Cystic Echinococcosis: Aspects of Immune Response, Immunopathogenesis and Immune Evasion from the Human Host

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1 16 Endocrine, Metabolic & Immune Disorders - Drug Targets, 2012, 12, Cystic Echinococcosis: Aspects of Immune Response, Immunopathogenesis and Immune Evasion from the Human Host Alessandra Siracusano 1,*, Federica Delunardo 2, Antonella Teggi 3 and Elena Ortona 2 1 Dipartimento di Malattie Infettive, Parassitarie ed Immunomediate, Istituto Superiore di Sanità, Viale Regina Elena 299, Roma; 2 Dipartimento di Biologia Cellulare e Neuroscienze, Istituto Superiore di Sanità, Viale Regina Elena 299, Roma; 3 Dipartimento di Malattie Infettive e Tropicali, Ospedale Sant Andrea, Università di Roma, Sapienza, Via di Grottarossa 1035, Roma Abstract: Cystic echinococcosis (CE) is a neglected infectious disease caused by the larval stage of Echinococcus granulosus. It constitutes a major public health problem in developing countries. During CE, the distinguishing feature of the host-parasite relationship is that chronic infection coexists with detectable humoral and cellular responses against the parasite. In order to establish successfully an infection, E. granulosus releases molecules that directly modulate the host immune responses favoring a strong anti-inflammatory response and perpetuating parasite survival in the host. In vitro and in vivo immunological approaches, together with molecular biology and immunoproteomic technologies provided us exciting insights into the mechanisms involved in the initiation of E. granulosus infection and the consequent induction and regulation of the immune response. Here, we review some of the recent developments and discuss how these observations helped to understand the immunology of E. granulosus infection in man. Although the last decade has clarified many aspects of host-relationship in human CE, establishing the full mechanisms that cause the disease require more studies. We need to define more clearly the events that manipulate the host immune response to protect the E. granulosus from elimination and minimizing severe pathology in the host. Keywords: Antigens, Echinococcus granulosus, immune evasion, immune response, immunomodulating molecules. INTRODUCTION Cystic echinococcosis (CE) is a human parasitic disease, caused by the larval stage of Echinococcus granulosus. Echinococcus cysts were known to Hippocrates, who spoken of livers full of water and mentioned the serious manifestations and consequences of rupture of hydatid cysts of the liver. The word echinococcus is of Greek origin and means hedgehog berry. Hydatid is also of Greek origin (hudatid, hudatis) and means a watery vesicle. The word hydatid also originates from the modern Latin word hydatis meaning a drop of water. Therefore, hydatid and cyst have the same meaning and the expression hydatid cyst is a pleonasm. The use of the term hydatid cyst in contemporary medicine is widespread and acceptable. A recent review clarifies some aspects concerning the hydatid cyst s nomenclature [1]. The persistence of these cysts is of interest to immunologists since, once fully formed, they are apparently unaffected by the host s immune response. The host parasite relationship is interactive and the outcome of infection depends on the balance achieved by the combination of the different variables involved with the host immunity and the parasite avoidance strategies. An understanding of the biological events occurring during infection is necessary to visualize the diverse immune stimuli to which the host is subjected and to define diagnostic and therapeutic tools. *Address correspondence to this author at the Dipartimento di Malattie Infettive, Parassitarie ed Immunomediate, Istituto Superiore di Sanità, Viale Regina Elena 299, Roma; Tel: ; Fax: ; alessandra.siracusano@iss.it - 3/12 $ This review highlights immunological advances, conducted in our laboratory, in immunity to infection and molecular and proteomic approaches in characterization of new immunomodulatory molecules that E. granulosus produces to exploit host immunity. Brief Notes on the Control of the Infection CE occurs worldwide; it affects primarily the rural areas and causes serious human morbidity due to the effect of the parasite larvae in the host [2, 3]. Recently, the World Health Organization included echinococcosis as part of a Neglected Zoonosis subgroup for its strategic plans for the control of neglected tropical diseases [4]. This disease is not of great concern to provincial, national or regional authorities within endemic zones for several causes. First, human echinococcosis is a non vector-borne zoonosis, which is not transmitted between humans, with animal host reservoirs (livestock, dogs, or