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Keywords:

  • giardiasis;
  • pathogenesis;
  • mucosal immunology;
  • small intestine;
  • epithelial cells

SUMMARY

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. CLINICAL ASPECTS
  5. LIFE CYCLE
  6. PATHOLOGY
  7. MODEL SYSTEMS TO STUDY GIARDIA INFECTIONS
  8. MUCOSAL DEFENCE MECHANISMS AGAINST GIARDIA
  9. REGULATION OF ANTI-GIARDIAL IMMUNE RESPONSES
  10. INTERACTIONS BETWEEN GIARDIA AND OTHER INTESTINAL MICROBES
  11. ACKNOWLEDGEMENTS
  12. REFERENCES

Giardia lamblia (syn. G. duodenalis or G. intestinalis), the causative agent of giardiasis, is one of the most common causes worldwide of intestinal infections in humans. Symptomatic infection is characterized by diarrhoea, epigastric pain, nausea, vomiting, and weight loss, yet many infections are asymptomatic. The protozoan, unicellular parasite resides in the lumen and attaches to the epithelium and overlying mucus layers but does not invade the mucosa and causes little or no mucosal inflammation. Giardiasis is normally transient, indicating the existence of effective host defences, although re-infections can occur, which may be related to differences in infecting parasites and/or incomplete immune protection. Mucosal defences against Giardia must act in the small intestinal lumen in the absence of induction by classical inflammatory mediators. Secretory IgA antibodies have a central role in anti-giardial defence. B cell-independent mechanisms also exist and can contribute to eradication of the parasite, although their identity and physiological importance are poorly understood currently. Possible candidates are nitric oxide, antimicrobial peptides such as Paneth cell α-defensins, and lactoferrin. Elucidation of the key anti-giardial effector mechanisms will be important for selecting the best adjuvants in the rational development of vaccination strategies against Giardia.


INTRODUCTION

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. CLINICAL ASPECTS
  5. LIFE CYCLE
  6. PATHOLOGY
  7. MODEL SYSTEMS TO STUDY GIARDIA INFECTIONS
  8. MUCOSAL DEFENCE MECHANISMS AGAINST GIARDIA
  9. REGULATION OF ANTI-GIARDIAL IMMUNE RESPONSES
  10. INTERACTIONS BETWEEN GIARDIA AND OTHER INTESTINAL MICROBES
  11. ACKNOWLEDGEMENTS
  12. REFERENCES

Infections with G. lamblia are a major public health problem worldwide, yet the interactions of this parasite with the host are only poorly understood and no effective vaccination strategies exist in humans. This review will briefly summarize salient aspects of the clinical and pathologic features of giardiasis and then focus on the mucosal defences involved in controlling and eradicating Giardia infection. The reader is referred to comprehensive recent reviews on the biology of Giardia and immune responses to the parasite (1,2).

CLINICAL ASPECTS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. CLINICAL ASPECTS
  5. LIFE CYCLE
  6. PATHOLOGY
  7. MODEL SYSTEMS TO STUDY GIARDIA INFECTIONS
  8. MUCOSAL DEFENCE MECHANISMS AGAINST GIARDIA
  9. REGULATION OF ANTI-GIARDIAL IMMUNE RESPONSES
  10. INTERACTIONS BETWEEN GIARDIA AND OTHER INTESTINAL MICROBES
  11. ACKNOWLEDGEMENTS
  12. REFERENCES

Giardia lamblia is one of the most common protozoal infections of the human intestine throughout the world and a leading cause of diarrhoeal disease. An estimated 200 million people have symptomatic giardiasis worldwide, with children under the age of five at particular risk (3,4). Infection rates of close to 100% have been reported for some developing countries, whereas infections are less common in developed countries, although incidence rates of up to 7% have been observed in some areas (3). Giardia is the most common cause of waterborne outbreaks of diarrhoeal disease in developed countries (5). A rising incidence of giardiasis has been noted for children in day-care centres, which has led to the designation of giardiasis as a ‘re-emerging’ infectious disease in the developed world (4). In addition, Giardia infection is a common cause of diarrhoeal disease in domestic animals (e.g. dairy calves, dogs, and cats) associated with substantial economic losses (4).

Symptomatic infection in humans, which is only a fraction (20–80%) of all stool positive Giardia infections (3,6), is characterized by diarrhoea, epigastric pain, nausea, vomiting, and weight loss. Symptoms typically occur 6–15 days after infection and last 2–4 days. Giardiasis is self-limiting in > 85% of cases, indicating that effective host defences exist, although chronic cases occur occasionally in the absence of apparent immunodeficiencies (3,6).

LIFE CYCLE

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. CLINICAL ASPECTS
  5. LIFE CYCLE
  6. PATHOLOGY
  7. MODEL SYSTEMS TO STUDY GIARDIA INFECTIONS
  8. MUCOSAL DEFENCE MECHANISMS AGAINST GIARDIA
  9. REGULATION OF ANTI-GIARDIAL IMMUNE RESPONSES
  10. INTERACTIONS BETWEEN GIARDIA AND OTHER INTESTINAL MICROBES
  11. ACKNOWLEDGEMENTS
  12. REFERENCES

The parasite has a relatively simple life cycle (Figure 1) consisting of two forms, the infectious cyst, which is resistant to many environmental factors, and the trophozoite, which colonizes the intestinal lumen and causes disease. Cysts, as shown in Figure 2, are ingested through contaminated drinking water or occasionally food, or are acquired by person-to-person contact (5). Excystation from cysts to trophozoites occurs in response to luminal host signals encountered in the stomach and duodenum, i.e. low pH followed by elevated pH and proteases, whereas encystation requires an elevated pH and bile (1,7). Giardia mostly colonize the proximal small intestine, although a significant proportion of patients also have ileal, colon, or stomach colonization (8). Trophozoites resist removal through the bulk flow in the intestinal lumen by proliferation and attachment to the mucus and intestinal epithelium (Figure 3), but they do not invade the mucosa. The non-invasive nature of the infection and close apposition to the epithelium (in an ‘off-shore’ location) are characteristic features of the infection, and are important in the consideration of host defences against the parasite.

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Figure 1. Life cycle of Giardia. Infectious cysts are ingested from faecally contaminated water or food and release trophozoites through excystation, which is induced by exposure to gastric acid in the stomach and digestive proteases in the proximal small intestine. Trophozoites are motile and proliferate and can attach to the intestinal epithelium and the overlying mucus layers. In response to the appropriate host signals (e.g. bile acids, alkaline pH), trophozoites differentiate to cysts (encystation), which are excreted in the stool.

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image

Figure 2. Giardia cysts in stool. Faecal pellets from adult mice infected with G. muris for 7 days (a) or uninfected controls (b) were collected and homogenized in isotonic saline. Cysts were enriched by centrifugation over a 1-m sucrose cushion. Photographs were taken with a phase-contrast microscope at original magnifications of 200 × (a, b) and 400 × (a, inset), respectively. Cysts are oblong in shape and highly refractile in appearance. The dark slender rods in the background are commensal bacteria.

