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and E.M. Fraser, ).F. Christie and M.W. Kennedy, unpublished). Something similar also appears to apply for T. muris 4. A more worrying finding from the T. muris system is that previous infection can abrogate adjuvant-assisted induction of antibody responses to certain parasite antigens4. Worse, vaccination can fail to protect previously infected mice; if mass vaccination against nematodes is going to work for human populations, then this phenomenon will have to be circumvented. Much more effort is currently being expended on cloning parasite antigens than on understanding and circumventing the limitations to immunization. If prevention of nematode diseases by immunological means has any future at all, then this imbalance will have to be redressed. That kind of research can

proceed now but it will be less easy to establish the scale and consequences of genetic diversity within the parasite populations themselves. If nematodes can develop resistance to the most powerful anthelmintics available, then for how long is a simple recombinant vaccine likely to be effective? References

I Wakelin, D. (1988) in Genetics of Resistance to Bacterial and Parasitic Infection (Wakelin, D. and Blackwell, J.M., eds), pp 153-224, Taylor & Francis 2 Keymer, A.E. et al. (I 990) Parasitology I 0 I, 69-73 3 Else,K.J.et aL (1990) Parasitology I 0 I, 61~7 4 Else, K.J. and Wakelin, D. (1990) Parasitology 100,479-489 5 Else, K.J. et al. (1990) Parasite Immunol. 12, 509-527 6 Kennedy,M.W. et oL (1987) Parasite ImmunoL 9, 269-273 7 Kennedy, M.W.etaLImmunology(inpress)

8 Pond, L., Wassom, D.L. and Hayes,C.E.(I 989) J. ImmunoL 143,4232-4237 9 Young, D.B. and Lamb,J.R.(1986) Immunology 59, 167-171 10 Haig, D. et al. (1989) Parasite Immunol. II, 463-477 I I Rittner, C. and Schneider,P.M.(1989) ImmunoL Today 10,401-403 12 M011er,U. etaL (1987)Nature 325,265-267 13 Young, R.A. (I 990) Annu. Rev. Immunol. 8, 401-420 14 Grencis, R.K. and Pinkerton, S. (1990) in Advances in Mucosal Immunology (MacDonald, T.T. et al., eds), pp 833-834, Kluwer Academic Publishers 15 Wassom, D.L., Krco, C.J. and David, C.S. (1987) Immunol. Today 8, 39-43 16 Varla-Leftherioti, M. et al. (1990) Tissue Antigens35, 60~3 17 Tomlinson, L.A. et aL (1989)J. ImmunoL 143, 2349-2356 Malcolm Kennedy is at the We//come Laboratories for Experimental Parasitology, University of Glasgow, Bearsden, Glasgow G61 I QH, UK.

Intestinal Protozoa and Epithelial Cell Kinetics, Structure and Function A. Buret, D.G. Gall, P.N. Nation and M.E. Olson Intestinal protozoa are not only common enteric pathogens in the tropics but also the high incidence of infection among immunocompromised patients in northern countries has evoked an increased interest in these parasitesl'2. Although enteric protozoa are a major cause of diarrhea and malabsorption in humanfl and other animals4,s, the pathophysiology of gut disturbances caused by them remains poorly understood. Clinical signs related to enteric protozoan disease commonly involve malabsorption, diarrhea, weight loss or retarded weight gain and anorexia. S in~=ethese infections are most prevalent and most severe in the young6-8, this may translate into considerable illness among children and significant loss to the agricultural economy where domestic animals are prone to infection. In this review we describe the effects of intestinal protozoan diseases on the structure, kinetics and function of absorptive intestinal cells and other epithelial cells, and correlate morphological injury with physiological alterations in the parasitized gut. Some of the interactions between immune responses and pathophysiology will ,be discussed, but in-depth disAndre Buret and Merle Olson are at the Department of Biological Sciences, Faculty of Sciences, University of Calgary, Calgary, Alberta T2N I N4, Canada, Grant Gall is atthe Gastrointestinal Research Unit, Department of Pediatrics, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N IN4, Canada and Nicholas Nation is at the Veterinary Pathology Branch, Alberta Agriculture, Edmonton, Alberta T6H 4P2, Canada. ~) 1990,ElsevierSciencePublishersLtd,(UK) 0169-4707/90/$02.00

