((155513 ..__*.. 94“.: 1"sL III- ,f: 1' II‘ IFI’ . II! 1'... ’O‘il" lpiljll'Igl' ‘. ' zit} ., ABSTRACT PERMEABILITY STUDIES ON TAENIID METACESTODES BY Stephen T. Hustead Host immunoglobulins of several different classes were detected within the bladder fluids of Taenia taeniaeformis, Taenia crassiceps, and Echinococcus granulosus. Radioiodinated proteins were taken up in vitro by larvae of both T. taeniaeformis and T. crassiceps and were shown to retain their physicochemical and antigenic character- istics. Rates of uptake were similar in the two species and were not related to the molecular weight of the proteins. Immunoglobulins were taken up both in vitro and in vivo by larvae of T. taeniaeformis. Absorbed immunoglobulins were shown to retain both antigen binding capacity and biologic functions associated with the Fc portion of the molecules. Not all cysts of E. granulosus contained detectable host proteins. This may be attributable to proteolysis within the bladder fluid, since uptake of 1125 occurred when hydatid cysts were exposed to labeled proteins in vitro, but rapid degradation of the labeled carrier led to the appearance of dialyzable fragments. Incubation in immune rat serum (IRS) was shown to increase the . 2 rate of absorption of I1 5 RNase—A but not 1125 BSA by larvae of Stephen T. Hustead T. taeniaeformis and T. crassiceps. This effect required a heat labile factor in serum, and partial activity could be restored in heat treated IRS (HI—IRS) by adding normal rat serum (NRS) as a source of complement (C'). In addition, the effectiveness of IRS in altering permeability was shown to be dependent on the available concentration of functional C'. Both live and dead larvae incu- bated in NRS rapidly depleted hemolytic C' levels in the surrounding medium. Immunoglobulin fractions from IRS separated by anion exchange chromatography and gel filtration were tested in the presence of excess complement for their ability to increase uptake of 1125 RNase-A. Enhanced permeability was observed in larvae incubated in each fraction. It appears that taeniid metacestodes are capable of absorbing a variety of proteins without demonstrable loss in their structural or functional integrity following transport. However, this absorptive capacity, which most likely accounts for the presence of host serum components within the bladder fluids, can be altered in vitro by incubating larvae in antibody and complement. The observation that larvae restore normal control with time suggests that they avoid this immunologic effector mechanism in vivo by interfering with complement function. PERMEABILITY STUDIES ON TAENIID METACESTODES BY Stephen T. Hustead 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 1976 Dedicated to Saundra Her Spirit has been my inspiration "Life is but a tiny piece of our soul's link with eternity" VKA ii ACKNOWLEDGEMENTS I wish to express my sincere appreciation to the Department of Microbiology and Public Health, Michigan State University, for the assistantship awarded to me during the course of my studies. The advice, encouragement, and careful consideration in all aspects of this work by my guidance committee, Drs. Donald Twohy, Gordon Carter, Richard Patrick, and Ralph Costilow, are gratefully acknowledged. I have particularly enjoyed the professional and social experiences with my fellow students, Drs. Antony Musoke, Roger Cook, Bruce Hammerberg, Wes Leid, Ashraf Ansari, Joseph Ayuya, Mr. John Picone, Ms. Anna Zajac, and Ms. Martha Calkins, which have led to lasting friendships, worldwide. I am extremely grate- ful for the expert technical assistance rendered by Ms. Marla Signs during each phase of this study. Above all I wish to express my sincere appreciation to Dr. Jeffrey F. Williams, my major advisor. His enthusiasm, encourage- ment, and personal concern shown each of his students are an inspiration to us all. iii TABLE OF CONTENTS INTRODUCTION 0 O O C O O O O O O O C I O O O O I 0 O O O O 0 LITERATURE REVIEW. 0 O O I C O O O O C C O O O O I O O O O C Biology of Taeniid Infections . . . . . . . . . . . . Parasites of Man . . . . . . . . . . . . . . . Taenia solium . . . . . . . . . . . . . Taenia saginata . . . . . . . . . . . . Parasites of Man and Laboratory Animals. . . . Echinococcus granulosus . . . . . . . . Parasites of Laboratory Animals. . . . . . . . Taenia taeniaeformis. . . . . . . . . . Taenia crassiceps . . . . . . . . . . . The Host-Parasite Relationship. . . . . . . . . . . . Immunologic Responses to Infection with Taeniid Parasites. . . . . . . . . . . . . . Proposed Mechanisms of Survival of Tissue Parasites. . . . . . . . . . . . . . . . . . Membrane Transport. . . . . . . . . . . . . . . . . . Theoretical Models of Small Molecule Transport Theoretical Models of Protein Transport. . . . Membrane Permeability Associated with the Cestoda . . Uptake of Sugars . . . . . . . . . . . . . . . Uptake of Amino Acids. . . . . . . . . . . . . Uptake of Macromolecules . . . . . . . . . . . REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . ARTICLE 1 - PERMEABILITY STUDIES ON TAENIID METACESTODES: I. UPTAKE OF PROTEINS BY LARVAL STAGES OF TAENIA TAENIAEFORMIS, TAENIA CRASSICEPS, ECHINOCOCCUS GRANULOSUS. . . . . . . . . . . . . ARTICLE 2 - PERMEABILITY STUDIES ON TAENIID METACESTODES: II. ANTIBODY MEDIATED EFFECTS ON MEMBRANE PERMEABILITY IN LARVAE OF TAENIA TAENIAEFORMIS AND TAENIA CRASSICEPS. . . . . . . . . . . . . . iv Page mmmwww 10 10 13 13 16 2O 21 23 25 25 27 28 31 42 69 LIST OF TABLES Table Page ARTICLE 1 l Uptake of homologous and.heterologous host antibody in vitro by larvae of Taenia taeniaeformis by indirect hemagglutination (IHA) and passive cutaneous anaphylaxis (PCA) . . . . . . . . . . . . . . . . . . . 58 125 . . 2 Uptake of I bov1ne serum albumin (BSA) by hydatid cysts of Echinococcus granulosus in vitro . . . 60 125 . 3 Uptake of I rat IgG2 by hydatid cysts of Echinococcus granulosus in vitro. . . . . . . . . . . . 61 Figure LIST OF FIGURES Page ARTICLE 1 1 Host immunoglobulins detected by immunoelectro- phoresis in the bladder fluids of T. taeniaeformis, T. crassiceps, and E. granulosus. . . . . . . . . . . . 52 Autoradioqraphy of bladder fluids from larvae of T. taeniaeformis (a) and T. crassiceps (b) incubated in 1125 labeled RNase-A, BSA, or rat IgGZ. Sup- plemented Eagle's medium (c) was assayed similarly and served as the control . . . . . . . . . . . . . . . 55 125 . Rates of uptake of I labeled protein by larvae of T. taeniaeformis, T. crassiceps, and E. granulosus incubated in vitro in Eagle's medium containing the labeled carriers. . . . . . . . . . . . . . . . . . . . 57 ARTICLE 2 1 Uptake of 1125 BSA (1a) or 1125 RNase—A (1b) by larvae of T. taeniaeformis and T. crassiceps following incubation at 37 C in IRS, HI-IRS, or NRS. Larvae were removed from serum preparations at 30 min intervals and exposed to radiolabeled carriers for 10 mins in each case . . . . . . . . . . . 75 Effect of incubation with live or dead larvae of T. taeniaeformis or T. crassiceps on hemolytic complement levels in NRS. Fifty larvae were added to 50 ml of Eagle's medium containing 20% NRS. One milliliter samples were removed at 30 min intervals and assayed by immune hemolysis. Cul- ture medium maintained without larvae but treated similarly served as the control . . . . . . . . . . . . 78 Limiting effect of complement on IRS mediated alterations in permeability of larvae of T. taeniae- formis and T. crassiceps to 1125 RNase-A. . . . . . . . 