Effects of mu, kappa or delta opioids administered by pellet or pump on oral Salmonella infection and gastrointestinal transit

European Journal of Pharmacology 534 (2006) 250 – 257 www.elsevier.com/locate/ejphar

Effects of mu, kappa or delta opioids administered by pellet or pump on oral Salmonella infection and gastrointestinal transit Pu Feng a,1 , Rahil T. Rahim a,1,2 , Alan Cowan b,d , Lee-Yuan Liu-Chen b,d , Xiaohui Peng a , John Gaughan c,d , Joseph J. Meissler Jr. a,d , Martin W. Adler b,d , Toby K. Eisenstein a,d,⁎ a

Department of Microbiology and Immunology, Temple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140, USA b Department of Pharmacology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA c Department of Physiology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA d Center for Substance Abuse Research, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA Received 6 October 2005; received in revised form 3 January 2006; accepted 20 January 2006 Available online 2 March 2006

Abstract Our laboratory has shown previously that subcutaneously implanted, slow-release morphine pellets markedly enhanced susceptibility to oral infection with Salmonella typhimurium. Further, morphine, kappa and delta opioid receptor agonists infused via osmotic minipumps were immunosuppressive. The present study compared morphine pellets to morphine pumps and also examined the differential effects of morphine versus U50,488H (kappa agonist), deltorphin II (delta2 agonist), and (D-Pen2, D-Pen5)-enkephalin (DPDPE, delta1 agonist), administered via Alzet® minipumps, on oral Salmonella infection and on gastrointestinal transit. The results show that all morphine-pelleted mice (26 / 26) had a marked increase in Salmonella burden in the Peyer's Patches, mesenteric lymph nodes and spleen. In comparison, only 8 / 20 mice receiving morphine by minipump at doses ranging from 1 to 25 mg/kg/day had any culturable Salmonella in their organs and the number of bacteria was very low. The level of Salmonella colonization correlated with blood morphine levels and gut transit measured using an intragastric charcoal meal. Morphine pellets inhibited gut transit by 38%, while mice receiving morphine by minipump at doses of 1 to 25 mg/kg/day showed only a dosedependent 7% to 17% inhibition. Mice receiving various doses of U50,488H or DPDPE had no culturable Salmonella in the three sites. Deltorphin II given by minipump resulted in a moderate level of Salmonella in the spleen. Deltorphin II and U50,488H (0.1 to 10 mg/kg/day) did not suppress gut transit. The present studies indicate that a predominantly mu opioid receptor agonist, morphine, given by slow-release pellet, potentiated Salmonella infection and inhibited gastrointestinal transit. In contrast, morphine in pumps slightly inhibited intestinal transit, but did not sensitize to Salmonella infection. A delta1 opioid receptor agonist did not sensitize to infection, and a delta2 and a kappa opioid receptor agonist had minimal effects on either parameter. © 2006 Elsevier B.V. All rights reserved. Keywords: Morphine; Opioid agonist; Infection; Intestinal transit; Osmotic minipump

1. Introduction There is continuing interest in elucidating the effects of drugs of abuse on the immune system and on resistance to infection. It ⁎ Corresponding author. Department of Microbiology and Immunology, Temple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140, USA. Tel.: +1 215 707 3585; fax: +1 215 707 7920. E-mail address: [email protected] (T.K. Eisenstein). 1 These two investigators contributed equally to the work. 2 Current address: Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine. 0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2006.01.048

is firmly established in a large literature that morphine and mu receptor agonists are immunomodulatory as measured by a variety of parameters of immune function (McCarthy et al., 2001; Wei et al., 2003). A few examples of the types of parameters measured include inhibition of antibody formation (Bussiere et al., 1992), inhibition of natural killer cell activity (Weber and Pert, 1989; Yeager et al., 1995), inhibition of responses to T cell mitogens (Bryant et al., 1987; Bayer et al., 1990; Lysle et al., 1993; Roy et al., 1998a), and alteration of macrophage activities including phagocytosis (Rojavin et al., 1993), production of reactive oxygen intermediates (Peterson et