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image

Figure 3. Histology of giardiasis. Normal adult mice were infected with G. muris cysts. After 7 days, the small intestine was removed, and paraffin sections were prepared and stained with haematoxylin/eosin. Trophozoites can be seen attached to the epithelium (a, arrows), or reside in the mucus layers overlying the epithelium (b, arrows). The lamina propria appears normal.

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PATHOLOGY

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. CLINICAL ASPECTS
  5. LIFE CYCLE
  6. PATHOLOGY
  7. MODEL SYSTEMS TO STUDY GIARDIA INFECTIONS
  8. MUCOSAL DEFENCE MECHANISMS AGAINST GIARDIA
  9. REGULATION OF ANTI-GIARDIAL IMMUNE RESPONSES
  10. INTERACTIONS BETWEEN GIARDIA AND OTHER INTESTINAL MICROBES
  11. ACKNOWLEDGEMENTS
  12. REFERENCES

Infections with G. lamblia in humans or mice, or with Giardia muris in mice, are remarkably devoid of typical histological features. Thus, despite the often dramatic symptoms of infection, in > 95% of patients with non-specific gastrointestinal complaints and Giardia-positive duodenal biopsies, the duodenal mucosa was considered ‘normal’, i.e. without inflammation or grossly altered epithelium (8). An example of this is shown in Figure 3 for mice acutely infected with G. muris. In some human cases, villus shortening is observed, and a small fraction of patients (3–4%) with Giardia-positive duodenal biopsies have mild duodenitis with infiltration of neutrophils and lymphocytes (8). In animal models of infection, a modest increase in mucosal mast cell numbers is observed (9,10). However, the host inflammatory response is not important for disease pathogenesis and diarrhoea in the vast majority of cases. This feature can be exploited clinically, as it suggests that diarrhoea in humans in the absence of mucosal inflammation should prompt a search for Giardia.

Despite the absence of gross histological signs, giardiasis is often accompanied by subtle ultrastructural and functional changes in the intestinal epithelium. For example, infection leads to a reduction in the height of brush border microvilli (11), which is paralleled by decreased expression and activity of several digestive enzymes located in the brush border (11,12). Interestingly, the microvillus alterations are not a direct consequence of the interaction between trophozoites and the intestinal epithelium. Instead, they are mediated by host factors, since severe-combined immunodeficient mice, which lack T and B cells, do not exhibit changes in microvillus height or digestive enzymes in response to Giardia infection compared to normal controls carrying the same infectious load (11).

MODEL SYSTEMS TO STUDY GIARDIA INFECTIONS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. CLINICAL ASPECTS
  5. LIFE CYCLE
  6. PATHOLOGY
  7. MODEL SYSTEMS TO STUDY GIARDIA INFECTIONS
  8. MUCOSAL DEFENCE MECHANISMS AGAINST GIARDIA
  9. REGULATION OF ANTI-GIARDIAL IMMUNE RESPONSES
  10. INTERACTIONS BETWEEN GIARDIA AND OTHER INTESTINAL MICROBES
  11. ACKNOWLEDGEMENTS
  12. REFERENCES

The characterization of host defences against a microbial pathogen requires the use of suitable experimental model systems, since clinical studies in infected patients rarely permit detailed mechanistic investigations and are often complicated by the occurrence of co-infections and other circumstances beyond control. It is important to acknowledge that few if any model systems, in vivo or in vitro, can reproduce all aspects of the human disease caused by a given pathogen. Furthermore, the choice of specific microbial strains and experimental models commonly requires compromises to achieve maximal clinical relevance and experimental feasibility and reproducibility. Nonetheless, selected aspects of the pathogenesis of human infections can often be reproduced and studied successfully in specific experimental models.

In the case of giardiasis, many studies have focused on murine models of infection to define immune responses. Normal adult mice can be infected readily with the naturally occurring murine parasite, G. muris, which resembles morphologically the human parasite, G. lamblia. Infection is limited to the small intestine and transient, and leads to sterilizing immunity in immunocompetent mice. This is similar to the self-limiting infection of most humans with G. lamblia, which makes G. muris a valuable model for defining immune defences against Giardia. However, G. muris is not a human parasite and, despite the morphological resemblance, the molecular similarities to G. lamblia are not well characterized at present. In addition, G. muris infection causes no overt diarrhoea or other clinical symptoms in adult mice and can thus not serve as a model to study disease mechanisms in giardiasis.

In contrast to G. muris, most strains of G. lamblia do not infect adult mice, with the exception of the GS/M-H7 strain (13). Developmental factors are probably important in the natural resistance of mice to G. lamblia, as suckling mice (3 days old) are readily infected but clear infection spontaneously by 17–20 days of age (14). Such factors could encompass many different aspects of intestinal function and are poorly understood, but are not likely to involve specific immune defences since even severe-immunodeficient adult SCID mice are not infectible with certain strains of G. lamblia (L.E., unpublished data). It is possible that the intestinal microbiota in normal mice might interfere with infection (15, see below), a hypothesis that could be tested in future studies by infecting germ-free mice with different G. lamblia strains. Alternatively, mice may not possess suitable receptors or other adaptations necessary for G. lamblia to establish infection. This concept is illustrated by the invasive enteric bacterium, Listeria monocytogenes, which binds to E-cadherin on human intestinal epithelial cells as a key step for invasion into the mucosa. A single amino acid difference in the murine E-cadherin prevents uptake of L. monocytogenes into mouse epithelial cells, rendering mice naturally resistant to oral infection (16). This can be overcome by engineering transgenic mice in which the human E-cadherin is expressed selectively in the intestinal epithelium (17). These mice are susceptible to oral L. monocytogenes infection (17). Thus, if the key molecular determinants of the interaction between G. lamblia and the murine host can be identified, it should be possible, in principle, to generate mutant mice that are susceptible to infection with diverse G. lamblia strains.

An important exception to the general lack of infectibility of adult mice with G. lamblia is the GS/M-H7 strain (13). This strain was originally isolated from a patient with severe diarrhoea and was subsequently shown to be diarrhoeagenic upon oral challenge of healthy volunteers (6). Thus, G. lamblia GS/M-H7 is fully virulent for humans, which makes it a clinically relevant model pathogen. The reasons for the differences between G. lamblia GS/M-H7 and other G. lamblia strains in regard to infectibility of adult mice are not clear, but will be important to elucidate in future studies as they might suggest new pharmacological targets for drug therapy. One caveat of studies with G. lamblia GS/M-H7 is that this strain belongs to the genetic assemblage group B, whereas most molecular studies of Giardia (including the Giardia genome project) are currently conducted with the WB strain of assemblage A (18,19). Therefore, these two strains are not closely related genetically within the species complex G. lamblia. Information obtained using one of these strains will have to be translated to other strains to be applicable, although future efforts to sequence the genome of the GS/M-H7 strain could alleviate this problem.

A relevant, albeit not widely used, animal model of giardiasis is the gerbil model. Adult gerbils can be infected with different G. lamblia strains (9,20). Infection is accompanied by disease symptoms, including diarrhoea and weight loss, which makes this model relevant for defining the mechanisms of Giardia-induced intestinal disease (21–23). However, the current dearth of genetic information in gerbils and of antibody reagents against defined gerbil proteins is a serious impediment for defining disease mechanisms in this model at the molecular level.

