Autoimmunity in the Mediation of Granulomatous Amoebic Encephalitis: Implications for Therapy

Acanthamoeba spp. are free-living amoebae that are ubiquitous in the environment. Most healthy individuals carry Acanthamoeba-reactive antibodies, suggesting constant exposure to amoebae. In spite of the high prevalence of the amoebae, the incidence of diseases caused by Acanthamoeba is very low. Non-opportunistically, Acanthamoebae can induce keratitis in healthy humans, but as an opportunistic pathogen, the amoebae can cause fatal encephalitis especially in immunocompromised individuals and treatments are often ineffective. Amoebic encephalitis is a life-threatening disease of the central nervous system (CNS) caused by free-living amoebae belonging to the genera Acanthamoeba, Balamuthia and Naegleria. Because they lack host-specificity, the ubiquitous amoebae can infect a wide range of species (Marciano-Cabral & Cabral, 2003, Schuster & Visvesvara, 2004). The diseases caused by Acanthamoeba spp. and Balamuthia spp. are generally termed ”granulomatous amoebic encephalitis” (GAE), whereas those caused by Naegleria spp. are called ‘primary amoebic meningioencephalitis (PAM)’(Marciano-Cabral & Cabral, 2003, Schuster & Visvesvara, 2004, Khan, 2006, da Rocha-Azevedo, et al., 2009). While Acanthamoebae induce illness mostly in immunocompromised individuals, Balamuthia spp. and Naegleria spp. can cause diseases in both immune-sufficient and immune-deficient individuals (Martinez & Visvesvara, 2001, Marciano-Cabral & Cabral, 2003, Schuster & Visvesvara, 2004, Khan, 2006, da Rocha-Azevedo, et al., 2009). Nevertheless, all of them can induce keratitis in healthy individuals, often in contact lens-wearers (Jones, et al., 1975, Martinez & Visvesvara, 1997, Marciano-Cabral & Cabral, 2003, da Rocha-Azevedo, et al., 2009). We recently discovered that A. castellanii contains mimicry sequence for immunodominant epitope of CNS myelin proteolipid protein (PLP), suggesting that exposure to A. castellanii can lead to the generation of autoimmune responses by antigenic mimicry. In this review, we discuss our understanding of the pathophysiology of Acanthamoeba-induced encephalitis, with a special emphasis on autoimmunity in mediation of the disease, and implications for therapy.

malnutrition (Martinez & Janitschke, 1985, Sell, et al., 1997, Marciano-Cabral & Cabral, 2003, Khan, 2006).Exacerbation of GAE lesions was reported in one patient undergoing treatment for cryoglobulinemia with a monoclonal antibody directed against CD20 which selectively depletes mature B cells (Meersseman, et al., 2007).Likewise, GAE can occur in patients with systemic lupus erythematosus, further emphasizing the importance of a compromised immune system for disease-predisposition (Koide, et al., 1998, Uschuplich, et al., 2004, Cha, et al., 2006).Since amoebic encephalitis is not a reportable disease, and diagnosis is often made postmortem, the number of cases documented in the literature does not reflect actual disease-incidence.One study has reported to have documented upto 500 cases of amoebic encephalitis worldwide (Sarica, et al., 2009).However, the recent availability of PCR-based detection of Acanthamoeba is greatly facilitating diagnosis (Schroeder, et al., 2001, Khan, 2006, da Rocha-Azevedo, et al., 2009, Maritschnegg, et al., 2011) and as a result, the number of cases reported in recent years show an increasing trend.