81 vi INTRODUCT ION Cysticercosis is characterized by infection with the larval stages of tapeworms belonging to the family Taeniidae. Although this group of diseases does pose a public health threat to man in endemic regions, they have become, above all, a major economic concern in most of the developing nations of the world, where well over 50 percent of the bovine carcasses show signs of infection. The legal sale of infected carcasses is prohibited, and thus cysticercosis has severely curtailed the growth of a profitable meat industry in these nations. No chemotherapeutic agents specific for larval tapeworms have been marketed to date. Recently some promising agents have emerged, and the results from preliminary field studies suggest that satisfactory drug therapy may be possible shortly. However, because of the lack of specific treatment, a variety of methods of control have been attempted, including extensive programs in health education and increased control and tighter restrictions on meat inspection processes, each with limited effectiveness. Pre- viously successful methods of control of other diseases were achieved following extensive investigation of the immunologic processes associated with infection. It is likely that such an approach is warranted with cysticercosis. However, at present even the basic biology of taeniid parasites is poorly understood, and must receive 1 2 more attention by trained investigators. The use of large domesti- cated animals in these studies is prohibited by cost, but infection with taeniid metacestodes does occur in small rodents and these parasite systems may serve as experimental models of cysticercosis. This study was designed to firstly identify the host immunoglobulin classes present in bladder fluid obtained from taeniid metacestodes, and then to conclusively demonstrate the origin of these macro- molecules. Further studies were concerned with examining the effects of the immune response on permeability. The literature review has been organized to provide the neces- sary background information considered crucial for the understanding of the work reported herein. The first section deals initially with the medically important taeniid parasites of man, and is followed by a comprehensive review of the experimental model systems used for investigative purposes. The second section deals with the immune responses associated with infection by taeniid organisms, including the current theories concerning concomitant immunity, a phenomenon frequently observed in helminth infections. The follow— ing section deals with membrane permeability and the theoretical models of small and large molecule transport. In the last section the mechanisms of transport associated with the Cestoda are reviewed in detail. LITERATURE REVIEW Biologypof Taeniid Infections In 1947 Stoll estimated that well over 40 million people in the world, particularly inhabitants of Asia, Africa, and the U.S.S.R., were infected with one of the two medically important species of the family Taeniidae: Taenia solium or Taenia saginata. Domestic animals, including the cow and the pig, are the primary intermediate hosts in these cyclo-zoonotic infections. Due to the increasing prevalence in food animals, taeniiasis has become an important economic problem affecting especially the less developed nations, which are prevented from exporting their contaminated meat products. In this section the biology of the two major human patho- gens is reviewed, followed by a detailed account of the experimental models used in laboratory investigations. Parasites of Man Taenia solium Taenia solium, commonly referred to as the "pork tapeworm", resides as an adult cestode in the lumen of the small intestine of man, and the pig serves as the intermediate host. Although the parasite is cosmopolitan in distribution, the highest prevalence of infection is recorded wherever man consumes raw or partially cooked pork. The most afflicted endemic areas currently include the Slavic 3 4 countries, Latin American countries, Mexico, and North China (Soulsby, 1968). Life Cycle MAN \ CYSTICERCUS EGG \\ (Cysticercus cellulosae) PIG MAN Man is the only definitive host and is readily infected by ingesting the cysticercus in inadequately prepared pork. However, humans are also susceptible to larval infection, commonly resulting from ingestion of eggs in contaminated food, or by the process of reverse peristalsis whereby eggs in the bowel are carried upwards to the duodenum and are stimulated to hatch. The ingested eggs hatch following disruption of the embryophore, composed of keratinized blocks. Hexacanth embryos surrounded by only a single limiting membrane are liberated into the intestine and become activated before penetrating the mucosa. Little is known regarding the hatching and activating conditions for T. solium. Following penetration the oncospheres are carried via the blood to the skeletal musculature where they develop into mature cysticerci, called Cysticercus cellulosae. The mechanism of penetration is not known, but several investigators have proposed that enzymatic digestion occurs during this process, leading to the development of clear refractile zones surrounding oncospheres observed in 5 histologic sections of the intestinal wall (Silverman and Maneely, 1955; Heath, 1971). Adult worms reside in the small intestine and may attain a length of several meters. The muscular scolex bearing four suckers and a double crown of prominent hooks functions to secure the worm to the mucosal surface. Large gravid segments are passed in the stool, and each may contain up to 40,000 eggs. Segments of T. solium, unlike those of T. saginata, are inactive in the feces, and eggs are not dispersed. This may perhaps explain the frequently acquired massive infections in pigs (Soulsby, 1968). Pathology. For the most part, infection with the adult worm is asymptomatic. Occasionally, mucosal irritation or intestinal obstruction occurs where the worm burden is heavy. Clinical problems more commonly result from infection with the larval stage, resulting from either ingestion of eggs or reverse peristalsis. Since the infected patient is a constant source of viable eggs, he is both a danger to himself and to those with whom he comes in contact. Cysticerci may be found in every organ of the body of man, but are primarily encountered in the subcutaneous tissues, the muscles, and the eye. Occasionally larvae develop in the brain. Surprisingly, little pathologic reaction occurs around the living parasite, but when the organism dies, intense tissue reaction follows and a variety of neurological symptoms may develop, including epilepsy, incoordination, transient paresis, meningoencephalitis, and failing vision (Faust et al., 1970). Taenia saginata Taenia saginata, referred to as the "beef tapeworm", has a world-wide distribution, occurring most commonly in Africa, Asia, and the U.S.S.R. In contrast to T. solium, infection with T. saginata is frequently encountered in the United States, particu- larly in the Southwest. Life Cycle CYSTICERCUS (Cysticercus bovis) ‘//////////GG CATTLE The life cycle of T. saginata is very similar to that of T. solium. The adult worm resides in the small intestine of man, who acquires the infection from the ingestion of raw or insufficiently cooked beef. The preference for very rare steak is one important feature of successful transmission in the United States. Embryonated eggs passed in the feces of man are ingested by cattle and result in the eventual development of infective cysticerci. Fortunately, man is not susceptible to the tissue phase of this parasite. The bladder form, termed Cysticercus bovis, is usually found in the intermuscular fascial layers surrounded by a connective tissue capsule. Larval viability is maintained from 4-6 months, at which time the cysticerci begin to degenerate and eventually die by the ninth month of infection. 7 Adults of T. saginata in man attain lengths exceeding twenty meters. The scolex bears four muscular suckers but, unlike T. solium, the armed rostellum is absent, and this alone may serve to differen- tiate the two species. Generally the gravid proglottids of T. saginata are longer than those of T. solium, and each may contain nearly 100,000 eggs. Patholggy. In View of the fact that T. saginata may reach lengths in excess of 20 meters, about three times as long as the entire human intestine, it is not difficult to understand that disruptive effects on normal digestive tract functions frequently occur following infection with this worm. Since the majority of cysticerci die by the ninth month of infection, little Clinical disease is manifested in infected cattle under normal conditions. The presence of cysticerci in beef car- casses results in a serious economic loss, particularly in developing nations. Successful treatment of infected persons is possible with various taeniicidal agents. Unfortunately, because of unsatisfactory control over meat inspection procedures, reinfection is inevitable. Until recently no chemotherapeutic agent specific for larval tape— worms was available. However, as a result of a number of intensive testing programs, primarily with laboratory models, some promising agents have emerged. Although the majority of these drugs are still in preliminary trial stages, successful drug therapy may become feasible in the future. 