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The effect of Giardia duodenalis filtrates on disaccharidases of the Caco-2 cell line Ochola, Beldinah Rachel 1997

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T H E EFFECT O F  GIARDIA DUODENALIS F I L T R A T E S I  O N DISACCHARIDASES O F T H E CACO-2 CELL L I N E |  by BELDLNAH RACHEL OCHOLA B.Sc, The University of London, UK, 1993  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Pathology and Laboratory Medicine, Faculty of Medicine  We accept this thesis as conforrning to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA May 1997 ® Beldinah Rachel Ochola, 1997  In  presenting  degree freely  at  this  the  thesis  in  University  of  partial  of  department  this or  publication of  thesis by  his  for  of  the  and study.  I further  scholarly purposes may  or  this thesis for  her  representatives.  of.  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  be  It  financial gain shall not  ^oi-Ht'al'ay^  J~<Z-k  that the  agree  permission.  Department  requirements  British Columbia, I agree  available for reference  copying  fulfilment  frUckdoA  is  for  an  Library shall make  that permission for granted  by the  understood  be  allowed  advanced  extensive  head  that  without  it  of  copying  my or  my written  ABSTRACT  Although Giardia duodenalis is one of the most common intestinal protozoan parasites world-wide, the pathogenesis of giardiasis is not understood. An attempt to correlate parasite excretory-secretory products with changes in the intestinal mucosa using an in vitro cell model was carried out to better understand this aspect of the pathogenesis.  Maltase activity and protein concentration obtained from a confluent  monolayer of Caco-2 cells after co-incubation with parasite filtrates were examined and compared. Appropriate controls were also assayed. Using this Caco-2 in vitro model, a significant reduction in both maltase activity and protein concentration (P<0.05) of Caco-2 cells was observed following co-incubation with the WB Giardia isolate filtrate. This effect was observed in three experiments carried out in triplicate. Reduction in maltase activity and protein concentration was as a result of co-incubating Caco-2 cells with WB parasite filtrate (absence of trophozoites) and was apparent within 48 hours of co-incubation of Caco-2 cells. Following Caco-2 monolayer co-incubation experiments with filtrates from four other Giardia parasite isolates (obtained originally from symptomatic and asymptomatic patients) a significant reduction (P<0.05) in protein concentration of Caco-2 cells was observed for one of the three (WH, symptomatic) isolates tested. No change in Caco-2 maltase was noted in this experiment.  Growth  curve analysis of various Giardia isolates was also carried out. Results showed that the isolates fell into two growth curve patterns (slow and fast growers). Differences in in vitro growth dynamics were found between the isolates.  Growth curve patterns,  however, did not predict either type of host source (symptomatic vs asymptomatic) or  ii  patterns in reduction of Caco-2 protein. Results of co-incubation of Caco-2 cells with WB filtrates are consistent with the hypothesis that the disaccharidase deficiency observed in human and animals with giardiasis may be associated with a direct effect produced by parasite excretory-secretory products on the intestinal brush border membrane.  Caco-2 cells are a useful in vitro model for the further study of the  pathogenesis of giardiasis.  iii  TABLE OF CONTENTS  ABSTRACT  ii  LIST OF FIGURES  vi  LIST OF TABLES  viii  ACKNOWLEDGEMENT 1  2  3  ix  BACKGROUND 1.1  Biology of Giardia spp  1  1.2  Taxonomy of the Genus Giardia  5  1.3  Epidemiology of Giardiasis  7  1.4  Clinical Presentation, Diagnosis and Therapy  10  1.5  Pathology of Infection  11  1.6  Pathophysiology of Infection  12  1.7  Phenotypic and Genotypic Parasite Variation  17  1.8  Models of Infection  22  1.9  Prekminary Disaccharidase Experiments  25  HYPOTHESIS AND OBJECTIVES 2.1  Hypothesis  2.2  Objectives  26 ...  26  MATERIALS AND METHODS 3.1  In Vitro  Growth Studies  27  3.2  Preparation of Parasite Filtrates  28  iv  TABLE OF CONTENTS- Continued  4  3.3  Caco-2 Cell Growth and Maintenance  29  3.4  Caco-2 Co-incubation Experiments  30  3.5  Measurements of Enzyme Levels  33  3.6  Statistical Analysis  36  RESULTS 4.1  Growth Studies  39  4.2  Co-incubation experiments  40  5  DISCUSSION  55  6  SUMMARY AND CONCLUSIONS  65  7  DIRECTION FOR FUTURE STUDIES  66  8  REFERENCES  67  v  L I S T  O F  F I G U R E S  Figure 1. Giardia rrophozoite  2  Figure 2. Giardia cyst  4  Figure3. Life Cycle of the Giardia Parasite  4  Figure 4. Proposed Mechanism for the Production of Diarrhea  21  Figure 5. Growth Curves of Various Parasite Isolates  44  Figure 6. Caco-2 Cell Monolayer Under Standard Conditions  45  Figure 7. Specific Maltase Activity of Caco-2 after serial inoculation  46  Figure 8. Maltase Activity of Caco-2 after serial inoculation with filtrates  47  Figure 9. Protein Content of Caco-2 after serial inoculation with filtrates  47  Figure 10. Specific Maltase Activity of Caco-2 after single inoculation (7 day monolayer)  48  Figure 11. Maltase Activity of Caco-2 after single inoculation (7 day monolayer)  49  Figure 12. Protein Content of Caco-2 after single inoculation (7 day monolayer)  49  Figurel3. Specific Maltase Activity of Caco-2 after single inoculation (10 day monolayer)  50  Figure 14. Maltase Activity of Caco-2 after single inoculation (10 day monolayer)  51  vi  Figure 15. Protein Content of Caco-2 after single inoculation (10 day monolayer)  51  Figure 16. Specific Maltase Activity of Caco-2 co-incubated with Various Parasite Isolates  52  Figure 17. Maltase Activity of Caco-2 co-incubated with Various Parasite Isolates  53  Figure 18. Protein Content of Caco-2 co-incubated with Various Parasite Isolates  54  vii  LIST O F T A B L E S  Table 1.  G. duodenalis Isolates  37  Table 2.  Summary of Experiments Carried Out  viii  38  ACKNOWLEDGEMENTS  Firstly, I would like to express my heartfelt gratitude to Dr. Isaac-Renton for her encouragement and guidance over the last two years or so. Her support was greatly appreciated as well as her remarkable efficiency! I would also like to thank my other committee members, Dr. Tom Beach, Dr. Dorovini-Zis, Dr. N . Kelly and Dr. W. Bowie for their encouragement, critical evaluation and comments of my work. In addition, I would like to thank both my former colleagues in the laboratory, Caleb Lee, Paulina Lee, and my present colleagues Lorraine Mclntyre, Loan Hoang and especially Anna Li for their assistance, patience, love and support both inside and outside the laboratory. It was a great pleasure always to work with all of you! Warmest and heartfelt thanks to Irene Ho for all her love and help, especially in typing out the corrections. Thanks too to Dr. C. Ong for her contribution to this project. Special thanks to Dr. N . Cimolai and Annette Castell of B.C. Children's Hospital for all their assistance in testing my many samples for Mycoplasma spp. I would also like to express my extreme gratitude to my family for all their continual and heartfelt support, love and encouragement.  ix  T H E EFFECT O F  GIARDIA DUODENALIS F I L T R A T E S I  O N DISACCHARIDASES OF T H E CACO-2 CELL L I N E |  CHAPTER 1 BACKGROUND  1.1  Biology of Giardia spp. The eukaryote Giardia was first described by Antony van Leeuwenhoek 300 years  ago.  Giardia are flagellated protozoan parasites in the class Zoomastigophorea and the  order Diplomonadida (Buret, 1994; Meyer, 1994). Studies show that they commonly infect the intestinal tract of many classes of vertebrates (Meyer, 1994). Giardia has two stages. Trophozoites (Figure 1) measure 12 to 15 urn in length and 5 to 9 um wide (Feely et al., 1990). pyriform with a large, rounded anterior.  Trophozoites are described as pear-shaped or They have a pair of functionally equivalent  (Kabnick and Peattie, 1991) nuclei, median bodies (internal microtubular structures of unknown function), and 4 pairs of flagellae. As well, they possess a distinctive structure which occupies most of the ventral surface, the sucking or adhesive disc. The ventral disc is morphologically identical between Giardia species and distinguishes this organism from other flagellates (Feely et al., 1990). It is surrounded at the anterolateral border by a cytoplasmic extension, the ventrolateral flange. Giardia do not possess mitochondria or a Golgi apparatus.  Scanning electron micrograph (SEM) of Giardia trophozoites. Ventrolateral flange (VLF) can be seen at the anterolateral border, also noticeable are flagellae (F). Bar equals 4  Cysts (Figure 2), the other stage of the parasite, are ovoid in shape, 8 to 18 urn in length and 5 to 15 pm wide (AWWA, 1995). They have between 2 to 4 nuclei, depending on cyst maturity. "Unassembled" trophozoite organelles are also present.  Cysts have a  wall that is comprised of filamentous structures interwoven and arranged in whorls. The amino acid N-acetylgalactosarnine is thought to be a major component of the cyst wall (Jarroll et al., 1989). Cysts are resistant to various environmental conditions, surviving for long periods oftimeoutside the host if they remain wet and cool farthing, 1993; Feely et al., 1990). In general they are known to survive a moderate range of temperatures. At some extremely low temperatures (as low as -13°C) their excystation ability is not destroyed. Boiling, however, effectively renders Giardia cysts non-viable. (Bingham et al., 1979). Giardia has a simple life cycle (Figure 3).  This parasite does not require an  intermediate host to survive (Kirkpatrick and Farrell, 1982).  The parasite alternates  between the two described asexual forms, trophozoites and cysts (Kirkpatrick and Farrell, 1982). Trophozoites are found in the upper t^o-thirds of the host small intestine and are associated with symptoms in the host (Meyer, 1994). They attach to the mucosal epithelial layer of the host by means of the ventral disc, and ingest particles from their milieu by pinocytosis. Binary fission of the trophozoite stage also takes place in the small intestine.  3  Figure 2:  Transmission electron micrograph (TEM) of a Giardia cyst. Fewer structures are visible in the cyst. Nuclei (N), outer cyst wall (OW) and inner cyst wall (TW) are easily detected.  Life Cycle of Giardia Species  Figure 3:  Giardia life cycle (adapted from Katz et al., 1982)  4  Encystation, the transition process from trophozoite to cyst, occurs in the intestinal tract. This process is promoted by the presence of high concentrations of bile, conjugated bile salts and high pH (Gillin et al., 1988). A single division of the two nuclei occurs within the cyst forming a quadrinucleate stage. Two separate organisms are then produced. Cysts passed in host fecal material are infectious immediately (Grant and Woo, 1977). If cysts are ingested by a new host, excystation occurs in the duodenum of a susceptible host. It has been suggested that the excystation process is accelerated on exposure to higher pH in the duodenum and pancreatic hydrolytic enzymes (Farthing, 1993; Bingham et al., 1979) although it is a poorly defined process (Meyer, 1994). Two binucleate trophozoites emerge or excyst through one end of the cyst wall and attach to the enterocytes of the upper small intestine.  1.2  Taxonomy of the Genus Giardia Despite the early description of Giardia trophozoites,  controversial.  