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al., 1987), and production of cytokines (Houghtling and Bayer, 2002) and chemokines (Wetzel et al., 2000). There are also papers demonstrating effects of exogenous opioids selective for kappa and delta receptors on the immune system (Portoghese et al., 1988; Taub et al., 1991; Arakawa et al., 1993; Radulovic et al., 1995; Shahabi and Sharp, 1995; House et al., 1996; Gavériaux-Ruff et al., 2001; Gómez-Flores et al., 2001; Rahim et al., 2001). An association between heroin addiction and increases in a variety of infections has been noted clinically (Hussey and Katz, 1950; Louria et al., 1967; Haverkos and Lange, 1990). Epidemiology studies on patients infected with HIV in the U.S. show that one-third are intravenous drug abusers (CDC, 2000). A limited number of laboratory experiments have demonstrated that morphine sensitizes mice to infection (Friedman et al., 2003), for example with Toxoplasma gondii (Chao et al., 1990) or Candida albicans (Tubaro et al., 1983). Our laboratory observed that mice given morphine by slowrelease, subcutaneously implanted pellets were greatly sensitized to oral infection with Salmonella typhimurium (MacFarlane et al., 2000). Further, we and others have found that morphine pellets induce sepsis in mice (Hilburger et al., 1997; Roy et al., 1999; Ocasio et al., 2004). Slow-release pellets were used in our studies to administer the drug continuously to avoid subjecting animals to the confounding variable of episodes of withdrawal. Use of these pellets is a standard pharmacological protocol (Cheney and Goldstein, 1971; Cerletti et al., 1976; Bryant et al., 1988). We have also examined the effect of administering opioids via osmotic pumps on immune responses, in order to be able to carry out dose–response studies (Rahim et al., 2001). We found that morphine, the kappa opioid receptor selective agonist U50,488H, and the delta2 opioid receptor agonist, deltrophin II, were all immunosuppressive, with U-shaped dose–response curves, when administered by minipumps implanted subcutaneously. The delta1 opioid receptor agonist, DPDPE, did not affect the immune parameter measured. Receptor selective antagonists (CTAP, nor-BNI, and naltriben), administered by implanting a second pump, blocked the immunosuppressive effects of morphine, U50,488H and deltorphin, respectively (Rahim et al., 2001). The current studies were undertaken to compare the sensitivity of mice to infection with orally administered Salmonella when they were treated with morphine, U50,488H, or deltorphin II given by minipump in the dose ranges shown in the previous study to be immunosuppressive. Activity was compared to that for morphine given by slowrelease pellets. These studies confirmed that implantation of a morphine pellet potently exacerbated oral Salmonella infection, but morphine given by pump, at doses which were immunosuppressive, had a substantially lesser effect. Kappa and delta1 opioid receptor agonists had no enhancing effect on Salmonella infection. A delta2 opioid receptor agonist resulted in moderate numbers of organisms in the spleen, but not in gut-associated lymphoid tissue. To examine the mechanism accounting for the differential effect of these opioid receptor agonists on altered susceptibility to oral Salmonella, their

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capacity to affect gut transit was tested. It was found that potentiation of Salmonella infection correlated with the extent of gut transit, as measured by movement of a charcoal meal through the intestinal tract. 2. Materials and methods 2.1. Mice Six week-old C3HeB/FeJ female mice were purchased from Jackson Laboratories (Bar Harbor, Maine) and housed in sterilized cages with mouse chow and water provided ad libitum. All mice were acclimated for a minimum of 1 week prior to being used in experiments. Experiments were conducted under protocols approved by the Animal Use Committee of Temple University. 2.2. Compounds Mu, kappa, and delta opioid receptor agonists were used. All opioids were provided by the National Institute on Drug Abuse (NIDA, Rockville, MD). These included 75 mg slow-release morphine pellets, 30 mg naltrexone pellets, and placebo pellets. In addition NIDA supplied morphine sulfate, trans-(±)-3, 4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide methane sulfonate (U50,488H), (D-Pen2, D-Pen5)enkephalin (DPDPE), and [D-Ala2]-deltorphin II. All compounds were dissolved in 0.9% pyrogen-free saline (Abbott Laboratories, North Chicago, IL) for delivery by minipumps. 2.3. Experimental design Mice were anesthetized with isoflurane and an area of the back was shaved. A 1 cm incision was made in the skin and mice were implanted s.c with a 75 mg slow-release morphine pellet, a placebo pellet, or a morphine plus a naltrexone pellet. Alternatively, mice were implanted with an Alzet® osmotic minipump (Model 1003D, Alza, Palo Alto, CA) filled with the desired opioid. Before implantation, pumps were filled with the test agent and then placed in a petri dish with sterile 0.9% saline at 37 °C for at least 4 h prior to implantation in order to prime the pumps. Controls received a saline minipump. Animals received the Salmonella orally while they were under the anesthesia, immediately following the pump or pellet implantation. To determine the number of Salmonella in extraintestinal sites, mice were sacrificed 48 h after inoculation and the desired tissues cultured. For analysis of morphine levels in the serum, blood samples were collected from uninfected individual animals in each group at the time of sacrifice. To determine the extent of gastrointestinal transit, opioid-treated (uninfected) animals were given a charcoal meal 48 h after drug administration. The choice of doses of opioids administered by minipump was based on a previous study from our laboratory in which the immunosuppressive capacity of the drugs was evaluated (Rahim et al., 2001). Morphine, U50,488H and deltorphin II all gave U-shaped immunosuppression dose–response curves,