8 Parasites of Man and Laboratory Animals Echinococcus granulosus Adults of Echinococcus granulosus inhabit the small intestine of a number of wild carnivores, particularly the dog. Man is a frequent accidental intermediate host, chiefly in the endemic regions of Africa, South America, Australia, and New Zealand, but transmis— sion normally occurs through a variety of herbivorous mammals. Life Cycle /DOG\ HYDATID CYST EGG ‘{//////////// SHEEP The minute adult tapeworm is seen only in dogs and other Canidae, and cannot develop in man. The worm measures between 3 and 6 mm and possesses a scolex, neck, and usually three proglottids: one immature, one mature, and one gravid segment. The ova released in the feces are indistinguishable from those of other taeniid species. Intermediate hosts, including domestic and wild animals, and man are infected by ingestion of the eggs. Eggs hatch and activate in the small intestine, penetrate the mucosa, and enter the blood stream to be carried to internal organs. The hydatid cyst consists of an external acellular and internal germinative membrane similar 9 to that of adult cestodes (Bortoletti and Ferretti, 1973). Brood capsules attached to the germinative layer develop internally. They may detach from this layer and, upon rupture of the cyst wall, they are expelled and develop into daughter cysts. Laboratory investigations with the larvae of E. granulosus have been limited to some extent by the high cost involved in maintaining adequate numbers of domestic herbivores. Inoculations of scolices or brood capsules have been used in attempts to propa— gate the organism in mice and rabbits (Deve, 1933, 1935), but more satisfactory growth rates have been attained by serial intraperi— toneal implantation of the cysts into rats and gerbils (Varela—Diaz et al., 1974). Pathology. Infection with the adult form is usually asympto— matic. The hydatid cyst grows slowly but steadily and may after years contain liters of fluid. The site of cyst development determines the rate of growth, but often serious functional impair» ment of vital structures results. This is of prime importance in the pathogenesis of hydatid disease. In addition, traumatic hydatid cyst rupture may provoke anaphylactic-like reactions which can be fatal. To date surgical intervention is the only available treatment, and this is possible only when cysts are located in operable sites. It is essential that dogs be prevented from feeding on animal car— casses in endemic regions if infection rates are to be limited in man and domestic animals. 10 Parasites of Laboratory Animals Taenia taeniaefbrmis The adult form resides in the small intestine of the cat and related carnivores, including the stoat, fox, and lynx, and is of cosmopolitan distribution. Rodents, chiefly rats and mice, serve as the intermediate hosts. Life Cycle CAT \ CYSTICERCUS EGG (Cysticercus fasciolaris) The bladderworm stage, Cysticercus fasciolaris, occurs in the livers of the intermediate hosts. The original account of larval development from the egg was provided in 1855 by K. G. F. R. Leuckhart, and further details were given by Raum in 1883. As with all taeniid ova, the oncospheres hatch in the intestine, penetrate the intestinal tissue, and enter the circulatory system. Unfortunately, the invasive mechanism is poorly understood. There is some evidence that carbohydrate complexes in the intestinal mucosa are broken down in the area of oncospheral penetration (Banerjee and Singh, 1969), but at the moment all proposed modes of invasion are clearly specula- tive. Each larva which reaches the liver becomes encapsulated by dense proliferative fibrous tissue. Mast cells and eosinOphils are ll irregularly distributed throughout the fibrous capsule in the cyst- wall (Varute, 1971). Within this cyst wall the larva develops to a segmented strobilate stage in which the scolex has evaginated, giving it the appearance of a small tapeworm. Upon ingestion by the definitive host the strobila and attached scolex develop within 6 weeks to an adult tapeworm. Pathology. Occasional digestive disturbance has been described. In addition, the scolex of the adult may become deeply embedded in the mucosa of the small intestine, leading to perforation. Although the cysticercus appear to be harmless even in large numbers, infec— tion is believed to predispose rats to the development of a variety of hepatic tumors, described by Bullock and Curtis (1920). Taenia crassiceps Adult Taenia crassiceps frequently inhabit the intestine of the red fox (Vulpus vulpes) in Europe and the arctic fox (Alopex lagopus invitus) in North America. The metacestode, Cysticercus longicollis, commonly infects small rodents and lemmings (Dicrostomyx groenlandicus) (Rausch, 1952; Freeman, 1962). Recently the first human infection was described when a cysticercus, surgically removed from the eye of a Canadian woman, was positively identified as T. crassiceps (Shea et al., 1973; Freeman et al., 1973). 12 Life Cycle FOX / (canidS) CYSTICERCUS EGG (Cysticercus longicollis) .\\\\(9) \\\\\\\\\\ MAN MICE (Small Rodents) In nature small rodents are infected by ingestion of the eggs passed in the feces of the fox or related Canids, who have previously eaten rodents infected with the developing metacestode. Typically the ingested eggs hatch to oncospheres in the intestine and then migrate to the subcutaneous tissues or the peritoneal cavity. Infec- tivity is achieved within 2 months. In the definitive host at least 5-6 weeks are necessary for development to a mature egg-producing adult to be completed. In the laboratory experimental infection has been achieved in the dog. The metacestode stage has been maintained in white mice by serial intraperitoneal transfer for generations, but there is an associated loss of infectivity for dogs (Freeman, 1962). Numerous investigators have attempted to explain the morphologic, reproductive, and antigenic abnormalities which have been ascribed to the non— infective or ORF strain (Mount, 1968; Fox et al., 1971). Data seem to substantiate the hypothesis that these features result from a genetic mutation in the ORF strain (Dorais and Esch, 1969; Smith et al., 1972). l3 Pathology. Other than a few reports of enteritis and digestive disturbances in dogs, little is known about the pathology of T. crassiceps in either the definitive or intermediate hosts. In the only reported human case partial visual function was restored upon surgical excision of the parasite from the retina. Interestingly, the family's pet dog was incriminated as the reservoir host pointing to yet another instance of taeniiasis as a public health problem. Perhaps the simplicity of maintaining this parasite in the laboratory combined with the recent report of human infection will arouse renewed interest in this rather neglected host-parasite system. The Host-Parasite Relationship In this portion of the literature review, a general discussion of the early and recent investigations in cestode immunology is presented with emphasis on the Taenia taeniaeformis system. This is followed by a detailed review of the mechanisms proposed to account for the phenomenon of concomitant immunity which character_ izes many helminth infections, including cysticercosis. Immunologic Resppnses to Infection with Taeniid Parasites Early investigations in cestode immunology began with the work of Miller (1931a), who demonstrated conclusively that rats infected with Taenia taeniaeformis are resistant to super-infection. Further experimental work definitively established the role of antibody in protectioh against this infection. Resistance was shown to be passively transferable with serum, and was transferred naturally 14 from mother to young (Miller and Gardiner, 1932, 1934; Miller, 1931b, 1932, 1935; Campbell, 1936, 1938a,b,c). Successful artificial immu- nization procedures were also developed (Miller, 1931b; Campbell, 1936). Immunity to T. taeniaeformis and T. crassiceps was successfully stimulated by implantation of live parasites or extracts (Miller, 1932; Freeman, 1962). Vaccination with extracts of T. pisiformis has also been shown to produce detectable immunity in rabbits (Kerr, 1934). However, a greater degree of immunity is generally established when animals are vaccinated with live material. Similar findings have been reported