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Ascaris suum, an Intestinal Parasite, Produces Yannick Goumon, Federico Casares, Stephen Pryor, Lee Ferguson, Bruce Brownawell, Patrick Cadet, Christos M. This information is current as Rialas, Ingeborg D. M. Welters, Dario Sonetti and George B. of September 27, 2021. Stefano J Immunol 2000; 165:339-343; ; doi: 10.4049/jimmunol.165.1.339

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Ascaris suum, an Intestinal Parasite, Produces Morphine1

Yannick Goumon,* Federico Casares,*† Stephen Pryor,* Lee Ferguson,† Bruce Brownawell,† Patrick Cadet,* Christos M. Rialas,* Ingeborg D. M. Welters,* Dario Sonetti,‡ and George B. Stefano2*

The parasitic worm Ascaris suum contains the morphine as determined by HPLC coupled to electrochemical -detection and by gas chromatography/mass spectrometry. The level of this material is 1168 ؎ 278 ng/g worm wet weight. Fur thermore, Ascaris maintained for 5 days contained a significant amount of morphine, as did their medium, demonstrating their ability to synthesize the opiate alkaloid. To determine whether the morphine was active, we exposed human monocytes to the material, and they immediately released nitric oxide in a -reversible manner. The anatomic distribution of morphine immunoreactivity reveals that the material is in the subcuticle layers and in the animals’ nerve chords. Furthermore, as deter- mined by RT-PCR, Ascaris does not express the transcript of the neuronal ␮ . Failure to demonstrate the expression of Downloaded from this receptor, as well as the morphine-like tissue localization in Ascaris, suggests that the endogenous morphine is intended for secretion into the microenvironment. The Journal of Immunology, 2000, 165: 339–343.

uccessful parasitism, in which the host survives for ex- that it might be using morphine to escape detection by the host’s tended periods, can be characterized as an equilibrium be- immune system. In this study, we report for the first time that A. tween the parasite and the host, more specifically between suum synthesizes morphine, thereby strengthening the common- S http://www.jimmunol.org/ the host’s immune system and the parasite’s ability to create a signal molecule hypothesis, i.e., using either similar or identical permissive microenvironment in situ. One mechanism that a par- host signaling to escape host immunosurveillance. asite may use to modify the host immune response is to down- regulate the host’s response (1–3). Capron and colleagues (4–7) suggested that parasites may communicate with their hosts via Materials and Methods common signaling molecules that diminish host immune surveil- Adult frozen A. suum were obtained from Carolina Biological Supply (Bur- lance. In this regard, morphine is generally acknowledged as an lington, NC). Live adult Ascaris were obtained from Josef Miller (Pine immune down-regulating agent (8). This finding is enhanced by Plains, NY) and were maintained in the laboratory for up to 6 days as described elsewhere in detail (23). Animals were examined on the fifth day

the fact that morphine is present in several mammalian tissues, by guest on September 27, 2021 for endogenous morphine levels. including brain and adrenal gland (9–20), supporting its role as a neural or inflammatory mediator. Recently, we have demonstrated that free-living and parasitic Extraction invertebrates produce several major opioid precursors, i.e., The extraction experiments using internal or external morphine standards , , and (21). were performed in different rooms to avoid morphine contamination of These mammalian-like opioid exhibit high sequence iden- Ascaris samples. Single-use siliconized tubes also were used to prevent the tity to their mammalian counterparts. For example, Mytilus adre- loss of morphine as well as contamination between assays (14). Tissues nocorticotropin has greater than 90% sequence identity with its were extensively washed with PBS buffer (three times, 1 min) to avoid an additional potential source of morphine contamination. Tissues were mammalian counterpart (21). We have also identified a tentative weighed, homogenized in 1 N HCl (1 ml/0.1 g), and then extracted with 5 morphine-like molecule in Schistosoma mansoni by way of radio- ml /isopropanol 9:1 (24). After 5 min, homogenates were cen- immunoassay (22). trifuged (3,000 rpm, 15 min). The supernatant was centrifuged twice (15 Given this and the fact that the pig intestinal parasite Ascaris min, 12,000 rpm, 4°C), dried, and dissolved in 0.05% trifluoroacetic acid (25). Samples were loaded on a Sep-Pak Plus cartridge (Waters, Milford, MA) and suum can live in its host for extended periods of time, we surmised eluted in water/acetonitrile/trifluoroacetic acid (89.95%:10%:0.05%, v/v/v). Samples were dissolved in buffer A before HPLC analysis (see below).

