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Biological characteristics of the growth hormone-like factor from plerocercoids of the tapeworm Spirometra mansonoides
Advances in Neuroimmunology Vol. 2, pp. 235-247, 1992 Printed in Great Britain. All rights reserved
0960-5428/92 $15.00 © 1992 Pergamon Press Ltd
Biological characteristics of the growth hormonelike factor from plerocercoids of the tapeworm
Spirometra mansonoides C. K i r k Phares D e p a r t m e n t of Biochemistry, University of Nebraska Medical Centre, 600 South 42nd Street, O m a h a , NB 68198-4525, U S A
Introduction There are a variety of defence mechanisms ranging from simple to very complex which enable animals to survive and reproduce in a world full of potentially lethal infectious agents. In order that an organism be a successful parasite in other animals, several significant barriers must be overcome. The parasite must gain access and enter a potential host and then survive an array of sophisticated and normally very effective host defense systems. For those parasites with multiple stages of their life-cycle, the potential to pass through several intermediate hosts and then 'find' an appropriate definitive host where reproduction can occur becomes very small. The biochemical mechanisms which enhance the odds that any parasite will succeed have been studied in only a very few of the many known parasite-host relationships. A phenomenon exhibited by the plerocercoid stage of the pseudophyllidean tapeworm Spirometra mansonoides may be an example of a very 'clever' mechanism to enhance survival. Plerocercoids of S. mansonoides release a substance which mimics some, but not all of the actions of mammalian growth hormone. The importance of growth hormone (GH) in regulation of overall body growth as well as carbohydrate, lipid and protein metabolism in vertebrates has long been established. More recently, GH has been shown to play a
stimulatory role in the regulation of immune function (Gala, 1991). By duplicating only some of the actions of GH, release of plerocercoid growth factor (PGF) in hosts results in alterations in growth, metabolism and in some cases, immune function. It is not unreasonable to suggest that PGF somehow enhances the survival of this tapeworm.
Discovery of the growth stimulating effect of plerocercoids J. F. Mueller (1937) is not only credited with the establishment of the genus Spirometra as being distinct from Diphyllobothrium but also with the serendipitous discovery of the growth enhancing phenomenon associated with plerocercoid infections of laboratory rodents (Mueller, 1963). Mueller's early work was primarily concerned with the taxonomy of Spirometra but he eventually became interested in establishment and maintenance of the entire life-cycle of S. mansonoides in his laboratory. His efforts to find a more manageable laboratory host than the most common natural hosts (frogs or snakes) for the tissue invading plerocercoid stage, resulted in his discovery that not only could laboratory mice serve as excellent maintenance hosts but that the presence of plerocercoids was associated with a marked
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increase in the size and weight of mice (Mueller, 1963). The observation that the plerocercoid infections were associated with accelerated growth was extended to hormonally-intact golden Syrian hamsters (MueUer, 1965), rats (Ruegamer and Phares, 1974) and lizards (Phares and Corkum, 1974). In addition to hormonally intact animals, rats with growth retardation induced by hormonal manipulation could be stimulated to grow when infected with plerocercoids. Hypothyroid (Mueller and Reed, 1968), hypophysectomised (Mueller, 1968) and castrated rats (Ruegamer and Phares, 1974) respond to the growth promoting effects of plerocercoids. The fact that plerocercoids stimulate growth in hypophysectomised (Hx) rats suggests that they produce and release a GH-like substance.
Actions of GH
Whereas a comprehensive review of the multiple actions of GH is inappropriate here, some of its activities must be mentioned. Normal production and secretion of GH from somatotroph cells in the anterior pituitary is an absolute requirement for normal growth of young mammals. The somatomedin hypothesis of growth suggests that after its release into the circulation GH stimulates the release of secondary hormones originally named somatomedins but now referred to as the insulin-like growth factors (IGF) which are directly responsible for growth. Insulin-like growth factor-1 (IGF-1) is thought to be the essential growth promoting substance. Recent data show that G H has direct growth promoting effects as well as its indirect effects on growth via stimulation of IGF-1 (Isaksson et al., 1987). In addition to its growth stimulating effects, GH is an important metabolic regulator. Preparations of GH from a variety of vertebrates as well as recombinant
GH exhibit multiple biological properties that are difficult to reconcile with a single mechanism of action. Growth hormone is not only anabolic but is diabetogenic producing hyperglycaemia, glucose intolerance, hyperinsulinaemia and insulin resistance (Kostyo, 1986). Furthermore, in the special circumstance of G H deficiency, exogenously administered GH produces transient insulin-like effects including hypoglycaemia, inhibition of lipolysis and stimulation of glucose oxidation in adipose tissue. In the past, investigators have questioned whether the complex biological activities are intrinsic characteristics of the GH molecule or if they are due to contaminants in GH preparations. However, it is now generally accepted that the growth promoting, diabetogenic and insulin-like activities are intrinsic properties of the GH molecule. Furthermore, there is a significant body of convincing evidence that there are multiple active sites in the GH molecule which account for its actions. An additional possibility exists that there may be several functionally distinct receptors for GH. In addition to the multivalent properties shared by all GHs, primate GHs express several unique characteristics as they are also lactogenic (prolactin-like), a characteristic not expressed by non-primate GHs. Lactogenic activity of primate GH stems from its ability to bind receptors for PRL with high affinity. Moreover, human GH (hGH) expressed potent growth promoting activities and other biological properties in all vertebrates whereas only hGH (or other primate GH) is biologically active in humans (or other primates). Biological effects associated with plerocercoid infections
Subsequent to the original observations of Mueller on growth promotion in experimental rodents, a number of reports followed which suggested that plerocercoids elabo-
Plerocercoid growth factor rate a GH-like substance. Steelman et al. (1970) reported that infection of Hx rats with plerocercoids produced a remarkable increase in body weight. The fact that these workers also found increases in tibial cartilage width and increases in somatomedin (IGF-1) in the infected rats suggests that the response was a normal GH-like growth response. Furthermore, they reported that injections of plasma from actively growing Hx rats infected with plerocercoids into uninfected Hx rats stimulated weight gains greater than that produced by 100 ~g of bovine GH each day. Garland et al. (1971) confirmed that serum from plerocercoid-infected Hx rats had high levels of GH-like activity as determined by an assay for sulphation factor activity (IGF1) but the serum had no immunoassayable rat GH. These data strongly suggest that a factor is produced by plerocercoids, released into the circulation and acts on sensitive tissues in a manner analogous to GH. Mueller (1970) demonstrated a quantitative relationship between the number of plerocercoids and the growth response in Hx rats and reported that one plerocercoid per rat could stimulate growth. Hypophysectomised rats with subcutaneous infections of plerocercoids (10 worms/rat) expressed biological responses similar to those observed with injections of 100 ~g/day of bovine GH (Steelman et al., 1971). These responses include increases in body, liver, thymus and kidney weights, as well as increases in epiphyseal cartilage width. The only consistent difference in organ weights of the experimental Hx rats in this study was seen in the epididymal fat pads. Plerocercoid-infected rats had significantly more stored fat than untreated or GHtreated animals. Normal golden hamsters are especially sensitive to the lipogenic effects of plerocercoid infection. Meyer et al. (1965) reported that plerocercoidinfected hamsters had a marked increase in carcass fat and Phares and Carroll (1978)
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found increases in fat pad weights and lipid content of serum and liver of infected hamsters. Insulin is distinctly lipogenic and it is possible that the presence of plerocercoids might result in an increase in insulin secretion which could account for the lipogenic response to pleroceroids. If this were the case, blood glucose levels should be lowered in infected animals. This is not the case as plerocercoid infections did not alter blood glucose levels of normal (Phares and Ai, 1982), Hx (Steelman et al., 1971) or diabetic rats (Salem and Phares, 1986). Furthermore, a marked increase in the weights of the epididymal fat pads of plerocercoid infected diabetic-Hx rats was observed (Phares and Carrol, 1978).
