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Ate Unt‘ 4‘" ‘pfi:y This is to certify that the thesis entitled PATHOGENESIS OF GASTROINTESTINAL HYPERPLASIA IN RATS INFECTED WITH TAENIA TAENIAEFORMIS presented by David Michael Blaies has been accepted towards fulfillment of the requirements for MASTERS Joyce mWY AND PUBLIC HEALTH Major professor Date August 10. 1981 0-7 639 OVERDUE FINES: 25¢ per day per item RETURNIKS LIBRARY MATERIALS : ________________._——-— Place in book return to remv: charge from circulat‘lon recon PAIHOGENESIS OF GASTROINTESTINAL HYPERPLASIA IN RATS INFECTED WITH TAENIA TAENIAEFORMIS by David Michael Blaies A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1981 ABSTRACT PATHOGENESIS 0F GASTROINTESTINAL HYPERPLASIA IN RATS INFECTED WITH TAENIA TAENIAEFORMIS BY David Michael Blaies Greatly enlarged stomachs and intestines are characteristic of chronic infections with the immature liver-dwelling parasitic tapeworm Taenia taeniaeformis. These severe hyperplastic changes were measured postmortem in.rats subjected to long-term infections with metacestodes. The resulting stomach weights in infected rats (> 16g) were markedly greater than the controls (ca. 2 g) after 63 days postinfection (DPI) and most reached their greatest masses after 80 DPI. The small bowels en- larged primarily due to growth of the villi and crypts, and serum gastrin levels rose dramtically in many infected rats but without a clear trend. Soon after infection, mucosal mast cells permeated the rat's intestinal lamina propria until 40-50 DPI when maximum levels were reached. Para- sitized rats generated nonuniformly great elevations in histamine con- centration throughout the small bowel, however, the histamine was nearly always distributed in a gradient increasing from the duodenum toward the ileum. Remarkably, no measureable changes in any of these parameters were produced if 10-40 large strobilocerci were surgically implanted in the peritoneal cavity. ACKNOWLEDGMENTS I must credit Dr. Forbes at Oakland University (Michigan) for first stimulating my interest in parasitology and to Dr. Jeffrey Williams who then had the patience to see me through three years of graduate study. Thanks to them both! ii HISTORICAL PERSPECTIVE "THE LACTEALS commence upon the inner surface of the intestines, and absorb, or such up the chyle, the milky—like fluid, formed from the digestive process, and from which the blood is renewed, and the general system built up, pouring the chyle, as before remarked, into the thoracic duct. And Dr. Gunn, in his "Domestic Physician" says that he thinks that it is a reverse action of the LACTEALS, in cholera, by which they pour back their contents in the intestines, or rather I should say, WANT OF ACTION, in not taking up the chyle, leaving it to be passed off in the milky and watery stools." From: Dr. Chase's Family Physician, Farrier, Bee-Keeper and Second Receipt Book, 1887, Chase Publishing Company; Toledo, Ohio. Entered according to Act of Congress, in the year 1885, by A.W. Hamilton, in the Office of the Librarian of Congress, at Washington, D.C. iii TABLE OF CONTENTS LIST OF TflLES . O O O O O C O O O O O O O O O O O O O O O O 0 LIST OF FIGURES O O O O O I O O O O O O O O O O O O O O O O O 0 LITERATURE REV IEW I O O O O O O O O O O O O O O O O O O O O O O I. II. III. IV. V. VI. VII. VIII. The tapeworm model . . . . . . . . . . . . . . . . . Pathology of Taenia taeniaeformis infected rats. . . . Mast cell characteristics. . . . . . . . . . . . . . . Heparin. . . . . . . . . . . . . . . . . . . . . . . . Histamine. . . . . . . . . . . . . . . . . . . . . . . a. Biochemistry . . . . . . . . . . . . . . . . . . b. Concentration in tissue and mast cells . . . . . IgE, mast cells, and parasitism. . . . . . . . . . . Histamine, hormones, and gastrointestinal function . . Research objectives. . . . . . . . . . . . . . . . . . LIST OF REFERENCES 0 I O O O O O O O O O O O O O I O O O O O O I CHAPTER 1 - GASTROINTESTINAL HYPERPLASIA IN RATS CHRONICALLY CHAPTER 2 INFECTED WITH TAENIA TAENIAEFORMIS: QUANTITATIVE PATHOLOGICAL AND HORMONAL CHANGES AND ATTEMPTS TO INDUCE THE SYNDROME WITH PARASITE PRODUCTS. . . . . - HYPERPLASTIC GASTROPATHY IN RATS INFECTED WITH TAENIA TAENIAFORMIS: HISTAMINE LEVELS IN GASTRIC TISSUES O I O O O O O O O O C C O O O O C I O O O 0 CHAPTER 3 - CHANGES IN HISTAMINE CONTENT AND MAST CELL DENSITY APPENDIX IN THE HYPERPLASTIC SMALL INTESTINES OF RATS IN- FECTED WITH TAENIA TAENIAEFORMIS. . . . . . . . . . A SIMPLIFIED METHOD FOR STAINING MAST CELLS WITH ASTRA BLUE 0 O O O O O I O O O O O O O O O O O O O 0 iv Page vi I" OWNNG‘FWH 12 14 17 24 59 71 9O Table LIST OF TABLES Page CHAPTER 3 Comparison of histamine levels in Taenia taeniaeformis infected and control rats . . . . . . . . . . . . . . . . 81 Histamine levels and mucosal mast cells/10 villus-crypt units for normal and infected animals . . . . . . . . . . 84 Figure CHAPTER 1 1 Stomach weights at necropsy of rats infected with Taenia taeniaeformis . . . . . . . . . . . . . . . . . . . . . . . 2 Glandular stomach from rat infected with Taenia taeniaeformis for 160 days (above) and normal age-matched CODEIOI (helm) o o o o o o o o o o o o o o o o o o o o o o 3 Changes in visceral organ weights in rats infected with Taenia taeniaeformis. . . . . . . . . . . . . . . . . . . . . 4 Changes in dimensions of small intestinal tissues in rats infected with Taenia taeniaeformis. . . . . . . . . . . . . . 5 Histological appearance of duodenum of rat infected with Taenia taeniaeformis at 160 DPI . . . . . . . . . . . . . . 6 Periodic acid-Schiff stained section of duodenum at 160 DPI . 