In addition to animal models, in vitro models have been used to characterize the interaction of G. lamblia with intestinal epithelial cells (24–26). Several readily available cell lines (e.g. Caco-2, T84) show relevant features of normal human intestinal epithelium, including polarization and tight junction formation, vectoral ion transport, and regulated production of inflammatory mediators. Most of these lines are of colonic origin, whereas Giardia typically reside in the upper small intestine. However, the colon can become colonized with trophozoites in a small fraction of infected patients (8), suggesting that giardial interactions with colonic epithelial cells occur in vivo and can be physiologically relevant. In addition, small intestinal and colonic epithelial cells have more functional and molecular similarities than differences, so that many results obtained in colonic epithelial cells are likely to apply to the small intestinal epithelium. In addition, a few studies have reported the development of human small intestinal epithelial cell lines (27), and they have been used for studying Giardia : epithelial cell interactions (25,28,29). Although these applications are potentially promising, these lines are not widely available at this time and their usefulness and relevance for studies of the human small intestinal epithelium has not been generally established.

MUCOSAL DEFENCE MECHANISMS AGAINST GIARDIA

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. CLINICAL ASPECTS
  5. LIFE CYCLE
  6. PATHOLOGY
  7. MODEL SYSTEMS TO STUDY GIARDIA INFECTIONS
  8. MUCOSAL DEFENCE MECHANISMS AGAINST GIARDIA
  9. REGULATION OF ANTI-GIARDIAL IMMUNE RESPONSES
  10. INTERACTIONS BETWEEN GIARDIA AND OTHER INTESTINAL MICROBES
  11. ACKNOWLEDGEMENTS
  12. REFERENCES

The elucidation of the key mucosal defences against Giardia is guided by two central features of the infection, the luminal localization of the parasite in the small intestine, and the lack of significant mucosal inflammation after infection. The former requires that effector molecules reach the lumen in sufficient quantities to be effective. The latter suggests that inflammatory mechanisms known to be important for eradication of many enteric pathogens, e.g. neutrophils for killing of Salmonella, are not likely to be important in anti-giardial host defence. Based on these considerations, the following sections focus on the importance of specific host effector mechanisms against Giardia. Elucidation of these mechanisms is not only important for understanding mucosal immune defence against this parasite, but also provides a crucial basis in the rational development of vaccination strategies for activating the most effective host defences against Giardia.

Secretory antibodies

Secretory antibodies of the IgA and IgM isotypes are attractive candidates for immune defence against Giardia, because they are secreted in large quantities into the intestinal lumen and their actions are antigen-specific. A number of studies have been performed to define the overall importance of B cells in clearing Giardia. The majority of reports suggest that B cells are indispensable for eradicating Giardia. For example, infections of humans with G. lamblia or of mice with G. muris result in the production of anti-giardial antibodies of the IgA, IgM, and IgG isotypes in mucosal secretions and serum, and specific antibody production correlates with giardial clearance (2,6,30–32). Such antibodies reach their targets in vivo, since anti-giardial IgA antibodies coat trophozoites in Giardia-infected mice (30). Mice depleted of B cells by treatment with anti-IgM antibodies and mice with X-linked immunodeficiency, which have a defect in B cell development and function, are unable to clear G. muris infection (33,34). Furthermore, B cell-deficient mice generated by gene targeting are unable to clear infections with G. muris or G. lamblia GS/M-H7 (35,36).

In contrast, other reports using animal models have suggested that B cells have only a limited role in anti-giardial immunity. For example, mice with B cell defects due to X-linked immunodeficiency can develop acquired immunity against secondary challenge with G. muris (37). A more recent study showed that B cell-deficient mice infected with either G. lamblia or G. muris controlled infection as well as normal litter-mate controls after 4 weeks, suggesting that B cells played a limited role, if any, in controlling acute G. lamblia infection in those studies (38). The reasons for the apparent discrepancy between these and other reports are not clear, but might relate to differences in the experimental design between the studies. For example, differences in the initial Giardia inocula could lead to different peak infectious loads. B cells might be more important for controlling higher infectious burdens (36), while they play a less important role in controlling lower initial and peak infectious loads (38). Other, more indirect factors could play a role. For example, the intestinal microbiota has been shown to play a role in determining susceptibility of mice to G. lamblia infection (15). The normal murine microbiota is likely to differ between different animal facilities, leading to differential innate susceptibility to Giardia and hence relative importance of B cells in giardial clearance. In any case, these data provide strong support for the existence of B cell-independent anti-giardial host defences.

Further support for the role of B cells in anti-giardial host defence comes from studies in mice genetically deficient for IgA production (36). These mice cannot eradicate infection with G. muris or G. lamblia, although they have a limited capacity to reduce the initial infectious load over extended periods (Figure 4). In contrast, mice deficient for secreted IgM show no defect in clearance of G. muris, indicating that IgM antibodies have no indispensable functions in anti-giardial host defence (36). These findings demonstrate that IgA is central for clearance of Giardia in the murine host, but also suggest the existence of IgA-independent, presently poorly defined host defences. Taken together, the preponderance of evidence in animal models suggests that B cells have a central role in anti-giardial host defence, but B cell-independent effector mechanisms also exist and contribute to host defence to a variable degree.

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Figure 4. Importance of IgA in anti-giardial host defence. Mice deficient for IgA (open bars) and normal litter-mate controls (closed bars) were infected orally with G. muris cysts (top panel) or G. lamblia GS/M-H7 trophozoites (bottom panel). Trophozoite numbers in the small intestine were determined at the indicated times after infection (week 1 after G. lamblia infection represents data points between 4 and 7 days). Data are mean ± SEM of 10 or more mice. Asterisks indicate values significantly different from those in litter-mate controls (P < 0·05). Results are a combination of data reported in reference (36) and additional, more recent data.

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Data from clinical studies on the incidence and severity of giardiasis in patients with selective immunodeficiencies have provided conflicting results. Patients with chronic variable immunodeficiency (CVID) have an increased susceptibility to Giardia infection (39). These patients show defects in B cell functions, but they also have T cell defects, so that the increase in Giardia prevalence cannot be attributed solely to a lack of antibodies. For patients with selective IgA deficiency, some studies found an increased incidence of infections while others did not (39–41). Selective IgA deficiency is usually defined by serum IgA levels of < 0·05 g/L (normal levels are 0·5–3·5 g/L), but many, if not most, IgA-deficient patients have low but detectable levels of serum IgA antibodies (40,42) and can produce IgA antibodies at near-normal levels in vitro (43,44). Therefore, most IgA-deficient patients are only relatively deficient and can still produce low levels of IgA, which may be sufficient for controlling G. lamblia infection. This is in contrast to IgA-deficient mice, which show a total loss of IgA production due to the deletion of key gene segments coding for IgA heavy chains (45). A simple interpretation of both clinical and experimental data is that IgA is required for clearance of Giardia, but IgA levels well below normal are sufficient. Alternative interpretations are possible, e.g. anti-giardial host defences in mice and humans differ substantially in regard to IgA dependency, or IgA-deficient patients but not mice can develop compensatory host defences against Giardia infection.

