Immunology of Entamoeba histolytica in Human and Animal Hosts

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1 REVIEWS OF INFECTIOUS DISEASES VOL. 4, NO.6. NOVEMBER-DECEMBER by The University of Chicago. All rights reserved /82/ $02.00 Immunology of Entamoeba histolytica in Human and Animal Hosts Dorothea Trissl From the Department of Biology and Chemistry, University of Osnabruck, Osnabriick, West Germany Although Entamoeba histolytica induces humoral and cellular immune responses in both human and animal hosts, there is no indication of postinfection immunity in humans; in contrast, several other mammals are protected by prior infection or immunization. The exacerbation of the disease by immunosuppression suggests a protective function of still-unknown defense mechanisms. Specific local and circulating antibodies are produced regularly during invasive amebiasis. Although serum antibodies, together with complement, are lytic to the trophozoites in vitro, the poor correlation of these antibodies with resistance contradicts a protective capacity in vivo. The parasite may evade harm by shedding antigen-antibody complexes from its surface. Demonstration of immediate-type skin reactions, elevated IgE titers, and specific anti amebic IgE suggests that anaphylaxis occurs. The function of the anaphylactic reaction in pathology and resistance remains to be studied. Delayed hypersensitivity parallels healing or resistance and is retarded in human hepatic amebiasis. This observation is consistent with a protective role of cell-mediated immunity. Amebiasis, the infection by the protozoan parasite Entamoeba histolytica, is a major health problem in many developing countries [1]. In temperate zones asymptomatic forms predominate, whereas in tropical countries severe forms of the disease are frequent [2, 3]. Although the assessment of epidemiologic data has been hampered by diagnostic problems [2], it is assumed that of the world's population is infected with E. histolytica [1] and that of those infected suffer from symptomatic amebiasis [4]. Clinical data illustrate the severity of the problem. For instance, it was reported in 1974 that amebiasis was the seventh most frequent cause of death in Guatemalan hospitals [5]. According to a 1970 publication, this disease was the sixth most frequent cause of death in Mexico [6]. A seroepidemiologic survey later showed that 6% of apparently healthy Mexicans were currently or had recently been infected with E. histolytica [7]. In the majority of infected persons, E. histolytica lives as a harmless commensal within the col- Received for publication August 30, 1981, and in revised form November 19, I am grateful to Drs. H. J. Bos and H. Kolb for their suggestions and valuable criticism; to Drs. I.-Y. Weiler, P. Schnetkamp, and H.-W. Trissl for reading the manuscript; and to Mr. U. Offermann for typing the manuscript. Please address requests for reprints to Dr. Dorothea Trissl, Department of Biology and Chemistry, P.O. Box 4469, University of Osnabruck, 4500 Osnabruck, West Germany. on. Its life cycle appears to be relatively simple compared with those of other parasites; sexual stages and intermediate hosts have not been found [8-11]. The vegetative form, the trophozoite, feeds on the intestinal contents and reproduces by binary fission. Under appropriate conditions, which are not well understood, the organism differentiates into the infective form, the cyst, which is excreted and may infect a new host via the oral route. After'passage through the stomach, excystation starts; during the following metacystic stages, the trophozoites develop again. This life cycle can be reproduced in vitro in the presence of bacteria [9]. Entamoeba histolytica has been cultivated in vitro in a variety of complex media that have been described in several detailed reviews [12-15]. Initially, the original intestinal bacterial flora (mixed cultures) or various single bacterial or protozoan species (monoxenic cultures) were used for cocultivation, but media for axenic cultivation (i.e., without other living cells) were later developed [16-18]. The axenic media have greatly facilitated the biochemical, pathological, and immunologic investigation of E. histolytica, but they have disadvantages as well: a loss of the encystation capacity of the amebas [19], a frequent decrease in the virulence of the organisms to animals [20-26], and a change in the inflammatory response of the hosts to the parasites [24, 26, 27]. The implications of these changes for the immune response must be taken into consideration. 1154

2 Immunology ofe. histolytica 1155 Although the life cycle of E. histolytica does not require an invasive stage, the trophozoite is able to invade the body by attacking the intestinal mucosa and by spreading from the lesions to the liver and to other organs. This capacity does not apply to all amebic strains; in humans as well as animal hosts, the degree of virulence may vary extremely, and the resulting disease may be asymptomatic or lethal [28-33]. Nevertheless, it is assumed that all strains of E. histolytica are potentially pathogenic [3, 24, 34, 35] and that an unknown stimulus converts the harmless commensal to the tissueattacking parasite. Considerable effort has been expended in attempts to identify this stimulus, but so far only influences of a variety of host and parasite factors on virulence have been detected [3, 34, 36-41]. A discussion of these factors is beyond the scope of this review. Several factors, such as the nutritional [37, 42] and hormonal [38] state of the host or the cytotoxicity [43-49] and the surface composition [48, 50-52] of the parasite may be suspected to influence the virulence of E. histolytica by modulating the immune response of the host. This modification would result in a mutual dependence of pathogenicity and immunology [3, 53]. The main consequences of invasion by E. histolytica are amebic colitis, dysentery, and liver abscesses, which all may lead to death. Given the severity of the disease, it is unfortunate that only scanty research is being done on the relationship of this parasite and its host. The lack of immunoparasitologic investigations is especially striking. In 1973 Kagan stressed that "when reviewing the literature on the immunology of protozoan infections, one is struck by the singular absence of any discussion of amebiasis" [53]. The last detailed description of the immunology of E. histolytica by Balamuth and Siddiqui in 1970 [54] showed that research has concentrated mainly on three fields: (1) the elaboration of serologic methods for diagnosis and epidemiologic study (2) the characterization of antigenic patterns of amebas, as revealed by immune sera; and (3) the comparison of different species and strains of Entamoeba with respect to their antigenic relationship. The final aim of any immunoparasitologic research, however, should be the development of effective therapeutic and immunoprophylactic measures. This goal will hardly be achieved without detailed knowledge of the immunologic aspects of the host-parasite relationship: protective mechanisms used by the host against the parasite and its products, immunopathologic changes caused by the parasite and its products, and immunosuppressive effects that enable the parasite to evade the defense mechanisms of the host. Since the last review, efforts have been 'made to attack such problems. These issues will be the main concern of this review. The description will concentrate on E. histolytica, with only occasional mention of related species. As will become evident, solid knowledge of the immunoparasitology of E. histolytica is scarce. We are still only beginning to understand the immunologic events that follow infection with this parasite. Definitions and Classifications The definitions and classifications recommended by the World Health Organization Expert Committee [1] will be used in this review. Amebiasis is defined as the condition of harboring the parasite E. histolytica, with or without clinical manifestations. Patients with asymptomatic amebiasis pass cysts, but do not show any symptoms. Symptomatic amebiasis may affect any organ, but intestinal amebiasis comprising amebic colitis, dysentery, ameboma, and appendicitis is most frequent. In most cases of extraintestinal amebiasis, the liver is afflicted by either acute non suppurative disease or abscess. A strain of E. histolytica is defined as a laboratory-established population of an isolate, the source of which is well documented. The Human Host: Pathology and Histopathology The pathologic changes in intestinal and extra intestinal amebiasis have been reviewed carefully [3, 5, 55, 56]. In summary, persons infected by E. histolytica may harbor the parasite without any intestinal or hepatic symptoms. At any time after infection, invasion may occur. In the intestine the disease varies from mild forms, with diffuse inflammation and only microscopically visible damage, to severe forms, with different degrees of ulceration. The erosion can proceed from the mucosa to the submucosa and finally can result in perforation of the intestinal wall. Then the trophozoites gain access to extraintestinal sites, especially the liver, where they give rise to socalled liver abscesses or discrete areas of necrosis.