cussion of intestinal immunity has recently been undertaken by other authors9. Epithelial cell kinetics Increased epithelial cell turnover can result from increased mitotic activity in the crypts and/or an increased migration rate of enterocytes along the villi. These events can be measured by metaphase accumulation 1°, radioautographic analysis following labelling with tritiated thymidine 11, or by bromodeoxyuridine uptake (Box 1)lz. These techniques have been used to show the increased rate of production of epithelial cells in the small intestinal crypts of mice and in human biopsy samples from cases of Giardia infection13-15. Bromodeoxyuridine uptake (Fig. 1) shows that increased epithelial turnover clearly is a feature of the infected gut in giardiasis. Similarly, increased epithelial turnover occurs in other protozoan infections, for example in chickens and lambs infected with Eimeria spp 16'17. In addition, increased numbers of mitotic enterocytes, indicating increased turnover, are a common histopathological finding in Cryptosporidium infections 18. Recent findings, in vitro 19, have shown that crypt cell hyperplasia is initiated by T-cell activation. In this study, fetal human small intestinal lamina propria T cells were activated in organ culture with the lectin pokeweed mitogen or with mitogenic monoclonal anti-CD3 antibodies. This T-cell activation was very

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Box 1. Methodologies for the Study of Epithefial Cell Kinetics Metaphase arrest technique. Metaphasic cells in experimental tissues are arrested by the pre-mortem intravenous injection of various agents, including colchicine, demecolcin and vincristine. The number of cells undergoing mitosis is determined under light microscopy from which the mitotic rate can be estimated. This technique is fairly quick and easy. However, its sensitivity is relatively low and it only provides information on mitotic indexes, not on epithelial migration rates in intestinal tissue.

[3H]Thymidine radioautography. Intravenously injected [3I-I]thymidine is incorporated into the DNA of replicating cells. Sections of tissue are cut, post-mortem, mounted on microscope slides and covered with a film of photographic emulsion in a dark room. After development for several days, the slides are developed to reveal staining. This technique is more sensitive than the metaphase arrest technique and allows assessment of epithelial migration rates; however, it is laborious and slow. Bromodeoxyuridine uptake. Intraperitoneally injected 5bromodeoxyuridine incorporates into newly synthesized DNA. Sections of tissue are cut, post-mortem, mounted on microscope slides, and incorporated bromodeoxyuridine is immunohistochemically identified with a mouse antibromodeoxyuridine monoclonal antibody. This technique is as sensitive as the [3H]thymidine technique and allows assessment of epithelial migration rates. Its advantages are that it is much less time-consuming, does not require the use of radioactive materials and can be completed under daylight conditions.

quickly (18h) followed by crypt hyperplasia, suggesting that activated T cells are mitogenic for epithelial cells. It has been suggested that the elongation of crypts is a direct result of an increase in the rate of enterocyte proliferation5'~9 and that crypt hypertrophy is the primary event in the development of villous

Fig. I. Ught micrographs of duodenal sections from an uninfected control (a) and from a gerbil infected (b) with a human Giardia lamblia isolate. The leading edge of bromodeoxyuridine (BrDU) uptake by epithelial cells is indicated (arrows) in both micrographs 30 h after inoculat/on of BrDU and shows increased epithelial turnover in the infected intestine. Note the presence of villous atrophy and crypt hyperplasia in the infected mucosa. Bar=50 lun.

