STRAIN VARIATION IN PATHOGENESIS AND IMMUNITY IN GIARDIA INFECTION

A Dissertation submitted to the Faculty of the

Graduate School of Arts and Sciences of Georgetown University

in partial fulfillment of the requirements for the degree of

Doctor of Philosophy in Biology

By

SHAHRAM SOLAYMANI-MOHAMMADI, M.S.P.H.

Washington, DC September 30 2011

ii

Copyright 2011 by SHAHRAM SOLAYMANI-MOHAMAMDI All Rights Reserved

iii

STRAIN VARIATION IN PATHOGENESIS AND IMMUNITY IN GIARDIA INFECTION

SHAHRAM SOLAYMANI-MOHAMMADI, M.S.P.H.

Thesis Advisor: STEVEN M. SINGER, Ph.D.

ABSTRACT

Infection with Giardia duodenalis (syn. Giardia lamblia, Giardia intestinalis) is one of the

most common intestinal protozoan infections in humans all around the world. Symptoms

associated with human giardiasis are diverse and infected patients may develop abdominal

cramps, nausea, and acute/subacute or chronic diarrhea, accompanied with malabsorption

syndrome. Transmission of the parasite to a new host happens via the ingestion of cyst

contaminated food and water. Human infections with G. duodenalis are distributed worldwide

with rates of infections between 2-5% in developed nations and 20-30% in the developing world.

Several reports have demonstrated variations in symptoms/ pathology among different Giardia

isolates. There is evidence supporting that a specific genotype of G. duodenalis (i.e. assemblage

A or B) is associated with symptomatic disease. These reports are sometimes contradictory. For

example, it was shown that 74% of isolates examined from symptomatic patients in Nepal were

assemblage B while 20% and 6% were assemblage A and mixed infections, respectively. In

contrast, others showed assemblage A to be predominantly associated with symptomatic disease

in Bangladesh. However, some reports do not find any correlation between the parasite genotype

and symptomatic disease. In order to evaluate the strain-dependent variability among the two

assemblages capable of infecting humans, we used representative strains of both assemblages to

infect mice. We then comparatively analyzed immune responses to the two stain of the parasite.

iv

Analyses showed that there were strain-dependent differences in the ability of a given strain to

induce pathological changes.

Using a mouse model of giardiasis, we examined the role of host immunity and pathogen

virulence in mediating disaccharidase deficiency postinfection. Mice were infected with two

strains, WB and GS, of the human parasite Giardia duodenalis. The levels of sucrase, maltase,

and lactase decreased in wild-type mice p.i. with the GS strain but not with the WB strain. Both

CD4-deficient and SCID mice failed to eliminate the infection and did not exhibit disaccharidase

deficiency. β2-Microglobulin knockout animals controlled infections similar to wild-type mice

but exhibited no decrease in disaccharidase activity. Analysis of cytokine production by spleen

and mesenteric lymph node cells showed production of IL-4, IL-10, IL-13, IL-17, IL-22, TNF-α,

and IFN-γ post-infection with both WB and GS, with IFN-g being the dominant cytokine for

both strains.

It has recently been hypothesized that most pathology induced during human giardiasis is, in

fact, because of the immune responses meant to eliminate the parasites. The structural proteins

ezrin and villin are among the most abundant proteins present in the intestinal brush border.

These proteins function as cross-linkers between the plasma membrane and the actin

cytoskeleton. We found that intestinal epithelial cells (IECs) underwent drastic cytoskeletal

changes following gut infection and that both expression and the distribution of the cytoskeletal

proteins were impacted by gut infection. We further demonstrated that the immense changes

observed following gut infections are driven by host adaptive immunity, and that the lack of

adaptive immune responses prevents hosts from exhibiting these changes. We also showed a

v

correlation between tyrosine-phosphorylation of villin and the presence of disaccharidase

deficiency in mice infected with the two strains of the parasite.

vi

I dedicate this dissertation to my mentor, Professor Steven M. Singer, who opened new doors in my scientific career in the field of Immunology

Many thanks,

to people in the Singer lab

vii

TABLE OF CONTENTS Chapter I Giardia duodenalis: the Double-Edged Sword of Immune Responses in Giardiasis Epidemiology of Giardiasis ............................................................................................. 2 Immune Responses that Control Infection .................................................................. 4 Pathology and the Immune Response during Giardiasis……………………………….12 Chapter II A Meta-analysis of the Effectiveness of Albendazole Compared with Metronidazole as

Treatments for Infections with Giardia duodenalis

2.1. Introduction………………………………………………………………………. .18 2.2. Methods 2.2.1. Data Source and Study Selection……………………………………… 22 2.2.2. Data Extraction and Analysis………………………………………….25 2.2.3. Assessment of Study Quality…………………………………………. 28 2.2.4. Sensitivity Analysis……………………………………………………29 2.2.5. Data Synthesis and Statistical Analysis……………………………….29 2.3. Results…………………………………………………………………...30 2.3.1. Publication Bias and Sensitivity Analysis…………………………….34

viii

2.3.2. Adverse Effects…………………………………………………………36 2.4. Discussion………………………………………………………………...37 Chapter III Host Immunity and Pathogen Strain Contribute to Intestinal Disaccharidase Impairment

following Gut Infection

3.1. Introduction…………………………………………………………………………44

3.2. Methods

3.2.1. Mice……………………………………………………………………………….46

3.2.2. Parasite and Infection Protocols………………………………………………….46

3.2.3. Disaccharidase Activity Assay…………………………………………………...47

3.2.4. Ex vivo Restimulation of Splenocytes and Mesenteric Lymph Node Cells……...47 3.2.5. Cytokine Analysis by ELISA…………………………………………………….48 3.2.6. Flow Cytometry…………………………………………………………………..48 3.2.7. Statistical Analyses……………………………………………………………….49 3.3. Results 3.3.1. CD8+ T Cells Are Required for Disaccharidase Deficiency but

Not Control of Infection………………………………………………………………..50

3.3.2. Cytokine Responses by Spleen and MLN Cells p.i……………………………...58 3.3.3. Disaccharidase Deficiency in WT Mice Is Strain-Dependent…………………..60 3.3.4. Cytokine Production by T Cells p.i……………………………………………...62

ix

3.3.5. CD4+ cells are the main cytokine-secreting T cells in mice infected

with WB and GS strains……………………………………………………………..62

3.4. Discussion……………………………………………………………………….65

Chapter IV Altered Positional Distribution of Intestinal Epithelial Cells and Intestinal

Cytoskeletal Remodeling Predispose Host to Gut Dysfunction

4.1. Introduction……………………………………………………………………...70

4.2. Methods…………………………………………………………………………..73

4.2.1. Mice…………………………………………………………………………….73

4.2.2. Parasites, Infection Protocols and Quantification of Parasite Burden…………73

4.2.3. Antibodies……………………………………………………………………....74

4.2.4. Immunofluorescence Microscopy………………………………………………74

4.2.5. In Vivo Epithelial Cell Proliferation Assay…………………………………….75

4.2.6. Western Blot Analysis………………………………………………………….75

4.2.7. Immunoprecipitation…………………………………………………………...76

4.2.8. Statistical Analysis……………………………………………………………...77

4.3. Results……………………………………………………………………………77

4.4. Discussion………………………………………………………………………...94

x

Chapter V 5. Conclusion……………………………………………………………………………99 References……………………………………………………………………………..106

1

Chapter I

Giardia duodenalis: the Double-Edged Sword of Immune

Responses in Giardiasis

Part of this Chapter was published in “Experimental

Parasitology”.

Solaymani-Mohammadi S, Singer SM. 2010. Giardia duodenalis:

The double-edged sword of immune responses in giardiasis.

Experimental Parasitology, 126 (3): 292-297.

2

1.1. Epidemiology of Giardiasis

Recent studies of Giardia have identified eight distinct genotypes within Giardia duodenalis,

only two of which, assemblages A and B, are capable of infecting humans [(reviewed in

Thompson (2009)]. These studies have led to a re-evaluation of the zoonotic potential of this

organism. Although parasites with both A and B genotypes can infect numerous mammalian

species in addition to humans, other genotypes appear to have more restricted host range.

Assemblages C and D, for example, are commonly found in dogs, but have yet to be reported in

humans. Thus, the idea that human transmission from dogs (and cats and livestock) to humans

needs to be reevaluated. Fortunately, new data are becoming available indicating that most cases

of giardiasis are due to anthroponotic spread, but zoonotic transmission can and does occur (Snel

et al., 2009).

Better understanding of the molecular epidemiology of Giardia will also require reanalysis of

studies of a commercially available vaccine for veterinary giardiasis (Olson et al., 2000). This

vaccine is essentially a mixture of lyophilized trophozoites of four parasite strains. Since these

strains can be grown in culture they are likely from assemblages A and B, but not the

assemblages commonly found in and restricted to cats (F), dogs (C and D) or livestock (E). Thus,

while some studies have shown some protection against experimental infections (Olson et al.,

1996; Olson et al., 1997; Olson et al., 2001), others failed to show such a protection against the

parasite (Stein et al., 2003; Anderson et al., 2004; Uehlinger et al., 2007). For example, in one

study vaccinated kittens had abnormal stools on fewer days, secreted fewer cysts, and had a

significantly higher weight gain in the post-challenge period (Olson et al., 1996). Conversely,

3

Stein and coworkers (2003) did not find any correlation between cats receiving three doses of a

Giardia vaccine and reduction in cyst shedding compared to unvaccinated kittens. New

veterinary vaccines will need to take into account the restricted host ranges of the different

genotypes and work around our inability to culture those other than types A and B. Potential

human vaccines will need to address the role of immune responses in contributing to pathology

and determining which responses are protective, as opposed to those which are merely present.

The factors determining the variability in clinical outcome in giardiasis are still poorly

understood (Buret, 2007). However, host factors (such as immune status, nutritional status and

age), as well as differences in virulence and pathogenicity of Giardia strains are recognized as

important determinants for the severity of infection (Haque et al., 2005). Numerous studies have

attempted to correlate the development of symptoms to the presence of either assemblage A or B

parasites. While individual studies often find a strong correlation between parasite genotype and

virulence, the answer comparing across studies is very unclear. For example, one study in Dutch

patients found assemblage A isolates solely in patients with intermittent diarrhea, while

assemblage B isolates were present in patients with persistent diarrhea (Homan and Mank,

2001). In contrast, Geurden et al. (2009) found that infections with assemblage B parasites were

commonly found in diarrhea patients, but that a high proportion of infections were with mixed

assemblages that might have interfered with previous analyses. This may be due to the fact that

assigning parasites to specific genotypes usually reflects alleles at loci such as glutamate

dehydrogenase, 18S RNA and triosephosphate isomerase (TPI) which are unlikely to be directly

associated with virulence. More effort, however, should be directed to understanding

4

mechanisms of virulence and identifying specific parasite virulence factors in order to

understand the relative contributions of both the host and the parasite to disease.

1.2. Immune Responses that Control Infection

The immune response to microbial pathogens, including Giardia sp., relies on both innate and

adaptive components. Although the actual host defense mechanisms responsible for controlling

Giardia infections are poorly understood, many studies have demonstrated the development of

adaptive immune responses as well as innate mechanisms in humans and other animals

(Roxström-Lindquist et al., 2006). Understanding the complex network of immune responses and

host–parasite cross-talk should assist us in identifying novel and common targets for the

therapeutic intervention of the infection (Solaymani-Mohammadi et al., 2010).

Epidemiological studies suggest that previous infection with Giardia leads to a reduced risk

of re-infection and to reduced development of overt symptoms in secondary infections. Analysis

of cases in an outbreak at a ski resort in Colorado showed that individuals residing in the

community for more than 2 years had a much lower risk of being affected than new residents

(Istre et al., 1984). Similarly, a community in British Columbia experienced two outbreaks 5

years apart and individuals affected in the first outbreak were much less likely to be ill during the

second outbreak (Isaac-Renton et al., 1994). Both studies suggest that previous exposure to

Giardia produces an immunity to disease. It is unclear in these studies whether prior exposure

actually prevented infection, or only if severe symptoms were avoided the second time.

Nonetheless, these findings suggest that development of an effective vaccine could be feasible. A

recent study in Brazilian children suggests that symptoms are less severe during re-infection,

5

consistent with the idea that previous exposure does not prevent infection, but does reduce the

pathology which can occur (Kohli et al., 2008). Additional studies in humans and animal models

are, however, needed to determine what types of immune responses mediate this protection.

Studies in animal models require careful interpretation. First, animal immune responses are

not always equivalent to that seen in humans. Additionally, many studies have utilized Giardia

muris, a rodent parasite that cannot be cultured, to analyze immune responses in mice. As

described below, however, G. muris may be resistant to immune mechanisms capable of killing

G. duodenalis. Studies of G. duodenalis are restricted by the inability of many strains of G.

duodenalis to colonize adult mice. Experiments with G. duodenalis in gerbils and neonatal mice

have also been performed, although immunologic analysis of these animals is difficult. In some

cases, understanding the host restriction of G. duodenalis may provide insights into the biology

of the host–parasite interaction. For example, certain bacterial flora have been shown to render

mice resistant to colonization (Singer and Nash, 2000b). Bacterial flora could inhibit Giardia

through direct action against the parasite. Studies in vitro showed that culture supernatants from

Lactobacillus johnsonii La1 significantly inhibited the proliferation of G. duodenalis

trophozoites in a strain-dependent manner (Pérez et al., 2001). Diverse animal species (and even

individuals) differ greatly in the dominant microbiota colonizing their intestinal mucosal

surfaces. Thus, the different microbiota could explain why some hosts are resistant to specific

parasitic infections. Host immune mechanisms play a key role in regulating the microbiota, e.g.

through expression of diverse sets of antimicrobial defensins by Paneth cells (Salzman et al.,

2007). Indeed, some Paneth cell defensins have also been show to kill G. duodenalis in vitro

6

(Aley et al., 1994). However, the effect of defensins on intestinal flora is bidirectional, and

changes in flora can also effect production of defensins (Ayabe et al., 2004). Further analysis of

the host–parasite–environment three-way interaction in giardiasis may provide novel means to

prevent this infection.

Several lines of evidence suggest that IgA antibodies contribute to protective immunity

against giardiasis. While most chronic infections have been reported in patients with no

underlying immune abnormality, patients with common variable immunodeficiency (CVID) and

Bruton’s X-linked agammaglobulinemia (XLA) are clearly prone to chronic giardiasis (Stark et

al., 2009). Patients with these syndromes both lack normal B cell function and reduced

production of IgG, suggesting the necessity of antibodies for control of the infection. However,

both CVID and XLA have additional defects in immune function and chronic Giardia infection

is not common in selective IgA deficiency, suggesting additional layers of complexity in host

immunity. As such, the rates of giardiasis in HIV-infected patients are higher than controls in

some studies (Angarano et al., 1997; Feitosa et al., 2001; Bachur et al., 2008), although it is still

unclear whether epidemiological factors rather than immunosuppression are responsible for these

differences (Stark et al., 2009).

A number of studies have used animal models of infection to help clarify the role of

antibodies in controlling Giardia infections. Snider et al. (1988) first showed that xid mice

(hypogammaglobulinemic mice with a defect in the same kinase that is altered in human XLA)

and wild-type mice treated with anti-IgM to deplete B cells developed chronic infections with G.

muris. However, when we used gene-targeted B cell deficient mice and G. duodenalis for

7

infections, no defect was apparent, suggesting alternate mechanisms exist to eradicate the

infection (Singer and Nash, 2000a). This contradiction has been resolved by studies directly

comparing G. muris and G. duodenalis infections in mice lacking the poly Ig receptor that cannot

transport IgA or IgM into the intestinal lumen (Davids et al., 2006). G. duodenalis infections

were controlled in the absence of antibodies, while G. muris infections became chronic. This

suggests that mice have additional mechanisms able to kill the human pathogen to which the

mouse species is resistant. Identification of these mechanisms in mice and their human

counterparts would therefore greatly facilitate development of effective vaccines or

immunotherapeutics.

The existence of antibody-independent mechanisms for Giardia elimination does not

necessarily mean that antibodies have no role. However, the ability of G. duodenalis to undergo

extensive variation of the surface coat antigens, called variant-specific surface proteins (VSPs),

likely delays the effectiveness of the antibody response (Nash, 1997). Mice deficient in the

cytokine interleukin (IL)-6 develop chronic infections with G. duodenalis (Bienz et al., 2003;

Zhou et al., 2003). In the first 2 weeks following infection, these mice make a strong IgA

response which reacts with only a small subset of parasites growing in vitro, suggesting a

response to a limited subset of parasite VSPs. In contrast, 8 weeks after infection IL-6 deficient

mice produce IgA reactive with all parasites from in vitro cultures, suggesting that the IgA

response now recognizes all possible VSPs, common epitopes on VSPs, or invariant antigens on

the parasite. The IL-6 deficient mice cleared their infections at this time point, indicating that

such broadly reactive antibodies could indeed confer protection (Zhou et al., 2007).

8

In contrast to B cells, T cells appear to be required for control of Giardia at all time points

post-infection. Nude mice, mice treated with anti-CD4 antibodies and mice lacking the T cell

receptor β gene all develop chronic infections with G. muris and/or G. duodenalis (Roberts-

Thomson and Mitchell, 1978; Stevens et al., 1978; Heyworth et al., 1987; Singer and Nash,

2000a). One role of T cells is to provide help for production of antibodies. These cells also

provide help for other aspects of the immune response, although it is unclear which of these are

essential for parasite control. Human studies have provided limited information about cytokine

levels in sera of patients. In one study, peripheral blood lymphocytes from naïve individuals

produced interferon (IFN)-γ in response to trophozoites (Ebert, 1999), and sera from infected

adults contained elevated levels of IL-5, IL-6 and IFN-γ (Matowicka-Karna et al., 2009). Mice

lacking either IL-4 or IFN-γ are able to control infections with kinetics similar to wild-type mice

(Singer and Nash, 2000a). We have recently shown that mice lacking TNF-α are delayed in

parasite elimination (Zhou et al., 2007). This is consistent with elevated TNF-α levels in sera of

infected children (Bayraktar et al., 2005). Altogether, additional studies are warranted to help us

better understand regulation of T cell development and the roles of these immune cells during

giardiasis.

Several recent studies have begun to examine how the innate immune system responds to

Giardia infection and how this might influence development of T cell responses. An early study

from the Kagnoff group examined the response of Caco2 cells to G. duodenalis parasites and

saw no change in the expression of numerous cytokines that were induced by other intestinal

pathogens (Jung et al., 1995). Roxström-Lindquist et al. (2005) used microarrays to reexamine

9

this interaction and discovered a strong response by a limited number of genes, including several

chemokines that might be expected to recruit dendritic cells and lymphocytes to the intestinal

mucosa. Unfortunately, studies in vivo have not determined the importance of these factors

during infection. Our lab has recently examined the interaction of dendritic cells with Giardia

and found that live parasites and parasite extracts activate these cells for antigen presentation, but

inhibit secretion of IL-12 while enhancing production of IL-10 (Kamda and Singer, 2009).

Inhibition of IL-12 production was partially mediated via IL-10, but could be more completely

reversed by inhibiting phosphoinositide 3-kinase (PI3K) with wortmannin. The host receptors

and parasite ligands involved in this inhibition remain to be identified. Interestingly, this

inhibition of IL-12 production could suggest that the parasite actively restricts the development

of severe inflammation and may contribute to the lack of pathology often seen during infections.

How dendritic cells and epithelial cells interact to determine the development of T cell responses

needs to be determined.

Several effector mechanisms have been proposed to eradicate Giardia infections [(reviewed

in Eckmann (2003)]. These include phagocytosis by macrophages, secretion of defensins, nitric

oxide (NO) and mucins by epithelial cells as well as the recruitment and activation of mucosal

mast cells. Macrophages have been shown to ingest and kill Giardia trophozoites in vitro, but a

role in vivo has not been demonstrated [reviewed by Smith (1985)]. Likewise, while some α-

defensins can lyse trophozoites in vitro (Aley et al., 1994), Eckmann (2003) reported that mice

lacking MMP-7, the protease which converts pro-defensin peptides to active defensins, had no

deficit in controlling G. muris infections. NO is cytostatic for G. duodenalis in vitro, but mice

10

lacking inducible nitric oxide synthase (iNOS) eradicate G. duodenalis infections normally

(Eckmann et al., 2000; Li et al., 2006; Andersen et al., 2006). Interestingly, the neuronal NOS

isoform did have a defect in parasite control, but this was more likely due to effects on intestinal

motility and not a direct effect on the parasite. The activity of mucins against the parasite has

also only been shown in vitro (Roskens and Erlandsen, 2002).

In contrast to these other effector mechanisms, a significant role for mast cells in controlling

Giardia has been demonstrated in vivo in several animal models. In an early study, mast cell

deficient (wf/wf) mice, unlike their wild-type littermates, developed chronic giardiasis after oral

administration of G. muris. Furthermore, the susceptible BALB/c mice injected with the anti-

histamine and anti-serotonin drug, cyproheptadine, also showed prolonged infections with G.

muris, implying some roles for mast cells in host’s ability to eliminate the parasite (Erlich et al.,

1983). A potential role for mast cells was suggested by Hardin et al. (1997), who examined

jejunal macromolecular transport, epithelial permeability, and mucosal and connective tissue

mast cell counts in Mongolian gerbils infected with G. duodenalis. In this study, mast cell counts

were correlated with epithelial permeability and antigen uptake, suggesting a link between

development of other immune responses and mast cell recruitment. This was further supported

by data from our laboratory showing that c-kitw/wv mice infected with G. duodenalis failed to

make parasite-specific IgA, mount a mast cell response, or to eliminate the infection (Li et al.,

2004). Anti-c-kit-treated C57BL/6 mice had normal IgA responses, lacked mast cell responses,

and failed to control the infection within 10 days. Hence, while the requirement for mast cells is

clear, their role in development of antibody responses is not. A recent study, however, found that

11

mast cells can help drive B cell switching to production of IgA (Merluzzi et al., 2010). We also

recently described an additional role for mast cells in mediating smooth muscle contractions and

contributing to changes in intestinal motility following infection (see below). It is likely that this

contributes to both protection and pathology in giardiasis.