Pathogenesis
The amoebae can gain entry into the CNS through two routes: migration via the olfactory neuroepithelium and/or blood (Fig. 1).Naegleria spp.tend to follow the former route.After penetrating the nasal mucosa, the amoebae pass through the cribriform plate and travel along the nerve fibers to the olfactory bulb in the cerebrum (Khan, 2007, Elsheikha & Khan).Alternatively, the amoebae that enter through nasal exposure go to the lungs, enter the blood stream, and reach the CNS possibly by disrupting the blood brain barrier (BBB).The hematogenous route also is a choice for amoeba that enter through the skin (Khan, 2007).However, the mechanism by which amoebae actually enter the CNS is not clearly elucidated.Although it is postulated that they enter through the cerebral capillary endothelium or choroid plexus, the former being the more widely accepted mechanism (Khan, 2003, Marciano-Cabral & Cabral, 2003, Khan, 2005b, Khan, 2006, da Rocha-Azevedo, et al., 2009).These two modes of entry lead to the localization of amoebae in the cerebrum and cerebrospinal fluid (CSF) respectively (Elsheikha & Khan, 2010).Regardless of route of entry, the amoebae have to cross the BBB, either paracellularly by damaging the tight junctions, or transcellularly, in which the integrity of the BBB is maintained (Khan, 2003, Khan, 2006, Khan, 2007).Disruption of the BBB is thought to be mediated by contact-dependent or contact-independent mechanisms (Khan, 2003, Khan, 2006, Khan, 2007).Contact-dependent mechanisms require attachment of the amoeba to the brain's microvascular endothelial cells through amoebic mannose-binding protein (mannose-BP) causing apoptosis of the endothelial cells, a phenomenon that depends on the phosphatidylinositol 3-kinase signaling pathway (Sissons, et al., 2005, Khan, 2006, Khan, 2007).In contrast, contact-independent mechanisms involve extracellular proteases secreted by Acanthamoeba, particularly serine proteases, and these enzymes destroy extracellular matrix proteins comprised of collagen (type I, III, and IV), elastin, and fibronectin (Khan, et al., 2000, Sissons, et al., 2005, Khan, 2007).Alternatively, infected immune cells, most importantly macrophages, may simply act as Trojan horses to carry the amoebae to the CNS (Khan, 2007).The pathogenicity of Acanthamoebae varies by species, depending on their inherent potential to tolerate temperatures, attachment to cellular surfaces, and induction of cytolysis (Marciano-Cabral & Cabral, 2003, Khan, 2006).The principal virulent factors are mannose-BP, Nicotinamide adenine dinucleotide (NADH)-dehydrogenase, GDP-mannose pyrophosphorylase and proteasomal ATPase (Marciano-Cabral & Cabral, 2003, Han, et al., 2006) and of these, the role of mannose-BP has been well-studied.As noted above, the amoebae use mannose-BP for cellular attachment, and the fact that only the infective stagetrophozoites but not cysts upregulates mannose-BP expression suggests that, this protein is critical for amoebic invasion (Garate, et al., 2006).However, once the infection is established, microglial cells produce inflammatory cytokines, such as tumor necrosis factor (TNF)-, interleukin (IL)-6, IL-1 and IL-1 and they can contribute to tissue damage (Benedetto & Auriault, 2002, Benedetto, et al., 2003, Marciano-Cabral & Cabral, 2003).

Immune responses to Acanthamoeba
The role of the immune system and immune defense mechanisms in protecting against Acanthamoeba has not been well characterized, but protection against amoebae appears to involve both innate and adaptive immune responses.Amoebae are extracellular organisms that lack a sialic acid coat or capsule, making them vulnerable to complement-mediated destruction (Bowers & Korn, 1968, Korn & Olivecrona, 1971, Khan, 2005a).Conversely, amoebae can evade immune mechanisms by binding to a C1q component, as shown in the case of A. culbertsoni, and the parasite-derived serine proteases can degrade IgG and IgA (Toney & Marciano-Cabral, 1998, Kong, et al., 2000, Na, et al., 2002, Marciano-Cabral & Cabral, 2003).Neutrophils, macrophages, and microglia can destroy amoebae, and their amoebicidal effects are mediated in part by respiratory burst and nitric oxide under the influence of IL-1 , IL-1 , TNF-and/or IFN- (Ferrante, 1991a, Ferrante, 1991b, Marciano-Cabral & Toney, 1998, Marciano-Cabral, et al., 2000, Benedetto & Auriault, 2002a, Benedetto & Auriault, 2002b, Dudley, et al., 2007, Khan, 2008).Affected patients, including healthy individuals upto 90%, carry the Acanthamoeba-reactive antibodies of IgM, IgG, and IgA isotypes with no significant differences between males (86.2%) and females (89.2%), indicating that humans are regularly exposed to Acanthamoeba and become sensitized with the amoebic antigens (Chappell, et al., 2001, McClellan, et al., 2002, Schuster, 2002, Khan, 2005a, Brindley, et al., 2009, da Rocha-Azevedo, et al., 2009).It has been reported that T cells from healthy individuals can react to Acanthamoeba antigens obtained from CSF and antigen-specific T cell clones capable of producing IFN-also have been derived (Tanaka, et al., 1994).Likewise, peripheral blood mononuclear cells from rheumatoid arthritis patients shows proliferative responses to A. polyphaga (Shadidi, et al., 2001).But the significance of these observations is not known.The fact that the individuals exposed to Acanthamoeba become seropositive and also carry antigen-specific IFN-secreting cells suggests that the host defenses might involve both antibody-and cellmediated immune responses but, this aspect requires additional investigations.

