WO1995024425A1 - Substances immunomodulatrices tirees de trichinella - Google Patents

Substances immunomodulatrices tirees de trichinella Download PDF

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Publication number
WO1995024425A1
WO1995024425A1 PCT/US1995/002991 US9502991W WO9524425A1 WO 1995024425 A1 WO1995024425 A1 WO 1995024425A1 US 9502991 W US9502991 W US 9502991W WO 9524425 A1 WO9524425 A1 WO 9524425A1
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substance
trichinella
cells
pseudospiralis
tpi
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PCT/US1995/002991
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English (en)
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George Stewart
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Board Of Regents, The University Of Texas System
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Priority to AU19876/95A priority Critical patent/AU1987695A/en
Publication of WO1995024425A1 publication Critical patent/WO1995024425A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
    • C07K14/4354—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates generally to the fields of immunotherapy and immunosuppression.
  • the invention is directed to the identification of novel substances with profound immuno- odulatory effects. More particularly, it concerns the identification of novel factors obtainable from the Trichinella sp. during mutually exclusive stages of its life cycle that directly exert an effect on T cells without countervailing side effects.
  • the invention further concerns the use of these novel substances and their analogues for immunotherapy and methods for identifying and purifying said components.
  • Trichinella sp. The phenotypically similar group of helminth parasites collectively known as the Trichinella sp. are responsible for widespread morbidity and mortality. Helminth parasites display a remarkable capacity to escape immunological destruction and may reside in their vertebrate hosts with relative disadvantageity, sometimes for the life of the host. Trichinella pseudospiralis is an especially interesting example of a metazoan parasite that employs several strategies for avoiding immune rejection. Unlike muscle larvae of a closely related species, Trichinella spiralis, muscle larvae of T. pseudospiralis are not enclosed by a collagenous capsule and, are exposed to host immunological effector elements. The life cycle of T. pseudospiralis begins with the consumption of vertebrate muscle containing infective larvae.
  • the intestinal phase of infection is initiated when larvae liberated from host muscle in the stomach enter the small intestine as preadults which mature to the adult stage within 24-48 hrs.
  • the parenteral phase of infection begins when adult female worms release newborn larvae which enter the lymphatics and blood vessels and are distributed throughout the body of the host. The newborn larvae enter striated muscle fibers and reside as intracellular muscle larvae. Unlike the muscle larvae of T. spiralis, those of T. pseudospiralis are not enclosed by a protective collagenous capsule in host muscle. Thus the life history of T. pseudospiralis has three distinct stages: 1) preadult/adult worms, 2) newborn larvae, and 3) muscle larvae.
  • T. pseudospiralis The ability of T. pseudospiralis to escape immunological attack is associated with a profound suppression of antigen-specific and nonspecific inflammatory responses.
  • Mice infected with T. pseudospiralis develop significantly elevated levels of plasma corticosterone that coincide with severe inhibition of delayed-type hypersensitivity (DTH) , contact hypersensitivity, and granuloma responses (Stewart, et al., (1985, 1988, 1989, 1991).
  • DTH delayed-type hypersensitivity
  • contact hypersensitivity contact hypersensitivity
  • granuloma responses The remarkable downregulation of host inflammatory responses in infected mice, in light of elevated serum IgE antibodies (Stewart, et al . , (1989)), suggests that T. pseudospiralis infection selectively regulates T cell- dependent immune responses.
  • T. pseudospiralis utilizes antigenic mimicry to avoid immunological recognition.
  • the muscle larvae of T. pseudospiralis express host-like asialo ganglio-N- tetraosylceramide (asialo GMl) antigen, which is believed to provide antigenic camouflage for the nonencapsulated larvae (Niederkorn, et al . , (1988), and Stewart et al . , (1989)).
  • asialo GMl asialo ganglio-N- tetraosylceramide
  • Trichinella pseudospiralis exists in three different stages during its stay in a vertebrate host.
  • the host response against this parasite would be expected to be very severe.
  • this conclusion is drawn from studies on other species of the genus which have identical life cycles and infect similar hosts. Although this parasite appears vulnerable to immunological destruction, it survives because the host does not respond in the expected fashion.
  • the present inventor conducted extensive studies of this organism to determine how it evades destruction by host immune response. The parasite suppresses the inflammatory response against itself and against any non-self organism or material present in the host during infection with T. pseudospiralis.
  • the newborn larval stage (which migrates systemically from the intestine to the muscle)
  • the muscle-stage parasite (which must exist for long periods of time in host muscle, in some cases for the life of the host) must both avoid non ⁇ specific inflammatory responses by the host.
  • the parasite continuously migrates within and between muscle fibers, causing low level damage to host tissues. Such activity would normally attract enormous numbers of host inflammatory cells.
  • the muscle larvae do not migrate and are enclosed within a capsule which affords them protection from immunological attack by the host.
  • T. spiralis an intense influx of inflammatory cells into infected host muscle occurs in response to the mechanical and chemical insults wrought by the encapsulated muscle larvae.
  • Trichinella pseudospiralis is accompanied by high levels of plasma corticosterone (Stewart, et al . , 1988). It is well documented that several key cytokines released by macrophages and T cells have the potential for modulating immune response through their secondary role as chemical communicators with the hypothalamic-pituitary-adrenal
  • HPA corticosteroid
  • cytokines operate to regulate host immune responses by their direct influence over the functions of immunological cells, they may also bring about a down-regulation of immune response indirectly through their influence over the HPA axis.
  • cytokines operate to regulate host immune responses by their direct influence over the functions of immunological cells, they may also bring about a down-regulation of immune response indirectly through their influence over the HPA axis.
  • the mechanisms responsible for this effect are unknown.
  • One of the possible explanations for the elevation of plasma corticosterone in animals infected with Trichinella pseudospiralis has been offered by Stewart et al . (1988) . It was proposed that the parasite may be inducing the release of at least one cytokine which feeds back on the HPA axis, bringing about an elevation in plasma corticosterone and a suppression of the non-specific inflammatory response.
  • T. spiralis is a strong inducer of the Th2 phenotype by, for example, inducing the secretion of IL-10 by T cells, thereby skewing the immune response toward the susceptible phenotype.
  • cytokines that ameliorate the effects of autoimmune diseases, such as rheumatoid arthritis, pemphigus, systemic lupus erythematosus, and others are highly envisioned.
  • the present invention in a general and overall sense, concerns novel T. pseudospiralis components applied to clinical and experimental immunology. It also seeks to overcome one or more of the drawbacks inherent in the prior art by providing methods for the purification of novel substances having cytokine activity and compositions for use in human immunotherapy with reduced side effects.
  • the present invention relates to the unexpected discovery of potent and selective immunomodulatory substances produced during infection with the parasite, T. pseudospiralis .
  • the present invention concerns two substances in the exudates of two different stages in the parasite's life cycle that modulate immunity.
  • the term "excretory/secretory”, (“excretory” or “secretory”), or “exudate” are used to define nematode parasite substances that are found in the extracts or supernatants derived from the helminth parasite Trichinella pseudospiralis .
  • These exuded products or substances may be derived from, for example, extracts of nematode infected tissue, the supernatants of nematode-containing media or humors, or by other methods commonly known to those of skill in the art, in light of the present disclosure.
  • Those of skill in the art can use the methods and examples described herein to identify and purify substances with activities similar to those described in the present invention from T. pseudospiralis or other members of the Trichinella sp. .
  • T. pseudospiralis exudes a substance that has interleukin-10-like activity.
  • the IL-10-like substance isolatable and purifyable from exuded products of the Trichinella sp. is hereinafter referred to as TP10.
  • TP10 has an approximate molecular weight of between 100 and 300kD in one form, and is highly specific in its effects. However, it is, of course, generally understood by those of skill in the art that the migration of a polypeptide can vary with different conditions of SDS/PAGE (Capaldi et al. , 1977). It will therefore be appreciated that under differing electrophoretic conditions, the molecular weight assignments quoted above may vary, for example molecular weight may vary due to the formation of multimeric complexes that have similar activity to the monomeric form.
  • TP10 is generally obtained from the preadult stage of T. pseudospiralis and directs the activation or "skewing" of T helper cells toward a differential ratio of T cell subsets called Th2 cells and away from the Thl phenotype.
  • Thl and Th2 describe distinct T cell phenotypes generally associated with inflammatory and B cell helper phenotypes, respectively, and are well known to those skilled and versed in the art. Thl cells promote inflammatory response and the bias induced by the parasite exudate against this subset is likely to cause the remarkable suppression of inflammation during the intestinal phase of infection, as disclosed in the present invention.