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with optimal immunosuppression observed at 1 mg/kg/day for morphine, 2 mg/kg/day for U50,488H, and 0.5 mg/kg/day for deltorphin II. DPDPE was not immunosuppressive. 2.4. Salmonella infection and determination of bacterial burden S. typhimurium, strain W118-2 (virulent), was used (Killar and Eisenstein, 1985). The oral LD50 under 4 h fasting conditions was 2 × 104 colony-forming units (cfu). The inoculum was prepared as described previously (Killar and Eisenstein, 1985). Salmonella were suspended in 0.2 ml of pyrogen-free 0.9% saline. Food, but not water, was withheld for 4 h before inoculation. Mice were then anesthetized with isoflurane and orally inoculated with a blunt-end feeding needle with W118-2 in a volume of 0.2 ml. Following inoculation, mice were implanted s.c. for 48 h with a pump dispensing the appropriate drug. Forty-eight hours following infection, animals from each group (usually 5) were sacrificed and three Peyer's Patches, a mesenteric lymph node, and the spleen of each animal were aseptically removed. Tissues from each mouse were individually homogenized separately using a Tekmar Tissumizer®, model SDT (Tekmar, Cincinnati). Peyer's Patches, mesenteric lymph nodes, and spleens were each suspended in 3 ml of sterile water. A 0.1 ml sample of homogenate or of an appropriate dilution was plated on Levine's eosin–methylene blue agar plates (Difco, Detroit). Plates were incubated at 37 °C overnight, and the number of Salmonella colonies counted. Select individual colonies were verified to be Salmonella by use of a semiautomated microbial identification system (Biomérieux Vitek, Hazelwood, MO) at the Clinical Microbiology Laboratory at Temple University. Data are expressed as colony forming units (cfu) per organ. 2.5. Determination of gastrointestinal transit time At 48 h post drug treatment (without infection), 8 mice per group were each given charcoal intragastrically through an 18 gauge feeding needle in a dosing volume of 0.2 ml per 10 g body weight. All animals were sacrificed 20 min after charcoal administration and the small intestine was excised from the pylorus to the ileocecal junction. The intestine was placed on a ruled template and distance travelled by the charcoal and total length of the intestine were measured. Charcoal transit was calculated as a percent of the total intestinal length. The 48 h time point was chosen because at that time we observed significant immunosuppression in the previous study (Rahim et al., 2001). 2.6. Determination of blood morphine levels Sera from 4 mice per group were collected 48 h after initiation of drug administration. Samples were pooled and drug levels were determined by GC–MS (gas chromatography–mass spectrophotometry) by National Medical Services (Willow Grove, PA).