for Hymenolepis nana in mice (Kerr, 1935; Hearin, 1941). Leid and Williams (1974a) extended these findings by demonstrat- ing that in the rat antibodies of a single immunoglobulin class (7Sy2a) are primarily responsible for the passive transfer of resistance to T. taeniaeformis. Experimental infection was then studied in the mouse system and again antibodies from one immuno- globulin class (7Syl) were shown to mediate resistance (Musoke and Williams, 1975a). It should be noted that mouse 7Sy1 immunoglobulins appear to have analogous biologic functions to rat 7872a immuno- globulins. In contrast, Musoke and Williams (in press) have recently demonstrated that immune serum fractions containing 7Syl and yM immunoglobulins from rats infected with T. taeniaeformis by intra— peritoneal implantation were most effective in passive transfer. Miller's (1935) finding that immunity to T. taeniaeformis could be transferred from mothers to their young was recently substantiated by Musoke, Williams, Leid, and Williams (1975). In addition, they demonstrated that the protective activity resided in the yA rich 15 fraction of immune colostrum. Although colostrum mediated transfer of resistance has been reported in T. hydatigena and T. ovis infec- tions, this appears to be the first report incriminating yA (Gemmell, Blundell-Hasell, and Macnamara, 1969; Rickard and Arundel, 1974). The mechanism of action of protective antibodies is unknown, but there is some evidence that immune destruction occurs prior to encystment and that the intestine plays a vital role in the acquired resistance to super-infection (Leonard and Leonard, 1941; Froyd and Round, 1960). Histological studies have revealed that epithelial cells may be lysed by penetrating oncospheres as they move into the lamina propria en route to the liver via the blood circulatory system (Banerjee and Singh, 1969; Heath, 1971). Recently Musoke and Williams (1975b) have presented experimental evidence demonstrat- ing the dependence of antibody on other humoral factors to achieve effective killing of invading oncospheres. They showed that prior to the 5th day of infection antibody mediated attack required an intact complement system in the host. The only other attempt to implicate complement in immunity to helminth infections was unsuc- cessful. Jones and Ogilvie (1971) failed to demonstrate the active involvement of complement in the phenomenon of worm expulsion by rats infected with Nippostrongylus brasiliensis. There have been few studies on the effects of antibody on the physiology and metabolism of either protozoan or metazoan para- sites. Incubation in immune serum apparently reduces oxygen consump- tion in Trypanosoma vivax and T. lewisi (Desowitz, 1956; Lincicome and Hill, 1965), disrupts protein and DNA synthesis in T. lewisi (Taliaferro and Pizzi, 1960), and inhibits amino acid incorporation 16 into protein by extracellular Plasmodium knowlesi (Cohen and Butcher, 1970). Murrell (1971) has presented some evidence that antibody- mediated complement dependent reactions were responsible for disrupting membrane permeability in Vitro in larvae of T. taeniaefbrmis. More recently Dean et a1. (1974) presented evidence that killing of schistosomula of Schistosoma mansoni by antibody and heat labile serum factors was greatly enhanced by the addition of polymorpho- nuclear leukocytes. Later they showed that although eosinophils and macrophages did not increase the rate of killing, they did react with schistosomula that had previously been damaged or killed by antiserum (Dean et al., 1975). It is becoming clear that in a number of parasitic infections acquired immunity may depend on the combined efforts of several immunological effector mechanisms, both humoral and cellular. Further studies characterizing these mechanisms are essential if the phenomenon of concomitant immunity is to be understood. Parasite induced inhibi- tion of effector systems should not be overlooked as a contributing factor in this process; Proposed Mechanisms of Survival of Tissue Parasites As advances were made in the understanding of cestode immunology a number of hypotheses were proposed to account for the resistance of the developing tissue phases in immune animals to inherent host defense mechanisms. Although concomitant immunity occurs in many helminthiases, this is a particularly perplexing aspect of taeniid metacestode infections since protective immunity is antibody mediated, yet l7 immunoglobulins appear to be present in and on the cystic larval forms surviving in the tissues. Chordi and Kagan (1965) first demonstrated that host-like proteins including immunoglobulins are present in hydatid cyst fluid. Further studies showed that immunoglobulins antigenically similar to host molecules could be extracted from cyst membranes (Varela-Diaz and Coltorti, 1973). In recent work attempts to determine the origin of these molecules have been inconclusive. Although their role in survival of the organsim is not at all clear, the fact that host or host—like plasma proteins are present is now well established and mechanisms proposed to explain the resistance of larval tissue phases in otherwise immune hosts must take this into account. Four major philosophies have evolved to explain con- comitant immunity as follows: A.) Molecular mimicry is the term applied to the occurrence of antigenic sharing between host and parasite as a result of evo- lutionary adaptation influenced by natural selection (Sprent, 1962; Dineen, 1963; Damian, 1964). Initially Sprent advanced the hypothesis that as both parasites and hosts adapt during evolutionary develop- ment they undergo reciprocal changes leading to the emergence of host specificity. It is postulated that as the host immune system evolved, those parasites were selected for an antigenic mosaic similar to that of the host. However, Dineen realized that following the principles of natural selection parasite antigens capable of stimu- lating resistance in the host would also be selected. In 1964 Damian was credited with unifying the general thoughts on this process of evolutionary adaptation and formally presenting it as a hypothesis of parasite survival. 18 B.) In the host induction hypothesis it is proposed that the parasite becomes able to synthesize macromolecules antigenidally identical to host components, and incorporate them into external surfaces (Capron et al., 1968). It has been pointed out that if a parasite were capable of responding to inductive stimuli by forma- tion of host components an extraordinary proportion of the genome would be required to code for such a variety of antigenic determinants. For example, the presence of donor and recipient host-associated immunoglobulins has been revealed in hydatid cysts transplanted from mice to guinea pigs (Varela-Diaz and Coltorti, 1972). For the host induction hypothesis to be valid the parasite would either have to be capable of reverse transcription and reverse translation, although the latter has never been demonstrated in any model system, or contain an unusually diverse genome comprised of genes coding for various serum proteins antigenically identical to those found in the array of possible hosts. Nevertheless, recent findings reported by Capron and his co-workers have been interpreted in support of this hypothesis. They demonstrated uptake in vitro of radiolabeled isoleucine and lysine by Schistosoma mansoni and incorporation into host-like antigens recovered from the surrounding culture medium (Bout, Capron, Dupas, and Capron, 1974). This strongly suggests that indeed similar genetic codes are present in the parasite genome. C.) Phenotypic adaptation differs from host induction in that host synthesized components are utilized and incorporated into external surfaces (Smithers and Terry, 1969). According to this hypothesis the invading parasite coats itself with antigenic determi- nants shed or actively cleaved from host cells. Host immune responses 19 are avoided since the parasite will antigenically resemble host tissue. Recent work with Schistosoma mansoni has provided most of the evidence in support of this view, but as yet conclusive and repeatable findings are lacking. In order to accept this hypothesis it is necessary to assume that parasites produce enzymes capable of cleaving membrane bound proteins and that their membranes contain receptors capable of binding these