Autoimmunity in the mediation of amoebic encephalitis
In our efforts to identify the disease-inducing microbial mimics for CNS myelin antigens, we recently identified a novel epitope from A. castellanii (Fig. 2; Massilamany, et al., 2010, Massilamany, et al., 2011).The epitope termed, ACA 83-95 is derived from rhodaneserelated sulfur transferase of Acanthamoeba.We tested the disease-inducing ability of ACA 83-95 in the mouse model of experimental autoimmune encephalomyelitis (EAE), which has been traditionally used to study the pathophysiology of multiple sclerosis (MS) in humans (Massilamany, et al., 2010, Massilamany, et al., 2011).(Massilamany, et al., 2010, Massilamany, et al., 2011) We verified the encephalitogenicity of mimicry epitope in both active immunization and adoptive transfer (AT) EAE protocols in autoimmune-prone SJL mice bearing the H-2 s haplotype.While EAE induction by active immunization requires administration of peptides emulsified in complete Freund's adjuvant (CFA), AT-EAE involves infusion of antigen-sensitized cells into naïve recipients (Miller & Karpus, 2007).The disease induction by active immunization essentially involves two phases: antigen-sensitization and effector T cell-expansion.On the contrary, the pathogenic potential of effector T cells is directly tested in AT-EAE protocol by transferring antigen-stimulated lymph node or spleen cells generated from previously immunized mice, thus eliminating the need to immunize naive recipients prior to disease induction.Figure 3a shows that SJL mice immunized with ACA 83-95 developed clinical signs of EAE reminiscent of disease induced by the cognate peptide PLP 139-151 (Tuohy, et al., 1989, Massilamany, et al., 2010, Massilamany, et al., 2011).Verification of these results in AT-EAE protocol clearly indicated that the disease-induction by ACA 83-95 requires the mediation of antigen-sensitized T cells (Fig. 3b, Fig. 4; Massilamany, et al., 2010, Massilamany, et al., 2011).EAE induction by adoptive transfer.Short-term T cell lines were derived from mice immunized with the indicated peptides and viable lymphoblasts were injected into naive SJL mice intraperitoneally, and the mice were monitored for signs of EAE and scored (Massilamany, et al., 2010, Massilamany, et al., 2011).Scoring scale: 0, healthy; 1, limp tail or hind limb weakness, but not both; 2, limp tail and hind limb weakness; 3, partial paralysis of hind limbs; 4, complete paralysis of hind limbs; 5, moribund or dead (Tuohy, et al., 1989, Massilamany, et al., 2010, Massilamany, et al., 2011).