Comparison of PGF to hGH
For several important reasons the characteristics of human G H warrant special attention. Not only does hGH possess growth promoting, anti-insulin/diabetogenic and insulin-like characteristics, but expresses distinct lactogenie activities as well. Furthermore, whereas exogenous hGH is biologically active in all vertebrate classes from fish to mammals, no subprimate G H is active in primates. Based on a variety of experiments it is possible to state that PGF expresses biological activities which are more similar to hGH than they are to any other vertebrate GH. For instance, Phares and Ruegamer (1973) reported that PGF had a dramatic stimulatory effect on growth of the mucosal epithelium of pigeon crop sac, a well established assay for lactogenie (PRL) activity (Nicoll, 1967). More recently, a cell line derived from a rat lymphoma (NB-2 cells) has been shown to have an absolute dependency on lactogenic hormones for growth (Lesniak and Roth, 1977). Human G H is a potent lactogen in this very sensitive assay. We have found that partially-purified PGF expresses mitogenic
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PRL-like activity in this cell line. Therefore, PGF expresses lactogenic activity similar to that reported for hGH. Another hGH-like property which is significant is the ability of PGF to express biological activity in a variety of vertebrates. In addition to examples of growth stimulation in mice, rats and hamsters and lactogenic activity in pigeons described above, PGF stimulated a growth increase in the lizard, Anolis carolinensis equivalent to 32% of the initial body weights while the untreated controls did not grow during the experimental period (Phares and Corkum, 1974). Perhaps the most convincing evidence that PGF has activity considered unique to hGH comes from experiments involving primates and human cells. Partially-purified PGF was injected into a Hx Rhesus monkey and was shown to increase production of somatomedin (IGF-1) and to decrease the concentration of blood urea nitrogen (Phares and Ruegamer, 1976). These are responses one would expect only with injections of a primate GH. In another experiment PGF was shown to displace hGH from its highly specific receptors on human IM9 cells (Phares and Watts, 1988). Additional evidence for the similarity between PGF and hGH comes from a study involving anti-hGH monoclonal antibodies (MAB). Antigenic mapping of hGH suggests that four distinct nonoverlapping regions are expressed (Ivanyi, 1982). The crossreactivity of PGF with four MABS, each of which describes a distinct epitope of hGH, was tested. The ability of PGF to displace 125IhGH from this series of MABS varied from nearly equal potency (~70%) to less than one percent the potency of the hGH standard (Phares and Booth, 1987). No nonhuman GH or PRL gave any degree of crossreactivity with any of the MABs (Ivanyi, 1982; Phares and Booth, 1987). Other evidence that PGF and hGH share molecular characteristics include the fact that cDNA for hGH gave a positive
hybridisation signal in Southern blots with genomic DNA from plerocercoids (Cox et al., 1990). Both the molecular hybridisation study and the hGH MAB study suggest that molecular similarity exists, however, the fact that the hGH cDNA hybridised with plerocercoid DNA only under relatively mild conditions and that PGF had an immunopotency of less than one percent that of hGH with one anti-hGH MAB strongly suggests that there are also important molecular differences between PGF and hGH. Whereas the similarities between PGF and hGH are remarkable and of great potential significance, the differences between these two substances with respect to both biological and molecular characteristics may be more interesting and of greater value.
Effects of PGF on the host's endocrine system
Whereas all of the body's systems, organs and tissues are not under obligatory regulation by hormones, essentially all are influenced by several hormones. Hormones regulate the metabolic activities of many tissues and have an important regulatory role in the immune system. If a parasite could manipulate a potential host's metabolic activities and/or its immune system in a manner which would allow the parasite to gain entrance and establish an infection without totally compromising the host's defence system, then this might be of selective advantage to the parasite. An important physiological and survival role for PGF in the biology of S. mansonoides may be dependent on the ability of PGF to alter the hormonal milieu of the plerocercoid host and therefore upset normal metabolic and immune regulation. A number of hormones influence the immune system, some like the adrenal cortical hormones suppress the immune system. Thyroid hormone and sex hormones influence both T- and B-cells
Plerocercoid growth factor responses (Peiropaoli et al., 1969). Prolactin and GH are involved in stimulating the immune system (Gala, 1991). The involvement of anterior pituitary hormones with the immune system had its origins from observations with Hx animals and in humans deficient in specific pituitary hormones. Hypophysectomy in rodents results in a suppression of immune function. Another consistent observation of anterior pituitary hormone deficiency is a decrease in thymus weight and function. Garland and Daughaday (1972) reported a decrease in pituitary weight and G H content as well as reduced serum GH in rats infected with plerocercoids of S. mansonoides. Tseng and Mueller (1977) studied cytological changes in the pituitaries of plerocercoidinfected rats and consistent with other reports, found a dramatic reduction in the weights of the pituitaries. They found no necrotic cells and no alterations in morphology of non-GH producing cells. However, the somatotrophs (GH producing cells) were markedly condensed. Glitzer and Steelman (1971) did not observe enhanced growth in young normal rats due to PGF but did report suppression of serum G H levels. Syrian hamsters are especially sensitive to the feedback regulation of G H by PGF as the pituitaries of control hamsters weighed two and a half times more and the serum contained four times more G H than did PGF-treated hamsters (Phares, 1982). Control of synthesis and secretion of hormones is complex and may involve regulation by homologous hormone, heterologous hormones, metabolic factors and other conditions. Most regulatory factors controlling G H secretion act via neural mechanisms. Somatostatin (GH release inhibiting hormone) is released from the hypothalamus in response to elevated serum levels of GH and IGF-1. Somatostatin acts directly upon the somatotrophs in the pituitary to inhibit both synthesis and release of GH. In addition to its regulatory effect at the level of the
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hypothalamus, IGF-1 also has a direct regulatory role on pituitary somatrophs. Other physiologically important negative regulators of G H levels include obesity, elevation of free fatty acids, increased glucocorticoids and hyperglycemia. As PGF does not affect serum glucose or increase free fatty acids, its mechanism of negative regulation of G H is presumably via its ability to mimic G H at the level of the hypothalamus and/or to increase the levels of IGF-1 in circulation. Although first identified as the release inhibiting hormone for GH, somatostatin also inhibits the production and release of thyroid stimulating hormone (TSH) therefore reducing the release of thyroid hormone. That PGF is recognised by GH-sensitive cells in the hypothalamus in feedback regulation is supported by the fact that thyroid hormone is also suppressed in PGF-treated animals (Phares, 1982; Sharp et al., 1982). The presence of PGF in animals results in a model with accelerated growth despite severe endogenous GH deficiency. If P G F mimics all of the activities of G H then PGFtreated animals should be representative of chronic G H excess (acromegaly). However, if PGF mimics only some of the multiple actions of G H then the model would be representative of G H excess with respect to some functions and of G H deficiency with respect to those actions not duplicated by PGF. The latter description is the model observed in PGF-treated animals. As previously stated, Hx and other GHdeficiency animals have reduced immune function. An obvious strategy for plerocercoids seeking to establish themselves in a new host would be to selectively suppress G H and not replace its stimulatory activity in the immune system. This was observed in rats as treatment with PGF resulted in transient suppression of the humoral immune response (Sharp et al., 1982). Interestingly, the humoral immune response was restored to normal with injections of thyroxine but not of G H in PGF-treated
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rats. It should be noted that suppression of the immune system due to hormonal manipulation by PGF is not observed in all hosts. Phares and Sharp (1991) reported that although plerocercoid-infected hamsters are deficient in GH and thyroxine their immune response was actually enhanced when compared to controls. Furthermore, PGF, like GH, stimulates growth of the thymus in Hx rats (Steelman et al., 1971; Phares et al., 1976). Whether the thymotropic action of PGF affects the immunological functions o f the thymus is not known. Therefore, it can be suggested that whereas all experimental animals tested respond to PGF all the responses are not uniformly predictable. These data are consistent with the concept that PGF mimics some but not all of the i actions of GH. There have long been questions concerning the roles that growth and GH play in sex~ual development. It has been unresolved as 'to whether GH itself or simply body growth are involved in the timing of puberty. In humans, GH deficiency is often accompanied by a delay of puberty onset (Rechler et al., 1987). Ramaley and Phares (1980) were able to demonstrate with PGF treatment that it is GH and not merely growth itself which is :essential for the normal timing of the onset of puberty in female rats. Confirmation of this observation was obtained in male rats (Ramaley and Phares, 1983). The influence of GH deficiency on the timing of puberty in rats has recently been confirmed by passive immunisation of rats to the GH releasing hormone to reduce circulating GH levels (Arsenijevic et al., 1991). The ability of PGF to reduce GH levels was used to study the controversial role of GH in the regulation of tissue receptors for PRL and estrogen. The inductive effect of GH on lactogenic receptors is suspected to be due to a direct somatogenic action and not to be associated with binding to the PRL receptor. Despite the fact that PGF (like hGH) binds to both somatogenic and lactogenic