  • a preferred embodiment of the invention is a purified Trichinella-derived substance (TP10) having certain immunomodulatory characteristics of IL-10. These characteristics include a Thl immunosuppressive effect.
  • Thl immunosuppression is defined as the inhibition or termination of an inflammatory immune response.
  • this substance is defined further as derived from Trichinella pseudospiralis , with a substance derived from a pre-adult larvae of Trichinella pseudospiralis being more preferred.
  • a related embodiment is a purified IL-10-like substance having the following characteristics; isolatable from pre-adult Trichinella exudates in a conditioned media; having a molecular weight of between 100-300 kD; and being immunosuppressive to T cells of the Thl phenotype.
  • conditioned media is used to describe a medium, extract, or humor, wherein the nematodes have been allowed to incubate and have exuded substances in accordance with the present invention.
  • purified as used herein, is intended to refer to a Trichi ⁇ ella-derived cytokine-like or cytokine inducing substance that is obtainable from Trichinella sp.
  • TP10 is derived or purifyable from the preadult stage of members of the Trichinella sp. .
  • TPi which is derived or purifyable from the newborn larvae of members of the Trichinella sp . .
  • TP10 and TPi are derived or purifyable from T. pseudospiralis .
  • a purified "TP10" or "TPi" therefore, refers to a substance free from the environment in which it may naturally occur in intact cells, e.g., free of infective Trichinella sp. organisms.
  • Another embodiment of the invention is a method of obtaining a purified IL-10-like substance (TP10) from Trichinella pseudospiralis comprising the steps of; obtaining Trichinella pseudospiralis organisms; incubating said organisms under conditions facilitating the exudation of a IL-10-like substance (TP10) , and purifying a Trichinella IL-10-like substance (TP10) from said exudate containing incubate by separating intact organisms or fragments thereof.
  • the purifying step is accomplished by separating intact organisms or fragments thereof through a 0.2 ⁇ m filter.
  • the purification step is accomplished by ultrafiltration with a molecular weight cut-off of between about 10 kDa to 100 kDa. In an even more preferred embodiment purification is accomplished by ultrafiltration with a molecular weight cut-off of about 10 kDa.
  • the present invention also comprises an antibody having specific binding affinity for an IL-10-like factor produced by a member of the Trichinella sp. .
  • a related embodiment of the invention is a method of purifying an IL-10-like substance (TP10) having the ability to stimulate T cells toward a Th2 phenotype and away from a Thl phenotype comprising; obtaining an exudate from freshly isolated or cultured nematodes, and retrieving an IL-10-like substance (TP10) from said exudate with an antibody having a specific binding affinity for the IL- 10-like substance in accordance with the present invention.
  • Newborn Trichinella pseudospiralis larvae exude a separate substance that stimulates host cells, e . g. , fibroblasts and T cells, to secrete large amounts of a molecule with potent cytokine-like activity.
  • the molecule released by fibroblasts and T cells stimulates proliferation of HT2 cells (IL-2 dependent cell line) in vi tro.
  • HT2 cells IL-2 dependent cell line
  • the released molecule does not cross-react in an ELISA for IL-2.
  • the release of both gamma interferon and IL-2 are dramatically increased in cultures of mouse spleen cells and mesenteric lymph node cells exposed to the parasite molecule in vitro.
  • the induced molecule acts as a potent Thl mitogen.
  • TPi The active cytokine-inducing substance, hereinafter referred to as TPi, can be isolated and purified from the parasite's exuded products.
  • TPi is characterized as having an approximate molecular weight of between 30 and 50 kD and which induces cells, such as fibroblasts and T cells, to release a cytokine-like molecule.
  • the release of elevated levels of this molecule induced by the products of the Trichinella sp. are likely to cause the adrenals to release increased amounts of corticosterone, raising host plasma levels of this steroid thereby inducing the systemic suppression of inflammatory responses to both self and non-self antigens, including the parasite.
  • TP10 may also be useful in the treatment of toxemias induced by bacterial infections in which tumor necrosis factor and/or IL-1, are generally elevated under the influence of Thl subset of lymphocytes. TP10 may also be used to skew the immune response away from a Thl phenotype to a Th2 phenotype, thereby enhancing Th2 functions such as B cell help. TPi, depending on the amount administered to animals, has different effects on the immune status of treated animals.
  • T. pseudospiralis when newborn larvae are deposited by adult female worms in the intestinal wall, release by the larvae of TPi has a local effect on immunological events in the intestine. This consists of an activation of T cells of the Thl subset in the mesenteric lymph nodes, which is accompanied by the development of expulsion of adult worms, an event necessary to protect the host from superinfection. Later, as newborn larvae are distributed systemically, an activation of T cells of the Thl subset is apparent in the spleens of infected animals, followed by a rise of Thl cell cytokines in host plasma and peritoneal washings.
  • TPi can, at low levels, induce activation of Thl cells
  • TPi may be used to stimulate release by non-lymphoid cells or undamaged T cells of the potent Thl cell mitogen, dramatically boosting Thl cell activation and proliferation and reversing the immunosuppression.
  • Trichinella derived substances refers to a composition of the present invention (TPIO or TPi) that has been subjected to fractionation to remove various non-active components, and which composition substantially retains its immunomodulatory activity.
  • TPIO or TPi composition of the present invention
  • purified this will refer to a composition in which TPIO or TPi cytokine-like activity is obtained by purifying the active fractions of exuded products away from non-active components, such as whole organisms.
  • a preferred embodiment of the invention is a purified Trichinella-derived substance eliciting release of an IL-2-like substance by eukaryotic cells.
  • the substance is defined further as being derived from Trichinella pseudospiralis , with purification from the newborn stage of Trichinella pseudospiralis being most preferred.
  • the IL-2-inducing substance may be further defined as: being isolatable from exudates of newborn Trichinella conditioned media, having a molecular weight of 30-50 kD, and being capable of inducing secretion of IL-2 by eukaryotic cells.
  • a related embodiment is a method of obtaining a purified IL-2-inducing substance comprising the steps of: obtaining Trichinella pseu ospiralis-organisms; incubating said organisms under conditions facilitating the exudation of an IL-2-inducing substance, and purifying a Trichinella derived IL-2-inducing substance from said incubate by separating intact organisms or fragments thereof.
  • the purifying step is accomplished by separating intact organisms through a 0.2 ⁇ m filter.
  • the purifying step is accomplished by ultrafiltration with a molecular weight cut-off of between about 10 kDa to 30 kDa.
  • the purifying step is accomplished by ultrafiltration with a molecular weight cut-off of about 10 kDa.
  • Another embodiment of the present invention is an antibody having specific binding affinity for a IL-2- inducing activity substance produced by a member of the Trichinella sp. .
  • a related embodiment is a method of purifying an IL-2-inducing substance able to stimulate eukaryotic cells to secrete an IL-2-like activity, comprising; obtaining an exudate from freshly isolated or cultured Trichinella and retrieving an IL-2-inducing substance from said exudate with an antibody having specific binding affinity for said IL-2-like inducing substance.
  • Various methods for quantifying the degree of purification of the cytokine-like substances of the present invention are known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the number of polypeptides within a fraction by SDS/PAGE analysis.
  • a preferred method for assessing the purity of a T. pseudospiralis cytokine-like fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial nematode extract or supernatant, and to thus calculate the degree of purity.
  • the actual units used to represent the inhibitory activity of TPIO and IL-2-inducing activity of TPi will, of course, be dependent upon the particular assay technique chosen to follow the purification.
  • the present inventor prefers to use an assay based upon the inhibition or immunosuppression of immune cell responses both in vi tro and in vivo.
  • Activity in the assay as used herein is defined as the immunosuppressive activity of TPIO required to cause a reduction in cytokine release by Thl cells.
  • the definition of a unit of activity would naturally vary. It may be assessed as, for example, the activity to other Thl-like cells or stimulation of Th2 cells.
  • the present inventor prefers to use an assay based upon: 1) secretion of a Thl-stimulating molecule by fibroblasts; 2) secretion of T cells in vi tro; or 3) stimulation of natural killer cells in vivo or in vi tro.
  • Activity in the assay is defined as the extent of stimulation required for cytokine release by Thl cells or release of Thl-stimulating molecule by NIH 3T3 cells in vi tro .
  • the definition of a unit of activity would naturally vary. It may be assessed as, for example, the activity to stimulate other cells capable of producing cytokines or by the stimulation of Thl cells.