7 Calcium deposition (black staining areas) in dilated duo- denal crypt by Kossa's silver deposition method . . . . . . . 8 Colonic mucosal thickening in rat at 80 DPI with Taenia taeniaeformis . . . . . . . . . . . . . . . . . . . . . . . . 9 Normal age-matched control. . . . . . . . . . . . . . . . . . 10 Changes in duodenal mucosal mast cell (MMC) counts in rats infected with Taenia_taeniaeformis (stippled bar) and normal age-matched controls (open bar). . . . . . . . . . . . 11 Serum gastrin levels at necropsy in rats chronically in- fected with Taenia taeniaeformis. . . . . . . . . . . . . . . CHAPTER 2 1 Distribution of gastric histamine levels in normal rats and rats infected with Taenia taeniaeformis . . . . . . . . . . . 2 The relationship between gastric histamine concentration and LIST OF FIGURES stomach weight in infected rats . . . . . . . . . . . . . . . vi Page 35 37 . 39 42 44 44 44 44 44 48 50 . 65 . 67 LIST OF FIGURES (CONTINUED) Figure A Page CHAPTER 3 1 An overall view of the changes in intestinal histamine concentration following oral administration of Taenia taeniaeformis eggs 0 o o o o o o o o o o o o o o o o o o o 78 2 A comparison of the histamine concentration elevations in the duodenum and ileum of rats infected with Taenia taeniaeformis O O O O O I O I O O I O O O O O O O O O O O 80 3 The total small bowel histamine values for infected rats (large dots) and normal rats (small dots) from the start of the investigation to 80 DPI. . . . . . . . . . . 83 APPENDIX 1 Duodenum from rat infected with Taenia taeniaeformis. . . 97 2 Liver from rat infected with Taenia taeniaeformis showing mast cells at periphery of host capsule . . . . . . . . . 97 vii LITERATURE REVIEW I. The Tapeworm Model The larval stages of Cyclgphylidean tapeworms of the genera Taenia and Echinococcus develop in muscular and visceral tissues of man and domesticated animals causing the diseases known as cysticercosis and hydatidosis, respectively. The parasites may lodge in vital organs causing serious health problems, or they may develop in the muscle tissues creating economic loss due to condemnation of domesticated animal carcases at the slaughter house or embargoes on the inter- national sale of meats from endemic countries. There are few satis- factory approaches to therapy, and the increasing prevalence in recent years has given rise to renewed interest in the immunology and pathology ‘of cestode infections. Taenia taeniaeformis in the rat shows many similarities in its life cycle and immunological characteristics to the medically and economically important tapeworms (Gemmell and Johnstone, 1977; Williams, 1979). In addition, I, taeniaeformis induces pathological and hormonal changes which appear similar to those of several important naturally occurring non-infectious human diseases (Cook and Williams, 1981; Cook et a1., 1981a, b). Examples include the cystic mucosal growth of the stomach as seen in patients with Menetrier's disease (Scully et a1., 1978), the thickened intestinal wall with mastocytosis that appears in Crohn's disease (Robbins, 1974), and hypergastrinemia comparable to that seen in the Zollinger-Ellison syndrome (Grossman et a1., 1961). The relationship 1 between these pathological changes and parasite survival and growth are not clear, but the suitability of this model for laboratory study pro- vides a good opportunity for research on the underlying mechanisms. Adults and larval stages are easy to maintain and none of the developing forms are infective to human beings. Cats are hosts of the adult tapeworms, and eggs of the parasite are collected from the feces of the cat, washed, then fed to rats via a gastric tube. In the stomach and intestine the keratinized blocks which form the outer surface of the eggs swell and fall off, leaving a series of oncospheral membranes surrounding the hexacanth (6-hooked) embryo. After entering the small intestine, the embryo undergoes a poorly understood process of acti- vation, in which the membranes are broken and the newly liberated oncosphere migrates into an intestinal villus. Penetration is achieved with the aid of enzymes secreted by the parasite and the oncospheral hooks (Heath, 1971). The oncosphere enters a venule in the mucosa and is then transported passively to its site of predilection in the liver. There, development into a larval tapeworm takes place within a con- nective tissue capsule. The work reported in this thesis concerns some aspects of the relationships between the gross pathologic changes in rodent cysti- cercosis and the mast cells which proliferate in affected tissues. The characteristics and the sequence of the pathological events after infection, the nature and functions of mast cells and their constituents, and the physiological regulation of gastrointestinal secretion and growth are reviewed in the following sections. II. Pathology of Taenia taeniaeformis Infection in Rats Pathological changes in rats with heavy infestations of T, taeniaeformis were first described by Bullock and Curtis (1930) and then Coleman and De Salva (1963). An account of the sequence of events has been developed by Cook et al. (1981a) with special reference to the liver, thymus, and lymph nodes. In the liver, fibroblasts rapidly proliferate in the capsule around the parasite at about 14 days post- infection (DPI). At this time, mast cells and plasma cells begin to permeate the capsule region. Hepatic mast cells increase in numbers reaching a peak at about 28 DPI and then decline. An acute thymic atrophy becomes grossly evident by 44 DPI. Cook and Williams (1981) described the dramatic changes in the stomach and small intestine of infected rats due to mucosal hyperplasia; the