taxonomy remains  Classification within the genus is currently based on trophozoite  morphological criteria, primarily the shape of median bodies observed by light microscopy. Using differences in internal (median body) morphology, Filice (1952) identified three groups.  These three Giardia groups have the same external morphological features  including 8 flagellae arranged in pairs in a similar pattern.  Two further groups were  recently described based on morphological differences observed by electron microscopy.  5  The five groups of Giardia currently recognized are: i)  Giardia agilis: Trophozoites are long and narrow and have long, teardrop-shaped median bodies  arranged parallel to the long axis of the body.  These isolates are generally found in  amphibians. ii)  Giardia maris: Trophozoites are pear-shaped and have two small, rounded, central median bodies.  These are isolates found in rodents, birds and reptiles. iii)  Giardia duodenalis (syn G. lamblia, G. intestinalis): Trophozoites in this group have been isolated from different hosts.  They are  generally distinguished by their median bodies (either one or two) resembling the claw of a claw hammer. They are found in a variety of mammalian host species including humans, reptiles and possibly rodents (Adam, 1991; Meyer and Jarroll, 1980). This is the parasite group of interest in the present project. Trophozoites from this group have been axenized and successfully established in vitro and therefore have been studied in greater detail (Sarafis and Isaac-Renton, 1993; Adam, 1991). iv)  Giardia ardeae: Trophozoites resemble those of G. duodenalis by light microscopy although they  have distinct characteristics including unique chromosomal banding patterns by pulsed field gel electrophoresis (Erlandsen et al., 1990). They are found in a variety of avian hosts including the Great Blue Heron (Ardea herodias) and the gray heron (Ardea cinerea).  6  v)  Giardia psittaci: Trophozoites are unique in that their ventral lateral flange is incomplete and hence  does not completely encircle the ventral adhesive disc.  Trophozoites do not possess a  marginal groove (Erlandsen and Bemrick, 1987). This strain is closely related to G. ardeae and was retrieved from budgerigars.  1.3  Epidemiology of Giardiasis The global impact of diarrheal diseases has been quantified to some extent by such  key indicators as mortality, morbidity and economic loss (Theilman and Guerrant, 1996). Diarrhea-inducing agents are major killers, particularly of small children, in the developing world (Hirschhorn and Greenough UI, 1991).  Even though most individual bouts of  diarrhea are not hfe-threatening, approximately 4 rnillion children world-wide under the age of five years (Hall, 1994) succumb annually to diarrheal diseases often compounding malnutrition and other chronic diseases (Theilman and Guerrant, 1996). Intestinal protozoan infections are among the most common infections world-wide (WHO/PAHO, 1991).  G. duodenalis, probably the most widespread of intestinal  protozoans (Hall, 1994; Adam, 1991; Farthing, 1990; Farthing, 1989; Gyorkos et al, 1987), is ranked as one of the ten most common human parasites (Buret, 1994; Schofield, 1985). Over 200 million cases of giardiasis are reported per year world-wide, clearly an underestimate even in countries where public health reporting systems are relatively reliable (Isaac-Renton, 1991 b). The ubiquity of Giardia may partly be due to a simple, efficient life cycle (Sarafis and Isaac-Renton, 1993), low infectious dose and broad host range.  7  There has been renewed interest in giardiasis (Isaac-Renton, 1991 a, b) even though the infection was considered of little clinical consequence three or four decades ago. Giardiasis appears to be more common in children than adults (Oyerinde et al., 1977). It is found throughout both temperate and tropical geographical areas world-wide. Its estimated prevalence is about 2-5% in the industrialized world and up to 20-30% in the developing world (Farthing, 1994).  Recent travel to developing countries is often associated with  acquiring this infection (Farthing, 1994) although transmission also occurs in both Canada and the United States.  In British Columbia alone, there are over 1000 new cases of  giardiasis reported annually making this one of the most common of the enteric reportable diseases in the province (Fisk, 1987). Parasite cysts survive well in a variety of  environments and therefore indirect  spread (by food or water) is common. Waterborne epidemics in North America and Europe are usually associated with inadequate treatment of surface drinking water and are well documented in British Columbia (Isaac-Renton, 1994; Fisk, 1987; Isaac-Renton, 1987). Parasite contamination of surface drinMng water supplies is ubiquitous and chlorination does not guarantee the destruction of cysts (Herwaldt et al., 1991; Farthing, 1989; Jephcott et al., 1986; Craun, 1984). Thus, since most surface drinking water supplies in British Columbia are not treated by multiple barriers, (including watershed or primary source protection, flocculation, filtration and disinfection of source water), drinking water is an important vehicle of spread in this province. Foodborne transmission is not common; three outbreaks have been identified to date (Adam, 1991). Person-to-person spread (via the fecal-oral route) is another route of transmission.  8  Infection is often found in children in day care centres, as well as in persons attending residential institutions and schools (Naiman et al., 1980; Keystone et al., 1978). Cysts may be transmitted during sexual activity (Owen 1984). Giardia is considered to be a highly infectious organism, with fewer than 100 viable cysts needed for transmission (Rendtorff, 1954). Parasites in the G. muris and G. agilis groups are not considered infectious to man at present (Isaac-Renton, 1993).  Some studies have shown that human-to-animal  transmission of G. duodenalis is possible (Erlandsen et al., 1988; Belosevic et al, 1983). Recently, it has been suggested that beaver-to-human transmission may occur (Erlandsen et al., 1988). However, there are no studies demonstrating the extent of cross-transmission of duodenalis type Giardia from one type of host to another (human-to-animal or animal-tohuman). Biological traits, including growth rates, host specificity, and virulence, cannot be assumed to correlate with biochemical traits (Isaac-Renton, 1993).  For example, it is  possible that there is a wide range or a narrow range of host specificity within this morphological (duodenalis) group. No markers of biological characteristics or activity have been reported to date.  9  1.4  Clinical Presentation, Diagnosis and Therapy The spectrum of symptomatology in giardiasis varies from acute, self-hmited  disease to chronic gastrointestinal symptoms (Gillon et al., 1982; Wolfe, 1978). Some patients are asymptomatic (Meyer and Jarroll, 1980; Hartong et al., 1979). The most frequent symptom of giardiasis is watery diarrhea although other symptoms such as abdominal pain, malaise, flatulence, weight loss, malabsorption and steatorrhea can occur (Meyer and Jarroll, 1980; Hoskins et al, 1967). 'Tanure-to-thrive" in infants has been reported  (Farthing et  al.,  1986;  Hartong  et  ah,  1979).  Patients  with  hyrxigammaglobulinernia (an immunodeficiency syndrome) often have a more severe form of disease (Khanna et al., 1988; Hartong et al., 1979; Hoskins et al., 1967). Laboratory confirmation of infection is based on examination of (Isaac-Renton, 1991 a, b).  fecal samples  Giardia cysts are detected using concentration methods and  microscopy of stained fecal preparations (Isaac-Renton, 1991 a). Examination of duodenal biopsy specimens and aspirated upper intestinal secretions may be useful in detecting some cases (Farthing, 1994). At present, laboratory methods are relatively labour-intensive and require trained technologists. Diagnostic kits for detection of Giardia antigen in feces using immunologically-based methods (Farthing, 1990; Green et al, 1985; Ungar et al, 1984) are now commercially available (Isaac-Renton, 1993). Nitroimidazole derivatives (e.g. metronidazole), acridine dyes (e.g. quinacrine) and nitrofurans (e.g. furazolidone) are the major classes of drugs useful in the treatment of giardiasis (Adam, 1991; Davidson, 1984). Metronidazole is the drug most commonly used by physicians although treatment failure rates of up to 20% have been reported (Davidson,  10  1984).  1.5  Pathology of Infection Clinical and pathological features of giardiasis vary. A spectrum of morphological  changes in mucosal architecture (by both electron and light microscopy) has been reported (Smith, 1985; Hartong et al., 1979). Examination of the proximal small intestinal mucosa by light microscopy frequently reveals normal histology despite documented infection (Farthing, 1992; Smith, 1985). Scanning and transmission electron micrographs suggest that trophozoites preferentially localize in "sheltered" areas (away from the flow of intestinal contents) such as crevices of the small intestinal villi (Khanna et al, 1988, Owen etal, 1979). The degree of pathology ranges from normal, to subtotal villous atrophy (Duncombe et al, 1978), to total absence of villi (Ferguson et al, 1980). When symptoms occur, it has been shown that severity correlates with the degree of damage in the small intestine (Khanna et al, 1988; Wright et al, 1977; Duncombe et al, 1978). During the course of infection, the jejunum may undergo villous atrophy and crypt hyperplasia (Buret et al, 1992, 1990; Duncombe et al, 1978). As infection progresses, villous atrophy may also be seen in the ileum (Gillon et al, 1982). Villous atrophy, has been assumed to be a major factor in the loss of the intestinal absorptive epithelial surface (Wright and Tomkins, 1977) and therefore a cause of diarrhea. Villous atrophy does not account for all of the disease since diarrhea can occur in its absence (Buret et al, 1992; Duncombe et al, 1978).  11  1.6  Pathophysiology of Infection Despite the high prevalence of giardiasis, the pathogenesis and the pathophysiology  of disease are not understood (Buret, 1994). Several disease-producing factors have been proposed. Aside from the mechanisms related to mucosal injury (described below), luminal pathogenetic mechanisms have been proposed. Bacterial overgrowth resulting in bile salt deconjugation and fat malabsorption (Tomkins et al., 1978) is one example of this. The presence of bile salts in the small intestine is known to promote trophozoite growth (Gillin et al., 1986; Keister, 1983) and therefore may be responsible for the trophism of Giardia for the small intestine. Another proposal has been that depletion of the bile salt pool by the parasite, particularly in chronic infections, could result in fat malabsorption by impairment of micellar solubilization of ingested fat, with reduction of the effectiveness of pancreatic lipases (Katelaris and Farthing, 1992). In addition, the decrease or absence of luminal enzymes from pancreatic exocrine secretion, such as trypsin and lipase (observed in some cases of giardiasis) could be related to the fact that sonicates of Giardia trophozoites and live trophozoites inhibit activity of these enzymes in vitro. Given the large pancreatic functional reserve of these enzymes, this mechanism, however, is unlikely to play a major part in disease (Seow et al., 1993; Katelaris et ah, 1991). The mechanisms of disease production in giardiasis are probably multifactorial (Katelaris and Farthing, 1992; Smith, 1985). Host and parasite factors are also both likely to be important. Possible host factors involved include the immune responses (peripheral and cell-mediated).  