If mast cells are needed for elimination of Giardia infection, then prior or concurrent

infection with intestinal helminthes that induce mastocytosis might be expected to prevent

infection. However, pre-infection with Trichinella spiralis led to an increase in the number of

trophozoites in the intestine and a delay in Giardia elimination, despite a large increase in mast

cell numbers and normal production of anti-Giardia IgA (von Allmen et al., 2006). Thus, while

mast cells are necessary for Giardia elimination, they are clearly not sufficient on their own.

Indeed, the strong TH2 environment created by T. spiralis infection may make the intestinal tract

more conducive to Giardia survival. Given that in most areas where giardiasis is endemic, the

overwhelming majority of inhabitants are infected with other parasitic protozoa and helminths as

well, additional study of the interactions between G. duodenalis and other infections is clearly

needed.

Murine models of infection have also been used to study possible new vaccine strategies for

giardiasis. A recombinant live attenuated vaccine, based on a gene segment of the VSP (VSPH7)

expressed in a Salmonella typhimurium vaccine strain, was evaluated for its potential to induce

local and systemic immune responses (Stager et al., 1997). The recombinant vaccine elicited

synthesis of serum IgG as well secretory IgA (sIgA) against both bacterial antigens and the VSP.

Vaccinated mice were never challenged with live parasites; however, several experiments have

12

examined the possibility of transmission-blocking vaccines that target cyst antigens. Larocque et

al. (2003) showed that oral immunization with recombinant cyst wall protein (CWP2) could

elicit IgA and mixed TH1/TH2 responses and reduce cyst shedding after live challenge with G.

muris cysts. Similarly, delivery of CWP2 expressed in Streptococcus gordonii or Lactococcus

lactis elicited high levels of antibodies in the vaccinated mice and also reduced cyst shedding

after challenge (Lee and Faubert, 2006a, b). Most recently, a DNA vaccine encoding CWP2

delivered to the intestinal mucosa via S. typhimurium led to production of antibody, a mixed

TH1/TH2 response and reduced cyst shedding compared to control mice (Abdul-Wahid and

Faubert, 2007). Analysis of vaccine efficacy in mice lacking elements of the immune system,

e.g. IL-4 deficiency, would be useful in determining which of the observed responses actually

contribute to the observed protection.

1.3. Pathology and the Immune Response during Giardiasis

Pathology in giardiasis is understood to arise in several ways. These include breakdown of the

epithelial barrier, defects in the epithelial brush border, secretion of chloride ions and

hypermotility of the intestinal smooth muscles. The role of parasite virulence factors and host

immune responses in contributing to these mechanisms is beginning to be clarified, although

much more remains to be understood.

Breakdown of the epithelial barrier has been shown to occur in human disease and animal

models of giardiasis (Hardin et al., 1997; Troeger et al., 2007; Zhou et al., 2007). This allows for

macromolecules and electrolytes to pass into the sub-mucosa, bypassing normal uptake by

13

epithelial cells. The paracellular flow of nutrients and electrolytes can contribute to nutrient

malabsorption by reducing electrochemical gradients needed for proper uptake and can cause

inflammation in some individuals, probably through activation of innate immune effectors like

macrophages. However, inflammation is often not observed in human giardiasis and does not

correlate well to symptomatic disease (Oberhuber et al., 1997). In humans, epithelial barrier

defects correlated with alteration in the level of expression of claudin-1, a component of the tight

junctions (Troeger et al., 2007). In tissue culture models, changes in another tight junction

protein, zona occludens (ZO)-1, as well as the cytoskeletal proteins actin and α-actinin have also

been demonstrated (Teoh et al., 2000; Buret et al., 2002). Giardia has also been shown to

increase epithelial permeability by inducing apoptosis in epithelial cells both in vivo and in vitro

(Chin et al., 2002; Troeger et al., 2007). Interestingly, the magnitude of the apoptotic response in

vitro is highly strain-dependent, although no specific parasite markers have been identified yet

which can induce apoptosis (Chin et al., 2002). Finally, excess ion secretion was recently

demonstrated ex vivo in biopsy material from giardiasis patients, suggesting that this too may

contribute to pathology during this infection (Troeger et al., 2007).

The excretion and/or secretion of virulence factors by the parasite has been speculated to be

responsible in part for the pathology induced during human giardiasis. Jiménez et al. (2004)

demonstrated that oral administration of excretory/secretory antigens from Giardia intestinalis

elicited a robust TH2 response (i.e. IgG1, IgG2, IgE) as well as moderate to profound histological

changes. They tentatively attributed these changes to be potentially due to the parasite’s

proteinase activity. Ringqvist et al. (2008) identified three major secreted proteins (ornithine

14

carbamoyl transferase, arginine deiminase, and enolase) released by Giardia. The release of

these proteins was trigged upon contact with host epithelial cells, and these secreted proteins

were able to impair host innate immune mechanisms, including the production of nitric oxide.

Another group has reported the presence of an excretory/secretory molecule in the parasite

that can induce fluid accumulation in ligated loops of rabbit ileum (Shant et al., 2002). A 58-kDa

enterotoxin purified from the excretory–secretory product of the parasite was able to decrease

host GTPase activity (Shant et al., 2004). In this study, activation of protein kinase A (PKA) as

well an increase in intracellular calcium ions were also observed after enterotoxin treatment of

mouse enterocytes. Subsequently, these authors demonstrated that purified 58-kDa enterotoxin

induced increased levels of phospholipase C gamma1, inositol triphosphate, and an upregulation

in the protein kinase C (PKC; Shant et al., 2005). An N-terminal peptide sequence for the

putative toxin has been reported. However, this peptide sequence is not represented in the virtual

proteome of the prototype assemblage A strain WB (Morrison et al., 2007). It is possible that this

peptide is encoded in a portion of the genome which has not been assembled, or that the WB

strain lacks this molecule. Further molecular characterization of this toxin would provide a

significant advance in developing tools to combat this disease.

The protective roles of intraepithelial lymphocytes (IEL) against parasitic infections have

been characterized in a number of protozoan and helminth infections (Guk et al., 2003; Moretto

et al., 2004; Little et al., 2005). In giardiasis, however, it was recently proposed that increased

numbers of IELs in patients with chronic disease in fact were associated with a malabsorption

syndrome including the impairment of the glucose/sodium uptake (Troeger et al., 2007).

15

Reduction in levels of some intestinal disaccharidases, sucrase, lactase, maltase, and

trehalase, have been shown to occur in human giardiasis (Levinson and Nastro, 1978) and in

experimental infections in mice (Gillon et al., 1982), rats (Cevallos et al., 1995), and gerbils

(Buret et al., 1991). In one study of G. muris infections in mice carried out by Daniels and

Belosevic (1995), disaccharidase deficiency during primary infection was strongly associated

with parasite number. In another study with the same model, the decrease in jejunal

disaccharidase activities correlated with a diffuse shortening of brush border microvilli (Buret et

al., 1990). Scott et al. (2000) showed that brush border damage and disaccharidase deficiency did

not develop in nude mice infected with G. muris. Adoptive transfer of CD8+, but not CD4+ T

cells, from infected wild-type mice into naïve recipients induced both symptoms, indicating that

immune responses are key to the brush border defects commonly observed following infection. It

still remains unclear how Giardia infection induces CD8+ T cell responses and how these cells

induce the damage. Interestingly, since CD8+ T cells are not required to control the infection

(Singer and Nash, 2000a), it is theoretically possible to induce protective immunity without

inducing nutrient malabsorption.

The final mechanism contributing to pathology is intestinal smooth muscle hypercontractility.

These muscular contractions contribute to the severe cramps associated with symptomatic

disease, but also increase the rate of transit of gut contents through the intestines reducing the

time available for nutrient uptake and water reabsorption. Recent work in our lab and others has

demonstrated that increased transit rates occur in wild-type mice following G. duodenalis and G.

muris infections, but not in SCID mice even though these mice have higher parasite loads (Li et

16

al., 2006; Andersen et al., 2006). Increased transit requires coordinated contractions and

relaxation of smooth muscle. Interestingly, nitric oxide (NO) production from the neuronal

isoform of nitric oxide synthase (NOS1) is required for enhanced transit. NO is normally an

inhibitor of muscle contractions in the enteric system. In contrast, we have shown that enhanced

muscular contractions occur following infection due to release of mast cell granule contents (Li

et al., 2007). Activation of muscular contractions could be triggered in organ baths by addition of

the hormone cholecystokinin (CCK), and this response was blocked by agents that interfered

with mast cell function. Given that Giardia parasites consume bile and that a normal

physiological function of CCK is to induce contraction of the gall bladder and release of bile into

the small intestine, it appears as though the parasite has evolved to co-opt the host’s normal

physiology in order to provide an essential nutrient for its growth. Moreover, variation in the

ability of different parasites to consume bile or in the host to produce CCK or recruit mast cells

following infection could help explain the variation in pathology seen in giardiasis.

Several studies have shown differences in pathology among Giardia isolates. There is

contradicting evidence showing that a specific genotype of G. duodenalis (i.e. assemblage A or

B) is associated with symptomatic disease. Singh et al. (2009), for example, showed that most

isolates examined from symptomatic patients in Nepal were assemblage B. In contrast, other

studies identified assemblage A to be associated with symptomatic disease (Haque et al., 2005).

Furthermore, Kohli et al. (2008) found no differences in the ability of assemblage A and B

parasites to cause symptomatic infections in children in Brazil. One possibility is that the

differences observed (if any) reflect variation within these genotypic classifications, especially

17

the presence or absence of specific virulence factors. Alternately, given that host immune

responses are, in part, responsible for pathological effects induced by G. duodenalis, it is

possible that different isolates may elicit host immune responses in different ways. For example,

Williamson et al. (2000) found distinct differences in the infection dynamics, histopathological

responses and serum antibody responses in a neonatal mouse model of infection with two

different strains of G. duodenalis (although the genotypes were not reported). Collection of

immune response data in parallel with patient symptoms and parasite genotypes may provide

answers to this difficult issue.

18

Chapter II

A Meta-analysis of the Effectiveness of Albendazole

Compared with Metronidazole as Treatments for Infections

with Giardia duodenalis

Part of this Chapter was published in “PLoS Neglected

Tropical Diseases”.

Solaymani-Mohammadi S, Genkinger JM, Loffredo CA, Singer SM.

2010. A meta-analysis of the effectiveness of albendazole compared

with metronidazole as treatments for infections with Giardia

duodenalis. PLoS Neglected Tropical Diseases, 4, e 682.

19

2.1. Introduction

Giardiasis in humans, caused by the protozoan parasite Giardia duodenalis (syn. G. lamblia,

G. intestinalis), is a common parasitic disease (Adam, 2001). The prevalence of infection is

commonly between 2-5% in the developed world and 20-30% in the developing and

underdeveloped countries (Farthing, 1994). Infection is initiated by ingestion of cysts in

contaminated drinking water and/or contaminated food (Adam, 2001). Ingested cysts release

trophozoites which colonize and replicate in the small intestine of the new host. G. duodenalis

does not invade the epithelial or deeper layers of the mucosa and propagation takes place on the

epithelial surface (Gillin et al., 1996).

The outcomes of Giardia infections vary significantly and the majority of infections are self-

limiting. Clinical manifestations range from a relatively asymptomatic phase marked by mild

nutrient malabsorption, to an ephemeral or persistent acute stage, with steatorrhea, intermittent

diarrhea, vomiting, malabsorption syndrome and weight loss, or to a subacute chronic phase that

can mimic gallbladder or peptic ulcer disease (Flanagan, 1992; Wolfe, 1992). Infections in

immunocompetent individuals are generally self-limited, suggesting the existence of effective

host defense mechanisms against the parasite (Kamda and Singer, 2009). Different diagnostic

methods are employed for the diagnosis of human giardiasis of which the most insensitive

method, direct stool microscopy is used routinely in developing countries where the disease is

endemic (Isaac-Renton, 1991; Wolfe, 1992).

20

Existing chemotherapy protocols recommend that patients should be treated if the parasite is

found, irrespective of the presence or absence of acute symptoms (Gardner and Hill, 2001).

However, some investigators question the usefulness of chemotherapy in infected people in

endemic areas due to the extremely high rate of reinfection, as high as 90% in some studies

(Sullivan et al., 1989; Saffar et al., 2005). Treatment preferences vary among clinicians and in

different locations. Several synthetic compounds (including metronidazole and other

nitroimidazole derivates such as albendazole, mebendazole, furazolidone, tinidazole, ornidazole)

are used in the treatment of giardiasis in humans. A single dose of tinidazole (2.0 g) has been

shown to have a clinical efficacy of 80-100% in different clinical trials (Andersson et al., 1972;

Jokipii and Jokipii, 1979) while the compliance is improved compared with other giardiasis

treatments. However, the high cost of tinidazole may restrict its use in mass chemotherapy

campaigns (Johnson, 2009) in developing and underdeveloped countries (e.g. $18 to $32 for a

single-dose of 2 g for the treatment of trichomoniasis). The most widely used treatment protocols

employ metronidazole given 3 times per day for 3-5 days (Bassily et al., 1970; Jokipii and

Jokipi, 1978; Kavousi, 1979; Gardner and Hill, 2001). Metronidazole is typically administered in

doses of 250 mg 3 times a day for 5–7 days for adults and 15 mg/kg 3 times a day for 5-7 days in

children. However, albendazole is typically given as a single dose of 400 mg/day for 3-5 days. In

recent years, therapeutic failure of metronidazole, the first-line drug of choice in giardiasis in

humans, has increasingly been reported from all around the world (Wright et al., 2003).

Metronidazole is prescribed widely for a wide range of non-parasitic infectious diseases;

overusing metronidazole as a treatment option for parasitic infections may increase the chances

of the development of clinically drug-resistant strains of Helicobacter pylori, an important cause

21

of gastric cancer in humans (Selgrad et al., 2009). Low compliance of patients with the current

metronidazole therapy protocols, the emergence of the metronidazole-resistant strains of the

parasite and other pathogens, and rapid reinfection of treated patients in the endemic areas are

additional reasons for considering alternative therapies (Lemée et al., 2000).

Treatment compliance is a key factor affecting the outcome of giardiasis. However,

compliance has been neglected in the literature (Shepherd and Boreham, 1986) and is therefore

not part of the current analysis. In one report on metronidazole use in patients with giardiasis,

treatment compliance was extremely poor because of missed doses, spillage, inaccurate

measuring implements, and poor adherence to the prescribed frequency and duration of

medication (Boreham et al., 1986). Common adverse reactions frequently reported with

metronidazole include metallic taste, nausea, vomiting, diarrhea, and epigastric discomfort

(Shepherd and Boreham, 1986) Moreover, its activity against the host's normal intestinal

microflora; its contraindication for children, pregnant and breastfeeding women; and its

carcinogenic and tumorigenic properties in animal models make it less than optimal for

widespread use (Gardner and Hill, 2001). Finding safer drugs with less toxicity and more

effective therapeutic properties and developing novel protocols (e.g. fewer doses and shortened

duration) to maximize the effects of existing drugs are, therefore, crucial for the field.

Albendazole has been used extensively for the treatment of a wide range of helminth parasites

including hookworms, Ascaris lumbricoides, Trichuris trichiura, Echinococcus sp. (Hemphill

and Müller, 2009) and Taenia sp. (Carpio, 2002) with few side effects (reviewed by Keiser and

Utzinger, 2008). The mechanism of action of albendazole differs from that of metronidazole.

22

While metronidazole affects electron transport of the parasite (Bharti et al., 2002), it is believed

that albendazole exerts its anti-giardial effects by interaction with tubulin of the Giardia

cytoskeleton (Reynoldson et al., 1991). Albendazole also has overt giardiacidal activity in vitro

(Cedillo-Rivera and Muñoz, 1992) as well as being able to resolve infections in a mouse model

of G. duodenalis infection (Reynoldson et al., 1991; Lemée et al., 2000). Using albendazole

against giardiasis in humans could potentially augment mass treatment programs, which are part

of helminth control campaigns, since most patients with Giardia are probably co-infected with

other parasitic agents. Altogether, the evidence suggests that albendazole could be considered as

a potential anti-giardial agent. Its lower toxicity, its relative insolubility and poor absorption

from the gut, and its lack of significant effects on the intestinal microflora could make

albendazole an ideal substitute for metronidazole. The aims of the current meta-analysis,

therefore, were first to address the effectiveness and second to assess the safety of albendazole

compared with metronidazole for the treatment of giardiasis in humans.

2.2. Methods

2.2.1. Data Source and Study Selection A literature search of the PubMed database (1966–February 2010), Scopus, EMBASE, the

Cochrane Controlled Trials Register (issue 4, 2009), LILIACS and the ISI Web of Science for

trials published before February 2010 was performed. The literature search used the following

terms: “giardiasis”, “metronidazole”, and “albendazole.” The abstracts of all selected articles

were read to identify the potentially eligible articles. A manual search was performed

systematically using the authors' reference files and reference lists from original

23

communications, selected books and review article (Gardner and Hill, 2001; Mank and Zaat,

2001). Language restriction was not applied. The contents of abstracts or full-text manuscripts

identified during our literature search were reviewed to determine whether they met the criteria

for inclusion. For inclusion, a study had to allocate the study participants randomly to study

groups (a prospective randomized clinical trial). Included studies had to compare the

effectiveness of albendazole with that of metronidazole in the treatment of giardiasis.

Figure 2.1 summarizes the trial selection process. Our search identified twenty-nine articles

for further consideration, of which only eight articles met the inclusion criteria. Major reasons

for exclusion of studies were duplicate publications from which only one article was selected

(Misra et al., 1995a; Misra et al., 1995b), animal models of infections (Reynoldson et al., 1991;

Lemée et al., 2000), studies of veterinary importance (Meyer, 1998), studies in vitro (Bernal-

Redondo et al., 2004), single-arm studies with no randomized control group (Reynoldson et al.,

1998), studies lacking a comparison between the effectiveness of albendazole with

metronidazole (Pungpak et al., 1996), review articles (Gardner and Hill, 2001; Mank TG, Zaat,

24

FIGURE 2.1. Flow diagram deciphering the article selection process for this meta-analysis

study. Individual searches do not add up to 56 as some of the same articles were retrieved

by multiple search engines

25

2001), studies with no clear randomization allocation procedure (Baqai et al., 2005), studies

using albendazole and metronidazole analogues (Navarrete-Vázquez et al., 2003) as well as the

studies showing the synergistic effects between albendazole and/or metronidazole with other

drugs (Cacopardo et al., 1995; Hanevik et al., 2008). Conference proceedings and unpublished

data were also not included. Included articles compared the effectiveness of albendazole with

that of metronidazole in the treatment of giardiasis (Hall and Nahar, 1993a, b; Dutta et al., 1994;

Romero-Cabello et al., 1995; Misra et al., 1995a; Rodríguez-García et al., 1996; Karabay et al.,

2004; Yereli et al., 2004; Alizadeh et al., 2006). Together these articles followed 900 patients

presenting with symptomatic and/or asymptomatic G. duodenalis infections. Among these 900

treated patients, 452 (50.2%) individuals were treated with albendazole whereas 448 (49.8%)

received metronidazole

2.2.2. Data Extraction and Analysis

Data were extracted independently by two reviewers (SSM and SMS) from the eight

randomized controlled trials (Table 2.1 and Table 2.2). Discrepancies were resolved by

discussion. Study characteristics recorded were as follows: 1) first author's name, year of

publication and country of origin; 2) description of the population; 3) number of participants; 4)

age and sex distribution of the participants; 5) number of participants in each arm; 6) clinical

profile (symptomatic, asymptomatic infections); 7) the follow-up period; 8) the outcome

measure; 9) study design; 10) type and dosage of the drugs; and 11) effectiveness range. The

primary outcome measure was parasitological cure defined as the absence of parasites

(trophozoites and/or cysts) in feces at the end of the treatment in at least two consecutive stool

26

microscopy examinations. Parasitological cure was considered necessary in order to evaluate the

effectiveness of the treatment.

TABLE 2.1. Characteristics of the randomized controlled trials included in the meta-

analysis

27

TABLE 2.2.  Follow-up, outcomes assessment and relative risk in the trials included in the meta-analysis.

The secondary outcome measure, clinical cure, was defined as the global improvement of

clinical symptoms, such as diarrhea, nausea/vomiting, transient abdominal pain and loss of

appetite, at the end of the follow-up period.

28

2.2.3.  Assessment of Study Quality

The quality of included reports was compared using the Jadad score which examines whether

there is randomization, blinding, and information on dropouts/withdrawals from the study (Jadad

et al., 1996). It also evaluates the appropriateness of randomization and blinding, if present. The

quality scale ranges from 0 to 5 points with a low-quality report earning score of 2 or less. A

study with a Jadad score ≥3 is considered to be of ample quality. The quality of parasitological

diagnostic methods was assessed by the scoring system utilized by Zaat et al. (1997). This

method evaluates whether techniques are sufficiently described and are adequate. Moreover, this

method evaluates the reproducibility of the parasitological examinations and the level of inter-

observer variation among methods (Table 2.3).

TABLE 2.3. Internal validity (methodological and parasitological) of included trials

29

2.2.4. Sensitivity Analysis

Three different methods were employed to perform sensitivity analysis of these trials. We

first excluded the trial in which the parasitological method employed was not clearly described

(Dutta et al., 1994). Second, the trials that utilized the most insensitive diagnostic methods, i.e.

direct stool microscopy, alone were excluded (Karabay et al., 2004; Alizadeh et al., 2006).

Finally, we excluded a trial that used the most sensitive parasitological methods (three methods

at the same time) (Yereli et al., 2004) and compared the results with the remaining trials which

used two parasitological methods.