atrophy 19. Increased epithelial cell turnover can also result in immature cells progressing along the villi and, during protozoan infection, villous epithelial cells may (but not always)2°'21 exhibit crypt cell characteristics, including a cuboidal shape, nuclear apolarity and disorganized, shortened brush-border microvilli5'2°'z2. In the absence of definitive evidence that these cells are physiologically immature, it cannot be concluded that their morphology represents immaturity rather than parasite-induced alterations. Normal enzyme levels and the mature anatomical appearance of enterocytes in Giardia-infected areas of the gut, although having an increased epithelial migration rate 2~,further question the possible enterocyte immaturity hypothesis. Thus, increased epithelial turnover during protozoan infections may result in increased loss of epithelial cells, owing to villous atrophy, and perhaps the appearance of immature enterocytes along the villi.

Loss of epithelial cells A wide range of host species has been shown to exhibit intestinal villous atrophy in response to protozoan infection (Table 1); humans are no exception and villous atrophy appears to be the most commonly reported pathological finding associated with this type of infection. Histopathological observations of the intestinal mucosa infected with various Table I. Occurrence of villous atrophy in a variety of hosts infected with enteric protozoan parasites Humans Cattle Sheep Swine Poultry Rodent Dog

Giardia Histomonas Cryptosporidium E/mer/a

Isospora

21,37a

21 5

1,62 18,38,58 63 22 39 5

53,58

64 17,22.58

65 66

alqumbersindicate selected references.

protozoa, including Sarcocystis (in humans), Toxoplasma (in felines) and Balantidium (in swine and humans) have failed to show any villous deformation caused by the organismsz3,z4. It appears that Balantidium is commonly a secondary infection, and as such, may occur in association with mucosal damage from another origin (Fig. 2). Description of mucosal architecture as normal during such infections may also reflect the lack of research data on these parasites rather than a true characteristic of the disease. Indeed, apparently normal villous architecture can occur in rodents and humans infected with Giardia, although it is more likely than Balantidium to cause villous atrophy 25,26. Interestingly, acute infection with Eimeria aceroulina results in elongation of the villi in the distal small intestine simultaneously with villous atrophy in the duodenum 16. We have recorded similar findings in primary Giardia lamblia infections where villous height in the ileum was significantly higher than in uninfected gerbils (Fig. 3), possibly compensating for the villous atrophy

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Fig. 2. Light micrograph of the colon from a hog infected with Balantidium coli (arrowheads). The parasite invaded the colonic tissuevia the mucosal ulcer illustrated on this micrograph. Haematoxylin and eosin. Bar=50 Ixm.

observed in the duodenum (Fig. 1). Since both E. acervulina and Giardia sp. colonize the upper small intestine, these findings also suggest that villous atrophy during infection is more likely to occur in these areas. Other awthors 13 have shown that villous hypertrophy occurs in the jejunum late during the course of murine gia:rdiasis and that jejunal villous elongation is accompanied by villous atrophy in the ileum. Such observations are consistent with the remarkable adaptive properties of the ileum, which has often been shown to compensate for alterations in the proximal small intestine21'26. There is growing evidence that cell-mediated immunity is a common factor in villous atrophy during parasitic infection. Despite heavy nematode infestation in T-cell-deficient (thymectomized) rats 27 and mice2s, in these studies the villi and crypts appeared largely normal; in contrast, villous atrophy has been observed in Cryptosporidium-infected Tcell-deficient nlice 29. Similarly, in graft-versus-host disease, where the small intestine is damaged solely by a cell-mediated immune reaction, experimental mice exhibit villous atrophy in the intestinal mucosa 1~. It has recently been shown that T-cellmediated villous atrophy may be secondary to crypt hyperplasia and can occur in the absence of damage to surface enterocytes19. Thus, although it appears that a local cell-mediated immune reaction may be a common mediator of villous atrophy in many small intestinal disorders, other factors appear to be involved. Besides being associated with villous atrophy, local T-cell activation also results in lymphokine release3°, increased expression of HLA-DR on the villous and crypt enterocytes3~, and increased numbers of intraepithelial lymphocytes3z, a common observation during protozoan infections. Explants of fetal small intestine containing T cells activated with the lectin pokeweed mitogen or with mitogenic