receptors it does not upregulate lactogenic receptors as treatment with hGH does. In fact, treatment of adult female rats with PGF resulted in nearly a three-fold reduction in the number of lactogenic receptors without any significant change in the binding affinity (Phares and Booth, 1986). Not only were PRL receptors reduced but the number of hepatic estrogen receptors was reduced by one half in the PGF-treated rats compared to controls (Phares and Booth, 1986). The reduction of estrogen receptors could result in a blunted response to estrogen in PGFtreated animals and may partially explain the delay in puberty onset described above and the regression of hormone-sensitive mammary tumours in rats treated with PGF (Phares, 1986). It is not known at this time if (or how) the disturbance of hormonal balance observed in plerocercoid-infected animals is of survival advantage to S. mansonoides. It is possible that the suppression of the immune response associated with the presence of PGF is important in the establishment of plerocercoid infections in new hosts. The ability of PGF to mimic the growth promoting effects of GH also may play an important role in the ability of plerocercoids to reach their final host and reproduce. The rationale for this is based on the fact that long-term exposure to elevated levels of GH results in acromegaly. This disease is associated with significantly increased incidences of a variety of disorders. The death rate for acromegalics is twice that of the normal population. The presence of plerocercoids and their unregulated production of PGF could produce complications similar to those observed in acromegaly. In support of this are data from aging studies with control and plerocercoid-infected mice which show a marked increase in the death rate of the infected mice after only six months of infection (Phares et al., 1990). In this same study, 50% of the plerocercoid-infected mice were dead by the 18th month of age whereas
Plerocercoid growth factor 50% of the age-matched controls were still alive at 30 months of age. Early death or diseases which might limit mobility would likely make the hosts of plerocercoids more vulnerable to predation by the carnivorous final hosts of S. mansonoides. Other biochemical effects of PGF
As cited above, early work by several groups of researchers clearly demonstrate that exposure to PGF via plerocercoid infection has distinct effects on carbohydrate and lipid metabolism. As further described above, the presence of PGF is associated with marked perturbations of endogenous hormones and hormone receptors. Without direct studies of the effects of PGF on metabolism, the results observed could be explained simply on the basis of increases or decreases in specific endogenous hormone function. It should be remembered that GH is not only growth promoting but elevates blood glucose levels, increases blood insulin levels, induces tissue insensitivity to insulin as well as to its own insulin-like activity, increases lipolysis and circulating free fatty acid levels and has other diabetogenic effects in normal animals and humans. In addition, under the special circumstance of GH deficiency, GH expresses transient insulin-like effects (Kostyo, 1986). Neither plerocercoid infections nor injections of partially-purified PGF have been reported to have any consistent and significant effect on blood glucose or insulin. However, PGF, like injections of GH, increased liver glycogen content but unlike GH, PGF had no effect on cardiac glycogen levels in Hx rats (Phares and Ai, 1982). Adipose tissue from animals chronically exposed to PGF (plerocercoid infection) had significantly elevated glucose oxidation rates (Salem and Phares, 1986). The results were essentially the same in intact and Hx rats, indicating the effects of PGF were not due to GH suppression. The basal glucose oxi-
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dation rate of adipose tissue from diabetic rats was stimulated by PGF but the tissue was not sensitive to insulin added in vitro. This suggests the possibility of a direct insulin-like effect of PGF. The acute effects of injections of PGF were compared to saline or insulin in intact male rats. The rats were injected intraperitoneally, sacrificed one half hour later and glucose and leucine metabolism were measured in adipose tissue and muscle. Both insulin and PGF stimulated dose-dependent increases in carbohydrate, lipid and protein metabolism but again PGF had no effect on blood glucose (Salem and Phares, 1987). These results suggest that PGF has intrinsic insulin-like activity in normal (non-GH deficient) and diabetic (insulin-deficient) rats and represents a dramatic difference from the actions of GH. It is well established that tissues from intact animals are not sensitive to the insulin-like actions of GH and this refractoriness is induced by GH itself (Goodman and Coiro, 1981). Salem and Phares (1987) conducted experiments with adipose tissue from normal male rats in vitro to determine if PGF expresses its insulin-like activity in the absence of any other hormone. As expected, hGH stimulated glucose oxidation only after an extended preincubation period to eliminate the refractoriness of the tissue to GH. Furthermore, hGH produced a dramatic lipolytic response in freshly removed adipose tissue. Neither insulin nor any dose of PGF had a lipolytic effect but both stimulated a lipogenic response in freshly removed and preincubated adipose tissue. Despite its obvious differences from GH and similarity to insulin, PGF does not bind the insulin receptor. Its effects are mediated through the same receptors which bind hGH. The sum of the anti-insulin actions of GH are responsible for its diabetogenic characteristic. Both highly purified natural and recombinant hGH have the ability to induce hyperglycaemia and insulin-resistance
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in man and overt diabetes in experimental animals. The genetically obese mouse (ob/ob) is extremely sensitive to the antiinsulin actions of GH and is the whole animal model used to test diabetogenicity (Kostyo et al., 1984). Affinity-purified PGF was first tested for its biological potency relative to hGH in a growth promoting assay in normal mice. The same preparations of PGF and hGH were then assayed for diabetogenicity in ob/ob mice. No dose of PGF was diabetogenic while the injections of hGH caused a significant increase in fasting blood glucose levels and a dramatic impairment of glucose tolerance (Salem and Phares, 1989).
in rabbit liver membranes consists of both somatogenic and lactogenic sites, we now know that PGF binds the same receptors. The ability to test PGF by R R A allows its quantification in terms of a hGH standard. Therefore, binding activity of PGF is expressed as equivalent (Eq) units of the hGH standard. The adaptation of a sensitive assay allowed for experiments intended to purify PGF from plerocercoid incubation media or from serum of plerocercoid-infected Hx rats. However, under the best circumstances, plerocercoid incubation media yields no more than 100 ngEq/ml. Efforts to concentrate the media results in unacceptable losses of activity making purification from this material prohibitive. Purification of trace quanIsolation of plerocercoid growth factor tities of PGF from serum is not pratical. No (PGF) binding or growth promoting activity can be Efforts to achieve final purification to al- obtained by homogenisation of whole worms low structural characterisation of PGF have or subcellular fractions of plerocercoids in not been fully successful. The fact that aqueous buffers. However, binding activities plasma from plerocercoid-infected Hx rats equivalent to more than 1000 ng of hGH/ml contains growth enhancing characteristics can be obtained by solubilisation of whole (Glitzer and Steelman, 1971; Garland et al., plerocercoids or membranes in the non-ionic 1971) suggests that the hormonally active detergent Triton-X 100 (Phares, 1984). With solubilised rabbit liver membrane resubstance is released by plerocercoids and it should be possible to collect and eventually ceptors for hGH purified over a hGH affinity purify this substance. Growth hormone-like column, Phares (1988) was able to construct activity was detected in media in which a hGH-receptor affinity column. Passage plerocercoids were incubated (Phares and of crude solubilised PGF over this column Ruegamer, 1973; Chang et al., 1973). These resulted in more than 1000-fold increases early studies were hampered by the need to in specific activity as determined by RRA. test for activity in the relatively insensitive Affinity-purified PGF stimulated a very draHx rat growth promoting assay. However, matic dose-dependent growth response in shortly after the development of the first Hx rats and had direct in vitro activity radioreceptor assay (RRA) (Tsushima and on adipose tissue from normal rats (Salem Friesen, 1973) Tsushima et al. (1974) re- and Phares, 1989). Despite the 1000-fold inported that PGF in incubation medium as crease in specific activity the affinity-purified well as in serum from infected Hx rats PGF was not homogenous but contained displaced 125IhGH from its receptors in a several distinct stained protein bands after manner comparable to the hGH standard. SDS-PAGE. Of the three major bands, one This report was very valuable for several had a MW of 22,000, identical to the major reasons. The R R A for hGH is at least form of hGH. However, there is no defini1000 times more sensitive than the Hx rat tive proof of the molecular characteristics of growth assay and as the receptors for hGH PGF.
Plerocercoid growth factor No PGF activity has been detected by any method in either the procercoid or adult stages of S. mansonoides (Phares et al., 1990). Distribution of PGF in the genus Spirometra
The genus Spirometra includes numerous forms from throughout the world. Many of the suggested spirometrids are known only in their larval forms. Morphological variations in the genus is continuous and it is essentially impossible to define species in any precise manner. All stages of S. mansonoides are known and this species can be defined with some distinction. A number of 'species' have been described from the Orient but these are believed to be variants of a single species, S. erinacei. Hirai and his colleagues in Japan have done extensive work with PGF from S. erinacei and found that growth is stimulated in several rodent models (Hirai et al., 1983). The differences in biological activities between the PGFs from S. erinacei (PGF-e) and S. mansonoides (PGFm) are of interest. Similar to the results obtained with plerocercoids of S. mansonoides, plerocercoids of S. erinacei stimulate growth in mice, increase IGF-1 levels (Shiwaku et al., 1986) and PGF-e displaces hGH from its receptors in rabbit liver membranes (Hirai et al., 1986). In contrast to PGF-m, PGF-e does riot stimulate growth of Syrian hamsters, normal or Hx rats (Hirai et al., 1983). Furthermore, the effects of PGF-e on carbohydrate and lipid metabolism are inconsistent with those of PGF-m (Hirai et al., 1983, 1987). In agreement with these differences, Mueller (1970) reported weak growth promoting effects of plerocercoids from an Australian spirometrid and no growth promoting effects in Hx rats from two additional oriental spirometrids. Mueller (1972) later reported no growth promoting activity in Hx rats for plerocercoids collected in Africa.
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Therefore, it is clear that production of a GH-like factor is not unique to S. mansonoides and it is also clear that speciation has resulted in evolutionary changes in the biological characteristics of PGF. It will be of great interest to compare the molecular characteristics of PGFs from a variety of geographical forms of Spirometra.