wildlife), that is not amenable to vector-based control nor to direct human-treatment approaches for case prevention, unlike say schistosomiasis, filariasis or malaria. Other reasons include: a) the disease in humans is chronic with very long asymptomatic periods so that health authorities fail to properly recognize the negative health impacts; b) the detection or diagnosis in humans is hard without access to expensive tools (e.g. ultrasound, Computerized Tomography Scan, serological tests); c) the treatment is very difficult and not always very effective (surgery or drugs); d) the occurrence is elevated in poor, often remote marginalized pastoral societies that may be ethnically/socio-culturally isolated from the general population; e) the burden of CE is therefore difficult to 2012 Bentham Science Publishers

2 The Host-Parasite Interplay in Human Cystic Echinococcosis Endocrine, Metabolic & Immune Disorders - Drug Targets, 2012, Vol. 12, No quantify; f) the disease is chronic in domestic animals so that livestock owners fail to recognize CE as an animal health or economic problem; g) dogs, that are the main carrier and spreader of the parasite, are asymptomatic. Finally, the control of CE is difficult and exacerbated by the requirement of cooperation between agricultural/veterinary services and medical authorities [5]. The Life Cycle and the Biology of the Hydatid Cyst The complex cycle of the parasite can explain the intricate host-parasite relationship. E. granulosus is a small tapeworm (rarely exceeding 7 mm in length) that lives firmly attached to the mucosa of the small intestine in definitive hosts, usually dogs, where the adult-stage reaches sexual maturity within 4 to 5 weeks. This is followed by the shedding of gravid proglottids (each containing several hundred eggs) and/or of released eggs in the feces of definitive hosts. Following ingestion of eggs by intermediate host (herbivorous species, rodents and humans) the oncosphere by lytic secretions goes through the intestinal mucosa and into the host circulatory system (venous and lymphatic) and reaches the liver, lungs, and other sites where cystic development begins. This process involves transformation of the oncospheral stage to reach the metacestode stage. In vitro 4-7 days are required for larvae to change into a typical bladder with a germinal layer. Subsequently, development time varies widely from one host species to another. In general, hydatid cysts increase in diameter from less than 1 to 5 cm each year. At the locations slow growing hydatid cysts develop, which may not be detected for months or years after the initial infection has occurred because the fate of the hydatid cysts is variable. The natural history of E. granulosus cysts and its clinical implications comprises various developmental stages. The initial stage, primary infection, is always asymptomatic. During this stage, small (<5 cm) well-encapsulated cysts develop in organ sites, where they induce no pathologic consequences, thus persisting without a noticeable. In humans, the hydatid cysts are localized in approximately two-thirds of cases in the liver and in about 20% in the lungs and less frequently in the kidneys, spleen, heart, and bone. Some 20-40% of patients have multiple cysts or multiple organ involvement. After an undefined incubation period lasting months or years, if cysts exert pressure on adjacent tissue and induce other pathologic events, the infection may became symptomatic. Cysts can also resolve spontaneously for various reasons, for example, when the cysts collapse, calcify or break into the bile duct or the bronchial tree thus discharging their content. Human hosts often tolerate a slowly growing hydatid cyst remarkably well. Patients with CE may come to clinical attention only when a large cyst mechanically alters body function; when allergic phenomena or other miscellaneous symptoms such as eosinophilia develop; or when the cyst accidentally ruptures thus triggering acute hypersensitivity reactions. Cysts or a cystic mass may also be discovered by chance during body scanning or surgery, or for other clinical complications [6]. The mature hydatid cyst consists of an inner germinal layer of cells supported externally by an acellular-laminated membrane of variable thickness. Tegumental cells of the germinal layer form a continuous syncytium that differentiates into numerous microvilli that project peripherally into the laminar layer toward the host tissues surrounding the hydatid cyst. Small secondary cysts called brood capsules bud internally from the germinative layer and, by asexual reproduction, produce multiple protoscoleces that can differentiate either into adult worms in the intestines of definitive hosts either into secondary hydatid cysts following