*Neuroscience Research Institute, State University of New York, Old Westbury, NY HPLC and electrochemical detection 11568; †Marine Sciences Research Center, State University of New York, Stony ‡ Brook, NY 11794; and Neurobiology Research Unit, Department of Animal Biology, HPLC separations were performed with two Acuflow Series VI and a C18 University of Modena, Modena, Unijet microbore column (Bioanalytical Systems, West Lafayette, IN). Received for publication January 3, 2000. Accepted for publication April 12, 2000. Morphine detection was performed with an amperometric detector LC-4C The costs of publication of this article were defrayed in part by the payment of page (Bioanalytical Systems) using a Unijet glassy carbon working electrode (3 charges. This article must therefore be hereby marked advertisement in accordance mm) and a 0.02 Hz filter (500 mV; range, 5 nA). The mobile phases were with 18 U.S.C. Section 1734 solely to indicate this fact. buffer A (10 mM ammonium bicarbonate, 10 mM ammonium chloride, 10 1 This work was supported by the following grants: National Institute of Mental mM sodium chloride, 0.2 mM EDTA (pH 5.0)) and buffer B (10 mM Health (NIMH) Career Opportunities Research Program 17138; National Institute on ammonium bicarbonate, 10 mM ammonium chloride, 10 mM sodium chlo- Abuse (NIDA) 09010; NIMH 47392, and the Research Foundation and Central ride, 0.2 mM EDTA, 50% acetonitile (pH 5.0) (24)). The injection volume Administration of the State University of New York and National Institutes of Health was 5 ␮l, and the flow rate was 70 ␮l/min. Conditions were: t ϭ 0, 0% Fogarty International 00045. P.C. is a NIDA Postdoctoral Fellow. buffer B; t ϭ 10 min, 10% buffer B; t ϭ 20 min, 30% buffer B; and t ϭ 25 2 Address correspondence and reprint requests to Dr. George B. Stefano, Neuro- min, 100% buffer B. Several HPLC purifications were performed between science Research Institute, State University of New York, College at Old Westbury, each sample to prevent residual morphine contamination remaining on the Old Westbury, NY 11568. E-mail address: [email protected] column. Furthermore, the fraction of blank chromatography corresponding

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 340 PARASITE MAKES MORPHINE

to the elution time of the morphine (flat line) was determined by gas chro- and incubated with anti-sheep rabbit IgG-FITC (Dako, Aarhus, Denmark; matography/mass spectrometry (GC/MS)3 analysis, confirming that no 1:40; 1 h; room temperature). morphine was remaining. Five nanograms of internal morphine standard Slides were rinsed in PBS and then dipped in 0.005% propidium iodide was first chromatographed (Fig. 1A) along with Ascaris morphine (Fig. for nuclei staining (1 min). After a final rapid rinse with PBS, sections were 1B), and then the same sample with 4 ng of morphine external standard was mounted in buffered glycerol (87.7% glycerol, 2.3% 1,4-diazalbicyclo added after the extraction (Fig. 1C). [2.2.2]octane, 10% Tris 20 mM (pH 8)), cover-slipped, and examined on a Zeiss microscope equipped for fluorescence with FITC excitation and bar- GC/MS rier filter combination. For comparison, we tested two other antisera, anti-adrenocorticotrophic The identity of the morphine detected by HPLC was further confirmed by hormone and anti-␤-endorphin (Biogenesis). Reaction specificity for each GC/MS using a modification of a method reported by Allen et al. (26). A technique was controlled by: 1) replacing the Ab with a nonimmune serum; fraction of the eluent corresponding to the HPLC morphine peak was col- 2) omitting the first antiserum; and 3) preincubating the primary Ab at 4°C lected in a 0.5-ml siliconized plastic vial, evaporated to dryness, and stored Ϫ for 24 h in the presence of excess Ag (morphine HCl 10 3 M). at Ϫ70°C. The sample was dissolved in 50 ␮l of 2% ammonia (in meth- anol) and transferred to 0.1-ml conical glass vials (Kimax, Alphuretta, GA) ␮ fitted with Teflon-lined septum liners. The sample was evaporated under Opiate receptor expression: isolation of total RNA nitrogen gas and then mixed with N,O-bis (trimethylsilyl) trifluoroacetim- Tissue samples, excised pedal ganglia of Mytilus as a