weight of the stomach increased up to 20 times normal and the intestines weighed up to 3 times normal. Their experiments with antrectomized rats demonstrated that the hyperplastic changes persisted in spite of reduced gastrin levels, indicating that hypergastrinemia was secondary to stomach growth. The growth is potentiated by an as yet unidentified factor or mechanism. Eosinophils in the peripheral blood and in the intestinal lamina propria were elevated during the course of infection, reaching a peak at 40 DPI and declining thereafter. The mast cell population in the small intestine increased significantly, with individual rats having over 10 times the normal mast cell level. The reason for the develop- ment of high numbers of intestinal mast cells in rats with hepatic cysticercosis is unknown. Local mast cell increases have been reported in the small intestine of rats infected with many different intestinal parasites (e.g., Nippostrongylus brasiliensis, Befus, 1979 ), and levels are known to rise in the skin of human patients infected with the filarial worms Loa loa, Wuchereria bancrofti, and Onchocerca volvulus (Fernex and Beyes, 1962; Fernex and Sarasin, 1962). However, their roles in the host-parasite relationship have not been clarified experi- mentally. III. Mast Cell Characteristics Mast cells, which were originally described by Paul Erlich (1879), are loosely defined as mononuclear tissue cells containing granules that stain metachromatically with Toluidine Blue 0. Because of the intense background staining with this dye, modern investigators use a variety of specific techniques to stain mast cells, but the variability of the results, particularly in mucosal tissues, is still troublesome. The cells are especially associated with skin, mesentery, lung, spleen, stomach, and intestine. They have been reported in all mammals, and even in amphibians such as frogs (Kapa and Csaba, 1972), although the quantity of mast cells in any tissue varies with the species. Mast cells, in general, are warehouses of inflammatory mediators such as histamine, serotonin (5-HT), slow reacting substance of anaphy- laxis (SRS-A), eosinophil chemotactic factor of anaphylaxis (ECF-A), and enzymes such as arylsulfatase. These mediators can be secreted from mast cells in specific response to immunological stimuli and non- specifically by complement anaphylatoxins, drugs, physical changes in the micro-environment, and chemical disruption. The membrane-bound granules themselves consist of sulfated glyco- saminoglycans, such as heparin, probably combined with histamine. Keller (1966) showed that only slight release of histamine occurred from rat mesenteric mast cells in the absence of granule extrusion in 33532, but that both are secreted simultaneously in 3332, In carefully con- trolled experiments, Uvnas (1972) used compound 48/80 to release hista- mine from free peritoneal mast cells. He determined that release was related to granule extrusion, but that some histamine from granules deep in the interior of the cell could also penetrate through the cell membrane by an independent mechanism. Enerback (1966a, b) first demonstrated that mast cells, formerly thought to be of one type, could be separated into two groups on the basis of their staining characteristics (granule-associated sulfated glycosaminoglycan type) and their reactivity towards compound 48/80. Connective tissue mast cells (CTMC) and peritoneal mast cells are degranulated by compound 48/80, but those of the intestinal or mucosal (MMC) type are not. Lindsay (1981) compared the reaction of mast cells in the liver and intestine of rats infected with I, taeniaeformis to CTMC from skin and tongue, by treatments with 48/80 compound and dexa- methasome. She was able to confirm that the two groups could be differ- entiated on the basis of drug reactivity and histochemical character- istics and that rat hepatic mast cells are largely of the MMC type. It should be borne in mind that until recently no such distinction was made; therefore, much of the older literature and many of the functional studies only apply to the readily accessible rat peritoneal and mesentery mast cells, not to MMC. I IV. Heparin Heparin, an important constituent of mast cells, was first found to be released from isolated dog liver after peptone shock (Rocha e Silva et a1., 1947); therefore, the name "heparin" has been derived from the Greek term "hepar" meaning liver. The heparin backbone (Jaques, 1979) is a polymer of glucuronic and uronic acid sugars, with an average of 3 sulfurous acid groups for every 2 sugar residues. This makes the molecule strongly negatively charged. The chondroitin sulfates have almost the same sugar backbone as heparin, but average only 1 sulfurous acid group per 2 sugar residues. Chondroitin sulfates are therefore less charged and, as a result, less active bio- logically. Chondroitin sulfates are generally present in cartilage and connective tissue. Although basophils and mast cells in all species have metachroma- tically staining granules (Jaques, 1975), some investigators point out that these may not always contain heparin. For example, recent studies by Orenstein et a1. (1978) show that guinea pig basophil granules, previously thought to contain heparin, actually are composed of 55% chondroitin sulfate A and C, 30-35% chondroitin sulfate B, and 15% heparan sulfate. No heparin was found. Tas (1977), by using micro- spectrophotometric analysiscfifthe metachromatic specturm of rat MMC granules after exposure to Toluidine Blue 0 dye, has determined that these granules do not contain heparin, as do the other mast cells in the rat (Yurt et a1., 