fragments in a similar configura- tion to self or host determinants. D.) Bound host antibody, a hypothesis similar to phenotypic adaptation, was formally presented by Varela-Diaz et a1. (1972) and supported in part by Rickard (1974). It is proposed that specific antibody produced against parasite antigens is responsible for neutralizing an effective immunologically mediated attack. Varela— Diaz et al. have proposed that two antibodies are involved, one neutral and one lethal. When both combine with the membrane bound antigenic determinants in juxtaposition, the resulting steric hindrance blocks the action of the lethal antibody. Rickard (1974) could not accept the simplicity of Damian's molecular mimicry model. Indeed parasites and hosts have adapted during evolution to ensure mutual survival. This is well documented by the frequently encountered phenomena of concomitant immunity observed in numerous larval helminth infections. However, animals infected with developing larval forms have been shown to be insuscep- tible to super~infection. Should such a regulatory mechanism be lacking, larval forms would accumulate with lethal consequences. Conversely, the development of complete resistance with rejection of all forms would endanger the survival of the entire parasite 20 population. In an attempt to explain each of these phenomena Rickard proposed that invading embryos do elicit a significant immune response fully capable of mediating resistance, but that they develop rapidly to an antibody resistant stage before this specific immunoglobulin is produced in sufficient amounts. He further con- tends that the developing forms become coated with a second specific antibody which blocks the induction of cell mediated responses. This one antibody, one antigen proposal is somewhat analogous to the situation encountered in tumor resistance whereby neoplastic cells coated with specific antibody avoid cell mediated immunity. To accept the feasibility of either of these hypotheses it will be necessary to demonstrate that developing tissue forms are capable of inducing formation of a variety of antibodies, some destructive, others neutral, or that other necessary humoral require- ments such as complement are rendered ineffective. Membrane Transport The circumstances whereby host or host-like proteins appear within the fluid-filled cavities of taeniid parasites are susceptible to experimental analysis both in vitro and in vivo, and form the basis of much of the research which is presented in this thesis. A considerable body of evidence has accumulated regarding the char- acteristics of membrane transport systems in general and of cestode membranes in particular, and the conclusions are appropriately reviewed at this point. Singer's fluid mosaic model of membrane structure is the cur- rently accepted theory describing membrane organization (Singer and 21 Nicholson, 1972) and it proposes that membranes consist of a con- tinuous phospholipid bilayer with randomly inserted protein molecules. Two major types of membrane proteins have been defined. Extrinsic proteins, such as spectrin of erythrocytes, can be removed from the membrane by increasing the salt concentration or upon addition of a metal ion chelating agent. Intrinsic proteins, on the other hand, can only be removed by organic solvents capable of destroying hydrophobic interactions. The model has numerous advantages in explaining observed phenomena. It permits for variations in lipid/ protein ratios, the observed random movement of lipids and proteins, and the varying half-lives of protein and lipid molecules. In this portion of the literature review the theoretical models of membrane transport will be described. Emphasis is placed on the current models of protein uptake. Theoretical Models of Small Molecule Transport Generally three specific types of transport phenomena are recognized to explain small molecule uptake. Simple diffusion is merely the movement of a solute due only to the kinetic energy of the molecules. Movement occurs from a regiOn of higher concentration to one of lower concentration. Usually in a diffusion system the rate of absorption versus the solute concentration will be a linear function. However} occasional anomalies do occur since the diffusion rate will depend to a certain extent on the physical and chemical properties of the membrane and particular solute in question. Dif- fusion requires no energy expenditure by the cell, and chemical stereospecificity is lacking. 22 A similar process termed facilitated diffusion also involves solute movement in relation to the prevailing concentration gradient. Again no energy is expended by the cell, but the process is stereo- specific. Chemicals of similar structure will disrupt or inhibit the process. In facilitated diffusion the rate of diffusion char- acteristically follows saturation kinetics. Active transport is identical to facilitated diffusion except that solute can be accumulated against a concentration gradient, and energy expenditure is required. The process is stereospecific and the rate of diffusion follows typical saturation kinetics. Included in this group of processes is the mechanism of co-transport, which involves carrier proteins in association with specific cations, and which has been used to account for transport of sugars through the villi of the small intestine. Specifically the binding of Na+ ion is believed to enhance or increase the affinity of the receptor for the sugar. As the receptor-Na+—sugar complex is moved into the cell the Na+ ion dissociates due to a decrease in ion concentration, thus reducing the carrier affinity for the sugar. With the transport of intact protein molecules numerous problems are encountered. Due to the size and molecular nature of membranes most transport processes are limited to small molecular weight substances and passage of large molecules or those of unusual conformational arrangement is prohibited. Consequently less con- ventional methods of transport have been postulated. 23 Theoretical Models of Protein Transport. Presently theorists recognize only one major method of protein absorption: pinocytosis, a process often termed cellular "drinking." Although similar to the process of phagocytosis carried out by many eukaryotic cell types, the actual mechanism of pinocytosis is poorly understood. The majority of studies have been carried out using amoebae, but recently significant contributions have been made in studies using erythroid cells, neural cells--especially at post- synaptic junctions, and fetal membrane and intestinal cells (Blanton et al., 1968; Waxman and Pappas, 1969; Hayward, 1967). A charged molecule binding to a specific membrane receptor is necessary to initiate the cellular process. It has been proposed that once binding has occurred complex internal processes are activated within the cell leading to complete vesicle formation utilizing an original membrane fragment. A controversial and even less under- stood process of macromolecular uptake termed trans-membranosis was proposed by Tanaka (1962) to explain the absorption of vital stains in the lymph node cells of mice. He was unable to observe the typical vesicle formation until dyes had completely penetrated the cell membrane. Particularly analogous to the question of uptake of plasma proteins by taeniid larvae is the transmission of immunity from mother to young. This involves immunoglobulin uptake by the yolk— sac membrane and through the neonatal intestine, and has been explored experimentally by Brambell and co—workers. Transmission of immunity occurs entirely before birth in the rabbit and early studies revealed that uptake occurred through the uterine lumen and 24 the yolk-sac, and not by way of the placenta (Brambell et al., 1949). Conversely, in the rat the greater part of immunity is transmitted after birth and throughout most of lactation (Halliday, 1955b). Brambell's group has shown conclusively that the small intestine of the suckling rat is capable of absorbing intact antibodies present in the colostrum and milk of the mother (Brambell, 1958, 1966, 1970; Rodewald, 1973). The process of uptake shows a high degree of selection for homologous IgG immunoglobulins which are transported intact (Halliday and Kekwick, 1960), whereas heterologous gamma globulin may undergo changes with complete loss of functional activity in the process (Brambell