Future studies with PGF
Parasitism is commonly associated with suboptimal or even halted growth of the host. Nutritional, hormonal and immunological consequences of parasitism may lead to diversion of nutrients away from supporting growth. There are a variety of examples where parasitism perturbs the hosts endocrine system including G H and IGF1. For instance, infections of cattle with the protozoan parasite, Sarcocystis cruzi are associated with increased somatostatin levels, decreased GH and IGF-1 levels which result in an inhibition of growth in calves (Elasser et al., 1988). There are immunocytochemical studies which show the presence of mammalian-like hormones in some parasites (Gustafsson et al., 1986). However, knowledge of the production and release of a hormone-like substance in sufficient abundance to dramatically stimulate normal growth and to seriously modulate the hormonal milieu, immune system and metabolic activities of the host is restricted to studies with plerocercoids and PGFs from S. mansonoides and S. erinacei. It is obvious that the substance responsible for mimicking G H must be purified and characterised to extend this area of study in a useful direction. This has not proven to be an easy task as success has not been achieved despite years of effort. Molecular studies with PGF and the genetic elements responsible for its production should reveal important information of interest to both parasitologists and endocrinologists. Questions as to the
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origins of P G F are very intriguing. Did P G F arise by 'normal' evolution or was a gene for a primate G H somehow transferred to Spirometra? Humans are very suitable hosts for plerocercoids and human infections occur at locations around the world (Swartzwelder et al., 1964). What evolutionary advantages does expression of P G F bestow on Spirometra? Are its hormonal activities essential or irrelevant to survival of the tapeworm? From a practical point of view, understanding the structure/function relationships of P G F could reveal what characteristics of the h G H molecule allow it to express its complex activities. Are there multiple receptors for G H or does G H have multiple active sites which can differentially activate a single receptor? A better knowledge of P G F could provide answers. Comparison of the structure of P G F and h G H may reveal a diabetogenic sequence of h G H and allow for its removal by genetic engineering and construction of a 'better' h G H .
References
Arsenijevic, Y., Wehrenberg, W. B., Conz, A., Eshkol, A., Sizonenko, P. C. and Aubert, M. L. (1989). Growth hormone (GH) deprivation induced by passive immunisation against rat GH-releasing factor delays sexual maturation in the male rat. Endocrinology 124:3050-3059. Chang, T. W., Raben, M. S., Mueller, J. F. and Weinstein, L. (1973). Cultivation of the sparganum of Spirometra mansonoides in vitro with prolonged production of sparganum growth factor. Proc. Exp. Biol. Med. 143:457-459. Cox, G. Stanley, Phares, C. K. and Schmidt, R. A, (1990). Molecular characterisation of the Spirometra mansonoides genome: renaturation kinetics, methylation and hybridisation to human cDNA probes. Biochim. Biophys. A cta 1049:134-144. Elsasser, T. H., Rumsey, T. S., Hammond,
A. C. and Fayer, R. (1988). Influence of parasitism on plasma concentrations of growth hormone, somatomedin-C and somatomedinbinding proteins in calves. J. Endocrinol. 116:191-200.
Gala, R. R. (1991). Prolactin and growth hormone in the regulation of the immune system. Proc. Exp. Biol. Med. 198:513-527. Garland, J. T. and Daughaday, W. H. (1972). Feedback inhibition of pituitary growth hormone in rats infected with Spirometra mansonoides. Proc. Soc. Exp. Biol. Med. 139:497-499. Garland, J. T., Ruegamer, W. R. and Daughaday, W. H. (1971). Induction of sulfation factor activity by infection of hypophysectomised rats with Spirometra mansonoides. Endocrinology 88:924-927. Gustafsson, M. K. S., Lehtonen, M. A. I. and Sundler, F. (1986). Immunocytochemical evidence for the presence of 'mammalian' neurohormonal peptides in neurones of the tapeworm Diphyllobothrium dendriticum. Cell Tissue Res. 243:41-49. Hirai, K., Shiwaku, K., Tsuboi, T., Torii, M., Nishida, H. and Yamane, Y. (1983). Biological effects of Spirometra erinacei plerocercoids in several species of rodents. Z. Parasitenkd. 69:489-499. Hirai, K., Tsuboi, T., Torii, M. and Nishida, H. (1987). Carbohydrate metabolism in intact golden hamsters infected with plerocercoids of Spirometra erinacei (Cestoda: Diphyllobothriidae). Parasitol. Res. 74:183-187. Ivanyi, J. (1982). Analysis of monoclonal antibodies to human growth hormone and related proteins. In: Monoclonal Hybridoma Antibodies: Techniques and Applications, eds. Hurrell J. G. R., p. 59. CRC Press. Boca Raton, FL. Kostyo, J. L. (1986). The multivalent nature of growth hormone. In: Human Growth Hormone, eds. Raiti, G. and Tolman, R., pp. 449-453. Plenum, New York. Kostyo, J. L., Gennick, S. E. and Sauder, S. E. (1984). Diabetogenic activity of native and biosynthetic human growth hormone in obese (ob/ob) mouse. Am. J. Physiol. 246:E356-E360. Lesniak, M. A. and Roth, J. (1977). Reactiv-
Plerocercoid growth factor ity of non-primate growth hormones and prolactins with human growth hormone receptors in cultured human lymphocytes. J. Clin. Endocrinol. Metab. 44:838-849. Mueller, J. F. (1937). A repartition of the genus Diphyllobothrium. J. Parasitol. 23:308-310. Mueller, J. F. (1963). Parasite-induced weight gain in mice. Ann. N Y Acad. Sci. 113:217-233. Mueller, J. F. (1968). Growth stimulating effect of experimental sparganosis in thyroidectomised and hypophysectomised rats and comparative activity of different species of Spirometra. J. Parasitol. 54:795-801. Mueller, J. F. (1970a). Quantitative relationship between stimulus and response in growthpromoting effect of Spirometra mansonoides spargana on the hypophysectomized rat. J. Parasitol. 56:840-842. Mueller, J. F. (1970b). Comparison of the growthpromoting effect of Spirometra mansonoides vs three Oriental forms in intact mice and hypophysectomized rats. J. Parasitol. 56:842-844. Mueller, J. F. (1972). Absence of sparganum growth factor in African Spirometra spp. J. Parasitol. 58:872-875. Mueller, J. F. (1974). The biology of Spirometra. J. Parasitol. 60:3-14. Norstedt, G., Wragge, O., Gustafsson, J. A. (1981). Multihormonal regulation of the estrogen receptor in rat liver. Endocrinology 108:1190-1196. Phares, C. K. (1982). The lipogenic effect of the growth factor produced by plerocercoids of the tapeworm, Spirometra mansonoides is not the result of hypothyrodism. J. Parasitol. 68:999-1003. Phares, C. K. (1984). A method for solubilisation of a human growth hormone analogue from plerocercoids of Spirometra mansonoides. J. Parasitol. 70:840-842. Phares, C. K. (1986). Regression of rat mammary tumours associated with suppressed growth hormone. Anticancer Res. 6:845-848. Phares, C. K. (1988). Use of receptor affinity chromatography in purification of the growth hormone-like factor produced by plerocercoids of the tapeworm Spirometra mansonoides. J. Recept. Res. 8:645-665. Phares, C. K. and Ai, N. D. (1982). Spirometra mansonoides: Effects of plerocercoid infec-
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tion on glycogen deposition in rats. J. Helminthol. 56:315-322. Phares, C. K. and Booth, B. J. M. (1986a). Reduction of lactogenic receptors in female hamster liver due to the hGH analogue produced by plerocercoids of the tapeworm, Spirometra mansonoides. Endocrinology 118:1102-1109. Phares, C. K. and Booth, B. J. M. (1986b) Suppression of receptors for prolactin and estrogen in rat liver due to treatment with the growth hormone analogue produced by the tapeworm Spirometra mansonoides. J. Recept. Res. 6:425-446. Phares, C. K. and Booth, B. J. M. (1987). Anti-human growth hormone antibodies crossreact with the growth hormone-like factor from plerocercoids of the tapeworm, Spirometra mansonoides. Endocrinology 121:1839-1844. Phares, C. K. and Carroll, R. M. (1977). A lipogenic effect in intact male hamsters infected with plerocercoids of the tapeworm, Spirometra mansonoides. J. Parasitol. 63:690-693. Phares, C. K. and Carroll, R. M. (1978). Comparison of the effects of the growth factor produced by Spirometra mansonoides and growth hormone in diabetic-hypophysectomized rats: Lipid composition. J. Parasitol. 64:401-405. Phares, C. K. and Carroll, R. M. (1984). Insulinlike effects on fatty acid synthesis in liver of hamsters infected with plerocercoids of the tapeworm, Spirometra mansonodies. J. Helminthol. 58:25-30. Phares, C. K. and Corkum, K. C. (1974). Effects of spirometrid plerocercoids on several species of lower vertebrates. Comp. Biochem. Physiol. 48A:525- 531. Phares, C. K. and Ruegamer, W. R. (1972). In vitro preparation of a growth factor from plerocercoids of the tapeworm, Spirometra mansonoides. Proc. Soc. Exp. Biol. Med. 142:374-377. Phares, C. K. and Ruegamer, W. R. (1973). Pigeon crop sac stimulation due to a factor produced by plerocercoids of the tapeworm, Spirometra mansonoides. Proc. Soc. Exp. Biol. Med. 143:147-151. Phares, C. K. and Ruegamer, W. R. (1976). Effects of growth factor from the tapeworm, Spirometra mansonoides, on a hypophysec-
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tomized Rhesus monkey. Proc. 58th Endocrine Soc. Mtg., 459, June, 1976. Phares, C. K. and Sharp, S. W. (1991). Immunoenhancememt associated with the growth hormone-like factor produced by the tapeworm Spirometra mansonoides. Life Sci. Adv. Exp. Clin. Endocrinol., in press. Phares, C. K. and Watts, D. J. (1988). The growth hormone-like factor produced by the tapeworm Spirometra mansonoides competitively inhibits the binding of human growth hormone to its receptors on IM9 cells. J. Parasitol. 74(5):896-898. Phares, C. K., Hofert, J. F. and Pettinger, C. L. (1976). Growth stimulation of lymphatic tissue by plerocercoid larvae of the tapeworm, Spirometra mansonoides. Gen. Comp. Endocrinol. 28L:103-106. Phares, C. K., Shaffer, J. L. and Heidrick, M. L. (1990). Characteristics, distribution and possible evolutionary importance of the human growth hormone-like factor from plerocercoids of the tapeworm genus Spirometra. In: Immune Recognition and Evasion: Molecular Aspects of Host-Parasite Interaction, eds. Van Der Ploeg, L., Cantor, C. R. and Vogel, H. J., pp. 149-161. Academic Press, San Diego, CA. Pieropaoli, W., Baroni, C., Fabris, N. and Sorkin, E. (1969). Hormones and immunological capacity. II. Reconstitution of antibody production in hormonally deficient mice by somatotropic hormone, thyrotropic hormone and thyroxine. Immunology 16:217-230. Ramaley, J. A. and Phares, C. K. (1980). Delay of puberty onset due to suppression of growth hormone. Endocrinology 106(6):1989-1993. Ramaley, J. A. and Phares, C. K. (1983). Delay of puberty onset in males due to suppression of growth hormone. Neuroendocrinology 36:321-329. Rechler, M. M., Nissley, S. P. and Roth, J. (1987). Hormonal regulation of human growth. N. Engl. J. Med. 316:941. Salem, M. A. M. and Phares, C. K. (1986). Some biochemical effects of the growth hormone analogue produced by plerocercoid larvae of the tapeworm, Spirometra mansonoides on carbohydrate metabolism of adipose tissue from normal, diabetic and hypophysectomized rat. J. Parasitol. 72(4):498--506.