1977; Robinson et a1., 1978). He concluded that they contain lower sulfated glycosaminoglycans of unknown chemical structure and function. V. Histamine A. Biochemistry In 1936, Werle discovered the DOPA decarboxylase enzymes in the kidney tissue of rabbits and guinea pigs. This was the only known system for endogenous formation of histamine, but in many animals such as cats, dogs, and man no DOPA decarboxylase could be detected (Waton, 1956). Since these animals withtu>obvious mechanism for histamine manu- facture showed evidence of endogenous stores, the amine was believed to be absorbed from gut contents, or in the case of other animals which has a recognizable amount of DOPA decarboxylase, body histamine stores were believed to be maintained by both endogenous production and ab- sorption (Kahlson and Rosengen, 1971). This led to the speculation that histamine was a vitamin. A change in concept followed the discovery of histidine decarboxy- lase (L-Histidine carboxy-Lyase) in the late 1950's. This enzyme, con- tained in mast cells, removes the number 1 carboxyl group from L- histidine, in the presence of the coenzyme pyridoxal phosphate, to form L-histamine and carbon dioxide (Rothschild and Schayer, 1959). Most endogenous histamine in mammals is now considered to be produced by this mechanism, with minor contributions from the DOPA decarboxylase enzymes (Beaven, 1978). The relationship between mast cells and histamine was first postu- lated by Riley (1953) and Riley and West (1953). This was based on the observed histamine release, along with heparin release, during peptone shock in the dog (Rocha e Silva et a1., 1947) and a known tendency of the skin mast cell lesions in human urticaria pigmentosa to form an itchy wheal and flare reaction upon mild trauma. This proposal was confirmed by Schayer (1956b) when he incubated 14C-labeled histidine with isolated rat peritoneal mast cells igugiggg. 14C-histamine was produced. After histamine has performed its function, there appear to be two methods of enzymatic degradation and one method of direct disposal. The first catabolic enzyme to be recognized was histaminase, now called diamine oxidase. A second enzyme system, discovered by Schayer (1966), deactivates histamine by the action of histamine-N-methyl transferase, which accepts a methyl group from S-adenosylmethionine (SAM), then attaches it to histamine, forming N-methyl histamine. This molecule may further be degraded to methyl imidazole acetic acid by the enzyme monoamine oxidase; however, its fate thereafter is unknown. Third, histamine may be disposed of by excretion in the urine of many mammals. Varying quantities of the total urinary histamine appear as free hista- mine in the rat (Gustafsson et a1., 1957) which, although present in males, is much more prominent in females. The remaining histamine in urine appears in a conjugated form, tentatively identified as acetyl histamine (Kahlson and Rosengren, 1971), although it is now thought to be largely of bacterial origin (Beaven, 1978). Extensive reviews of histamine metabolism or catabolism may be found in Kahlson and Rosengren (1971), Beaven (1978) and Maslinski (1973). B. Concentration in tissue and mast cells There are three methods for the determination of histamine. The oldest is the biological assay which measures the contraction of guinea pig ileum or rat uterus in the presence of histamine in vitro. An improved method for histamine determination (Beaven et a1., 1972) uti- lized N-methyltransferase to attach a 14C-methyl group from SAM. The radioactively labeled N-methyl histamine is then quantitated in a liquid scintillation spectrometer. This procedure will detect as little as 0.1 ng histamine. The analysis method developed by Shore et al. (1959) has been well accepted among investigators. In this procedure, the histamine must be . extracted with butanol and heptane, then condensed with o-phtalaldehyde (OPT). May et a1. (1970) have adapted the technique to small volumes of blood cells for clinical allergy testing. An extensive review of the methods and data concerning OPT-induced histamine fluorescence may be L— found in Hankanson et al. (1972). Attempts have been made to quantitate the histamine in mast cells and animal tissue from several sources. Paterson et a1. (1976), using cells that were dispersed from tissue enzymatically, report levels of '1.0-2.8 pg/mast cell from human lung and 1.0-2.0 pg/mast cell from rat lung. Mast cells isolated from dog stomach fundus by 8011 et a1. (1979) contain about 2.5 pg histamine per cell. Higher amounts have been re- ported by Austin and Humphrey (1963) and Enerbach and Wingren (1980) for rat peritoneal mast cells. In their reports, levels ranges from 15-40 pg/mast cell. Similarly, Veilleux and Cantin (1976), using cytofluor- escent techniques, have determined that there is less histamine in duo- denal mast cells than in the CTMC of the trachea or urinary bladder. Since peritoneal mast cells and CTMC contain substantially more histamine than mucosal mast cells, this may be a further indication of differences between the two types. 