et al., 1961). A hypothesis concerning the mechanism of transmission in the small intestine, which seems to be consistent with most of the observations, was proposed by Brambell (1958) and supported by Rodewald (1973). Uptake is believed to take place by pinocytosis. Vesiculation occurs between the bases of the microvilli at the apical pole of the cell. Each vesicle, which is derived or was at least in continuity with the cell membrane, has a limited number of specific immunoglobulin receptors. Attachment to these receptors protects the protein from lysosomal degradation. Experimental findings have shown that only a fraction of the protein absorbed is transmitted to the circulation intact. In fact, further studies indicate that as protein concentration increases a greater percentage of the absorbed protein is degraded while the amount released from the cell remains constant, suggesting that receptors do become saturated. Presumably these receptors have the greatest affinity for homologous gamma globulin, and since the Fc portion alone is 25 readily transmitted, it is believed to be the critical determinant. Release to the cellular interstitial fluid supposedly occurs through a process of reverse pinocytosis, similar to that followed by the endoplasmic reticulum in secretion of newly synthesized protein. Membrane Permeability Associated with the Cestoda Of the four major groups of helminth parasites the Cestoda have been studied most extensively. Unlike the trematodes and nematodes, cestodes lack a digestive system, and therefore all life-sustaining nutrients must be absorbed through the external tegument. Ultrastructurally the parenchymal covering is in the form of microvilli, distinctly resembling the mammalian intestinal brush border. This modification functions to increase the absorptive surface area. Considerable work has been concerned with the uptake of sugars and amino acids by a number of larval and adult cestodes in hopes of gaining a basic understanding of the biochemistry and physiology of these parasites. Unfortunately, until the recent demonstration by Chordi and Kagan (1965) that intact "host-associated" proteins exist in hydatid fluid, very little work was done concerning the phenomena of macromolecule 'uptake. In this section the investiga- tions in three major areas of study concerning membrane permeability and transport in the Cestoda will be reviewed. Uptake of Sugars Since the demonstration that specific cations are necessary for sugar transport through the villi of the small intestine in mammals, considerable evidence has accumulated indicating that mediated 26 processes are responsible for sugar transport in numerous cell types in higher animals. Recent work in lower metazoans, including tape- worms, suggests that similar mechanisms occur at this level. In 1966 von Brand and Gibbs first demonstrated Na+-dependent glucose transport in Taenia taeniaeformis. Subsequently identical findings were published for Hgmenolepis diminuta (Dike and Read, 1971; Read.et al., 1974), Calliobothrium verticillatum (Fisher and Read, 1971; Pappas and Read, 1972a), Hymenolepis microstoma (Pappas and Freeman, 1975), and for larvae of Taenia crassiceps (Pappas et al., 1973a). Their collective findings strongly suggest that glucose transport is inhibited in all cell types by the presence of phlorizin or ouabain in the medium. An apparent contradiction to this hypothesis was reported for the adult blood fluke, Schistosoma mansoni by Isseroff 9t a1. (1972). They proposed that there may not be a common sugar transport system for flatworms as there appears to be in vertebrates. However, this has apparently been refuted by- Uglem and Read (1975), who were able to demonstrate sensitivity to both of these inhibitors, further substantiating the similar nature of sugar transport in a wide variety of cell types. Recently some interesting findings have been reported concerning the uptake of other hexoses. Fructose has been shown to enter larvae of T. crassiceps by diffusion, while galactose appears to be taken up by a combination of diffusion and a mediated process. In addition inhibitor studies indicate that glucose and galactose are mutually competitive inhibitors (Pappas et al., 1973a). Clearly studies involving a number of parasites have contributed significantly to the understanding of sugar transport. Because of 27 their ease of maintenance and value as a laboratory model, helminth parasite systems will continue to be used in future studies concerning the in vivo and in vitro processes of sugar absorption, particularly at the molecular level. Uptake of Amino Acids A great deal of work has been published concerning amino acid absorption in a variety of cestode species. The early experiments exploring the possibility of mediated transport mechanisms were carried out using Hymenolepis diminuta (Daugherty, l957a,b; Daugherty and Foster, 1958). Since then the transport mechanisms for a large number of amino acids have been described for H. citelli (Senturia, 1964) . Calliobothri'um verticillatum (Read et a1. , l960a,b) , and Taenia crassiceps (Haynes and Taylor, 1968; Hayes, 1970; Pappas and Read, 1973). Each of the three classical mechanisms of membrane transport has been incriminated at one time or another to explain the mode of uptake for specific amino acids. Often the solute concentration appears to determine whether uptake will occur through diffusion or by a combination of diffusion and a mediated process, e.g., methionine, phenylalanine, and lysine uptake by larvae of T. crassiceps (Pappas and Read, 1973). Uptake of other amino acids occurs solely through active transport processes, e.g., methionine uptake by H. diminuta (Pappas et al., 1974). It should be emphasized that the amount of data accumulated pertaining to amino acid transport in cestodes is far too vast and complex to be adequately covered in this review. Instead the basic considerations will be discussed. In general, research findings 28 reveal that the amino acid transport systems of the Cestoda are similar in many respects. Each species possesses more than one mechanism of uptake. Their transport systems appear to differ from the generalized mammalian systems in two respects. Firstly, they appear to lack stereospecificity. While the mammalian system has a much greater affinity for L-amino acids, cestode systems appear to absorb each equally, or occasionally even demonstrate a high affinity for D~isomers (Arms and Coatis, 1973). Secondly, cestode tran8port systems apparently lack the mechanism of ion-coupled active transport. As with sugar absorption, the mammalian transport system frequently utilizes the coupling of amino acids to the move~ ments of cations (Pappas et al., 1974). The co-transported ions are maintained at low intracellular levels through an active extru- sion process often termed "pumping." The organic solute and ions move in relation to the prevailing concentration difference in the co—transported ion, and since this ion is actively removed transport will continue and solute will be accumulated. Uptake of Macromolecules Since the demonstration of host-associated proteins in hydatid fluid and the realization of the significance of this finding to the understanding of the phenomenon of concomitant immunity associ- ated with cestode infections numerous investigators have sought to explain the mechanism of uptake. Most workers agree that there appear to be only two possible sources of proteins which accumulate in the bladder fluid of taeniid metacestodes.) They are either syn- thesized in the parenchyma by the parasite and leaked into or stored 29 in the fluid, or they are of host origin. Unfortunately results from studies designed to determine the origin have been conflicting and inconclusive. Rothman (1967), for example, sought to explain observed colloid transport in adult Hymenolepis diminuta. Unable to find evidence of pinocytosis he turned to a previously proposed process of trans— membranosis described by Tanaka (1962). However, his results were effectively refuted by Lumsden and his co-workers (1970) and Rothman himself admitted that he had erroneously interpreted his electron microscopic photographs. Later Esch and Kuhn (1971) described significant uptake of l4chhlorella protein by T. crassiceps. They associated this uptake with the presence of canals observed in sections of the larval membrane by light microscopy. However, they failed to demonstrate that the proteins were maintained intact following transport, and the tegumentary canals have not been detected by others. In 1973 Pappas and Read questioned these results when they were unable to demonstrate uptake of inulin, an uncharged molecule similar in molecular weight to small proteins, by larvae of T. crassiceps. Because of the discrepancy in findings they indicated that further study was necessary but that their data suggested that intact pro- tein uptake by larvae of T. crassiceps was highly improbable. 