rupture of a cyst in the intermediate hosts. In intermediate hosts, protoscoleces develop exclusively in fertile cysts. In bovine infected with E. granulosus the cystic form induces IgGs that cross the tegument and plasma membrane present between the laminar and the germinal layers of the cyst. In this last structure, IgGs recognize specific antigens involved in the cell proliferation process and/or in the differentiation mechanisms leading to protoscolex formation. This antigen-antibody interaction may inhibit cell proliferation and/or cell differentiation involved in the formation of buds and protoscoleces and may induce apoptosis leading to cyst infertility [7]. The hydatid liquid is clean and clear, as well as the clean water from its natural source, containing secretions from both the parasite and host and all the elements from the inner wall of the cyst, named hydatid sand [1]. It has an identical composition to that of the host s serum (Na, K, Cl, CO 2, a density between and 1.015, alkaline ph) and some proteins that confer antigenic properties. Through the slow evolution of a cyst, several events can occur into the cyst: the death of the parasite due to dysfunction of the germinal membrane (detachment or aging), the cyst s wall fissure due to detachment of membranes or micro traumatisms, the transformation of scoleces into vesicles (vesiculation) attempting to preserve the specie. These new vesicles, called offspring or daughter vesicles, live into the hydatid fluid and have the same constitution as well the same mission of the vesicle mother. Therefore, in this way, protoscoleces may evolute into either a new cyst or an adult parasite. The hydatid fluid is the main factor responsible for the antigenic stimulation, but the germinal layer of the cyst is a barrier against immune competent cells of the host. Therefore, it is necessary to have damages in the germinal layer, like fissures or rupture, to get an antigenic stimulation. When this antigenic stimulation occurs, there is a continuous elevation of the immunologic values for an indeterminate time [1]. This elevation also happens after the cyst manipulation (surgery, puncture, etc.). In addition to this physical barrier, the parasite has probably evolved other immune evasion strategies. The long-term survival of the hydatid indicates the existence of protection mechanisms against host immune response. Host-parasite Relationship During parasite infection, the human host utilizes several innate and acquired protective mechanisms. However, not all of these responses are protective. In fact, defensive immunity is the exception rather than the rule after a specific parasitic infection. Successful survival of parasites has dictated that parasites have developed several mechanisms of evasion from host protective mechanisms to ensure their propagation. Either parasites evade their host s immune responses by simple mechanisms such as intracellular location or by changing their antigenic structure or altering the host

3 18 Endocrine, Metabolic & Immune Disorders - Drug Targets, 2012, Vol. 12, No. 1 Siracusano et al. responses in a way that favors their survival [8]. During CE, the distinguishing feature of the host-parasite relationship is that chronic infection coexists with detectable humoral and cellular responses against the parasite. Because of the expression of different antigens during the various evolution stages, the human host responds independently to antigenic stimuli of the invading oncosphere, the metacestode in transformation from the oncosphere, and finally, the mature metacestode (larva) [9]. E. granulosus, could use two mechanisms to subvert the host immune response: passive escape, in which the parasite, by developing into a hydatid cyst, avoids the damaging effects of an immune response, and immunomodulation, through which the parasite actively interacts with the host immune system to reduce the impact of a host response [10]. Human clinical research is complementary to animal studies for understanding host-parasite interactions better and is necessary for designing new therapeutic approaches to CE [10-12]. Circulating Antibodies as Immunological Marker During CE Although E. granulosus induces a strong humoral response in humans and sera from individuals with CE contain abundant circulating IgG, IgM, IgA and IgE antibodies to E. granulosus antigens, none of these antibodies is associated with protection [13]. Contrary to the detection of specific antibodies that represents the confirmatory diagnostic test in combination with ultrasound, the analysis of IgG subclass, that vary during the outcome of the disease, has proved useful in follow-up [14]. Because IgG4, a subclass normally associated with prolonged, chronic infections, is neither cytophilic