positive control (32), ␮ ide (20–50 l) catalyzed with 1% trimethylchlorosilane (90°C, 30 min). and all body sections of Ascaris were homogenized in Tri-Reagent (Mo- ϭ This reaction yields a ditrimethylsilyl derivative of morphine (m.w. lecular Research Center, Cincinnati, OH) containing 1-bromo-3-chloropro- 429). The reaction conditions chosen resulted in higher yields than did pane (0.1 ml/ml Tri-Reagent) using a Polytron homogenizer. The homog- reactions conducted at lower temperature (70°C) or for shorter times. The enates were stored at room temperature for 5 min to allow complete derivatized morphine was analyzed on a Varian Saturn III GC/MS dissociation of nucleoprotein. The samples were vortexed vigorously (15 equipped with a 30-m (0.25 mm internal diameter and 0.25 ␮m) DB-5 s), kept at room temperature for 7 min, and then centrifuged (15 min, Downloaded from capillary column (J & W Scientific, Folsom, CA). Two-microliter injec- 12,000 ϫ g). The aqueous phase was transferred to a fresh tube, and the tions were made in splitless mode at an initial column temperature of RNA was precipitated with isopropanol, washed with 75% ethanol, air- 180°C, and after 2 min the column temperature was heated at a rate of dried, and resuspended in water. RNA was analyzed on a 1% denaturing 15°C/min up to 300°C. Morphine was detected at 10 min; injection port agarose gel, and purity was determined spectrophotometrically. temperature was 275°C. Mass spectrum indicated major ions (base peak ϩ ϩ depended on instrument tune conditions) at 429 (M ) and 414 (M-CH3 ). RT-PCR of total RNA Analyses of samples were conducted using a selected ion storage method,

in which mass windows (Ϯ 2 atomic mass units) around ions 429 and 414 First-strand cDNA synthesis was performed using random hexamers (Life http://www.jimmunol.org/ were collected. Morphine identity was confirmed by the retention time, Technologies, Gaithersburg, MD). Three micrograms of total RNA isolated peak shape, and comparison of parasite-derived morphine to authentic mor- from pedal ganglia as positive control or Ascaris tissue was denatured at phine standards injected. 95°C and reverse transcribed at 42°C for 1 h using Superscript II RNase H-RT (Life Technologies). Seven microliters of the reverse transcription Bioassay of Ascaris morphine: NO release product was added to the PCR mix containing specific primers for the ␮ gene and Taq DNA polymerase (Life Technologies). The Human peripheral monocytes (Long Island Services, Melville, NY) PCR reaction was denatured at 95°C for 5 min before 30 cycles at 95°C for were isolated using the Accurate Scientific (Westbury, NY) monocyte kit 1 min, 57°C for 1 min, 72°C for 1 min, and then an extension step cycle and were washed as previously described (27–29). We used monocytes to at 72°C for 10 min. PCR products were analyzed on a 2% agarose gel measure the biological activity of Ascaris-derived morphine because we (Sigma, St. Louis, MO) and visualized by ethidium staining.

have demonstrated that they contain stereospecific opiate alkaloid-selective by guest on September 27, 2021 The ␮-specific primers used in the PCR reactions amplified a 441-bp receptors that are coupled to morphine and NO release that is naloxone fragment starting at map position 896 (primer M1, 5Ј-GGTACTGG reversible (27, 28). GAAAACCTGCTGAAGATCTGTG-3Ј) and at map position 1336 (prim- NO release from the incubated monocytes (107 cells/chamber) was mea- er M4, 5Ј-GGTCTCTAGTGTTCTGACGAATTCGAGTGG-3Ј). This seg- sured directly using an NO-specific amperometric probe (World Precision ment of the gene encodes the third extracellular loop of the receptor that is Instruments, Sarasota, FL) (30, 31). Briefly, the cells were placed in a important for ␮ selectivity. In addition, primers for the internal superfusion chamber in 1 ml PBS. The probe was positioned 15 ␮m above control gene G3PDH (forward primer, 5Ј-ACCACAGTCCATGCCAT the cell surface by a micromanipulator (Zeiss-Eppendorff, Suwanee, GA) CAC; reverse primer, 5Ј-TCCACCACCCTGTTGCTGGTA) were used to attached to the stage of an inverted microscope (Diaphot; Nikon, Melville, amplify a 451-bp fragment by RT-PCR from Ascaris total RNA. NY). The