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monoclonal anti-CD3 antibodies have been found to contain the lymphokines IL-2 and gamma interferon29. However, on their own these lymphokines did not induce changes in mucosal morphology29and although the presence of gamma interferon, released by activated T cells, can explain the increased expression of HLA-DR on epithelial cells3°, it remains to be shown whether any other lympholdnes released by activated T cells may be responsible for mucosal alterations in vivo. Whether such an induction is direct, or occurs via interaction with lamina propria cells, also remains unknown. Infection of rats 33 and lambs 34 with Eimeria is characterized by a substantial increase in the number of intestinal mucosal mast cells, which in rats have been shown to respond to parasitism by releasing a potent collagen IV protease (rat mast cell protease II) 35. Type IV collagen is found exclusively in the epithelial basement membranes which are important in providing structural support for the overlaying enterocytes. The immunopathophysiological role played by mediators released from mast cells during epithelial cell loss observed during protozoan infections and other intestinal disorders deserves further study. Loss of epithelial cells is a complex event, potentially involving increased epithelial turnover, epithelial damage caused by host-immune response factors, direct parasite-induced damage and other factors. Loss of epithelial cells is also associated with villous fusion and direct enterocyte sloughing, both of which have been observed in infections with Eimeria 16 and Cryptosporidium 18. Transmission electron microscopy has revealed the presence of desmosomal junctions between enterocytes of adjacent villi36 but the mechanisms of fusion remain unclear. Several authors have suggested that epithelial loss

a

b

Fig. 3. Light micrographs of the ileal mucosa from uninfected Mongolian gerbils (a) and gerbils infected with Giardia lamblia (b). In the ileum, infection results in villous hypertrophy and crypt hyperplasia. Note also the increased goblet cell (arrowheads) numbers in the intestinal mucosa from infected animals. Periodic acid SchilT's reagent. Bar=50 lain.

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Fig. 4. Transmission electron micrographs of microvillous brush-border alterations caused by two intestinal protozoan parasites. (a) Giardia muris trophozoites in the duodenal lumen of an infected mouse. Note the slight shortening or bending of epithelial microvilli (arrows) where the edge of the ventral adhesive disk of the parasite penetrates the brush-border or where an adjacent organism applies pressure on the epithelium. Bar=2 gin. (b) Cryptosporidium sp. within the ileal brush-border of an infected calf. Total effacement of microvilli can be s e e n at the sites of parasite attachment. Bar=2 gin.

associated with villous atrophy, fusion and enterocyte sloughing is the basis for diarrhea, particularly in giardiasis 25'37 and coccidiosis38'39. However, clinical signs have also been shown to occur in infected hosts where only minimal or no mucosal abnormalities are evident by light microscopy4°. This evidence clearly suggests that epithelial cell loss alone cannot explain the clinical signs observed in gastrointestinal infection. The changes in villous structure described here also occur in many other intestinal disorders of parasitic, bacterial, viral, allergenic or genetic origin. Such evidence suggests that these pathological changes are part of a nonspecific host response in which mediators translate a wide variety of challenges into a common response.

Increased epithelial permeability Increased epithelial permeability in protozoan infections can have serious pathophysiological consequences. During coccidial infections the per-