nor complement fixing, is non functional, and binds weakly to receptors for the Fc portion of immunoglobulins, it may help the parasite to evade the host immune response [15]. Moreover, parasitespecific IgG4 antibodies can inhibit IgE-mediated degranulation of effector cells reducing allergic pathology in the host [16]. Given that the first studies of IgG subclass antibody responses in advanced human CE indicated a switch from predominant IgG1 response to IgG4 in CE patients with progressive disease, the peculiar role of IgG4 during CE has been extensively studied and IgG4 actually are considered as immunological markers during CE [17]. In agreement with these studies, we found that albendazoletreated patients, who exhibited a good therapeutic and clinical response to treatment, had significantly lower levels of serum IgG4 antibodies, than poor responders or nonresponders; whereas IgG1 antibody levels showed a reverse trend [18, 19]. Later we confirmed the presence of higher IgG4 and IgE in patients with progressive disease and higher IgG1 and IgG3 in patients with stable disease [14]. Because these observations and data obtained from mass screening in various countries suggested a correlation of distinct IgG subclass antibody responses with the host-parasite relationship [20], we focused our study on the characterization of molecules involved in evading mechanisms. Before describing these molecules it is important to evaluate multiple factors, including the prevailing cytokine environment and changes in cytokine profiles, influencing the IgG subclass expression and the outcome of host-e. granulosus interactions. Cytokine Production and Immune Modulation by E. granulosus The immune response that occurs during an infectious disease is characterized by plasticity in both its nature and its magnitude. This feature provides an important advantage that permits the immune system to tailor its defense strategy to particular groups of infectious pathogens. Th1 and Th2 cells are not pre-committed phenotypes but rather, represent endpoints of a multistep differentiative process whereby a common precursor population acquires a distinct cytokine secretion profile [21]. A key question is how the distinct pathogens that encounter the immune system can influence this differentiation decision. During CE, the evidence concerning antibody levels of IgG4 and IgE isotypes and frequent eosinophilia, suggested that the immune response to established E. granulosus infection is Th2 dominated and that Echinococcus antigens modulate polarized T-cells. Immunological studies conducted in our laboratory, showing high in vitro production of parasite antigen-driven IL-4, IL- 5, IL-6, IL-10 and IFN- by PBMC isolated from patients with CE, confirmed that the human immune response to E. granulosus infection is predominantly regulated by Th2 cell activation but also by the Th1 (or Th0) cell subset [18, 19]. Data obtained in E. granulosus experimental infection supported the hypothesis that early IL-10, secreted by B cells in response to non-proteic antigens, may favor parasitesurvival and the establishment of a polarized type-2 cytokine response [22]. Recent findings suggested that IL-4/IL-10 impairs the Th1 protective response and allows the parasite to survive in hydatid patients [23]. Evidences underlining crucial role of cytokines in the host-parasite relationship come from studies on parasitedriven cytokine production in a large number of albendazoletreated patients with CE. PBMC from patients who responded to chemotherapy produced high amounts of IFN- (Th1-derived) whereas PBMC from patients who did not respond produced IL-4 and IL-10 (Th2-derived). We later confirmed this finding in a molecular study by detecting IL- 12 p40 mrna in 86% of successfully treated patients at the end of chemotherapy. PBMC from patients in whom therapy failed, expressed weakly IL-4 mrna before therapy, and strongly thereafter; PBMC from patients who responded to therapy expressed higher IFN- and TNF- mrna values than patients who did not [24]. Finally, T-cell-lines from a patient with an inactive cyst had a Th1 profile, whereas T- cell lines derived from patients with active and transitional cyst had mixed Th1/Th2 and Th0 clones [25]. In accordance with the influence of parasite antigens and defined cytokines on heavy chain immunoglobulin switching and the production of one or a few specific antibody isotypes, we found that PBMC from seronegative patients produced no parasite antigen driven-il-5 and scarce IL-4 and IL-10 [26]. We suggested that during CE the seronegativity occurs because host or parasite factors or both preclude Th2 cell activation thus limiting or preventing production of IL-5, the cytokine that has a critical role in immunoglobulin expression.