3 1156 Trissl The histopathologic features of amebic lesions are of special interest with regard to the immunology of E. histolytica, since the host cells that infiltrate the lesions may give a clue as to the immunologic mechanisms involved. Early studies of amebiasis, mainly done with autopsy material, led to the general opinion that inflammation is mild or absent in amebic lesions that are free of pathogenic bacteria and that the degree of inflammation does not correlate with the vigorous necrotic processes [3, 5, 38, 55, 57, 58]. In accordance with this opinion, an impairment of inflammatory reactions is indicated by some recent experiments. Inhibition of monocyte chemotaxis by E. histolytica [59] and reduced phagocytic activity of peripheral polymorphonuclear leukocytes from patients with amebiasis [60] have been observed. On the other hand, inflammatory processes do occur [55], as indicated by elevated titers of complement [61, 62] and leukocytosis [63-67]. From biopsy studies it is known that acute intestinal amebiasis is accompanied by generalized hemorrhagic inflammation of the mucosa, with pronounced edema and infiltrations by neutrophils and eosinophils [65, 68-71]. Also in hepatic amebiasis the viable liver tissue surrounding an abscess can be infiltrated by neutrophils and eosinophils [72, 73]. Furthermore, in chronic amebiasis and in ameboma, chronic and allergic types of inflammation with infiltrates of lymphocytes, histiocytes, and eosinophils have been described [74-77]. One reason for the different histopathologic results may be the cytopathogenicity of E. histolytica itself [43-49]. According to Griffin [70], inflammatory cells adjacent to amebas are rapidly destroyed and are thus difficult to identify. Consequently, infiltrating cells are found at some distance from the trophozoites, separated by viable tissue or by layers of necrotic cells [71, 73]. Degenerated inflammatory and tissue cells are found in exudates [57, 78]. Thus, inflammation is induced by E. histolytica, but is mild in comparison with that seen in bacterial infections and is obscured by necrosis evoked by the cytotoxic amebas or other conditions. The role of the immune system in the inflammatory response to E. histolytica has not been studied, but (as will be discussed later) early antibody production and retarded delayed hypersensi- tivity are in accordance with the observed cellular infiltrates. Antigens of E. histolytica The elucidation of antiamebic immune reactions is complicated by the large number of antigenic components that may evoke an equally large number of different immune responses. The successful mass cultivation ofe. histolytica in axenic medium [16-18] has provided the basis for separating these antigens and studying their immunologic characteristics. The antigens used at present are mainly whole cells and homogenates, in addition to soluble and particulate fractions. Most studies are concerned with antigens detectable by serologic methods, whereas little work has been done on antigens eliciting cellular immune responses. Trophozoite Surface Antigens Among the antigens of E. histolytica, those exposed on the surface are of special interest since they may induce effector mechanisms lethal to the parasite. Such antigens have been demonstrated by the immobilization of trophozoites by immune sera [79], by the surface binding of fluorescencelabeled immune sera [80], by the abrogation of surface binding after absorption of immune sera with trophozoites [81], and by antibody-mediated lysis of trophozoites by complement [82-85]. Surface antigens have been shown to contain carbohydrates, in that they. bind to concanavalin A (Con A) [86]; however, it has not yet been demonstrated whether these antigens are identical to the glycoproteins isolated from amebic plasma membranes [87]. In studies with Entamoeba invadens, an ameba pathogenic for reptiles, antigenic glycoproteins have been shown to be liberated from the surface by incubation with Con A [88]. On the other hand, carbohydrate-free membrane antigens of E. invadens [89] and carbohydrate-free membrane proteins of E. histolytica [90] have been described. Although the existence of antigens on the surface of trophozoites has been proved unambiguously, there is some doubt about their origin. Serum and medium components adhering to the amebic surface give rise to a humoral response

4 Immunology ofe. histolytica 1157 upon immunization [91, 92]. Thus, it remains to be proved that the antigens found on the amebic surface are produced by E. histolytica itself. The role of surface antigens in the immune response of the host is unclear. From the distribution of antigens ofe. histolytica and E. invadens in different fractions, it has been concluded that the number of antigens is larger and the level of antigenicity and immunogenicity higher in cytoplasmic vesicle fractions than in the plasma membrane fraction [81, 89]. Thus, the maj or stimulus for antibody production by the host would be provided by intracellular immunogens of the disrupted amebas rather than by the surface of the intact trophozoites. Low immunogenicity of live amebas then might be responsible for the failure of the host to prevent invasion by the parasite [81]. This interpretation depends on the reliable identification ofinternal and surface antigens, a goal that has not yet been achieved. Complications arise from the continuous turnover of membranes by endo- and exocytosis, the periodic expression of surface antigens in vitro [93], the unusual distribution of marker enzymes in amebas [87, 94], and the vesicularization of plasma membranes during antigen preparation. Nevertheless, support for this interpretation is offered by the observation that a low percentage of human immune sera contain antibodies to surface antigens [79, 86, 95]. On the other hand, there is no doubt that antibodies binding to the amebic surface and killing the parasite in vitro are produced [79, 82, 96, 97]. The development of a serodiagnostic method based on the immobilization reaction was possible only because of the high frequency of immobilizing antibodies in symptomatic amebiasis [96]. Thus, a low level of surface immunogenicity alone seems to be too simple an explanation for the evasion of the host's response by the amebas. Possible escape mechanisms will be discussed in more detail later. Trophozoite Homogenates Homogenates and extracts of whole amebas [54, 98, 99] are the antigen preparations most frequently used for serodiagnosis, for the analysis of immune sera, and for immunization. The complexity of these preparations is displayed by numerous precipitin bands in gel diffusion and in one- and two-dimensional immunoelectrophoresis [ ]. An extensive comparison of amebic antigens and human immune sera from different parts of the world has revealed at least 14 precipitating antigens [104, 106]. The similarity of bands suggests that different strains of E. histolytica - pathogenic as well as nonpathogenic - have a common antigenic "backbone." The frequency of individual bands, however, varies widely with the individual responses of the host. Trophozoite Fractions Amebic homogenates have been fractionated by the usual biochemical methods. Column chromatography [98, 105, ] has generally allowed the separation of three to four peaks. Indirect HA (lha), CF, and precipitating activity reside mainly in the high-molecular-weight peaks [98, 105, 108, 109, 114, 117]. Furthermore, protective immunity to E. histolytica infection in hamsters and guinea pigs can be induced most effectively by the fractions of high molecular weight [114, 116, 118]. Particulate fractions isolated by differential centrifugation [87, 90, 115] induce protective immunity in hamsters [115] and blast transformation of lymphocytes from sensitized humans [119]. The macrophage migration inhibition factor (MIF) of peripheral blood cells from sensitized individuals is induced effectively by an amebic fraction isolated by polyacrylamide gel electrophoresis [120]. Amebic constituents, such as an acid proteinase [121] and a cytotoxin [46, 122], induce antibody production [46, 121, 123, 124]. An inhibitory effect of antibodies to the parasitic cytotoxin on the pathology of E. histolytica is to be expected from their inhibition of cytotoxicity in vitro [46, 123, 124]. Antigens released from live amebas may playa role in the pathology of amebiasis by toxic reactions or by the formation of immune complexes. Early attempts to find such antigens by immunoprecipitation of culture medium were unsuccessful [125]. On the other hand, the cytotoxin seems to be released into the medium [126]. Other potentially antigenic constituents, including a glycosidase [127] and a microexudate that trails behind the amebas on the culture substrate in vitro [128], are released. An exudate similar to that seen in vitro may account for the amebic antigens found on