meability of the intestinal mucosa increases, resulting in a significant loss of plasma proteins. This does not appear to occur in other protozoan infections, probably because of the degree of invasiveness of Coccidia, which can cause severe enterocyte sloughing. In chickens infected with Eimeria acavulina or E. tenella, the most severe epithelial damage is seen in areas of heavy colonization 5,22, ie. the duodenum and caecum. However, there are interspecific differences, and E. acervulina shows significantly impaired carotenoid absorption in the caecum4x, whereas there is no indication of caecal malabsorption in E. tenella-infected chicks fed labelled carotenoid diets. Both infections result in a reduction in levels of plasma carotenoids and in increased concentrations of the pigment in the caecum. This suggests that low plasma protein levels are primarily associated with malabsorption in E. acervulina infections during the acute phase of infection, whereas they appear to be directly caused by leakage when the caecum is damaged by E. tenella. Recent studies made in vivo and in vitro have shown that wounding and resealing of cell membranes occurs in normal, undisturbed enterocytes as well as in mechanically injured ones 42. In normal gastrointestinal epithelia, cell wounding can be caused by chemical injury from ingested substances, mechanical abrasion from luminal contents or even tears resulting from gut motility. Although the capacity for reseating preserves epithelial integrity, frequent wounding and resealing may also provide another route for molecular traffic across the epithelium. Recent reports 43 indicate that Entamoeba and a number of other parasites contribute to cellular damage by the production of pore-forming proteins. This will not only result in lysis of the target cells, but also will contribute to leakage through the epithelium. Whether protozoan infections impair the resealing capacity of enterocytes or cause increased mucosal permeability by the release of pore-forming proteins remains to be determined.

Brush-border injuries In cryptosporidiosis, electron microscopy reveals the total effacement of microvilli in areas of parasite attachment (Fig. 4) and although it has been recently shown that brush-border interruption can be repaired through intrinsic mechanisms44, temporary microvillous injury can have clinical repercussions 45. In giardiasis, where brush-border abnormalities may only be visible as localized shortening and bending of individual microvilli (Fig. 4) 20'46, marked and diffuse brush-border enzyme deficiencies result largely because of a loss of brush-border surface area (Fig. 5) 26'47 . S i m i l a r l y , epithelial dysfunction during bacterial enteritis 4s and intestinal anaphylaxis 49 appears to correlate with diffuse loss of brush-border surface area. As for villous atrophy, diffuse loss of brushborder surface area may be a generalized host response to a variety of intestinal insults but the mediators involved remain unknown.

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Intestinal brush-border enzyme deficiencies can be easily demonstrated during protozoan disease and are probably the most commonly reported abnormalities of gut epithelial physiology. Disaccharidase impairments have been observed in humans 21'37 and rodents during primary and secondary infections of Giardia 13"21'5° but these return to normal levels of activity after treatment or immune-elimination of the parasite 21'26. Brush-border enzyme deficiencies have also been observed in infections with Eimeria 7 and Cryptosporidium '5~ and here, too, enzyme activities return to normal values after recovery5x. The degree of impairment can be directly related to the number of parasites present since maximal disaccharidase deficiencies occur in the intestinal regions most heavily coloniTed. Impairment of enzyme activity can lead to nutrient malabsorption as well as osmotic diarrhea. Enzyme impairment does not solely result from villous atrophy because areas of the small intestine infected with Giardia may exhibit normal villous height simultaneously with disaccharidase deficiency26, and although villous atrophy may contribute to this impairment, reduced microvillous surface area appears to be the limiting factor for disaccharidase deficiencies.

Epithelial transport abnormalities Entamoeba histolytica stimulates intestinal chloride and fluid secretion52, mediated by serotonin (5-hydroxytryptamine) contained within the parasite, which results in diarrhea and mucosal injury s2. In the jejunum and ileum of piglets infected with Cryptosporidium, glucose-stimulated Na + and water absorption are significantly impaired s3. The absorption of a variety of substances has been measured in a number of protozoan infections. The malabsorption of D-xylose, glucose and amino acids has also been well documented in acute Eimeria infections5,2z. D-Xylose absorption has been found to be normal in some patients with giardiasis46 but impaired in others zS, and experimental results using

a

b

Fig. 5. Transmission electron micrographs, both at same magnification, from the duodenum of a noninfected gerbil (a) and the same tissue from a gerbil infected with Giardia lamblia (b), illustrating the diffuse brush-border shortening cau'sed by the infection. Bar= I I~m.