  • the muscle tissue is homogenized, e . g. , in 1% pepsin/HCl for isolation of larvae, and the resulting homogenate is incubated at 37°C for 1 hr. Incubates are allowed to sediment and the larvae are washed twice, sedimented, placed in glass incubation chambers in medium as described herein and incubated for 48-72 hr at 37°C. Larvae are removed by centrifugation at 1000 rpm, and the supernatant is passed through a 0.22 ⁇ m filter, and concentrated to 5X using a lOkD membrane in an ultrafiltration apparatus.
  • TPi cytokine-inducing substance in accordance with one embodiment of the present invention one isolates adult worms from the intestines of rats infected with T. pseudospiralis isolated from mouse muscle by conventional methods (e . g. , Pepsin-HCl) . Briefly, the entire small intestine of each infected animal is removed, washed, and adult worms allowed to migrate out of the intestinal wall into sterile saline.
  • the preferred animals for raising nematodes are rats, mice, and rabbits. Intestines and debris are removed by passing the worm suspension through a series of sieves and worms are washed three times by centrifugation.
  • the isolated worms are placed in medium and incubated at 37°C for 24 hr in a water bath, allowing adult worms to release newborn larvae. Following incubation, adult worms are removed by passing the worm suspension through a series of sieves. Recovered newborn larvae are washed 5 times in fresh medium and incubated in fresh medium for 48 hr at 37°C. Following incubation, newborn larvae are removed by centrifugation, and excretion/secretion (ES) products are obtained and purified as described for TP10.
  • ES excretion/secretion
  • Trichinella pseudospiralis derived compositions have been subjected to fractionation to remove various non-active substances such as other cell components.
  • Various techniques suitable for use in further purification are well known to those of skill in the art. These include, for example, precipitation with ammonium sulphate, PEG, antibodies, and the like or by heat denaturation, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques.
  • a specific example presented herein is the purification of the Trichinella pseudospiralis cytokine-like substance using concentration by ultrafiltration and by chromatographic separation on a
  • the preferred purification method disclosed herein contains several steps and represents the best mode presently known by the inventors for purification of either TPIO or TPi. This method is currently preferred as it results in the purification, as assessed by immunosuppression for TPIO, and stimulation of Thl cells for TPi, in yields sufficient for further characterization and use.
  • This preferred mode of purification involves the execution of certain purification steps in the order described herein. However, as is generally known in the art, it is understood that the order in which the various purification steps are conducted may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a purified material.
  • Trichinella derived cytokine-like substances there is no general requirement that the Trichinella derived cytokine-like substances always be provided in their most purified state. Indeed, it is contemplated that less purified material, that is nonetheless enriched in their respective activities relative to the natural state, will have utility in certain embodiments. These include, for example, the production of specific polyclonal and monoclonal antibodies for use in the complete characterization of the Trichi-ella-derived cytokine-like substances. These immuno-reagents will be useful for the development of kits for use in the rapid identification of different members of the Trichinella sp. .
  • Partially purified fractions for use in such embodiments may be obtained by subjecting a pre-adult worm extract (for TPIO) or a newborn extract (for TPi) or supernatant derived by incubating said organisms to one or a combination of the steps described above.
  • biologically functional equivalent of TPIO or TPi is used herein to refer to purified substances from nematodes having substantially the same biologic activity of TPIO or TPi as described herein. Thus, from the studies on related organisms presented herein, it can readily be seen that equivalents from other parasites having substantially the same biologic activity are encompassed.
  • the present invention includes an antibody that is immunoreactive with polypeptide TPIO or TPi.
  • An antibody can be a polyclonal or a monoclonal antibody. In a preferred embodiment, an antibody is a monoclonal antibody.
  • Means for preparing and characterizing antibodies are well known in the art (See, e.g. , Antibodies "A Laboratory Manual , E. Howell and D. Lane, Cold Spring Harbor Laboratory, 1988) .
  • Another embodiment of the invention is a method of early prognosis of Trichinella pseudospiralis infection comprising: identifying a human subject suspected of being infected with Trichinella; and assaying a blood sample for the presence of TPIO or TPi.
  • a pharmaceutical composition comprising the TPIO or TPi substance, dispersed in a pharmaceutically acceptable carrier. This substance is useful, as an improved and novel composition for suppressing an immune response comprising treating eukaryotic cells with an effective amount of a pharmaceutical composition; wherein said cells are contacted with the TPIO or TPi substances, or biological equivalents thereof.
  • contacted is used to describe the close juxtaposition of an effective amount of TPIO, TPi, or functional equivalents thereof, sufficient to elicit their respective biologic functions.
  • a related embodiment is a method for altering the immune response of an animal comprising the following steps; preparing a pharmaceutical composition comprising the TPIO or TPi substances dispersed in a pharmacologically acceptable carrier; and administering to an animal a therapeutically effective amount of said substance.
  • a more preferred embodiment is a method for treating an animal comprising; identifying an animal with a need for immunosuppression, administering to an animal a therapeutically effective amount of TPIO or TPi substances dispersed in a pharmaceutically acceptable carrier, wherein the immune response being suppressed is a graft-versus-host response, an autpimmune response, or an allergic response.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like that do not produce an allergic or similar untoward reaction when administered to a human.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • compositions that contains a protein or proteoglycan as an active ingredient are well understood in the art.
  • such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.
  • the preparation can also be emulsified.
  • a proteoglycan can be formulated into a composition in a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.
  • aqueous solutions For parenteral or intravenous administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 mL of isotonic NaCl solution and either added to lOOOmL of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example,
  • TPi and TP10 have very specific effects on the host's immune response, that is, they do not appear to exhibit all the broad effects attributed to their respective mammalian counterparts.
  • the mammalian counterpart being IL-10; and for TPi the mammalian counterpart being the secretion of mammalian Thl cytokines gamma interferon and IL-2.
  • the low morbidity and mortality experienced by animals infected with T. pseudospiralis suggests that TP10 and TPi in the host do not induce the side-effects seen in clinical treatment of patients with IL-10 and Thl cytokine therapy.
  • the specific nature of action for these two molecules would also be of advantage in their application in immunological research.
  • TP10 may be applied in any in vivo or in vi tro system to suppress the activation and development of the Thl subset of T cells (cellular immune responses in general and inflammation specifically) .
  • TPi is a potent inducer of Thl- activating substance by cells capable of manufacturing this material. Release of Thl cytokines induces T cell activation, a required initial step in mounting an immune response.
  • TPi can be used to boost activation of T cells which orchestrate host immunological reaction to non-self materials.
  • TPi The anti-inflammatory effects of TPi come into play when the extended duration of Thl cytokine release reaches a critical point, whereby the host considers its immune response to be "out-of-control" and some of these cytokines assume their role as communication molecule between the immune system and the HPA axis. At this time they begins to feed back on the HPA axis and increase release of corticosteroids which suppress the runaway immune reaction.
  • the present invention includes a showing that T. spiralis (all isolates tested) may release materials with activity similar to TPIO.
  • the muscle stage of all forms of T. spiralis are enclosed within a collagenous capsule with protects them from immunological destruction by the host immune response. Thus, they do not require so much of a systemic suppression of inflammatory response for their survival as does the unencapsulated muscle stage of T. pseudospiralis .
  • plasma corticosterone levels in mice infected with this parasite are elevated, albeit at a much lower level than that seen in mice infected with T. pseudospiralis .
  • FIG. 5 Establishment of muscle larvae of TS in a host (the Chinese hamster) showing strong resistance to TS in animals infected with either TS alone or with TS and TP (TP + TS) .
  • TS differed significantly from TP + TS, P ⁇ 0.05.
  • FIG. 9 Peroxidase activity measured from enriched eosinophil preparations.
  • a specific inhibitor of eosinophil peroxidase (3-amino-l,2,4-triazole) was used in controls to measure only eosinophil-specific peroxidase (ESPO) .
  • Change in absorbance at 492 nm was recorded every 6 sec for 3 min. Data are presented as Vmax (Mean OD/min) .
  • NO Ag no parasite antigen
  • 750C bovine serum albumin
  • FIG. 13 The effects of TP preadult ES on the amounts of IL-2, interferon gamma, IL-4, IL-5 and IL-10 released by spleen cells recovered from uninfected mice (UI) and incubated in the presence of either Con A or TP preadult ES. Significantly lower levels of IL-2, interferon gamma and IL-5 were released by spleen cells from UI mice stimulated with TP preadult ES than by those primed with Con A. Data from 3 separate studies in each of which 5 replicates of cells were tested. P ⁇ 0.05.
  • FIG. 14 Mesenteric lymph node cells were recovered from uninfected mice (UI) and incubated in the presence of Con A or TP preadult ES and the amounts of IL-2, interferon gamma, IL-4, IL-10 and IL-5 released were measured. The amount of IL-2 released by TP ES- stimulated cells was drastically reduced compared to Con A primed cells (P ⁇ 0.05). The concentrations of the other cytokines was similar. Data from 3 separate studies in each of which 5 replicates of cells were run.