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ARTICLE 1 PERMEABILITY STUDIES ON TAENIID METACESTODES: I. UPTAKE OF PROTEINS BY LARVAL STAGES OF TAENIA TAENIAEFORMIS, TAENIA CRASSICEPS, ECHINOCOCCUS GRANULOSUS Permeability Studies on Taeniid Metacestodes: I. Uptake of proteins by larval stages of Taenia taeniaeformis, Taenia crassiceps, Echinococcus granulosus SHORT TITLE: Protein Uptake by Taeniid Metacestodes. S. T. Hustead and J. F. Williams Department of Microbiology and Public Health Michigan State University East Lansing, MI 48824 42 43 ABSTRACT Host immunoglobulins of several different classes were detected within the bladder fluids of Taenia taeniaeformis, Taenia crassiceps, and Echinococcus granulosus. Radioiodinated proteins were taken up in vitro by larvae of both T. taeniaeformis and T. crassiceps and were shown to retain their physicochemical and antigenic character- istics. Rates of uptake were similar in the two species and were not related to the molecular weight of the proteins. Immunoglobulins were taken up both in vitro and in vivo by larvae of T. taeniaeformis. Absorbed immunoglobulins were shown to retain both antigen binding capacity and biologic functions associated with the Fc portion of the molecules. Not all cysts of E. granulosus contained detectable host proteins. This may be attributable to proteolysis within the bladder fluid, since uptake of 1125 occurred when hydatid cysts were exposed to labeled proteins in vitro, but rapid degradation of the labeled carrier led to the appearance of dialyzable fragments. We conclude that taeniid metacestodes are capable of absorbing a variety of proteins, and that these macromolecules can retain their structural and functional integrity following transport. This absorptive capacity accounts for the presence of host serum components within bladder fluids. INTRODUCTION The fluid-filled bladders of taeniid metacestodes of several species have been shown to contain proteins physicochemically and antigenically identical to those present in host plasma (Chordi and 44 Kagan, 1965; Esch, 1964; Coltorti and Varela-Diaz, 1972). A number of hypotheses have been put forward to account for their presence (Damian, 1964; Capron et al., 1968; Coltorti and Varela-Diaz, 1972) but experimental investigation of the phenomenon has produced con- flicting results. Coltorti and Varela-Diaz (loc. cit.) were able to demonstrate species specific determinants on immunoglobulin molecules in hydatid cysts of Echinococcus granulosus from a variety of hosts, and proposed that macromolecules entered the cysts by means of simple diffusion. Esch and Kuhn (1971) reported that larvae of Taenia crassiceps were able to take up whole Cl4 Chlorella protein and tentatively linked their findings to the occurrence of membranous pores which had been observed by light microscopy. However, Pappas and Read (1973) disputed the existence of pores and after failing to demonstrate uptake of intact Cl4 inulin, concluded that passage of entire macromolecules into larvae of T. crassiceps was very improbable. An impressive degree of acquired immunity to infection by taeniid larvae has been shown to develop, and a conclusive demonstra- tion of the origin of host or host-like proteins present in the bladder fluid of larvae growing in immune animals is essential if the mechanism of concomitant immunity in these infections is to be understood. In an attempt to characterize the relationship between permeability and the immune response, we have identified immuno- logically the host serum proteins present in cyst fluids of several taeniid metacestodes maintained in the rat and mouse, and report here 45 on the uptake of intact heterologous and homologous host proteins in vitro and in vivo. MATERIALS AND METHODS Maintenance of Parasites The strain of Taenia taeniaeformis used in this investigation was maintained in Spartan (Spb:[SD]Br) rats and domestic cats as described by Leid and Williams (1974). The KBS strain of T. crassiceps was originally obtained through the generosity of Dr. G. W. Esch, Wake Forest University, Winston-Salem, North Carolina. The larvae were maintained in Carworth CFl mice, and propagated by serial intraperitoneal injections of larvae as described by Freeman (1962). Hydatid cysts of E. granulosus were surgically implanted into the peritoneal cavity of rats and allowed to develop for periods ranging from 10 to 15 months (Varela-Diaz et al., 1974). Reagents Proteins Crystalline bovine serum albumin (BSA), bovine gamma globulins (BGG), and human gamma globulins (HGG) were purchased from Sigma Chemical Company (St. Louis, MO). A standard preparation of ribo- nuclease A (RNase-A) was obtained from Pharmacia Fine Chemicals (Piscataway, N.J.). Rat IgG immunoglobulins were isolated from 2 normal rat serum by DEAE-cellulose chromatography (Leid and Williams, 1974). Four to five milligram quantities of rat IgG BSA, and RNase-A 2! were trace labeled with 1125 by a slight modification of the 46 procedure of Helmkamp et a1. (1960). Counting was performed in a Packard scintillation counter, and the values are expressed as counts per minute per microliter (cpm/ul) unless otherwise indicated. Antisera Anti-whole rat serum (anti WRS), and specific antisera for each immunoglobulin class in the rat were prepared as described by Leid and Williams (1974). Anti-mouse IgM and IgA were purchased from Meloy Laboratories, Springfield, Virginia. Rabbit anti-mouse IgG was prepared by immunization with a fraction purified by DEAE chromatography of normal mouse serum globulins precipitated with 40% ammonium sulphate. Sheep anti-rabbit IgG was similarly prepared. Rabbit antisera were prepared against BSA and RNase-A by inoculating antigen solutions emulsified with an equal volume of Freund's complete adjuvant (FCA, Difco, Detroit). Injections of 0.5 ml portions were given intramuscularly in each hind leg, and 0.2-0.25 ml portions were inoculated subcutaneously in two sites along the back. Each rabbit received approximately 70 ug of antigen. Similar preparations were given as booster inoculations at 21 days and the rabbits were bled out 10 days later. Anti-BGG and anti-HGG were prepared by sensitizing rats with a 1:1 suspension of antigen diluted in phosphate buffered saline (PBS), and homogenized in FCA. All animals received 250 ug of protein in the form of injections of 0.1 ml of the emulsion in each hind foot pad. Serum was harvested 14 days later. 47 Experimental Techniques Immunoelectrophoresis (IEP) and Double Immunodiffusion (DID) Immunoelectrophoresis and double immunodiffusion were performed following the method described by Leid and Williams (1974). Polyacrylamide Gel Electrophoresis (PAGE) Polyacrylamide gel electrophoresis was performed following the method of Weber and Osborn (1969). A 1% solution of aniline blue black was used to stain separated protein bands. All standard pro- tein solutions were assayed for purity by PAGE prior to iodination. Radioimmunoelectrophoresis (RIE) and Autoradiography The presence of radioiodinated protein was detected in bladder fluid samples by radioimmunoelectrophoresis. Immunoelectrophoresis of the concentrated fluids was performed as described above. Two plexiglass plates were used to secure the dried, unstained IEP slides in position on the Kodak No—screen X-ray film. Exposure times were varied depending on the sample source, but never exceeded one half-life. The film was processed according to routine X-ray film development procedures. Control slides were included in order to detect any pressure induced artifacts. Indirect Hemagglutination (IHA) The procedure for indirect hemagglutination was a slight modi- fication of that described by Stavitsky (1954). HA titers were determined using disposable microtiter plates and micro-diluters (Cooke Engineering