5 1158 Trissl various cell types in human amebic liver abscesses [129] and may be related to a lectin involved in agglutination and phagocytosis of red blood cells by trophozoites [52, 130]. Cyst Antigens Only indirect information on cyst antigens is available. In one study [131] the reactivity of E. invadens cyst antigens and that of E. histolytica trophozoite antigens with human immune sera were shown to be correlated. This result indicates cross-reactivity of antigens not only from different species but also from different stages of the life cycle. Nevertheless, part of the sera from patients with intestinal amebiasis contained antibodies to the cyst exclusively. Thus, there are stage-specific antigens of the luminal cyst that are able to induce a systemic antibody response. These antigens have not been characterized. The Immune Response in Humans The Humoral Response Serodiagnosis. Serologic tests have been studied extensively with the aim of establishing reliable methods for diagnosing amebiasis [54, 98, 101, 104, ]. Because current knowledge has been reviewed repeatedly [54, 117, 168, 169], this topic will be treated only briefly here. Serum antibodies can be demonstrated by commonly used serologic techniques [101, 104, ] and by the ameba-specific immobilization test [96]. With the use of axenic antigen, a high degree of specificity can be obtained [98, 148, 154, 155, 163]. A comparison of different tests shows that the sensitivity is usually highest in IHA tests, moderate in precipitation tests, and lowest in CF and immobilization tests [98, 136, , 160, 163]. Table 1 shows some data on serodiagnosis by IHA. The large variations in results reflect problems in the comparison of data obtained with different antigen preparations and with different criteria for the reading of tests and the classification of patients. Standardization of such parameters would be desirable. Circulating and local antibodies. As can be seen in table 1, serum antibodies are produced regularly in symptomatic amebiasis, but frequently also in asymptomatic amebiasis. There is a general correlation between the stage of infection and the presence, titers, and immunoelectrophoretic patterns of antibodies. Acute invasive amebiasis generally provokes high serum titers and complex patterns, whereas asymptomatic or previously symptomatic amebiasis leads to lower (if any) titers and to fewer precipitation bands [30, 147, 156]. This statement may not hold true for all individual sera, but in endemic areas high antibody titers are seen more frequently in symptomatic than in asymptomatic patients [156, 157]. A low percentage of false-negative results are obtained in intestinal and hepatic amebiasis and may be due in part to a later onset of antibody production [145, 170]. False-positive results are found more frequently in endemic areas [156, 160, 171], probably because of a history of amebiasis in the population. In follow-up studies titers of antibodies have been shown to decrease but to persist at a low level for years [148, 156, 160, 162, 172, 173]. In uninfected newborns antibodies derived from the mother [174] disappear within three months [175]. Thus, prolonged persistence of antibodies after recovery may depend on the undetected persistence of antigen or infection. Such a situation may account for the frequent absence of intestinal infections in cases of amebic liver abscess [38, 176, 177]. It is generally assumed that serum antibodies to E. histolytica appear only after invasion by the parasite [54, 168, 178]. The relatively high frequency of positive sera in asymptomatic patients who pass cysts (up to [179]) (table 1) is thought to result from subclinical invasion. If so, the results only reflect the insolvable problem of differentiating between low-grade invasive and exclusively luminal amebiasis. However, the possibility of serum antibody production prior to invasion by the parasite, as is seen with other intestinal antigens [180, 181], should not be excluded. Since systemic tolerance also may result from intestinal immunization [180, 181], this question requires careful studies. Sensitive indicators of a local humoral response are locally secreted coproantibodies. Several studies have demonstrated the release of such antibodies in intestinal amebiasis [l82-185a]. A predominance of antibodies of the IgA class is indicated by a higher incidence of HA than of CF antibodies [183] and by the preferential precipitation of antibodies with antisera to IgA [185]. In

6 Immunology ofe. histolytica 1159 Table 1. Serum antibodies in amebiasis, as measured by indirect HA. Percentage of positive sera from persons in indicated group Symptomatic amebiasis Reference Asymptomatic amebiasis Intestinal Hepatic Control Significant titer" : : : : I 1: : : : : NOt : <I 1: : I: 18 Significant titer is the lowest titer considered to be positive. t NO = not done. contrast to serum antibodies, coproantibodies persist for only a short time [185]. Since invasive and luminal amebiasis have not been differentiated in these studies, the contribution of luminal antigens to the induction of antibody synthesis remains to be evaluated. The finding of serum antibodies to the exclusively luminal cyst [131] argues in favor of a response to luminal E. histolytica. Although neither the function of coproantibodies to the ameba nor the existence of local cellular immune responses has been studied, considerable influence of the gut-associated immune system [186, 187] on the immunology as well as on the pathology of E. histolytica may be suspected. Immunoglobulin classes. In areas endemic for E. histolytica, studies on the serum levels of the major immunoglobulin classes have been performed repeatedly [61, 62, 173, 188, 189]. Such studies are complicated by the elevation of immunoglobulin levels due to other parasitic infections. Nevertheless, levels of IgG seem to be elevated in amebiasis, whereas those of IgM and IgA are less affected (table 2). The intimate involvement of IgG is indicated by the rapid fall of its concentration during chemotherapy [173]. Levels of IgG do not increase in pregnant women [173], who are known to be especially prone to amebic invasion [ ]. Ravi et al. [62] did not find increased levels of IgG in patients with amebiasis, but did find increased levels of IgM and IgA. This result may have been due to differences in the times ofsampling, since as in other parasitic infections [193], the highest concentrations of IgG are found late after onset of symptoms [61]. It is unknown whether the rise of IgG levels in amebiasis is due only to specific antibodies to E. histolytica, At least part of the increase can be attributed to the specific response [131, 195]. Passive cutaneous anaphylactic, IHA, CF, immunoprecipitating, and immobilizing activities reside in the IgG fraction [194, 195]. Since human immune sera induce passive cutaneous anaphylaxis (PCA) in guinea pigs [195], heterocytotropic antibodies of the IgGl, IgG3, or IgG4 subclass must be in- Table 2. Serum immunoglobulin levels in amebiasis. Result with sera from patients in indicated category" Intestinal amebiasis Hepatic amebiasis Reference IgG IgM IgA IgG IgM IgA NO NO NO t NO NO NO Results are expressed as percentages of values in normal controls. NO = not done. t Controls in this study were asymptomatic but passed cysts of Entamoeba histolytica.

7 1160 Trissl volved. In addition to IgG, specific IgM and IgA antibodies have been detected [ ]. Quantitative data on the proportion of immunoglobulin which is nonspecific and that which is specifically directed against the amebas are of interest in view of the high synthesis of nonspecific immunoglobulin noticed in several other protozoan infections [ ]. In these cases it has been assumed that nonspecific immunoglobulin is produced as a result of polyclonal stimulation of lymphocytes by mitogenic constitutents of the parasites and that exhaustion of the available immune cell pool then results in immunodepression [ ]. An activity mitogenic to human as well as murine lymphocytes has also been detected in E. histolytica [ ]. Stimulation of immunoglobulin synthesis, however, has not been demonstrated in vitro. In vivo studies have not been done. The role of antibodies in amebiasis. Antibodies have been shown to be protective against a variety of extracellular parasites other than amebas. In amebiasis, however, the epidemiologic evidence argues against effective protection. On the one hand, the highest antibody titers are found particularly in symptomatic amebiasis [156, 157]; on the other hand, reinfection is frequent in spite of the presence of antibodies [156]. In addition, amebas from a dysenteric stool have been observed to be refractory to lysis by the patient's own high-titer immune serum [210], although cultured amebas can by lysed readily. (See discussion of complement and antibodies.) The obvious ineffectiveness of antibodies detected by the usual serologic techniques does not rule out the possibility that protective antibodies do exist, perhaps in the form of antibodies to masked antigens or to antigens that are not permanently expressed and thus do not induce antibodies regularly; alternatively, minor subpopulations, the titers of which do not parallel total antibody titers, may be protective [211]. Furthermore, cells cooperating with antibodies may be the limiting factors in antibody-dependent cellular effector mechanisms. In view of the regular presence of serum antibodies, it has been postulated that the formation of soluble immune complexes may be involved in the pathogenesis of amebiasis [53]. However, the available evidence speaks against the induction of a systemic immune complex disease. Complement levels in sera of patients with amebic liver abscesses are increased rather than lowered [61, 62]. A reported decrease was restricted to C3 in the presence of normal Clq levels [212L a situation indicating activation of complement via the alternate pathway. Nephrotic injuries have not been described. Soluble immune complexes, which could not be demonstrated earlier [3, 213] have been detected recently in a few sera [214, 214a]. In addition, some immune complexes studied have been interpreted to be complexes of antiamebic antibodies and anti-antibodies [215]. However, the absence of amebic antigen was not unambiguously proven under the conditions used. The finding of amebic antigen in addition to antibodies to E. histolytica in immune sera [216, 217] may be a further indication of the formation of antigen-antibody complexes that need not necessarily lead to immune damage, but may be involved in antigen elimination and immunoregulation. In hepatic amebiasis, antibodies to E. histolytica have been detected directly in the pus of hepatic abscesses [62, 170,218]. The abscesses contain also amebas [55, 129], amebic antigens [129, 217, 219, 220], and complement [62]. Thus, CF immune complexes may form locally and give rise to an Arthus-like reaction. The finding of much lower titers of complement in the abscesses than in serum [62] argues for strong complement activation by either the classic antibody-dependent pathway [82, 221, 222] or the alternate pathway [83, 84]. A consequence may be infiltration by neutrophils, which has been observed in hepatic amebiasis [72, 73]. Together with the cytotoxic effects of the amebas, this event could contribute to the necrotic process, which has much in common with other immune complex-induced injuries [223]. Immediate-type hypersensitivity and 19B. Immediate-type hypersensitivity (ITH) to E. histolytica antigen occurs frequently in sensitized persons [142, 171, 224, 225]. This finding has led to the hypothesis that local anaphylactic reactions are involved in the pathology of intestinal amebiasis [53]. As in helminthic infections [ ], the binding of amebic antigen to mast cells via IgE antibodies could lead to degranulation, to release of vasoactive substances, and to an increase in intestinal permeability. Invasion by E. histolytica could thus be facilitated. Experimental support for such a mechanism is expected to come from studies on IgE and immune and inflammatory