2.2.5. Data Synthesis and Statistical Analysis

We identified eight randomized, controlled trials that reported data on the comparison of the

effectiveness of albendazole with metronidazole in the treatment of giardiasis in humans. The

inconsistency across trials was calculated using the I2 statistic; results range between 0% (i.e., no

observed heterogeneity) and 100% (Higgins and Thompson, 2002). High values reflect

increasing heterogeneity. Publication bias was assessed by means of funnel plots (Egger et al.,

1997). Relative risks (RRs) were calculated for each study outcome separately based on

information presented in articles (i.e. the percentage of people exhibiting parasitological cure in

both groups relative to the percentage of people continuing to shed cysts during the follow up

period); the pooled RRs and 95% confidence intervals (CIs) were estimated by using the inverse-

variance random-effects method (DerSimonian and Laird, 1986). Although there is no standard

description, an I2 statistic greater than 20% suggests heterogeneity while an I2 statistic greater

than 50% usually is considered to represent significant heterogeneity (Higgins and Thompson,

30

2002). The statistical package Review Manager Software 5 (Cochrane Collaboration, Oxford,

UK) was used for analyzing the data.

2.3. Results

Table 2.1 and Table 2.2 summarize the characteristics of the randomized clinical trials

(RCTs) included in the meta-analysis. Studies were conducted in areas that are endemic for

giardiasis in humans, including Iran (Alizadeh et al., 2006), Turkey (Karabay et al., 2004; Yereli

et al., 2004) Mexico (Romero-Cabello et al., 1995; Rodríguez-García et al., 1996), India (Dutta

et al., 1994; Misra et al., 1995a), and Bangladesh (Hall and Nahar, 1993a, b). Only one study

(Hall and Nahar, 1993a, b) was rated as having good methodological quality based on a Jadad

score of 3 (see Table 2.3). However, because of the difficulty of comparing different treatment

protocols, one would rarely expect to achieve a high Jadad score of 3 or greater. Only two

studies (Hall and Nahar, 1993a, b; Alizadeh et al., 2006) had at least one blinded outcome

measurement (parasitological cure); whereas the other 6 trials were open label RCTs, allocating

patients to albendazole and metronidazole groups randomly. Patients included in one of the

groups in all trials were given albendazole. The dosages of albendazole ranged from 10 mg/kg

sid for 5 days (Yereli et al., 2004) to 400 mg/d for 5 days in most trials; lengths of therapy

ranged from a single dose for 1 day to 5 days. Metronidazole dosage ranged from 22.5 mg/day

(Misra et al., 1995a) to 1500 mg/day (Karabay et al., 2004), and the treatment course varied from

5 to 7 days. In six studies, subjects had symptomatic and/or asymptomatic giardiasis while the

clinical status of patients in one study (Dutta et al., 1994) was unclear. It is likely that the

overwhelming majority, if not all, of the cases included in the study of Hall and Nahar (1993a, b)

31

were asymptomatic cyst-passers, since the general population in an urban slum in Dhaka,

Bangladesh was screened. The post-treatment follow-up differed across the studies from 10 days

(Hall and Nahar, 1993a, b; Alizadeh et al., 2006) to 21 days (Dutta et al., 1994; Misra et al.,

1995a; Romero-Cabello et al., 1995). Loss of follow-up did not occur in six studies whereas two

articles (Misra et al., 1995a; Alizadeh et al., 2006) reported withdrawals and/or dropouts. In one

study (Misra et al., 1995a), only 18/32 children in the albendazole group and 16/32 children in

the metronidazole group finished the study, while in the other study, of 60 patients in each arm

15 from the albendazole group and 9 from the metronidazole group failed to complete the course

of medication (Alizadeh et al., 2006). Side effects from metronidazole therapy did not appear to

influence the treatment outcome, since low compliance in the latter study was reported to be due

to difficulties in returning to the study clinic rather than to side effects of the treatments.

The included studies implemented different diagnostic procedures alone or in combination

with other parasitological methods. As seen in Table 2.2, Yereli et al. (2004) applied three

different parasitological methods at the same time (a parasitological assessment score of 13 out

of 15). Misra et al. (1995a), Romero-Cabello et al. (1995), and Nahar and Hall (1993a, b)

employed two different parasitological methods at the same time, Rodríguez-García et al. (1996)

used the Faust's concentration method whereas Alizadeh et al. (2006) and Karabay et al. (2004)

utilized the least sensitive parasitological method, conventional direct stool microscopy, for

measuring the outcomes. In all studies, the absence of detectable G. duodenalis trophozoites

and/or cysts in the stool microscopy during the follow-up period was required to declare the

patients cured; five studies measured the outcomes solely based on parasitological parameters

32

(Hall and Nahar, 1993a, b; Rodríguez-García et al., 1996; Karabay et al., 2004; Yereli et al.,

2004; Alizadeh et al., 2006), while three studies applied both parasitological and clinical

parameters for measuring the outcomes (Dutta et al., 1994; Misra et al., 1995a; Romero-Cabello

et al., 1995). Treatment effects were evaluated as relative risks (RR), estimates that were

calculated for each study individually based on the incidence of undetectable infections among

those taking metronidazole compared to the incidence of undetectable cases among those taking

albendazole. Study-specific RRs were combined using a random-effects model. The study-

specific RRs were weighted by the inverse of the sum of their variance and the estimated

between-studies variance component (DerSimonian and Laird, 1986). This method calculates the

mean difference between the treatment and control groups, with SEM for the difference. There

was no statistically significant heterogeneity among these studies using the random effects model

(χ2 = 11.91, 8 degrees of freedom, P = 0.16). An I2 of 33% in the current meta-analysis suggests

moderate heterogeneity. Results demonstrated no differences between the effectiveness of

albendazole compared with metronidazole for treatment of infections with G. duodenalis (RR,

0.97; 95% CI, 0.93 to 1.01). When analysis was restricted to trials with a Jadad score of less than

3 (seven trials, 617 patients), inconsistency between the trials was low (I2 = 0%), although the

overall estimates remained almost constant (RR, 0.99; CI, 0.96 to 1.03). Individual analyses of

the eight studies demonstrated that three studies (Rodríguez-García et al., 1996; Yereli et al.,

2004; Alizadeh et al., 2006), showed a relative risk of greater than 1 for being cured after

albendazole therapy (Figure 2.2). These differences showed that albendazole produced more

apparent cures compared with metronidazole. In one study (Dutta et al., 1994), the relative risk

was 1 indicating no differences between the effectiveness of albendazole and metronidazole.

33

However, four studies (Hall and Nahar, 1993a, b; Misra et al., 1995a; Romero-Cabello et al.,

1995; Karabay et al., 2004), showed a relative risk less than 1 (ranging from 0.89 to 0.96)

indicating that metronidazole was more effective (Figure 2.2). As illustrated in figure 2.2, the

95% confidence intervals for these studies overlapped to a large degree, suggesting that

albendazole and metronidazole are equally effective for treatment of giardiasis

FIGURE 2.2. Forest plot showing the effects of albendazole and metronidazole on human

giardiasis

Further examination of the two-phase study carried out by Hall and Nahar (1993a, b) showed

that by increasing the duration of therapy with albendazole from 400 mg/d for 3 days (in the first

phase) to 400 mg/d for 5 days (in the second phase) the RR increased from 0.89 to 0.94. This

suggests that when duration of treatment is similar there is less of a difference between

albendazole and metronidazole therapy. Additionally, the efficacy of albendazole in the

34

treatment of giardiasis increased in the second phase of the same study (94.1%) compared with

the first phase of the trial (87.8%), implying the need for using albendazole for longer periods of

time.

2.3.1. Publication Bias and Sensitivity Analysis

Publication bias was examined using a funnel plot. Figure 2.3 plots the funnel plot of the

treatment effects estimated from individual studies on the x-axis (RR) and the standard error of

these estimates on y-axis (S.E [log RR]). This analysis shows that the included studies were

almost evenly distributed around the vertical axis, providing no evidence of publication bias.

35

Figure 2.3. Funnel plots of included studies

To explore further the possibility of heterogeneity due to the use of different outcome

measures, we confined our analysis to trials which used the least sensitive methods to detect

parasites and then to those that used the most sensitive methods (three methods at the same

time). Similarly, we performed an analysis restricted to those studies with clearly defined

outcome measures. As seen in Table 2.4, the overall estimates were equal and the confidence

intervals were comparable among these restricted data sets, as well as with the combined meta-

analysis values.

36

TABLE 2.4. Sensitivity-analysis of the effect of the quality of methods implemented for the

measurement of parasitological cure

2.3.2. Adverse Effects

In six studies, side effects related to therapy were absent or were less prominent in the

patients receiving albendazole. Only in one study (Hall and Nahar, 1993a, b) were the reported

side effects more evident in patients in the albendazole group compared with those in the

metronidazole group (40 cases vs. 7 cases; P<0.005). Overall, metallic taste and anorexia were

the most commonly observed side effects in patients treated with metronidazole, while loose

stools and abdominal pain were more frequent among patients receiving albendazole. Most side

effects were transient and no trials were discontinued because of severe adverse effects. In Yereli

37

et al. (2004), no side effects were reported in patients treated with either albendazole or

metronidazole. The report by Rodríguez-García et al. (1996) does not mention if treated children

showed any side effects. In order to perform a safety analysis, the two latter studies were

excluded from the analysis. Hall and Nahar (1993a, b) reported the adverse effects of a two-stage

trial, and these were treated as a single trial. Considering all side effects together, 61 of 373

(16.3%) patients treated with albendazole and 82 of 371 (22.1%) of patients treated with

metronidazole experienced at least a single side effect. The estimated summary RRs showed that

individuals treated with albendazole had a lower risk of adverse effects (RR 0.36) compared with

those who took metronidazole, but with a wide confidence interval (95% CI, 0.10, 1.34) that

included the null value.

2.4. Discussion

The major finding of this analysis is that when albendazole was given as a single dose of 400

mg/day for 5 days it was as effective as metronidazole in the treatment of giardiasis in humans.

Additionally, albendazole had statistically the same safety profile as metronidazole.

Metronidazole has been widely used to treat giardiasis in humans (Jokipii and Jokipii, 1978;

Jokipii and Jokipii, 1979; Bulut et al., 1996; Saffar et al., 2005; Hanevik et al., 2008), and often

causes side effects such as nausea, metallic taste, dizziness and headache (Gardner and Hill,

2001). In addition, this drug is a known mutagen in bacteria (Cantelli-Forti et al., 1983; De Méo

et al., 1996), it is genotoxic to human cells (Reitz et al., 1991; Elizondo et al., 1996) and it has

been shown to be carcinogenic in animal models (Krause et al., 1985; Bendesky et al., 2002).

However, there is no evidence showing metronidazole is also carcinogenic in humans (Bendesky

38

et al., 2002). Typically, metronidazole is administered in doses of 250 mg 3 times a day for 5-7

days for adults and 15 mg/kg 3 times a day for 5-7 days in children. Some clinicians tend to use

single-dose regimens, while others like to administer higher dosages for an extended period of

time. The latter is problematic in developing countries, as medications are frequently purchased

in quantities which represent less than a single day's dose and effective therapies of short

duration are preferable (Hossain et al., 1982). The need for an extended period of time for the

treatment of giardiasis again may in part explain the frequent side effects associated with

metronidazole therapy. Extended treatment with albendazole also appears to be more effective

than shorter duration protocols. However, the once per day regimen would still be preferable to

the three times per day required for metronidazole therapy.

A further complication when using metronidazole therapy to treat giardiasis is that the

consumption of alcohol should be avoided by patients during systemic metronidazole therapy

and for at least 24 hours after completion of treatment (Tillonen et al., 2000; Jang and Harris,

2007). Taking metronidazole and alcohol may result, rarely, in a disulfiram-like reaction (nausea,

vomiting, flushing, and tachycardia). It should be noted that the consumption of alcohol by

patients was not monitored in any of the studies considered in the current meta-analysis. Alcohol

uptake could potentially explain side effects in some patients receiving metronidazole. The lack

of placebo-controlled trials makes it difficult to attribute the existence and severity of side effects

to either of these two drugs. However, one study that did not meet our inclusion criteria

suggested that patients receiving a placebo control presented with minimal side effects (Saffar et

39

al., 2005). Together, these limitations can potentially restrict the use of metronidazole, in the

treatment of giardiasis in humans.

In the trials included in the current meta-analysis, only one study (Alizadeh et al., 2006)

clearly described the inclusion of both adults and children (2–53 yr), while other studies

exclusively included either only adults (Karabay et al., 2004) or only children (Hall and Nahar,

1993a, b; Dutta et al., 1994; Misra et al., 1995a; Romero-Cabello et al., 1995; Rodríguez-García

et al., 1996; Yereli et al., 2004). The inclusion of different age-groups potentially allows us to

assess the effectiveness of treatment and to ascertain the extent to which side effects occur in

different age-groups. Similarly, including patients with diverse clinical presentations (i.e.

asymptomatic, symptomatic; acute, subacute, chronic) in clinical trials could give us the

opportunity to evaluate the effect(s) of a given chemotherapy agent/protocol on patients with

different clinical manifestations. From the information presented in the articles, it seems that

only three articles (Romero-Cabello et al., 1995; Rodríguez-García et al., 1996; Yereli et al.,

2004) included both symptomatic and asymptomatic patients, although the clinical status of

patients who participated in the study of Hall and Nahar (1993a, b) and Dutta et al. (1994) was

not clear. Including patients from different age-groups and with different clinical presentations in

future studies would allow investigators to analyze whether albendazole has a differential effect

that correlates to the disease clinical profile and/or age of the patient.

Our analysis suggests that the study designs typically used for evaluating these drugs could be

improved. Open-label trials may be suitable for comparing two extremely similar treatments to

verify which one is more effective. Only Alizadeh et al. (2006) and Hall and Nahar (1993a, b)

40

used an adequate protocol for concealing the treatment protocol while determining the

parasitological outcome. The six other trials either did not specify or were insufficient in using

blinded observers to determine the outcome. Since albendazole and metronidazole may produce

certain side effects specific to each drug and since these two drugs may be available in different

forms, the use of homogenous therapy regimens and/or using blinded studies may be warranted

in future clinical trials.

Several factors may influence the effectiveness of a particular therapy. Nutritional and

physiological conditions such as pregnancy and immunodeficiency could potentially alter the

effectiveness of a specific drug as shown for other parasitic diseases (Lwin et al., 1987;

Bjorkman, 1988; Mohan et al., 1999; Cravo et al., 2001). Individuals with “pre-existing”

nutritional and physiological (pregnant women) complications were excluded in only two studies

among those we have analyzed (Dutta et al., 1994; Karabay et al., 2004). Dutta et al. (1994)

excluded children having grade I and II malnutrition, patients with acute febrile illness and those

on long term drug therapy; while Karabay et al. (2004) excluded pregnant women and patients

with fever from the study. In future studies, it would be desirable to include only patients with no

known nutritional, physiological or immunological problems.

Resistance of G. duodenalis strains to metronidazole and other drugs has been reported both

in vitro and in vivo (Upcroft and Upcroft, 1993; Gardner and Hill, 2001; Müller et al., 2008).

Misra et al. (1995a) reported a 100% cure rate in groups treated with either metronidazole or

albendazole, while the other seven reported an effectiveness of 72.7–100% for metronidazole

and 77–97% for albendazole. At least part of the so-called “failure-to-treat” cases might be

41

attributed to the presence of “drug-resistant” strains, a mechanism to which none of the studies

referred as a potential reason for treatment failure. The use of different combinations of

albendazole with other anti-parasitic agents in future studies may be desirable in order to

minimize the risk of the emergence of drug-resistant strains. However, the design of placebo-

controlled double blinded clinical trials may help us to better understand the most appropriate

regimen(s) and the most suitable chemotherapy protocols.

Some limitations in the current analysis should be considered before making definitive

conclusions. First, the small number of trials and patients included in the current analysis (8

studies, 900 patients) led to wide confidence intervals that rendered some of the results

inconclusive (Matthaiou et al., 2008). Second, publication bias is constantly a potential pitfall in

meta-analyses. While we did not try to trace unpublished data for the current meta-analysis, our

analysis failed to detect any suggestion of such bias (Figure 2.3). Third, heterogeneity among

studies is another potential limitation to our meta-analysis. It might be argued that differences in

the methods used for measuring the outcome of treatment could result in differences in the

reported parasitological cure rates, as some combined methods are more sensitive than others. As

seen in Table 2.4, the effect sizes remained fairly constant in these analyses, suggesting that

heterogeneity due to diverse outcome measures probably did not adversely affect our analyses.

Performing repeated microscopy-based stool examinations on at least two consecutive occasions

is sensitive enough to detect up to 95% of infections (Naik et al., 1978; Gordts et al., 1985). This

could potentially explain why we did not see any difference among studies employing diverse

42

methods since all the studies required at least two consecutive negative stool examinations

before considering the patients cured.

The high rate of side effects from metronidazole therapy for giardiasis, combined with the

global emergence of resistant strains, led us to consider the effectiveness of alternative

treatments. This meta-analysis revealed that albendazole cures Giardia infections with the same

effectiveness as metronidazole. However, we were not able to show conclusively, due to

limitations of the sample size, that its toxicity profile is more favorable than metronidazole.

Therefore, we conclude that larger, double-masked, randomized controlled trials of albendazole

and metronidazole with uniform outcome measures are needed to shed light on this important

clinical question.

43

Chapter III

Host Immunity and Pathogen Strain Contribute to

Intestinal Disaccharidase Impairment following Gut

Infection

Part of this Chapter was published in “Journal of

Immunology”.

Solaymani-Mohammadi S, Singer SM. 2011. Host immunity and

pathogen strain contribute to intestinal disaccharidase impairment

following gut infection. The Journal of Immunology, 187 (7): 3769-

3775.

44

3.1. Introduction

The brush border (BB) lining the surface of the small intestinal epithelium provides the major

interface for nutrient absorption. Reduction in the surface area of the microvilli is linked with

impaired levels of disaccharidase enzymes such as sucrase and lactase that are essential for

proper digestion and absorption of sugars (Farthing, 1997). Several infectious agents, such as

rotavirus (Jourdan et al., 1998) and HIV (Lim et al., 1995; Taylor et al., 2000), as well as

noninfectious causes including celiac disease (Murray et al., 2001), iron deficiency (Lanzkowsky

et al., 1982; West et al., 2005), and vitamin A deficiency (Reifen et al., 1998) are reported to be

linked with decreased intestinal disaccharidase activity. Reduced BB surface area has been

documented in human and murine giardiasis (McDonnell et al., 2003; Scott et al., 2004).

Reduced disaccharidase activity has been previously reported in murine, human, and gerbil

models of infection with Giardia muris and Giardia duodenalis (Buret et al., 1990; Buret et al.,

1992); infected gerbils exhibited symptomatic disease, and reduced BB enzymes were evident

from day 10 postinfection (p.i.) and onward.

G. duodenalis has eight distinctive genotypes, of which only two genotypes (assemblages A

and B) infect humans (Thompson, 2009). Several reports have demonstrated variations in

symptoms and pathology resulting from infections with different Giardia isolates. There is

controversy surrounding the impact of a given assemblage of G. duodenalis (i.e., assemblage A

or B) on symptomatic disease and intestinal pathology. For example, Singh et al. (2009) showed

that 74% of isolates examined from symptomatic patients in Nepal were assemblage B, whereas

20 and 6% were assemblage A and mixed infections, respectively. As such, a strong correlation

was found between assemblage B and symptomatic disease, whereas all asymptomatic

45

individuals harbored assemblage A (Al-Mohammed, 2010). In contrast, different studies found

assemblage A to be predominantly associated with symptomatic disease in Bangladesh (Haque et

al., 2005). However, some reports did not find any correlation between the parasite genotype and

symptomatic disease. For instance, Kohli et al. (2008) did not find any differences in the ability

of either assemblage A or B to cause symptomatic infections in children in Brazil. One

possibility is that the differences observed reflect variation within these genotypic classifications,

especially the presence or absence of specific virulence factors. Alternatively, given that host

immune responses are, in part, responsible for pathology induced by G. duodenalis, it is possible

that different isolates may elicit different immune effectors of the host immune responses in

different ways (Solaymani-Mohammadi, S., and S.M. Singer. 2010).

Studies in vitro and in vivo showed differences in the ability of different Giardia isolates to

induce apoptosis and mucosal damage, whereas other strains did not affect the gut integrity. For

instance, the NF and S2 strains of G. duodenalis induced enterocyte (EC) apoptosis in cell

culture, whereas other strains such as WB or PB did not (Chin et al., 2002). Likewise, studies in

a neonatal rat model validated mucosal damage to be strain dependent (Cevallos et al., 1995).

This may support the idea that the pathology observed in human giardiasis is, in part, strain-

dependent.

We performed this study to address to what extent host immunity and parasite strain mediate

the development of disaccharidase impairment in a murine model of gut infection. Analyses

showed that murine hosts that lack a functional adaptive immune response did not exhibit

impaired enzymatic activity despite considerable parasite burdens and the host’s inability to

resolve infections with strain GS. Also of interest was the finding that mice infected with the WB

46

strain of the parasite did not manifest any sign of intestinal enzyme deficiency, although spleen

cell cytokine production was similar p.i. with either strain.

3.2. Methods

3.2.1. Mice

Six- to 8-wk-old female C57BL/6J, SCID (B6.CB17-Prkdcscid/SzJ), β2- microglobulin (β2m)–

deficient (B6.129P2-B2mtm1Unc/J), and CD4 knockout (B6.129S2-Cd4tm1Mak/J) mice were

purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were housed at the

Georgetown University Animal Care Facility, and all experiments were carried out in accordance

with guidelines approved by the Georgetown University Animal Care and Use Committee in

compliance with the National Institutes of Health guidelines.

3.2.2. Parasites and Infection Protocols

The GS/M-83-H7 (ATCC 50581) and the WB clone-C6 (ATCC 50803) strains of G.

duodenalis were used in these experiments. Parasites were axenically grown in TYI-S-33 media

supplemented with adult bovine bile, L-cysteine, ascorbic acid, and antibiotics (all from Sigma-

Aldrich, St. Louis, MO). Forty-eight hours before infection, mice were given antibiotics in

drinking water ad libitum: neomycin oral solution (1.4 mg/ml; Durvet, Blue Spring, MO),

ampicillin (1 mg/ml; Sigma-Aldrich), and vancomycin (1 mg/ml; Hospira, Lake Forest, IL)

(Singer and Nash, 2000). Mice were gavaged with 106 trophozoites in 0.1 ml PBS (pH 7.4).