10 VI. ,Ig§,_Mast Cell§,_and Parasitism Investigating the question of the mechanism of histamine release in rats, Mots (1957) used horse serum sensitization to cause anaphylactic shock, and anaphylatoxin as a histamine releasing agent, to study mast cell secretion. He theorized that there were antibodies on the surface of mast cells and that the cells would react to the antigen or anaphy- latoxin by releasing granule contents and histamine into the blood stream of rats. Mots and Da Silva (1960) reported further definition of the granule extrusion phenomenon in gitgg with rat peritoneal mast cells that had been sensitized with horse serum. Highly reactive cells were washed thoroughly and the release of chemical mediators upon exposure to horse serum provided indirect evidence for the occurrence of specific anti- body on the mast cell surface. Later, Mota (1964a, b) discovered a mast cell-sensitizing antibody which is now considered to be reagin or immunoglobulin E (IgE) in most species. IgE is the major sensitizing antibody involved with allergic re— actions of all types (Austen et a1., 1976; Snider, 1978), and is also one of the hallmarks of antibody response to parasites in all mammalian species. It appears, for example, in response to infection with N, brasiliensis (Ishazaka et a1., 1976), Fasciola hepatica (Day et a1., 1971) and Schistosoma mansoni in rats (Ogilvie et a1, 1966; Rousseaux- Prevost et a1., 1977), in monkeys infected with S, mansoni, and in sheep infected with Trichostrgngylus colubriformis (Ogilvie, 1964). There is a high correlation between elevated levels of serum IgE and infections with Ascaris lumbricoides and/or Necator americanus in human patients (Turner et a1., 1979). 11 A cardinal feature of T, taeniaeformis infections in the rat (Leid and Williams, 1974b) and T, pisiformis infections in the rabbit (Leid and Williams, 1975) is the production of circulating IgE antibody. Leid (1977) demonstrated indirect evidence for mast cell—associated IgE by the antigen-induced release of histamine ig_vitro from peritoneal cells and lung fragments of rats infected with T, taeniaeformis. However, it now appears that not all IgE is on the mast cell surface. Mayrhofer et a1. (1976) established the relationship between intracellular IgE and MMC in rats experimentally infected with N, brasiliensis, and Lindsay (1981) has confirmed this for T, taeniaeformis. By using immunofluo- rescence techniques, IgE can be detected both on the surface of and within MMC, these large numbers of IgE-containing mast cells develop in the duodenum by 42 days postinfection with T, taeniaeformis in rats (Lindsay, 1981). Murray et a1. (1971) speculated that stimulation of intestinal mast cells sensitized with.specific IgE antibodies to N, brasiliensis, leads to increased worm expulsion due, in a large part, to hyper-permeability of the intestine. Mast cell stabilizing drugs such as cortisone, which prevent vasoactive amine release, were found to slow the worm expulsion. These authors suggest that increased permeability in the intestinal epithelium allows a faster movement of antiworm antibodies into the gut lumen. Befus et a1. (1979) measured the histamine concentration of the intestine in conjunction with MMC counts in N, brasiliensis—infected rats. Levels in normal rats averaged 3.4 MMC/VCU but rose to 58 MMC/VCU at 19 DPI with a strongly corresponding (r - 0.88) rise in histamine concentration from 0.5 ug/g to 23.3 ug/g after only ninteen days. 12 Rothwell et a1. (1974) showed that direct infusion of histamine into the gut of guinea pigs caused expulsion of the enteroparasite I, colubriformis. A close association has been established between obser- vations of increased worm expulsion and increased levels of histamine and 5—HT in the guinea pig intestine (Jones et a1., 1974; Jones et a1., 1978). Hypothetically, if histamine is intimately involved with worm expulsion from.the gut, antihistamines should slow or stop the reaction. Rothwell et a1. (1978) tested this hypothesis. Live larvae worms trans— planted into immune animals were not rejected if the recipient animals were first treated with the antiallergic drug, CI 47,917, or the anti- histamine, mepyramine. Although Musoke et a1. (1978) demonstrated that 1. taeniaeformis oncospheres exposed to histamine in _\_r__i_v_o_ and in 1393; show reduced viability and infectivity in rats, the results were varia- ble and the potential for mast cell and histamine influences on sus— ceptibility to this infection remains unknown. Many authors have speculated on the role mast cells play in resis- tance to parasites (wakelin, 1978; Ogilvie, 1974; Dvorak and Dvorak, 1972). It has generally been proposed that vasoactive amines are the most important components, but even though the vasoactive effects of histamine are well known, there has been no unifying theory on the re- lationship between histamine release from mast cells and either micro— environmental modulation or direct parasite death/expulsion in_vivo. VII. Histamine, Hormones and Gastrointestinal Function Many hormones affect stomach function, such as secretin, glucagon, vasoactive intestinal peptide (VIP), gastric inhibitory peptide (GIP), cholecystokinin (CCK), and gastrin; only the latter has a proposed dual 13 role as a secretion stimulator and as a growth hormone. This peptide hormone and its analogs, such as the synthetically produced pentagastrin, apparently regulate acid release (Walsh and Grossman, 1975a, 1975b; Sewaga et a1., 1979; Tani et a1., 1979). Code (1977) suggests that gastrin stimulated acid production in the stomach is controlled by a simple pH feedback system, whereby a low pH inhibits acid secretion and gastrin stimulation. There is now strong evidence for the role of histamine as a common final mediator in acid secretion [for comprehensive reviews see Code, 1977, or Waton, 1971], because H histamine receptor blocking drugs, 2 such as metiamide and cimetidine, inhibit acid release (Tani et a1., 1979; Sewaga et a1., 1979). The gastric histamine—containing cells are of two types. The first is the granulated mast cell such as that iso- lated fromdog stomach fundus by 8011 et a1. (1979). These cells are in the mucosal layer and are similar in