Possible parasite factors involved include direct mechanical damage  caused by trophozoite ventral discs (Wright, 1979) or damage by trophozoite toxic  12  products. Any proposed models of pathogenesis must be consistent not only with the wide range of clinical presentations (Farthing, 1993), but also with the role of both host and parasite. It is possible that there is a cascade of abnormalities that occurs together and impairs the intestinal absorptive ability of the host (Katelaris and Farthing, 1992). Many reports (Katelaris and Farthing, 1992; Buret et al., 1990; Hartong et al., 1979; Hoskins et al., 1967) suggest that the mucosal injury associated with the presence of Giardia trophozoites is the prime abnormality resulting in the main symptom of giardiasis, diarrhea. Recent attention has focused on functional and morphological damage to small bowel enterocytes (Katelaris and Farthing, 1992). Host factors that may contribute to the disease process include the following. Studies have found that patients with hypogammaglobuhnaemia have higher parasite loads and sustain more severe mucosal damage than do individuals with normal immune systems (Khanna et ah, 1988; Hartong et al., 1979). The reason for this is not known. It has also been observed that the presence of a protease capable of activating a mannose-binding lectin, is present on the surface of Giardia trophozoites.  This lectin may promote  attachment of the parasite to enterocytes (Lev et al, 1986), localizing trophozoites in the small intestine prior to mechanical attachment by its ventral disc (Buret, 1994; Katelaris and Farltiing, 1992). There is also growing evidence to support the concept that intestinal Tcells, possibly activated as a result of this lectin stimulation, may play a role in the pathogenesis of villous atrophy in human enterocytes in vitro (MacDonald and Spencer, 1988). Experiments in T-cell depleted (athymic) mice infected with G. muris have shown that when T-cells are added in vivo, intestinal pathology is significantly worsened (Roberts-  13  Thompson and Mitchell, 1978). Parasite factors are also undoubtedly important in the pathogenesis of disease. It is possible that these factors play an important role in initiating a cascade or a number of different pathophysiological events. Diarrhea may be due to two basic pathophysiological mechanisms, impaired intestinal absorption or enhanced secretion (Clark and Sears, 1996). It is possible that both pathophysiological mechanisms occur together in some disorders. Deficiency of disaccharidases (particularly lactase) due to brush border damage, may lead to an "osmotic" type of diarrhea after the ingestion of milk or milk products (Ferguson et al., 1980). This is an example of diarrhea secondary to impaired absorption. Alteration of the enterocyte sodium transport system may result in net secretion of both sodium and water, i.e. a secretory diarrhea (Ferguson et al., 1980). This is an example of diarrhea secondary to enhanced secretion. Both pathophysiological mechanisms may contribute to the diarrhea in giardiasis. In this project, we have chosen to focus on brush border damage. The intestinal brush border is situated at the luminal pole of intestinal columnar epithelial cells or enterocytes (Ferguson et ah, 1980) and is a functional organelle. It is the location of various disaccharidases and for the sodium dependent transport of glucose and galactose.  In fact, over 22 enzymes and 19 transport or binding functions have been  localized to the brush border (Holmes and Lobley, 1989).  It consists structurally of  numerous, finger-like apical projections or microvilli, which cover a transverse fibrillar meshwork or the terrninal web (Holmes and Lobley, 1989).  Ultras tructurally and  functionally, the brush border may be considered as two different substructures, the surface (microvillous) membrane and the underlying brush border cytoskeleton. Each has its own  14  unique molecular composition and function (Lentze, 1995; Holmes and Lobley, 1989). The structural organization of the brush border is important to understanding disaccharidase functions.  The microvillous membrane comprises a lipid bilayer, the  external (luminal) face of which is covered by a carbohydrate-rich 'fuzzy coat' or glycocalyx (Ito, 1969). Brush border enzymes, glycoproteins with globular structures, are found attached to the external surface of this membrane by a small (2-5 kDa) anchoring segment (Holmes and Lobley, 1989) embedded in the lipid bilayer (Semenza, 1986). The cytoskeleton, consisting of the microvillous core and the terminal web, serves as a support for the architecture of the entire apical pole of the enterocyte. It may play an active role in regulating the uptake of some nutrients into the cell and in control of paracellular permeability (Holmes and Lobley, 1989). Hydrolysis of macronutrients in the gastrointestinal tract is an essential prerequisite for all mammals (Lentze, 1995). The primary functions of the intestinal brush border relating to terminal hydrolysis and absorption of nutrients (Mohammed and Faubert, 1995 b; Holmes and Lobley, 1989) requires specific enzymes such as disaccharidases and peptidases. Maldigestion and malabsorption of carbohydrates occurs following mucosal damage or dysfunction of the brush border.  Primary alteration in either structural or  functional organization of the brush border membrane may be due to either a congenital or acquired absence or inactivity of digestive enzymes. This is thought to result in impaired digestion and absorption of carbohydrates (Holmes and Lobley, 1989; Tietz et al., 1986). The congenital (primary) disorder manifests as either as a low or an absence in enzyme (particularly lactase) activity at birth. Some congenital disaccharidase level disorders are a  15  result of an inherited autosomal recessive trait as is the case in glucose-galactose malabsorption (Tietz et al, 1986). Pathophysiological changes in brush border function are responsible for acquired lactase deficiency (Tietz et al., 1986). Secondary malabsorption, a common finding in infections such as giardiasis, is considered to be caused by structural or functional damage to the brush border membrane with subsequent reductions in disaccharidase levels (Holmes and Lobley, 1989; Tietz et al., 1986).  Reduction in  disaccharidase activities are maximal when diarrhea and villous morphological abnormalities are most pronounced (Parthing, 1993; Duncombe et al,  1978).  This area of small  intestinal pathophysiology, however, still requires clarification. Symptoms of diarrhea in giardiasis may be due to the osmotic effects of higher than normal amounts of undigested oligosaccharides in the small intestine. It has been shown that the severity of symptoms relates to the degree of malabsorption of these undigested oligosaccharides which in turn relates to the degree of brush border damage or dysfunction (Tietz et al, 1986). While it is now clear that trophozoites are not invasive (Parthing, 1993; Chavez et al, 1986; Duncombe et al, 1978), brush border microvilli appear to be damaged where trophozoites attach.  Severe flattening and blunting of microvilli with the loss of  intracellular organellar organization of columnar cells has also been observed (Vinayak and Naik, 1992). It is important to note that this shortening of the brush border microvilli and loss of the brush border surface area is a diffuse lesion, appearing in areas removed from trophozoite attachment as well as at sites of attachment (Buret et al, 1992). While it has been suggested that the disaccharidase deficiency may be due to damage caused by the parasite's ventral disc (Gillon et al, 1982), this seems unlikely however, since the  16  relatively mild insult to the surface epithelium by the ventral suction disc is not sufficient enough to account for the observed diffuse decrease in brush border height (Katelaris and Farthing, 1992).  1.7  Phenotypic and Genotypic Parasite Variation Phenotypic and genotypic variation in the G. duodenalis group has been well  described (Thompson and Meloni, 1993). A variety of biochemical methods have been used to distinguish between isolates of the duodenalis groups. Methods include iso-enzyme analysis (Andrews et ah, 1989; Meloni et al., 1988), restriction fragment length polymorphism analysis (Meloni et al., 1989; Nash et al., 1985), chromosome pattern analysis (Sarafis and Isaac-Renton, 1993; Upcroft et ah, 1989; Adam et al., 1988), and eridonuclease mapping (Nash et ah, 1985). These studies indicate that morphologically indistinguishable Giardia isolates may vary biochemically, antigenically and genetically (Andrews et al., 1989). Despite these observations, no parasite virulence markers have been found and no correlation between biochemical-antigenic patterns and clinical presentation has been made. Several studies describe a group of cysteine-rich proteins variant surface proteins (VSPs) present on the surface of trophozoites which exhibit antigenic variation (Aggarwal et al, 1989; Nash, 1989; Adam et al, 1988; Nash et al, 1988).  Giardia trophozoites  undergo surface antigenic variation in in vitro cultures, in humans, and in animal models (Nash, 1992; Udezulu et al, 1992; Aggarwal and Nash, 1988).  Change in  antigenic composition has been shown to occur extremely frequently (Nash et al., 1990  17  b) although different isolates have been shown to vary in the rate of antigenic variation (Nash et al, 1990 b), the proportion of trophozoites within any population that expresses a particular antigen (Nash et al., 1990 c; Pimenta et ah, 1991), as well as the range of antigen epitopes the trophozoites express (Nash et al., 1990 a; Nash et al., 1988). G. duodenalis produces multiple proteases or proteinases (Williams and Coombs, 1995). Many of these differ in substrate preferences. The major proteases of Giardia trophozoites are of the cysteine type based on the fact that these enzymes are inhibited by certain reagents, iodoacetamide (which are known to inhibit cysteine type proteases), for example (Hare et al., 1989). The large numbers of proteolytic activities and substrate specificities of these proteases, indicate that the enzymes are probably involved in many different aspects of trophozoite development, pathogenesis or metabolism (Williams and Coombs, 1995). Parasite excretory-secretory products have been characterized and are probably the VSPs (Nash et al., 1983) noted above. Furthermore, Giardia isolates were observed to release these excretory-secretory (E-S) products into the surrounding culture medium (Nash et al., 1983).  Two basic types of microbial metabolites, primary and  secondary, have been described (Madigan et ah, 1997). Primary metabolites are produced during the exponential growth phase of micro-organisms, more or less simultaneously with micro-organism and are generally similar in all cells (Madigan et al., 1997). Secondary metabolites are produced near the end of the exponential growth phase, frequently at or near the stationary phase (Madigan et al,  1997).  Secondary  metabolites are also more frequently produced when organisms are propagated in vitro,  18  production occurring after cells and primary metabolites are formed by either conversion of primary metabolite to secondary metabolite, or further growth substrate to secondary metabolite (Madigan et al,  1997).  Alcohol is a typical primary  metabolite formed during the fermentation process of yeast and certain bacteria. Lactamases are examples of secondary metabolites produced during the stationary growth phase of Streptomyces.  