system was calibrated daily by adding potassium nitrite to a solution of , resulting in the liberation of a known quantity of NO (World Precision Instruments). Baseline levels of NO release were Results determined by evaluation of NO release in PBS. Cells were stimulated with the respective purified morphine-like material, and the concentration of NO To determine whether the parasitic worm A. suum utilized the opi- gas in solution was measured in real time with the DUO 18 computer data oid alkaloid morphine, this compound was identified and quanti- acquisition system (World Precision Instruments). The amperometric probe fied, and then the worms were analyzed for the presence of mor- was equilibrated for at least 12 h in PBS before being transferred to the phine receptors. Morphine was identified in Ascaris extracts by superfusion chamber containing the cells, and manipulation of the cells was performed only with glass instruments. To evaluate NO release, the cells reversed-phase HPLC using a gradient of acetonitrile after liquid were exposed to morphine as indicated. and solid extraction (Fig. 1). All experiments were carefully per- formed to prevent exogenous morphine contamination (see Mate- Morphine immunohistochemistry rials and Methods). The morphine extracted from Ascaris (Fig. Adult specimens of A. suum were obtained from Italcarni (Carpi, Italy) 1B) was identical with the major peak for the morphine internal immediatly after the slaughter of parasitized pigs. The worms were fixed (Fig. 1A) and external (Fig. 1C) standard. This finding was repli- either in phosphate-buffered formalin (4% paraformaldehyde in 0.1 M (pH cated in five other worms. The linearity of the detection of the 7.4)) for8hat4°Corinamixture of glutaraldehyde/picric acid/acetic acid (GPA; 1:3 ϩ 1%) overnight and then for 48 h at 4°C. Specimens were HPLC technique at low concentrations of morphine substantiates rinsed in 70% ethanol, dehydrated, embedded in paraffin, and sectioned (8 the sensitivity of this method for determining morphine levels (Fig. ␮m). Morphine was localized by indirect immunofluorescence using a 1B, inset). polyclonal Ab with minimal cross-reactivity to (Biogenesis, The concentration of morphine was determined using the Chro- Bournemouth, U.K.). Briefly, rehydrated sections were washed in PBS (0.9% NaCl, 0.01 M sodium phosphate buffer (pH 7.4)), preincubated in matogram Report (Bioanalytical Systems) and extrapolated from 5% normal serum for 30 min, and then incubated overnight at 4°C in a the peak area calculated for the internal standard. The average moist chamber with the primary Ab. Sections were then washed with PBS concentration of morphine in the six samples was 1168 Ϯ 278 ng/g wet weight, and after 5 days in culture it was 41 Ϯ 15 ng/g wet 3 Abbreviations used in this paper: GC/MS, gas chromatography/mass spectrometry; weight. In the worm incubation medium (nine adult worms in 1.48 MOR-IR, immunoreactive morphine. L) changed daily, on the fifth day morphine also was present at 725 The Journal of Immunology 341

nolocalization (data not shown). There was no staining in sections in which the primary antiserum was substituted with normal serum. We next determined whether tissues from Ascaris have opiate receptors that would allow them to utilize the morphine they con- tain. We used RT-PCR to amplify a fragment of the coding region of the ␮ opiate receptor from Ascaris and also one from Mytilus as a positive control (Fig. 3). Using ␮-specific primers, we isolated a transcript of the expected size for the ␮ receptor (441 bp) from Mytilus as positive contol (Ref. 32 and Fig. 3, lane 2) but not from Ascaris (Fig. 3, lane 3). This was not due to a lack of mRNA from Ascaris, because we were able to amplify a 451-bp mRNA corresponding to G3PDH from the worm (34). Sequence analysis of the Mytilus PCR product demonstrated that the ␮ receptor fragment exhibited 95% sequence identity with the human brain ␮ opiate re- ceptor (32). Failure to demonstrate the ␮ receptor in Ascaris tissues suggests that this material is intended for secretion into the microen- vironment as suggested by the MOR-IR localization. Downloaded from