8 Immunology oje. histolytica 1161 cells in intestinal lesions. Although such studies have been initiated, however, no conclusions can be drawn as yet. A major problem arises from helminthic infections, which are frequently endemic in the same regions as amebiasis. Helminths are known to induce extremely high levels of specific as well as nonspecific IgE and to potentiate IgE responses to unrelated antigens [ ]. Consequently, it is difficult to evaluate whether IgE is induced by E. histolytica alone or only in connection with helminthic infections. Early studies disclosed various degrees of skin reactivity to amebic antigen in patients with amebiasis and in controls [ ]. The discrepancies may have arisen from nonspecific, nonimmune reactions caused by the use of antigen concentrations that were too high and of heterogeneous antigens extracted from feces, liver abscesses, and mixed cultures. However, such reactions can now be avoided by the use of low concentrations of axenic antigen [236]. Immediate-type hypersensitivity is found in most patients with symptomatic amebiasis and in various proportions of patients with asymptomatic infection, but rarely in controls [142, 171, 224, 225] (table 3). Because this response seems to persist for a long time, control groups in endemic areas may also show positive reactions [171, 224]. In order to reveal the class of antibodies mediating ITH, PCA experiments were performed [142, 195]. The heat lability of the skin-sensitizing activity argues in favor of IgE as the antibody class [195]. On the other hand, sera of only a few sensitized persons had the capacity to induce PCA in monkeys [142], which are known to be sensitive to human 19B. This failure may have been due to a lower level of sensitivity of monkey than of human skin [237]. Performance of Prausnitz Kuestner tests could clarify this point. Unfortunately, direct measurements of specific antiamebic IgE in patients with symptomatic amebiasis are still lacking, but in endemic areas low titers have been found in sera of persons with mixed infections of helminths and amebas [238, 239]. Furthermore, Revoltella et al. demonstrated antiamebic IgE in the sera of persons with mixed infections of helminths, Entamoeba coli (a nonpathogenic ameba), and E. histolytica [240]. Statistical analysis revealed a complex relationship between specific and total levels of IgE and parasitic loads. Levels of specific IgE antibody to Table 3. Immediate-type skin reactions in amebiasis. Percentage of persons in indicated group with ITH* Symptomatic amebiasis Asymptomatic Reference amebiasis Intestinal Hepatic Control 142 ND ND 91 ND ND ND 6 * ITH = immediate-type hypersensitivity; ND = not done. all parasites studied were proportional to total levels of 19B. In addition, a correlation was noticed between the titers of IgE antibody to E. histolytica and those to E. coli and between the titers of IgE antibody to E. histolytica and those to two intestinal helminths. These correlations could be explained by partial cross-reactivity and by correlated infection rates. Other factors must playa role in the induction of antiamebic IgE, however, since not one serum contained antiamebic IgE in the absence of anthelminthic IgE. In addition, increasing helminthic loads were associated with increasing levels of specific antiamebic 19B. However, the reverse - an increase in levels of anthelminthic IgE with an increase in E. histolytica load-was not found. This fact could mean that nonspecific induction of antiamebic IgE by the helminths indeed plays a role. On the other hand, an increase in the permeability of the intestinal epithelium due to mechanical or anaphylactic damage by the helminths [228, 241] could have a similar effect: an increased uptake of amebic antigen and an increased synthesis of systemic antiamebic 19B. Furthermore, the conclusions of Revoltella et al. [240] depend on the reliable determination ofthe parasite load in feces, which is difficult in amebiasis [242]. If helminths did exacerbate amebiasis, a high rate of symptomatic amebiasis would be expected in persons who harbor helminths in addition to E~ histolytica. This pattern has been noticed frequently [67, ]. Although the rate of infection by E. histolytica has been found not to be increased significantly in the presence of intestinal helminths [248], the rate of invasion would be a more relevant criterion. The levels of total IgE in sera of patients with

9 1162 Trissl symptomatic amebiasis were studied carefully by several workers [189, 249, 250]. Patients with symptomatic amebiasis but without helminths had IgE levels up to 10 times higher than those of control groups [189, 249]. An increase of IgE from normal to elevated levels was observed early after onset of symptoms [249]. Since normal levels of IgE were observed in control groups infected by intestinal protozoa other than E. histolytica, a solely helminthic origin of the IgE increase seems improbable [249]. On the other hand, normal IgE levels have been reported in Brazilian patients with amebiasis [250]. In other protozoan infections normal levels are regularly found [249, 251, 252]. Thus, the subject remains controversial. Further studies in populations free of helminths, like those of Saskatchewan in Canada [253], might be useful in determining the influence of helminths on IgE levels in amebiasis. Although levels of IgE have not yet been measured in patients of that region, immediate-type skin reactions to amebic antigen are frequent [171]. Until now, IgE has been sought in serum only. Since this antibody class is preferentially produced near mucosal surfaces and in regional lymph nodes [ ] and since local and systemic levels of IgE are not correlated [256, 257], an evaluation of the local IgE response in amebiasis is important. However, neither IgE-producing cells nor local IgE levels have been studied, and information on the target cells of IgE - the mast cells and basophils - is missing. This research gap may be due to the difficulties of detecting these cell types by commonly used fixation and staining techniques [258]. However, reactions induced by the binding of IgE to mast cells may be indicated by eosinophils and their reaction products, the Charcot-Leyden crystals [56, 65, 68, 70, 71, 74, 78, 259]. The possible contribution of helminthic infection to the recruitment of eosinophils cannot be excluded [78]. Delayed Hypersensitivity Delayed hypersensitivity (DH) to E. histolytica has been measured by delayed skin reactions in vivo and by MIF production of peripheral blood cells in vitro. In addition, blast transformation, as detected by ph]thymidine incorporation, has been used as an in vitro correlate of antiamebic DH. Although this reaction frequently parallels Table 4. Cellular immune reactions in amebiasis. Reaction of persons in indicated groupt Intestinal Hepatic Reference Test amebiasis amebiasis Control 142 OH OH OH NO OH NO 8, OH NO 16,64 NO 120 MIF MIF NO 13, 53 < BT NO BT NO BT NO BT OH delayed hypersensitivity (skin reaction); MIF = macrophage migration inhibition factor; and BT = blast transformation. t OH results are expressed as the percentage of persons with positive reactions; MIF results as the mean percentage of migration inhibition; and BT results as the transformation index (cpm in the presence of antigen/cpm in the absence of antigen). When two values are given, the first is that obtained at the time of admission to the hospital and the second is that obtained after cure. NO = not done. DH, it is not always correlated but is rather an expression of cell proliferation in general. Delayed hypersensitivity to E. histolytica antigens is induced only under certain conditions. In endemic areas some patients generate an erythematous skin reaction hr after intradermal injection of amebic antigen [142, 200, ]. This reaction is accompanied by a typical infiltration of skin sites by mononuclear cells [260, 262]. Table 4 shows that the percentage of responding persons is rather low. Large variations have been found in blast transformation and MIF production (table 4) [119, 120, 200, 261, 263, 264]. The discrepancies may be technical in nature, since, for instance, different antigen preparations have been used and one amebic fraction has been shown to be more potent than commonly used amebic extracts [120]. On the other hand, a phenomenon that is most relevant to the evasion of the immune defenses by the parasite may be responsible: retarded onset of DH [200, 262]. The authors found that results of skin tests and tests for MIF production were negative shortly after the onset of symptoms and before the initiation of therapy, but became positive after patients were