It is reasonable to consider that E-S products are  secondary trophozoite metabolites. It is possible that these metabolites cause damage to the small intestine either directly by a cytopathologic effect or indirectly by inducing a host immune response. In summary, there appears to be a number of pathophysiological and pathogenic factors that may be considered as important. They may be complementary or independent, acting in a multifactorial process at biochemical, immunological and genetic levels (Figure 4). We propose that the pathology of giardiasis is associated with the E-S products acting directly or indirectly on the small intestine brush border. Mucosal injury, most severe in the upper small intestine, is characterized by a diffuse shortening of epithelial microvilli (occurring independently of any alteration in height of mucosal villi) with loss of brush border surface area as shown in Figure 4. Previous studies have reported that culturefiltratesof Giardia trophozoites damaged fibroblasts cultured in vitro (Radulescu et al, 1980). This observation is consistent with the action of a trophozoite toxin or toxic E-S parasite product. This project focused on possible consequences of parasite E-S products on the activity of a brush border enzyme, maltase, using a Caco-2 cell monolayer model.  19  To study the pathogenesis of giardiasis, it is necessary to identify well-characterized parasite isolates that may differ in their ability to produce damage under controlled experimental conditions (Cevallos et al., 1995). Differences in virulence of G. duodenalis isolates have previously been characterized by infectivity rates, differences between Giardia isolates' innate ability to infect a new host (Visvesvara et al., 1988), or maximal parasite load in the intestine (Aggarwal et al., 1983).  20  Figure 4: Summary of proposed mechanisms involved in the production of diarrhea in giardiasis due to excretorysecretory products of Giardia trophozoites  GIARDIA COLONIZATION (small intestine of host)  EXCRETORY-SECRETORY PRODUCTS  INDIRECT DAMAGE  DIRECT DAMAGE  VILLOUS ATROPHY eg. Decrease in absorptive epithelial  \  MICROVILLOUS INJURY eg. Diffuse injury to epithelial brush border  INCREASED SECRETION eg. N a +  HOST IMMUNE RESPONSES TO STRUCTURAL ANTIGENS  i DISACCHARIDASE DEFICIENCY^  DIARRHEA  T CELLS cytotoxic cells  21  B CELLS lymphokines  OTHERS Mast cells  1.8  Models of Infection Various experimental models simulating human giardiasis have been developed to  assess the duration of infection, the severity of infection, and other aspects of the pathogenesis of disease. Both in vivo (humans and animal, Buret et al., 1991) and in vitro models (Favennec et al., 1991, 1990; Duncombe et al., 1978) have been used to describe enterocyte enzyme levels in giardiasis.  The following is an overview important to  understanding the rationale for the experimental model chosen for these experiments.  a)  Animal Models The small intestinal pathophysiology in giardiasis has been studied using two animal  models, mice (outbred female Swiss albino, CF-1) and Mongolian gerbils (Meriones unguiculatus). Reproducible patterns of pathology with intestinal villous atrophy and impairment of weight gain were described using the mouse model (Robert-Thomson et al., 1976).  G. maris cysts were used in these experiments.  Mongolian gerbils (Meriones  unguiculatus) may be infected with G. duodenalis cysts obtained from humans (Buret et al., 1991; Belosevic et al., 1989, 1983). Experiments using Meriones infected with humansource G. duodenalis have shown that the duration of infection and the pattern of cyst excretion are similar to that found in human infections.  Most studies relating to  pathogenesis, however, have used the murine model. Mucosal abnormalities in murine giardiasis have been quantitated by microscopic examination of mucosal architecture (villous heights, crypt depths and cell proliferation in the crypts). Brush border disaccharidase enzymes were measured and abnormalities were  22  found after infection with Giardia cysts (Buret et al, 1990; Gillon et al., 1982; Ferguson et al., 1980), infection with Giardia trophozoites (Buret et al., 1991; Khanna et al., 1988) Or infection with soluble extracts of trophozoites (Daniels and Belosevic, 1992).  It was  observed that enzyme activities decreased as the parasite load increased. This is consistent with the idea that reduction of brush border activity was associated with the presence of the parasite (Khanna et al., 1988; Gillon et al., 1982; Ferguson et al., 1980).  In other  experiments, mice infected with G. duodenalis showed more severe brush-border enzyme deficiencies when immunosuppressed (Khanna et al., 1988), demonstrating that both host and parasite factors are involved in producing disaccharidase deficiencies. We chose not to use an animal model for these experiments for several reasons including the inherent biological variation well known to occur in such systems.  b)  Cell Monolayers The human colon carcinoma celltines,Caco-2 and HT-29, have gained attention in  recent years as models for studies of intestinal cell function (Rousset, 1986). The Caco-2 cell line is the most widely used model since these cells spontaneously express a high degree of differentiation under standard culture conditions (glucose plus serum) in the absence of specific inducers of differentiation (Rousset, 1986; Pinto et al., 1983). This is in contrast to HT-29 celltinewhich does not differentiate in the absence of specific inducers. Most other cultured celltinesoriginating from normal small intestine and colon have been shown to be unreliable in their ability to differentiate into functional intestinal cells (Quaroni et al., 1979).  23  The time required for differentiation of Caco-2 cells is more rapid compared to that required by that of the FfT-29 cell line. Onset of Caco-2 cell differentiation usually starts on day 7 and is finished by day 20 (Pinto et al., 1983).  This differentiation time  (exponentially dividing crypt cells are undifferentiated and differentiation is defined by cessation of division during crypt to villous migration) mimics that found in the human small intestine (Rousset, 1986; Pinto et al., 1983). Despite the fact that the Caco-2 cell Une was originally derived from malignant large bowel cells, a number of investigations (Hauri et al., 1985; Rousset et al., 1985; Pinto et al., 1982) have shown that these cells exhibit small intestinal enterocyte functions. Caco-2 cells have been used for drug screening assays (Favennec et al., 1992) and intestinal epithelial permeability studies (Hidalgo et al., 1989). G. duodenalis trophozoite adherence on Caco-2 cells (Katelaris et ah, 1995; Magne et ah, 1991; Favennec et al., 1990) has been described. Observations regarding a possible cytopathogenic effect of trophozoites (Katelaris et ah, 1995; Favennec et al., 1992; Favennec et al., 1991; Favennec et ah, 1990) have also recently been reported in a Caco-2 model. In one study (Favennec et al,  1991) Giardia trophozoites produced a direct  cytopathogenic effect on Caco-2 cells with subsequent impairment of disaccharidase activities. It was also reported in this brief communication that cell damage varied with the strain and the number of trophozoites used (Favennec et al., 1991).  Caco-2 levels of  saccharase (sucrose-isomaltase) and alkaline phosphatase apparently decreased following exposure to trophozoites, but there was no effect obseved on the lactase level. Follow-up of these preliminary results were never published and significant experimental details are  24  lacking from this intriguing study.  1.9  Preliminary Disaccharidase Experiments  Previous work carried out in this laboratory (Anna Li and Dr. C.S. Ong) included assays of disaccharidase (sucrase, lactase and maltase) levels from the small intestine of Mongolian gerbils infected with G. duodenalis. In these experiments, gerbils were sacrificed at various time intervals post-inoculation with G. duodenalis trophozoites. The small intestine of infected and control gerbils were removed, the mucosa scraped, homogenized, centrifuged and the resulting supernatant assayed for disaccharidase activity. The highest disaccharidase level assayed was maltase. Further experiments were carried out to determine which disaccharidase (maltase, lactase and sucrase) was expressed in the highest amounts in a confluent monolayer of Caco-2 cells. Since maltase levels were the highest of the three enzymes (data not shown) tested, we chose to assay for maltase activity in all subsequent experiments.  25  CHAPTER 2 HYPOTJHESIS A N D OBJECTIVES  2.1 Hypothesis The hypothesis of this project is that, under some circumstances, trophozoites of Giardia duodenalis produce excretory-secretory (E-S) products that damage the small intestinal microvilli. This damage is marked by a decrease in levels of associated brush border disaccharidases. This damage, or a cascade of pathological events starting with this damage, would theoretically produce or contribute to the major clinical symptom of giardiasis, watery diarrhea.  2.2 Objectives While the overall goal of this thesis is to understand the pathogenesis of giardiasis, the specific aims are: a) to compare growth rates of G. duodenalis isolates, b) to develop an experimental model that would provide a reproducible assay of the pathological effect of E-S parasite products, c) to test the effects of E-S products of in vitro grown Giardia trophozoites.  26  CHAPTER 3 MATERIALS AND METHODS  3.1  Trophozoite In Vitro Growth Studies Seven Giardia isolates retrieved from asymptomatic and symptomatic patients were  selected for growth experiments (Table 1). Trophozoites retrieved from cryopreservation (Isaac-Renton et al 1993) were sub-cultured in 13 X 125 mm screw-cap borosilicate testtubes. Approximately 15 ml of filter-sterilized TYI-S-33 medium (Keister, 1983) was used in each culture tube. Trophozoite cultures were maintained at 37°C and examined daily by light microscopy (X100, Nikon TMS Type 104) until sufficient numbers of trophozoites were obtained.  Growth was maintained until as many confluent (maximal trophozoite  adherence to walls of tubes) culture tubes as necessary for each growth experiment (one set for each of the seven isolates, each set consisting of 3 tubes) were available. To obtain the required inoculum of trophozoites for each experiment, organisms were dislodged from test tube walls by chilling on ice for 15 min. Tubes were inverted 3 times and centrifuged at 1400 X g for 10 min (DEC Centra 8R). Pooled pellets of trophozoites were enumerated using an electronic cell counter (Haematology Series Cell Counter Model 13; Baker Instruments Corp., Allentown, PA.), the required inoculum calculated and the appropriate volume of trophozoites resuspended in fresh TYI-S-33 medium to form the stock suspension. Seven sets of 3 tubes (one set for each isolate tested), each with 15 ml of TYI-S-33 culture medium were inoculated with 0.5 ml of the stock suspension to give a final  27  trophozoite concentration of 5 X 10 /ml. All cultures were incubated at 37°C for 7 days 4  and examined daily by light microscopy. At 24 hour intervals, the characteristics (adherent abilities of trophozoites, duration of monolayer formation and clumping) of the three tubes per isolate were noted, and then trophozoites from each tube were harvested as described above. Trophozoites in each pellet from respective tubes were resuspended in a known volume and counted in the same manner. Growth curves were then plotted (number of trophozoites/ml at each time interval). All growth experiments were done in duplicate for each isolate.  