Antibiotic use was maintained for the duration of the infection. To quantify parasite loads at

different times p.i., we euthanized mice and discarded the first 3 cm of the small intestine

(pylorus to ligament of Treitz). The next 2-cm section of the duodenum was removed, opened

47

longitudinally, and minced in 4 ml ice-cold PBS (pH 7.4). Tissues were kept on ice for 15 min,

and the numbers of trophozoites were counted using a hemocytometer.

3.2.3. Disaccharidase Activity Assay

Intestinal disaccharidase activity was measured using the method originally developed by

Dahlqvist (1968) with some modifications (Belosevic et al., 1989; Daniels, and Belosevic, 1992).

In brief, jejunal segments of ~10 cm in length were removed, and homogenates were prepared in

Milli-Q water supplemented with protease inhibitor mixture III (Calbiochem, La Jolla, CA).

Protein concentrations for each sample were determined by the Bradford method (Bio-Rad

Laboratories, Hercules, CA) with BSA as standard. The relevant substrates (56 mM) were

prepared in maleic buffer (0.1 M; pH 6.0) as previously described (Daniels and Belosevic, 1995).

After initial incubation of the intestinal lysates with relevant substrates for 1 h at 37°C, 300 ml

Tris-glucose oxidase-peroxidase was added to each well. The samples were incubated at 37°C

for another hour. The glucose release was measured at 450 nm using a microplate reader (BioTek

Instrument, Winooski, VT) with D-glucose as standard. Disaccharidase activity was expressed as

nanomole glucose produced per milligram of total protein per minute.

3.2.4. Ex vivo Restimulation of Splenocytes and Mesenteric Lymph Node Cells

G. duodenalis trophozoites were axenically grown in TYI-S-33 media as described earlier.

Confluent tissue flasks were kept on ice for 30 min, and detached trophozoites were washed five

times with ice-cold PBS (pH 7.4). Whole-cell lysate was prepared using repeated freeze–thaw

cycles (5x), stored at 280°C, and protein concentration of each sample was measured by the

Bradford method (Bio-Rad Laboratories, Hercules, CA). Spleens and mesenteric lymph nodes

48

(MLNs) from uninfected and infected mice were removed aseptically, and cells were isolated by

mechanical disruption. RBCs were lysed in cold isotonic NH4Cl lysis buffer (155 mM NH4Cl, 10

mM KHCO3, and 100 mM EDTA, pH 7.4), and the remaining cells were washed twice with ice-

cold PBS. A total of 5x106 cells were cultured in duplicate in 1 ml RPMI 1640 (Invitrogen)

supplemented with 10% FBS (Hyclone, Logan, UT), 1 mM glutamine (Life Technologies-BRL),

50 mM 2-ME, and 100 mg/ml antibiotics/antimycotic (Sigma-Aldrich). Cells were seeded in

24-well plates (Corning Costar Corporation, Cambridge, MA), were stimulated with 100 mg/ml

relevant Giardia extract, and were incubated at 37°C containing 5% CO2. Culture supernatants

were harvested 48 h after stimulation and kept at -20°C until further analysis. In some

experiments, anti-mouse CD4 IgG (clone RM4-5; BioLegend, San Diego, CA) was added to

cultures at a concentration of 20 mg/ml to block activation of CD4+ T cells. Control wells

received PBS alone.

3.2.5. Cytokine Analysis by ELISA Culture supernatants from splenocytes were diluted with an appropriate sample diluent, and

cytokines were measured in duplicate by a sandwich ELISA method. Supernatants from MLN

cultures were measured undiluted. Mouse ELISA kits for TNF-α, IFN-γ, and IL-10 (BD-OptEIA,

San Diego, CA), IL-4 (SouthernBiotech, Birmingham, AL), and IL-13 and IL-22 (eBioscience,

San Diego, CA), as well as IL-17 (DuoSet; R&D Systems, Minneapolis, MN) were used.

3.2.6. Flow Cytometry

For flow cytometry, MLNs and spleens were collected in HBSS supplemented with 5% FBS

(Hyclone) and 25 mM HEPES and strained through a 70-µm nylon membrane (BD Falcon).

Live/dead cell labeling was performed using a LIVE/DEAD Fixable Yellow Dead Cell Stain Kit

49

(Invitrogen) for 45 min at 4°C in the dark according to the manufacturer’s instructions. Cells

were incubated in HBSS supplemented with 5% FBS and 25 mM HEPES to reduce nonspecific

binding. Cells (106/sample) were stained with anti–CD3-PerCP (clone 145-2C11), anti-CD4-PE

(clone RM4-5), and anti–CD8-allophycocyanin (clone 53-6.6; all from Bio-Legend) for 45 min

at 4°C and were fixed with 1% paraformaldehyde. Cells were analyzed using a Becton Dickinson

FACSAria (BD Biosciences) and FACS Express Version 4.0 software (DeNovo Software, Los

Angeles, CA).

3.2.7. Statistical Analyses Data were analyzed using GraphPad Prism 5 software (GraphPad, San Diego, CA) and

expressed as mean 6 SEM. For statistical analyses, a two-tailed Mann-Whitney U test was used,

and p ˂ 0.05 was considered statistically significant.

50

3.3. Results

3.3.1. CD8+ T Cells Are Required for Disaccharidase Deficiency but Not Control of

Infection

To investigate the role of different T cell subsets in control of infection and in mediating

intestinal pathology, we used a murine model system. We infected adult mice with the GS strain

of G. duodenalis (Byrd et al., 1994). Wild-type (WT) mice and mice lacking all adaptive

immune responses (SCID), CD4+ T cells only (CD4-/-), or CD8+ T cells (β2m-/-) were infected

and parasite loads were enumerated at days 5 and 18 p.i. Results showed that all mice were

heavily infected at day 5, and that WT mice eliminated almost all parasites by day 18 p.i. (Fig.

3.1A). In SCID and CD4-/- mice, however, parasite loads were even higher at day 5 p.i. and

significant parasite elimination was not seen by day 18 (Fig. 3.1B, 3.1D), indicating the vital role

of host immunity in clearing this infection. In contrast, β2m-/- mice eliminated parasites with

essentially the same kinetics as WT mice (Fig. 3.1C), indicating that although CD4+ T cells are

required to control this infection, CD8+ T cells are not.

51

FIGURE 3.1. CD4+ T cells are required for the clearance of infections in a mouse model of

disaccharidase deficiency. C57BL/6J (A), SCID (B), β2m-/- (C), and CD4-/- (D) mice were

infected on day 0 with the GS strain of G. duodenalis. Mice were euthanized on the

indicated days p.i., and parasite loads in the small intestine were enumerated as described

in Materials and Methods. Each bar indicates the mean ± SEM of four mice per time point.

Data are representative of two independent experiments.

s

52

Disaccharidase deficiency has been documented p.i. and after other inflammatory conditions

(Kvietys, 1999; West et al., 2005). We hypothesized that infection of WT C57BL/6J mice with

G. duodenalis would result in disaccharidase deficiency as previously shown for the murine

species of Giardia, G. muris (Buret et al., 1990). To test this hypothesis, we measured

disaccharidase enzyme activity in the jejunum of infected mice. We found that the levels of

sucrase, maltase, and lactase, decreased 40, 29, and 37%, respectively, on day 5 p.i. with the GS

strain of the parasite (Fig. 3.2A–C). The decrease in trehalase levels was not statistically

significant (Fig. 3.2D). All enzyme levels returned to normal by day 18 when the hosts had

cleared the infections.

53

FIGURE 3.2. Disaccharidase enzymes are depressed in the intestines of WT C57BL/6J

mice after gut infections. Disaccharidase activity assays were performed on jejunal

homogenates from mice infected in Fig. 1. Levels of sucrase (A), maltase (B), lactase (C),

and trehalase (D) were determined using the appropriate disaccharide. Data are shown as

mean and SEM for four mice per group. *p ˂ 0.05 by Mann–Whitney U test versus

uninfected mice. Data are representative of three independent experiments. ns, not

significant.

54

We next investigated whether host immunity could mediate the depression of intestinal

enzyme activity after gut infections. To test this, we infected SCID mice with the GS strain of

the parasite and measured disaccharidase activities in the jejunum. Although infection in SCID

mice resulted in increased parasite burdens and failure to eliminate the infection by the host (Fig.

3.1B), no significant impairment of intestinal enzymes was observed (p ˂ 0.05; Fig. 3.3A-D).

These findings suggest that disaccharidase impairment is mediated by host immunity.

 

55

FIGURE 3.3. Lack of adaptive immunity prevents SCID mice from exhibiting impaired

intestinal disaccharidase activity p.i. Sucrase (A), maltase (B), lactase (C), and trehalase (D)

activities were measured in jejunal homogenates from SCID C57BL/6J mice infected with

106 trophozoites of the GS strain or uninfected SCID mice. Data represent means and SEM

for four mice per group. Data are representative of two independent experiments.

56

Having established that disaccharidase impairment is immune-mediated, we examined

specific immune cell populations that could contribute to disaccharidase impairment during

Giardia infection. In β2m-/- mice infected with Giardia, there were no significant changes in the

levels of sucrase activity at day 5 p.i. (p ˂ 0.05; Fig. 3.4A). Similarly, no significant changes

were observed in the activity levels of maltase, lactase, and trehalase 5 d p.i. compared with

uninfected animals (Fig. 3.4B–D). Results showed that the absence of CD4+ T cells in infected

mice did not induce lower enzymatic levels compared with uninfected controls (Fig. 3.4A-D).

These findings clearly suggest that the pathology seen in the intestine during the Giardia

infections is T cell dependent.

 

57

FIGURE 3.4. Lack of CD8+ T cells prevents mice from exhibiting impaired intestinal

disaccharidase activity p.i. β2m-/- and CD4-/- mice were orally inoculated with 106

trophozoites of the GS strain of G. duodenalis. Sucrase (A), maltase (B), lactase (C), and

trehalase (D) activities were measured in jejunal homogenates from mice infected for 5 or

18 d or uninfected mice. Data represent means and SEM for four mice per group. Data are

representative of two independent experiments.

58

3.3.2. Cytokine Responses by Spleen and MLN Cells p.i.   Our results suggest that T cell responses are required for elimination of infection and

induction of disaccharidase deficiency. We were therefore interested to determine whether the

proportion of CD4+ and CD8+ T cells would change p.i. and to determine the cytokines being

produced by T cells. Flow cytometry analysis showed that the composition of CD4+ and CD8+ T

cells in the spleen and MLNs remained constant in mice infected with the GS strain of G.

duodenalis 5 and 7 d p.i. compared with uninfected mice (Fig. 3.5A, 3.5B). Analysis of cytokine

production by cells from spleens and MLNs of mice infected with the GS strain showed that the

peak of cytokine production occurred at day 7 p.i., and that the levels of cytokines produced by

splenocytes were generally higher than the levels of cytokines produced by MLN cells (Fig.

3.5C–H). IFN-γ was the dominant cytokine produced by both splenocytes and MLN cells in

response to parasite Ags, followed by IL-10. Lesser amounts of TNF-α, IL-4, IL-17, and IL-22

were also detected.

59

FIGURE 3. 5. Cytokine production by cells from spleens and MLNs of WT mice infected

with the GS strain of G. duodenalis. WT mice were infected with the GS strain of G.

duodenalis and euthanized at days 5 and 7 p.i. The proportions of CD4+ and CD8+ cells in

spleens (A) and MLNs (B) were determined in uninfected and infected mice during the

course of infection. Production of IFN-γ (C), TNF-α (D), IL-4 (E), IL-10 (F), IL-17 (G), and

IL-22 (H) by spleens and MLNs of infected mice was determined using ELISA. Cell

viability after 48 h of stimulation was about 50 and 70% in stimulated and unstimulated

preparations, respectively. Data presented are means and SEM for four mice per time

point.

60

3.3.3. Disaccharidase Deficiency in WT Mice Is Strain-Dependent Human giardiasis can cause a wide range of clinical symptoms ranging from asymptomatic

infections to severe diarrhea and cramps, which can result in maldigestion and stunted growth,

especially in pediatric patients (Farthing, 1997). It has been suggested that distinct assemblages

of G. duodenalis differ in their capacity to cause symptomatic disease. We therefore asked

whether the changes we observed in C57BL/6J mice infected with the GS strain of the parasite,

the prototype for assemblage B, would occur during infection with the prototype strain for

assemblage A. Because initial studies indicated that mouse infections with strain WB, the

prototype for assemblage A, were unsuccessful (Byrd et al., 1994), we developed a revised

protocol for mouse infections to allow us to work with this strain. To determine the kinetics of

infection and disaccharidase activity p.i. in mice with both the GS and WB strains, we infected

WT mice with each strain of G. duodenalis separately, and the infection and disaccharidase

activity kinetics were measured at days 5, 7, 10, 14, and 18 p.i. Parasitological examinations

revealed substantial colonization of the C57BL/6J mouse intestine by the WB strain of G.

duodenalis. Although parasite burdens in the intestines of mice infected with the WB strain were

generally slightly lower compared with mice infected with the GS strain of the parasite, the

differences in parasite burdens between these strains were not statistically significant (Fig. 3.6A).

In contrast, our analyses showed that the WB strain of G. duodenalis was not able to induce

disaccharidase deficiency, whereas WT mice infected with the GS strain exhibited enzyme

impairment p.i.; the reduced enzyme activity in the GS-infected mice started 5 d p.i., remained

low 7 d p.i., and returned to normal levels by day 18 p.i. (Fig. 3.6B). These data suggest a strain

dependent basis for disaccharidase impairment.

61

FIGURE 3.6. Infection and disaccharidase kinetics and cytokine production profile in WT

mice infected with WB and GS strain of G. duodenalis.WTC57BL/ 6J mice were infected

with 106 trophozoites of either the WB or GS strain. Mice were euthanized at 5, 7, 10, 14,

and 18 d p.i. Parasite burdens (A) and disaccharidase activities (B) were determined at

each time point for both strains (A). Spleen cells were re-stimulated in vitro with Giardia

extracts prepared from the same strain used for infection, and supernatants were collected

after 48 h. Levels of IFN-γ (C), TNF-α (D), IL-4 (E), IL-10 (F), IL-13 (G), IL-17 (H), and

IL-22 (I) were measured by ELISA. Intestines and splenocytes from individual mice were

assayed in duplicate, and the data presented are means and SEM for four mice per time

point (*p ˂ 0.05, **p ˂ 0.005, compared with uninfected mice). Data are representative of

three independent experiments.

62

3.3.4. Cytokine Production by T Cells p.i.

We next sought to determine whether T cell cytokine responses were significantly different

p.i. between these two strains. Cytokine production by spleen cells from mice infected with each

strain of the parasite was measured ex vivo at six different time points (i.e., days 0, 5, 7, 10, 14,

18). Our analysis showed that both strains of the parasite elicited robust cytokine production in

response to parasite extract, with detectable levels of IFN-γ, TNF-α, IL-4, IL-10, IL-13, IL-17,

and IL-22 being produced at day 7 p.i. with either strain (Fig. 3.6C–I). IFN-γ was the dominant

cytokine secreted p.i. with both parasite strains. An average of 5000 and 2500 pg/ml was

observed at 7 d p.i. in mice infected with the WB and GS strains, respectively (Fig. 3.6C).

Interestingly, the levels of all cytokines assayed in mice infected with the WB strain of G.

duodenalis were somewhat higher than those infected with the GS strain of the parasite.

3.3.5. CD4+ cells are the main cytokine-secreting T cells in mice infected with WB and GS

strains

Considering the essential roles of CD4+ T cells in resolving the intestinal infection in our

model, we were interested in finding to what extent CD4+ cells contributed to the production of

cytokines. WT mice were infected with either WB or GS and euthanized on day 7. Parasite

burdens were similar to what was observed in previous experiments (Fig. 3.7A). Splenocytes

were stimulated in vitro with Giardia extract, and CD4+ responses were blocked with an anti-

CD4 IgG. Analyses of supernatants showed that CD4+ T cells were the major cytokine-secreting

cells p.i. with either strain WB or GS. Blocking CD4 significantly reduced the production of

IFN-γ, IL-4, IL-13, IL-17, and IL-22 (Fig. 3.7B, 3.7D, 3.7F–H). CD4+ T cell involvement in the

63

production of TNF-α was only 30 and 34% for WB and GS strains, respectively (Fig. 3.7C),

consistent with our previous findings that dendritic cells contributed to the secretion of TNF-α

during infection with G. duodenalis (Kamda and Singer, 2009). The production of IL-10 in mice

infected with each strain was reduced drastically (87.5 and 37.5% for WB and GS strains,

respectively), and it seemed that non-CD4 sources continued to secrete IL-10 in mice infected

with the GS strain of the infection despite CD4 blockade (Fig. 3.7E).

64

FIGURE 3.7. CD4+ cells are the main cytokine-secreting T cells in mice infected with WB

and GS strains. WT mice were infected with either WB or GS, and parasite burdens were

determined 7 d p.i. (A). Splenocytes from the WB- and GS-infected mice were collected and

stimulated in duplicate in vitro with the homologous Giardia extract in the presence or

absence of an anti-mouseCD4 Ab. Production of IFN-γ (B),TNF-α (C), IL-4 (D), IL-10 (E),

IL-13 (F), IL-17 (G), and IL-22 (H) was determined using ELISA. Data represent means

and SEM for four mice per group (*p ˂ 0.05, **p ˂ 0.005).

65

3.4. Discussion

The major findings of this study are that levels of disaccharidase enzymes such as sucrase,

maltase, and lactase decline significantly after murine infections with the GS strain of the human

parasite G. duodenalis. We also showed that in the absence of adaptive immunity in SCID mice,

there was no reduction of intestinal enzyme activity. Infections in CD4-/- and β2m-/- mice further

showed that this impairment was an immune-based event requiring both CD4+ and CD8+ T cells.

The results of this study also revealed an important difference in the ability of different pathogen

strains to induce intestinal enzymatic deficiency in the mouse model of human giardiasis;

C57BL/6J mice infected with the GS strain of the parasite displayed enzymatic deficiency,

whereas mice infected with the WB strain of the parasite did not.

Similar to results from previous studies, our results indicate that CD4+ T cells are required for

parasite elimination, whereas CD8+ T cells are not required. Interestingly, although β2m-/-

eliminate infections normally, they fail to exhibit disaccharidase deficiency. Scott et al. (2004)

showed that CD8+ T cells from MLNs of G. muris-infected mice could induce disaccharidase

deficiency after adoptive transfer into nude recipients. Our results are consistent with an

important role for CD8+ T cells in causing disaccharidase deficiency. Our results further suggest

that CD4+ T cells have a role in inducing disaccharidase deficiency because no disaccharidase

deficiency was seen in CD4-/- mice. This role may be indirect, however; for example, CD4+ T

cells may be necessary to generate an effector CD8+ T cell population. In summary, these results

suggest that immunotherapies or vaccines that promote CD4+ T cell responses without

66

generating CD8+ T cell responses could lead to protection without resulting in disaccharidase

deficiency and contributing to nutrient malabsorption.

Different mechanisms have been regarded as the cause of disaccharidase deficiency p.i. and

after other intestinal insults. These include direct damage to the epithelium caused by the

parasite, changes in gene expression within epithelial cells, and altered rates of EC maturation

p.i. (Farthing, 1997). Our results clearly exclude direct damage as a cause of disaccharidase

deficiency in this model because no changes in disaccharidase levels were seen in SCID or CD4-

/- mice despite having prolonged infections with very heavy parasite burdens. Although our data

suggest that CD8+ T cells are important in mediating pathology, they do not discriminate

between specific changes in gene expression (i.e., reduced transcription of the isomaltase or

lactase/phlorizin hydrolase genes) within epithelial cells or more global changes in epithelial cell

biology (i.e., an increased proportion of immature ECs). Indeed, changes in the maturation of

epithelial cells can involve both transcriptional and posttranscriptional regulation of

disaccharidase enzymes (Reifen, et al., 1998; Zaiger, et al., 2004).

Different isolates of G. duodenalis have been shown to differ in their abilities to trigger

pathological changes in vitro. For example, some strains of the parasite (i.e., NF and S2) were

able to induce apoptosis in a non-transformed epithelial cell line (SCBN), whereas other strains

(i.e.,WB and PB) could not (Chin et al., 2002). A more recent study showed that the WB strain

could actually facilitate caspase-dependent apoptosis in the epithelial cell line HCT-8 (Panaro et

al., 2007). Consistent with previous reports describing important biological differences between

WB and GS (Solaymani-Mohammadi and Singer, 2010), we found that the induction of

disaccharidase deficiency and the dynamics and kinetics of cytokine production in mice infected

67

with each strain were distinct. Whether these differences in cytokine response are connected with

the absence of disaccharidase deficiency p.i. with strain WB, however, remains to be determined.

To our knowledge, this study is the first to describe the cytokines produced by CD4+ T cells

in response to Giardia infection. Spleen cells and MLNs stimulated ex vivo with Giardia extracts

produced IFN-γ, TNF-α, IL-4, IL-10, IL-13, IL-17, and IL-22. Blocking CD4 eliminated

production of almost all of these cytokines, indicating that CD4+ T cells are the likely source of

these cytokines. The lone exception was TNF-α, which we have previously shown can be

produced in small amounts by dendritic cells stimulated with Giardia extracts (Kamda and

Singer, 2009). Previous work in the G. muris model was unable to demonstrate cytokine

production in response to parasite Ags, although some changes in mitogen-driven cytokine

production were noted (Venkatesan et al., 1996).

Among the cytokines produced, IFN-γ was the most abundant. However, previous work using

GS infection in adult mice lacking IFN-γ suggested that parasite elimination could occur

normally in the absence of this cytokine. In contrast, anti–IFN-γ treatment of C57BL/10 mice

resulted in prolonged infections with G. muris (Venkatesan et al., 1996). There may be

differences in the effector mechanisms that are needed to eliminate the different pathogens. For

example, NO, antimicrobial peptides, IgA, bile salts, and intestinal hypermotility have all been

shown to participate in parasite clearance, and it is likely that significant redundancy results in

the absence of strong effects after elimination of single cytokines or pathways.

Interestingly, WB-infected mice exhibited two distinct cytokine peaks, at 7 and 14 d p.i.