ACA 83-95 induces cytokine responses that favor CNS autoimmunity
One of the hallmarks of CNS autoimmunity is the production of T helper (Th)1 and Th17 cytokines.Although both Th1 and Th17 cells could contribute to EAE pathogenicity, it appears that the ratio between the two subsets of T cells determines the severity of EAE.Predominance of Th17 over Th1 cells exacerbates inflammation and infiltration into the CNS (Bettelli, et al., 2007, Stromnes, et al., 2008).Furthermore, it has been proposed that Th1 cells enter non-inflamed CNS tissues and initiate inflammation, then facilitate the entry of Th17 cells (O'Connor, et al., 2008).Flow cytometrically, we verified Th1 (IL-2, and IFN-) and Th17 (IL-17A, IL-17F and IL-22) and Th2 (IL-4 and IL-10) cells, which mediate pro-and antiinflammatory effects respectively by intracellular staining.As expected, ACA 83-95 induces predominantly Th1 and Th17 cytokines similar to that induced with the cognate peptide, PLP 139-151 (Fig. 7; Massilamany, et al., 2010, Massilamany, et al., 2011).Based on these data, we expect that animals infected with A. castellanii will show the generation of pathogenic PLP-reactive T cells.Fig. 7. ACA 83-95 from A. castellanii induces cytokines similar to that of PLP 139-151.Lymph node cells obtained from mice immunized with PLP 139-151, or ACA 83-95 were stimulated with the corresponding peptides for two days and then maintained in IL-2 medium.Viable lymphoblasts were harvested on day 4 and stimulated briefly with phorbol 12-myristate 13acetate and Ionomycin.After staining with anti-CD4 and 7-AAD, intracellular staining was performed using cytokine antibodies and frequencies of cytokine-secreting cells were then determined by flow cytometry in the live (7-AAD -) CD4 subset.Shown are the frequencies of cytokine-secreting cells corresponding to Th1 (IL-2 and IFN-), Th2 (IL-4 and IL-10) and Th17 (IL-17A, IL-17F and IL-22) subsets (Massilamany, et al., 2010, Massilamany, et al., 2011).

Murine Acanthamoeba granulomatous encephalitis as a disease model of MS
Acanthamoeba spp.can cause choriomeningitis and destructive encephalomyelitis in mice and monkeys (Culbertson, et al., 1958, Culbertson, et al., 1959).The mouse model is widely used to study the pathogenesis of GAE (Kim, et al., 1990, Janitschke, et al., 1996, Marciano-Cabral, et al., 2001, Gornik & Kuzna-Grygiel, 2005, Khan, 2009).Intranasal inoculations of mice with A. castellanii produce subacute to chronic granulomatous encephalitis accompanied by rhinitis and pneumonitis (Martinez, et al., 1975, Kim, et al., 1990, Janitschke, et al., 1996, Gornik & Kuzna-Grygiel, 2005).Clinically, GAE in mice is manifested by respiratory distress, pneumonia, head tilt, circling, twirling, seizures, and limb paresis (Culbertson, et al., 1959, Culbertson, 1961, Culbertson, et al., 1966, Martinez, et al., 1975, Kim, et al., 1990, Janitschke, et al., 1996, Gornik & Kuzna-Grygiel, 2005).Histologically, infiltrations consist of microglia, histiocytes and lymphocytes around capillaries, suggestive of formation of foreign body granuloma (Martinez, et al., 1975, Janitschke, et al., 1996, Gornik & Kuzna-Grygiel, 2005).Electron microscopic studies reveal swelling and disintegration of dendrites, astrocytes, oligodendrocyte disruption, and disassociation of the myelin sheath along swollen axon cylinders (Martinez, et al., 1975).In our studies with ACA 83-95-induced autoimmune encephalomyelitis, in spite of the presence of mononuclear cells (MNC), the dominance of plasma cells and giant cells was absent.Instead, the histologic disease resembled typical PLP 139-151-induced EAE (Sobel, et al., 1990, Massilamany, et al., 2010, Massilamany, et al., 2011), suggesting that naturally occurring GAE may involve the mediation of multiple factors and different cell types, which may reflect the host's response to living organisms in situ.To date, there are no reports to indicate that autoimmune response is a component of disease pathogenesis in GAE, and our data with ACA 83-95induced autoimmune encephalomyelitis provide compelling evidence to test this possibility.This notion is further supported by the fact that humans affected with A. castellanii infection can never be treated successfully (Marciano-Cabral & Cabral, 2003).Pathogens that primarily infect the CNS can induce autoimmune responses secondarily.As previously discussed, A. castellanii is a pathogen of the CNS that causes granulomatous inflammation of the brain and spinal cord.We propose that A. castellanii can induce myelinreactive T cells by two mechanisms in infected mice (Fig. 8).(a) Molecular mimicry.Upon exposure to the parasites, the immune system recognizes parasite-derived mimic of PLP, generating T cells in the periphery, which then migrate into the CNS and cause inflammation.In support of this theory, we have demonstrated that the mimicry epitope from A. castellanii, ACA 83-95, can induce clinical signs of autoimmune encephalomyelitis in the adjuvant protocol of inducing CNS autoimmunity in SJL mice (Massilamany, et al., 2010, Massilamany, et al., 2011).(b) Epitope spreading.A. castellanii causes granulomatous inflammation in the brain and spinal cord, which can lead to the local release of myelin antigens and prime T cells.Alternatively, the newly released myelin antigens are carried by antigen-presenting cells to the draining lymph nodes and prime T cells, which, in turn, migrate back into the CNS and further aggravate inflammation.Similar events have been earlier demonstrated in the case of murine pathogen, TMEV (Miller, et al., 2001, Olson, et al., 2004, McMahon, et al., 2005).MS is a disease of the CNS characterized by inflammation and infiltration of MNC and the loss of myelin sheath encapsulating the axons (Noseworthy, et al., 2000, Sospedra & Martin, 2005).Autoimmune responses to myelin antigens have been implicated in MS pathogenesis and this requires the mediation of autoreactive T cells and B cells, but the mechanisms by which the disease is initiated are unknown (Kerlero de Rosbo, et al., 1993, Sospedra & Martin, 2005).Although genetic susceptibility is a major predisposing factor, exposure to environmental microbes such as viruses and bacteria have been suspected in the initiation of autoimmune diseases.In support of the latter, exacerbations of MS attacks or temporal alterations in the disease course have been linked primarily to exposure to virus infections such as Epstein Barr virus and Human Herpes virus-6, but the clinical evidence remains elusive (Cirone, et al., 2002, Pohl, 2009, Salvetti, et al., 2009).The current dogma is that MS does not appear to follow Koch's postulates in that no single organism appears to trigger it; rather, exposure to multiple organisms might be critical for MS predisposition (Sospedra & Martin, 2005).The fact that ACA contains mimicry epitope for PLP, one of the candidate autoantigens implicated in MS pathogenesis, suggests that ACA infection can potentially lead to the generation of PLP reactive T cells and predispose to MS.

