, H. Schulz-Key, P.T. Soboslay, Determinants for resistance and susceptibility to microfilaraemia in Litomosoides sigmodontis filariasis, Parasitology 122 (2001) 641–649. [81] P.T. Soboslay, C.M. Dreweck, W.H. Hoffmann, C.G. Lüder, C. Heuschkel, H. Gorgen, M. Banla, H. Schulz-Key, Ivermectin-facilitated immunity in onchocerciasis. Reversal of lymphocytopenia, cellular anergy and deficient cytokine production after single treatment, Clin. Exp. Immunol. 89 (1992) 407–413. [82] L.H. Elson, A. Days, M. Calvopina, W. Paredes, E. Araujo, R.H. Guderian, J.E. Bradley, T.B. Nutman, In utero exposure to Onchocerca volvulus: relationship to subsequent infection intensity and cellular immune responsiveness, Infect. Immun. 64 (1996) 5061–5065. [83] C. Steel, T.B. Nutman, CTLA-4 in filarial infections: implications for a role in diminished T cell reactivity, J. Immunol. 170 (2003) 1930– 1938. [84] M. Yazdanbakhsh, P.G. Kremsner, R. van Ree, Allergy, parasites, and the hygiene hypothesis, Science 296 (2002) 490–494. [85] C.A. Akdis, K. Blaser, Role of IL-10 in allergen-specific immunotherapy and normal response to allergens, Microbes Infect. 11 (2001) 891–898. [86] J.E. Allen, P. Loke, Divergent roles for macrophages in lymphatic filariasis, Parasite Immunol. 23 (2001) 345–352. [87] P. Loke, M.G. Nair, J. Parkinson, D. Guiliano, M. Blaxter, J.E. Allen, IL-4 dependent alternatively-activated macrophages have a distinctive in vivo gene expression phenotype, BMC Immunol. 3 (2002) 7–17. [88] S. Gordon, Alternative activation of macrophages, Nat. Rev. 3 (2003) 23–35. [89] A.J. MacDonald, P.S. Turaga, C. Harmon-Brown, T.J. Tierney, K.E. Bennett, M.C. McCarthy, S.C. Simonek, P.A. Enyong, D.W. Moukatte, S. Lustigman, Differential cytokine and antibody responses to adult and larval stages of Onchocerca volvulus consistent with the development of concomitant immunity, Infect. Immun. 70 (2002) 2796–2804. [90] X. Zang, A.K. Atmadja, P. Gray, J.E. Allen, C.A. Gray, R.A. Lawrence, M. Yazdanbakhsh, R.M. Maizels, The serpin secreted by Brugia malayi microfilariae, Bm-SPN-2, elicits strong, but shortlived, immune responses in mice and humans, J. Immunol. 165 (2000) 5161–5169. [91] E.H. Johnson, M. Irvine, P.H. Kass, J. Browne, M. Abdullai, A.M. Prince, S. Lustigman, Onchocerca volvulus: in vitro cytotoxic effects of human neutrophils and serum on third-stage larvae, Trop. Med. Parasitol. 45 (1994) 331–335.

[92] M. Haarbrink, G.K. Abadi, W.A. Buurman, M.A. Dentener, A.J. Terhell, M. Yazdanbakhsh, Strong association of interleukin-6 and lipopolysaccharide-binding protein with severity of adverse reactions after diethylcarbamazine treatment of microfilaremic patients, J. Infect. Dis. 182 (2000) 564–569. [93] H.F. Cross, M. Haarbrink, G. Egerton, M. Yazdanbakhsh, M.J. Taylor, Severe reactions to filarial chemotherapy and release of Wolbachia endosymbionts into blood, Lancet 358 (2001) 1873–1875. [94] K.M. Pfarr, K. Fischer, A. Hoerauf, Involvement of Toll-like receptor 4 in the embryogenesis of the rodent filaria Litomosoides sigmodontis, Med. Microbiol. Immunol. (Berlin) 192 (2003) 53–56. [95] A. Jolodar, P. Fischer, D.W. Büttner, N.W. Brattig, Wolbachia endosymbionts of Onchocerca volvulus express a putative periplasmic HtrA-type serine protease, Microbes Infect., 6 (2004) in press. [96] S. Varadaradjalou, F. Féger, N. Thieblemont, N.B. Hamouda, J.-M. Pleau, M. Dy, M. Arock, Toll-like receptor 2 (TLR2) and TLR4 differentially activate human mast cells, Eur. J. Immunol. 3 (2003) 899–906. [97] R.M. Maizels, N. Gomez-Escobar, W.F. Gregory, J. Murray, X. Zang, Immune evasion genes from filarial nematodes, Int. J. Parasitol. 31 (2001) 889–898. [98] R.M. Maizels, M.L. Blaxter, A.L. Scott, Immunological genomics of Brugia malayi: filarial genes implicated in immune evasion and protective immunity, Parasite Immunol. 23 (2001) 327–344. [99] G. Wildenburg, E. Liebau, K. Henkle-Dührsen, Onchocerca volvulus: ultrastructural localization of two glutathione S-transferases, Exp. Parasitol. 88 (1998) 34–42. [100] K. Henkle-Dührsen, A. Kampkotter, Antioxidant enzyme families in parasitic nematodes, Mol. Biochem. Parasitol. 114 (2001) 129–142. [101] A. Schönemeyer, R. Lucius, B. Sonnenburg, N. Brattig, R. Sabat, K. Schilling, J. Bradley, S. Hartmann, Modulation of human T cell responses and macrophage functions by onchocystatin, a secreted protein of the filarial nematode Onchocerca volvulus, J. Immunol. 167 (2001) 3207–3215. [102] S. Hartmann, R. Lucius, Modulation of host immune responses by nematode cystatins, Int. J. Parasitol. 33 (2003) 1291–1302. [103] J.L. Mpagi, K.D. Erttmann, N.W. Brattig, The secretory Onchocerca volvulus protein OvS1/Ov20 exhibits the capacity to compete with serum albumin for the host’s long-chain fatty acids, Mol. Biochem. Parasitol. 105 (2000) 273–279. [104] A.R. Mawson, M. WaKabongo, Onchocerciasis-associated morbidity: hypothesis, Trans. R. Soc. Trop. Med. Hyg. 96 (2002) 541–542. [105] W. Harnett, M.R. Deehan, K.M. Houston, M.M. Harnett, Immunomodulatory properties of a phosphorylcholine-containing secreted filarial glycoprotein, Parasite Immunol. 21 (1999) 601–608. [106] D.V. Pastrana, N. Raghavan, P. FitzGerald, S.W. Eisinger, C. Metz, R. Bucala, R.P. Schleimer, C. Bickel, A.L. Scott, Filarial nematode parasites secrete a homologue of the human cytokine macrophage migration inhibitory factor, Infect. Immun. 66 (1998) 5955–5963. [107] N. Gomez-Escobar, E. Lewis, R.M. Maizels, A novel member of the transforming growth factor-beta (TGF-beta) superfamily from the filarial nematodes Brugia malayi and B. pahangi, Exp. Parasitol. 88 (1998) 200–209.