Discussion The present study demonstrates for the first time the presence of morphine in A. suum and its expression under presumed nonstimu- lated (or basal) conditions. Animals maintained in the laboratory

for 5 days also had morphine present and were secreting the ma- http://www.jimmunol.org/ terial into the medium, demonstrating their ability to synthesize FIGURE 1. Purification of morphine from Ascaris. Morphine was iso- this material. Unlike the free-living mollusk Mytilus edulis (32), lated by HPLC. Running conditions: range, 5 nA; filter, 0.02 Hz; potential, Ascaris does not express ␮ opiate receptor transcripts. In Ascaris, 500 mV; flow rate, 500 ␮l/min; A buffer: 1 mM ammonium bicarbonate, 10 mM ammonium chloride, 10 mM sodium chloride, 0.2 mM EDTA (pH MOR-IR was found in the epidermis of the animal. These two 5.0); B buffer: same as A buffer but with 50% acetonitrile. Gradient: t ϭ 0, pieces of data suggest that, in Ascaris, morphine is secreted into 0% buffer B; t ϭ 10 min, 10% buffer B; t ϭ 20 min, 30% buffer B; t ϭ 25 the microenvironment where it could be used as a signaling mol- min, 100% buffer B. A, Five nanograms of morphine internal standard. B, ecule. We hypothesize that the morphine helps the parasite to Standard curve of low morphine concentrations. C, Ascaris extract. D, evade the immunosurveillance machinery of the host and thereby Ascaris extract ϩ 4 ng morphine external standard. E, Ascaris incubation increase the stability of its microenvironment and ensure its sur- by guest on September 27, 2021 medium. vival. This strategy appears to also ensure the survival of the ova. Furthermore, the morphine Ascaris displays the same effect as nat- ural morphine on the NO release of the monocyte, an effect an- ng/L, demonstrating that the worm was synthesizing and secreting tagonized by the naloxone. This result suggests that the morphine the material (Fig. 1E). from Ascaris can act on immune cells to enhance its survival. The morphine in the HPLC fractions was further analyzed by Opioid peptides and their precursors are present in free-living GC/MS (Fig. 2A). Again, the morphine fraction isolated from A. and parasitic invertebrates (21). These free-living invertebrates suum was identical with synthetic morphine according to retention contain complex and highly specific opioid and opiate receptor time, peak shape, and comparison to authentic morphine. Mor- sites (32, 35–37) involved in dopaminergic and NO processes (31, phine quantification was confirmed by this method. Additionally, 38). In these animals, the tissues that produced these signaling the identity of morphine in the worm incubation medium, noted molecules have the appropriate receptors to utilize them. For ex- above, was confirmed by GC/MS (data not shown). ample, in Mytilus, we recently demonstrated both morphine and a To determine whether the morphine was active, we used a clas- ␮ opiate receptor subtype that exhibited 95% sequence identity sic bioassay for morphine, i.e., the ability of the compound of with the human neuronal ␮ opiate receptor (32). We had surmised, interest to release NO from human monocytes in a naloxone-re- based on the Mytilus model, that the signaling material was used versible manner (28). Incubation of human monocytes with mor- within that tissue. However, Ascaris makes morphine without a phine from Ascaris resulted in the immediate release of NO that corresponding receptor, suggesting that it is secreted for “external” was antagonized by naloxone (Fig. 2B). signaling, i.e., host immune down-regulation (8, 39). This hypoth- We next analyzed the anatomic distribution of morphine in A. esis is supported by our immunohistochemistry results, which suum by immunofluorescence using a morphine-specific Ab (Ref. demonstrated the presence of this material in the epidermis of the 33 and Fig. 2, C and D). Immunoreactive morphine (MOR-IR; animal. green fluorescence) was found in subcuticle layers among collag- Complementing the present study are reports that Schistosoma enous structures, in fiber-like structures in the hypodermis, in the synthesize a morphine-like compound inhibiting immunocytes in a radial intercellular spaces between longitudinal contracting