The mechanisms by which IgA exerts its anti-giardial functions are not well understood, but are likely to involve ‘immune exclusion’ (e.g. immobilization or detachment of trophozoites from the intestinal epithelium or the mucus layer) rather than direct killing. Anti-giardial IgA antibodies have been reported not to kill G. muris trophozoites in the presence or absence of complement (46), although one study suggested that anti-giardial IgA may exert cytotoxic effects on G. lamblia (47).

A number of antigens have been characterized that are recognized by anti-giardial antibodies, although only a small number of studies have focused specifically on antigens recognized by anti-giardial IgA, rather than other, functionally less relevant isotypes. Moreover, although at least 20 antigenic polypeptides ranging from 14 to 125 kDa have been identified in crude extracts of trophozoites, relatively few of these have been characterized at the molecular level (2). The best-described giardial antigens are members of the family of variant-specific surface proteins (VSPs), which constitute a major fraction of surface proteins in trophozoites (48). At least 150 genes coding for different VSPs are estimated to exist in the giardial genome, of which only one is generally produced per trophozoite (49). However, in a population of trophozoites, multiple different VSPs are expressed simultaneously and at different times during the infection (48,50). VSPs are major antigenic targets but their high variability during the course of infection suggests that antibodies against specific VSPs may have a limited role in mediating eradication of Giardia from the host, since the emergence of trophozoites expressing ‘new’ VSPs would not be prevented by such antibodies. Although much has been learned about the expression of VSPs and their role as antigenic targets, their function has remained unclear. Several other giardial antigens have been identified at the molecular level, including heat shock proteins, giardins, tubulin, and chitin (2). Some of these are intracellular proteins, which raises doubts about their importance as relevant antigenic targets and rather suggests that the development of a specific antibody response might be secondary to trophozoite lysis in the host.

Nitric oxide

In addition to secretory antibodies, other effector mechanisms are likely to operate in mucosal defence against Giardia. Such defences, like secretory antibodies, have to reach the intestinal lumen in sufficient quantities to exert their effects on the parasite. This suggests that antimicrobial products synthesized by epithelial cells in the small intestine are good candidates. One of these is nitric oxide (NO), which has antimicrobial actions on a wide range of bacterial and parasitic pathogens (51,52) and has multiple other functions, including a role in neurotransmission and regulation of mucosal barrier integrity and vascular tone in the intestine (53). NO is produced enzymatically from arginine through the action of NO synthase (NOS), which exists in three isoforms, neuronal NOS (nNOS/NOS1), inducible NOS (iNOS/NOS2), and endothelial NOS (eNOS/NOS3). In many cell types, the expression of iNOS is inducible by cytokines and microbial products, and iNOS is the major NOS isoform expressed by intestinal epithelial cells (54,55). Expression of iNOS in these cells can either be constitutive, e.g. in mouse ileum (56) and isolated normal human duodenocytes (57), or inducible in vivo during intestinal inflammation (58) or in vitro by cytokines such as interferon-γ (54) or in response to infection with invasive bacteria (55,59). In polarized intestinal epithelial cells, the stable NO end products, nitrite and nitrate, are preferentially detected at the apical side (26,55), suggesting that Giardia could be a relevant target for epithelial cell-derived NO and its metabolites. The underlying mechanisms for apical NO release are not known, but might be related to the preferential localization of iNOS at the apical side of polarized epithelial cells underneath the cell membrane (56,60).

Consistent with a role of epithelial cell-produced NO as a potential anti-giardial effector molecule, NO was found to inhibit proliferation of G. lamblia trophozoites in vitro, but not to kill them (26). Thus, NO was cytostatic rather than cytotoxic for trophozoites in these studies. Another report suggested that NO can kill trophozoites in vitro (61), although that study used minimal media (i.e. buffered salt solution) for culture, which may have exaggerated the results, as such media do not support prolonged viability and growth of trophozoites even in the absence of NO. In addition, NO also inhibited excystation and encystation of G. lamblia in vitro (26). These data show that NO exhibits complex inhibitory effects on growth and differentiation of G. lamblia in vitro (as depicted schematically in Figure 5). Inhibition of growth and excystation would be expected to reduce the number of trophozoites in the intestinal lumen, whereas inhibition of encystation might have the opposite effect, i.e. increased numbers of trophozoites in the lumen. The net outcome of these opposing effects is difficult to predict at this time. Growth inhibition may be important for the infected host, because local trophozoite growth is probably crucial for the ability of G. lamblia to establish and maintain infection of the proximal small intestine. In contrast, inhibition of encystation by NO could reduce the formation and passing of infectious cysts and, thereby, transmission to other potential hosts.

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Figure 5. Model of the role of NO in the interactions between Giardia and intestinal epithelium. Intestinal epithelial cells produce NO enzymatically from arginine through the action of inducible NO synthase (iNOS). NO is released predominantly at the apical side of the cells, where it can inhibit growth of G. lamblia trophozoites, as well as encystation and excystation. On the other hand, apical arginine is required for epithelial NO production but is efficiently taken up by trophozoites and used to generate ATP. As a by-product of this reaction, ornithine is released, which competitively inhibits arginine uptake by the intestinal epithelium. Both mechanisms reduce the availability of arginine for the epithelium and thus inhibit NO production.

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Evaluation of the potential importance of NO in controlling Giardia infection is complicated by the observation that trophozoites can suppress epithelial NO production in vitro. Thus, co-culture of G. lamblia trophozoites with human intestinal epithelial cells was shown to strongly inhibit inducible NO production (26). This was not due to impaired iNOS expression, but rather caused by highly effective arginine consumption by the parasite, which limited the availability of this substrate for NO production by epithelial NOS (26). Addition of sufficient exogenous arginine reversed the Giardia induced inhibition of epithelial NO production (26). Arginine is an important source of energy for the parasite (62). Consistent with this, G. lamblia possesses a highly efficient arginine transporter system, which has a comparable substrate affinity to the mammalian arginine transporter but a 10- to 20-fold higher maximal transport capacity (63–65). Therefore, G. lamblia may have an advantage over the host in taking up arginine. This is functionally relevant since epithelial NO production appears to depend largely on apical but not basolateral or intracellular arginine availability (26). These observations demonstrate that G. lamblia has strategies to counteract epithelial NO production as a potential host defence mechanism. This places NO at the centre of several, mutually inhibitory interactions between the parasite and the host intestinal epithelium (Figure 5).

Little is known about the importance of NO in controlling Giardia infection in vivo. One report suggested that NO plays no role in clearance of G. muris in the murine host, as treatment of infected mice with the NOS inhibitor, L-NMMA, did not affect trophozoite numbers in the first 12 days after infection (10). These negative data do not completely rule out an anti-giardial function of NO in vivo. It is possible that NO production was not completely inhibited by pharmacological means in that study, or that NO might exert anti-giardial activity at later times after infection. However, NO may simply not have anti-giardial actions during the course of infection in normal mice. This would suggest that the in vitro observed NO-mediated giardial growth inhibition does not occur in vivo, or that insufficient amounts of NO are produced by epithelial cells to exert those effects. In regard to the latter, it is possible that constitutive epithelial NO production may be low under non-inflamed conditions in the small intestine, similar to those observed after Giardia infection in normal mice. However, epithelial NO production can be increased during small intestinal inflammation (57). Infections with a number of other pathogens of the small intestine, such as Trichinella spiralis, are characterized by mucosal inflammation, and co-infections with multiple pathogens are common in developing countries (66). Under these conditions, increased epithelial iNOS expression caused by one pathogen may help to control infection with another pathogen, such as G. lamblia. This concept is illustrated, for example, by the observation that infection of mice with T. spiralis, which causes mucosal inflammation and increased NO production in the small intestine (67), interferes with subsequent Giardia infection (68).