10 Immunology ofe. histolytica 1163 discharged from the hospital [200, 262]. The results of serologic tests, however, were positive from the beginning [200]. From the available data, it is not clear whether the initial unresponsiveness was specific for amebic antigen. The unrelated antigens streptokinase-streptodornase and purified protein derivative, to which a high percentage of persons are sensitized, elicited a normal DH reaction in patients with amebiasis at the time of admission to the hospital [200, 260, 262]. Furthermore, the polyclonal stimulation of lymphocytes by the mitogen phytohemagglutinin (PHA) was normal in patients with intestinal and hepatic amebiasis [197]. These results argue in favor of a specific unresponsiveness to amebic antigen. In contrast, a nonspecific impairment in hepatic amebiasis is indicated by a reduced blastogenic response of blood cells to the T-cell mitogens Con A and PHA [261, 264, 264a]. The corresponding cellular basis could be the lowered number of responding T cells in blood [ a], which was correlated with the reduced blastogenic response to PHA and to amebic antigen [264]. Reduced T-cell numbers and blastogenic responses have not been measured in other studies, however [119, 262]. In summary, it seems justified to assume that, in comparison with the antibody response, DH is retarded, but that it does develop during recovery from amebiasis. The mechanism of retardation is still not known and may be specific or nonspecific. As pointed out by Phillips and Colley [266], in severely ill persons immunologic mechanisms may work under suboptimal conditions, thus producing a delayed response. Furthermore, malnutrition retards the DH reaction [267]. A compartmentalization of specifically activated immune cells at local sites simulates an unresponsiveness if measured in peripheral blood cells [268]. Two mechanisms by which E. histolytica may induce immunodepression are supported by some experimental evidence: (1) The cytopathogenic and phagocytic activity of trophozoites [43-52] may lead to an elimination of immune cells. (2) The mitogenicity of amebic constituents, which affects T rather than B cells [208, 209], may result in an exhaustion in the supply of the required cells. Apart from these mechanisms, several others (e.g., antigenic competition and lymphocyte suppression) are assumed to induce immunosuppression in other parasitic infections [205] but have not been studied in amebiasis. In addition, the regular presence of antibodies suggests that antagonistic effects of humoral and cellular immunity may playa role [ ]. Autoimmune Reactions Intestinal amebiasis has many aspects in common with ulcerative colitis [259, 273, 274], of which a prominent feature is autoimmunity to intestinal tissue antigens. In fact, in some patients with amebic dysentery, circulating antibodies to colon antigens have been detected [275, 276]. The finding that these antibodies are mainly confined to patients with chronic amebiasis [276] indicates that prolonged exposure to the parasite may evoke this response. One study disclosed the production of circulating antibodies to human liver during hepatic amebiasis [277]. The titers of these antibodies paralleled those of antibodies -to E. histolytica. Studies on the significance of antihepatic antibodies have been restricted to the demonstration of such antibodies in rabbits immunized with amebic homogenates [277]. The possibility cannot be excluded, however, that liver antigens in the amebic culture medium are responsible for the production of these antibodies [91, 92]. The role of autoimmunity in amebiasis remains unknown. An involvement ofautoimmunityin tissue injuries has not been demonstrated. Thus, there is no experimental support for autoimmunity as a cause of pathologic events in amebiasis. Treatment with corticosteroids, which usually is beneficial in diseases with a similar etiology, may have fatal consequences in amebiasis [190, ]. Effector Mechanisms in Vitro Research during the last decade has disclosed the great diversity of anti parasite effector mechanisms [ ]. Few of these mechanisms have been studied and found to be reactive against E. histolytica in vitro. Their effectiveness in vivo remains to be demonstrated. Complement and antibodies. E. histolytica activates complement, as do a variety of other parasites [288]. Normal human serum inhibits the growth of amebas in culture [221] and kills trophozoites in vitro [289]. These effects may be

11 1164 Trissl attributable to the activation of the alternate pathway of complement [83, 84]. In the presence of specific immune serum, the classical antibodydependent pathway of complement is activated; the results are rapid death of trophozoites and more effective inhibition of cultural growth [82-85, 221, 222]. Cytotoxicity is abolished by absorption of immune sera with live trophozoites or with amebic extracts [290]. Recent experiments in hamsters have yielded preliminary evidence of protection against amebic invasion by complement in vivo [291]. Decomplementation by cobra venom factor resulted in an exacerbation of hepatic lesions in hamsters. Although complement may exert its protective effect in this situation by killing the parasite directly via the classic or alternate pathway, indirect effects - via recruitment of inflammatory cells or the recently detected solubilization of immune complexes [292, 293] - are possible as well. Under some circumstances antibody may actually interfere with antibody-induced lysis by complement. Initial experiments have shown that trophozoites become immobile and spherical in the presence of heat-inactivated immune sera but regain their motility after some time (79, 95]. IgO antibodies are responsible for these complementindependent changes [96, 194, 294]. In recent years these reactions have been studied in detail. Cross-linking agents like antibodies and Con A bind to the amebic surface and induce the redistribution of surface components into patches and dense caps [51, 93, 97, 128]. The complexes are released from the cell surface as membranecontaining caps, as amorphous sheets, or as soluble material [97, 295, 296]. In addition, part of the bound material is internalized [296, 297]. Cap formation requires a critical antibody concentration [97] that is not reached in all immune sera, since cross-linking of antibodies by anti-antibodies is sometimes necessary [93]. In the case of Con A, increasing concentrations result in the release of amorphous material instead of caps [295]. Repeated exposure of amebas to antiserum within hours results in repeated capping [97]. If amebas are cultivated in the presence of antiserum, they lose their susceptibility to immobilization [298] and to antibody-dependent lysis by complement [299]. The mechanism of this process, which may help the amebas to evade damage by antibodies, has not been elucidated. Besides the parasite-protecting effects of antibodies, adverse effects on the parasite have also been observed [221, 222]. Cultural growth is partially inhibited by heat-inactivated immune sera as well as by the immunoglobulins isolated from such sera. These inhibitory effects may be based on an inhibition of feeding, since the phagocytosis ofred blood cells is inhibited by immune sera [300]. In summary, ameba trophozoites may be lysed by complement or by antibody plus complement, but may escape from lysis by shedding antibodies from the surface. The relative expression of these three types of reaction in the presence of various concentrations of antibody and complement has not been evaluated. This information would be relevant to the situation in vivo, where - except on secretory surfaces [301]-antibody and complement are present together. Cellular mechanisms. Studies on peripheral blood cells of patients with amebiasis have uncovered some evidence that cell-dependent mechanisms may operate against E. histolytica. In one study [302] amebas were readily killed by lymphocytes of patients with cured amebic liver abscess; in contrast, normal cells or cells from patients with acute amebiasis were not cytotoxic, but were phagocytosed by the trophozoites. A lymphotoxin-like substance [303] may have been involved, since contact between cells was not necessary for cytotoxocity and supernatants of lymphocytes that had been incubated with amebic antigen were equally effective. Similar cytotoxicity was found by other investigators [304], but the general occurrence of this mechanism against E. histolytica has not been established. Antibody-dependent cellular cytolysis may play a role in amebiasis. An adhesion of trophozoites to leukocytes of immune blood has been observed [305]. Furthermore, red blood cells coated with amebic antigen and tagged with StCr are lysed by human immune serum and mononuclear cells from normal blood [304]. On the other hand, direct damage to trophozoites has not been shown so far; Stemberger found that stcr-tagged target amebas were not lysed by immune serum plus complement or by immune serum plus normal blood cells [304]. The reason for this finding is not clear, since complement-dependent lysis has been demonstrated repeatedly by other investigators. The evasion mechanisms described earlier may playa role in this situation.

12 Immunology ofe. histolytica 1165 An immunologically nonspecific mechanism, which nevertheless may be under immunologic control, was studied by Guerrant et al. [306]. Human neutrophils killed E. histolytica in a process independent of oxygen, complement, or specific antibodies; however, only amebas of low virulence were killed, whereas those of high virulence were resistant. Further characterization of cells and soluble factors involved in cell-dependent killing of amebas will be necessary for the elucidation of the mechanisms. Immunoresistance, Acquired Immunity, and Evasion As described earlier, humoral and cellular immune reactions to E. histolytica are undoubtedly induced in humans. However, it is questionable whether these reactions can cause protection. As in infectious diseases in general, it is difficult to prove acquired immunity from epidemiologic data. In the analysis of older surveys, problems arise from difficulties in reliable diagnosis at a time when serologic methods were not available [2]. A further difficulty involves the gathering of data on appropriate control groups. Two aspects of the protective capacity of the human immune system in amebiasis should be considered separately: (1) the ability of the host to respond to an infection by restricting invasion by the parasite, and (2) the ability of the host to develop an immunologic memory that will more effectively hinder a second invasion. Concerning the first point, Kagan has stressed that E. histolytica normally lives as a harmless commensal within the intestine and that the defense mechanisms of the host are seldom breached [3]. The majority of infected persons do not become seriously ill, and, in the minority who do, the disease usually remains restricted to the intestine. If this resistance rests on an immunologic basis, the invasiveness of E. histolytica presumably must be enhanced in situations of reduced immunocompetence. Examples that support this view will now be described. Corticosteroids are known for their immunosuppressive and antiinflammatory effects. Although a controlled study of amebiasis in corticosteroid-treated persons has not been done, some relevant information is available. Several authors have suspected that these compounds provoke an increase in the virulence of E. histolytica since asymptomatic and undiagnosed amebiasis is dramatically exacerbated during corticosteroid treatment [190, , ]. Similarly, hormonal changes may influence resistance during pregnancy, when a variety of immunologic functions are impaired [310, 311]. Lower serum levels of total immunoglobulins have been measured in pregnant than in nonpregnant women with intestinal amebiasis [173]. On the other hand, a high frequency of specific serum antibodies to E. histolytica [312] and a high mortality due to amebiasis [191, 192] indicate an enhancement of the invasiveness of E. histolytica in pregnant women. In Nigeria, pregnancy, more than any other condition, has been shown to predispose to colonic amebiasis [190]. Infants are also especially susceptible to fulminating forms of amebiasis [56, 67, 190, 313, 314]. The generally high level of susceptibility of infants to infectious diseases is ascribed to the immaturity of the immune system [315]. Finally, malnutrition, which may influence a variety of immune as well as nonimmune functions [267, 316], is highly correlated with symptomatic amebiasis. For instance, malnutrition was found in conjunction with of cases of amebiasis in an autopsy study [5] and in more than 90% of patients with amebic liver abscesses [67, 317]. In all of these situations, parameters indepen.dent of the immune system are obviously influenced. In addition, however, impairment of the host's protective mechanisms in these conditions conceivably may alter the balanced relationship between the host and the parasite, with the change favoring the parasite. The protective immune mechanisms by which the invasion of E. histolytica may be restricted are not known. At present, it is unclear whether the effector mechanisms that kill amebas in vitro are equally effective in vivo. The high frequency of invasion and reinfection despite high antibody titers [156, 157] are evidence against effective protection by antibodies. The shedding of antigen-antibody complexes may result in the parasite's evasion of complement lysis. Although material resembling such complexes has been found in intestinal lesions [219, 318], it cannot be differentiated from antigens released from dead or live amebas. A masking of surface antigens by host material, as is found in schistosomiasis [266], is possible since