Furthermore, all of the cytokines tested were produced at higher levels in mice infected with the

WB strain of G. duodenalis than in mice infected with the GS strain. This may explain why mice

68

infected with the WB strain of G. duodenalis showed consistently lower parasite burdens (Fig.

6A). Alternately, the differences in levels of cytokines produced could reflect different levels of

immunogenicity associated with the two strains. In mice infected with either strain, marked

cytokine production at day 7 p.i. seems to correlate with the peak of parasite burden within the

small intestine. In mice infected with the GS strain of G. duodenalis, cytokine production by

spleen T cells was observed at high levels on day 7 p.i. and at lower levels on day 10 p.i.,

whereas spleen cells from the WB-infected mice exhibited two distinct peaks of cytokine

production. Potential explanations for the differences in cytokine production kinetics could be

attributed to different patterns of antigenic variation between the strains or to different patterns of

T cell migration into and out of the spleen p.i.

Disaccharidase deficiency can result from numerous situations such as intestinal infection or

inflammation. Disaccharidase deficiency can contribute to diarrhea and nutrient malabsorption in

these situations. Our results clearly indicate an important role for host T cell responses in

contributing to this pathology. This study also marks important advances in our understanding of

giardiasis. To our knowledge, it is the first report to directly show cytokine production by CD4+

T cells p.i. It also establishes a model for direct, controlled comparisons among different parasite

strains to examine their ability to stimulate immune responses and to reproduce aspects of the

clinical syndrome seen in human disease.

69

Chapter IV

Altered Positional Distribution and Migration of Intestinal

Epithelial Cells and Intestinal Cytoskeletal Remodeling

Predispose Host to Gut Dysfunction.

In Preparation for submission to Cell Host & Microbe

70

4.1. Introduction

Intestinal epithelium structural integrity is a crucial factor for proper gut function and

absorption. The brush border surface provides the major interface for nutrient absorption and

reduction in the surface area of the microvilli is linked with reduced levels of enzymes such as

sucrase and lactase that are necessary for proper digestion and absorption of sugars (Farthing,

1997). Damaged intestinal epithelial cells could result in reduced intestinal enzymes, disposing

infected individuals to malnutrition. The structural proteins villin and ezrin are among the most

abundant proteins present in the intestinal brush border.

4.1.1. Villin

Villin is an actin-binding protein, is present in gastrointestinal, renal and urogenital epithelial

cells; villin is an extraordinarily versatile actin modifying protein in that it has the ability to

polymerize and depolymerize actin (Khurana and George, 2008) as well as sever, nucleate,

bundle and cap actin filaments. Unlike gelsolin, another protein in the same superfamily, villin

has the ability to assemble actin filaments as well (Wang et al., 2008). These studies provided a

molecular mechanism for villin’s role in regulating epithelial cell plasticity related to cell injury

(Wang et al., 2008). It has been previously indicated that fragments containing the COOH-

terminus fragment of the protein have anti-apoptotic activity while the N-terminal-containing

fragments of villin have pro-apoptotic functions through regulating the activation of caspase 3

and caspase 9 (Tomar et al., 2006; Wang et al., 2008). It has been hypothesized that as intestinal

epithelial cells migrate from the crypt to the villus tip to be shed into the lumen, villin is cleaved

to generate pro-apoptotic fragments, thus enhancing and/or assisting the process of cell extrusion

from the gastrointestinal epithelium (Khurana and George, 2008). A correlation between villin

71

deficiency and increased apoptosis has been noted in several inflammatory diseases of the

gastrointestinal tract. For example, a decrease in the levels of villin expression in enterocytes

from patients with inflammatory bowel disease relative to healthy controls has been reported

(Kersting et al., 2004) and villin-null mice have higher levels of apoptosis compared to their

wild-type littermates that correlates with the severity of colitis induced by dextran sodium sulfate

(DSS)-treatment (Wang et al., 2008). Additionally, differed villin expression has been

implicated as a useful marker to predict the prognosis and the progress of different carcinomas

(Bacchi and Gown, 1991; Savera et al., 1996; Zhang et al., 1999).

Tyrosine phosphorylation of villin regulates the villin/F-actin association, and tyrosine-

phosphorylated villin molecules have lower affinity for actin filaments (Zhai et al., 2001). Upon

phosphorylation, villin molecules show different affinity for several ligands (Khurana and

George, 2008); tyrosine phosphorylation of villin streamlines its ability to cut actin filaments at

extremely low concentrations of Ca2+ (Kumar et al., 2004), suggesting that tyrosine

phosphorylation of villin may be responsible for the efficient severing of actin filaments in vivo.

However, tyrosine-phosphorylated villin promotes cell migration and induces cytoskeletal

changes including the redistribution of F-actin to the cell perimeter and loss of stress fibers,

suggesting that phosphorylated villin induced actin cytoskeletal remodeling is a crucial step in

cell migration (Tomar et al., 2004).

4.1.2. Ezrin

Ezrin functions as a cross-linker between the plasma membrane and the actin cytoskeleton.

Evidence suggests that ezrin participates in a wide range of biological functions such as signal

72

transduction, cell division, cell growth, cytoskeletal organization and morphogenesis (Osawa et

al., 2009). Ezrin also plays an important role in both microvillus formation and tight junction

integrity, key elements in proper nutrient absorption (Berryman et al., 1993). Given that ezrin is

a major element of the microvilli formation and a linker between actin filaments and brush

border proteins, conformational changes in ezrin could influence microvilli integrity drastically.

4.1.3. Hypothesis

In this chapter, we examined the hypothesis that functional changes in the intestinal

epithelium that occur following infection are due to immune mediated alterations in the epithelial

cell cytoskeleton. We investigated the expression level, localization, and post-translational

modifications of villin and ezrin as well as any involvement of host immune responses in these

changes using a gut infection model. Using assemblages A and B of G. duodenalis as a model

system, we provide evidence indicating that the cytoskeletal proteins are considerably impacted

following gut infection, and these changes may result in altered intestinal integrity and organ

dysfunction observed. Also, we provide evidence suggesting roles for villin as an anti-apoptotic

and homeostatic protein following gut infection. Furthermore, experiments using knockout

animals demonstrated that ezrin modification was CD4+-dependent, while villin modification

required both CD4+ and CD8+ T cells.

73

4.2. Methods

4.2.1. Mice

Six- to eight-week old female WT C57BL/6 mice, SCID mice, β2m-/- (B6.129P2-B2mtm1Unc/J),

and CD4-/- (B6.129S2-Cd4tm1Mak/J) mice were purchased from The Jackson Laboratories (Bar

Harbor, ME). Mice (n= 4/group/time point) were housed at the Georgetown University Animal

Care Facility. All experiments were carried out in accordance with guidelines approved by the

Georgetown University Institutional Animal Care and Use Committee in compliance with the

National Institutes of Health guidelines.

4.2.2. Parasites, Infection Protocols and Quantification of Parasite Burden

The GS/M-83-H7 (ATCC# 50581) and the WB clone-C6 (ATCC# 50803) strains of G.

duodenalis were used in these experiments. Parasites were axenically grown in TYI-S-33 media

supplemented with adult bovine bile, L-cysteine, ascorbic acid, and antibiotics (all from Sigma-

Aldrich, St. Louis, MO). Mice were gavaged with 106 trophozoites in 100 µl phosphate-buffered

saline (pH 7.4). To equally mice with different strains of G. duodenalis mice were given

antibiotics in drinking water ad libitum: neomycin oral solution (1.4 mg/ml; Durvet, Blue Spring,

MO), ampicillin (1 mg/ml; Sigma-Aldrich) and vancomycin (1 mg/ml; Hospira, Inc., Lake

Forest, IL) for 48 h before parasite inoculation and during the entire infection period. After

euthanizing mice, the first 3 cm of the small intestine (pylorus to ligament of Trietz) were

discarded. The next two cm section of the duodenum were removed, opened longitudinally, and

minced in 4 ml of ice-cold PBS (pH 7.4). Tissues were kept on ice for 15 minutes, and the

numbers of trophozoites were counted on a hemocytometer.

74

4.2.3. Antibodies

The following antibodies were used for immunoblotting, immunopercipitation and IHC:

mouse monoclonal anti-ezrin (sc-58758), rabbit polyclonal anti-villin (sc-28283; Santa Cruz

Biotechnology, Santa Cruz, CA), rabbit polyclonal against the N-terminal region of villin (a gift

from Dr. Seema Khurana), rabbit polyclonal anti-GAPDH (sc-25778), anti-calpain-1 large

subunit (no.2556; Cell Signaling), rabbit polyclonal anti-p-ezrin (Thr567; Cell Signaling), anti-

phospho-tyrosine (PY-20; sc-508), peroxidase-conjugated anti-rabbit IgG and anti-mouse IgG

secondary antibodies (A0545 and A4416; both from Sigma, St Louis, USA), goat anti-rabbit

IgG-FITC, and goat anti-mouse IgG-TRITC (Southern Biotech, Birmingham, AL).

4.2.4. Immunofluorescence Microscopy

Intestinal tissues for immunohistochemistry (IHC) were fixed in 10% formalin in PBS and

subsequently embedded in paraffin as described previously (Chapter 3). Tissue sections (5-µm

thick) were deparaffinized in xylene, followed by hydration through graded ethanol solutions. In

order to unmask the antigens, the slides were treated in sodium citrate buffer (pH 6.0; Vector

Laboratories, Burlingame, CA) at 120°C for 20 min in a pressure cooker. Subsequently, the

slides were blocked for 1 h at room temperature in a blocking solution (10% goat serum, 0.05%

Tween 20 and 1% BSA in PBS). After probing with primary antibody, the sections were

mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA) and a

coverslip. Samples were analyzed with a Zeiss Axioplan 2 fluorescence microscope (Zeiss, Jena,

Germany).

75

4.2.5. In Vivo Epithelial Cell Proliferation Assay

Gender and age-matched WT mice C57BL/6 mice (n = 4/time point) were injected

intraperitoneally with 5-bromo-2'-deoxyuridine (BrdU; Sigma) dissolved in DMSO at a

concentration of 50 mg/kg of body weight (1mg/mouse) 2 hr before sacrifice. Control littermates

(n = 4) received sham (DMSO) injections intraperitoneally. Mice were sacrificed, and tissue

sections (5 µm) of paraffin-embedded duodenal, jejunal, and ileal tissues were deparaffinized

and stained for the presence of BrdU-containing nuclei using a BrdU In-Situ Detection Kit

according to manufacturer’s instruction (BD Pharmingen, San Diego, CA). BrdU-labeled

enterocytes were counted at day 0, 5, and 18 p.i., and the positional distribution of IECs was

determined as previously described (Cliffe et al., 2005). The proportion of BrdU-containing

enterocytes was measured at high magnification under light microscopy. Intestinal epithelial cell

proliferation was expressed as the percentage of BrdU-labeled enterocytes per 100 crypt cells,

and at least 20 full-length, well-oriented duodenal crypts per animal were examined.

4.2.6. Western blot analysis

Fifteen cm of the small intestine were homogenized in 1 ml of tissue protein extraction

reagent (T-PER; Pierce, Rockford, IL) supplemented with protease inhibitor cocktail III

(Calbiochem, La Jolla, CA). Tissue lysates were centrifuged at 14000 g for 5 min, and

supernatants were collected and used for further analysis. Protein concentration of each sample

was determined using a Bradford assay (Bio-Rad, Hercules, CA). Equal amounts of each sample

were loaded with 2× Laemmli sample buffer and were separated by gradient 4-12% SDS-PAGE

(Invitrogen) in the MOPS buffer system (Invitrogen). Proteins were transferred to a PVDF

membrane (Millipore, Bedford, MA) by standard techniques and then probed with relevant

76

antibodies. Primary antibodies were detected by using peroxidase-conjugated secondary

antibodies and ECL reagents (Amersham Bioscience), and signals recorded on X-ray film

(Kodak Biomax; Kodak, Rochester, NY). Protein abundance was quantified using ImageJ

software (NIH); protein abundance were normalized to uninfected animals and presented relative

to results obtained with the control sample set as 1.0.

4.2.7. Immunoprecipitation

Five hundred µg of the intestinal homogenates were pre-cleared with 20 µL of Protein A/G

Plus-Agarose (sc-2003) for 1 h at 4°C. Agarose beads were removed by centrifugation, and villin

polyclonal antibodies were added at a concentration of 4 µg/500 µg of homogenate and

incubated with agitation for 6 h at 4°C. Subsequently, 20 µL of fresh agarose beads were added

and incubated for another 2 h at 4°C. During IP, agarose bead complexes were collected by

centrifugation and washed three times with ice-cold NP40 lysis buffer (250 mM NaCl, 5 mM

HEPES, 10% v/v glycerol, 0.5% NP40, 2 mM EDTA (pH 8.0) supplemented with 100 µM

NaVO4 and 1 mM NaF, and protease inhibitor cocktail III). Immunocomplexes were eluted by

boiling in 2× Laemmli sample buffer, resolved by gradient 4-12% SDS-PAGE (Invitrogen) in

the MOPS buffer system (Invitrogen) and transferred overnight at 4°C as described earlier

(Chapter 3).

77

4.2.8. Statistical Analysis

Differences between samples were evaluated with the Mann-Whitney U test; P values <0.05

were considered significant.

4.3. Results

Proteolysis of Villin in Wild-Type Mice Following Gut Infection Is Immune-Mediated

To investigate the impact of gut infection on the regulation of villin following gut infection,

wild-type mice were analyzed at days 0, 5, and 18 p.i. (Fig. 4.1A) by Western blotting using

antibodies against epitopes in the C-terminal region of the protein. Results showed that villin was

proteolyzed following infection at day 18 p.i., and that several C-terminal products

(approximately 87-kD, 54-kD, and 41-kD) were detected. The total level of villin (native protein

and proteolyzed products) was upregulated about 2.5 times by densitometry analysis at day 18

while total proteins levels at day 5 remained unchanged (Fig. 4.1A). The upregulation of villin

could be explained by the existence of a compensatory mechanism in the infected intestine in

immediate response to villin proteolysis at day 18. Since post-translational changes in villin were

detected later in the course of G. duodenalis infection at which time point parasites were barely

detectable in the small intestine, we hypothesized that these changes could be triggered by the

host immune responses. To test this, the intestinal tissues of SCID mice infected with the parasite

were analyzed by Western blot at 0, 5, and 18 days p.i. Results demonstrated that in the absence

of adaptive immunity, no villin proteolysis was seen despite the high numbers of parasites

present in the intestine (Fig. 4.1B). Furthermore, no villin upregulation was observed. We then

were interested in the specific cell-types driving these changes. CD4-/- and β2m-/- mice were

infected and analyzed. No villin proteolysis was observed either in CD4-/- or β2m-/- mice, and

78

levels of villin were unchanged during the course of infection (Fig. 4.1C, Fig. 4.1D). These

findings suggest that upregulation and cleavage of villin require both CD4+ and CD8+ T cells. In

order to determine the impact of pathogen strain on the cytoskeletal changes we observed during

the infection of WT mice with the GS strain of G. duodenalis, we infected WT C57BL/6 mice

with the WB strain of the parasite. Our findings showed that no apparent villin proteolysis

occurred in WT mice following infection with the WB strain (Fig. 4.1E). This is consistent with

the absence of disaccharidase deficiency following WB infection and the induction of

disaccharidase deficiency following GS infection. Finally, we also examined villin in kidneys

from mice infected with GS and found that no upregulation or cleavage occurred at a remote site,

indicating and that villin proteolysis is a site-specific event (Fig. 4.1F).

Together with our previous report (Solaymani-Mohammadi and Singer, 2011), we concluded

that the discrepancies observed between the two strain could partially explain the dissimilar

nature of the clinical symptoms and malnutrition syndrome in infected individuals with different

strains.

79

Figure 4. 1. Villin proteolysis following infection. WT (a), SCID (b), β2m-/- (c), and CD4-/-

mice (d) were infected with the GS strain of G. duodenalis. Jejunal homogenates were

analyzed by Western blot for post-translational modifications in villin using a polyclonal

antibody against the protein. GAPDH was used as a loading control. Villin modifications

were tested in mice infected with the WB strain (e) of the parasite. Renal villin in WT mice

infected with the GS strain was also analyzed (f). Each figure is representative of four

animals / time point.

80

Anti-apoptotic Role of Villin Following Gut Infection

We next examined the possible pro-apoptotic role of villin and whether we could see any

cleaved products containing the N-terminal region of the protein by using a rabbit polyclonal

antibody raised against the N-terminus of the protein. No N-terminal-containing cleaved

products were detected in WT, SCID, CD4-/- and β2m-/- mice (Fig. 4.2A-D), suggesting a

potential role for villin as an anti-apoptotic protein following infection. These findings indicate

that the proteolysis of villin following gut infections generates anti-apoptotic signals that could

counterbalance pathological effects of immune response to the parasite, maintaining the

homeostatic balance in the infected intestine.

81

FIGURE 4.2. Analysis of N-terminal-containing villin fragments following G. duodenalis

infection. WT (a), SCID (b), β2m-/- (c) and CD4-/- mice were infected with the GS strain of

the parasite, and jejunal homogenates were analyzed using an antibody against a peptide in

the N-terminal portion of villin by Western blot. GAPDH was used as a loading control.

Each figure is representative of four animals / time point.

a b

c d

82

Tyrosine Phosphorylation of Villin Occurs Following Gut Infection with GS but not WB

Tyrosine phosphorylation of villin promotes cell migration and cell movement and can induce

cytoskeletal changes including the redistribution of F-actin to the cell perimeter and loss of stress

fibers, suggesting that tyrosine phosphorylation of villin is a crucial step in promoting cell

migration (Tomar et al., 2004). We next asked whether villin was tyrosine-phosphorylated

following gut infection, and if phosphorylation required by host adaptive immunity.

Immunoprecipitation analysis showed that tyrosine-phosphorylated villin was more abundant at

day 5 after infection of wild-type mice with the GS strain. Using densitometry, we found that

phosphorylated villin was upregulated 4.1 times at day 5 p.i. compared with uninfected animals

(Fig. 4.3A). Moreover, villin phosphorylation was not seen following infection with the WB

strain or after GS infection in SCID mice (Fig. 4.3B, 4.3.C).

83

Figure 4.3. Tyrosine-phosphorylation of villin. Villin was immunoprecipitated from jejunal

homogenates of WT mice infected with the GS strain of G. duodenalis (a) or WB strain (b).

Immunoprecipitated proteins were analyzed for tyrosine phosphorylation by Western blot

and levels of phosphorylated protein were normalized to levels of total villin by

densitometry. Tyrosine-phosphorylation of villin was also examined in SCID mice infected

with the GS strain of the parasite (c). Each figure represents 4 animals / time point.

84

Infection with G. duodenalis increases both proliferation and migration of enterocytes

Based on data represented in figure 4, we hypothesize that tyrosine-phosphorylation of

villin following infection with G. duodenalis will cause increased IECs proliferation/movement

resulting in altered immature/mature IECs. Immature IECs do not express sucrase/maltase, and

lactase which, in turn, this predisposes infected people with enzyme deficiency (see Chapter V).

We therefore asked if infection with the parasite would increase IECs proliferation/migration.

WT mice were infected with the GS strain of the parasite and mice were injected with 5-bromo-

2'-deoxyuridine (BrdU) 2 hr before sacrifice. Staining for BrdU-positive IECs by a biotinylated

anti-BrdU antibody revealed a significant increase in the rate of cell proliferation on day 5 p.i.

(Fig. 4.4a). Further positional studies showed that IECs in WT mice infected with the GS strain

move up luminally at a faster pace compared with uninfected animals (Fig. 4.4b). Together, these

data suggest that infection with parasite accelerates both proliferation and migration of IECs.

This likely can be responsible for the disaccharidase deficiency seen during infection.

85

FIGURE 4.4. WT mice were infected with the GS strain of the parasite and IEC

proliferation was measured by BrdU incorporation during a two-hour pulse of BrdU. BrdU

positive cells were visualized using IHC and light microscopy and the percentage of cells in

each crypt that were labeled was determined (a). The positional distribution of BrdU + cells

IECs was then calculated (b). Each figure represents 4 animals / time point.

86

FIGURE 4.4. Continued

Villin Is Redistributed in IECs Following Gut Infections

To analyze whether gut infection also induced villin redistribution, we performed an

immunohistochemistry (IHC) analysis on formalin-fixed intestinal sections at three time points

p.i. Analyses showed that in uninfected mice enterocytes displayed strong apical decoration (the

brush border) as well as diffuse cytoplasmic labeling. In infected mice, however, villin primarily

87

was localized near the apical plasma membrane and baso-lateral membranes with loss of villin

expression at the brush border and redistribution of villin to the cytoplasm. Additionally, weak,

diffuse labeling was seen in the cytoplasm of enterocytes in infected mice (Fig. 5.4). Given that

we had 2.5 times more villin at day 18 p.i., it seemed that the changes in the pattern of villin

could be a consequence of villin redistribution as well as stimulation of protein synthesis.

88

Figure 4.5. WT mice were infected with the GS strain, and villin distribution was

determined by IHC during the course of infection at day 5 (middle panel) and day 18

(lower panel) compared with uninfected mice (upper panel). Each panel is representative of

4 mice/time point.

The actin-binding protein, ezrin, is cleaved following gut infection: ezrin cleavage is CD4+-

dependent

Because the linker protein ezrin is also intimately involved in the formation of microvilli, we

next investigated whether there were changes in the post-translational modification and protein

expression levels of ezrin following gut infection, and any possible involvement of host immune

responses. Our findings demonstrated total ezrin expression levels were not changed

significantly following gut infections in wild-type mice. The full-length ezrin, however, was

89

almost completely cleaved by day 18 p.i., while a constant full-length/cleaved protein ratio was

observed at days 0 and 5 p.i. The cleavage process yielded a major fragment of 55-kD (ezrin

p55; Fig. 4.6A). Cleaved products were also seen in uninfected animals to a lesser degree. This

suggests that cleavage could be part of the protein’s post-translational regulation, and that gut

infections accelerate the turn-over process of protein degradation/synthesis, pushing the

equilibrium between native/cleaved proteins towards the cleaved defective proteins.