Antimicrobial peptides

Peptides and proteins with antimicrobial activity are highly conserved in evolution and appear to have an important role in innate host defence at mucosal surfaces. The defensins are one major class of antimicrobial peptides. They can be divided into two major families, α-defensins and β-defensins. α-defensins are produced by neutrophils and specialized epithelial cells, the Paneth cells, in the small intestinal crypts (69). Humans express at least two family members, human defensin (HD) 5 and HD6, in Paneth cells, whereas mice produce 20 family members, which are collectively termed cryptdins. α-defensins are stored as inactive proforms in granules in the Paneth cells. In mice, Paneth cell stimulation induces proteolytic processing through the matrix metalloproteinase (MMP)-7, and release of bioactive cryptdins into the lumen of the small intestinal crypt (70). Mice deficient in MMP-7 cannot produce bioactive cryptdins (71). In contrast to α-defensins, several β-defensins are ubiquitously expressed throughout the epithelium in the small intestine and colon, as well as other mucosal surfaces. In humans, β-defensin (HBD)-1 is expressed constitutively in epithelial cells, whereas HBD-2 is normally expressed at only low levels, but its expression can be strongly up-regulated by several proinflammatory mediators and infection with invasive bacteria (72). Another class of antimicrobial peptides are the cathelicidins, in which a cathelin domain is linked to a peptide that has antimicrobial activity. The only known cathelicidin in humans is LL-37/hCAP18, which has a restricted distribution in the intestinal tract, as its expression is limited to the differentiated surface and upper crypt cells in the colon (73). LL-37/hCAP-18 is not expressed in the small intestinal epithelium. Based on the distribution of the different antimicrobial peptides, it is conceivable that Paneth cell α-defensins and the constitutively expressed β-defensins (e.g. HBD-1) could have functions in anti-giardial host defence. In contrast, the inducible β-defensins such as HBD-2, or the cathelicidin, LL-37/hCAP18, are not likely to be relevant for controlling Giardia infection, as they are either not induced in the absence of inflammation or appear not to be expressed in the small intestine under any circumstances.

The potential importance of antimicrobial peptides in killing Giardia trophozoites has been investigated in vitro (74). This study found that cryptdins 2 and 3 killed trophozoites effectively, whereas cryptdins 1 and 6 did not, suggesting a high degree of specificity for killing despite the extensive sequence similarities between cryptdins (74). The cytotoxic activity of cryptdin 2 was lost in the presence of physiological concentrations of sodium chloride, calcium, or magnesium, which is characteristic of many defensins. This raises the question whether these molecules would be active in the intestinal lumen, given the abundant yet variable presence of these ions in the diet. However, other studies have suggested that the salt sensitivity of cryptdins may be an artifact of testing them as purified molecules, rather than in conjunction with other components normally present in Paneth cell secretions (75). In any case, elucidation of the structure/function relationship for cryptdin-dependent killing of G. lamblia might reveal new approaches for designing anti-giardial drugs, particularly since antimicrobial peptides are generally acid resistant and would be expected to be active luminally after oral administration.

No reports have been published on the physiological significance of antimicrobial peptides in controlling and clearing Giardia infection in vivo. Our laboratory found in preliminary studies that MMP-7 deficient mice, which cannot produce bioactive Paneth cell α-defensins (70), were able to control and eradicate G. muris as well as normal litter-mate controls (Figure 6). Interestingly, initial infectious load was significantly lower in these mice compared to normal controls. This could suggest that Paneth cell α-defensins affect giardial numbers indirectly by controlling the abundance and/or composition of the intestinal microbiota in the small intestine, which can inhibit giardial colonization (see below) (15). The apparent lack of inhibitory effects of Paneth cell defensins on giardial numbers may not be surprising in light of reports that G. lamblia cannot induce the release of these molecules in isolated small intestinal crypts (75). These results also suggest that Giardia might be affected by Paneth cell products under conditions when they are effectively released, such as co-infection with stimulatory enteric microbes (75). This possibility remains to be tested.

image

Figure 6. Giardia muris infection of MMP-7 deficient mice. Adult MMP-7 deficient mice (○), which cannot produce bioactive Paneth cell defensins, and normal litter-mate controls (•) were infected orally with 104G. muris cysts. The small intestine was removed at the indicated times after infection, chilled on ice, opened longitudinally, and agitated vigorously to release attached trophozoites. The number of viable trophozoites was determined in a counting chamber using a phase-contrast microscope. Data are means ± SEM of 6–8 mice for each point. The asterisk indicates a value significantly different from the control at the same time point (P < 0·05 by rank sum test). The grey line represents the detection limit of the assay.

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Other effector mechanisms

In addition to α-defensins, Paneth cells release two other major products, lysozyme and secretory phospholipase A (76), but neither of them has been tested for their anti-giardial activity. Another antimicrobial protein is lactoferrin, which is produced mainly by mammary epithelial cells and present in the breast milk. This protein and its N-terminal peptides were shown to kill G. lamblia trophozoites in vitro, which probably contributes to the protection of nursing infants from giardiasis (77). Lactoferrin can also be produced by epithelial cells in the small intestine in humans (78,79). This suggests that it might participate in mucosal defence against Giardia after weaning, but this has not been investigated.

Although most studies have focused on specific molecules with antimicrobial functions, earlier reports some 15 years ago had suggested that host effector cells might be directly involved in Giardia killing. In vitro studies showed that G. lamblia trophozoites can be ingested by human monocytes/macrophages, and then killed through an oxidative mechanism, although with an effectiveness of less than one trophozoite per macrophage (80). For such a mechanism to be functionally relevant in vivo, macrophages must be present in the lumen where the trophozoites reside. Consistent with that, different leucocyte subsets can transmigrate across the epithelium into the intestinal lumen. For example, in mice a modest number (2 × 105/mouse) of leucocytes can be recovered from the small intestinal lumen, although this number did not differ between G. muris infected and control mice (81). More importantly, the vast majority of these cells were lymphocytes, with only a small percentage being macrophages (81,82). Given that peak infectious loads in the murine host are typically > 107 trophozoites/mouse (Figures 4 and 6), this suggests that luminal macrophages are vastly outnumbered by trophozoites during the course of infection and thus not likely to be relevant for Giardia eradication. Furthermore, although greater numbers of T cells are present in the lumen, classical cytotoxic CD8 T cells are not important for clearance of Giardia, as shown in T cell subset ablation experiments (83). Taken together, little convincing evidence exists to suggest that direct leucocyte-mediated killing of trophozoites in the intestinal lumen is a physiologically relevant mucosal defence mechanism in giardiasis.