  • FIG. 16 Competitive inhibition of IL-10 detection by antisera from T. pseudospiralis-infected mice.
  • FIG. 17 IL-2 secretion by mesenteric lymph node and spleen cells recovered from TP-infected mice on different days postinfection and stimulated in vi tro with Con A. Each bar represents the mean of data from 4 separate studies in each of which 4 replicates of cell cultures were analyzed. IL-2 secretion on day 10 postinfection was significantly greater than that for controls and for days 7, 17 and 24 postinfection (P ⁇ 0.01) .
  • FIG. 18 Hypersecretion of IL-2 detected in T. pseudospiralis -infected muscle by in situ hybridization.
  • A Normal mouse diaphragm muscle and
  • B Diaphragm muscle from a mouse following infection with T. pseudospiralis (50 days) . Note the strong reaction in connective tissue elements of infected muscle and the absence of such reaction in similar locations within uninfected muscle.
  • the nematode parasites of mammals stimulate dramatic inflammatory cell mobilization and recruitment to sites occupied by the parasite.
  • T. pseudospiralis the inflammatory response is almost completely abrogated.
  • Ameliorated immune responses may be explained by a lack of parasite antigen immunogenicity, masking of the parasite's antigens, antigenic mimicry, or suppression of host cellular responses.
  • the intestinal phase of infection with T. pseudospiralis is only the first of three phases in the parasite's life cycle to which the host must respond.
  • the second stage begins around day 6 postinfection when newborn larvae are shed by adult female worms in the small intestine where they penetrate the lamina intestinal, invade neighboring tissues, and enter the lymphatics and vasculature.
  • the migration of newborn larvae coincides with a profound suppression of DTH, granuloma formation and contact hypersensitivity responses.
  • the dramatic elevation in plasma corticosterone which accompanies this down-regulation of cellular immunity in the T.
  • pseudospiralis-infected host is believed to be due to either the elaboration of parasite-derived factor(s) which act directly on the pituitary or adrenals and thereby induce corticosteroid release, or to the larvae stimulating the release of neuroendocrine-active cytokines by host cells. Since parasite ES have no effect on ACTH release by pituitary cells or on corticosteroid release by adrenal cells in vitro
  • T. spiralis is a strong inducer of the Th2 phenotype by, for example, inducing the secretion of IL- 10 by T cells, thereby skewing the immune response toward the susceptible phenotype.
  • the present application discloses novel substances that have immunomodulatory capabilities and are isolatable from parasitic nematodes, such as Trichinella pseudospiralis .
  • parasitic nematodes such as Trichinella pseudospiralis .
  • All parasites of vertebrates must evade host immune response in order to survive. However, by virtue of the site it occupies in the host, this parasite should elicit, as do other closely related parasites, a violent immune response by the host.
  • T. pseudospiralis must employ far-reaching and effective immunoevasive strategies to insure its survival. Its most pressing requirement in this regard is to evade host inflammatory response, an obstacle which the host presents to every stage in the parasite's life cycle.
  • the inventor has identified and isolated the parasite components responsible for such evasion and has assessed the applicability of these components in the control of vertebrate inflammatory response, both specifically and systemically.
  • the present invention takes advantage of the newly discovered substances that the parasite uses to control the host inflammatory response.
  • the novel substances of the present invention are, for example, widely applicable to a wide variety of medically important autoimmune diseases seen in humans. This applicability is based on the strong anti- inflammatory, and immunomodulatory effects exerted by TPIO and TPi.
  • the present invention discloses the elaboration of two novel parasite-derived components that directly and indirectly regulate T cell responses in murine hosts infected with T. pseudospiralis .
  • One component, TPIO is secreted or exuded by the preadult stage of
  • a second component (TPi) is produced by newborn larvae and induces hypersecretion by host lymphoid and non-lymphoid cells of a potent cytokine-like substance that dramatically stimulates cells of the Thl subset of lymphocytes to hypersecrete their characteristic profile of cytokines, including IL-2, interferon gamma and IL-3, resulting in downregulation in systemic cellular immunity.
  • cytokines including IL-2, interferon gamma and IL-3
  • the T. pseudospiralis nematode is parasitic in a wide variety of mammals (including several species of commonly employed laboratory animals) in which it invades deep tissues and survives for the life of the host.
  • the present inventor characterized the surprising lack of inflammation in response to tissue invasion by this parasite (Stewart et al . , 1985). This paucity of host cellular response has been documented both histologically, and chemically by assaying for inflammatory cell marker enzymes.
  • This paucity of host cellular response has been documented both histologically, and chemically by assaying for inflammatory cell marker enzymes.
  • the parasite's extensive migratory activities within a variety of host tissues there is little morbidity or mortality associated with even massive infections (Stewart, 1989) .
  • TPIO is excreted by the preadult worm stage in the life cycle of the parasite T. pseudospiralis .
  • TPIO is very similar to native mammalian interleukin 10 (IL-10) in terms of its ability to shut down a Thl response (Thl cells promote and orchestrate inflammatory cellular immunity) .
  • IL-10 native mammalian interleukin 10
  • Thl response Thl cells promote and orchestrate inflammatory cellular immunity
  • TPIO may be used in the same clinical settings that IL-10 is used, as is apparent, for example, from the in vivo examples disclosed herein.
  • TPIO is very specific in its effects, shutting down the Thl response in a highly effective manner, while not impacting on unrelated aspects of host immunological responsiveness. In this sense, TPIO enjoys an advantage over native IL-10, which has broader effects including some undesired or harmful effects on host immunity.
  • IL-10 is also used routinely in the basic research and biomedical research laboratories. TPIO is useful for similar applications and for use as a therapeutic agent.
  • TPi a substance that is a powerful inducer of release by vertebrate lymphoid and non- lymphoid cells of a cytokine-like substance which stimulates Thl cells to proliferate and release copious amounts of their characteristic cytokines.
  • TPi this parasite-derived substance
  • IL-2 interferon gamma levels
  • the activity of TPi can be measured in terms of its ability to stimulate fibroblasts and T cells to release a cytokine-like substance which caused proliferation of HT2 cells, an IL-2 dependent cell line, or by activation and proliferation of Thl cells.
  • the ability of this parasite-induced molecule to stimulate HT2 cell proliferation suggests that it may be similar in some ways to IL-2.
  • the TPi-induced molecule released by T cells or fibroblasts showed an HT2 proliferation response as much as 20,000 times greater than controls.
  • fibroblasts were exposed in vi tro to live newborn larvae of T. pseudospiralis , these cells released material that induced HT2 proliferation at levels 500,000 times that seen with controls.
  • the potency of TPi in inducing release of this IL-2-like activity by host cells is no less than phenomenal.
  • cDNA was recovered from 3T3 cells following 48 hr exposure to TPi and then used in a polymerase chain reaction for IL-4 IL-6, IL-7, GM-CSF, gamma interferon, IL-12, and IL-2. Results were negative for all cytokines tested.
  • Preadult ES was fractionated by application to a liquid chromatography system (LC100, Millipore Corp.) using an anionic DEAE column. Elution was at (pH 8) with a gradual increase in concentration of NaCl over a 30 min period from 10% to 100% of a 1M NaCl solution. A total of 10 major peaks were eluted from the column. Preadult ES fractions were tested for their ability to inhibit cytokine (IL-2, IL-3 and interferon gamma) release by HDK-1 cells. Dramatic reductions in all three cytokines below controls were seen with 4 out of the 10 peaks.
  • cytokine IL-2, IL-3 and interferon gamma
  • TPi of the present invention has been used by the present inventor in routine research to greatly boost natural killer cell activity in vivo and to promote growth of IL-2-dependent cell lines.
  • TPi has numerous applications in basic and biomedical research settings in immunology.
  • TPi will also be useful in clinical settings.
  • TPi should be especially helpful in the treatment of patients with certain immunocompromising conditions such as AIDS, radiation-induced or chemotherapy-induced suppression of host immunity, some immunosuppressive forms of cancer, and the like.
  • the strong specificity of activity associated with these parasite-derived substances will limit any diverse effects usually seen with therapy employing native cytokines which often have a broad range of activities, some of which are detrimental.
  • the present invention also concerns the effect of TPi on a model for T cell-mediated immune response in vivo .
  • the enteric response of the host to infection with a parasite closely related to T. pseudospiralis was dramatically augmented in mice injected with TPi. This demonstrated, along with augmentation of in vivo natural killer cell activity, that this substance directly influences IL-2-inducible immune responses in the intact vertebrate host.