mus- naloxone-reversible manner (22). In this report, we also demon- cles underlying the skin, in the spaces encircling the inner big strated that in worms maintained in the laboratory the endogenous bodies of muscle cells, and in the nerve chords. Additionally, the morphine-like levels were low and that, upon exposure to human animal’s ova appear to be surrounded by this material in the ex- immunocytes, a significant increase in the worm’s opiate level tracellular space. Different fixation methods gave the same results. occurred, suggesting a feedback process. In the present report we Staining for adrenocorticotrophic hormone and ␤-endorphin were also note a significant drop in morphine levels in animals main- negative, further confirming the specificity of the morphine immu- tained in the laboratory. However, its high level in the incubation 342 PARASITE MAKES MORPHINE Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 2. Characterization of Ascaris morphine. A, GC/MS spectrum of two morphine standards (200 pg and 400 pg) and the morphine material collected during HPLC analysis of Ascaris. B, Bioassay of morphine-like activity extracted from Ascaris. Ascaris extracts stimulate NO release from human monocytes. Top, A total of 500 ␮lofAscaris whole body extract was added to a solution containing purified human monocytes (107 cells/ml) (arrowhead). This resulted in NO release. Bottom, Naloxone (first arrowhead; 10Ϫ6 M) was added to another batch of cells under similar conditions before the addition of morphine extract (second arrowhead). This treatment blocked the Ascaris morphine-stimulated NO release, demonstrating that the Ascaris morphine material is opiate alkaloid in character. C and D, Immunocytochemical localization of morphine in Ascaris. C, Ascaris body wall region incubated with nonimmune serum shows a complete lack of positive immunoreactivity. D, MOR-IR in Ascaris suum. Transverse section of the body wall. Cuticule (a) MOR-IR is localized in the fibrous layer under the cuticle (b), in fibers running in the hypodermis (c) and in the intercellualr spaces between the contractile parts of myoepithelial cells (d) (bar ϭ 20 microns). E, Ascaris ova incubated with nonimmune serum shows a complete lack of positive immunoreactivity. F, Morphine-like immunoreactivity immediately outside the ova (bar ϭ 50 microns). medium indicates that as this material is made it is secreted into the These Ascaris morphine data also suggest that morphine may immediate environment. In this regard, if morphine were not syn- play a role in gastrointestinal regulation. In human history it has thesized in the worm, by day 5 it should not be detected because widely been accepted that represents the body’s attempt to its tissue half-life is about 2 h and it cannot be detected in mam- flush out toxins, including parasitic worms. The Persian physician malian and invertebrate tissues after 24 h (Ref. 40 and our unpub- Avicenna prescribed morphine for , anemia, and diarrhea lished observations). thousands of years ago (41). Thus, Ascaris may secrete this opiate The Journal of Immunology 343

16. Weitz, C. J., L. I. Lowney, K. F. Faull, G. Feistner, and A. Goldstein. 1986. Morphine and codeine from mammalian brain. Proc. Natl. Acad. Sci. USA 83: 9784. 17. Bianchi, E., C. Alessandrini, M. Guarna, and A. Tagliamonte. 1993. Endogenous codeine and morphine are stored in specific brain neurons. Brain Res. 627:210. 18. Bianchi, E., M. Guarna, and A. Tagliamonte. 1994. Immunocytochemical local- FIGURE 3. Identification of ␮ opioid receptor mRNA transcript. Total ization of endogenous codeine and morphine. Adv. Neuroimmunol. 4:83. RNA isolated from these tissues was reverse transcribed, PCR amplified, 19. Guarna, M., C. Neri, F. Petrioli, and E. Bianchi. 1998. Potassium-induced release of endogenous morphine form rat brain slices. J. Neurochem. 70:147. and run on agarose gels. Lane 1, G3PDH housekeeping gene (expected 20. Tagliamonte, A., M. Guarna, and E. Bianchi. 