REGULATION OF ANTI-GIARDIAL IMMUNE RESPONSES

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. CLINICAL ASPECTS
  5. LIFE CYCLE
  6. PATHOLOGY
  7. MODEL SYSTEMS TO STUDY GIARDIA INFECTIONS
  8. MUCOSAL DEFENCE MECHANISMS AGAINST GIARDIA
  9. REGULATION OF ANTI-GIARDIAL IMMUNE RESPONSES
  10. INTERACTIONS BETWEEN GIARDIA AND OTHER INTESTINAL MICROBES
  11. ACKNOWLEDGEMENTS
  12. REFERENCES

The production and delivery of effector molecules against Giardia is likely to be regulated by a network of cells and mediators that are not directly involved in killing the parasite. Studies in mice lacking specific populations of cells, due to genetic deficiency or antibody-mediated ablation, have shown that CD4 αβ T cells are required for clearing giardial infection (38,83), whereas CD8 T cells and natural killer cells play no role (83,84). The key mediators produced by CD4 αβ T cells are not known, but are likely to comprise one or several cytokines important for regulating mucosal IgA responses or epithelial production and delivery of anti-giardial effector molecules. A possible candidate might be interferon-γ, which is produced by CD4 T cells and can up-regulate epithelial NO production, although mice lacking this cytokine appear to clear infection normally (38). Recent studies have suggested that IL-6, a multifunctional cytokine with IgA regulatory activity, is needed for clearance of G. lamblia in mice (85,86). Although this cytokine is not only produced by CD4 T cells but by many other cell types, the discovery of its physiological importance for controlling Giardia represents one of the first examples of a defined mediator with indirect, regulatory involvement in anti-giardial host defence.

INTERACTIONS BETWEEN GIARDIA AND OTHER INTESTINAL MICROBES

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. CLINICAL ASPECTS
  5. LIFE CYCLE
  6. PATHOLOGY
  7. MODEL SYSTEMS TO STUDY GIARDIA INFECTIONS
  8. MUCOSAL DEFENCE MECHANISMS AGAINST GIARDIA
  9. REGULATION OF ANTI-GIARDIAL IMMUNE RESPONSES
  10. INTERACTIONS BETWEEN GIARDIA AND OTHER INTESTINAL MICROBES
  11. ACKNOWLEDGEMENTS
  12. REFERENCES

Giardia is not the only microbe in its natural niche in the small intestine, since the intestinal tract is colonized by commensal bacteria and potentially other pathogenic microbes. This suggests that interactions between Giardia and other microorganisms occur and might play a role in the pathogenesis of Giardia infections. For example, one study showed that commensal bacteria can determine susceptibility and resistance of the murine host to G. lamblia infection (15). Mice from one commercial vendor were found to be more resistant to infection with G. lamblia GS/M-H7 than isogenic mice from a different vendor, and this resistance could be transferred by housing animals together (15). Furthermore, treatment of mice with the non-absorbable antibiotic neomycin made both groups of mice equally susceptible to infection. These findings raise the possibility that probiotic therapy may be useful in preventing infection or as an adjunct for treating infection (15), and might provide an explanation for differences in the infectibility of different strains of adult mice with various G. lamblia isolates. A possible mechanism for such bacteria-mediated infection resistance is suggested by another study, in which probiotic Lactobacilli were shown to release a low-molecular weight, heat-sensitive factor that inhibits proliferation of G. lamblia trophozoites (87). Although the molecular identity of this factor has not been established, a further analysis might suggest alternative therapeutic strategies for treating giardiasis.