Salem, M. A. M. and Phares, C. K. (1987). Insulin-like effects in the rat of the purified growth factor from Spirometra mansonoides plerocercoids. Proc. Soc. Exp. Med. BioL 185:31-38. Salem, M. A. M. and Phares, C. K. (1989a). The growth factor from plerocercoid larvae of the tapeworm, Spirometra mansonoides stimulates growth but is not diabetogenic. Proc. Soc. Exp. Med. Biol. 191:187-192. Salem, M A. M. and Phares, C. K. (1989b). In vitro insulin-like actions of the growth factor from the tapeworm, Spirometra mansonoides. Proc. Soc. Exp. Med. Biol. 190:203-210. Sharp, S. E., Phares, C. K. and Heidrick, M. L. (1982). Immunological aspects associated with suppression of hormone levels in rats infected with plerocercoids of Spirometra mansonoides (Cestoda). J. Parasitol. 68(6):993- 998. Shiwaku, K., Hirai, K., Torii, M. and Tsuboi, T. (1983a). Effects of Spirometra erinacei plerocercoids on the growth of Snell dwarf mice. Parasitology 87:447-453. Shiwaku, K., Hirai, K., Tsuboi, T. and Torii, M. (1986). Enhancement of serum somatomedin activity and cartilage mitotic activity in Snell normal and dwarf mice infected with Spirometra erinacei plerocercoids. Jpn. J. Parasitol. 35:4ll-417. Steelman, S. L., Morgan, E. R., Cuccaro, A. J. and Glitzer, M. S. (1970). Growth hormonelike activity in hypophysectomized rats implanted with Spirometra mansonoides spargana. Proc. Soc. Exp. Biol. Med. 133:269-273. Steelman, S. L., Glitzer, M. S., Ostlind, D. A. and Mueller, J. F. (1971). Biological properties of the growth hormone-like factor from the plerocercoid of Spirometra mansonoides. Recent Prog. Hormone Res. 27:97-120. Swartzwelder, J. C., Beaver, P. C. and Hood, M. W. (1964). Sparganosis in southern United States. A m J. Trop. Med. Hyg. 13:43-48. Tseng, M. T. and Mueller, J. F. (1977). Suppression of somatotrops in rats bearing spargana of Spirometra mansonoids, an ultrastructural study. J. Parisitol. 63:168-169. Tsuboi, T. and Hirai, K. (1986). Lipid metabolism in golden hamsters infected with plerocercoids of Spirometra erinacei (Cestoda: Pseudophyllidea). Parasitology 93:143-151.
Plerocercoid growth factor l~sushima, T. and Friesen, H. G. (1973). Radioreceptorassay for growth hormone. J. Clin. Endocrinol. Metab. 37:344-348. Tsushima, T., Friesen, H. G., Chang, T.
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W., Raben, M. S. (1974). Identification of sparganum growth factor by a radioreceptor assay for growth hormone. Biochem. Biophys. Res. Commun. 59:1062-1068.
0960-5428/92 $15.00 © 1992 Pergamon Press Ltd
Biological characteristics of the growth hormonelike factor from plerocercoids of the tapeworm
Spirometra mansonoides C. K i r k Phares D e p a r t m e n t of Biochemistry, University of Nebraska Medical Centre, 600 South 42nd Street, O m a h a , NB 68198-4525, U S A
Introduction There are a variety of defence mechanisms ranging from simple to very complex which enable animals to survive and reproduce in a world full of potentially lethal infectious agents. In order that an organism be a successful parasite in other animals, several significant barriers must be overcome. The parasite must gain access and enter a potential host and then survive an array of sophisticated and normally very effective host defense systems. For those parasites with multiple stages of their life-cycle, the potential to pass through several intermediate hosts and then 'find' an appropriate definitive host where reproduction can occur becomes very small. The biochemical mechanisms which enhance the odds that any parasite will succeed have been studied in only a very few of the many known parasite-host relationships. A phenomenon exhibited by the plerocercoid stage of the pseudophyllidean tapeworm Spirometra mansonoides may be an example of a very 'clever' mechanism to enhance survival. Plerocercoids of S. mansonoides release a substance which mimics some, but not all of the actions of mammalian growth hormone. The importance of growth hormone (GH) in regulation of overall body growth as well as carbohydrate, lipid and protein metabolism in vertebrates has long been established. More recently, GH has been shown to play a
stimulatory role in the regulation of immune function (Gala, 1991). By duplicating only some of the actions of GH, release of plerocercoid growth factor (PGF) in hosts results in alterations in growth, metabolism and in some cases, immune function. It is not unreasonable to suggest that PGF somehow enhances the survival of this tapeworm.
Discovery of the growth stimulating effect of plerocercoids J. F. Mueller (1937) is not only credited with the establishment of the genus Spirometra as being distinct from Diphyllobothrium but also with the serendipitous discovery of the growth enhancing phenomenon associated with plerocercoid infections of laboratory rodents (Mueller, 1963). Mueller's early work was primarily concerned with the taxonomy of Spirometra but he eventually became interested in establishment and maintenance of the entire life-cycle of S. mansonoides in his laboratory. His efforts to find a more manageable laboratory host than the most common natural hosts (frogs or snakes) for the tissue invading plerocercoid stage, resulted in his discovery that not only could laboratory mice serve as excellent maintenance hosts but that the presence of plerocercoids was associated with a marked
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increase in the size and weight of mice (Mueller, 1963). The observation that the plerocercoid infections were associated with accelerated growth was extended to hormonally-intact golden Syrian hamsters (MueUer, 1965), rats (Ruegamer and Phares, 1974) and lizards (Phares and Corkum, 1974). In addition to hormonally intact animals, rats with growth retardation induced by hormonal manipulation could be stimulated to grow when infected with plerocercoids. Hypothyroid (Mueller and Reed, 1968), hypophysectomised (Mueller, 1968) and castrated rats (Ruegamer and Phares, 1974) respond to the growth promoting effects of plerocercoids. The fact that plerocercoids stimulate growth in hypophysectomised (Hx) rats suggests that they produce and release a GH-like substance.