both ultrastructure and compound 48/80 responsiveness to MMC of the intestinal lamina propria. The second type is the enterochromaffin cell, first identified in rat stomach by Thunberg (1966). In other species few or no mast cells appear in stomach mucosa and the precise cellular location of histamine stores is unknown (Beaven, 1978). Gastrin and pentagastrin are also growth-stimulating agents for the stomach and intestines. Crean et a1. (1969) reported excessive acid output and mucosal growth in the fundus of the stomach after massive administration of pentagastrin, but saw only a smaller acid increase and no trophic effect after the administration of histamine. Gastrin-like hormone stimulation of DNA synthesis was found to be independent of acid secretory activity ig_vivo because it occurred in the presence of the 14 histamine Hz-blocker metiamide. The tropic action, however, was antago- nized and drastically reduced in the presence of secretin. Histamine alone did not stimulate DNA synthesis in the mucosa (Johnson and Guthrie, 1974). The gastrin—like hormones only have a direct effect on the mucosa, not on the underlying smooth muscle or connective tissue (Johnson, 1976). For example, a direct proliferative response to pentagastrin has been reported for human and rat gastric mucosa in tissue culture, but normal fibroblastic outgrowth was inhibited (Miller et a1., 1973). There are two areas of the gastrointestinal tract that do not undergo growth in the presence of gastrin. Either with injection of pentagastrin or in hypergastrinemic conditions of human Zollinger-Ellison syndrome there is a growth of the gut exclusive of the antral mucosa and esophagus (Mak and Chang, 1976), which seems to be characteristic for this hormone. From a physiological point of view, hormones do not stimulate or influ- ence the gland from which they originate; most of the gastrin-producing G cells are located in the antral region of the stomach. Gastrin is now the only known trophic gastrointestinal hormone. Although histamine has been shown to be associated with tumor growth and wound healing (Kahlson and Rosengren, 1971), it has never been shown to exert a trophic effect, by itself, on gut mucosa or underlying tissue. VIII. Research Objectives The manner in which the development of I, taeniaeformis in the liver influences the rate of growth of the stomach and intestine, the circulating level of gastrin and the proliferation of MMC is not clear. However, the connection established between parasite burden and the 15 degree of hypergastrinemia and mast cell increases suggests a dose-de- pendent interaction between these elements. A clarification of their respective roles may come from further work in the directions initiated by the research reported here. Specifically, the issues addressed are as follows: Can pathological and hormonal changes be induced in non-infected rats experimentally with parasite-derived products? The results of Cook and Williams (1981) suggest that some factor, circulating between parabiotic rats, may be responsible for the gastrointestinal enlargement and mucosal mast cell population increase. If so, the parasite may produce a hormone-like substance which acts to influence growth, similar to that produced by Spirometra mansonoides (Steelman et a1., 1971). How long do the gastrointestinal changes persist? Studies conducted by Cook et al. (1981b) indicate that gastrin levels are erratically elevated in infected rats from 60 up to 100 DPI, but, because the re- lationship between gastrointestinal architecture, gastrin, and continued parasitism thereafter is unknown, it is difficult to predict long term effects. Quantitative studies are required over the extended course of infection. Are there changes in stomach histamine levels which reflect or correlate with changes in gastrin levels, gastric pH, and stomach weight? Histamine is normally involved with stomach function, and some changes in histamine levels might be expected in the pathologically affected organs, but there is no information available on this question in experimental cysticercosis. Finally, what is the relationship between the intestinal mastocy- tosis and histamine levels in I, taeniaeformis-infected rats? There are 16 reports from other host-parasite systems, such as N, brasiliensis and T, spiralis in the rat, that histamine is implicated in expulsion of the worms. However, these worms live in the intestinal lumen. Although there is evidence of MMC proliferation in the intestinal lamina propria of rats with T, taeniaeformis, it is not known if they contain or re- lease histamine. The difference in timing of appearance of MMC in cysticercosis and nippostrongylosis, and the unique sustained high levels of MMC in the former justify an investigation of the histamine content, as a prelude to experimental work on characterization of mast cell secretory functions and responsiveness to parasite antigens and secre- tions. LIST OF REFERENCES LIST OF REFERENCES Austin, K.F., Humphrey, J.H. 1963. lg_vitro studies of the mechanism of anaphylaxis. Adv. Immunol., 3: 3. 