3.2  Preparation of Filtrates Obtained from Trophozoites Grown In Vitro The same Giardia strains used in the growth studies (Table 1), were retrieved from  the same cryopreserved stock and used to prepare parasite filtrates containing (possible excretory-secretory products). Trophozoites were retrieved from -135°C aliquots, DMSO washed off and the pellet inoculated into fresh TYI-S-33 broth medium.  Following  retrieval, an aliquot from each culture tube was also inoculated into brain-heart infusion broth (BHI) and onto sheep blood agar plates (BAP) for incubation at 37°C (3 days) to rule out fungal or bacterial contamination.  Tubes with freshly retrieved trophozoites were  incubated for an hour at 37°C on an incline of 45° then non-attached trophozoites discarded with the growth medium and new TYI-S-33 medium added.  Culture tubes were re-  incubated at 37°C for at least one week, with daily examination of tubes, until sufficient numbers of confluent tubes forfiltrateexperiments were available . Cultures, at late log growth phase (actively growing trophozoites) were used for filtrate preparation as follows.  28  Trophozoites were concentrated and counted as described previously to give a final concentration of 2xl0 /ml in the stock tube. Fresh culture tubes of 15 ml of TYI-S-33 8  medium were then inoculated with 0.5 ml of the stock suspension to give a final concentration of 1 X 10 trophozoites/ tube (test culture filtrates). Uninoculated, control 8  tubes of TYI-S-33 were also set up and all tubes (test and control) were incubated at 37°C for 24 hours. Test and control tubes were chilled on ice for 15 min and centrifuged at 1400 X g for 10 min. Supematants of the spent medium from both test and control culture tubes were withdrawn aseptically,filteredseparately through low protein-binding 0.22 um sterile disposable filters (Millex-GV, Millipore, Bedford, MA.) and stored at -70°C until assayed.  3.3  Caco Cell Culture Growth and Maintenance 2  Caco-2 cells (BF strain), originally established from a moderately well differentiated colon adenocarcinoma by Pinto et al. (1983) were kindly provided by Dr. B. Finlay (Biotechnology Laboratory and Departments of Microbiology and Biochemistry, University of British Columbia). After retrieval from cryopreservation, cells were grown in 25 cm tissue culture flasks.  3  Filter-sterilized, supplemented/complete cell culture medium  (CMEM) containing minimal essential medium (MEM, Gibco, Burlington, Ont., tt 4101500EF), 10% (v/v) of heat-inactivated (56°C, 30 min) fetal bovine serum and 1% antibiotic/antimycotic mix containing 10,000 units/ml penicillin G, 10,000 ug/ml streptomycin and 25 ug/ml amphotericin B (Gibco, Burlington, Ont.) was placed aseptically  29  into each flask.  If MEM was over one month old, a supplement of 1% L-glutamine  (Gibco, Burlington, Ont.) was added. Once cells were adapted to in vitro growth and had attained a monolayer status (around day 7) as determined by inverted microscopy (Nikon Diaphot), they were transferred to 150 cm tissue culture flasks. Cells were harvested from flasks by removal of spent medium, rinsing with Hank's balanced salts solution (HBSS, Gibco, Burlington, Ont.) and addition of 0.25% trypsin in 0.53 mM EDTA (Gibco, Burlington, Ont.) for 10 min. All cell cultures were maintained at 37°C in a 10% C 0 atmosphere, for 7 days with 2  medium changes every 3 days. All culture flasks were examined daily for evidence of fungal contamination. On day 7, cells were divided and subcultured (1:1) between 2 flasks to increase the total number of cells. Caco-2 cell passage numbers were kept between 2140 throughout all experiments.  3.4  Co-Incubation Experiments: Caco-2 and Giardia Trophozoite Filtrates  a)  Serial Inoculation of Caco-2 Cell Monolayer with Filtrates Serial inoculation experiments were based on previous work carried out in this  laboratory (personal communications, Dr. C. S. Ong, Dept. of Pathology and Laboratory Medicine, University of British Columbia). For each experiment, 1 X 10 Caco-2 cells 5  were initially seeded into 150 cm tissue culture flasks and cultured for 7 days (37°C, 5% 3  CO2) until the necessary number of confluent flasks were available. When the appropriate number of Caco-2 cells were grown, all tissue culture flasks were inoculated from the same stock suspension of Caco-2 cells.  On day 7 post-seeding, spent CMEM from all  30  tissue cultureflaskswas replaced with either fresh CMEM only (growth controlflasks),or equivolume mixtures of CMEM plus WB test filtrate, or CMEM plus TYI control filtrate. All flasks were incubated (37°C, 5% C0 ) for a further 5 days with appropriate 2  media (CMEM/test, CMEM/TYT control, CMEM only) changes every 2 days. On day 12, an aliquot of media from each culture flask used was inoculated onto BAP and the plate incubated (37°C) to rule but bacterial or fungal contamination.  Cells from flasks with  similar media conditions were harvested with 0.25% trypsin in 0.53 mM EDTA, the cells pooled and a trypan blue exclusion test for cell viability carried out. Cells were washed 3 times with sterile ice cold saline and enumerated using an electronic cell counter after centrifugation (4°C) at 450 X g for 10 min. The appropriate dilution to give 1 X 10  7  cells/ml in 2.5 mM EDTA was made. Aliquots of cells were stored at -70°C until analysis. Both the disaccharidase assay and Lowry protein assay were carried out on Caco-2 cell sonicates originally co-incubated with either CMEM/test, CMEM/TYT or CMEM (as described later). All samples were tested in triplicate for both disaccharidase and Lowry assays. Two separate experiments were carried out.  b)  Single Inoculation of Caco-2 Cell Monolayer With Filtrates In these experiments, 1 X 10 Caco-2 cells were seeded into each 150 cm tissue 5  3  cultureflaskand cultured (37°C, 5% CO2) as described above until the necessary number of confluent flasks were obtained. Once sufficient cell numbers were available, the required number of new flasks were seeded from the same cell suspension. On either day 7 or 10 post-seeding, spent CMEM from eachtissueculture flask was replaced with either fresh  31  CMEM only (growth control flasks), or equivolume mixtures of either CMEM plus test filtrate, or CMEM plus TYI control filtrates. Cells were incubated (37°C, 5% CO2) for 48 hours then an aliquot from each flask tested for bacterial or fungal contamination as described. Cells were harvested and frozen (-70°C) until analysis. Both the disaccharidase and Lowry assays were carried out in triplicate on Caco-2 sonicated cells following inoculation with either each a filtrate or a control. A 48 hour co-incubation experiment comparing maltase activity and protein concentration of a day 7 and a day 10 Caco-2 cell monolayer (using the same batch of test and control filtrates) was also carried out. In this experiment, a large number of Caco-2 cells were cultured to allow for co-incubation of filtrates and growth control (CMEM) on both a day 7 and a day 10 monolayer.  All tissue culture flasks were  inoculated from the same stock of Caco-2 cells. On day 7 the Caco-2 monolayer was co-incubated with filtrates, harvested 48 hours later (as described above) and the cells frozen until sonication and testing by the disaccharidase and Lowry assays. Three days later, the remaining flasks were co-incubated with the same batch of filtrates for 48 hours, the Caco-2 cells harvested (as above), and frozen (-70°C) until assayed. Disaccharidase and Lowry assays for each culture filtrate and controls (sonicated preparations) were carried out in triplicate. Cells harvestedfromboth the day 7 and day 10 experiments were assayed at the same time.  32  3.5  Measurement of Caco-2 Cell Monolayer Disaccharidase Levels  a)  Reagents Maltose, maltase, glucose oxidase, peroxidase, o-dianisidine and Triton X-100 were  obtained from Sigma (Mississauga, Ontario), Trizma base from Bio Rad Labs (Canada) and glucose was obtained from Fisher (Canada). Sodium maleate buffer was prepared by adding 1.16 g maleic acid (BDH, Canada) to 17 ml sodium hydroxide (1 M) and making the volume up to 100 ml with distilled water and pH adjusted to 6.4. The disaccharidase substrate (maltose, 200 mg) was made up at 0.056 M concentration in sodium maleate buffer (10 ml). The TRIS-glucose oxidase (TGO) reagent was prepared by adding 0.3 ml glucose oxidase, 0.5 ml of 1 mg/ml peroxidase, 0.5 ml of O-dianisidine solution (100 mg O-dianisidine to 10 ml distilled water) and 1 ml of detergent solution (10 ml Triton X-100 in 40 ml of 95% ethanol) to 100 ml of 0.5 M (pH 7.0) TRIS buffer (61 g Trizma base, 85 ml 5 M HC1, made up to 1000 ml with distilled water).  b)  Maltase Control: Assay Optimization A test control for the disaccharidase assay was developed. In these experiments, a  series of concentrations of maltase (Sigma, Mississauga, Ontario) were tested to detennine an optimal concentration at which optical density readings fell within the limits of the glucose standard curve. In all subsequent disaccharidase assays a dilution of 1 in 200 of the control maltase in distilled water was used as the concentration of the test control. Graphic records of both control maltase activity as well as maltase concentration were recorded and used to determine interassay variation (data not shown).  33  c)  Disaccharidase Assay Prehminary experiments in this study investigated various methods of Caco-2 cell  disruption.  Methods investigated included freeze-thaws (2X, methanol/dry ice  mixture), ultrasonication (3 pulses of 5 sec each on ice), and homogenisation (batterypowered tissue grinder, 3 min on ice) of harvested Caco-2 cells (data not shown). It was found that ultrasonication released more Caco-2 maltase than the other two methods and therefore, this method was used to disrupt cells in all further experiments. Maltase activity was thus determined after a partial purification of brush border membranes. At thetimeof maltase analysis, cell suspensions were thawed, ultrasonicated on ice by 3 pulses of 5 sec each of ultrasonic treatment (Ultrasonic Processor, 600 watts model dual output, John's Scientific Inc. Canada) with 30 sec intervals using a probe with an output of 20 kHz. The sonicate was centrifuged (20 min, 4°C, 950 X g in a microfuge (Eppendorf 5415)) and the supernatant containing the brush border membranes collected. Maltase activity was determined using the glucose oxidase assay method (Dahlqvist, 1964). The assays were carried out in duplicate. A glucose standard series (25, 50, 75 and 100 /xg glucose), reagent blanks and maltase control (1 in 200 dilution, see above) were run in parallel. The assay .was performed by adding 0.1 ul of appropriately diluted supernatant sample to 0.1 pi of disaccharidase substrate buffer and incubated for 30 min in a shaking water bath at 37°C.  Samples were diluted in distilled water. Following incubation, the  enzymic reaction was stopped by immediately immersing tubes in a boiling water bath (2 min).  Tubes of blanks with the same composition were set up and were immersed in  boiling water for 2 min, immediately after mixing enzyme with substrate tubes. TGO  34  reagent (3 ml) was added to all tubes (blanks and test) and incubated for a further hour at 37°C in the shaking water bath. The addition of TGO stops the production of glucose and forms a coloured product depending upon the amount of glucose present.  