13 1166 Trissl such medium components as human and bovine serum proteins absorb strongly to the amebic surface [91, 92]. The finding of a more prominent surface coat on trophozoites from intestinal and hepatic lesions than on those from cultures [ ] may be an additional indication of absorbed host material. A low level of immunogenicity of amebic surface antigens [81, 89], as discussed before, is probably not responsible, since antibodies to surface antigens are in fact induced. Information on cell-dependent effector mechanisms is too scanty for evaluation of their role in the restriction of amebic invasion. The retarded induction of DH, MIF, and cytotoxic lymphocytes may indicate that E. histolytica can inhibit cellular immunity and thus escape its effects. Different opinions exist about the ability of humans to manifest an immunologic memory that results in resistance after infection. Most authors question the development of protective immunity in amebiasis [3, 39, 54, ], but others assume that severe invasive amebiasis does result in immunity [ ]. Unfortunately, controlled epidemiologic studies are not available. Beaver et al. [329] found that two of three persons who had spontaneously recovered from an intestinal infecton with nonpathogenic E. histolytica-like amebas were susceptible to reinfection one year later. Krupp concluded from serologic and epidemiologic studies that in intestinal amebiasis reinfection occurs frequently within months after recovery [156]. Furthermore, the recurrence of liver abscesses within months or years has been observed [326, 330, 331]. The low percentage of recurrence of amebic liver abscesses in a Mexican hospital (0.30/0) has been taken as an indication of protective immunity [326]. If this point of view were correct, the probability of a control group's contracting a first amebic liver abscess would be much higher than the probability of their contracting a second abscess. This does not seem to be the case; a frequency of first abscess of 0.1 % in Mexico City can be estimated from data presented by Kagan [3]. Furthermore, results of studies on the age dependence of symptomatic amebiasis do not support the hypothesis of protective immunity. In endemic areas, where reinfection may occur constantly, protective immunity would be expected to lead to a decrease in the frequency and/or the severity of infectious diseases with age. Such a mechanism has been assumed to operate in other parasitic diseases [266, ]. The contrary is found with regard to amebiasis in several highly endemic regions. Morbidity and mortality due to E. histolytica increase up to the age of years [190, 335, 336] and years [5, 55], respectively. This pattern has been attributed to hormonal influences [38]. No matter what the underlying mechanism, however, such increases in the frequency of severe disease with age argues against effective immune resistance acquired as a result of E. histolytica infection. The Immune Response in Animals Animal Models Many of the questions arising from immunologic studies of clinical amebiasis may be better approached by studies in animal models. Very few such studies have been done so far, perhaps partially because the animal models, while suitable for studies of pathogenicity and chemotherapy, are not satisfactory for immunoparasitologic studies. Required characteristics of a good model for such studies would be (1) that the animal can be infected easily by the natural route, (2) that the reaction of the animal to infection is similar to that of humans, and (3) that the characteristics of the immune system of the animal are well known. These criteria are only partially fulfilled by the animal systems commonly used to study the immunoparasitology of E. histolytica. Amebiasis is essentially a disease of humans; as a zoonosis, it is rare [337, 338]. Natural infections with E. histolytica in monkeys and pigs are asymptomatic; those in dogs and rats can be symptomatic [337, 338]. In those animal systems used for experimental infections, the degree of lesion formation depends mainly on the strain of E. histolytica, the type of culture, the age of the animals, and the route of infection. As is summarized in table 5, three main animal systems have been used: the intracecal infection of young rats [373] and guinea pigs [357] and the intrahepatic infection of hamsters [342] by E. histolytica trophozoites. In the absence of a reliable measure of amebic infection in live animals [373, 375], cecal or hepatic criteria including the presence or absence of amebas, the size and number of lesions, and the appearance of the tissue and the intestinal con-

14 Immunology ofe. histolytica 1167 Table 5. Virulence of pathogenic Entamoeba histolytica in animal models. Host, age or stage Type of culture Route of infection* Virulence] Reference(s) Hamster Lactating or weanling Axenic/mixed ic High 339,340 Lactating or weanling Axenic/mixed ip High 340, 341 Newborn to adult Axenic/mixed ih High 23, 24, 115, 116, 340, Guinea pig Lactating to adult Monoxenic/mixed ic High 32, 114, 118, Lactating Axenic ic None 22 Newborn Axenic ic High 369 Adult Monoxenie/mixed ih Low Rat Adult Mixed ie/rectally Low 373, 374 Weanling to young Mixed ie/rectally High 28, 29, 373, Mouse Adult Monoxenie/mixed ic Medium to high 356, Adult Axenic/monoxenie ih Low 384 * ie = intracecal; ih = intrahepatic. t Virulence was assessed by the frequency of lesion formation. tents [28, 341, 373] must be assessed at necroscopy. The reactions of rats are the most similar to those of humans; in these animals infections with strains of E. histolytica from symptomatic patients are more severe than those with strains from asymptomatic patients [29-31, 377]. Guinea pigs react more vigorously, developing lesions during infection with strains from asymptomatic patients [32, 33]. The lesions induced in guinea pigs resemble those found in biopsy material from patients with intestinal amebiasis [68-71]. These lesions consist mainly of necrotic tissue surrounded by edematous and hyperemic areas and are infiltrated by neutrophils, which in necrotic areas show degranulation and degeneration [ ]. Amebas migrate from intestinal lesions to the liver, from which trophozoites have been cultivated up to three weeks after intracecal infection [358, 359, 385]. In guinea pigs, in spite of the presence of amebas in the liver, abscesses do not develop [358, 359, 370, 385] and can be induced only in animals with long-standing intestinal infections [386]. In contrast, hamsters are susceptible to the formation of liver abscesses after intracecal [339], ip [341], and direct intrahepatic infection [23, 342, 344, 387]. Since naturally occurring liver abscesses are characteristically free of bacteria [388], the use of axenic amebas is essential in experimental intrahepatic infection. Axenic intrahepatic infection usually induces only atypical granulomatous inflammation [26, 27, 344], whereas intrahepatic infection with virulent bacteria-associated amebas results in typical necrotic abscesses, with scarce inflammation [24, 27, 343, 348]. Only by infection of newborn animals [352, 389] or by infection with a liver-passaged strain [26] has it been possible to induce progressive necrotic liver lesions with axenic E. histolytica. Experimental animals are not infected by the oral (natural) route because cysts are rarely available in sufficient numbers; rather they are infected artificially by intracecal or intrahepatic injection of trophozoites after laparotomy. Thus, in addi tion to developing an infection, animals may suffer from surgical complications and may develop lesions at the site of injection [390]. Intrahepatic infection has the additional disadvantage of bypassing the local immune system of the gut, with its complex influences on.local.as well as systemic immune responses [180, 181, 391]. These responses are, however, essential aspects of the pathology and immunology of amebiasis. The importance of this point is emphasized by the finding that the level of virulence of E. histolytica in intracecal infections is not correlated to its level in intrahepatic infections [20, 392] and that axenic strains that are able to invade the liver [23] are not able to invade the intestine [22]. Because it is desirable to attain a high rate of lesion formation within a short time, the animals usually infected are young, weanling, or even newborn, and the doses of trophozoites used are lethal [352, 369, 373, 374]. The relatively incompetent immune system of such animals does not allow the