1995. Endogenous morphine and transcript size is 451 bp). Lane 2, ␮ opioid receptor transcript in Mytilus codeine as possible physiological ligands of ␮-opiate receptors. In Drug Addic- pedal ganglia (expected size, 441 bp). Lane 3, lack of ␮ receptor transcript tion and Related Clinical Problems. I. Maremmani and A. Tagliamonte, eds. in Ascaris. Lane 4, 100-bp DNA markers. Both negative and positive Springer-Verlag, Vienna, p. 27. strands were sequenced; however, only the sequence from the negative 21. Stefano, G. B., and M. Salzet. 1999. Invertebrate opioid precursors: evolutionary conservation and the significance of enzymatic processing. Int. Rev. Cytol. 187: strand is shown in lane 2. The tissues were washed extensively with PBS 261. to limit any contamination. 22. Leung, M. K., C. Dissous, A. Capron, H. Woldegaber, O. Duvaux-Miret, S. C. Pryor, and G. B. Stefano. 1995. Schistosoma mansoni: the presence and potential use of opiate-like substances. Exp. Parasitol. 81:208. 23. Bowman, J. W., C. A. Winterrowd, A. R. Friedman, D. P. Thompson, alkaloid to diminish its “flushing” from the host because a sec- R. D. Klein, J. P. Davis, A. G. Maule, K. L. Blair, and T. G. Geary. 1995. Nitric oxide mediates the inhibitory effects of SDPNFLRFamide, a nematode FMRF- ondary effect on gastrointestinal motility is to induce . amide-related neuropeptide, in Ascaris suum. J. Neurophysiol. 74:1880. In conclusion, opioid processes appear to have evolved much 24. Grieco, D., A. Porcellini, E. V. Avvedimento, and M. E. Gottesman. 1996. Re- Downloaded from earlier than previously thought. Opiate alkaloid signaling appears quirement for cAMP-PKA pathway activation by M phase-promoting factor in the transition from mitosis to interphase. Science 271:1718. to have been conserved during evolution and used to enhance the 25. Macarthur, H., M. B. Mattammal, and T. C. Westfall. 1995. A new perspective longevity of the host and parasite. Clearly, this represents an im- on the inhibitory role of nitric oxide in sympathetic neurotransmission. Biochem. portant strategy for enhancing the odds for survival and, thus, for Biophys. Res. Commun. 216:686. simultaneously preserving the message. 26. Allen, D. L., K. S. Scott, and J. S. Oliver. 1999. Comparison of solid-phase extraction and supercritical fluid extraction for the analysis of morphine in whole blood. J. Anal. Toxicol. 23:216. Acknowledgments 27. Stefano, G. B., A. Digenis, S. Spector, M. K. Leung, T. V. Bilfinger, http://www.jimmunol.org/ M. H. Makman, B. Scharrer, and N. N. Abumrad. 1993. Opiatelike substances in We thank Danielle Benz, Ireen Khan, Mazen Madhoun (National Institute an invertebrate, a novel opiate receptor on invertebrate and human immunocytes, of Mental Health-Career Opportunities Research Program Fellows), and and a role in immunosuppression. Proc. Natl. Acad. Sci. USA 90:11099. Yun Su Lee for technical assistance. 28. Magazine, H. I., Y. Liu, T. V. Bilfinger, G. L. Fricchione, and G. B. Stefano. 1996. Morphine-induced conformational changes in human monocytes, granulo- cytes, and endothelial cells and in invertebrate immunocytes and microglia are References mediated by nitric oxide. J. Immunol. 156:4845. 29. Stefano, G. B., P. Melchiorri, L. Negri, T. K. Hughes, and B. Scharrer. 1992. 1. Capron, A., J. P. Dessaint, M. Capron, J. H. Ouma, and A. E. Butterworth. 1987. (D-Ala2)- I binding and pharmacological evidence for a special sub- Immunity towards schistosomes: progress toward vaccine. Science 238:1065. type of ␦ opioid receptor on human and invertebrate immune cells. Proc. Natl. 2. Capron, A., and J. P. Dessaint. 1988. Parasitism, a model of cell sociology. News Acad. Sci. USA 89:9316. by guest on September 27, 2021 Physiol. Sci. 3:75. 30. Stefano, G. B., A. Hartman, T. V. Bilfinger, H. I. Magazine, Y. Liu, F. Casares, 3. Capron, A., and J. P. Dessaint. 1989. Molecular basis of host-parasite relation- and M. S. Goligorsky. 1995. Presence of the ␮3 opiate receptor in endothelial ship: toward the definition of protective antigens. Immunol. Rev. 112:27. cells: coupling to nitric oxide production and vasodilation. J. Biol. Chem. 270: 4. Duvaux-Miret, O., C. Dissous, J. P. Guatron, E. Pattou, C. Kordon, and 30290. A. Capron. 1990. The helminth Schistosoma mansoni expresses a peptide similar 31. Liu, Y., D. Shenouda, T. V. Bilfinger, M. L. Stefano, H. I. Magazine, and to human ␤-endorphin and possesses a POMC-related gene. New Biol. 2:93. G. B. Stefano. 