The resultant  product was measured at 420 nm against a substrate-free blank, in a spectrophotometer (UV-160 A recording spectrophotometer, Shimadzu Corp. Kyoto, Japan). Protein content of the sonicated cell supernatant (10 Caco-2 cells/ml) was assayed by the method of Lowry 7  et al. (1951). Activities of maltase (from 10 Caco-2 cells/ml) were thus expressed as 7  enzyme units per gram of total cellular proteins (specific activity) and this quantity was calculated from the following formula:  OD sample OD standard  n  X  concentration of X standard  10 3  On  1000  = no. of moles glucose produced/ mole substrate consumed (maltose n=2)  10 = 1/0.1 ml, correction for volume of sample added 180= molecular weight of glucose p  180  X  =totalprotein concentration (mg/ml)  df = dilution factor  35  3.6  Statistical Analysis All values (cell monolayer protein content, maltase units and specific maltase  activity) are expressed as mean values± SD. Means were compared by one-way analysis of variance (ANOVA, Statistical Analysis System, SAS 1994), except for the growth curves which are graphic plots of means values. All data represent a minimum of n=3 for each data point. Differences were considered significant at a level of P< 0.05.  36  Table 1.  Summary of the characteristics of G. duodenalis isolates used in Caco-2 and growth rate experiments.  Strain Designation  Lab Name*  Geographic Origin  Host  Clinical Presentation  ATCC 30957/11  WB  Afghanistan  human  diarrhea  Vanc/96/UBC/128  BH  Canada  human  diarrhea  Vanc/90/UBC/43  WH  Canada  human  diarrhea  Vanc/93/UBC/70  OV  Canada  human  asymptomatic  Vanc/87/UBC/8  L  Canada  human  asymptomatic  Vanc/89/UBC/59*  Woof  Canada  dog  diarrhea  Vanc/90/UBC/42*  Jab  Canada  human  diarrhea  ^designations used in this report * not tested in Caco-2 assay  37  Table 2.  Summary of experiments carried out.  Type of I'AperinK'iit  Number of Number uf n?l>lica<cs/c\periiiicnt experiments  Isolates  Growth Curves  3  2  Interassay Variation  2  6  WB/BH/WH/OV/L/ Woof/Jab Not applicable  Serial Inoculation  3  2  WB  Single Inoculation a) Day 7  3  3  WB  3  2  L/WH  3  2  WB  b) Day 10  38  CHAPTER 4 R E S U L T S  4.1  Growth Studies The isolates tested in these experiments showed variability in their ability to  establish and grow in culture following retrieval from cry©preservation.  Differences  were demonstrated in growth rates as well as in the less quantifiable observations such as trophozoite ability to adhere to culture tube walls, duration of monolayer formation, the speed which the parasite monolayer is formed as well as how fast trophozoites clump. A starting concentration of 0.5 X 10 Giardia trophozoites per ml was sufficient 5  to initiate cultures of all isolates (WB, Jab, L and Woof) except BH, WH and OV. These three isolates repeatedly required inoculation with a 10-fold increased concentration from the stock tube before the trophozoites began to divide in TYI culture medium. Growth curves are represented graphically in Figure 5.  It appears that the  isolates fall into two groups based on the observed curves. The slower growing group (BH, Jab and Woof) have a less pronounced exponential phase of growth.  The  remaining isolates (WH, WB, OV and L) have an exponential phase with a steeper slope (Figure 5).  39  4.2  Co-Incubation Experiments: Caco-2 Cell Monolayer Model At the end of all co-incubation experiments, Caco-2 cell monolayers remained  confluent. The cells appeared intact with normal morphology when examined by light microscopy.  Caco-2 cultures were characterized by a monolayer consisting of  polygonal cells with well-defined boundaries (Figure 6) as well as randomly distributed domes. Domes, which are comprised of cells growing over each other, were observed to increase both in number and size as the cell layer matured. The activity of Caco-2 disaccharidases has been reported previously (Jumarie and Malo, 1991; Favennec et ah, 1990; Pinto et al., 1983) as units per gram of brush border proteins (termed specific activity). Since it was noted in the present study that changes in protein concentration may mask enzyme catalytic activity, and since specific activity depends on both maltase activity and protein concentration, we reported, separately, maltase activity, protein concentration and the specific maltase activity.  a)  Serial Inoculation of Caco-2 Cell Monolayer with Trophozoite Filtrates Caco-2 cells were observed to be between 85-95% viable following co-  incubation with Giardia filtrates as indicated by the trypan blue-exclusion test. There was no difference between the specific activities of 7 day old Caco-2 monolayers coincubated serially with WB filtrates compared to those incubated with TYI control filtrates (harvested on day 12). There was however, a significant difference (P<0.05, ANOVA) between specific activity of the Caco-2 growth control cells (CMEM) and the Caco-2 cells co-incubated serially with either WB or TYI filtrate (Figure 7).  40  Maltase activity after co-incubation with either WB or TYI control filtrates, showed a significant decrease (/ <0.05, ANOVA) when compared to Caco-2-CMEM >  maltase activity (Figure 8) and when compared to each other. As seen in Figure 9, it was also observed that the protein concentration of Caco-2 cells co-incubated with WB filtrate was significantly lower (P<0.05, ANOVA) than for either TYI control or Caco-2-CMEM growth control (which were similar to one another).  b)  Single Inoculation of Caco-2 Cell Monolayer with Trophozoite Filtrates The previous experiment (serial inoculation of Caco-2 with filtrates) was  modified and repeated to determine how quickly the previously observed decline in maltase activity and protein concentration occurred. Instead of two consecutive coincubations of filtrates on a 7 day Caco-2 monolayer (section a), one 48 hour coincubation was carried out also on a 7 day old monolayer. No difference in specific activities between Caco-2 cells co-incubated with WB or TYI filtrates and Caco-2CMEM control (Figure 10) was observed. Maltase activity, however, was observed to be significantly reduced (P<0.05, ANOVA) for Caco-2 cells co-incubated with TYI filtrate in comparison to Caco-2-CMEM growth control. As for serial co-incubation, maltase activity of cells co-incubated with WB filtrate was found to be significantly lower (P<0.05, ANOVA) than either TYI control or Caco-2-CMEM control (Figure 11).  Again, protein concentration of Caco-2 cells co-incubated with WB filtrate was  observed to be significantly reduced (P<0.05, ANOVA) compared to both TYI or CMEM controls (Figure 12).  41  In this of experiments, the specific activity (U/g) and maltase activity (U) of Caco-2 co-incubated with filtrates, were observed to be lower than in the previous experiments (protein concentrations were similar). This was probably due to the fact that Caco-2 cells were harvested on day 9 compared to monolayers harvested on day 12 in the previous experiments (following serial inoculation with filtrates). A 48 hour coincubation experiments was therefore performed on a day 10 monolayer to test the effect of cell age on maltase activity.  No difference between the specific maltase  activity of TYI control and WB test filtrates (Figure 13) was observed. Both specific activity of TYI and WB, however, were significantly lower (P<0.05, ANOVA) than the Caco-2-CMEM growth control.  Maltase activity (Figure 14) and protein  concentration (Figure 15) for both WB test and TYI control filtrates, however, were not significantly different  (P<0.05,  ANOVA) from the CMEM growth control.  Furthermore, maltase levels measured following the 48 hour co-incubation, with the more mature (day 12) Caco-2 monolayer was not significantly different (P<0.05, ANOVA) to that measured for the less mature (day 7) Caco-2 monolayer. None of the levels were as high as those observed in the first experiments (serial inoculation of Caco-2 with filtrates, section a). L, WB and WH were grown in different TYI batches.  In follow up  experiments, L and WB were grown and filtrates collected using the same batch of TYI media whilst WH was grown in a separate TYI batch.  There was no significant  difference (P<0.05, ANOVA) between specific maltase activity of test filtrates and TYI control filtrates for all isolates except WH. In the repeat experiment, the specific  42  activity for L , WB and WH were similar compared to one another and to growth control CMEM (Figure 16). Maltase activity of WB and WH were similarly decreased, whilst it was similar for L and CMEM in the first experiment. In repeat experiments, maltase activity was similar for all isolates and CMEM. Also, no difference were observed between maltase activity of Caco-2 cells co-incubated with test or TYI control filtrates for all the isolates, in the first set of experiments (Figure 17). The protein concentration of Caco-2 cells co-incubated with L and WB parasite filtrates were similar to each other as well as to CMEM growth control. However, the protein content of Caco-2 cells following co-incubation with WH was significantly different (P<0.05, ANOVA) in comparison to the remaining parasite isolate filtrates as well as the CMEM growth control. Furthermore, there was no significant difference (P<0.05, ANOVA) between test or TYI control filtrates of L although a significant difference (P<0.05, ANOVA) was noted between test filtrate and TYI control of WH and WB.  In the second run, there was no significant difference (P<0.05, ANOVA)  between L , WB and WH parasite filtrates and corresponding TYI filtrate, as well as between both filtrates and CMEM growth control (Figure 18).  43  60  Hours of incubation period  Fig 5: Comparison of growth curves for different G. duodenalis isolates. Results are the mean of 2 separate experiments with trophozoites counted in triplicate for each isolate studied. 44  Figure 6:  S E M of a Caco-2 cell monolayer (courtesy of Dr. C.S. Ong, Dept. of Pathology and Laboratory Medicine, University of British Columbia). The brush border membrane (BBM) and tight junctions (TJ) between adjacent cells are visible. Bar equals 10 um  45  80  70  60  f  50  >  '•g  40  re o  20  10  CMEM  TYI  WB  filtrates  Figure 7: Specific maltase activity of 7 day Caco-2 after serial inoculation with W B or TYI filtrates. There was a significant difference * (P<0.05) between CMEM and both other groups. Results are means of 2 separate experiments carried out in triplicate.  46  250  CMEM  WB  TYI filtrates  Figure 8: Maltase activity of 7 day Caco-2 after serial inoculation with WB or TYI filtrates. There was a significant difference * (P<0.05) between all groups. Results are means of 2 separate experiments carried out in triplicate.  CMEM  WB  TYI filtrates  Figure 9: Protein concentration of 7 day Caco-2 after serial inoculation with WB or TYI filtrates. There was a significant difference * (P<0.05) between WB and both other groups. Results are means of 2 separate experiments carried out in triplicate.  47  CMEM  Figure"!0: Specific maltase activity of 7 day Caco-2 after single inoculation with WB filtrate. Results are means of 2 separate experiments carried out in triplicate.  48  80 70 60  5  >.50  > 140 01 Ul  5  — $  30 20  4-  1  10 +  ^ ^ - "I CMEM  WB  TYI filtrates  Figure 11: Maltase activity of 7 day Caco-2 after single inoculation with WB or TYI filtrate. There was a significant difference * (P<0.05) between all groups. Results are means of 2 separate experiments carried out in triplicate.  CMEM  WB  TYI filtrates  Figure 12: Protein concentration of 7 day Caco-2 after single inoculation with WB or TYI filtrate. There was a significant difference * (P<0.05) between WB and both other groups. Results are means of 2 separate experiments carried out in triplicate.  49  Figure 13: Specific maltase activity of 10 day Caco-2 after single inocluation with WB or TYI filtrate. There was a significant difference* (P<0.05) between CMEM and both other groups. Results are means of 2 separate experiments carried out in triplicate.  