REFERENCES

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. CLINICAL ASPECTS
  5. LIFE CYCLE
  6. PATHOLOGY
  7. MODEL SYSTEMS TO STUDY GIARDIA INFECTIONS
  8. MUCOSAL DEFENCE MECHANISMS AGAINST GIARDIA
  9. REGULATION OF ANTI-GIARDIAL IMMUNE RESPONSES
  10. INTERACTIONS BETWEEN GIARDIA AND OTHER INTESTINAL MICROBES
  11. ACKNOWLEDGEMENTS
  12. REFERENCES
  • 1
    Adam RD. Biology of Giardia lamblia. Clin Microbiol Rev 2001; 14: 447475.
  • 2
    Faubert G. Immune response to Giardia duodenalis. Clin Microbiol Rev 2000; 13: 3554.
  • 3
    Flanagan PA. Giardia– diagnosis, clinical course and epidemiology. A review. Epidemiol Infect 1992; 109: 122.
  • 4
    Thompson RC. Giardiasis as a re-emerging infectious disease and its zoonotic potential. Int J Parasitol 2000; 30: 12591267.
  • 5
    Slifko TR, Smith HV & Rose JB. Emerging parasite zoonoses associated with water and food. Int J Parasitol 2000; 30: 13791393.
  • 6
    Nash TE, Herrington DA, Losonsky GA & Levine MM. Experimental human infections with Giardia lamblia. J Infect Dis 1987; 156: 974984.
  • 7
    Gillin FD, Reiner DS & McCaffery JM. Cell biology of the primitive eukaryote Giardia lamblia. Annu Rev Microbiol 1996; 50: 679705.
  • 8
    Oberhuber G, Kastner N & Stolte M. Giardiasis, a histologic analysis of 567 cases. Scand J Gastroenterol 1997; 32: 4851.
  • 9
    Hardin JA, Buret AG, Olson ME, Kimm MH & Gall DG. Mast cell hyperplasia and increased macromolecular uptake in an animal model of giardiasis. J Parasitol 1997; 83: 908912.
  • 10
    Venkatesan P, Finch RG & Wakelin D. A comparison of mucosal inflammatory responses to Giardia muris in resistant B10 and susceptible BALB/c mice. Parasite Immunol 1997; 19: 137143.
  • 11
    Scott KG, Logan MR, Klammer GM, Teoh DA & Buret AG. Jejunal brush border microvillous alterations in Giardia muris-infected mice: role of T lymphocytes and interleukin-6. Infect Immun 2000; 68: 34123418.
  • 12
    Nain CK, Dutt P & Vinayak VK. Alterations in enzymatic activities of the intestinal mucosa during the course of Giardia lamblia infection in mice. Ann Trop Med Parasitol 1991; 85: 515522.
  • 13
    Byrd LG, Conrad JT & Nash TE. Giardia lamblia infections in adult mice. Infect Immun 1994; 62: 35833585.
  • 14
    Hill DR, Guerrant RL, Pearson RD & Hewlett EL. Giardia lamblia infection of suckling mice. J Infect Dis 1983; 147: 217221.
  • 15
    Singer SM & Nash TE. The role of normal flora in Giardia lamblia infections in mice. J Infect Dis 2000; 181: 15101512.
  • 16
    Lecuit M, Dramsi S, Gottardi C, Fedor-Chaiken M, Gumbiner B & Cossart P. A single amino acid in E-cadherin responsible for host specificity towards the human pathogen Listeria monocytogenes. EMBO J 1999; 18: 39563963.
  • 17
    Lecuit M, Vandormael-Pournin S, Lefort J et al. A transgenic model for listeriosis: role of internalin in crossing the intestinal barrier. Science 2001; 292: 17221725.
  • 18
    Thompson RC, Hopkins RM & Homan WL. Nomenclature and genetic groupings of Giardia infecting mammals. Parasitol Today 2000; 16: 210213.
  • 19
    McArthur AG, Morrison HG, Nixon JE et al. The Giardia genome project database. FEMS Microbiol Lett 2000; 189: 271273.
  • 20
    Leitch GJ, Udezulu IA, He Q & Visvesvara GS. Effects of protein malnutrition on experimental giardiasis in the Mongolian gerbil. Scand J Gastroenterol 1993; 28: 885893.
  • 21
    Buret A, Hardin JA, Olson ME & Gall DG. Pathophysiology of small intestinal malabsorption in gerbils infected with Giardia lamblia. Gastroenterology 1992; 103: 506513.
  • 22
    Deselliers LP, Tan DT, Scott RB & Olson ME. Effects of Giardia lamblia infection on gastrointestinal transit and contractility in Mongolian gerbils. Dig Dis Sci 1997; 42: 24112419.
  • 23
    Astiazaran-Garcia H, Espinosa-Cantellano M, Castanon G, Chavez-Munguia B & Martinez-Palomo A. Giardia lamblia: effect of infection with symptomatic and asymptomatic isolates on the growth of gerbils (Meriones unguiculatus). Exp Parasitol 2000; 95: 128135.
  • 24
    Katelaris PH, Naeem A & Farthing MJ. Attachment of Giardia lamblia trophozoites to a cultured human intestinal cell line. Gut 1995; 37: 512518.
  • 25
    Teoh DA, Kamieniecki D, Pang G & Buret AG. Giardia lamblia rearranges F-actin and α-actinin in human colonic and duodenal monolayers and reduces transepithelial electrical resistance. J Parasitol 2000; 86: 800806.
  • 26
    Eckmann L, Laurent F, Langford TD et al. Nitric oxide production by human intestinal epithelial cells and competition for arginine as potential determinants of host defense against the lumen-dwelling pathogen Giardia lamblia. J Immunol 2000; 164: 14781487.
  • 27
    Pang G, Buret A, O'Loughlin E, Smith A, Batey R & Clancy R. Immunologic, functional, and morphological characterization of three new human small intestinal epithelial cell lines. Gastroenterology 1996; 111: 818.
  • 28
    Chin AC, Teoh DA, Scott KG, Meddings JB, Macnaughton WK & Buret AG. Strain-dependent induction of enterocyte apoptosis by Giardia lamblia disrupts epithelial barrier function in a caspase-3-dependent manner. Infect Immun 2002; 70: 36733680.
  • 29
    Buret AG, Mitchell K, Muench DG & Scott KG. Giardia lamblia disrupts tight junctional ZO-1 and increases permeability in non-transformed human small intestinal epithelial monolayers: effects of epidermal growth factor. Parasitology 2002; 125: 1119.
  • 30
    Heyworth MF. Intestinal IgA responses to Giardia muris in mice depleted of helper T lymphocytes and in immunocompetent mice. J Parasitol 1989; 75: 246251.
  • 31
    Daniels CW & Belosevic M. Serum antibody responses by male and female C57Bl/6 mice infected with Giardia muris. Clin Exp Immunol 1994; 97: 424429.
  • 32
    Snider DP & Underdown BJ. Quantitative and temporal analyses of murine antibody response in serum and gut secretions to infection with Giardia muris. Infect Immun 1986; 52: 271278.
  • 33
    Snider DP, Gordon J, McDermott MR & Underdown BJ. Chronic Giardia muris infection in anti-IgM-treated mice. I. Analysis of immunoglobulin and parasite-specific antibody in normal and immunoglobulin-deficient animals. J Immunol 1985; 134: 41534162.
  • 34
    Snider DP, Skea D & Underdown BJ. Chronic giardiasis in B-cell-deficient mice expressing the xid gene. Infect Immun 1988; 56: 28382842.
  • 35
    Stager S & Muller N. Giardia lamblia infections in B-cell-deficient transgenic mice. Infect Immun 1997; 65: 39443946.
  • 36
    Langford TD, Housley MP, Boes M et al. Central importance of immunoglobulin A in host defense against Giardia spp. Infect Immun 2002; 70: 1118.
  • 37
    Skea DL & Underdown BJ. Acquired resistance to Giardia muris in X-linked immunodeficient mice. Infect Immun 1991; 59: 17331738.
  • 38
    Singer SM & Nash TE. T-cell-dependent control of acute Giardia lamblia infections in mice. Infect Immun 2000; 68: 170175.
  • 39
    Lai Ping So A & Mayer L. Gastrointestinal manifestations of primary immunodeficiency disorders. Semin Gastrointest Dis 1997; 8: 2232.
  • 40
    Burrows PD & Cooper MD. IgA deficiency. Adv Immunol 1997; 65: 245276.
  • 41
    Strober W & Sneller MC IgA deficiency. Ann Allergy 1991; 66: 363375.
  • 42
    Prince HE, Norman GL & Binder WL. Immunoglobulin A (IgA) deficiency and alternative celiac disease-associated antibodies in sera, submitted to a reference laboratory for endomysial IgA testing. Clin Diagn Lab Immunol 2000; 7: 192196.
  • 43
    Briere F, Bridon JM, Chevet D et al. Interleukin 10 induces B lymphocytes from IgA-deficient patients to secrete IgA. J Clin Invest 1994; 94: 97104.
  • 44
    Marconi M, Plebani A, Avanzini MA et al. IL-10 and IL-4 co-operate to normalize in vitro IgA production in IgA-deficient (IgAD) patients. Clin Exp Immunol 1998; 112: 528532.