2.7. Statistical analyses Differences in bacterial burden in various organs were determined using the Wilcoxon rank order test. Intestinal transit data were analyzed by a mixed model analysis of variance followed by pairwise comparisons using the Dunn–Bonferroni method adjusting for multiple comparisons and random experimental effects. Differences were considered significant for P values ≤ 0.05. 3. Results 3.1. Comparison of effect of morphine pellets and morphine administered by osmotic minipump on sensitization of mice to oral Salmonella infection To examine the effect of morphine on bacterial burden in Salmonella-infected animals, mice were orally inoculated with Salmonella at the time they were implanted with either a morphine pellet or an osmotic minipump infusing morphine sulfate at various doses. At 48 h following drug treatment, mice receiving a morphine pellet had a significant number of Salmonella in Peyer's Patches, mesenteric lymph nodes and spleen, with high levels in Peyer's Patches (median = 1 × 106 cfu) (Fig. 1). In contrast, mice receiving morphine by minipump had very low levels of colonization, with only 8 of the 20 mice receiving any dose of morphine by this delivery method demonstrating culturable Salmonella. The number of organisms per site was below 10 for the mesenteric lymph nodes and spleen, and only 2 animals were colonized in Peyer's Patches, with less than 100 organisms each. 3.2. Administration of a kappa opioid receptor agonist by osmotic minipump does not sensitize mice to oral Salmonella infection Groups of mice were infused with various doses of the kappa opioid receptor selective agonist, U50,488H, to determine if a kappa compound has the capacity to potentiate Salmonella infection. For comparison and as a positive control, another group of mice was implanted with morphine pellets. U50,488H administered by minipump did not increase bacterial burden in Salmonella-infected mice (Fig. 2). Only one mouse of the 15 animals given U50,488H had Salmonella at any of the sites cultured. In comparison, Salmonella burdens in Peyer's Patches, mesenteric lymph nodes and spleens of morphinepelleted were similar to those in Fig. 1, showing that the Salmonella given to the U50,488H-treated animals were of comparable pathogenicity to those used in the morphine experiments. 3.3. Effect of administration of delta opioid receptor agonists by osmotic minipump on oral Salmonella infection To test the effect of delta opioid receptor agonists on oral Salmonella infection, mice were implanted with osmotic

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pellet. None of the mice receiving DPDPE had any increase in Salmonella in the sites which were cultured (Fig. 4). 3.4. Morphine pellets and morphine in osmotic minipumps, but not kappa or delta opioid receptor agonists in pumps, inhibit intestinal transit in mice Studies were done to see if there was a correlation between potentiation of oral Salmonella infection and decrease in intestinal transit. In the first experiments, morphine pellets and morphine minipumps were compared. Animals were fed a

Fig. 1. Effect of morphine administered by pellet or osmotic minipump on Salmonella colonization after oral inoculation. Mice were orally inoculated with Salmonella (2.1 × 104 CFU/mouse) and implanted with either a morphine pellet or an osmotic minipump infusing morphine sulfate at various doses. After 48 h the PP, MLN and spleen were isolated and homogenized. Bacterial colonization was assayed in EMB agar plates. Dashes are median values for each group. Numbers in parentheses are the number of animals with undetectable levels of Salmonella in that tissue.

minipumps infusing various doses of deltorphin II (delta2) or DPDPE (delta1) and then orally inoculated with Salmonella. Mice receiving morphine pellets were again used as a positive control. In animals given deltorphin II, all 5 had Salmonella culturable from the spleen, with the bacterial burden being about a log lower than mice receiving the morphine pellet (Fig. 3). In contrast, in Peyer's Patches and mesenteric lymph nodes of mice receiving deltorphin, only 2 / 40 were colonized, and the number of Salmonella was a log lower than that observed for animals given a morphine

Fig. 2. Effect of the kappa opioid receptor agonist U50,488H administered by osmotic minipump on oral Salmonella infection. Mice were orally inoculated with Salmonella (1.9 × 104 CFU/mouse) and implanted with either a morphine pellet as positive control or an osmotic minipump infusing U50,488H at various doses. After 48 h the PP, MLN and spleen were isolated and homogenized. Bacterial colonization was assayed in EMB agar plates. Dashes are median values for each group.

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deltorphin II showed no inhibition of gastrointestinal transit when compared to mice receiving a saline pump (Table 1). 3.5. Serum levels of morphine in drug-treated mice Differences between the effects of morphine administered by pellet and morphine administered by minipump could be due to the pharmacokinetics of the drug. Serum levels of morphine in pelleted mice were compared to levels in serum of mice

Fig. 3. Effect of the delta2 opioid receptor agonist, deltorphin II, administered by osmotic minipump on oral Salmonella infection. Mice were orally inoculated with Salmonella (1.9 × 104 CFU/mouse) and implanted with an osmotic minipump infusing deltorphin II at various doses. As a control, mice were implanted with a morphine pellet. After 48 h the PP, MLN and spleen were isolated and homogenized. Bacterial colonization was assayed in EMB agar plates. Dashes are median values for each group.