Clinical signs and histology
Acanthamoeba-induced encephalitis is often overlooked (Schuster & Visvesvara, 2004, da Rocha-Azevedo, et al., 2009), partly due to the rarity of Acanthamoeba infections and a lack of familiarity and diagnostic tools.However, when diagnosed, it is difficult to differentiate PAM from GAE because symptoms overlap between each other (da Rocha-Azevedo, et al., 2009).PAM is initially manifested by severe headache, rhinitis, nausea, and fever followed by anosmia, seizures, stiff neck, diplopia, and coma, finally leading to death (Marciano-Cabral & Cabral, 2003, da Rocha-Azevedo, et al., 2009).Histologically, brains contain inflammatory infiltrates comprised of neutrophils, eosinophils, and macrophages (Martinez & Janitschke, 1985, Marciano-Cabral & Cabral, 2003, da Rocha-Azevedo, et al., 2009).In contrast, symptoms of GAE are diverse in that a wide range of clinical manifestations can be expected.These include headache, rise in intracranial pressure, abnormal gait or ataxia, diplopia, stiff neck, confusion, behavioral changes, hemiparesis, cranial nerve palsies, seizures, photophobia, and anorexia; more than 90% of individuals affected with GAE tend to die (Marciano-Cabral & Cabral, 2003, Khan, 2006).The histologic disease is characterized by hemorrhagic or necrotic encephalitis, edema of the brain accompanied by the presence of focal lesions around the cerebrum, cerebellum, and corpus callosum.Cellular infiltrations in histological sections include multinucleated giant cells, plasma cells, polymorphonuclear cells and mononuclear cells (Martinez & Visvesvara, 1997, da Rocha-Azevedo, et al., 2009) and HIV patients can develop granulomas within the CNS possibly due to low CD4 T cell count (Marciano-Cabral & Cabral, 2003, Cha, et al., 2006, Khan, 2006).In some patients, organs other than brain such as skin, liver, lungs, kidneys, prostate glands, lymph nodes, pancreas, and adrenals can also be affected.