In order to investigate the role of host immunity in mediating the ezrin cleavage, we

examined the levels and cleavage of this protein following infection in SCID mice. We observed

no increase in cleavage of ezrin during the course of infection in spite of massive infection

burdens detected in these mice, suggesting that the ezrin cleavage/synthesis is under the control

of host adaptive immunity (Fig. 4.6B). Next, we sought to determine the specific cell types

mediating these events. CD4-/- and β2m-/- mice were infected and analyzed for the cleavage of

ezrin. Analyses showed that no increase in ezrin cleavage occurred in CD4-/- animals, while β2m-

/- mice exhibited a cleavage pattern identical to WT littermates (Fig. 4.6C and Fig. 4.6D). Thus,

the cleavage of ezrin is regulated in a CD4+-T cell dependent manner. This differs from

regulation of villin. While both CD4+ and CD8+ T cells are required for villin proteolysis, ezrin

cleavage is a CD4+-dependent phenomenon, but does not require CD8+ T cells. We also

examined the cleavage of ezrin following infection with a different strain of the parasite. Ezrin

was cleaved following WB infection in WT mice in a manner similar to WT mice infected with

the GS strain (Fig. 4.6E). This suggests that cleavage of ezrin is unlikely to be directly related to

the induction of disaccharidase deficiency since it occurs in β2m-/- mice infected with GS and in

WT mice infected with WB, two scenarios in which disaccharidase deficiency does not occur

90

Figure 4.6. WT mice were infected with the GS strain of the parasite and jejunal

homogenates were examined for post-translational modifications in ezrin using Western

blot using a monoclonal antibody against the protein. Ezrin cleavage in WT (a), SCID (b),

β2m-/- (c), and CD4-/- (d) mice following infection with the GS strain of G. duodenalis. Ezrin

is also cleaved in mice infected with WB strain of the parasite (e). GAPDH was used as a

loading control. Each figure is representative of 4 mice / time point.

e

91

µ-calpain is activated in wild-type mice, but not in SCID mice, following gut infection

Ezrin serves as a substrate for calpain proteases, and calpain I targets ezrin specifically as part

of the protein regulation process yielding a specific 55-kD cleaved product (Yao et al., 1993).

Next, we asked whether µ-calpain was activated following gut infection. To test this, we used a

polyclonal antibody enabled to detect both inactive (pre-autolytic µ-calpain; about 80-kD) and

the active (post-autolytic µ-calpain; 76-kD) forms of µ-calpain. The native protein is inactive

(pre-autolytic µ-calpain), and upon activation, the activated fragment (post-autolytic µ-calpain)

is processed to a 76-kD fragment representing the active form of µ-calpain. Results of the

Western blot analysis showed that µ-calpain was activated at day 18 p.i., and densitometry

analysis indicated that the active form of µ-calpain was about 3.5 times more abundant than that

observed in uninfected animals (Fig. 4.7A). These findings correlate with cleaved ezrin observed

at day 18 p.i. We next sought the role of host adaptive immunity in mediating these events. In

SCID mice, however, most µ-calpain was in its inactive form (the 80-kD protein) consistent with

lack of ezrin cleavage in these mice (Fig. 4.7B). Again, we concluded from the lack of ezrin

cleavage following infection in SCID mice that the µ-calpain of host origin was likely

accountable for the post-translational modifications of ezrin following infection.

92

FIGURE 4.7. WT mice were infected with the GS strain of G. duodenalis, and jejunal

homogenates were tested for calpain using an antibody capable of detecting both active and

inactive forms of calpain. Calpain I, µ-calpain, is activated in WT mice (a) following

infection with the GS strain of G. duodenalis but not in SCID mice infected with the same

strain of the parasite (b). Each figure is representative of 4 mice/time point.

Localization of Total Ezrin and Phospho-Ezrin Is Altered following Gut Infection

It is well-recognized that increased cell motility, i.e. cell movement and cell migration,

requires ezrin cleavage. Ezrin cleavage is associated with the extension of microvilli and the

formation of surface lamellipodia. Ezrin phosphorylation on Thr567 was often found to correlate

with extensive long microvillar projections (Zhou et al, 2005). Additionally, it has been shown

that ezrin cleavage happens during wound healing and subsequent cell migration (Wang et al.,

2005; Bach et al., 2005). Also, it was explicitly shown that in cancers, for example in

medulloblastoma cells, the high expression of ezrin promotes filopodia formation and in vitro

93

invasion and that ezrin was primarily localized to filopodia, concluding that ezrin plays an

important role in medulloblastoma adhesion, migration, and invasion (Osawa et al., 2009).

A monoclonal antibody against the major phosphorylation site of ezrin (Thr567) showed that

localization of ezrin and p-ezrin altered during the course of infection with significantly

reduced/redistributed ezrin and phospho-ezrin on day 5 p.i. (Fig. 4.8).

FIGURE 4.8. Using paraffin-embedded tissue and IHC, the localization of total and

phopho-ezrin (p-ezrin) was determined in the intestines of mice infected with the GS strain

of the parasite for 0, 5 or 18 days. Apical localization of ezrin (upper panel) and p-ezrin

(lower panel) decreases in intestinal epithelial cells following infection of WT mice with GS

strain of G. duodenalis. Each panel represents 4 mice/time point.

94

DISCUSSION

We have examined the effect of intestinal infection on IEC cytoskeleton and on the dynamics

of actin-binding proteins at different phases following the infection course. We have found that

gut infections induce drastic cytoskeletal changes in IECs and that these changes are mediated by

host adaptive immunity. The actin-severing protein, villin, was proteolyzed in WT mice

following gut infection, and these changes were not observed in SCID, β2m-/-, and CD4-/- mice.

We concluded that these events are driven by host immunity. We also showed that in WT mice

villin redistributed to the cytoplasm of IECs following gut infection, while villin localization

remained apical in uninfected littermates. We also found that the proteolysis of villin was strain-

dependent and no protein proteolysis was detected in WT mice infected with the WB strain of G.

duodenalis; the proteolysis of villin was tissue-specific, and renal protein was intact. The N-

terminal-containing fragments of proteolyzed villin did not accumulate in infected intestines,

while fragments containing the C-terminal part of the protein were detected by Western blotting,

suggesting an anti-apoptotic role for villin following gut infections (Tomar et al., 2004). We also

demonstrated that villin is tyrosine-phosphorylated following gut infection at day 5 p.i., and that

the tyrosine phosphorylation of villin was not observed in SCID mice. Similarly, we observed

post-translational changes in another actin-binding protein, ezrin following gut infection in WT

mice. Native ezrin was completely cleaved by day 18 p.i. in an immune-dependent process. In

contrast to villin, the ezrin cleavage was not strain-dependent and the WB strain of G. duodenalis

induced the protein cleavage by day 18 p.i. similar to the GS strain. We found that the protease

95

µ-calpain was activated on day 18 p.i., and it could be responsible for the cleavage of ezrin.

Likewise, the localization of both total and phosphorylated ezrin changed at day 5 p.i.

Host cell cytoskeletal remodeling has been reported following gut infection (Carabeo et al.,

2002; Walker et al., 2008). Several entropathogens including Salmonella enterica serovar

Typhimurium and Shigella flexneri alter cytoskeleton architecture at the site of bacterial

attachment; changes in the actin cytoskeleton following enteric infections occur by the activation

of Rho GTPases and tyrosine kinases and by directly modifying actin dynamics which facilitate

bacterial invasion (Dunn and Valdivia, 2010). Furthermore, some enteric pathogens manipulate

the host actin cytoskeleton to enter IECs and to disseminate from cell to cell (Athman et al.,

2005). Manipulation of the host cytoskeletal architecture is also seen in other intestinal

pathogens; attachment of the protozoan parasite Cryptosporidium parvum triggers changes in the

microvillar structures in the vicinity of parasitophorous vacuole containing the parasite (Elliott et

al., 2000; Elliott et al., 2001).

Villin proteolysis has been reported to be associated with infection with the causative agent of

amebic colitis, Entamoeba histolytica, via the parasite’s cysteine proteinase activity and the

inhibition of cysteine proteinases in trophozoites by specific inhibitors prevented villin

proteolysis (Lauwaet et al., 2003). These authors also showed that cysteine proteinases of enteric

origin were likely not responsible for the proteolysis. The results of the current study, however,

showed the lack of villin proteolysis in absence of host adaptive immunity in SCID mice in spite

of massive infection burdens in these hosts (data not shown). Based on these observation, we

concluded that a proteinase of host origin (not a proteinase of parasite origin) is likely

responsible for the post-translational events in villin following infection.

96

It previously was shown for gelsolin, a protein in the villin superfamily, that the caspase-3-

generated fragments of the protein had the ability to sever actin filaments in a Ca2+-independent

manner (Kothakota et al., 1997), consequently promoting apoptosis while the full-length protein

severs actin in a Ca2+-dependent manner (Kwiatkowski et al., 1989; Weeds et al, 1991). The

actin-severing activity of gelsolin resides in the NH2-terminal region, residues 1 to 160

(Kwiatkowski et al., 1989). On the other hand, if the caspase-3-generated fragment of gelsolin

contains the NH2-terminal fragment, it has the ability to depolymerize actin filaments in vivo and

subsequently induces apoptosis. Conversely, the overexpression of the full-length protein as well

as the COOH-terminal fragment had no effect on actin filaments (Kothakota et al., 1997).

Likewise, it has been demonstrated that the N-terminal fragment of villin has pro-apoptotic

functions (Tomar et al., 2006), and it has been hypothesized that as intestinal epithelial cells

migrate from the crypt to the villus tip and are ready to be shed into the lumen, villin is cleaved

to generate pro-apoptotic fragments thus enhancing and/or assisting the process of cell extrusion

from the gastrointestinal epithelium (Khurana and George, 2008). We found that the fragments

containing the C-terminal portion of villin accumulated following enteric villin proteolysis while

fragments containing the NH2 portion of protein did not. These findings demonstrated an

important role for villin as an anti-apoptotic and homeostatic protein following infection in vivo

as shown before in vitro (George and Khurana, 2008) and indicate that the proteolysis of villin

following gut infections generates anti-apoptotic signals that can counterbalance the parasite

deleterious effects, maintaining the homeostatic balance in the infected intestine.

Tyrosine kinases have been demonstrated to be important for intestinal epithelial cell

migration (Reiske et al.,1999). It has been shown that villin is phosphorylated in vitro by c-src

97

kinase (Zhai et al., 2002), as well as in migrating cells in vivo (Tomar et al., 2004). In the current

study, densitometry analysis indicated that in WT mice villin was tyrosine-phosphorylated at day

5 p.i. and the levels of phosphorylated villin were approximately 4 times more compared with

uninfected animals; levels of tyrosine-phosphorylated villin returned to pre-infection levels on

day 18 p.i. In SCID mice, however, no changes were seen in the levels of tyrosine-

phosphorylated villin during the course of infection, suggesting a role for host’s adaptive

immunity in mediating these changes. Tyrosine phosphorylation of villin regulates the villin/F-

actin association, and the tyrosine-phosphorylated villin molecules have lower affinity to bind to

F-actin filaments (Zhai et al., 2001). Villin has the ability to cut actin filaments at high

concentrations of Ca2+ (100-200 µM) in vitro. Instead, the tyrosine-phosphorylated villin can

sever actin filaments at much lower nanomolar concentrations of Ca2+ ion (Kumar et al., 2004),

suggesting that tyrosine phosphorylation of villin may be responsible for the efficient serving of

action filaments in vivo.

Facilitated rates of epithelial cell turnover following intestinal infections have been reported

as a defensive mechanism implemented by the host to expel intestinal helminth parasites (Cliffe

et al., 2005). Increased levels of tyrosine-phosphorylated villin observed following gut infection

may be an immediate response of the host to the peak of infection at day 5 p.i. Tyrosine-

phosphorylated villin can, in turn, results in an increase in the IECs movement, resulting in the

extrusion of lodged parasites. This may result in an imbalanced immature/mature IECs ratio, and

can lower the levels of intestinal enzymes and may account for the decreased intestinal enzymes

observed during intestinal infections (Buret et al., 1995; Solaymani-Mohammadi and Singer,

2011).

98

Calpains are calcium-dependent enzymes which are ubiquitously found in almost all tissues

and organs. Calpain’s proteolytic activities have been shown to be involved in pathogenesis of a

wide array of diseases such as spinal cord injuries, diabetic neuropathy, calcium-dependent

disorders (i.e. cerebral ischemia and Alzheimer’s disease), chronic heart failure, and renal

ischemia (Takahashi et al., 2006). Furthermore, calpain has been shown to mediate the cleavage

of cytoskeletal proteins during cell death caused by E. histolytica (Lee et al., 2011).

In a few instances, it has been shown that bacterial pathogens are able to induce ezrin

phosphorylation followed by proteolytic cleavage resulting in defective organ function. The

Helicobacter pylori CagA, for example, has been linked to src-mediated phosphorylation

(Selbach et al., 2004) inducing a conformational change that renders it susceptible to cleavage by

proteases like calpains (Zhou et.al, 2005). In the current study, we found that a µ-calpain of

enteric origin is likely responsible for ezrin cleavage and that G. duodenalis redistributes ezrin/p-

ezrin localization. Given that ezrin is a major element of the microvilli formation and a linker

between actin filaments and brush border proteins, post-translational changes in ezrin could

severely influence the microvilli integrity, and this could account for abnormalities seen during

gut infections.

In conclusion, this study indicated that gut infections induced drastic cytoskeletal changes in

an immune-driven manner, and these changes can lead to organ dysfunction and lack of

intestinal integrity following gut infection. A comprehensive understanding of the host-parasite

interaction following gut infections, including identification of the pathogen-induced structural

changes, actin-interacting proteins, and signaling pathways involved would be a substantial

advance in our knowledge of the immunopathology seen during gut infections.

99

Chapter V

Conclusion: What we have learned so far

This dissertation examined the role of strain variation in pathogenesis and immunity of human

giardiasis. In chapter II, we show that albendazole is equally as effective as metronidazole in the

treatment of human giardiasis. It has been shown before that there are strain variations in the

levels of susceptibility of a given strain of G. duodenalis to metronidazole as the first-line drug

for the treatment of giardiasis in humans. For instance, one study found that WB C6 strain of the

parasite was resistant to both nitazoxanide and to metronidazole; different expression of genes

between drug-resistant strains and sensitive strains of Giardia were speculated as the source of

the drug resistance (Müller et al., 2007; Müller et al, 2008). In contrast, no significant variations

in the susceptibility of different Giardia strains to different drugs were reported (Bénéré et al.,

2011). The high rate of side effects from metronidazole therapy for giardiasis, combined with the

global emergence of resistant strains, led us to consider the effectiveness of alternative

treatments such as albendazole for the treatment of human giardiasis. The results of this analysis

are presented in Chapter II. In our meta-analysis comparing the effectiveness of albendazole and

metronidazole as treatments for human giardiasis, Giardia-infected patients were not clustered

based on the strains with which they were infected (Solaymani-Mohammadi et al., 2010). In this

100

Chapter of the dissertation, we hypothesize that at least part of the so-called “failure-to-treat”

cases might be attributed to the presence of “drug-resistant” strains, a mechanism to which none

of the studies referred as a potential reason for treatment failure. To overcome this, we

recommend the use of different combinations of albendazole with other anti-parasitic agents in

future studies to minimize the risk of the emergence of drug-resistant strains. Furthermore, the

design of placebo-controlled double-blinded clinical trials in which human subjects are infected

with known Giardia strain may help us to better understand the most appropriate regimen(s) and

the most suitable chemotherapy protocols in different parts of the world where drug-resistant

strains exist.

In Chapter III of the dissertation, we examined the impact of the disease triangle elements

(susceptible host, pathogen and favorable environment) in a mouse model of human giardiasis.

Specifically in this Chapter, first we examined the role of host factors in mediating mucosal

damage observed in a mouse model of human giardiasis. We found that hosts lacking adaptive

immunity, SCID, did not develop signs of disaccharidase deficiency while WT counterparts

developed mucosal damage characterized by disaccharidase deficiency. Furthermore, our results

showed that mice lacking CD4 T-cells did not show signs of enzyme deficiency although they

could not clear the infection by day 18. Likewise, CD8-deficient hosts did not show any signs of

enzyme deficiency although they managed to clear the infection similar to WT mice. From these

observations, we conclude that host factors can play key roles in reciprocal interplay between

host and parasite but likely are not the only factors determining the fate of the disease.

101

Later in this Chapter, we tested the role of pathogen factors and their impacts on the outcome

of giardiasis in a mouse model of infection. Mice were infected with either the GS or WB strains

of the parasites and the parameters of epithelial damage (i.e. disaccharidase activity) were

measured. Our analysis revealed distinct strain variation in the induction of disaccharidase

deficiency evidenced by depressed intestinal enzymatic activity at day 5 p.i. While WT mice

infected with the WB strain, representing assemblage A, did not show any signs of decreased

enzymatic activity, WT counterparts infected with the GS strain, representing assemblage B,

displayed considerable disaccharidase deficiency. Likewise, variations were observed in host

immune responses to each strain of the parasite: WT mice infected with the WB strain showed a

robust, biphasic response to parasite extract, however, mice infected with the GS strain

constantly showed lower levels of cytokine production, representing a mono-phasic pattern.

From these results we conclude that pathogen factors are, in part, involved in determining the

outcome of giardiasis.

In this dissertation, we used both WB and GS strain of G. duodenalis in order to infect mice.

We were capable of successfully and substantially infecting C57BL/6J mice with both strains of

G. duodenalis. Infections of WT C57BL/6J with the GS strain were earlier reported (Byrd et al.,

1994). By revising existing infection protocols and adding antibiotics to drinking water of

animals, we were able, for the first time, to infect WT C57BL/6J mice with the WB strain of the

parasite. Subsequently, using antibiotics in drinking water of animals allowed us to infect mice

consistently with each strain and this subsequently enabled us to examine the behavior of each

strain within the intestines of infected hosts in parallel. Altogether, the need to change host gut

102

microbiota in order to facilitate infections may reflect a pivotal role for environmental factors, as

another element of the disease triangle. As a result of the current dissertation, efforts are now

underway in the lab to use additional strains from these assemblages to infect mice.

In Chapter IV of this dissertation, the association between cytoskeletal changes and

disaccharidase deficiency were tested. Specifically, we looked at two actin-binding proteins as

markers of intestinal integrity (villin and ezrin) and the ability of Giardia infection to induce

post-translational changes in these two proteins. Consistent with our findings in Chapter III, we

found host factors to be important in mediating cytoskeletal/epithelial changes observed

following giardiasis. Lack of adaptive immunity prevented mice from displaying cytoskeletal

changes following gut infection whereas immunocompetent mice showed considerable

cytoskeletal changes following infection. Our finding also ruled out the notion that epithelial

changes are caused by the direct effects of the parasite attachment since no changes were

observed in heavily-infected SCID mice. Of the utmost interest in this Chapter was the

observation that the ability of a given parasite strain to induce phosphorylation of the actin-

binding protein villin correlated with the ability of that strain to induce disaccharidase

deficiency. We clearly showed that the ability of a given Giardia strain to induce elevated levels

of tyrosine-phosphorylated villin at day 5 p.i. correlated with enzyme deficiency. We

hypothesized in this Chapter that parasite colonization of the gut provokes T-cell responses

which lead to tyrosine phosphorylation of villin. It has previously been shown that tyrosine-

phosphorylation of villin is one of the initial steps in cell proliferation and cell migration and that

103

it is controlled by IL-2 via Janus kinase 3 (Kumar et al., 2007). Based on our hypothesis,

phosphorylated villin, in turn, re-organizes the actin cytoskeleton and facilitate IEC proliferation

and migration in intestinal crypts. Increased IEC movement subsequently could result in an

increased immature/mature IEC ratio. Immature IECs are not fully functional and accordingly

express lower levels of disaccharidase enzymes (Fig 5.1).

Figure 5.1. Proposed model for regulation of disaccharidase expression following gut

infection (See text for explanation).

Our findings demonstrated a correlation between the ability of the GS strain of G. duodenalis

to induce tyrosine-phosphorylation of villin and disaccharidase deficiency at day 5 p.i. In

104

contrast, our findings also showed that the WB strain neither caused tyrosine-phosphorylation of

villin nor disaccharidase deficiency (Table 5.1). By performing BrdU incorporation studies, we

further found that that infection with the GS strain significantly increased the rate of IEC

proliferation at day 5 p.i. The concordance of disaccharidase deficiency, tyrosine-

phosphorylation of villin and increased rate of IEC proliferation at day 5 p.i. in the GS-infected

WT mice may strengthen our hypothesis that a villin-dependent increase in IEC proliferation

would be likely, in part, responsible for the enzyme impairment.

In the context of gut infections, cytoskeletal remodeling in IECs can adversely impact the

structural integrity and the absorptive capacity of the intestine and may play critical roles in the

immunopathology observed during many infectious and non-infectious intestinal conditions.

Cytoskeletal proteins, however, may be considered as targets for the development of new

therapeutics against giardiasis in human in future.

105

Table 5.1. Summary of changes in cytoskeletal markers following infections with different

strains of G. duodenalis and in different hosts. The ability of the pathogen strain to induce

tyrosine-phosphorylation of villin at day 5 p.i. correlates with disaccharidase deficiency.

106

REFERENCES

Abdul-Wahid A, Faubert, G (2007) Mucosal delivery of a transmission-blocking DNA vaccine

encoding Giardia lamblia CWP2 by Salmonella typhimurium bactofection vehicle. Vaccine, 25:

8372–8383.

Adam RD (2001) Biology of Giardia lamblia. Clinical Microbiology Reviews, 14: 447–475.

Aley SB, Zimmerman M, Hetsko M, Selsted MD, Gillin FD (1994) Killing of Giardia lamblia

by cryptdins and cationic neutrophil peptides. Infection and Immunity, 62: 5397–5403.

Alizadeh A, Ranjbar M, Kashani KM, Taheri MM, Bodaghi M (2006) Albendazole versus

metronidazole in the treatment of patients with giardiasis in the Islamic Republic of Iran. Eastern

Mediterranean Health Journal, 12: 548–554.

Al-Mohammed HI (2011) Genotypes of Giardia intestinalis clinical isolates of gastrointestinal

symptomatic and asymptomatic Saudi children. Parasitology Research, 108: 1375-1381.