15 1168 Trissl investigation of normal immune responses, and the long-term effects of E. histolytica on the immune system cannot be studied. However, amebiasis and parasitic infections in general are frequently chronic diseases in which both the host and the parasite survive and the parasite is continuously exposed to the immune system of the host [286]. The most suitable host for long-term studies in amebiasis is the rat, which has been shown to be infected and invaded for years [375]. Of the animals used for the study of amebic infections, hamsters have been investigated the least in terms of basic immunologic charcteristics; guinea pigs and rats have been well studied in this respect, and mice have been studied by far the most carefully. In many recent immunoparasitologic studies, mice have been used because of this detailed knowledge of their immune system and the availability of numerous inbred strains with distinct immunologic characteristics [193, 205, 393, 394]. The successful intracecal infection of mice with E. histolytica [356, 381, 382] opens up the possibility of using these advantages for the immunoparasitologic study of amebiasis as well. In conclusion, rats are currently the most suitable animals for immunologic studies of amebiasis because (1) natural infection and invasion occur, (2) the virulence of different strains of E. histolytica in rats is similar to that in humans, (3) long-standing intestinal infections can be induced, and (4) the characteristics of the immune system are relatively well known. However, future studies, especially in mice and rats, may one day provide us with a more convenient model in which the use of weanling animals, surgical infection methods, and bacteria-associated E. histolytica is avoided. Immune Reactions Antibody production. The production of antibody during infection and after immunization has been monitored by the usual serologic techniques in hamsters and rabbits [41, 353, 355, 395, 396]. As in humans, invasion results in the synthesis of antibody in parallel with abscess formation [41, 353, 355]. In intrahepatically infected hamsters, titers of serum antibody rise from nine days up to four weeks after infection [41]. Animals that develop the largest abscesses usually have the highest titers of serum antibody [355], and those that are resistant to abscess formation have low titers [41, 353]. These primary responses to E. histolytica infection thus do not indicate a protective function of antibodies. Unfortunately, the kinetics of secondary responses to E. histolytica infection in immunized animals have not been studied, but normal primary and secondary responses of IgM and IgG are induced by immunization of rabbits with trophozoites in complete Freund's adjuvant (CFA) [395, 396]. The production of secretory or anaphylactic antibodies has not been studied in experimental amebiasis. Cellular immune reactions. Reports on cellular immune reactions in animals with amebiasis are still scarce. The results are seldom clear, perhaps partly because of complications with bacteria-associated amebas. The production of MIF was studied in peritoneal cells and spleen cells of intrahepatically infected hamsters [349, 397]. Cells responding to amebic antigen were found for several weeks after infection, but a clearcut temporal relation corresponding to the initial depression seen in humans was not apparent. In these experiments, strong splenomegaly was detected [397]; this effect is also observed occasionally in human amebiasis [398]. Splenomegaly accompanies the growth of liver abscesses in hamsters [116], but the cells involved-as well as their function in pathology or immune protection- have not been investigated. A protective effect of cellular immune reactions is indicated by the experiments of Karapetian et al. [374]. Peritoneal cells of adult rats that were resistant to amebic intestinal infection [373, 374] produced high concentrations of MIF after intracecal infection. In contrast, MIF activity was suppressed in animals that were susceptible to invasion after sensitization with amebic antigen [374]. (Sensitization will be discussed later.) Cellular reactions to E. histolytica are also likely to occur during transient liver infection of guinea pigs, although in this situation the influence of bacteria from amebic cultures is unknown [372]. In intrahepatically infected animals, blast transformation parallels the appearance and disappearance of lesions. In contrast, MIF is detected only when the lesions are healing. The immunization of guinea pigs with amebic antigen in CFA results in DH, with delayed skin reactions, typical cell infiltration at the site of antigen injection, and MIF production [399]. Adjuvant does not seem to be

16 Immunology ofe. histolytica 1169 necessary for such a DH reaction; similar reactions can be elicited in mice immunized intradermally with trophozoites alone (author's unpublished observation). Results of a study of mesenteric mast cells and eosinophils in intracecally infected mice [381] give some support to the hypothesis that anaphylactic reactions are involved in amebic infections [53]. Ulceration of the intestine, which became apparent on day 9 after infection, was paralleled by a considerable increase in numbers of both mesenteric mast cells and blood eosinophils. In addition, degranulation of mast cells was more marked in infected than in control animals. Protection by active immunization. In contrast to the unsettled question of acquired immunity in humans, protection of various animals against E. histolytica has been achieved repeatedly [ , 118, , 350, 354, ]. This success may be due to a higher level of resistance in animals, in which natural invasion is seldom encountered. Protection against intestinal infections has been studied in dogs, rats, and guinea pigs. In early experiments, dogs were infected intrarectally with E. histolytica from intestinal aspirates [400, 401]. Most of the dogs became refractory to further infections for a period of weeks to months. On the other hand, only poor protection was observed in rats immunized with amebic extracts [403]. Guinea pigs were rendered immune to intracecal infection by immunization with whole trophozoites [402] or with soluble extracts and fractions of amebas in CFA [114, 118]. A fraction of high molecular weight was most effective and resulted in complete resistance [114, 118]. Serum antibody titers measured after immunization and prior to infection were not correlated with protection [114, 118]. The possible protective function of cellular immune reactions induced under similar conditions [399] has not been studied. Resistance to direct intrahepatic infection can also be achieved in the hamster system. Methods of immunization have included the prior intrahepatic infection (cured by metronidazole) and the injection of whole trophozoites, soluble extracts, and various fractions [115, 116, , 350, 354]. As in guinea pigs, a correlation of the presence of antibody with protection has not been observed [116, 346]. Protection by passive immunization. The re- suits of only a few studies on passive immunization have been published [348, 378, 401]. The findings indicate that partial protection is possible, but conclusions with respect to the mechanisms cannot be drawn. In early experiments with a small number of dogs, animals immunized with whole blood from immune donors contracted an infection less frequently (30070) than control animals (85070) [401]. Protection was short-lived; some dogs were susceptible to infection one to two months later. Partial protection was also attained in some hamsters via immunization with massive amounts of human immune serum [348]. Seven days after infection, the immunized animals developed small atypical abscesses with granulomatous inflammation. In comparison, the infected control animals developed heavy necrotic lesions. Trophozoites were found in the livers of both groups. Thus, necrosis was inhibited by immunization. It would be interesting to know the effect of normal serum alone in this situation, since complement seems to be protective [291]. If, on the other hand, antibodies are responsible for the reduction in necrosis, then a transfer of homologous hamster immune serum would have a similar protective effect. These experiments have not yet been reported. Recently, Ishaq et al. demonstrated that adoptive transfer of spleen cells from intracecally infected rats can confer partial protection to syngeneic recipients [378]. The cecal scores ofimmunized and infected animals were about one third as high as those of control animals. Since spleen cells usually contain all cell types necessary for immune responses, no conclusion as to the mechanism of protection can be drawn. Sensitization to amebic invasion. Animals can be sensitized to invasion by and infection with E. histolytica under certain conditions. An immunologic basis-e.g., immunosuppression, tolerance, or allergy induction - may be postulated, but few data support this view. Guinea pigs usually resistant to the formation of liver abscesses develop heavy lesions when immunized with amebic antigen or infected intracecally for long periods [386, 392]. Sensitization to intestinal infection and invasion, with liver abscesses in some animals, can also develop under these c;ircumstances [363, 367]. The presence of immobilizing antibodies in this situation indicates a sensitizing rather than a protective role for an-