Diagnosis
Acanthamoeba-induced encephalitis is not routinely suspected, the disease can be misdiagnosed as neurocysticercosis; viral, rickettsial, fungal, and bacterial meningitis; toxoplasmosis; and brain tumors (Schuster & Visvesvara, 2004, khan, 2005b).Serologically, detection of Acanthamoeba-reactive antibodies gives an indication of amoebic exposure at a population level (Cursons, et al., 1980, Cerva, 1989, Khan, 2006).However, definitive diagnosis requires the demonstration of amoebic trophozoites or cysts in biological samples.Examination of wet-mount smears prepared from CSF or methanol-fixed smears stained with Giemsa-Wright permit identification of amoebic trophozoites.While evaluating wetmount smears, careful consideration should be given to differentiate trophozoites from macrophages because of their close morphological resemblance to each other (Cleland, et al., 1982, Lalitha, et al., 1985, Singhal, et al., 2001).In addition to CSF, trophozoites can also be detected in bronchoalveolar lavage fluid from patients with respiratory distress (Newsome, et al., 1992).Detection of trophozoites in fixed tissue sections prepared from brains is usually performed using hematoxylin and eosin and trichrome stainings (Newsome, et al., 1992).In contrast, amoebic cysts in brain tissues are detected using calcofluor white staining (Silvany, et al., 1987).Alternatively, periodic acid-Schiff's stain and Gomori-methenamine silver stain can be used to stain tissue sections in which, cysts appear red whereas tissues appear black in color (Marciano-Cabral & Cabral, 2003).Other specialized techniques employed to demonstrate the presence of amoeba are transmission electron microscopy and immunofluorescent or immunoperoxidase staining (Willaert & Stevens, 1976, Stevens, et al., 1977, McKellar, et al., 2006, Guarner, et al., 2007).However, because most Acanthamoeba spp.are antigenically related, the use of immunohistochemical techniques does not permit identification by species.To identify structural brain lesions, computed tomography and magnetic resonance imaging are widely used (Sell, et al., 1997, Kidney & Kim, 1998).These evaluations can reveal changes such as multifocal areas of signal intensities or ring-like lesions or low-density areas indicating occupying mass of tumor or abscess (Martinez, et al., 1977, Martinez, et al., 1980, Ofori-Kwakye, et al., 1986, Matson, et al., 1988, khan, 2005b, Khan, 2008, da Rocha-Azevedo, et al., 2009).The regions of the brain that are usually affected are midbrain, basal areas of the temporal and occipital lobes, and the posterior fossa (Seijo Martinez, et al., 2000, Marciano-Cabral & Cabral, 2003, Khan, 2006).Molecularly, PCR amplification of the 18S rDNA using sequence-specific primers is currently used as a quick and reliable method of diagnosis (Schroeder, et al., 2001, Khan, 2006, da Rocha-Azevedo, et al., 2009, Maritschnegg, et al., 2011).Hematologically, pleocytosis accompanied by lymphocytosis, neutrophilia, hypoglycemia and hyperprotenemia may be seen in patients with GAE (Marciano-Cabral & Cabral, 2003).In addition, Acanthamoeba can be isolated from clinical specimens by plating the samples on non-nutrient agar plates coated with E. coli or Enterobacter aerogenes (Schuster, 2002, Khan, 2006, da Rocha-Azevedo, et al., 2009).