  • T. pseudospiralis employs several different immunoevasive strategies to insure its survival.
  • Initial efforts related to the specific mechanism by which the parasite successfully infects a host included analyses of the functional status of specific cell types assigned to play an effector role in nematode infections.
  • To quantitate the effects of T. pseudospiralis ES products on specific cell populations the oxidative burst in neutrophils and eosinophils was determined and found to be suppressed during infection with this parasite. Likewise, eosinophil peroxidase levels were greatly affected.
  • Thl cells are known to up-regulate inflammatory response.
  • the parasite could manipulate inflammatory response by preventing the activation of Thl cells. If the parasite were able to control T cell activation, it could accomplish this in a number of ways.
  • Nematodes exude a wide variety of substances which are collectively referred to as excretory/secretory products (ES) .
  • ES excretory/secretory products
  • the ES of r. pseudospiralis are strong candidates for participation in this parasite's ability to modulate host response since crude extracts from the parasite dramatically suppress chemotactic response and oxidative burst in neutrophils from uninfected animals.
  • this same material alters the pattern of cytokine release by spleen and lymph node cells from uninfected animals.
  • a finding of the present invention is also that the closely related parasite Trichinella spiralis may also elaborate substances with similar effects to those shown for TPIO and TPi, albeit at apparently much lower levels that T. pseudospiralis .
  • Trichinella TPIO and TPi modification and changes may be made in the structure of the Trichinella TPIO and TPi and still obtain a substance having like or otherwise desirable characteristics.
  • certain amino acids may be substituted for other amino acids in protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen- binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence (or, of course, its underlying DNA coding sequence) and nevertheless obtain a protein with like or even countervailing properties (e.g., antagonistic v. agonistic) . Thus various changes may be made in the sequence of the Trichinella cytokine-like protein substances or peptides without appreciable loss of their biological utility or activity.
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte & Doolittle, J. Mol. Biol. , 157:105-132, 1982) . It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity.
  • Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2) ; literally glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • the relative hydropathic character of the amino acid determines the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, cytokines, antibodies, antigens, and the like. It is known in the art that an amino acid may be substituted by another amino acid having a similar hydropathic index and still obtain a biological functionally equivalent protein. In such changes, the substitution of amino acids whose hydropathic indices are within ⁇ 2 is preferred, those which are within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ⁇ 1); glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ⁇ 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
  • an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein.
  • substitution of amino acids whose hydrophilicity values are within ⁇ 2 is preferred, those which are within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • the present invention contemplates an antibody that is immunoreactive with a component of the invention.
  • An antibody can be a polyclonal or a monoclonal antibody. In a preferred embodiment, such an antibody is a monoclonal antibody.
  • Means for preparing and characterizing antibodies are well known in the art (See, e.g. , Antibodies "A Laboratory Manual , E. Howell and D. Lane, Cold Spring Harbor Laboratory, 1988) .
  • a polyclonal antibody is prepared by immunizing an animal with an active or deactivated immunogen comprising a component of the present invention and collecting antisera from that immunized animal.
  • an animal used for production of anti-antisera is a goat, rabbit, mouse, rat, hamster or guinea pig. Because of the relatively large blood volume of rabbits and goats, a rabbit or a goat is a preferred choice for production of polyclonal antibodies.
  • Antibodies both polyclonal and monoclonal, specific for the Trichinella derived substance or component of the present invention may be prepared using conventional immunization techniques, as will be generally known to those of skill in the art.
  • a composition containing antigenic epitopes of the Trichinella substances can be used to immunize one or more animals, such as a rabbit or mouse, which will then proceed to produce specific antibodies against TPIO or TPi as the case may be.
  • Polyclonal antisera may be obtained, after allowing time for antibody generation, simply by bleeding the animal and preparing serum samples from the whole blood.
  • Hybridomas which ' produce monoclonal antibodies to the selected antigens are identified using standard techniques, such as ELISA and Western blot methods.
  • Hybridoma clones can then be cultured in liquid media and the culture supernatants purified to provide the TPIO or TPi-specific monoclonal antibodies.
  • monoclonal antibodies to the desired TPIO or TPi antigen of Trichinella can be used in both the diagnosis and treatment of Trichinosis infections.
  • Trichinella derived substances may be utilized in other useful applications.
  • their use in immunoabsorbent protocols may be useful in purifying native or recombinant TPIO or TPi species or variants thereof.
  • both poly- and monoclonal antibodies against TPIO or TPi may be used in a variety of embodiments. They may be used in inhibition studies to analyze the effects of blocking the actions of the components released during a Trichinella infection in cells or animals.
  • Anti-TPIO or -TPi antibodies will also be useful in immunolocalization studies to analyze the distribution of TPIO and TPi during various cellular events, for example, to determine developmental expression during the different stages of the nematodes life cycle, and their correlation with pathogenicity.
  • a particularly useful application of such antibodies is in purifying native or recombinant TPIO or TPi, for example, using by using an antibody affinity column. The operation of all such immunological techniques will be known to those of skill in the art in light of the present disclosure.
  • the active compounds may also be administered parenterally or intraperitoneally.
  • Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for parenteral use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • a coating such as lecithin
  • surfactants for example, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus ny additional desired ingredient from a previously sterile-filtered solution thereof.
  • the active substances of the present invention may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard or soft shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with food.
  • the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Such compositions and preparations should contain at least 0.01% of active compound.
  • the percentage of the compositions and preparations may, of course, be varied.
  • the amount of active substances in such therapeutically useful compositions is such that a suitable dosage will be obtained.
  • the tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring.
  • a binder as gum tragacanth, acacia, cornstarch, or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin may be added or a flavor
  • any material may be present as coatings or to otherwise modify the physical form of the dosage unit.
  • tablets, pills, or capsules may be coated with shellac, sugar or both.
  • a syrup of elixir may contain the active compounds sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compounds may be incorporated into sustained- release preparation and formulations.
  • TPi and TPIO have very specific effects on the host's immune response, that is, they do not appear to exhibit the broad effects attributed to their mammalian counterparts (IL-2-like activity and IL-10) . Furthermore, the low morbidity and mortality experienced by animals infected with the Trichinella pseudospiralis parasite further corroborate that these two materials do not induce the side-effects observed in the clinical treatment of patients with IL-10 and IL-2-like activity.
  • TPi is a potent inducer of production/release of IL-2-like activity by cells of the vertebrate capable of manufacturing this cytokine ( e . g. , fibroblasts and T cells) .
  • the release of the IL-2-like molecule follows T cell activation, the required initial step in mounting an immune response.
  • TPi can be used just like IL-2 to boost activation of T cells which orchestrate host immunological reaction to non-self materials.
  • the anti-inflammatory effects of TPi come into play when the duration of IL-2 release reaches a critical point and the neuro-immunologic feedback mechanism is activated to control IL-2 release. Therefore TPi may play a role as a communication molecule between the immune system and the HPA axis.
  • the feedback response from the HPA axis increases the release of corticosteroids which suppress the runaway immune reaction.
  • Trichinella pseudospiralis derived products were surprising, given the previous studies conducted on Trichinella spiralis and other helminth parasites. Delayed-type hypersensitivity responses were assessed to antigens unrelated to the parasite in animals infected with T. pseudospiralis and found that the host response to three different unrelated antigens: cotton string (FIG. 1); tumor cell antigens (FIG. 2) ; and trinitrochlorobenzene (FIG. 3) . These responses were dramatically suppressed (Stewart et al . , 1991) in the presence of a T. pseudospiralis infection.
  • T. pseudospiralis The immunosuppressive capacity of T. pseudospiralis was also examined by comparison with the dynamic interactive effects of immune-associated events due to infection with another parasite.
  • Hosts infected with T. pseudospiralis were challenged with the closely related nematode parasite, Trichinella spiralis, a helminth known to induce dramatic inflammation at several host sites (Stewart et al . , 1985). Inflammation at all sites that normally exhibit a response to T. spiralis was dramatically suppressed as determined by histochemical and chemical means.
  • the success of T. spiralis as measured in terms of number of muscle larvae, increased by 57% in hosts concurrently infected with T. pseudospiralis compared to those with T. spiralis alone.
  • RESULTS 1 Heterologous Antigen Assay.
  • UI had a significantly greater inflammatory response than TP infected animals, P ⁇ 0.05. Histological analyses confirmed these chemical ' data (not shown). Inflammation was measured in terms of a marker enzyme for granulocytes (myeloperoxidase) . This technique was applied to assess inflammatory response in studies examining enteritis, myositis and granuloma formation.