4 The Host-Parasite Interplay in Human Cystic Echinococcosis Endocrine, Metabolic & Immune Disorders - Drug Targets, 2012, Vol. 12, No Collectively our findings indicated that in CE a strong Th2 response correlates with susceptibility to disease (active cyst), whereas a Th1 response correlates with protective immunity (inactive cyst) and that Th1 and Th2 responses coexist. Recently the role of DCs in the immunity of CE and in the host-parasite relationship has been evaluated. Immature DCs are bone marrow-derived cells present in most nonlymphoid tissues, where they exhibit a potent capability to capture and process antigens. Inflammatory mediators or microbial agents promote the migration of DCs into the secondary lymphoid organs. As they migrate, DCs mature, lose their Ag-capture ability and gain an increased capacity to prime T cells. DC-parasite interactions are pivotal in triggering and regulating parasite-induced immunity. DC function is itself modulated during parasitic infection for the mutual benefit of the host and of the parasite [27, 28]. E. granulosus hydatid fluid modulates DC differentiation and cytokine secretion [29]. Recently, we have demonstrated that E. granulosus hydatid fluid impairs monocyte precursor differentiation into immature DCs rendering them unable to mature when stimulated with lipopolysaccharides. The parasite modulates also sentinel DC maturation, priming them to polarize lymphocytes into Th2 cells [30]. Collectively, these cellular findings establish that E. granulosus can directly influence the components of host cellular response, T lymphocytes and DCs. Because E. granulosus inhabits immunocompetent hosts for prolonged periods it is not surprising that it should possess modulator molecules that remodel host responses to enhance its survival. Immunomodulating Molecules: Role of Antigen B and of Other New Antigens In this section, we examine individual E. granulosus components, in the context of particular roles in immune evasion (Table 1). Of the various proteins isolated from hydatid fluid and characterized, the principal E. granulosus immunomodulant antigen is AgB [31]. Because it can modulate both innate and adaptive host immune responses, AgB plays a prominent role in the immunomodulatory mechanisms that E. granulosus uses to develop, progress and cause chronic disease [9]. To survive in host tissues the parasite must be able to adapt metabolically to the host microenvironment and plentiful AgB in hydatid cyst fluid probably guarantees parasite survival. AgB is a 160 kda thermostable lipoprotein that resists boiling for 15 min without losing antigenicity. AgB consists of a regularly spaced group of molecules with apparent molecular sizes of 8, 16, 24 and 32 kda that became asymptotically less numerous as their molecular size increased [32]. Molecular studies show that E. granulosus AgB is encoded by a multigene family having at least five gene loci (B1-B5), each consisting of several minor variants grouped into two clusters: EgAgB1/B3/B5 and EgAgB2/B4 [33-37]. In contrast with previous data, showing that E. granulosus strains differ in the types of genomic and transcribed EgAgB sequences, a recent study demonstrated that each of the genes is identical in both larval and adult E. granulosus isolates collected from two geographical areas (different continents) [38, 39]. The E. granulosus AgB gene family comprises at least 10 unique genes in five subclasses which are differentially expressed. In fact, DNA alignment comparisons with AgB sequences deposited in GenBank databases showed that each gene in the gene family is highly conserved within E. granulosus. Quantitative PCR analysis revealed that the genes were differentially expressed in different life-cycle stages of E. granulosus with EgAgB3 expressed predominantly in all stages. These findings are fundamental for determining the expression and the biological function of AgB. Table 1. Principal Echinococcus granulosus antigenic molecules identified and characterized. Antigen Name References antigen 5 Ag5, ex antigen 4 Capron et al., 1967, SIMEP, Lyon, antigen B AgB, ex antigen 5 Williams et al., 1971, Am. J. Trop. Med. Hyg., 20, Eg95 Eg95 Lightowlers et al., 1996, Parasite Immunol., 18, EgA31 EgA31 Fu et al., 1999, Mol. Biochem. Parasitol., 102, elongation factor 1 / EgEF-1 / Margutti et al., 1999, Parasite Immunol., 21, Cyclophilin EA21 Ortona et al., 2002, Clin. Exp. Immunol., 128, EpC1 EpC1 Li et al., 2003, J. Infect. Dis., 15, 188, Echinococcus granulosus heat shock protein 70 Eg2HSP70 Ortona et al., 2003, Parasite Immunol., 25, Echinococcus granulosus Tegumental antigen EgTeg Ortona et al., 2005, Clin. Exp. Immunol., 142, Echinococcus granulosus Thioredoxin peroxidase EgTPx Salinas et al., 1998, Exp. Parasitol., 90, Eg19 Eg19 Delunardo et al., 2010, Acta Trop., 113, Echinococcus granulosus heat shock protein HSP20 Vacirca et al., 2011, Parasite Immunol., 33, regg5 regg5 Li et al., 2004, Biol. Proced. Online, 6,