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Histamine and the parietal cell. Am. J. Dig. Dis., 16: 921-938. Williams, J.F. 1979. Recent advances in the immunology of cestode infections. J. Parasitol., 65: 337-349. Yurt, R.W., Leid, R.W., Austen, K.F., Silbert, J.E. 1977. Native heparin from rat peritoneal mast cells. J. Biol. Chem., 252: 518-521. CHAPTER I GASTROINTESTINAL HYPERPLASIA IN RATS CHRONICALLY INFECTED WITH TAENIA TAENIAEFORMIS: QUANTITATIVE PATHOLOGICAL AND HORMONAL CHANGES AND ATTEMPTS TO INDUCE THE SYNDROME WITH PARASITE PRODUCTS by David M. Blaies and Jeffrey F. Williams 24 ABSTRACT Hyperplastic pathological changes in the gastrointestinal tract and serum gastrin levels were measured at post-mortem in rats given long—term infections with metacestodes of Taenia taeniaeformis. Stomach weights were greater than in controls from the beginning of the experiment at 63 days postinfection (DPI) onward, and generally reached highest levels (> 16g) in the more chronically infected rats (up to 368 DPI). Gross and histological evidence of papillary hyperplasia of gastric mucosal cells increased with duration of infection. Intestinal weights also increased with time, but to a much lesser degree than those of the stomachs. These changes were sustained throughout the period of study, and were associated with significant elongation of villi and increased mucosal crypt depth, especially in the duodenum. In the most chroni- cally infected rats multiple cystic dilatations of crypts occurred, and within them accumulated masses of mucus and necrotic cells often became calcified. Villi, sometimes over 1 mm in length, became club-like and frequently fused together. Mucosal mast cell (MMC) numbers in the small intestine were significantly higher than in controls throughout in- fection. Hyperplastic changes were detected inconsistently in the colonic mucosa, but there were no effects on the esophagus. Splenomegaly was a constant finding in infected rats, which also showed an earlier onset of thymic atrophy than normal age-matched controls. Serum gastrin levels were elevated in all affected rats at 5 months postinfection, but by the end of the first year some animals had normal gastrin levels, even though they showed severe hyperplastic gastroenteropathy at necropsy. Attempts to induce changes in gastric and intestinal weights, MMC numbers 25 26 or serum gastrin by the serial inoculation of extract or $2 vitro pro- ducts of I, taeniaeformis strobilocerci were unsuccessful. Surgical implantation of strobilocerci intraperitoneally also produced no measur- able changes in these parameters. The results suggest that the hyper- plastic stimulus provided by I, taeniaeformis is sustained for many months, and that there is a gradient of responsiveness in gastrointestin- al tissues, declining from the glandular stomach mucosa posteriorly. ! The stimulus may be augmented by endogenous gastrin, but this hormone is not necessary for maintenance of hyperplastic changes. Our failure to . induce changes $g_vivo artificially suggests that more subtle criteria, perhaps involving 12_vitro influences on gastric or intestinal cell a, turnover, will be necessary to establish if the parasite exerts its effects on host cells directly or indirectly. 27 INDEX DESCRIPTORS Taenia taeniaeformis (Cestoda: Taeniidae); gastric hyperplasia; intestinal hyperplasia; mastocytosis; gastrin; intraperitoneal implan- tation. ii; I- INTRODUCTION Rats infected with the hepatic metacestodes of Taenia taeniaeformis develop severe hyperplastic changes in the stomach and intestine which become grossly evident by 45 days postinfection (DPI) (Cook and Williams, 1981). The degree of hyperplasia is extraordinary, especially in the stomach which may reach twenty times the normal weight. Accompanying E. microscopic changes over the first 70 DPI include cystic gastric mucosal ) hyperplasia, increases in intestinal villar length and intense mast cell proliferation in the intestinal lamina propria. The gastric luminal pH becomes elevated, and circulating levels of the hormone gastrin rise abruptly during the first two months of infection; however, hypergastri- nemia is not a prerequisite for induction of the lesions (Cook et a1., 1981). The mechanism whereby these changes are brought about is unknown, but similarities to some of the pathological and hormonal alterations caused by gastrointestinal nematodiasis (e.g., ostertagiasis,[Anderson et a1., 1976]; trichinellosis,[Castro et a1., 1976]) are striking and suggestive of common underlying pathogenetic processes. The physical separation of larvae of I, taeniaeformis from the sites of hyperplasia, combined with the observation that all the lesions can be produced in noninfected rats by parabiotic union to parasitized animals (Cook et a1., 1981), indicate that chemical mediators may be involved. Furthermore, concomitant onset of a complex of uncontrolled mucosal cell proliferation, aberrant gastric acid secretion and hypergastrinemia suggests that the normal regulatory and homeostatic mechanisms of gastrointestinal growth and secretion are being compromised at a fundamental level. 28 29 We have extended our characterization of this parasite-induced gastroenteropathy and report here on the nature of mucosal and hormonal abnormalities in rats from 2 to 12 months PI, and on our attempts to stimulate gastrointestinal and hormonal changes in normal rats with inoculations of parasite-derived extracts or products, and by surgical implantation of larvae. We found a progressive distortion of mucosal architecture in affected tissues, over the long term, and persistence of E! severe pathologic changes even when circulating gastrin eventually de— i clined to the normal level. We were unable to induce pathological or . i hormonal changes either by parenteral administration of parasite products or by prolonged exposure to live parasites in the peritoneal cavity. MATERIALS AND METHODS Experimental infection: The characteristics of the strain of T, taeniaeformis and procedures for the routine maintenance of the parasite in our laboratory have been described recently by Williams et a1. (1981). Pathologgcal changes in chronically infected rats: Pathological changes were measured