  • the entire small bowel was removed from the animal, rinsed out with 20 ml of sterile saline using a 20 cc syringe, split lengthwise, rinsed in sterile saline, the entire mucosa was removed and homogenized in 2 ml of sterile saline with 30 strokes of a glass homogenizer on ice.
  • Myeloperoxidase activity and total protein was measured in this homogenate.
  • the implanted string and surrounding granulation tissue were removed from their subcutaneous location and treated as above for homogenization and measurement of myeloperoxidase activity and total protein.
  • Measurement of myositis or ear inflammation involved removal of the entire diaphragm, pectoralis and/or masseter muscles or ear followed by homogenization (50 strokes) and measurement of myeloperoxidase activity and total protein as above in the homogenate.
  • Myeloperoxidase activity was measured as described by Charniga et al . (1981) .
  • the effects of host sex on enteric response to infection with Trichinella spiralis were added 1 ml of 20mM aqueous guaiacol (Sigma Chemical Company, St.
  • Controls included measurement of inflammation in the unchallenged left ear of the same animals, in ears from animals not sensitized and not challenged, and in ears from animals either challenged but not sensitized, or sensitized but not challenged. Histological analyses of ears from all groups confirmed chemical data.
  • the P91 mastocytoma a mutagenized derivative of P815 mastocytoma (H-2d) , was originally obtained from Dr. T. Boon (Ludwig Cancer Institute, Brussels, Belgium) .
  • MEM Dulbecco's modified Eagle's minimal essential medium
  • GIBCO heat-inactivated fetal calf serum
  • a third group of similarly measured animals were infected with TS alone but received daily intramuscular (im) injections of 0.5 mg cortisone acetate suspended in 0.1 ml of sterile saline from the day of infection until the completion of the study (0.5 mg CORT) .
  • Controls infected with TS received daily injections of sterile saline only and did not differ from TS alone.
  • TS T. spiralis
  • TP + TS TP + TS
  • Male Chinese hamsters Crisetulus griseus were 4-5 weeks old when used as hosts (Cytogen Research and Development, West Roxbury, MA) .
  • FIG. 6 demonstrates the effects of Trichinella sp. coinfection on granulocyte marker enzyme activity (inflammation) in diaphragm and pectoralis muscle from Chinese hamsters (a host normally resistant to infection with T. spiralis) infected with T. spiralis alone (TS) or concurrently infected with T. spiralis and T. pseudospiralis (TP + TS) .
  • FIG. 7 demonstrates the effects of TP infection on oxidative burst as measured from enriched neutrophil preparations.
  • Neutrophil preparations were collected by conventional methods from the peritoneal spaces of uninfected (UI) or TP-infected animals (TP) 12 hr following intraperitoneal injection with 2 ml of 0.2% glycogen in sterile saline.
  • UI showed significantly more oxidation that TP, P ⁇ 0.05.
  • Negative controls consisted of incubates as above with 30 ⁇ g of superoxide dismutase (Sigma) added to inhibit superoxide anion-dependent reactions. The reaction was started by the addition of 0.1 ml of 1.2 mM ferricytochrome c (Sigma) and 0.1 ml of opsonized zymosan at 10 g/ml. Opsonized zymosan was prepared by suspending zymosan (Sigma) in sterile saline at concentration of 50mg/ml with incubation of 1 volume of this preparation with 4 volumes of fresh, nonheat- inactivated, mouse serum from uninfected mice for 30 min at 37°C.
  • zymosan was collected by centrifugation and resuspended in sterile saline at 10 mg/ml for use in these studies. Following activation of granulocytes by exposure to opsonized zymosan for 10 min at 37°C, the reaction was stopped by placing the tubes containing granulocytes in an ice bath, and promptly centrifuging the tubes at 400 x g at 4°C for 10 min. Absorbance of triplicate samples of the supernatants of these granulocyte suspensions at 550 nm was determined in an Array 3000 Spectrophotometer. Oxidative burst is presented in terms of micromoles of cytochrome c reduced/million cells per hr.
  • Eosinophil Peroxidase Activity was measured from enriched eosinophil preparations.
  • a specific inhibitor of eosinophil peroxidase (3-amino-l,2,4-triazole) was used in controls to measure only eosinophil-specific peroxidase (ESPO) .
  • the assay is based on the oxidation of o-phenylenediamine by ESPO in the presence of H 2 0 2 .
  • Eosinophils were activated by incubation in the presence of cytochalasin B and PMA prior to measurement of ESPO. Change in absorbance at 492 nm was recorded every 6 sec for 3 min.
  • Vmax Mean OD/min
  • Spleen Cell Activation Splenocytes were recovered under sterile conditions by conventional methods from uninfected or on day 17 postinfection from T. pseudospiralis-infected (TP) 6-8 wk old female C57/BL6 mice (Jackson Labs, Bar Harbor, ME) and stimulated during 24 hr incubations at 37°C under 5% CO 2 with Con A (5 ⁇ g/well of 96-well plates containing 10 6 splenocytes/well) and supernatants were analyzed for IL-2 (using the HT2 IL-2 dependent cell proliferation assay) , gamma interferon, IL-4, IL-5 and IL-10 (measured by ELISA) .
  • IL-2 using the HT2 IL-2 dependent cell proliferation assay
  • gamma interferon IL-4, IL-5 and IL-10
  • Neutrophil chemotaxis was assessed using enriched neutrophil preparations recovered from the peritoneum of uninfected mice 9 hr following intraperitoneal injection of 2 ml of 0.2% oyster glycogen (Sigma) suspended in RPMI 1640 containing 20% normal mouse serum and 2% antibiotic/antimycotic (streptomycin [10,000 ug/ml] - penicillin [10,000 units/ml-fungizone [amphotercin B, 25 ⁇ g/ml] ; GIBCO) .
  • the bottom well of the blind well chamber (Nucleopore Corp., Pleasanton, CA) was filled with 0.2 ml of a solution containing the solution to be tested for chemotactic potential.
  • a membrane with 3 ⁇ m pores was placed over the bottom chamber of the well.
  • the plastic top chamber was screwed in place over the membrane to form the upper chamber of the well.
  • the top chamber was filled with 0.8 ml of neutrophil suspension (10 6 cells/0.8 ml).
  • Blind wells were incubated at 37°C under 5% C0 2 for 90 min. following incubation, the top chamber was removed and the membrane fixed in methanol and stained in 0.3% aqueous Toluidine Blue for 1.5 min, rinsed in deionized water and placed on a glass slide to air dry over night.
  • T. pseudospiralis (TP) preadult ES might directly impact cytokine release by T cells
  • spleen cells (FIG. 13) and mesenteric lymph node cells (MLN; FIG. 14) were recovered by conventional methods from uninfected 6-8 wk- old, female C57/BL6 mice (Jackson Labs) and exposed form 24 hr in vi tro to either Con A or to TP preadult ES.
  • the amounts of the cytokines IL-2, gamma interferon, IL-4, IL-5 and IL-10 were measured in culture supernatants. Data were obtained from 3 separate studies with 5 replicates for each group. Analysis of spleen cell culture supernatants revealed that exposure to TP preadult ES was accompanied by release of significantly lower levels of IL-2, gamma interferon, and IL-5 than was evident in cultures exposed to Con A.
  • mice infected with T. spiralis remained uninjected (UINJ) or were injected each day for the first 9 days following infection with either partially purified TP preadult ES (TPIO) (designated as PAD ES) , medium only (MEDIJC) .
  • FIG. 15 demonstrates that TPIO-injected mice showed a dramatic reduction in enteritis when compared to uninjected control mice or medium-injected mice. P ⁇ 0.05.
  • TPIO displayed some of the activities of mammalian IL-10, the parasite molecule was undetectable by a standard ELISA for IL-10.
  • T. pseudospiralis which should contain antibodies against TPIO, was tested for its ability to bind to rIL-10 and inhibit its detection by an ELISA specific for IL-10, some cross-reactivity between TPIO and IL-10 was demonstrated. Mean percent inhibition by 100 ⁇ l of serum from mice infected for 45 days with T.
  • T. pseudospiralis TP10 elaborated by intestinal stage of T. pseudospiralis inhibits enteritis. Intestinal inflammation on day 9 postinfection measured as mean myeloperoxidase activity ⁇ S.D. ( ⁇ Moles H 2 0 2 decomposed/min/mg protein) in the small intestines of 6-8 wk-old female ICR Swiss albino mice infected with T. spiralis .
  • Myeloperoxidase activity in the small intestines of mice injected with preadult ES differed significantly (P ⁇ 0.05) from either control group.
  • NBL ES The ES of stage 2 in the life cycle of the parasite (newborn larvae) are referred to hereafter as NBL ES.