Andersson T, Forssell J, Sterner G (1972) Outbreak of giardiasis: effect of a new antiflagellate

drug, tinidazole. British Medical Journal, 2: 449–451.

Andersen YS, Gillin FD, Eckmann L (2006) Adaptive immunity-dependent intestinal

hypermotility contributes to host defense against Giardia spp. Infection and Immunity, 74: 2473-

2476.

107

Anderson KA, Brooks AS, Morrison AL, Reid-Smith RJ, Martin SW, Benn DM, Peregrine AS

(2004) Impact of Giardia vaccination on asymptomatic Giardia infections in dogs at a research

facility. The Canadian Veterinary Journal, 45: 924-930.

Angarano G, Maggi P, Di Bari MA, Larocca AM, Congedo P, Di Bari C, Brandonisio O,

Chiodo F (1997) Giardiasis in HIV: a possible role in patients with severe immune deficiency.

European Journal of Epidemiology, 13: 485-487.

Athman R, Fernandez MI, Gounon P, Sansonetti P, Louvard D, Philpott D, Robine S (2005)

Shigella flexneri infection is dependent on villin in the mouse intestine and in primary

cultures of intestinal epithelial cells. Cellular Microbiology, 7: 1109-1116.

Ayabe T, Ashida T, Kohgo Y, Kono T (2004) The role of Paneth cells and their antimicrobial

peptides in innate host defense. Trends in Microbiology, 12: 394-398.

Bacchi CE, Gown AM (1991) Distribution and pattern of expression of villin, a gastrointestinal-

associated cytoskeletal protein, in human carcinomas: a study employing paraffin-embedded

tissue. Laboratory Investigation, 64: 418-424.

Bach LA, Gallicchio MA, McRobert EA, Tikoo A, Cooper ME (2005) Effects of advanced

glycation end products on ezrin-dependent functions in LLC-PK1 proximal tubule cells. Annals

of the New York Academy of Science, 1043: 609-616.

108

Bachur TB, Vale JM, Coêlho IC, Queiroz TR, Chaves Cde S (2008) Enteric parasitic infections

in HIV/AIDS patients before and after the highly active antiretroviral therapy. The Brazilian

Journal of Infectious Disease, 12: 115-122.

Baqai R, Zuberi SJ, Qureshi H, Ahmed W, Hafiz S (2001) Efficacy of albendazole in giardiasis.

Eastern Mediterranean Health Journal, 7: 787-790.

Bassily S, Farid Z, Mikhail JW, Kent DC, Lehman JS (1970) The treatment of Giardia lamblia

infection with mepacrine, metronidazole and furazolidone. Journal of Tropical Medicine and

Hygiene, 73: 15-18.

Bayraktar MR, Mehmet N, Durmaz R (2005) Serum cytokine changes in Turkish children

infected with Giardia lamblia with and without allergy: effect of metronidazole treatment. Acta

Tropica, 95: 116-122.

Belosevic M, Faubert GM, MacLean JD (1989) Disaccharidase activity in the small intestine of

gerbils (Meriones unguiculatus) during primary and challenge infections with Giardia lamblia.

Gut, 30: 1213-1219.

Bendesky A, Menéndez D, Ostrosky-Wegman P (2002) Is metronidazole carcinogenic? Mutation

Research, 511: 133-144.

Bénéré E, VAN Assche T, Cos P, Maes L (2011) Variation in growth and drug susceptibility

among Giardia duodenalis assemblages A, B and E in axenic in vitro culture and in the gerbil

model. Parasitology, 138: 1354-1361.

109

Bernal-Redondo R, Martínez-Méndez LG, Mendoza-Chavez A, Velasco-Perales D, Chavez-

Munguia B (2004) Evaluation of the in vitro effect of albendazole, metronidazole and

nitazoxanide on viability and structure of Giardia lamblia cysts. Journal of Submicroscopic

Cytology and Pathology, 36: 241-245.

Berryman M, Franck Z, Bretscher A (1993) Ezrin is concentrated in the apical microvilli of a

wide variety of epithelial cells whereas moesin is found primarily in endothelial cells. Journal of

Cell Sciences, 105: 1025-1043.

Bharti N, Husain K, Gonzalez Garza MT, Cruz-Vega DE, Castro-Garza J, Mata-Cardenas BD,

Naqvi F, Azam A (2002) Synthesis and in vitro antiprotozoal activity of 5-nitrothiophene-2-

carboxaldehyde thiosemicarbazone derivatives. Bioorganic and Medicinal Chemistry Letters, 12:

3475-3478.

Bienz M, Dai WJ, Welle M, Gottstein B, Müller N (2003) Interleukin-6-deficient mice are highly

susceptible to Giardia lamblia infection but exhibit normal intestinal immunoglobulin A

responses against the parasite. Infection and Immunity, 71: 1569-1573.

Bjorkman A (1988) Interactions between chemotherapy and immunity to malaria. Progress in

Allergy, 41: 331-356.

Boreham PFL, Benrimoj S, Ong M, Craig M, Shepherd RW, Hill D (1986) A compliance study

in pediatrics patients receiving treatment for giardiasis. Australian Journal of Hospital

Pharmacy, 16: 138-142.

110

Bulut BU, Gülnar SB, Aysev D (1996) Alternative treatment protocols in giardiasis: a pilot

study. Scandinavian Journal of Infectious Disease, 28: 493-495.

Buret A, Gall DG, Olson ME (1990) Effects of murine giardiasis on growth, intestinal

morphology, and disaccharidase activity. The Journal of Parasitology, 76: 403-409.

Buret A, Gall DG, Olson ME (1991) Growth, activities of enzymes in the small intestine, and

ultrastructure of microvillous border in gerbils infected with Giardia duodenalis. Parasitology

Research, 77: 109-114

Buret A, Hardin JA, Olson ME, Gall DG (1992) Pathophysiology of small intestinal

malabsorption in gerbils infected with Giardia lamblia. Gastroenterology: 103: 506-513.

Buret AG, Mitchell K, Muench DG, Scott KG (2002) Giardia lamblia disrupts tight junctional

ZO-1 and increases permeability in non-transformed human small intestinal epithelial

monolayers: effects of epidermal growth factor. Parasitology, 125: 11-19.

Buret AG (2007) Mechanisms of epithelial dysfunction in giardiasis. Gut, 56: 316-317.

Byrd, L.G., J.T. Conrad, and T.E. Nash. 1994. Giardia lamblia infections in adult mice. Infection

and Immunity, 62: 3583-3585.

Cacopardo B, Patamia I, Bonaccorso V, Di Paola O, Bonforte S, Brancati G (1995) Synergic

effect of albendazole plus metronidazole association in the treatment of metronidazole-resistant

giardiasis. La Clinica terapeutica, 146: 761-767. [In Italian].

111

Cantelli-Forti G, Aicardi G, Guerra MC, Barbaro AM, Biagi GL (1983) Mutagenicity of a series

of 25 nitroimidazoles and two nitrothiazoles in Salmonella typhimurium. Teratogenesis,

Carcinogenesis and Mutagenesis, 3: 51-63.

Carabeo RA, Grieshaber SS, Fischer E, Hackstadt T (2002) Chlamydia trachomatis induces

remodeling of the actin cytoskeleton during attachment and entry into HeLa cells. Infection

and Immunity, 70: 3793-803.

Carpio A (2002) Neurocysticercosis: an update. Lancet Infectious Disease, 2: 751-762.

Cedillo-Rivera R, Muñoz O (1992) In vitro susceptibility of Giardia lamblia to albendazole,

mebendazole and other chemotherapeutic agents. Journal of Medical Microbiology, 37: 221-224.

Cevallos A, Carnaby S, James M, Farthing JG (1995) Small intestinal injury in a neonatal rat

model of giardiasis is strain dependent. Gastroenterology, 109: 766-773.

Chin AC, Teoh DA, Scott KG, Meddings JB, Macnaughton WK, Buret AG (2002) Strain-

dependent induction of enterocyte apoptosis by Giardia lamblia disrupts epithelial barrier

function in a caspase-3-dependent manner. Infection and Immunity, 70: 3673-3680.

Cliffe LJ, Humphreys NE, Lane TE, Potten CS, Booth C, Grencis RK (2005) Accelerated

intestinal epithelial cell turnover: a new mechanism of parasite expulsion. Science, 308: 1463-

1465.

112

Cravo P, Culleton R, Hunt P, Walliker D, Mackinnon MJ (2001) Antimalarial drugs clear

resistant parasites from partially immune hosts. Antimicrobial Agents and Chemotherapy, 45:

2897–2901.

Dahlqvist A (1968) Assay of intestinal disaccharidases. Analytical Biochemistry, 22: 99-107.

Daniels CW, Belosevic M (1992) Disaccharidase activity in the small intestine of susceptible and

resistant mice after primary and challenge infections with Giardia muris. American Journal of

Tropical Medicine and Hygiene, 46: 382-390.

Daniels CW, Belosevic M (1995) Disaccharidase activity in male and female C57BL/6 mice

infected with Giardia muris. Parasitology Research, 81: 143-147.

Davids BJ, Palm JE, Housley MP, Smith JR, Andersen YS, Martin MG, Hendrickson BA,

Johansen FE, Svärd SG, Gillin FD, Eckmann L (2006) Polymeric immunoglobulin receptor in

intestinal immune defense against the lumen-dwelling protozoan parasite Giardia. The Journal

of Immunology, 177: 6281-6290.

De Méo M, Vanelle P, Bernadini E, Laget M, Maldonado J, Jentzer O, Crozet MP, Duménil G

(1992) Evaluation of the mutagenic and genotoxic activities of 48 nitroimidazoles and related

imidazole derivatives by the Ames test and the SOS chromotest. Environmental and Molecular

Mutagenesis, 19: 167-181.

DerSimonian R, Laird N (1986) Meta-analysis in clinical trials. Controlled Clinical Trials, 7:

177-188.

113

Dunn JD, Valdivia RH (2010) Uncivil engineers: Chlamydia, Salmonella and Shigella alter

cytoskeleton architecture to invade epithelial cells. Future Microbiology, 5: 1219-1232.

Dutta AK, Phadke MA, Bagade AC, Joshi V, Gazder A, Biswas TK, Gill HH, Jagota SC (1994)

A randomised multi-centre study to compare the safety and efficacy of albendazole and

metronidazole in the treatment of giardiasis in children. Indian Journal of Pediatrics, 61: 689-

693.

Ebert EC (1999) Giardia induces proliferation and interferon gamma production by intestinal

lymphocytes. Gut, 44: 342-346.

Eckmann L, Laurent F, Langford TD, Hetsko ML, Smith JR, Kagnoff MF, Gillin FD (2000)

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. The

Journal of Immunology, 164: 1478-1487.

Eckmann L (2003) Eckmann, Mucosal defences against Giardia. Parasite Immunology, 25: 259-

270.

Egger M, Davey Smith G, Schneider M, Minder C (1997) Bias in meta-analysis detected by a

simple, graphical test. British Medical Journal, 315: 629-634.

Elizondo G, Gonsebatt ME, Salazar AM, Lares I, Santiago P, Herrera J, Hong E, Ostrosky-

Wegman P (1996) Genotoxic effects of metronidazole. Mutagenesis Research, 370: 75-80.

114

Erlich JH, Anders RF, Roberts-Thomson IC, Schrader JW, Mitchell GF (1983) An examination

of differences in serum antibody specificities and hypersensitivity reactions as contributing

factors to chronic infection with the intestinal protozoan parasite, Giardia muris, in mice. The

Australian Journal of Experimental Biology and Medical Science, 61: 599-615.

Elliott DA, Clark DP (2000) Cryptosporidium parvum induces host cell actin accumulation

at the host-parasite interface. Infection and Immunity, 68: 2315-2322.

Elliott DA, Coleman DJ, Lane MA, May RC, Machesky LM, Clark DP (2001)

Cryptosporidium parvum infection requires host cell actin polymerization. Infection and

Immunity, 69: 5940-5942.

Farthing MJG (1994) Giardiasis as a disease, R.C.A. Thompson, J.A. Reynoldson, A.J.

Lymbery, Editors , Giardia: From Molecules to Diseases, CAB International, Oxon , pp. 15-37.

Farthing MJ (1997). The molecular pathogenesis of giardiasis. Journal of Pediatric

Gastroenterology and Nutrition, 24: 79-88.

Feitosa G, Bandeira AC, Sampaio DP, Badaró R, Brites C (2001) High prevalence of giardiasis

and strongyloidiasis among HIV-infected patients in Bahia, Brazil. The Brazilian Journal of

Infectious Diseases, 5: 339-344.

Flanagan PA (1992) Giardia–diagnosis, clinical course and epidemiology. A review.

Epidemiology and Infection, 109: 1-22

115

Gillon J, Al Thamery D, Ferguson A (1982) Features of small intestinal pathology (epithelial cell

kinetics, intraepithelial lymphocytes, disaccharidases) in a primary Giardia muris infection. Gut,

23: 498-506.

Gordts B, Hemelhof W, Van Tilborgh K, Retore P, Cadranal S, Butzler JP (1985) Evaluation of

a new method for routine in vitro cultivation of Giardia lamblia from human duodenal fluid.

Journal of Clinical Microbiology, 22: 702-704.

Gardner TB, Hill DR (2001) Treatment of giardiasis. Clinical Microbiology Reviews, 14: 114-

128.

Geurden T, Levecke B, Cacció SM, Visser A, De Groote G, Casaert S, Vercruysse J, Claerebout

E (2009) Multilocus genotyping of Cryptosporidium and Giardia in non-outbreak related cases

of diarrhoea in human patients in Belgium. Parasitology, 136: 1161-1168.

Gillin FD, Reiner DS, McCaffery JM (1996) Cell biology of the primitive eukaryote Giardia

lamblia. Annual Review of Microbiology, 50: 679-705.

Guk SM, Yong TS, Chai JY (2003) Role of murine intestinal intraepithelial lymphocytes and

lamina propria lymphocytes against primary and challenge infections with Cryptosporidium

parvum. The Journal of Parasitology, 89: 270-275.

Hall A, Nahar Q (1993a, b) Albendazole as a treatment for infections with Giardia duodenalis in

children in Bangladesh. Transactions of the Royal Society of Tropical Medicine and Hygiene,

87: 84-86.

116

Haque R, Roy S, Kabir M, Stroup SE, Mondal D, Houpt ER (2005) Giardia assemblage A

infection and diarrhea in Bangladesh. The Journal of Infectious Diseases, 192: 2171-2173.

Hardin JA, Buret AG, Olson ME, Kimm MH, Gall DG (1997) Mast cell hyperplasia and

increased macromolecular uptake in an animal model of giardiasis. The Journal of Parasitology,

83: 908-912.

Hanevik K, Mørch K, Eide GE, Langeland N, Hausken T (2008) Effects of

albendazole/metronidazole or tetracycline/folate treatments on persisting symptoms after Giardia

infection: a randomized open clinical trial. Scandinavian Journal of Infectious Disease, 40: 517-

522.

Hemphill A, Müller J (2009) Alveolar and cystic echinococcosis: towards novel

chemotherapeutical treatment options. Journal of Helminthology, 83: 99-111.

Heyworth MF, Carlson JR, Ermak TH (1987) Clearance of Giardia muris infection requires

helper/inducer T lymphocytes. The Journal of Experimental Medicine, 165: 1743-1748.

Higgins JP, Thompson SG (2002) Quantifying heterogeneity in a meta-analysis. Statistics in

Medicine, 21: 1539-1558.

Homan WL, Mank TG (2001) Human giardiasis: genotype linked differences in clinical

symptomatology. International Journal for Parasitology, 31: 822-826.

117

Hossain MM, Glass RI, Khan MR (1982) Antibiotic use in a rural community in Bangladesh.

International Journal of Epidemiology, 11: 402-405.

Isaac-Renton JL (1991) Laboratory diagnosis of giardiasis. Clinics in Laboratory Medicine, 11:

811-827

Isaac-Renton JL, Lewis LF, Ong CS, Nulsen MF (1994) A second community outbreak of

waterborne giardiasis in Canada and serological investigation of patients. The Transactions of

the Royal Society of Tropical Medicine and Hygiene, 88: 395-399.

Istre GR, Dunlop TS, Gaspard GB, Hopkins RS (1984) Waterborne giardiasis at a mountain

resort: evidence for acquired immunity. American Journal of Public Health, 74: 602-604.

Jadad AR, Moore RA, Carroll D, Jenkinson C, Reynolds DJ, Gavaghan DJ, McQuay HJ (1996)

Assessing the quality of reports of randomized clinical trials: is blinding necessary? Controlled

Clinical Trials, 17: 1-12.

Jang GR, Harris RZ (2007) Drug interactions involving ethanol and alcoholic beverages. Expert

Opinion on Drug Metabolism and Toxicology, 3: 719-731.

Jiménez JC, Fontaine J, Grzych JM, Dei-Cas E, Capron M (2004) Systemic and mucosal

responses to oral administration of excretory and secretory antigens from Giardia intestinalis.

Clinical and Diagnostic Laboratory Immunology, 11: 152-160.

118

Johnson G (2009) STEPS New Drug Review: Tinidazole for trichomoniasis and bacterial

vaginosis. American Family Physician, 79: 102-103.

Jokipii L, Jokipii AM (1978) Comparison of four dosage schedules in the treatment of giardiasis

with metronidazole. Infection, 6: 92-94.

Jokipii L, Jokipii AM (1979a) Single-dose metronidazole and tinidazole as therapy for giardiasis:

success rates, side effects, and drug absorption and elimination. Journal of Infectious Diseases,

140: 984-988.

Jokipii L, Jokipii AM (1979b) Single-dose metronidazole and tinidazole as therapy for

giardiasis: success rates, side effects, and drug absorption and elimination. Journal of Infectious

Diseases, 140: 984-988.

Jourdan N, Brunet JP, Sapin C, Blais A, Cotte-Laffitte J, Forestier F, Quero AM, Trugnan,

Servin AL (1998) Rotavirus infection reduces sucrase-isomaltase expression in human intestinal

epithelial cells by perturbing protein targeting and organization of microvillar cytoskeleton.

Journal of Virology, 72: 7228-7236.

Jung HC, Eckmann L, Yang SK, Panja A, Fierer J, Morzycka-Wroblewska E, Kagnoff MF

(1995) A distinct array of pro-inflammatory cytokines is expressed in human colon epithelial

cells in response to bacterial invasion. The Journal of Clinical Investigation, 95: 55–65.

Kamda JD, Singer SM (2009) Phosphoinositide 3-kinase-dependent inhibition of dendritic cell

interleukin-12 production by Giardia lamblia. Infection and Immunity, 77: 685-693.

119

Karabay O, Tamer A, Gunduz H, Kayas D, Arinc H, Celebi H (2004) Albendazole versus

metronidazole treatment of adult giardiasis: An open randomized clinical study. World Journal

of Gastroenterology, 10: 1215-1217.

Kavousi S (1979) Giardiasis in infancy and childhood: a prospective study of 160 cases with

comparison of quinacrine (Atabrine®) and metronidazole (Flagyl®). American Journal of

Tropical Medicine and Hygiene, 28: 19-23.

Keiser J, Utzinger J (2008) Efficacy of current drugs against soil-transmitted helminth infections:

systematic review and meta-analysis. JAMA, 299: 1937-1948.

Kersting S, Bruewer M, Schuermann G, Klotz A, Utech M, Hansmerten M, Krieglstein CF,

Senninger N, Schulzke JD, Naim HY, Zimmer KP (2004) Antigen transport and cytoskeletal

characteristics of a distinct enterocyte population in inflammatory bowel diseases. American

Journal of Pathology, 165: 425-437.

Kothakota S, Azuma T, Reinhard C, Klippel A, Tang J, Chu K, McGarry TJ, Kirschner MW,

Koths K, Kwiatkowski DJ, Williams LT (1997) Caspase-3-generated fragment of gelsolin:

effector of morphological change in apoptosis. Science, 278: 294-298.

Khurana S, George SP (2008) Regulation of cell structure and function by actin-binding proteins:

villin's perspective. FEBS Letter, 582: 2128-2139.

Kohli A, Bushen OY, Pinkerton RC, Houpt E, Newman RD, Sears CL, Lima AA, Guerrant RL

(2008) Giardia duodenalis assemblage, clinical presentation and markers of intestinal

120

inflammation in Brazilian children. Transactions of the Royal Society of Tropical Medicine and

Hygiene, 102: 718-725.

Krause JR, Ayuyang HQ, Ellis LD (1985) Occurrence of three cases of carcinoma in individuals

with Crohn's disease treated with metronidazole. American Journal of Gastroenterology, 80:

978-982.

Kumar N, Tomar A, Parrill AL, Khurana S (2004) Functional dissection and molecular

characterization of calcium-sensitive actin-capping and actin-depolymerizing sites in villin.

Journal of Biological Chemistry, 279: 4536-4546.

Kumar N, Mishra J, Narang VS, Waters CM (2007) Janus kinase 3 regulates interleukin 2-

induced mucosal wound repair through tyrosine phosphorylation of villin. Journal of Biological

Chemistry, 282: 30341-30345.

Kvietys PR (1999) Intestinal physiology relevant to short-bowel syndrome. European Journal of

Pediatric Surgery, 9: 196-199.

Lanzkowsky P, Karayalcin G, Miller F (1982) Disaccharidase levels in iron deficient rats at birth

and during the nursing and post-weaning periods: response to iron treatment. Pediatrics

Research, 16: 318-323.

Larocque R, Nakagaki K, Lee P, Abdul-Wahid A, Faubert GM (2003) Oral immunization of

BALB/c mice with Giardia duodenalis recombinant cyst wall protein inhibits shedding of cysts.

Infection and Immunity, 71: 5662-5669.

121

Lauwaet T, Oliveira MJ, Callewaert B, De Bruyne G, Saelens X, Ankri S, Vandenabeele P,

Mirelman D, Mareel M, Leroy A (2003) Proteolysis of enteric cell villin by Entamoeba

histolytica cysteine proteinases. Journal of Biological Chemistry, 278: 22650-22656.

Lee P, Faubert G (2006a) Faubert, Expression of the Giardia lamblia cyst wall protein 2 in

Lactococcus lactis. Microbiology, 152: 1981-1990.