Actions of GH
Whereas a comprehensive review of the multiple actions of GH is inappropriate here, some of its activities must be mentioned. Normal production and secretion of GH from somatotroph cells in the anterior pituitary is an absolute requirement for normal growth of young mammals. The somatomedin hypothesis of growth suggests that after its release into the circulation GH stimulates the release of secondary hormones originally named somatomedins but now referred to as the insulin-like growth factors (IGF) which are directly responsible for growth. Insulin-like growth factor-1 (IGF-1) is thought to be the essential growth promoting substance. Recent data show that G H has direct growth promoting effects as well as its indirect effects on growth via stimulation of IGF-1 (Isaksson et al., 1987). In addition to its growth stimulating effects, GH is an important metabolic regulator. Preparations of GH from a variety of vertebrates as well as recombinant
GH exhibit multiple biological properties that are difficult to reconcile with a single mechanism of action. Growth hormone is not only anabolic but is diabetogenic producing hyperglycaemia, glucose intolerance, hyperinsulinaemia and insulin resistance (Kostyo, 1986). Furthermore, in the special circumstance of G H deficiency, exogenously administered GH produces transient insulin-like effects including hypoglycaemia, inhibition of lipolysis and stimulation of glucose oxidation in adipose tissue. In the past, investigators have questioned whether the complex biological activities are intrinsic characteristics of the GH molecule or if they are due to contaminants in GH preparations. However, it is now generally accepted that the growth promoting, diabetogenic and insulin-like activities are intrinsic properties of the GH molecule. Furthermore, there is a significant body of convincing evidence that there are multiple active sites in the GH molecule which account for its actions. An additional possibility exists that there may be several functionally distinct receptors for GH. In addition to the multivalent properties shared by all GHs, primate GHs express several unique characteristics as they are also lactogenic (prolactin-like), a characteristic not expressed by non-primate GHs. Lactogenic activity of primate GH stems from its ability to bind receptors for PRL with high affinity. Moreover, human GH (hGH) expressed potent growth promoting activities and other biological properties in all vertebrates whereas only hGH (or other primate GH) is biologically active in humans (or other primates). Biological effects associated with plerocercoid infections
Subsequent to the original observations of Mueller on growth promotion in experimental rodents, a number of reports followed which suggested that plerocercoids elabo-
Plerocercoid growth factor rate a GH-like substance. Steelman et al. (1970) reported that infection of Hx rats with plerocercoids produced a remarkable increase in body weight. The fact that these workers also found increases in tibial cartilage width and increases in somatomedin (IGF-1) in the infected rats suggests that the response was a normal GH-like growth response. Furthermore, they reported that injections of plasma from actively growing Hx rats infected with plerocercoids into uninfected Hx rats stimulated weight gains greater than that produced by 100 ~g of bovine GH each day. Garland et al. (1971) confirmed that serum from plerocercoid-infected Hx rats had high levels of GH-like activity as determined by an assay for sulphation factor activity (IGF1) but the serum had no immunoassayable rat GH. These data strongly suggest that a factor is produced by plerocercoids, released into the circulation and acts on sensitive tissues in a manner analogous to GH. Mueller (1970) demonstrated a quantitative relationship between the number of plerocercoids and the growth response in Hx rats and reported that one plerocercoid per rat could stimulate growth. Hypophysectomised rats with subcutaneous infections of plerocercoids (10 worms/rat) expressed biological responses similar to those observed with injections of 100 ~g/day of bovine GH (Steelman et al., 1971). These responses include increases in body, liver, thymus and kidney weights, as well as increases in epiphyseal cartilage width. The only consistent difference in organ weights of the experimental Hx rats in this study was seen in the epididymal fat pads. Plerocercoid-infected rats had significantly more stored fat than untreated or GHtreated animals. Normal golden hamsters are especially sensitive to the lipogenic effects of plerocercoid infection. Meyer et al. (1965) reported that plerocercoidinfected hamsters had a marked increase in carcass fat and Phares and Carroll (1978)
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found increases in fat pad weights and lipid content of serum and liver of infected hamsters. Insulin is distinctly lipogenic and it is possible that the presence of plerocercoids might result in an increase in insulin secretion which could account for the lipogenic response to pleroceroids. If this were the case, blood glucose levels should be lowered in infected animals. This is not the case as plerocercoid infections did not alter blood glucose levels of normal (Phares and Ai, 1982), Hx (Steelman et al., 1971) or diabetic rats (Salem and Phares, 1986). Furthermore, a marked increase in the weights of the epididymal fat pads of plerocercoid infected diabetic-Hx rats was observed (Phares and Carrol, 1978).
Comparison of PGF to hGH
For several important reasons the characteristics of human G H warrant special attention. Not only does hGH possess growth promoting, anti-insulin/diabetogenic and insulin-like characteristics, but expresses distinct lactogenie activities as well. Furthermore, whereas exogenous hGH is biologically active in all vertebrate classes from fish to mammals, no subprimate G H is active in primates. Based on a variety of experiments it is possible to state that PGF expresses biological activities which are more similar to hGH than they are to any other vertebrate GH. For instance, Phares and Ruegamer (1973) reported that PGF had a dramatic stimulatory effect on growth of the mucosal epithelium of pigeon crop sac, a well established assay for lactogenie (PRL) activity (Nicoll, 1967). More recently, a cell line derived from a rat lymphoma (NB-2 cells) has been shown to have an absolute dependency on lactogenic hormones for growth (Lesniak and Roth, 1977). Human G H is a potent lactogen in this very sensitive assay. We have found that partially-purified PGF expresses mitogenic
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PRL-like activity in this cell line. Therefore, PGF expresses lactogenic activity similar to that reported for hGH. Another hGH-like property which is significant is the ability of PGF to express biological activity in a variety of vertebrates. In addition to examples of growth stimulation in mice, rats and hamsters and lactogenic activity in pigeons described above, PGF stimulated a growth increase in the lizard, Anolis carolinensis equivalent to 32% of the initial body weights while the untreated controls did not grow during the experimental period (Phares and Corkum, 1974). Perhaps the most convincing evidence that PGF has activity considered unique to hGH comes from experiments involving primates and human cells. Partially-purified PGF was injected into a Hx Rhesus monkey and was shown to increase production of somatomedin (IGF-1) and to decrease the concentration of blood urea nitrogen (Phares and Ruegamer, 1976). These are responses one would expect only with injections of a primate GH. In another experiment PGF was shown to displace hGH from its highly specific receptors on human IM9 cells (Phares and Watts, 1988). Additional evidence for the similarity between PGF and hGH comes from a study involving anti-hGH monoclonal antibodies (MAB). Antigenic mapping of hGH suggests that four distinct nonoverlapping regions are expressed (Ivanyi, 1982). The crossreactivity of PGF with four MABS, each of which describes a distinct epitope of hGH, was tested. The ability of PGF to displace 125IhGH from this series of MABS varied from nearly equal potency (~70%) to less than one percent the potency of the hGH standard (Phares and Booth, 1987). No nonhuman GH or PRL gave any degree of crossreactivity with any of the MABs (Ivanyi, 1982; Phares and Booth, 1987). Other evidence that PGF and hGH share molecular characteristics include the fact that cDNA for hGH gave a positive
hybridisation signal in Southern blots with genomic DNA from plerocercoids (Cox et al., 1990). Both the molecular hybridisation study and the hGH MAB study suggest that molecular similarity exists, however, the fact that the hGH cDNA hybridised with plerocercoid DNA only under relatively mild conditions and that PGF had an immunopotency of less than one percent that of hGH with one anti-hGH MAB strongly suggests that there are also important molecular differences between PGF and hGH. Whereas the similarities between PGF and hGH are remarkable and of great potential significance, the differences between these two substances with respect to both biological and molecular characteristics may be more interesting and of greater value.