  • stage 4 preadult worms
  • PAD ES The ES of stage 4 in the life cycle of the parasite (newborn larvae) are referred to hereafter as NBL ES.
  • TPIO a molecule that mimics in activity the mammalian cytokine, IL-10.
  • IL-10 is a substance released by mammalian T cells that, among a broad range of other functions, controls the development of the T cell subset destined to orchestrate the entire immune response the host will mount, suppressing the activation of Thl cells (the T cell subset that promotes cellular response) and promoting the development of Th2 cells.
  • mice infected with T. spiralis were injected ip with PAD ES to see if TP 10 would alter host response to a different parasite. Intestinal inflammations in mice injected with medium alone or PAD ES were compared with that seen in uninfected mice (FIG. 15) .
  • Exposure of (HDK-1 DNAX Research Institute) T cells (a Thl type of T cell) in vi tro to PADES inhibits the release of IL-2, IL-3 and ⁇ -IFN (cytokines released by Thl cells; Table 1) .
  • Table 1 Exposure of (HDK-1 DNAX Research Institute) T cells (a Thl type of T cell) in vi tro to PADES inhibits the release of IL-2, IL-3 and ⁇ -IFN (cytokines released by Thl cells; Table 1) .
  • T. pseudospiralis is modulating the host's immune response at this very basic level, and by using a molecule that mimics IL-10, is directing host reaction in a manner that best promotes its own survival as well as that of the host.
  • TPIO host IL-10
  • Commercially prepared monoclonal antibody to mammalian IL-10 DNAX Research Institute
  • serum from hosts infected with T. pseudospiralis does cross react with recombinant mammalian IL-10 and interfere with the ELISA for IL-10 (See Table 2 and FIG. 16) .
  • HDK-1 CD4+ Thl cell
  • Cytokine assays were performed on supernatants derived from cultures of HDK-1 cells following 24 hr exposure to antigen-presenting cells (x-irradiated Balb/c spleen cells [3000 rads] ) and antigen (KLH) and either (1) rIL-10 at 40 units/well (positive controls) , (2) 50 ⁇ l of medium alone. IFN- ⁇ and IL-3 were measured by two-site sandwich ELISA (Mosmann, et al . (1989, and (1987)). IL-2 was assayed using the method of Cherwinski et al. (1987). Preadult and adult ES were generated as described in Example 2. Data are presented as mean percent inhibition of cytokine secretion. Standard deviation and n are presented for each mean. Preadult ES and rIL-10 differed significantly from controls (P ⁇ 0.01). Table 3. Preadult ES Inhibit Thl Cytokine Release
  • Table 3 are data from a typical study in which the effects of preadult ES on cytokine release by a Thl cell line were assessed.
  • TPIO in PAD ES showed greater potency for suppressing Thl cytokine release than did mammalian rIL-10 (200 IU/ml) , significantly lowering the release of gamma interferon, IL-2 and IL-3.
  • Cytokines were assessed as indicated above.
  • the first indication of systemic modulations in cytokine patterns occurs during the newborn larval phase of infection (days 6-12 postinfection) , as found when mesenteric lymph node (MLN) cells and spleen cells isolated from T. pseudospiralis-infected mice were examined.
  • IL-2 secretion by MLN cells and spleen cells recovered from infected mice was increased by almost 225% and 450% respectively by day 10 postinfection, as shown in FIG. 17.
  • Blood for cytokine analysis was obtained by cardiac puncture from uninfected mice and from mice infected with T. pseudospiralis for 10 days was treated according to conventional methods for recovery of serum. Peritoneal exudate for analysis of cytokines was obtained from mice infected for 10 days with T.
  • 3T3 murine fibroblasts were exposed in vi tro to newborn larval ES and the supernatant was examined for IL-2 using an HT2 (IL-2 dependent) cell line. 3T3 murine fibroblasts were maintained in complete DMDM (see above) at 37°C under 5% C0 .
  • NBL ES Trichinella pseudospiralis newborn larval ES
  • AD ES adult ES
  • PAD ES preadult ES
  • NBL live newborn larvae
  • NK natural killer
  • TPi directed the stimulation of in vivo NK cell activity, as shown in Table 6, of mice injected with supernatants from 3T3 murine fibroblasts stimulated with newborn larval ES.
  • Pulmonary NK cell activity was assessed by an in vivo clearance assay with NK-sensitive tumor target cells (YAC-1 lymphoma) .
  • 51 Cr-labeled tumor cells (10 6 ) were intravenously injected into groups of treated and untreated mice.
  • % enhanced clearance [1 - (mean cpm in test lungs/mean cpm in control lungs)] x 100. The data were normalized for each pair of test and control groups so that controls were designated as producing 0% enhanced clearance (i.e. background level) .
  • mice were untreated (Naive) , injected mtraperitoneally daily for 2 days with either 0.5 ml of supernatant from 3T3 cultures not exposed to newborn larval ES (Medium Only) or 0.5 ml of supernatant from 3T3 cultures exposed for 48 hr to newborn larval ES (100 ⁇ g parasite protein/ml of culture medium) .
  • IL-2 Secretion Assay To measure IL-2 secretion by lymphoid cells during the newborn larval phase of infection with T. pseudospiralis , spleen and MLN cells were collected from noninfected (control) and infected female C57/BL6 mice (Jackson Labs, Bar Harbor, ME) on days 6, 10, 17 or 24 following infection with T. pseudospiralis .
  • IL-2 secretion is represented as the mean percent of normal control. Each bar represents the mean of 16 separate samples. IL-2 detected in culture supernatant of spleen cells and MLN cells recovered from infected mice on day 10 was significantly greater than that for controls and for days 7, 17 and 24 postinfection (P ⁇ 0.01) as shown in FIG. 17.
  • T. spiralis A number of hosts are highly resistant to infection with T. spiralis .
  • the Chinese hamster the basis for resistance to this parasite is an extremely rapid and intense inflammatory response mounted during the early stages of infection.
  • T. pseudospiralis suppressed the inflammatory response normally mounted by this host. This provided levels of success for T. spiralis infection normally seen only in the most permissive of hosts (FIG. 5) .
  • Trichinella pseudospiralis IL-10-like factor TPIO
  • TPi IL-2-like activity inducing factor
  • mice infected with the muscle stage of this parasite were skinned and eviscerated, homogenized in a Waring blender in 250 ml/mouse of 1% pepsin-1% HCl solution at high speed for two 15 sec bursts.
  • the resulting homogenate was incubated in beakers at 37°C for 1 hr in a water bath shaker. Incubates were allowed to sediment for 20 min at room temperature and the supernatant removed by aspiration.
  • the larvae were washed twice with PBS by sedimentation and forced through 16 layers of cheesecloth using a stream of PBS.' The larvae were placed in glass incubation chambers containing RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) , 2% antibiotic/antimycotic, L L glutamine and 10 mM HEPES buffer (approximately 100,000 larvae/50 ml of medium) and incubated for 48-72 hr at 37°C in a serological water bath. Fetal calf serum may be left out for incubations of 24-48 hr. Larvae were removed by centrifugation at 1000 rpm for 5 min and the supernatant containing the ES was retained.
  • FCS fetal calf serum
  • HEPES 10 mM HEPES buffer
  • TP10 is defined as freed from whole or viable T. pseudospiralis .
  • the purification of a Trichinella pseudospiralis inducer of IL-2-like activity inducing factor, as described in the preferred embodiments and with the characteristics described in Example 1 may be isolated in the following manner.
  • T. pseudospiralis larvae were isolated from mouse muscle by methods described by Stewart et al . (1990).
  • Intestines and debris were removed by passing the worm suspension through a series of sieves and the worms were washed three times with saline-l%AA by centrifugation.
  • adult worm suspensions were passed through a Whatman #4 filter pad mounted in a filter holder with a mild suction applied.
  • Worms and debris adherent to the filter pad were separated by floating the filter pad worm-side down in a shallow bowel containing PBS-1% AA to a depth of 2 cm and placed in a 37°C serological water bath for 30 min to allow the worms to migrate off the filter pad, leaving debris behind.
  • the isolated worms were washed twice in RPMI 1640 supplemented as indicated above (except that 30% FCS was used and approximately 25,000 worms were incubated in each 50 ml of medium) .
  • Adult worms were incubated in the above medium in covered glass dishes for 24-72 hr for release of newborn larvae (NBL) .
  • NBL newborn larvae
  • worm suspensions were pooled and passed through a 325 mesh brass sieve (Baxter, Grand Prairie, TX) to remove adult worms.
  • NBL were washed 5 times in RPMI 1640 supplemented as indicated above (except that 30% FCS was used and approximately 25,000 worms were incubated for an additional 48 hr.