50  80 70 -60 --  5  >>50 > 140 o> (A  ~30 re E  20 + 10 CMEM  WB  TYI filtrates  Figure 14: Maltase activity of 10 day Caco-2 after single inoculation with WB filtrate. Results are the mean of 2 separate experiments carried out in triplicate.  1 4 c o  t  3 +  OI CJ  c  o o  c •  'cu  o  a.  CMEM  WB  TYI filtrates  Figure 15: Protein concentration of 10 day Caco-2 after 48 hours co-incubation with WB filtrate. Results are means of 2 separate experiments carried out in tripicate.  51  60 • TEST, 1st run HTYI control, 1st run in TEST, 2nd run • TYI control, 2nd run  o>  40  WB  CMEM  WH  filtrates  Figure 16: Comparison of specific maltase activity of various parasite isolates after 48 hour co-incubation on 7 day monolayer; 'significant difference (P<0.05) between that group and CMEM of the same run.  52  120  100  TEST, 1st run TYI control, 1st run HI TEST, 2nd run ED TYI control, 2nd run  WB  CMEM  WH  filtrates  Figure17: Comparison of maltase activity of 7 day Caco-2 after 48 hours co-incubation with various parasite isolate filtrates; 'significant difference (P<0.05) between that group and CMEM of same run.  53  • TEST, 1st run ffl TYI control, 1st run • Test, 2nd run  filtrates  Figure 18: Comparison of protein concentration of 7 day Caco-2 after 48 hours co-incubation with various parasite isolate filtrates; 'significant difference between that group and CMEM of the same run.  54  CHAPTER 5 DISCUSSION AND CONCLUSIONS  Although both host and parasite factors probably play a role in pathogenesis, these experiments focused on the parasite aspect in the microbial-host interaction. Previous  studies have  demonstrated  that  Giardia isolates differ both  biochemically and biologically (Nash et al., 1987). Such differences may contribute to the variable clinical features of giardiasis (Nash et al., 1987) observed in humans. The different in vitro growth curve patterns observed in the present experiments are consistent with a variable biological activity.  Two isolates obtained originally from  symptomatic hosts (WB, WH) and two from asymptomatic persons (OV, L) were more rapidly growing than three other isolates (BH, Woof, Jab) also obtained from symptomatic hosts. This is consistent with previous studies (Binz et al., 1992) which showed that genetically different isolates of Giardia differ in such fundamental biological parameters as growth rates and pH requirements. There did not appear to be a correlation between growth patterns of isolates retrieved from symptomatic as compared to asymptomatic hosts.  Also, there did not appear to be a correlation  between patterns of trophozoite growth rate and the Caco-2 effect. Growth rates may be influenced by environmental conditions such as minor variations in the composition of the culture medium (Madigan et al., 1997). Specific gene(s) coding for enzymes that enable Giardia trophozoites to adapt to new environmental conditions may be found is some isolates but not in others. The rapidlygrowing (WH, WB, OV, L) isolates adapted more quickly to in vitro conditions and attained maximal growth in a relative shorter period of time compared to the other slower-growing (BH, Jab, Woof) isolates.  Due to difficulties in trying to establish  some of the slow growing isolates in culture as well as keeping the trophozoites alive  55  over a prolonged time period, no filtrates for use in the Caco-2 cell model were prepared for the Woof or Jab isolates. Primary metabolites are usually formed during the exponential in vitro growth phase of micro-organisms. They are more or less produced simultaneously with microorganism and are generally similar in all cells (Madigan et ah, 1997).  Secondary  metabolites are produced near the end of the exponential growth phase, frequently at or near the stationary phase (Madigan et ah, 1997).  It is known that secondary  metabolites may vary considerably from one organism to another (Madigan et ah, 1997).  Since each secondary metabolite is usually produced by relatively few  organisms, and is also dependent upon optimal in vitro growth conditions such as the composition of the medium (Madigan et ah, 1997), demonstration of their activity may be difficult.  As secondary metabolites are seemingly not essential for growth and  reproduction (Madigan et ah, 1997), growth rates do not appear to be related to production of putative secondary metabolites.  It is possible that a putative toxic  parasite excretory-secretory (E-S) product is a secondary metabolite and therefore the kinetics of growth, rather than the absolute growth rate of the micro-organism, may be important. E-S products, also known as variant-specific surface proteins (VSPs), are released from the surface of trophozoites into the culture medium (Nash et al., 1983). Depending on the particular VSP and the parasite isolate, it has been shown that 70% of the major trophozoite surface antigen was released into the culture medium within 24 (Nash et ah, 1983) to 32 hours (Papanastasiou et ah, 1996).  Many of the  characteristics of Giardia VSPs and their antigenic variation are unusual (Nash, 1992) as judged by DNA sequences from both genomic and complementary DNA (cDNA) clones (Adam et ah, 1988). The DNA sequences of five VSPs reveal a family of cysteine-rich proteins (Nash, 1992) that vary in size from 30-200 kDa or more. These VSPs are, for the most part, antigenically distinct. VSPs cover the entire surface of the  56  trophozoite including the flagella (Nash, 1992; Pimenta et al., 1991; Nash et al., 1990a).  We postulated that trophozoite E-S products (possibly VSPs) have a toxic  effect on Caco-2 cells. Proteases that alter intestinal brush border enzymes have been described for microbes such as Bacteroides fragilis, Clostridium perfringens and Streptococcus fecalis (Williams and Coombs, 1995; Riepe et ah, 1980; Jonas et al., 1978). Following binding to the brush border surface, microbial proteases and/or phospholipases, act on the enterocyte membrane, degrading or releasing microvillar enzymes from the membrane (Lobley, 1991).  Levels of protease secretion depend on the bacteroides  species as well as on the bacterial in vitro growth conditions. Protease activity was observed in the cell-free medium only after Bacteroides reached a stationary phase of in vitro growth in a minimally defined media (Riepe et ah, 1980). The potential of these proteases to damage the enterocyte membrane is greater than that of pancreatic proteases in equivalent amounts (Riepe et al., 1980).  Riepe et al. (1980) also  demonstrated that secreted proteases could destroy human brush border disaccharidases. It has been postulated that this leads to maldigestion and diarrhea (Lobley, 1991). Williams and Coombs (1995) reported that one strain of G. duodenalis produced at least eighteen different proteases with high activity and with multiple substrate preferences.  These Giardia proteases were predominantly of the cysteine type, but  some serine, aspartic and aminopeptidases activities were noted.  It is possible that  some VSPs are proteases which affect intestinal brush border enzymes.  The  relationship between VSPs, proteases and E-S products is not yet understood. It has been proposed that the decrease in disaccharidase levels observed in some human cases of giardiasis (Mohammed and Faubert, 1995 a; Vinayak and Naik, 1992) is as a result of a diffuse shortening of the intestinal brush border with a subsequent decrease in cell surface area (Buret et al., 1992). Disaccharidase deficiencies have also been demonstrated in gerbils {Meriones unguiculatus). In these studies, animals 57  previously infected with G. duodenalis, were challenged with soluble extracts of trophozoites resulting in a significant reduction in disaccharidase activity comparable to that induced by challenge with live trophozoites (Belosevic et al., 1989). The small intestinal pathology in giardiasis is diffuse, often distant from the location of parasites. We hypothesized that the presence of toxic E-S products, possibly proteases or VSPs, secreted from Giardia trophozoites, play a role in the pathology of giardiasis by damaging enterocyte brush border enzymes. Caco-2 cells, a human colon carcinoma cell line, differentiate spontaneously in vitro and exhibit both structural and functional differentiation (Pinto et al., 1983). Favennec et al., (1991) showed that live Giardia trophozoites incubated on a Caco-2 monolayer appeared to have a cytopathogenic effect.  Decreased Caco-2 sucrase and  alkaline phosphatase levels were also observed in these experiments. Preliminary experiments in this study using a Caco-2 model demonstrated that maltase levels increased in these cells (data not shown) as they matured from day 7 to 12.  In another study (Pinto et ah, 1983) maximal activities of sucrase, alkaline  phosphatase and aminopeptidase were noted at day 19 when cells were post-confluent and mature. In the present experiments we were unable to duplicate the maltase levels described by Katelaris et al. (1995) despite repeated attempts and various methods of Caco-2 cell disruption as previously described.  Experiments using this Caco-2 cell  model and parasite filtrates, showed a significant decrease (P<0.05, ANOVA) in both maltase activity and protein concentration following serial co-incubation with WB filtrate (no live parasites).  It was also noted that only maltase levels (not protein  concentration) were also affected by the control (TYI parasite broth media) filtrate. However, maltase levels after WB filtrate co-incubation, were significantly lower (P<0.05, ANOVA) than control (TYI) filtrate.  In these experiments, Caco-2 cells  appeared viable by trypan blue exclusion after co-incubation with filtrates.  58  It was  therefore postulated that a cytopathological action was occurring in this experiment rather than Caco-2 cell death. The effect was apparent as early as 48 hours following co-incubation of filtrates on a day 7 Cacor2 monolayer (Figures 11 and 12). Decreases in maltase activity and protein concentration, and maltase activity only, of harvested Caco-2 cells following co-incubation with WB filtrate and TYI filtrate respectively, may be explained in at least three ways. Due to the fact that TYI is not the optimal cell medium for Caco-2 growth, this medium has a detrimental effect on Caco-2 cell metabolism. The TYI filtrate effect on Caco-2 cells may also change Caco-2 protein (disaccharidase) configuration following binding of a TYI component (undefined media) to an allosteric site (not the active site). This binding by TYI could thus change the 3-dimensional structure of the enzyme, resulting in loss of activity. Alternatively, TYI medium could contain proteases (such as those found in the pancreas; TYI contains pancreatic digest of casein) that destroy or release Caco-2 membrane-attached disaccharidases. A WB filtrate effect on Caco-2 cells would therefore be in addition to that of the TYI medium effect. Thus the significant decrease in maltase levels observed (levels of Caco-2 cells co-incubated with WB filtrates compared to levels of cells co-incubated with TYI filtrate) would be related to the additional presence of toxic parasite product (E-S products) in the parasite filtrate. Putative parasite E-S products may have two toxic effects on Caco-2 cells, or in  vivo  host enterocytes.  disaccharidases.  If they are proteases, they may destroy membrane bound  They may also act as cytotoxic molecules affecting the Caco-2 cell  membrane with leakage of intracellular material. Damage to Caco-2 membranes rather than damage to disaccharidases alone is consistent with the observation that the decreased protein cell concentration which was observed only during co-incubation with WB parasite filtrate, not with TYI filtrate.  