Co., Alexandria, VA). Reactions were read after 48 incubation at room temperature for 4 hrs. Sheep red blood cells (SRBC) used in this procedure were collected directly into Alsever's solution and stored at 4 C. Homologous Passive Cutaneous Anaphylaxis (PCA) Homologous passive cutaneous anaphylaxis was performed following a slight modification of the procedure described by Ogilvie (1967). Rats were shaved and 0.1 ml quantities of serum or larval bladder fluid were injected intradermally (i.d.) along the back. Two hours later rats were challenged intravenously with a 1:1 solution con- taining ECG and a 1% solution of brilliant blue R (Bio Rad., Cali- fornia). Reactions were read 15-30 minutes after challenge. When- ever doubtful responses occurred the skin was removed and viewed from the underside. Experimental Procedures Larvae of T. taeniaefbrmis, 48-63 days old, were dissected free of liver cysts into chilled Eagle's medium. Groups of 50-75 larvae were washed 3X in distilled water, 3X in sterile water, and 3X in sterile normal saline. Fluid samples to be analyzed by IEP for host serum components were immediately procured by bladder puncture, and concentrated 2— to 5-fold by using polyethylene glycol (Carbowax, Union Carbide). Other larvae were transferred to sterile flasks containing 20 ml of Eagle's medium at 37 C for a pre-incubation period of one hour. Uptake experiments were initiated by the addition of iodinated proteins to culture media. Incubations were terminated by removal 49 of individual larvae. Each larva was washed 3X in normal saline, blotted dry, and the bladder fluid collected in 5 mm x 40 mm test tubes. After the prescribed incubation period in each experiment the fluids obtained from a minimum of 5 larvae were pooled and the levels of radioactivity were determined. Fluid volumes were measured using either 10 ul or 50 ul Hamilton syringes. Pooled bladder fluid samples were also concentrated using polyethylene glycol and analyzed immunoelectrophoretically. Washed and dried IEP slides were examined for radioactive bands by autoradiography. After 3-4 months of infection the larvae of T. crassiceps were removed from the peritoneal cavity and transferred to Eagle's medium. Only advanced larvae with well-formed bladders were selected for use in the experiments. The incubation procedures and methods of bladder fluid collection were identical to those outlined for T. taeniaeformis. Hydatid cysts removed from the peritoneal cavity 10 to 15 months after transplantation were immediately washed as described for T. taeniaefbrmis larvae and then transferred to sterile incuba- tion flasks containing 30-50 m1 of Eagle's medium at 37 C. Fluid samples were obtained by puncture with a 30 gauge needle and 1.0 ml syringe. Otherwise the experiments were carried out as described for T. taeniaeformis. At times it was necessary to maintain all three species over- night in Eagle's medium at 4 C before use in uptake experiments. Larvae of T. crassiceps and E. granulosus were stored under these conditions for periods exceeding two weeks without demonstrable loss of infectivity following transplant into mice and rats 50 respectively. In order to show that larvae had not degenerated during the course of our in vitro studies representative specimens of T. crassiceps and E. granulosus were reimplanted in animals at the end of a typical experiment. All these organisms successfully established in their respective hosts. We did not test the infec- tivity of larvae of T. taeniaeformis after maintenance in vitro because at this age (48-62 d) their ability to infect cats is questionable (Hutchinson, 1958). However, more advanced larvae (100-120 d) which had been maintained in vitro under similar cir- cumstances for 24 hrs were able to infect cats and produce adult tapeworms equally as well as fresh worms obtained directly from rat livers. RESULTS The results of immunoelectrophoretic analyses of bladder fluids of each of the three species of taeniid metacestodes are shown in Figure l. A number of host associated antigenic determinants, including albumin and a variety of globulins, were revealed with antisera to whole host serum. When class specific antisera against rat immunoglobulins were employed IgG IgG and IgM but not IgA 1' 2' were detected in bladder fluids of both T. taeniaeformis and E. granulosus. In bladder fluid from larvae of T. crassiceps mouse IgG, IgM, and IgA were present. Fluid samples from individual hydatid cysts of E. granulosus did not always contain detectable host associated components. Preliminary results from studies in which larvae had been incubated in radioiodinated protein solutions indicated that 51 Figure 1. Host immunoglobulins detected by immunoelectro- phoresis in the bladder fluids of T: taeniaeformis, T. crassiceps, and E. granulosus. An array of host serum components were revealed in bladder fluid (3X) concentrated, obtained from larvae of T. taeniae- formis (a) and E. granulosus (b) by reaction against anti-whole rat serum (a-WRS). Rat IgGl, Ing, and IgM were detectable by reaction against antisera specific for each of these immunoglobulin classes. Bladder fluid from larvae of T. crassiceps (c) was analyzed similarly. Again an array of host serum components were revealed by reaction against anti-whole mouse serum (a-WMS). Mouse IgG, IgM, and IgA were detectable by use of antisera specific for each of these immunoglobulin classes. \ 52 23 03 03 a musmwm 53 . 125 . . substantial uptake of I had occurred. In order to determine if uptake of intact protein molecules was involved we employed RIE analysis. Figure 2 shows the results of RIE tests with bladder fluids (5x concentrated) from larvae maintained for 4 hrs in Eagle's 25 culture medium supplemented with I1 labeled rat IgG BSA, or 2, RNase—A. In samples from T. taeniaeformis and T. crassiceps radio- active bands were detected which corresponded in position to those of the parent molecules. In a number of cases considerable cpm/ml were observed in individual hydatid fluid samples but radioactive bands were detected in RIE on only one occasion. In View of the fact that the labeled protein standards dif- fered markedly in molecular weight we studied the rates at which they appeared in bladder fluid in order to determine if uptake of 1125 was influenced by the molecular weight of the carrier. Larvae were incubated in isotopically labeled protein solutions in Eagle's medium as described above, and bladder fluid samples were taken at 30 min intervals for the first two hours, and then after 4, 8, and 24 hrs of incubation. Larvae of T. taeniaeformis and T. crassiceps showed remarkably similar rates of uptake for each protein (Figure 3), although there was considerable variation in uptake rates for individual parasites. In pools of hydatid cyst fluid levels of radioactivity were consistently low, and did not increase notably over the duration of the experiment. Although I125 labeled proteins were detected within the bladder fluid of taeniid metacestodes further evidence was required in order to demonstrate the transport of functionally intact macro- molecules. For this purpose we examined the appearance of 54 Figure 2. Autoradiography of bladder fluids from larvae of T. taeniaeformis (a) and T. crassiceps (b) incubated in 1125 labeled RNase-A, BSA, or rat Ing. Supplemented Eagle's medium (c) was assayed similarly and served as the control. Larvae were incubated in Eagle's culture medium supplemented with the labeled carriers for 4 hrs at 37 C. Bladder fluid was collected and concentrated (5X) prior to being assayed by RIE and autoradiography for the presence of the iodinated protein. N musmflm omH 4mm auwmmzm 56 Figure 3. Rates of uptake of I125 labeled protein by larvae of T. taeniaeformis (* -*), T. crassiceps(Q-Q), and E. granulosus (O-O), incubated in vitro in Eagle's medium containing the labeled carriers. Seventy-five larvae were incubated at 37 C in Eagle's culture medium supplemented with 1125 labeled rat IgG (56,000 cpm/ml), BSA (64,000 cpm/ml) or RNase-A (47,000 cpm/ml). During the first 2 hrs, 5 larvae were removed every 30 mins, washed extensively in saline and their bladder fluids collected and pooled. The levels of radioactivity were assayed in a gamma scintillation counter. Additional samples were collected similarly after 4, 8, and 24 hrs of incubation. 57 maxing-All: m w (.ommzm .2 wmzumzfillll a a .4 f {P T fT