  • 45
    Harriman GR, Bogue M, Rogers P et al. Targeted deletion of the IgA constant region in mice leads to IgA deficiency with alterations in expression of other Ig isotypes. J Immunol 1999; 162: 25212529.
  • 46
    Heyworth MF. Relative susceptibility of Giardia muris trophozoites to killing by mouse antibodies of different isotypes. J Parasitol 1992; 78: 7376.
  • 47
    Stager S, Gottstein B, Sager H, Jungi TW & Muller N. Influence of antibodies in mother's milk on antigenic variation of Giardia lamblia in the murine mother–offspring model of infection. Infect Immun 1998; 66: 12871292.
  • 48
    Singer SM, Elmendorf HG, Conrad JT & Nash TE. Biological selection of variant-specific surface proteins in Giardia lamblia. J Infect Dis 2001; 183: 119124.
  • 49
    Nash TE & Mowatt MR. Characterization of a Giardia lamblia variant-specific surface protein (VSP) gene from isolate GS/M and estimation of the VSP gene repertoire size. Mol Biochem Parasitol 1992; 51: 219227.
  • 50
    Muller N & Gottstein B. Antigenic variation and the murine immune response to Giardia lamblia. Int J Parasitol 1998; 28: 18291839.
  • 51
    Fang FC. Perspectives series: host/pathogen interactions. Mechanisms of nitric oxide-related antimicrobial activity. J Clin Invest 1997; 99: 28182825.
  • 52
    Brunet LR. Nitric oxide in parasitic infections. Int Immunopharmacol 2001; 1: 14571467.
  • 53
    Wallace JL & Miller MJ. Nitric oxide in mucosal defense: a little goes a long way. Gastroenterology 2000; 119: 512520.
  • 54
    Salzman A, Denenberg AG, Ueta I, O'Connor M, Linn SC & Szabo C. Induction and activity of nitric oxide synthase in cultured human intestinal epithelial monolayers. Am J Physiol 1996; 270: G565G573.
  • 55
    Witthoft T, Eckmann L, Kim JM & Kagnoff MF. Enteroinvasive bacteria directly activate expression of iNOS and NO production in human colon epithelial cells. Am J Physiol 1998; 275: G564G571.
  • 56
    Hoffman RA, Zhang G, Nussler NC et al. Constitutive expression of inducible nitric oxide synthase in the mouse ileal mucosa. Am J Physiol 1997; 272: G383G392.
  • 57
    Murray IA, Daniels I, Coupland K, Smith JA & Long RG. Increased activity and expression of iNOS in human duodenal enterocytes from patients with celiac disease. Am J Physiol Gastrointest Liver Physiol 2002; 283: G319G326.
  • 58
    Singer II, Kawka DW, Scott S et al. Expression of inducible nitric oxide synthase and nitrotyrosine in colonic epithelium in inflammatory bowel disease. Gastroenterology 1996; 111: 871885.
  • 59
    Salzman AL, Eaves-Pyles T, Linn SC, Denenberg AG & Szabo C. Bacterial induction of inducible nitric oxide synthase in cultured human intestinal epithelial cells. Gastroenterology 1998; 114: 93102.
  • 60
    Islam D, Veress B, Bardhan PK, Lindberg AA & Christensson B. In situ characterization of inflammatory responses in the rectal mucosae of patients with shigellosis. Infect Immun 1997; 65: 739749.
  • 61
    Fernandes PD & Assreuy J. Role of nitric oxide and superoxide in Giardia lamblia killing. Braz J Med Biol Res 1997; 30: 9399.
  • 62
    Edwards MR, Schofield PJ, O'Sullivan WJ & Costello M. Arginine metabolism during culture of Giardia intestinalis. Mol Biochem Parasitol 1992; 53: 97103.
  • 63
    Knodler LA, Schofield PJ & Edwards MR. l-arginine transport and metabolism in Giardia intestinalis support its position as a transition between the prokaryotic and eukaryotic kingdoms. Microbiology 1995; 141: 20632070.
  • 64
    Pan M, Malandro M & Stevens BR. Regulation of system y+ arginine transport capacity in differentiating human intestinal Caco-2 cells. Am J Physiol 1995; 268: G578G585.
  • 65
    Mailliard ME, Stevens BR & Mann GE. Amino acid transport by small intestinal, hepatic, and pancreatic epithelia. Gastroenterology 1995; 108: 888910.
  • 66
    Torres ME, Pirez MC, Schelotto F et al. Etiology of children's diarrhea in Montevideo, Uruguay: associated pathogens and unusual isolates. J Clin Microbiol 2001; 39: 21342139.
  • 67
    Hogaboam CM, Collins SM & Blennerhassett MG. Effects of oral L-NAME during Trichinella spiralis infection in rats. Am J Physiol 1996; 271: G338G346.
  • 68
    Roberts-Thomson IC, Grove DI, Stevens DP & Warren KS. Suppression of giardiasis during the intestinal phase of trichinosis in the mouse. Gut 1976; 17: 953958.
  • 69
    Ouellette AJ & Bevins CL. Paneth cell defensins and innate immunity of the small bowel. Inflamm Bowel Dis 2001; 7: 4350.
  • 70
    Ayabe T, Satchell DP, Pesendorfer P et al. Activation of Paneth cell α-defensins in mouse small intestine. J Biol Chem 2002; 277: 52195228.
  • 71
    Wilson CL, Ouellette AJ, Satchell DP et al. Regulation of intestinal α-defensin activation by the metalloproteinase matrilysin in innate host defense. Science 1999; 286: 113117.
  • 72
    O'Neil DA, Porter EM, Elewaut D et al. Expression and regulation of the human β-defensins hBD-1 and hBD-2 in intestinal epithelium. J Immunol 1999; 163: 67186724.
  • 73
    Hase K, Eckmann L, Leopard JD, Varki N & Kagnoff MF. Cell differentiation is a key determinant of cathelicidin LL-37/human cationic antimicrobial protein 18 expression by human colon epithelium. Infect Immun 2002; 70: 953963.
  • 74
    Aley SB, Zimmerman M, Hetsko M, Selsted ME & Gillin FD. Killing of Giardia lamblia by cryptdins and cationic neutrophil peptides. Infect Immun 1994; 62: 53975403.
  • 75
    Ayabe T, Satchell DP, Wilson CL, Parks WC, Selsted ME & Ouellette AJ. Secretion of microbicidal α-defensins by intestinal Paneth cells in response to bacteria. Nat Immunol 2000; 1: 113118.
  • 76
    Porter EM, Bevins CL, Ghosh D & Ganz T. The multifaceted Paneth cell. Cell Mol Life Sci 2002; 59: 156170.
  • 77
    Turchany JM, Aley SB & Gillin FD. Giardicidal activity of lactoferrin and N-terminal peptides. Infect Immun 1995; 63: 45504552.
  • 78
    Tedeschi A, Tuccari G, Magazzu G, Arena F, Ricciardi R & Barresi G. Immunohistochemical localization of lactoferrin in duodenojejunal mucosa from celiac children. J Pediatr Gastroenterol Nutr 1987; 6: 328334.
  • 79
    Mason DY & Taylor CR. Distribution of transferrin, ferritin, and lactoferrin in human tissues. J Clin Pathol 1978; 31: 316327.
  • 80
    Hill DR & Pearson RD. Ingestion of Giardia lamblia trophozoites by human mononuclear phagocytes. Infect Immun 1987; 55: 31553161.
  • 81
    Heyworth MF, Owen RL, Seaman WE, Schaefer FW 3rd & Jones AL. Harvesting of leukocytes from intestinal lumen in murine giardiasis and preliminary characterization of these cells. Dig Dis Sci 1985; 30: 149153.
  • 82
    Heyworth MF, Owen RL & Jones AL. Comparison of leukocytes obtained from the intestinal lumen of Giardia-infected immunocompetent mice and nude mice. Gastroenterology 1985; 89: 13601365.
  • 83
    Heyworth MF, Carlson JR & Ermak TH. Clearance of Giardia muris infection requires helper/inducer T lymphocytes. J Exp Med 1987; 165: 17431748.
  • 84
    Heyworth MF, Kung JE & Eriksson EC. Clearance of Giardia muris infection in mice deficient in natural killer cells. Infect Immun 1986; 54: 903904.
  • 85
    Zhou P, Li E, Zhu N, Robertson J, Nash T & Singer SM. Role of Interleukin-6 in the control of acute and chronic Giardia lamblia infections in mice. Infect Immun 2003; 71: 15661568.
  • 86
    Bienz M, Dai WJ, Welle M, Gottstein B & Muller N. Interleukin-6-deficient mice are highly susceptible to Giardia lamblia infection but exhibit normal intestinal immunoglobulin A responses against the parasite. Infect Immun 2003; 71: 15691573.
  • 87
    Perez PF, Minnaard J, Rouvet M et al. Inhibition of Giardia intestinalis by extracellular factors from Lactobacilli: an in vitro study. Appl Environ Microbiol 2001; 67: 50375042.