59  During co-incubation of a 10 day old Caco-2 monolayer with WB filtrate, no significant difference (P<0.05, ANOVA) was observed between specific enzyme activity (enzyme units per gram of total cellular proteins) of control TYI filtrate and test WB filtrate, both of which, however, were significantly lower (P<0.05,ANOVA) than CMEM, the growth control (Figure 13). There was also no significant difference in maltase activity or protein concentrations between growth control/TYI control and test filtrates.  One explanation for this is that 10 day Caco-2 monolayers are  functionally more differentiated than 7 day old cells (Pinto et al., 1983). It is possible that cell repair systems are therefore working at optimal levels with maximal brush border turnover rates in effect. Overall turnover rates of brush border proteins are known to be rapid (half-life approximately 18 hours).  in vivo  Rates of turnover for  disaccharidases are even more rapid (ti =11.5 hours); larger brush border proteins /2  such as these enzymes generally have shorter half-lives than other, smaller brush border components (Alpers and Tedesco, 1975).  It has been observed that the damage to  gastric mucosal cells is followed by rapid epithelial cell migration (called 'restitution'). In this process, adjacent living gastric mucosal cells are rapidly mobilized to restore the injured site to full function once the injurious agent is removed (Ito et al., 1984). Also, the reparability of a cell, is a function of its stage of growth at the time of injury (Petin and Petin, 1995). Therefore, in our 10 day Caco-2 model, it is possible that the detachment and/or the destruction of maltase by putative toxic E-S products, are repaired by Caco-2 cell mechanisms (new disaccharidase molecules synthesized and inserted into the membrane surface). If the brush border membrane itself was injured, it is possible that it too could be repaired more quickly in an older, 10 day cell monolayer. A WB effect (decrease in both maltase activity and protein concentration) was observed (Figures 8 and 9) on Caco-2 cells harvested at day 12 only after progressive co-incubations with the parasite filtrate starting on a 7 day old cell monolayer. 60  Consequently, it is plausible that if WB filtrate were producing a cytopathologic effect, it would not be detected in the more mature 10 day monolayer after a single, 48 hour co-incubation. In such a dynamic cell model, it is also conceivable that cytopathologic effects need to be cumulative in order to be observed and a single 48 hour coincubation did not produce enough damage to be detectable in this model. Continuous (serial co-incubation) injury in an immature monolayer (day 7) might affect or overwhelm cell repair mechanisms resulting in the decreases in both maltase level and protein concentration observed in the first experiments (serial inoculation of Caco-2 cell monolayer with filtrates; Figures 8 and 9). The effect of the mechanisms described above on Caco-2 cells may therefore explain the differences seen following both the serial co-inocubation with filtrates and a single inocubation both on a day 7 old monolayer and not on a day 10 monolayer. Maltase levels obtained from Caco-2-CMEM growth control cells from both the single inoculation (day 7 or day 10 monolayer) were similar. Growth control maltase levels from a 10 day old Caco-2 monolayer (single inoculation) was lower than that from a 7 day old monolayer (serial inoculation) even though, in both cases, maltase was measured in 12 day old cells.  This observed change (decreased growth control  Caco-2 cell maltase levels) could be a result of several things: (i)  in vitro  growth  conditions selected a sub-population of Caco-2 cells that did not retain its initial phenotypic properties, (ii) contamination of Caco-2 cells by  Mycoplasma spp.  (in this  set of experiments, it cannot be ruled out). These parasitic bacteria may have caused a decrease in Caco-2 maltase levels (observed in the CMEM control).  Mycoplasma  known to have profound effects on all parameters of cell function, growth and metabolism (Tully, 1996; Barile and Rottem, 1993). If parasite factors play a major part in the pathogenesis of giardiasis, it might be hypothesized that isolates from symptomatic hosts (WH, WB) would produce an effect in this Caco-2 model, a decrease in Caco-2 protein concentration and/or maltase 61  is  activity. The isolate from asymptomatic host (L), however, would not have any effect in this cell model. In our experiments testing different isolates, no difference was seen between the maltase activity of Caco-2 cells co-incubated with test filtrates and corresponding TYI filtrates from symptomatic and asymptomatic Giardia isolates. Thus, the observed decrease in maltase activity observed after co-incubation of cells with WB and WH filtrates (in comparison to CMEM growth control) is due to an effect from the TYI broth as discussed previously.  There was no difference observed  between protein concentration of Caco-2 cells co-incubated with L filtrate and from its corresponding TYI control filtrate, however, a difference was seen following coincubation WB and WH (Figure 18). Protein concentration of Caco-2 cells with WB filtrates (and TYI control) was not significantly different from the C M E M control as in previous experiments (serial inoculation and single inoculation of Caco-2 cell monolayers with trophozoite filtrates). We did not demonstrate a significant difference in protein concentration between Caco-2 cells following co-incubation with WB filtrate and growth control cells (CMEM) in this set of experiments (Figure 18). However, I believe that from these experiments, the effect of putative parasite E-S products are on the Caco-2 cell protein content, with a secondary effect on maltase activity. Further experiments using different in vitro Giardia culture conditions more likely to induce secondary metabolism, were not possible due to time constraints. Conditions under which the filtrate containing E-S products are collected (point in the growth phase cycle after stationary phase has been reached) should be studied. The serum batch used to prepare both CMEM and in some instances TYI, was different between experiments. Thus Caco-2 cells cultured using different batches of serum may not function in the same way (Martin, 1994; Bifulco and Schaefer, III, 1992). It has been reported that serum previously giving acceptable results failed to do so again after having been frozen (-20°C) and thawed once (Visvesvara, 1980). 62  While Giardia  trophozoites tested in these experiments were grown as per standard laboratory protocol using relatively consistent in vitro culture conditions, subtle differences in the "health" of isolates grown under "identical" culture conditions was observed by light microscopy.  It is possible that differences in the undefined TYI media (or serum  batches) occurred leading to differing trophozoite metabolism. This may explain why in some experiments a WB effect (decreased protein concentration and maltase activity) was observed and in other experiments it was not. It is known that a prolonged cultivation in vitro reduces the pathogenicity (Phillips et al., 1972) of many microbes. Therefore, production of putative toxic E-S products or proteinases could be reduced on prolonged culture.  Differences in  biological activity between parasite strains may induce differences in proteinases produced (Hare et al., 1989). As well, proteinases or possibly secondary metabolites may not be expressed, or may be expressed at undetectable levels. Mixed infections occur in nature (Binz et al., 1992; Andrews et al., 1989) and it is possible that in vitro cultivation selects for specific biotypes (Nash et al., 1985). Furthermore, some strains of Giardia have a growth advantage over others in vitro (Camaby et al., 1991). Therefore, in the experiments carried out, it is possible that clonal selection occurred in vitro and that the prevalent population of trophozoites in the culture stopped producing toxic E-S products. Phenotypic changes due to genotypic mutations (Upcroft et al., 1989) may also occur under in vitro culture conditions. Genotypic mutation events could result in alteration of proteinase or E-S product expression, and hence the change in effect on Caco-2 cell monolayers. All Caco-2 cells during repeat experiments (testing the different parasite isolate filtrates on Caco-2 cells, Figures 16,17,18) were found to be contaminated by Mycoplasma spp. The presence of these parasitic bacteria on the Caco-2 cell surface could possibly prevent binding of E-S products thus preventing these molecules from  63  exerting an observable effect. It is also possible that Mycoplasma alter the Caco-2 cells phenotypically i.e. changing maltase or protein levels. Although the results obtained thus far are from a limited number of experiments, if substantiated by further analysis, they would suggest that further studies be carried out, not the least of which would be isolation and characterization of Giardia E-S products. Isolation of E-S products could be carried out by fractionation of the supernatants harvested from trophozoites in the late log phase of growth and compared to TYI medium. Alternatively, Giardia trophozoites could be grown up in a minimal medium and filtrates tested, to exclude the effects of TYI on the Caco-2 monolayer.  64  SUMMARY AND CONCLUSIONS  The clinical features and the pathology of giardiasis in infected hosts varies. While the diarrhea associated with infection by small intestinal protozoan, Giardia is likely to have multifactorial origins, both parasite and host factors are probably involved.  The present experiments attempted to address possible parasite virulence  factors in the light of this knowledge. It appears that G. duodenalis infection causes a mild injury to the brush border in the host small intestine. This injury is expressed in terms of impaired activities of disaccharidases as well as in diffuse loss of microvillous surface area along the entire small intestine. Results from the present study are not inconsistent with the presence of a parasite excretory-secretory product, possibly a VSP or protease causing a cytopathologic effect on enterocytes.  The results of the experiments and the  conclusions drawn have been discussed. Work with the Caco-2 cell monolayer model, which requires a period of up to at least 7 days before a monolayer is established and membrane-bound enzymes expressed, is very labour intensive.  Nevertheless, Caco-2  cells appear to be a useful in vitro model for the further study of mechanisms of pathogenesis in giardiasis.  65  POSSIBLE DEFECTIONS FOR FURTHER STUDIES  Results of these experiments suggest several areas for future study.  •  Work with WB isolate should be repeated using optimal Caco-2 cell conditions (7 day old Caco-2 cells co-incubated with parasite filtrate for a period of 48 hours) in a Mycoplasma-frtQ cell line.  •  If a reproducible effect is observed, filtrates from the other parasite isolates should be tested.  •  Different points in stationary phase should be analyzed to provide a better understanding the kinetics of this interaction (when in the growth phase maximal levels of secondary metabolites assumed to be E-S products are produced).  •  If an E-S product effect is clearly demostrated, these products should be isolated and characterized further.  66  CHAPTER 6 REFERENCES 1.  Adam, R.D. (1991). The Biology of Giardia spp. Microbiol. 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