in a group of 38 female Spartan (Spb: [SD]) rats (Spartan Research Animals, Haslett, Michigan) given 1000-2000 eggs orally and killed at intervals over 63-231 DPI, as specified in the results. Twenty-six age-matched normal rats served as controls. The animals were killed by exposure to CO vapor and exsan- 2 guinated; wet weights were then recorded for the liver, spleen, thymus, stomach and small intestine. Tissue samples from the stomach, duodenum, jejunum and ileum were fixed in formalin and stained with haematoxylin eosin. Tissues from each small intestinal segment were also fixed in Carnoy's solution and stained with Astra blue by the method of Blaies and Williams (1981). Samples of esophagus and colon were taken from some groups, but were not routinely included because these tissues were not generally affected even in rats showing severe gastroenteropathy. Quantitative evaluation of small intestinal changes was based on measurement of villar length, crypt depth, and thickness of the smooth muscle layer and its overlying connective tissue at 5 sites on a complete transverse section of duodenum, jejunum and ileum from each rat. In the colon the thickness of the mucosal layer and the height of the rugae were measured. Only the overall thickness of the esophageal wall was measured. Mucosal mast cell (MMC) counts per villus crypt unit (VCU) in the small 30 31 intestine were made on the Astra blue-stained sections, as described by Cook and Williams (1981). Arithmetic mean values for all variables were computed for each rat, and for the purpose of statistical comparison of trends over the course of infection, the rats were considered to form two groups: those killed 40-150 DPI, and the remainder killed 151-240 DPI. Data from each group were analyzed by Student's "t" test. Gastrin levels in chronically infected rats: Serum gastrin levels were examined in a second group of 46 chroni— cally infected rats killed 155-368 DPI. Control samples were drawn from 12 normal uninfected female rats aged 166-223 days. Gastrin was measured by radio-immunoassay with a commercial kit (Beckton-Dickinson). Fresh wet weights for stomach and small intestine were recorded for all rats. Attempts to induce hyperplasia by parenteral inoculations or surgical implants: Stomach and small intestine weights, MMC counts and serum gastrin levels at necropsy were recorded in two series of rats in which we attempted to induce changes by either serial inoculations of parasite- derived products or surgical implantation of live parasites into the peritoneal cavity. Saline soluble extracts (SS) of strobilocerci and products from worms maintained i2 giggg (IVP) for 24 hours were prepared as described by Ayuya and Williams (1979). Protein estimations were made by the Lowry method. Subcutaneous inoculations of SS or IVP were given twice daily for 3 weeks to groups of 8 twenty-eight day old female Spartan rats. Each dose contained at least 0.5 mg (protein) SS or 0.05 mg IVP. Age-matched control groups were given twice daily inoculations of 0.15 M NaCl, or 1000 eggs of T, taeniaeformis orally on Day 0, or no treatment. All groups were killed after 21 days. 32 Strobilocerci were extracted from hepatic cysts and implanted intra- peritoneally into groups of 6 rats as described by Musoke and Williams (1976). Recipients received either 10 or 40 parasites and were sacri- ficed after 70 and 21 days, respectively. Gross organ weights, MMC counts and serum gastrin levels were compared with values obtained from age-matched uninfected and infected control groups killed in parallel. Gastrin levels in these rats were measured in the laboratory of Dr. Lenard Lichtenberger, Department of Physiology, The University of Texas, Houston, using the RIA procedure described by Cook et a1. (1981). RESULTS Chronic patholggical changgg: Stomach wet weights from the first (38) and second (46) groups of chronically infected rats are shown in Figure 1. From 63 DPI onwards infected rats generally had larger stomachs than the controls; those which did not always had low numbers of viable parasites (liver weight 30 g or below). However, some rats which had few parasites developed . . -,—..~.—-g-— _ u—T enlarged stomachs. The range for stomach weights in all normal unin- fected rats was 0.8-2.25 g. There was a trend toward increasing stomach weight with duration of infection but there were many exceptions. Some individuals developed very heavy stomachs relatively early in in- fection. Histopathologically, the gastric mucosa in all affected rats showed severe cystic hyperplastic changes, with occasional hyperemic papillary_outgrowths into the lumen, up to 1.5 cm in diameter as shown in Figure 2. These were more pronounced in rats killed in the later stages of infection (151-240 DPI). Splenic weights of infected rats in the first group exceeded those of controls at all ages (Figure 3). Thymic weights declined abruptly in infected rats in the early stages, but did not differ from controls in the 151-240 DPI period (Figure 3). Small intestinal weights exceeded those of controlsanzall stages of infection, but the extent of the change was markedly less than that seen in the affected stomachs. Intestinal hyperplasia persisted but did not increase significantly after 151 DPI. Quantitative aspects of histopathological changes in the small in- testines of 36 infected rats are shown in Figure 4. The hyperplastic changes were manifested in all 3 segments of the small bowel, but were 33 FIGURE 1 Stomach weights at necropsy of rats infected with Taenia taeniaeformis. In normal rats the average was 1.5 i 0.08 g. 34 00¢ ZOECMuZ. thm w>>Cm 18' 1s- 14- 12» 1oL al- a 4L 2r 1.5- m<<