Effects of PGF on the host's endocrine system
Whereas all of the body's systems, organs and tissues are not under obligatory regulation by hormones, essentially all are influenced by several hormones. Hormones regulate the metabolic activities of many tissues and have an important regulatory role in the immune system. If a parasite could manipulate a potential host's metabolic activities and/or its immune system in a manner which would allow the parasite to gain entrance and establish an infection without totally compromising the host's defence system, then this might be of selective advantage to the parasite. An important physiological and survival role for PGF in the biology of S. mansonoides may be dependent on the ability of PGF to alter the hormonal milieu of the plerocercoid host and therefore upset normal metabolic and immune regulation. A number of hormones influence the immune system, some like the adrenal cortical hormones suppress the immune system. Thyroid hormone and sex hormones influence both T- and B-cells
Plerocercoid growth factor responses (Peiropaoli et al., 1969). Prolactin and GH are involved in stimulating the immune system (Gala, 1991). The involvement of anterior pituitary hormones with the immune system had its origins from observations with Hx animals and in humans deficient in specific pituitary hormones. Hypophysectomy in rodents results in a suppression of immune function. Another consistent observation of anterior pituitary hormone deficiency is a decrease in thymus weight and function. Garland and Daughaday (1972) reported a decrease in pituitary weight and G H content as well as reduced serum GH in rats infected with plerocercoids of S. mansonoides. Tseng and Mueller (1977) studied cytological changes in the pituitaries of plerocercoidinfected rats and consistent with other reports, found a dramatic reduction in the weights of the pituitaries. They found no necrotic cells and no alterations in morphology of non-GH producing cells. However, the somatotrophs (GH producing cells) were markedly condensed. Glitzer and Steelman (1971) did not observe enhanced growth in young normal rats due to PGF but did report suppression of serum G H levels. Syrian hamsters are especially sensitive to the feedback regulation of G H by PGF as the pituitaries of control hamsters weighed two and a half times more and the serum contained four times more G H than did PGF-treated hamsters (Phares, 1982). Control of synthesis and secretion of hormones is complex and may involve regulation by homologous hormone, heterologous hormones, metabolic factors and other conditions. Most regulatory factors controlling G H secretion act via neural mechanisms. Somatostatin (GH release inhibiting hormone) is released from the hypothalamus in response to elevated serum levels of GH and IGF-1. Somatostatin acts directly upon the somatotrophs in the pituitary to inhibit both synthesis and release of GH. In addition to its regulatory effect at the level of the
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hypothalamus, IGF-1 also has a direct regulatory role on pituitary somatrophs. Other physiologically important negative regulators of G H levels include obesity, elevation of free fatty acids, increased glucocorticoids and hyperglycemia. As PGF does not affect serum glucose or increase free fatty acids, its mechanism of negative regulation of G H is presumably via its ability to mimic G H at the level of the hypothalamus and/or to increase the levels of IGF-1 in circulation. Although first identified as the release inhibiting hormone for GH, somatostatin also inhibits the production and release of thyroid stimulating hormone (TSH) therefore reducing the release of thyroid hormone. That PGF is recognised by GH-sensitive cells in the hypothalamus in feedback regulation is supported by the fact that thyroid hormone is also suppressed in PGF-treated animals (Phares, 1982; Sharp et al., 1982). The presence of PGF in animals results in a model with accelerated growth despite severe endogenous GH deficiency. If P G F mimics all of the activities of G H then PGFtreated animals should be representative of chronic G H excess (acromegaly). However, if PGF mimics only some of the multiple actions of G H then the model would be representative of G H excess with respect to some functions and of G H deficiency with respect to those actions not duplicated by PGF. The latter description is the model observed in PGF-treated animals. As previously stated, Hx and other GHdeficiency animals have reduced immune function. An obvious strategy for plerocercoids seeking to establish themselves in a new host would be to selectively suppress G H and not replace its stimulatory activity in the immune system. This was observed in rats as treatment with PGF resulted in transient suppression of the humoral immune response (Sharp et al., 1982). Interestingly, the humoral immune response was restored to normal with injections of thyroxine but not of G H in PGF-treated
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rats. It should be noted that suppression of the immune system due to hormonal manipulation by PGF is not observed in all hosts. Phares and Sharp (1991) reported that although plerocercoid-infected hamsters are deficient in GH and thyroxine their immune response was actually enhanced when compared to controls. Furthermore, PGF, like GH, stimulates growth of the thymus in Hx rats (Steelman et al., 1971; Phares et al., 1976). Whether the thymotropic action of PGF affects the immunological functions o f the thymus is not known. Therefore, it can be suggested that whereas all experimental animals tested respond to PGF all the responses are not uniformly predictable. These data are consistent with the concept that PGF mimics some but not all of the i actions of GH. There have long been questions concerning the roles that growth and GH play in sex~ual development. It has been unresolved as 'to whether GH itself or simply body growth are involved in the timing of puberty. In humans, GH deficiency is often accompanied by a delay of puberty onset (Rechler et al., 1987). Ramaley and Phares (1980) were able to demonstrate with PGF treatment that it is GH and not merely growth itself which is :essential for the normal timing of the onset of puberty in female rats. Confirmation of this observation was obtained in male rats (Ramaley and Phares, 1983). The influence of GH deficiency on the timing of puberty in rats has recently been confirmed by passive immunisation of rats to the GH releasing hormone to reduce circulating GH levels (Arsenijevic et al., 1991). The ability of PGF to reduce GH levels was used to study the controversial role of GH in the regulation of tissue receptors for PRL and estrogen. The inductive effect of GH on lactogenic receptors is suspected to be due to a direct somatogenic action and not to be associated with binding to the PRL receptor. Despite the fact that PGF (like hGH) binds to both somatogenic and lactogenic
receptors it does not upregulate lactogenic receptors as treatment with hGH does. In fact, treatment of adult female rats with PGF resulted in nearly a three-fold reduction in the number of lactogenic receptors without any significant change in the binding affinity (Phares and Booth, 1986). Not only were PRL receptors reduced but the number of hepatic estrogen receptors was reduced by one half in the PGF-treated rats compared to controls (Phares and Booth, 1986). The reduction of estrogen receptors could result in a blunted response to estrogen in PGFtreated animals and may partially explain the delay in puberty onset described above and the regression of hormone-sensitive mammary tumours in rats treated with PGF (Phares, 1986). It is not known at this time if (or how) the disturbance of hormonal balance observed in plerocercoid-infected animals is of survival advantage to S. mansonoides. It is possible that the suppression of the immune response associated with the presence of PGF is important in the establishment of plerocercoid infections in new hosts. The ability of PGF to mimic the growth promoting effects of GH also may play an important role in the ability of plerocercoids to reach their final host and reproduce. The rationale for this is based on the fact that long-term exposure to elevated levels of GH results in acromegaly. This disease is associated with significantly increased incidences of a variety of disorders. The death rate for acromegalics is twice that of the normal population. The presence of plerocercoids and their unregulated production of PGF could produce complications similar to those observed in acromegaly. In support of this are data from aging studies with control and plerocercoid-infected mice which show a marked increase in the death rate of the infected mice after only six months of infection (Phares et al., 1990). In this same study, 50% of the plerocercoid-infected mice were dead by the 18th month of age whereas
Plerocercoid growth factor 50% of the age-matched controls were still alive at 30 months of age. Early death or diseases which might limit mobility would likely make the hosts of plerocercoids more vulnerable to predation by the carnivorous final hosts of S. mansonoides. Other biochemical effects of PGF
As cited above, early work by several groups of researchers clearly demonstrate that exposure to PGF via plerocercoid infection has distinct effects on carbohydrate and lipid metabolism. As further described above, the presence of PGF is associated with marked perturbations of endogenous hormones and hormone receptors. Without direct studies of the effects of PGF on metabolism, the results observed could be explained simply on the basis of increases or decreases in specific endogenous hormone function. It should be remembered that GH is not only growth promoting but elevates blood glucose levels, increases blood insulin levels, induces tissue insensitivity to insulin as well as to its own insulin-like activity, increases lipolysis and circulating free fatty acid levels and has other diabetogenic effects in normal animals and humans. In addition, under the special circumstance of GH deficiency, GH expresses transient insulin-like effects (Kostyo, 1986). Neither plerocercoid infections nor injections of partially-purified PGF have been reported to have any consistent and significant effect on blood glucose or insulin. However, PGF, like injections of GH, increased liver glycogen content but unlike GH, PGF had no effect on cardiac glycogen levels in Hx rats (Phares and Ai, 1982). Adipose tissue from animals chronically exposed to PGF (plerocercoid infection) had significantly elevated glucose oxidation rates (Salem and Phares, 1986). The results were essentially the same in intact and Hx rats, indicating the effects of PGF were not due to GH suppression. The basal glucose oxi-
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dation rate of adipose tissue from diabetic rats was stimulated by PGF but the tissue was not sensitive to insulin added in vitro. This suggests the possibility of a direct insulin-like effect of PGF. The acute effects of injections of PGF were compared to saline or insulin in intact male rats. The rats were injected intraperitoneally, sacrificed one half hour later and glucose and leucine metabolism were measured in adipose tissue and muscle. Both insulin and PGF stimulated dose-dependent increases in carbohydrate, lipid and protein metabolism but again PGF had no effect on blood glucose (Salem and Phares, 1987). These results suggest that PGF has intrinsic insulin-like activity in normal (non-GH deficient) and diabetic (insulin-deficient) rats and represents a dramatic difference from the actions of GH. It is well established that tissues from intact animals are not sensitive to the insulin-like actions of GH and this refractoriness is induced by GH itself (Goodman and Coiro, 1981). Salem and Phares (1987) conducted experiments with adipose tissue from normal male rats in vitro to determine if PGF expresses its insulin-like activity in the absence of any other hormone. As expected, hGH stimulated glucose oxidation only after an extended preincubation period to eliminate the refractoriness of the tissue to GH. Furthermore, hGH produced a dramatic lipolytic response in freshly removed adipose tissue. Neither insulin nor any dose of PGF had a lipolytic effect but both stimulated a lipogenic response in freshly removed and preincubated adipose tissue. Despite its obvious differences from GH and similarity to insulin, PGF does not bind the insulin receptor. Its effects are mediated through the same receptors which bind hGH. The sum of the anti-insulin actions of GH are responsible for its diabetogenic characteristic. Both highly purified natural and recombinant hGH have the ability to induce hyperglycaemia and insulin-resistance
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in man and overt diabetes in experimental animals. The genetically obese mouse (ob/ob) is extremely sensitive to the antiinsulin actions of GH and is the whole animal model used to test diabetogenicity (Kostyo et al., 1984). Affinity-purified PGF was first tested for its biological potency relative to hGH in a growth promoting assay in normal mice. The same preparations of PGF and hGH were then assayed for diabetogenicity in ob/ob mice. No dose of PGF was diabetogenic while the injections of hGH caused a significant increase in fasting blood glucose levels and a dramatic impairment of glucose tolerance (Salem and Phares, 1989).