  • TPi Purified TPi is defined as freed from whole or viable T. pseudospiralis .
  • Trichinella spiralis The closely related parasite, Trichinella spiralis, also appears to elaborate at least one substances with effects similar to those shown for TP10 and TPi.
  • Many of the Inventor's studies on T. pseudospiralis (TP) have been comparative in nature, with T. spiralis (TS) run in parallel as a control infection. In practically all cases, TS appears to employ immunoevasive strategies similar to those used by TP. However, TP consistently has a much greater influence over targeted immunological functions in the host. Examples from past studies by the Inventor would include: 1) suppression of inflammation to unrelated antigen (e.g., cotton string implants; Stewart et al . , 1985); 2) host pleural natural killer cell activity (Niederkorn et al .
  • TS The adult worm of TS residing in the gut of the host bowel generally displays much greater reproductive potential in most hosts than does TP. Yet TS is much more pathogenic than is TP. However, TS induces significantly higher levels of enteritis in its hosts than does TP. If TS were to exert the same degree of control over the development of enteritis displayed by TP, it would extend the duration of its adult phase and increase its fecundity (enteritis has a direct negative effect on the longevity and fecundity of adult Trichinella; (Stewart, G.L., et al . (1982)) which would cause an increase in morbidity and mortality associated with the stage of muscle invasion. Indeed, administration of cortisone to TS-infected mice at levels equivalent to those reached naturally during the course of infection with TP causes a dramatic increase in morbidity and mortality from both the intestinal and muscle phases of infection with TS.
  • Trichinella Nevertheless, both encapsulated and unencapsulated forms of Trichinella appear capable of modulating immunity by mechanisms similar to those described in this document.
  • compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the composition, methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Abstract

Le parasite helminthique Trichinella pseudospirallis élabore deux substances immunomodulatrices des lymphocites T. La phase intestinale du parasite libère une substance de cytokine analogue à l'interleukine-10, TP10, supprimant directement les réponses des lymphocites T auxiliaires Th1 et amortissant l'inflammation au niveau du site d'infection dans l'intestin grêle. Les larves tout juste nées envahissant les tissus élaborent une substance, TPi, laquelle fait générer aux cellules lymphoïdes et non lymphoïdes d'énormes quantités d'activité de type interleukine 2. L'activité de type IL-2 induite par TPi provoque la prolifération spectaculaire d'une lignée cellulaire dépendante de IL-2 (cellules HT2), ainsi qu'une augmentation au niveau de la libération de cytokine par des cellules du sous-ensemble Th1 (IL-2, interféron gamma and IL-3). Pendant le déroulement normal de l'infection dûe à T. pseudospiralis, TPi est libéré initiallement localement dans l'intestin grêle de l'hôte et provoque l'activation spectaculaire de cellules Th1 dans les ganglions mésentériques, avec la génération d'une entérite grave expulsant les vers adultes. La répartition dans l'organisme des larves venant de naître (la source de TPi), est accompagnée d'une augmentation spectaculaire des niveaux de plasma de cytokines de Th1 (essentiellement IL-2), lesquelles ont une rétroaction sur l'axe hypothalamique-pituitaire-surénal provoquant une augmentation significative de la corticostérone du plasma et une dépression systémique de l'immunité cellulaire.
PCT/US1995/002991 1994-03-11 1995-03-10 Substances immunomodulatrices tirees de trichinella WO1995024425A1 (fr)

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WO1996029082A1 (fr) * 1995-03-23 1996-09-26 Governors Of Dalhousie College And University Procede permettant de prolonger la survie d'une greffe allogenique d'organe
US5958671A (en) * 1996-04-23 1999-09-28 Presidents And Fellows Of Harvard College Methods and compositions for regulating T cell subsets by modulating transcription factor activity
US6090413A (en) * 1996-03-25 2000-07-18 Lee; Timothy D. Process of prolonging organ allograft survival
EP1041994A1 (fr) * 1997-12-31 2000-10-11 University of Iowa Research Foundation Utilisation d'agents biologiques parasitaires permettant de prevenir et de lutter contre les maladies auto-immunes
US6537810B1 (en) 1996-04-23 2003-03-25 President And Fellows Of Harvard College Methods for regulating T cell subsets by modulating transcription factor activity
WO2005000215A2 (fr) 2003-06-23 2005-01-06 The Regents Of The University Of Colorado Methodes de traitement de la douleur
WO2006130581A2 (fr) 2005-05-31 2006-12-07 Avigen, Inc. Methodes d'administration de genes
WO2011015819A3 (fr) * 2009-08-03 2011-04-21 Moredun Research Institute Collecte de parasites

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BIOLOGICAL ABSTRACTS, Volume 91, Number 7, issued 01 April 1991, HONG et al., "Serological Cross Reactions Between Saline Extract of Trichinella Spiralis Muscle Larvae and Human Sera Infected With Trematodes", page 874, Abstract No. 76503; & CHUNG-ANG JOURNAL OF MEDICINE, Volume 15, No. 2, issued 1990, pages 197-208. *

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996029082A1 (fr) * 1995-03-23 1996-09-26 Governors Of Dalhousie College And University Procede permettant de prolonger la survie d'une greffe allogenique d'organe
US6090413A (en) * 1996-03-25 2000-07-18 Lee; Timothy D. Process of prolonging organ allograft survival
US6967077B1 (en) 1996-04-23 2005-11-22 President And Fellows Of Harvard College Methods and compositions for regulating T cell subsets by modulating transcription factor activity
US6399322B1 (en) 1996-04-23 2002-06-04 President And Fellows Of Harvard College Methods and compositions for regulating T cell subsets by modulating transcription factor activity
US6537810B1 (en) 1996-04-23 2003-03-25 President And Fellows Of Harvard College Methods for regulating T cell subsets by modulating transcription factor activity
US5958671A (en) * 1996-04-23 1999-09-28 Presidents And Fellows Of Harvard College Methods and compositions for regulating T cell subsets by modulating transcription factor activity
EP1041994A1 (fr) * 1997-12-31 2000-10-11 University of Iowa Research Foundation Utilisation d'agents biologiques parasitaires permettant de prevenir et de lutter contre les maladies auto-immunes
JP2001527048A (ja) * 1997-12-31 2001-12-25 ユニヴァーシティー オブ アイオワ リサーチ ファンデーション 自己免疫疾患の予防および抑制のための寄生生物剤の使用
EP1041994A4 (fr) * 1997-12-31 2003-02-19 Univ Iowa Res Found Utilisation d'agents biologiques parasitaires permettant de prevenir et de lutter contre les maladies auto-immunes
US6764838B2 (en) 1997-12-31 2004-07-20 University Of Iowa Research Foundation Use of parasitic biological agents for prevention and control of autoimmune diseases
JP4733830B2 (ja) * 1997-12-31 2011-07-27 ユニヴァーシティー オブ アイオワ リサーチ ファンデーション 自己免疫疾患の予防および抑制のための寄生生物剤の使用
JP2011016840A (ja) * 1997-12-31 2011-01-27 Univ Of Iowa Research Foundation 自己免疫疾患の予防および抑制のための寄生生物剤の使用
EP1749534A3 (fr) * 1997-12-31 2007-02-21 University Of Iowa Research Foundation Utilisation d'agent biologiques parasitaires permettant de prévenir et de lutter contre les maladies auto-immunes
US7250173B2 (en) 1997-12-31 2007-07-31 University Of Iowa Research Foundation Use of parasitic biological agents for prevention and control of autoimmune diseases
EP2255820A1 (fr) * 1997-12-31 2010-12-01 University Of Iowa Research Foundation Utilisation d'agents biologiques parasitaires permettant de prévenir et de lutter contre les maladies auto-immunes
EP2258841A1 (fr) 2003-06-23 2010-12-08 The Regents of the University of Colorado Procédés de traitement de la douleur
WO2005000215A2 (fr) 2003-06-23 2005-01-06 The Regents Of The University Of Colorado Methodes de traitement de la douleur
WO2006130581A2 (fr) 2005-05-31 2006-12-07 Avigen, Inc. Methodes d'administration de genes
EP2816118A1 (fr) 2005-05-31 2014-12-24 The Regents of the University of Colorado, A Body Corporate Procédés pour administrer des gènes
WO2011015819A3 (fr) * 2009-08-03 2011-04-21 Moredun Research Institute Collecte de parasites
AU2010280524B2 (en) * 2009-08-03 2015-09-24 Moredun Research Institute Parasite harvesting
AU2010280524C1 (en) * 2009-08-03 2015-12-17 Moredun Research Institute Parasite harvesting

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