379

mice infected with Giardia lamblia have shown that malabsorption of glucose, glycine and L-alanine occurs 54. However, similar infections in rats have failed to confirm the occurrence of glucose malabsorption but do show that a Na+-K+ATPase deficiency develops55. To determine whether or not malabsorption occurs in a certain infection, it is vital to document pathological changes throughout the length of the small bowel, including parasite-free regions, because of the compensatory capacity of certain areas of the gut for impairment of function in other regions5.

Altered goblet cell population Mucus, secreted from goblet cells, may provide nonspecific protection against some pathogens s6. Increased goblet cell populations have been reported in mice57 and rats 56 infected with Eimeria, and in humans and gerbils infected with Giardia (Fig. 3)2°. It seems reasonable to suggest that increased mucus secretion may help in cleating a lumen-dwelling organism like Giardia. In Eimeria infections, goblet cell hyperplasia also correlates with resistance to the pathogen 56, but not in cryptospotidiosis58. More recently, it has been shown that the lectin-mediated adherence ofEntamoeba histolytica to rat and human colonic mucins is associated with an inhibition of parasite attachment to colonic epithelial cells59. Although this mechanism may prevent the organism from invading the intestinal mucosa, specific binding of the amoebic lectin to mucin may also facilitate luminal colonization by the parasite. M cells M cells are found within the epithelial lining of Peyer's patches (a secondary lymphoid organ). These cells are involved in the processing of antigens found in the gut lumen and are important in mobilizing gut-associated lymphoid tissue against infectious agents. Cryptosporidium organisms have been observed within the cytoplasm of M cells and it has been suggested that endocytic transport mechanisms within M cells may be involved in antigen presentation 6° and may explain why cryptosporidiosis is a self-limited disease in immunocompetent hosts. Although transepithelial transport of antigens may initiate protective mechanisms against the infections, it may also promote a potentially pathogenic inflammatory response. The net effect of Cryptosporidium translocation by M cells on the parasite and/or on the host remains unclear. Although Giardia muris has been found within macrophages in the Peyer's patches of nude mice 61, it still is not understood whether the trophozoites gained access to macrophages via M cells, or whether this was solely the result of direct transepithelial phagocytosis. Little is known of the precise role of M cells in processing protozoan antigens and the effect of infection on the morphology and kinetics of M cells remains to be clarified.

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Conclusions Although enterocytes can undoubtedly be injured by a pathogen, they can also be lost altogether, and various factors can contribute to epithelial cell loss during gut infections, including (1) a decreased rate of cell production in the crypts being unable to compensate for normal physiological loss of cells from the villi, (2) an increased rate of cell loss but rate of production remaining normal, (3) an increased rate of cell loss and decreased production or (4) increased rate of cell loss with insufficiently increased production. Morphological changes are associated with the various mechanisms of enterocyte loss and may include villous shortening, blunting and/or atrophy, decreased villus:crypt ratio and crypt hyperplasia. The precise changes that occur in any particular infection are determined by various factors, such as the age and immunocompetence of the host, history of previous intestinal damage, the presence of concurrent infections and the stage of the disease at which the intestine is examined. Therefore, the mucosal architecture associated with protozoan infections in the gut can show a spectrum of variation 22'25'62. Enteric protozoan infections can be the cause of physiological deficiencies directly correlating with loss of rnucosal surface in common with many other instances of intestinal insult. Clearly enteric protozoan infections result in various morphological and physiological abnormalities in the host intestine, and several areas of study need further investigation. The paucity of information is particularly striking in the study of the cytokines leading to mucosal injury and in the field of fluid, electrolyte and nutrient transport of the parasitized gut. Additional evidence may lead to a better understanding of the pathophysiology of diarrhea, a ubiquitous symptom of a number of gastrointestinal disorders.

Acknowledgements This work was supported by the Swiss National Scientific Research Foundation,the Natural Sciencesand EngineeringResearchCouncil of Canada, the Alberta Heritage Foundation for Medical Researchand the MedicalResearchCouncil of Canada.

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