in rabbit liver membranes consists of both somatogenic and lactogenic sites, we now know that PGF binds the same receptors. The ability to test PGF by R R A allows its quantification in terms of a hGH standard. Therefore, binding activity of PGF is expressed as equivalent (Eq) units of the hGH standard. The adaptation of a sensitive assay allowed for experiments intended to purify PGF from plerocercoid incubation media or from serum of plerocercoid-infected Hx rats. However, under the best circumstances, plerocercoid incubation media yields no more than 100 ngEq/ml. Efforts to concentrate the media results in unacceptable losses of activity making purification from this material prohibitive. Purification of trace quanIsolation of plerocercoid growth factor tities of PGF from serum is not pratical. No (PGF) binding or growth promoting activity can be Efforts to achieve final purification to al- obtained by homogenisation of whole worms low structural characterisation of PGF have or subcellular fractions of plerocercoids in not been fully successful. The fact that aqueous buffers. However, binding activities plasma from plerocercoid-infected Hx rats equivalent to more than 1000 ng of hGH/ml contains growth enhancing characteristics can be obtained by solubilisation of whole (Glitzer and Steelman, 1971; Garland et al., plerocercoids or membranes in the non-ionic 1971) suggests that the hormonally active detergent Triton-X 100 (Phares, 1984). With solubilised rabbit liver membrane resubstance is released by plerocercoids and it should be possible to collect and eventually ceptors for hGH purified over a hGH affinity purify this substance. Growth hormone-like column, Phares (1988) was able to construct activity was detected in media in which a hGH-receptor affinity column. Passage plerocercoids were incubated (Phares and of crude solubilised PGF over this column Ruegamer, 1973; Chang et al., 1973). These resulted in more than 1000-fold increases early studies were hampered by the need to in specific activity as determined by RRA. test for activity in the relatively insensitive Affinity-purified PGF stimulated a very draHx rat growth promoting assay. However, matic dose-dependent growth response in shortly after the development of the first Hx rats and had direct in vitro activity radioreceptor assay (RRA) (Tsushima and on adipose tissue from normal rats (Salem Friesen, 1973) Tsushima et al. (1974) re- and Phares, 1989). Despite the 1000-fold inported that PGF in incubation medium as crease in specific activity the affinity-purified well as in serum from infected Hx rats PGF was not homogenous but contained displaced 125IhGH from its receptors in a several distinct stained protein bands after manner comparable to the hGH standard. SDS-PAGE. Of the three major bands, one This report was very valuable for several had a MW of 22,000, identical to the major reasons. The R R A for hGH is at least form of hGH. However, there is no defini1000 times more sensitive than the Hx rat tive proof of the molecular characteristics of growth assay and as the receptors for hGH PGF.
Plerocercoid growth factor No PGF activity has been detected by any method in either the procercoid or adult stages of S. mansonoides (Phares et al., 1990). Distribution of PGF in the genus Spirometra
The genus Spirometra includes numerous forms from throughout the world. Many of the suggested spirometrids are known only in their larval forms. Morphological variations in the genus is continuous and it is essentially impossible to define species in any precise manner. All stages of S. mansonoides are known and this species can be defined with some distinction. A number of 'species' have been described from the Orient but these are believed to be variants of a single species, S. erinacei. Hirai and his colleagues in Japan have done extensive work with PGF from S. erinacei and found that growth is stimulated in several rodent models (Hirai et al., 1983). The differences in biological activities between the PGFs from S. erinacei (PGF-e) and S. mansonoides (PGFm) are of interest. Similar to the results obtained with plerocercoids of S. mansonoides, plerocercoids of S. erinacei stimulate growth in mice, increase IGF-1 levels (Shiwaku et al., 1986) and PGF-e displaces hGH from its receptors in rabbit liver membranes (Hirai et al., 1986). In contrast to PGF-m, PGF-e does riot stimulate growth of Syrian hamsters, normal or Hx rats (Hirai et al., 1983). Furthermore, the effects of PGF-e on carbohydrate and lipid metabolism are inconsistent with those of PGF-m (Hirai et al., 1983, 1987). In agreement with these differences, Mueller (1970) reported weak growth promoting effects of plerocercoids from an Australian spirometrid and no growth promoting effects in Hx rats from two additional oriental spirometrids. Mueller (1972) later reported no growth promoting activity in Hx rats for plerocercoids collected in Africa.
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Therefore, it is clear that production of a GH-like factor is not unique to S. mansonoides and it is also clear that speciation has resulted in evolutionary changes in the biological characteristics of PGF. It will be of great interest to compare the molecular characteristics of PGFs from a variety of geographical forms of Spirometra.
Future studies with PGF
Parasitism is commonly associated with suboptimal or even halted growth of the host. Nutritional, hormonal and immunological consequences of parasitism may lead to diversion of nutrients away from supporting growth. There are a variety of examples where parasitism perturbs the hosts endocrine system including G H and IGF1. For instance, infections of cattle with the protozoan parasite, Sarcocystis cruzi are associated with increased somatostatin levels, decreased GH and IGF-1 levels which result in an inhibition of growth in calves (Elasser et al., 1988). There are immunocytochemical studies which show the presence of mammalian-like hormones in some parasites (Gustafsson et al., 1986). However, knowledge of the production and release of a hormone-like substance in sufficient abundance to dramatically stimulate normal growth and to seriously modulate the hormonal milieu, immune system and metabolic activities of the host is restricted to studies with plerocercoids and PGFs from S. mansonoides and S. erinacei. It is obvious that the substance responsible for mimicking G H must be purified and characterised to extend this area of study in a useful direction. This has not proven to be an easy task as success has not been achieved despite years of effort. Molecular studies with PGF and the genetic elements responsible for its production should reveal important information of interest to both parasitologists and endocrinologists. Questions as to the
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origins of P G F are very intriguing. Did P G F arise by 'normal' evolution or was a gene for a primate G H somehow transferred to Spirometra? Humans are very suitable hosts for plerocercoids and human infections occur at locations around the world (Swartzwelder et al., 1964). What evolutionary advantages does expression of P G F bestow on Spirometra? Are its hormonal activities essential or irrelevant to survival of the tapeworm? From a practical point of view, understanding the structure/function relationships of P G F could reveal what characteristics of the h G H molecule allow it to express its complex activities. Are there multiple receptors for G H or does G H have multiple active sites which can differentially activate a single receptor? A better knowledge of P G F could provide answers. Comparison of the structure of P G F and h G H may reveal a diabetogenic sequence of h G H and allow for its removal by genetic engineering and construction of a 'better' h G H .
References
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Salem, M. A. M. and Phares, C. K. (1987). Insulin-like effects in the rat of the purified growth factor from Spirometra mansonoides plerocercoids. Proc. Soc. Exp. Med. BioL 185:31-38. Salem, M. A. M. and Phares, C. K. (1989a). The growth factor from plerocercoid larvae of the tapeworm, Spirometra mansonoides stimulates growth but is not diabetogenic. Proc. Soc. Exp. Med. Biol. 191:187-192. Salem, M A. M. and Phares, C. K. (1989b). In vitro insulin-like actions of the growth factor from the tapeworm, Spirometra mansonoides. Proc. Soc. Exp. Med. Biol. 190:203-210. Sharp, S. E., Phares, C. K. and Heidrick, M. L. (1982). Immunological aspects associated with suppression of hormone levels in rats infected with plerocercoids of Spirometra mansonoides (Cestoda). J. Parasitol. 68(6):993- 998. Shiwaku, K., Hirai, K., Torii, M. and Tsuboi, T. (1983a). Effects of Spirometra erinacei plerocercoids on the growth of Snell dwarf mice. Parasitology 87:447-453. Shiwaku, K., Hirai, K., Tsuboi, T. and Torii, M. (1986). Enhancement of serum somatomedin activity and cartilage mitotic activity in Snell normal and dwarf mice infected with Spirometra erinacei plerocercoids. Jpn. J. Parasitol. 35:4ll-417. Steelman, S. L., Morgan, E. R., Cuccaro, A. J. and Glitzer, M. S. (1970). Growth hormonelike activity in hypophysectomized rats implanted with Spirometra mansonoides spargana. Proc. Soc. Exp. Biol. Med. 133:269-273. Steelman, S. L., Glitzer, M. S., Ostlind, D. A. and Mueller, J. F. (1971). Biological properties of the growth hormone-like factor from the plerocercoid of Spirometra mansonoides. Recent Prog. Hormone Res. 27:97-120. Swartzwelder, J. C., Beaver, P. C. and Hood, M. W. (1964). Sparganosis in southern United States. A m J. Trop. Med. Hyg. 13:43-48. Tseng, M. T. and Mueller, J. F. (1977). Suppression of somatotrops in rats bearing spargana of Spirometra mansonoids, an ultrastructural study. J. Parisitol. 63:168-169. Tsuboi, T. and Hirai, K. (1986). Lipid metabolism in golden hamsters infected with plerocercoids of Spirometra erinacei (Cestoda: Pseudophyllidea). Parasitology 93:143-151.
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W., Raben, M. S. (1974). Identification of sparganum growth factor by a radioreceptor assay for growth hormone. Biochem. Biophys. Res. Commun. 59:1062-1068.