17 1170 Trissl tibodies to surface antigens [363]. A suppression of MIF activity in adult rats immunized with amebic extracts has been shown to coincide with an increased susceptibility to intestinal invasion by amebas [374]. On the basis of available data, the conditions resulting sometimes in protection and sometimes in sensitization are difficult to understand; however, local and systemic immune responses have been found to influence each other in a complex way [186, 391]. Depending on the type and concentration of immunogen, the use of adjuvant, the immunization schedule, and the route of administration, either tolerance or responsiveness may result. The variation of such parameters in amebic infections may lead to a better understanding of the phenomenon of sensitization and its role in the induction of invasion. Experiments have shown that the susceptibility of mice to intracecal infection can be increased by concomitant infections with intestinal helminths like Schistosoma mansoni and Syphacia obvelata [383, 404]. This observation is in accordance with the frequent coincidence of intestinal helminthic infection and invasive amebiasis in humans [67, , , 247]. Besides mechanical damage, local hypersensitivity may facilitate invasion by E. histolytica. However, data supporting this view are not available. Mice have been sensitized to intracecal infection with massive amounts of egg white [379]. This result was ascribed to the induction of tolerance by the heterologous antigen, but immunologic studies were not performed to prove this assumption. Infections in immunologically incompetent animals. In accordance with experiences with clinical amebiasis, an exacerbation of experimentally induced infections by a variety of immunosuppressive measures indicates the relevance of the immune response in resistance. However, since most studies reported so far have been preliminary in character, the mechanisms of protection remain unknown. Reports of fulminating amebiasis in corticosteroid-treated persons [282, 371, 376, 405, 406] prompted several studies in animals. Amebic lesions were clearly exacerbated by hydrocortisone in intrahepatically infected hamsters and guinea pigs [371, 406]. Contradictory results were obtained in studies of intestinal infections in rats and guinea pigs [282, 376, 405]. It was concluded that corticosteroids do not facilitate invasion by E. histolytica, but rather exacerbate existing lesions [376]. After splenectomy in hamsters and rats [ ], the virulence of the nonpathogenic ameba Entamoeba hartmanni [408], that of an attenuated amebic strain [407], and that of a virulent amebic strain [409] are increased. X irradiation causes increased rates of infection [410] and deeper ulceration [376] in rat intestines. T-cell depletion by thymectomy and treatment with antilymphocyte serum results in an increase in the virulence of E. histolytica [ ]. Experiments on the latter topic emphasize the importance of T cell-dependent immune responses in resistance to invasion by E. histolytica and provide a starting point for more detailed studies of the numerous host-vs.-parasite reactions influenced by T cells [412]. Conclusions One of the central problems in amebiasis is the question of whether or not protective immunity is induced. Controlled studies comparing the invasion rates and the reinvasion rates in humans have not been reported, but the available epidemiologic data speak against protection: in endemic areas, reinfection is frequent and the morbidity and mortality due to amebiasis increase with age. In contrast, several mammals that are rarely invaded by E. histolytica under natural conditions can be protected by previous infection as well as by immunization. Evaluation of the defense mechanisms that protect these animals may lead to a better understanding of immune reactions in humans with amebiasis. Immunologic studies are complicated by the extremely complex antigen mixtures (as opposed to well-defined antigens) that must be used. The mixtures consist mostly of whole amebic homogenates and soluble extracts. Efforts to obtain more potent and less heterogeneous antigens by fractionation have been initiated. Amebic ulceration is exacerbated by immunosuppressive measures like thymectomy, splenectomy, X irradiation, and treatment with corticosteroids or antilymphocyte serum. An intact immune system is therefore necessary for restriction of inva-

18 Immunology ofe. histolytica 1171 sion by the parasite. The involvement of T cells is indicated by experiments in animals; the results of these studies probably also apply to humans. Specific serum antibodies - mainly of the IgG class - are produced regularly in response to symptomatic amebiasis. These antibodies are valuable in diagnosis and in epidemiologic studies. As has been shown in experiments in vitro, antibodies together with complement are cytotoxic to amebas. However, the serum antibodies do not seem to be protective against infection in vivo because (1) their titers are not correlated with protection, but rather with invasion, and (2) reinfection is possible in - or even enhanced by - their presence. It is not known whether these observations apply also to local antibodies found in the intestine and in liver pus. The function of the latter antibodies in pathology and protection has yet to be evaluated. Possible mechanisms by which parasites can escape the lytic effects of immune sera are the shedding of antigen-antibody complexes from the surface and the masking of surface antigens by host molecules. Although it has been suggested that low immunogenicity of surface antigens may be responsible for evasion, antibodies to these antigens are in fact produced. The coincidence of the development of lesions with an increase in the titer of serum antibodies raises the question of whether antibodies are involved in the pathologic effects of amebiasis. These effects may result from tissue injuries induced by immune complexes or by anaphylactic antibodies. Symptoms of a systemic immune complex disease have not been found. On the other hand, the local situation - especially in the livermay be different in that the low titers of complement in the presence of both antigen and antibody may result from depletion by immune complexes. In addition, complement activation via the alternate pathway is possible. An Arthus-like reaction, together with the lytic effects of the amebas themselves, may contribute to the observed necrosis. The frequent finding of immediate skin reactions in humans suggests that antibodies of the IgE class playa role in human amebiasis. Specific antiamebic IgE has been demonstrated, and high levels of total IgE in serum have been reported by some (but not all) authors. Thus, doubt remains about whether IgE is induced by E. histolytica itself or by concomitant helminthic infections, which are frequently endemic in the same regions as amebiasis. The occurrence of anaphylactic reactions is supported by a study on mast cells and eosinophils in mice. The role of these reactions in pathology and resistance remains to be studied. Investigations on cellular immunity suggest that healing and resistance are more closely related to cell-mediated than to humoral immune reactions, although the effector mechanisms are unknown. In adult rats resistance to amebic invasion is correlated with the production of MIF. Furthermore, DH and the production of MIF are retarded in humans with acute hepatic amebiasis, but do appear after recovery. The specificity of this immunodepression is controversial. Some experimental evidence has.been obtained for two mechanisms by which amebas may induce immunodepression: (1) Immune cells may be eliminated by cytotoxicity and phagocytosis. (2) The supply of immune cells may be exhausted by the mitogenic effect of amebas. The inhibition of cellular immune reactions may make possible the escape of E. histolytica from the defense mechanisms of the host. References 1. World Health Organization Expert Committee. Amoebiases. WHO Technical Report Series no. 421:1-52, Elsdon-Dew, R. The epidemiology of amoebiasis. Adv. Parasitol. 6:1-62, Kagan, I. G. Pathogenicity of E. histolytica. Arch. Invest. Med. (Mex.) 5(Suppl. 2): , Albach, R. A., Booden, T. Amoebae. In J. P. Kreier [ed.]. Parasitic protozoa. Vol. 2. Academic Press, New York, 1978, p Castro, H. F. Anatomic and pathological findings in amebiasis; report of 320 cases. In C. A. Padilla y Padilla, and G. M. Padilla [ed.]. Amebiasis in man. Charles C Thomas, Springfield, Ill., 1974, p, Flores-Barroeta, F., Saavedra-Shimidzu, R., Velasco Aviles, F. Invasion de Entamoeba histolytica a diversos organos y tejidos en sujetos humanos. Arch. Invest. Med. (Mex.) I(Suppl. 1):SI29-S146, Gutierrez, G., Ludlow, A., Espinosa, G., Herrera, S., Munoz, 0., Rattoni, N., Sepulveda, B. National serologic survey. Search for antibodies against Entamoeba histolytica in Mexico. In B. Sepulveda, and L. S. Diamond [ed.]. Amibiasis: proceedings of the International Conference on Amebiasis. Instituto Mexicano del Seguro Social, Mexico City, 1976, p Dobell, C. Researches on the intestinal protozoa of monkeys and man. I. General introduction. Parasitology 20: , Dobell, C. Researches on the intestinal protozoa of mon-

19 1172 Trissl keys and man. II. Description of the whole life-history of Entamoeba histolytica in cultures. Parasitology 20: , McConnachie, E. W. The morphology, formation, and development of cysts of Entamoeba. Parasitology 59: 41-53, II. Sadun, E. H. Life cycles of common parasites of medical importance. In S. Cohen, and E. Sadun [ed.]. Immunology of parasitic infections. Blackwell Scientific Publications, Oxford, 1975, p Neal, R. A. The in vitro cultivation of Entamoeba histolytica. In A. E. R. Taylor [ed.]. Problems of in vitro culture. Blackwell Scientific Publications, Oxford, 1967, p Taylor, A. E. R., Baker, J. R. The cultivation of parasites in vitro. Blackwell Scientific Publications, Oxford, 1968, p Singh, B. N. Pathogenic and nonpathogenic amoebae. Macmillan, London, 1975, p Diamond, L. S. Axenic cultivation of Entamoeba histolytica: progress and problems. Arch. Invest. Med. (Mex.) II(Suppl. 1):47-54, Diamond, L. S. Axenic cultivation of Entamoeba histolytica. 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