Treatment
The low degree of therapeutic success in treating amoebic encephalitis is due in part to the fact that immunocompromised individuals are most often affected, and the disease outcome thus depends on successful treatment of underlying causes.Furthermore, Acanthamoeba infections tend to escape early diagnosis due to the lack of both awareness and diagnostic tools.Nonetheless, if diagnosed early, the disease can be treated successfully (Marciano-Cabral & Cabral, 2003, Schuster & Visvesvara, 2004, Khan, 2006, Khan, 2008, Matin, et al., 2008, Elsheikha & Khan, 2010, Akpek, et al., 2011).Various treatment regimens have been reported in the literature, but there are no reports to indicate that Acanthamoeba infections can be treated with a single drug; rather, a combination of multiple drugs is used (Table 1).These include ketoconazole, fluconazole, flucytosine, sulfa-trimethoprim, amphotericin B, pentamidine isothionate, azithromycin, itraconazole and rifampicin.Currently, to enhance BBB-permeability, soluble analogs of the most effective drugs are being tested (Khan, 2006).Likewise, experimental attempts also are being made to use non-viral plasmid DNAs encoding anti-sense RNA sequences for virulence factors of amoebae which can block their entry into the CNS (Elsheikha & Khan, 2010).Based on our data (Massilamany, et al., 2010, Massilamany, et al., 2011), we propose that amoebic encephalitis might involve mediation of autoimmunity, but this hypothesis needs to be tested experimentally in animal models and clinically in GAE patients.Proving that autoimmunity is a component of GAE provides a basis for exploring treatment modalities directed toward autoimmunity in patient subjects.

Conclusion
In spite of high prevalence, the diseases induced by Acanthamoebae are extremely low.
Although amoebic encephalitis is more commonly seen in immunocompromised individuals, the disease can occur in immunocompetent healthy individuals (Marciano-Cabral & Cabral, 2003, Schuster & Visvesvara, 2004).Our discovery that A. castellanii contains mimicry epitope for PLP indicates that exposure to Acanthamoeba can accompany autoimmunity through the generation of self-reactive T cells.Acanthamoebae are free-living organisms that are ubiquitous in the environment, leading to constant exposure.It is possible that such coexistence can help microbes acquire some of the genetic elements of their hosts as an evasive mechanism for survival.Alternatively, exposure to such organisms could lead to a break in self-tolerance as a result of antigenic mimicry in genetically susceptible individuals who potentially carry pathogenic autoreactive T cell and B cell repertoires.Further research is required to address these hypotheses, proving which creates opportunities to also target therapy toward autoimmunity in patients affected with GAE.

Fig. 1 .
Fig. 1.Proposed routes of entry of Acanthamoeba into the CNS.Under favorable conditions, cysts become trophozoites which are the infective stages of Acanthamoeba.Upon nasal exposure, trophozoites can reach CNS hematogenously via lungs or through olfactory neuroepithelium.The amoeba that gains entry through the skin can also reach CNS hematogenously.However, actual entry into the brain tissue involves crossing the BBB which appears to be mediated through either contact-dependant mechanisms by inducing apoptosis of the endothelial cells or contact-independent mechanisms via destruction of extracellular matrix by amoebic proteases.The micrographs of Acanthamoeba trophozoites and cysts were kindly provided by Dr. Francine Marciano-Cabral, Virginia Commonwealth University, VA, USA

Fig. 3 .
Fig. 3. ACA 83-95 induces autoimmune encephalomyelitis similar to that induced by PLP 139-151.(a) EAE induction by active immunization.SJL mice were immunized with peptides emulsified in CFA, and pertussis toxin was administered on day 0 and 2 postimmunization and the mice were monitored for clinical signs of EAE and scored.(b)EAE induction by adoptive transfer.Short-term T cell lines were derived from mice immunized with the indicated peptides and viable lymphoblasts were injected into naive SJL mice intraperitoneally, and the mice were monitored for signs of EAE and scored(Massilamany, et al., 2010, Massilamany, et al., 2011).Scoring scale: 0, healthy; 1, limp tail or hind limb weakness, but not both; 2, limp tail and hind limb weakness; 3, partial paralysis of hind limbs; 4, complete paralysis of hind limbs; 5, moribund or dead(Tuohy, et al., 1989, Massilamany, et al., 2010, Massilamany, et al., 2011).

Fig. 8 .
Fig. 8. Proposed mechanisms for the induction of CNS autoimmunity in mice infected with A. castellanii.(a) Molecular mimicry.Peripherally, the immune system can recognize mimicry epitope of PLP, and generates cross-reactive T cells, which then migrate into the CNS and cause inflammation.(b) Epitope spreading.Granulomatous CNS inflammation induced by Acanthamoeba can lead to the release of myelin antigens and prime T cells locally or peripherally and the de novo generated myelin-reactive T cells can further aggravate CNS Conversely, mice can remain infected and clinically normal but, crossreactive cells can still be generated by mimicry.