Lee P, Faubert GM (2006b) Oral immunization of BALB/c mice by intragastric delivery of

Streptococcus gordonii-expressing Giardia cyst wall protein 2 decreases cyst shedding in

challenged mice. FEMS Microbiology Letters, 265: 225-236.

Lee YA, Kim KA, Shin MH (2011) Calpain mediates degradation of cytoskeletal proteins

during Jurkat T-cell death induced by Entamoeba histolytica. Parasite Immunology, 33:

349-356.

Lemée V, Zaharia I, Nevez G, Rabodonirina M, Brasseur P, Ballet JJ, Favennec L (2000)

Metronidazole and albendazole susceptibility of 11 clinical isolates of Giardia duodenalis from

France. Journal of Antimicrobial Chemotherapy, 46: 819-821.

Levinson JD, Nastro LJ (1978) Giardiasis with total villous atrophy. Gastroenterology, 74: 271-

275.

122

Li E, Zhou P, Petrin Z, Singer SM (2004) Mast cell-dependent control of Giardia lamblia

infections in mice. Infection and Immunity, 72: 6642-6649.

Li E, Zhou P, Singer SM (2006) Neuronal nitric oxide synthase is necessary for elimination of

Giardia lamblia infections in mice. The Journal of Immunology, 176: 516-521.

Li E, Zhao A, Shea-Donohue T, Singer SM (2007) Mast cell-mediated changes in smooth

muscle contractility during mouse giardiasis. Infection and Immunity, 75: 4514-4518.

Lim SG, Menzies IS, Nukajam WS, Lee CA, Johnson MA, Pounder RE (1995) Intestinal

disaccharidase activity in human immunodeficiency virus disease. Scandinavian Journal of

Gastroenterology, 30: 235-241.

Little MC, Bell LV, Cliffe LJ, Else KJ (2005) The characterization of intraepithelial

lymphocytes, lamina propria leukocytes, and isolated lymphoid follicles in the large intestine of

mice infected with the intestinal nematode parasite Trichuris muris. The Journal of Immunology,

175: 6713-6722.

Lwin M, Targett GA, Doenhoff MG (1987) Reduced efficacy of chemotherapy of Plasmodium

chabaudi in T cell-deprived mice. Transactions of the Royal Society of Tropical Medicine and

Hygiene, 81: 899-902.

Mank TG, Zaat JO (2001) Diagnostic advantages and therapeutic options for giardiasis. Expert

Opinion in Investigational Drugs, 10: 1513-1519.

123

Matthaiou DK, Panos G, Adamidi ES, Falagas ME (2008) Albendazole versus praziquantel in

the treatment of neurocysticercosis: A meta-analysis of comparative trials. PLoS Neglected

Tropical Diseases, 2: e194.

Matowicka-Karna J, Dymicka-Piekarska V, Kemona H (2009) IFN-gamma, IL-5, IL-6 and IgE

in patients infected with Giardia intestinalis. Folia Histochemica et Cytobiologica, 47: 93-97.

McDonnell PA, Scott KG, Teoh DA, Olson ME, Upcroft JA, Upcroft P, Buret AG (2003)

Giardia duodenalis trophozoites isolated from a parrot (Cacatua galerita) colonize the small

intestinal tracts of domestic kittens and lambs. Veterinary Parasitology, 111: 31-46.

Merluzzi S, Frossi B, Gri G, Parusso S, Tripodo C, Pucillo C (2010) Mast cells enhance

proliferation of B lymphocytes and drive their differentiation toward IgA-secreting plasma cells.

Blood, 115: 2810-2817.

Meyer EK (1998) Adverse events associated with albendazole and other products used for

treatment of giardiasis in dogs. Journal of the American Veterinary Medicine Association, 213:

44-46.

Misra PK, Kumar A, Agarwal V, Jagota SC (1995a) A comparative clinical trial of albendazole

versus metronidazole in children with giardiasis. Indian Pediatrics, 32: 779-782.

Misra PK, Kumar A, Agarwal V, Jagota SC (1995b) A comparative clinical trial of albendazole

versus metronidazole in giardiasis. Indian Pediatrics, 32: 291-294.

124

Mohan K, Sam HH, Stevenson MM (1999) Therapy with a combination of low doses of

interleukin-12 and chloroquine completely cures blood-stage malaria, prevents severe anemia,

and induces immunity to reinfection. Infection and Immunity, 67: 513-519.

Moretto M, Weiss LM, Khan IA (2004) Induction of a rapid and strong antigen-specific

intraepithelial lymphocyte response during oral Encephalitozoon cuniculi infection. The Journal

of Immunology, 172: 4402-4409.

Morrison HG, McArthur AG, Gillin FD, et al (2007). Genomic minimalism in the early

diverging intestinal parasite Giardia lamblia. Science, 317: 1921-1926.

Müller J, Ley S, Felger I, Hemphill A, Müller N (2008) Identification of differentially expressed

genes in a Giardia lamblia WB C6 clone resistant to nitazoxanide and metronidazole. Journal of

Antimicrobial Chemotherapy, 62: 72-82.

Müller J, Sterk M, Hemphill A, Müller N (2007) Characterization of Giardia lamblia WB C6

clones resistant to nitazoxanide and to metronidazole. Journal of Antimicrobial Chemotherapy,

60: 280-287.

Murray IA, Smith JA, Coupland K, Ansell ID, Long RG (2001) Intestinal disaccharidase

deficiency without villous atrophy may represent early celiac disease. Scandinavian Journal of

Gastroenterology, 36: 163-168.

125

Naik SR, Rau NR, Vinayak VK (1978) A comparative evaluation of three stool samples, jejunal

aspirate and jejunal mucosal impression smears in the diagnosis of giardiasis. Annals of Tropical

Medicine and Parasitology, 72: 491-492.

Nash TE (1997) Antigenic variation in Giardia lamblia and the host’s immune response.

Philosophical Transactions of The Royal Society of London. Series B, Biological Science, 352:

1369-1375.

Navarrete-Vázquez G, Yépez L, Hernández-Campos A, Tapia A, Hernández-Luis F, Cedillo R,

González J, Martínez-Fernández A, Martínez-Grueiro M, Castillo R (2003) Synthesis and

antiparasitic activity of albendazole and mebendazole analogues. Bioorganic and Medicinal

Chemistry, 11: 4615-4622.

Oberhuber G, Kastner N, Stolte M (1997) Giardiasis: a histologic analysis of 567 cases.

Scandinavian Journal of Gastroenterology, 32: 48-51.

Olson ME, Morck DW, Ceri H (1996) The efficacy of a Giardia lamblia vaccine in kittens.

Canadian Journal of Veterinary Research, 60: 249-256.

Olson ME, Morck DW, Ceri H (1997) Preliminary data on the efficacy of a Giardia vaccine in

puppies. The Canadian Veterinary Journal, 38: 777-779.

Olson ME, Ceri H, Morck DW (2000) Giardia vaccination. Parasitology Today, 16: 213-217.

126

Olson ME, Hannigan CJ, Gaviller PF, Fulton LA (2001) The use of a Giardia vaccine as an

immunotherapeutic agent in dogs. The Canadian Veterinary Journal, 42: 865-868.

Osawa H, Smith CA, Ra YS, Kongkham P, Rutka JT (2009) The role of the membrane

cytoskeleton cross-linker ezrin in medulloblastoma cells. Neuro-Oncology, 11: 381-393.  

Panaro MA, Cianciulli A, Mitolo V, Mitolo CI, Acquafredda A, Brandonisio O, Cavallo P

(2007) Caspase-dependent apoptosis of the HCT-8 epithelial cell line induced by the parasite

Giardia intestinalis. FEMS Immunology and Medical Microbiology, 51: 302-309.

Pérez PF, Minnaard J, Rouvet M, Knabenhans C, Brassart D, De Antoni GL, Schiffrin EJ (2001)

Inhibition of Giardia intestinalis by extracellular factors from Lactobacilli: an in vitro study.

Applied and Environmental Microbiology, 67: 5037-5042.

Pungpak S, Singhasivanon V, Bunnag D, Radomyos B, Nibaddhasopon P, Harinasuta KT (1996)

Albendazole as a treatment for Giardia infection. Annals of Tropical Medicine and Parasitology,

90: 563-565.

Rastegar-Lari A, Salek-Moghaddam A (1996) Single-dose secnidazole versus 10-day

metronidazole therapy of giardiasis in Iranian children. Journal of Tropical Pediatrics, 42: 184-

185.

Reifen R, Zaiger G, Uni Z (1998) Effect of vitamin A on small intestinal brush border enzymes

in a rat. International Journal for Vitamin and Nutrition Research, 68: 281-286.

127

Reiske HR, Kao SC, Cary LA, Guan JL, Lai JF, Chen HC (1999) Requirement of

phosphatidylinositol 3-kinase in focal adhesion kinase-promoted cell migration. Journal of

Biological Chemistry, 274: 12361-12366.

Reitz M, Rumpf M, Knitza R (1991) DNA single strand-breaks in lymphocytes after

metronidazole therapy. Arzneimittelforschung, 41: 155-156.

Reynoldson JA, Behnke JM, Gracey M, Horton RJ, Spargo R, Hopkins RM, Constantine CC,

Gilbert F, Stead C, Hobbs RP, Thompson RC (1998) Efficacy of albendazole against Giardia

and hookworm in a remote Aboriginal community in the north of Western Australia. Acta

Tropica, 71: 27-44.

Reynoldson JA, Thompson RC, Meloni BP (1991) In vivo efficacy of albendazole against

Giardia duodenalis in mice. Parasitology Research, 77: 325-328.

Ringqvist E, Palm JE, Skarin H, Hehl AB, Weiland M, Davids BJ, Reiner DS, Griffiths WJ,

Eckmann L, Gillin FD, Svärd SG (2008) Release of metabolic enzymes by Giardia in response

to interaction with intestinal epithelial cells. Molecular and Biochemical Parasitology, 159: 85-

91.

Roberts-Thomson and Mitchell IC, Mitchell GF (1978) Giardiasis in mice. I. Prolonged

infections in certain mouse strains and hypothymic (nude) mice. Gastroenterology, 75: 42-46.

128

Rodríguez-García R, Aburto-Bandala M, Sánchez-Maldonado MI (1996) Effectiveness of

albendazole in the treatment of giardiasis in children. Boletín Médico del Hospital Infantil de

México, 53: 173-177. [In Spanish].

Romero-Cabello R, Robert L, Muñoz-García R, Tanaka J (1995) Randomized study comparing

the safety and efficacy of albendazole and metronidazole in the treatment of giardiasis in

children. Revista Latinoamericana Microbiologia, 37: 315-323. [In Spanish].

Roskens H, Erlandsen SL (2002) Inhibition of in vitro attachment of Giardia trophozoites by

mucin. The Journal of Parasitology, 88: 869-873.

Roxström-Lindquist K, Ringqvist E, Palm D, Svärd SG (2005) Giardia lamblia-induced changes

in gene expression in differentiated Caco-2 human intestinal epithelial cells. Infection and

Immunity, 73: 8204-8208.

Roxström-Lindquist K, Palm D, Reiner D, Ringqvist E, Svärd SG (2006) Giardia immunity – an

update. Trends in Parasitology, 22: 26-31.

Saffar MJ, Qaffari J, Khalilian AR, Kosarian M (2005) Rapid reinfection by Giardia lamblia

after treatment in a hyperendemic area: the case against treatment. Eastern Mediterranean

Health Journal, 11: 73-78.

Salzman NH, Underwood MA, Bevins CL (2007) Paneth cells, defensins, and the commensal

microbiota: a hypothesis on intimate interplay at the intestinal mucosa. Seminars in Immunology,

19: 70-83.

129

Scott KG, Logan MR, Klammer GM, Teoh DA, Buret AG (2000) Jejunal brush border

microvillous alterations in Giardia muris-infected mice: role of T lymphocytes and interleukin-6.

Infection and Immunity, 68: 3412-3418.

Scott KG, Yu LC, Buret AG (2004) Role of CD8+ and CD4+ T lymphocytes in jejunal mucosal

injury during murine giardiasis. Infection and Immunity, 72: 3536-3542.

Selbach M, Moese S, Backert S, Jungblut PR, Meyer TF (2004) The Helicobacter pylori CagA

protein induces tyrosine dephosphorylation of ezrin. Proteomics, 4: 2961-2968.

Selgrad M, Kandulski A, Malfertheiner P (2009) Helicobacter pylori: diagnosis and treatment.

Current Opinion in Gastroenterology, 25: 549-556.

Shant J, Bhattacharyya S, Ghosh S, Ganguly NK, Majumdar S (2002) A potentially important

excretory–secretory product of Giardia lamblia. Experimental Parasitology, 102: 178-186.

Shant J, Ghosh S, Bhattacharyya S, Ganguly NK, Majumdar S (2004) The alteration in signal

transduction parameters induced by the excretory–secretory product from Giardia lamblia.

Parasitology, 129: 421-430.

Shant J, Ghosh S, Bhattacharyya S, Ganguly NK, Majumdar S (2004) Mode of action of a

potentially important excretory–secretory product from Giardia lamblia in mice enterocytes.

Parasitology, 131: 57-69.

130

Shepherd RW, Boreham PFL (1986) Recent advances in the diagnosis and management of

giardiasis. Scandinavian Journal of Gastroenterology Supplement, 169: 60-64.

Singer SM, Nash TE (2000a) T-cell-dependent control of acute Giardia lamblia infections in

mice. Infection and Immunity, 68: 170-175.

Singer SM, Nash TE (2000b) The role of normal flora in Giardia lamblia infections in mice. The

Journal of Infectious Diseases, 181: 1510-1512.

Singh A, Janaki L, Petri WA, Houpt ER (2009) Giardia intestinalis assemblages A and B

infections in Nepal. American Journal of Tropical Medicine and Hygiene, 81: 538-539.

Smith PD (1985) Pathophysiology and immunology of giardiasis. Annual Review of Medicine,

36: 295-307.

Snel J, Baker MG, Kamalesh V, French N, Learmonth J (2009) A tale of two parasites: the

comparative epidemiology of cryptosporidiosis and giardiasis. Epidemiology and Infection, 137:

1641-1650.

Snider DP, Skea D, Underdown BJ (1988) Chronic giardiasis in B-cell-deficient mice expressing

the xid gene. Infection and Immunity, 56: 2838-2842.

Solaymani-Mohammadi S, Genkinger JM, Loffredo CA, Singer SM (2010) A meta-analysis of

the effectiveness of albendazole compared with metronidazole as treatments for infections with

Giardia duodenalis. PLoS Neglected Tropical Diseases, 4: e682.

131

Solaymani-Mohammadi S, Singer SM (2011) Host immunity and pathogen strain contribute to

intestinal disaccharidase impairment following gut infection. The Journal of Immunology, 187:

3769-3775.

Stager S, Gottstein B, Müller N (1997) Systemic and local antibody response in mice induced by

a recombinant peptide fragment from Giardia lamblia variant surface protein (VSP) H7

produced by a Salmonella typhimurium vaccine strain. International Journal for Parasitology,

27: 965-971.

Stark D, Barratt JL, van Hal S, Marriott D, Harkness J, Ellis JT (2009) Clinical significance of

enteric protozoa in the immunosuppressed human population. Clinical Microbiology Reviews,

22: 634-650.

Stein JE, Radecki SV, Lappin MR (2003) Efficacy of Giardia vaccination in the treatment of

giardiasis in cats. Journal of American Veterinary Medicine Association, 222: 1548-1551.

Stevens DP, Frank DM, Mahmoud AA (1978) Thymus dependency of host resistance to Giardia

muris infection: studies in nude mice. The Journal of Immunology, 120: 680-682.

Sullivan PS, DuPont HL, Arafat RR, Thornton SA, Selwyn BJ, el Alamy MA, Zaki AM (1989)

Illness and reservoirs associated with Giardia lamblia infection in rural Egypt: the case against

treatment in developing world environments of high endemicity. American Journal of

Epidemiology, 127: 1272-1281.

132

Takahashi M, Tanonaka K, Yoshida H, Koshimizu M, Daicho T, Oikawa R, Takeo S (2006)

Possible involvement of calpain activation in pathogenesis of chronic heart failure after

acute myocardial infarction. Journal of Cardiovascular Pharmacology, 47: 413-421.

Taylor C, Hodgson K, Sharpstone D, Sigthorsson G, Coutts M, Sherwood R, Menzies I, Gazzard

B, Bjarnason I (2000) The prevalence and severity of intestinal disaccharidase deficiency in

human immunodeficiency virus-infected subjects. Scandinavian Journal of Gastroenterology,

35: 599-606.

Teoh DA, Kamieniecki D, Pang G, Buret AG (2000) Giardia lamblia rearranges F-actin and

alpha-actinin in human colonic and duodenal monolayers and reduces transepithelial electrical

resistance. The Journal of Parasitology, 86: 800-806.

Thompson RC, Reynoldson JA, Mendis AH (1993) Giardia and giardiasis. Advances in

Parasitology, 32: 71-160.

Thompson RC (2009) Echinococcus, Giardia and Cryptosporidium: observational studies

challenging accepted dogma. Parasitology, 136: 1529-1535.

Tillonen J, Väkeväinen S, Salaspuro V, Zhang Y, Rautio M, Jousimies-Somer H, Lindros K,

Salaspuro M (2000) Metronidazole increases intracolonic but not peripheral blood acetaldehyde

in chronic ethanol-treated rats. Alcoholism Clinical and Experimental Research, 24: 570-575.

133

Tomar A, George S, Kansal P, Wang Y, Khurana S (2006) Interaction of phospholipase C-

gamma-1 with villin regulates epithelial cell migration. Journal of Biological Chemistry, 281:

31972-1986.

Troeger H, Epple HJ, Schneider T, Wahnschaffe U, Ullrich R, Burchard GD, Jelinek T, Zeitz M,

Fromm M, Schulzke JD (2007) Effect of chronic Giardia lamblia infection on epithelial

transport and barrier function in human duodenum. Gut, 56: 328-335.

Uehlinger FD, O’Handley RM, Greenwood SJ, Guselle NJ,Gabor LJ, Van Velsen CM, Steuart

RF, Barkema HW (2007) Efficacy of vaccination in preventing giardiasis in calves. Veterinary

Parasitology, 146: 182-188.

Upcroft JA, Upcroft P (1993) Drug resistance and Giardia. Parasitology Today, 9: 187-190.

Venkatesan P, Finch RG, Wakelin D (1996) Comparison of antibody and cytokine responses to

primary Giardia muris infection in H-2 congenic strains of mice. Infection and Immunity, 64:

4525-4533.

von Allmen N, Christen S, Forster U, Gottstein B, Welle M, Müller N (2006) Acute trichinellosis

increases susceptibility to Giardia lamblia infection in the mouse model. Parasitology, 133: 139-

149.

Walker ME, Hjort EE, Smith SS, Tripathi A, Hornick JE, Hinchcliffe EH, Archer W, Hager

KM (2008) Toxoplasma gondii actively remodels the microtubule network in host cells.

Microbes and Infection, 10: 1440-1449.

134

Wang H, Guo Z, Wu F, Long F, Cao X, Liu B, Zhu Z, Yao X (2005) PKA-mediated protein

phosphorylation protects ezrin from calpain I cleavage. Biochemical and Biophysical Research

Communications, 333: 496-501.

Wang Y, Srinivasan K, Siddiqui MR, George SP, Tomar A, Khurana S (2008) A novel role for

villin in intestinal epithelial cell survival and homeostasis. Journal of Biological Chemistry, 283:

9454-9464.

West AR, Oates PS (2005) Decreased sucrase and lactase activity in iron deficiency is

accompanied by reduced gene expression and upregulation of the transcriptional repressor PDX-

1. American Journal of Physiology, Gastrointestinal and Liver Physiology, 289: 1108-1114.

Williamson AL, O’Donoghue PJ, Upcroft JA, Upcroft P (2000) Immune and pathophysiological

responses to different strains of Giardia duodenalis in neonatal mice. International Journal for

Parasitology, 30: 129-136.

Wolfe MS (1992) Giardiasis. Clinical Microbiology Review, 5: 93-100.

Wright JM, Dunn LA, Upcroft P, Upcroft JA (2003) Efficacy of antigiardial drugs. Expert

Opinion on Drug Safety, 2: 529-541.

Yao X, Thibodeau A, Forte JG (1993) Ezrin-calpain I interactions in gastric parietal cells.

American Journal of Physiology, 265: 36-46.

135

Yereli K, Balcioğlu IC, Ertan P, Limoncu E, Onağ A (2004) Albendazole as an alternative

therapeutic agent for childhood giardiasis in Turkey. Clinical Microbiology and Infection, 10:

527-529.

Zaat JO, Mank TG, Assendelft WJ (1997) A systematic review on the treatment of giardiasis.

Tropical Medicine and International Health, 2: 63-82.

Zaiger G, Nur T, Barshack I, Berkovich Z, Goldberg I, Reifen R (2004) Vitamin A exerts its

activity at the transcriptional level in the small intestine. European Journal of Nutrition, 43: 259-

266.

Zhai L, Zhao P, Panebra A, Guerrerio AL, Khurana S (2001) Tyrosine phosphorylation of

villin regulates the organization of the actin cytoskeleton. Journal of Biological Chemistry,

276: 36163-36167.

Zhang PJ, Harris KR, Alobeid B, Brooks JJ (1999) Immunoexpression of villin in

neuroendocrine tumors and its diagnostic implications. Archive of Pathology and Laboratory

Medicine, 123: 812-816.

Zhou P, Li E, Zhu N, Robertson J, Nash TE, Singer SM (2003) Role of interleukin-6 in the

control of acute and chronic Giardia lamblia infections in mice. Infection and Immunity, 71:

1566-1568.

Zhou P, Li E, Shea-Donohue T and Singer SM (2007) Tumour necrosis factor alpha contributes

to protection against Giardia lamblia infection in mice. Parasite Immunology, 29: 367-374.

136

Zhou R, Zhu L, Kodani A, Hauser P, Yao X, Forte JG (2005). Phosphorylation of ezrin on

threonine 567 produces a change in secretory phenotype and repolarizes the gastric parietal cell.

118: 4381-4391.