5 20 Endocrine, Metabolic & Immune Disorders - Drug Targets, 2012, Vol. 12, No. 1 Siracusano et al. Native AgB binds hydrophobic ligands as many proteins from other Cestodes. Because these proteins are immunogenic and some are involved in lipid detoxification, transport and metabolism with their fatty acid binding properties, AgB could be involved in the process of parasite survival in the host microenvironment [40]. How can AgB modulate host immune response? It acts directly on innate and acquired host-immunity. First, distinct studies showed that the 12 kda subunit of AgB is a serine protease inhibitor with strong chemoattractant activity and with the ability to inhibit human neutrophil chemotaxis without altering either random migration or oxidative metabolism [41, 42]. In agreement with the negative immunomodulatory role suggested for AgB on human neutrophils, when accidentally released hydatid fluid activates neutrophils, AgB could act as an interference antigen allowing the released protoscoleces to develop into secondary cysts [43]. Because AgB induces synthesis of specific IgE and IgG4 (Th2), we investigated the role of AgB in acquired immunity by evaluating AgB-driven Th1 and Th2 cytokine production by PBMC from patients with CE [42, 44]. Patients PBMC stimulated with AgB produced IL-4, IL-13 and low IFN- concentrations, but did not produce IL-12. This Th2 polarization was more evident in patients with active disease, in whom the stimulus with AgB increased the imbalance observed in cultures from patients with inactive disease. Then, AgB expresses distinct immunodominant T-cell epitopes able to stimulate each T-cell subset [25]. Finally, AgB modulates sentinel DC maturation, priming those to polarize lymphocytes into an exclusive Th2 response that benefits the parasite (IL-4 expression). Our data provide a rationale for this polarization by showing that if AgB encounters immature DCs, it suppress IL-12p70 production by inducing the immunoregulatory cytokine IL-10. AgB reduces lipopolysaccharide-induced production of IL-12p70 but not of IL-6, providing further evidence that it actively modulates DC responsiveness in a manner favouring a Th2 outcome [30]. All these data suggest that AgB directly immunomodulates the host immune response by inhibiting polymorphonuclear cell chemotaxis and indirectly by skewing the Th1:Th2 cytokine ratio towards a preferentially Th2 polarization associated with chronic CE disease. In a series of molecular studies designed to identify new allergenic targets in CE, we screened an E. granulosus cdna library with IgE from patients with CE who had acute cutaneous allergic manifestations and identified three conserved constitutive proteins: EgEF-1 /, EA21, and Eg2HSP70 [45-48]. Almost two of these proteins appear to have immunomodulant propriety. The EgEF-1 / intervenes in immunomodulation because continues to be released into the hydatid fluid after the protoscoleces degenerate; in fact, we found a higher percentage of antibodies specific against EgEF-1 / in patients with CE who had inactive cysts than in patients with active cysts [46]. Because we found that a high percentage of sera from patients with CE without allergic manifestations had IgG4 antibodies specific to EA21 whereas patients with allergic manifestations showed IgE specific to EA21 we suggested that in CE, as in other parasitic diseases, IgG4 apparently acts to block pathogenic processes, minimizing severe pathology in the host [47]. Regarding Eg2HSP70, this antigen seems to elicit IL-4 production not through its intrinsic ability but by strengthening, the generalized Th2 polarization previously established [45]. Later, we screened an E. granulosus cdna library with IgG4 from patients with active disease and with IgG1 from patients with inactive disease. By screening with IgG4 from patients with active disease, we obtained two proteins. The first is present on the protoscolex tegument and on the germinal layer of cyst wall (EgTeg) and the second protein has 19.0 kda (Eg19) [49, 50]. EgTeg is an immunomodulatory molecule that, as AgB, contributes to chronic infection by inhibiting chemotaxis and inducing IL-4-and IgG4 [49]. Regarding Eg19 reactivity, the percentage of total IgG, IgG1 and IgG4-positive sera were significantly higher in sera from patients with active disease and cyst in multiple sites than from patients with inactive disease and cyst in the liver. Because anti- Eg19 antibody concentration decreased over the course of treatment in sera from patients with cured disease, our data, confirming the presence of antigens inducing both IgG1 and IgG4 during active CE, suggest that Eg19 might be a marker of disease status [50]. By screening the E. granulosus cdna library with IgG1 from patients with inactive disease, we obtained a EgTPx. Because EgTPx seems to have an unclear role in immunomodulation, further researches are necessary to clarify precisely how EgTPx intervenes in immune evasion and whether anti-egtpx antibodies can be used to counteract larval survival and development [51]. Recently, seeking biomarkers reflecting disease development in cystic CE, we used a proteomic approach linked to immunologic characterization for the identification of new antigens. Two-dimensional gel electrophoresis (2- DE) of sheep hydatid fluid, followed by immunoblot analysis with sera from patients with distinct phases of disease, enabled us to identify by mass spectrometry HSP20 as a potential marker of active CE. Immunoblot analysis revealed anti-hsp20 antibodies in a statistically significant higher percentage of sera from patients with active disease than in sera from patients with inactive disease. Anti-HSP20 antibody levels significantly decreased over the course of pharmacological treatment in sera from patients with cured disease, relative to sera from patients with progressive disease [52]. This proteomic approach emphasizes the presence of a large number of antigenic proteins associate to parasite immunoevasion during the development of the disease and highlights the difficulty in understanding the host-parasite relationship. Recent studies identified by proteomic analysis and mass spectrometry numerous proteins that E. granulosus larval stage expresses and releases during intermediate host infection. The host proteins identified and the proteins produced by the parasite provide new insights into the intriguing host-parasite interactions. Further immunological studies should investigate the role and possibly the immunomodulatory effects of these proteins [53, 54].