1996. Morphine stimulates nitric oxide release from invertebrate 5. Duvaux-Miret, O., G. B. Stefano, E. M. Smith, C. Dissous, and A. Capron. 1992. microglia. Brain Res. 722:125. Immunosuppression in the definitive and intermediate hosts of the human parasite ␮ Schistosoma mansoni by release of immunoactive neuropeptides. Proc. Natl. 32. Cadet, P., and G. B. Stefano. 1999. Mytilus edulis pedal ganglia express opiate ␮ Acad. Sci. USA 89:778. receptor transcripts exhibiting high sequence identity with human neuronal 1. 6. Duvaux-Miret, O., G. B. Stefano, E. M. Smith, L. Mallozzi, and A. Capron. 1992. Mol. Brain Res. 74:242. Proopiomelanocortin-derived peptides as tools of immune evasion for the human 33. Sonetti, D., L. Mola, F. Casares, E. Bianchi, M. Guarna, and G. B. Stefano. 1999. trematode Schistosoma mansoni. Acta Biol. Hung. 43:281. Endogenous morphine levels increase in molluscan neural and immune tissues 7. Duvaux-Miret, O., G. B. Stefano, E. M. Smith, and A. Capron. 1992. Neuroim- after physical trauma. Brain Res. 835:137. munology of host parasite interactions: proopiomelanocortin derived peptides in 34. Kochman, M., J. Golebiowska, T. Baranowski, J. R. Dedman, D. W. Fodge, and the by Schistosoma mansoni. Adv. Neuroimmunol. 2:297. B. G. Harris. 1975. Studies on enzymes from parasitic helminths. V. Purification 8. Stefano, G. B., B. Scharrer, E. M. Smith, T. K. Hughes, H. I. Magazine, and characterization of glyceraldehyde 3-phosphate dehydrogenase from Ascaris T. V. Bilfinger, A. Hartman, G. L. Fricchione, Y. Liu, and M. H. Makman. 1996. suum muscle. Comp. Biochem. Physiol. 52:301. Opioid and opiate immunoregulatory processes. Crit. Rev. Immunol. 16:109. 35. Kream, R. M., R. S. Zukin, and G. B. Stefano. 1980. Demonstration of two 9. Donnerer, J., G. Cardinale, J. Coffey, C. A. Lisek, I. Jardine, and S. Spector. classes of opiate binding sites in the nervous tissue of the marine mollusc Mytilus 1987. Chemical characterization and regulation of endogenous morphine and edulis: positive homotropic cooperativity of lower affinity binding sites. J. Biol. codeine in the rat. J. Pharmacol. Exp. Ther. 242:583. Chem. 255:9218. 10. Donnerer, J., K. Oka, A. Brossi, K. C. Rice, and S. Spector. 1986. Presence and 36. Stefano, G. B., R. M. Kream, and R. S. Zukin. 1980. Demonstration of stereospe- formation of codeine and morphine in the rat. Proc. Natl. Acad. Sci. USA 83: cific opiate binding in the nervous tissue of the marine mollusc Mytilus edulis. 4566. Brain Res. 181:445. 11. Cardinale, G. J., J. Donnerer, A. D. Finck, J. D. Kantrowitz, K. Oka, and 37. Stefano, G. B., and B. Scharrer. 1981. High affinity binding of an S. Spector. 1987. Morphine and codeine are endogenous components of human analog in the cerebral ganglion of the insect Leucophaea maderae (Blattaria). cerebrospinal fluid. Life Sci. 40:301. Brain Res. 225:107. 12. Donnerer, J., G. Cardinale, J. Coffey, C. A. Lisek, I. Jardine, and S. Spector. 38. Stefano, G. B., and B. Scharrer. 1996. The presence of the ␮3 opiate receptor in 1987. Chemical characterization and regulation of endogenous morphine and invertebrate neural tissues. Comp. Biochem. Physiol. 113C:369. codeine in the rat. J. Pharmacol. Exp. Ther. 242:583. 13. Goldstein, A., R. W. Barrett, I. F. James, L. I. Lowney, C. Weitz, 39. Eisenstein, T. K., and M. E. Hilburger. 1998. Opioid modulation of immune L. I. Knipmeyer, and H. Rapoport. 1985. Morphine and other from beef responses: effects on phagocyte and lymphoid cell populations. J. Neuroimmunol. brain and adrenal. Proc. Natl. Acad. Sci. USA 82:5203. 83:36. 14. Weitz, C. J., L. I. Lowney, K. F. Faull, G. Feister, and A. Goldstein. 1986. 40. Reisine, T., and G. W. Pasternak. 1996. Opioid and antagonists. In The Morphine and codeine from mammalian brain. Proc. Natl. Acad. Sci. USA 83: Pharmacological Basis of Therapeutics. J. G. Hardman and L. E. Limbird, eds. 9784. McGraw–Hill, New York, p. 521. 15. Weitz, C. J., K. F. Faull, and A. Goldstein. 1987. Synthesis of the skeleton of 41. Merlin, M. D. 1984. On The Trail of Ancient . Associated Univer- morphine molecule by mammalian . Nature 330:674. sity Press, London.