6 The Host-Parasite Interplay in Human Cystic Echinococcosis Endocrine, Metabolic & Immune Disorders - Drug Targets, 2012, Vol. 12, No Table 2. In vitro and in vivo immunoregulatory effect of Echinococcus granulosus on human host immune responses to parasite invasion. In Vitro Antigen Cell Population Action Effect AgB, EgTeg Neutrophils Chemotaxis inhibition, H 2O 2 reduction Immune evasion AgB Monocytes Impaired differentiation (CD1a) Immune evasion AgB Immature dendritic cells Elevated IL-10, IL-6 and TNF- production no IL-12 production Immunomodulation: polarization of dendritic cells differentiation towards Th2 priming EgEF-1 /, EA21, Eg2Hsp70, Hydatid fluid T-helper lymphocytes Elevated IL-4, IL-13 production Low IFN- production No IL-12 production Immunomodulation: exploitation of Th activation eliciting a non protective Th2 response In Vivo AgB, EgTeg, EgEF-1 /, EA21, Eg19, EgTPx B lymphocytes Specific IgG4 production Immune evasion:igg4 are neither cytophilic antibodies nor complement activators and can block IgE-mediated immunity EgEF-1 /, EA21 B lymphocytes Specific IgE production Allergic reaction In conclusion, in patients with CE we found an highly Th2-polarized microenvironment where besides AgB, EgTeg and EgEF-1 /, several other parasite molecules could elicit Th2 production. All these antigens can allow the chronic disease modulating the host immune response not only through their intrinsic ability but also by strengthening the generalized Th2 polarization previously established (Table 2). CONCLUDING REMARKS Although the last decade has clarified many aspects of host-relationship in human CE, establishing the full mechanisms that cause the disease require more studies. We need to define more clearly the events that manipulate the host immune response to protect the E. granulosus from elimination and minimizing severe pathology in the host. According to the 'hygiene hypothesis', the decreasing incidence of infections in western countries and more recently in developing countries is at the origin of the increasing incidence of both autoimmune and allergic diseases. Recent studies demonstrate that treatment or prevention of immune-mediated disease with immunomodulating molecules is consistent, showing that helminthes can protect a host from developing autoimmune disease [55]. Future studies understanding the mechanisms of E. granulosus immune regulation, will potentially uncover novel compounds that alter inflammatory responses, and will address the myriad of questions surrounding their potential for clinical application. Recent experimental evidence suggests that parasites can not only evade immune responses actively but also exploit the hormonal microenvironment within the host to favor their establishment and growth [56]. The benefit for parasites of hormonal exploitation is so great that they have evolved structures similar to the steroid and protein hormone receptors expressed in upper vertebrates that can bind to the hormonal metabolites synthesized by the host. This strategy is exemplified by Schistosoma mansoni and Taenia crassiceps that respond to adrenal steroids and sexual steroids, respectively [56]. Understanding how the host endocrine system can, under certain circumstances, favor the establishment of a parasite, and characterizing the parasite hormone receptors that are involved might aid the design of hormonal analogs and drugs that affect the parasite exclusively. Future investigations will allow us to integrate these new studies with previous results to recognize the extreme complexity of the host-parasite interaction for the development or improvement of CE diagnosis, treatment, and control strategies. CONFLICT OF INTEREST The Authors declare no conflict of interest related to this work. ABBREVIATIONS Ag = Antigen AgB = E. granulosus Antigen B CE = Cystic Echinococcosis DCs = dendritic cells E. granulosus = Echinococcus granulosus EA21 = E. granulosus cyclophilin Eg19 = E. granulosus 19 kda EgEF-1 / = E. granulosus Elongation Factor 1 / EgTeg = E. granulosus Tegumental antigen EgTPx = E. granulosus thioredoxin peroxidase HSP20 = Heat shock protein 20

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