charcoal meal 20 min before being sacrificed (48 h post pellet or pump implantation). Charcoal transit along the gastrointestinal tract was significantly decreased (− 38%) in morphine-pelleted mice, in comparison with mice receiving a placebo pellet (Fig. 5). Similarly, mice receiving morphine by minipump at the 25 mg/kg/day dose showed a statistically significant inhibition of gut transit compared to mice implanted with a saline pump (~ 17%). The effect of the morphine pellet was opioid receptor mediated as naltrexone blocked the inhibition of transit (data not shown). Mice implanted with minipumps infusing U50,488H or

Fig. 4. Effect of the delta1 opioid receptor agonist, DPDPE, administered by osmotic minipump on oral Salmonella infection. Mice were orally inoculated with Salmonella (1.9 × 104 CFU/mouse) and implanted with an osmotic minipump infusing DPDPE (delta1) at various doses. As a control, mice were implanted with a morphine pellet. After 48 h the PP, MLN and spleen were isolated and homogenized. Bacterial colonization was assayed in EMB agar plates. Dashes are median values for each group.

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Table 2 Concentration of morphine in mouse serum Drug Placebo pellet Morphine pellet Saline pump Morphine pump 1 mg/kg/day Morphine pump 10 mg/kg/day Morphine pump 25 mg/kg/day a b c

Fig. 5. Effects of morphine administered by pellet or osmotic minipump on gastrointestinal transit in mice. Mice were implanted with either a morphine pellet or an osmotic minipump infusing morphine sulfate for 48 h. Animals were then orally fed charcoal 20 min before being sacrificed. The results are expressed as the percent of the total small intestinal length travelled by the charcoal. The data represent the mean + standard error of two separate experiments. **P b 0.05 vs. placebo pellet. *P = 0.05 vs. saline pump.

receiving morphine by minipump. Serum levels of mice receiving morphine pellets were approximately 2.7 μg/ml (free) and 22 (total) μg/ml (Table 2). In contrast, serum levels in mice receiving a morphine pump were an order of magnitude lower (free or total). 4. Discussion S. typhimurium is a natural pathogen of mice, and is used as a murine model of typhoid fever (Eisenstein and Sultzer, 1983). The natural route of infection is via the gastrointestinal tract where it invades Peyer's Patches and travels via the mesenteric lymph nodes into the lymphatics, thoracic duct, and the venous circulation (Carter and Collins, 1974). Organisms are trapped in the liver and spleen where they take up residence and multiply intracellularly in macrophages (Mackaness et al., 1966). In humans this pathogen causes gastroenteritis and is usually confined to invasion of intestinal epithelial cells, but does not routinely penetrate to become systemic (Pegues et al., 1995). The present results confirm the previous study from our laboratory showing that 75 mg slow-release morphine pellet Table 1 Effect of kappa and delta opioid agonists given in osmotic minipumps on gastrointestinal transit in mice Dose (mg/kg/day)

Saline 0.1 0.5 2.0 2.5 10.0

Percent of gut length travelled by charcoal ± S.E.M.a U50,488H

Deltorphin II

49.7 ± 5.3 55.5 ± 6.8 53.8 ± 8.2 48.4 ± 5.3 – 49.9 ± 3.6

49.5 ± 4.6 53.2 ± 5.2 53.6 ± 6.8 – 53.2 ± 5.7 51.9 ± 5.7

a Data represent mean values from a total of 7–8 mice tested per group in 2 experiments.

Free μg/mla ND 2.7 ND ND 0.2 0.1

c

Total μg/mlb ND 22 ND ND 0.5 0.7

Unconjugated morphine (glucuronides removed). Unconjugated and conjugated morphine. ND = none detected.

implantation is a potent enhancer of oral Salmonella infection (MacFarlane et al., 2000). If the results of all experiments presented in the current paper are pooled, animals given morphine pellets had a median log10 bacterial burden of 6.1 in the Peyer's Patches, 4.1 in the mesenteric lymph nodes, and 2.5 in the spleen. In contrast, mice receiving a placebo pellet (MacFarlane et al., 2000) or a saline pump had no culturable Salmonella in these tissues, showing that enhanced movement of these bacteria out of the gastrointestinal tract is not due to the brief anesthesia and surgery needed to implant the opioid delivery system. The lack of effect of morphine administered by pump was unexpected, as this mode of drug delivery resulted in immunosuppression over a dose range of 0.3 to 2 mg/kg/day 48 h after pump implantation (Rahim et al., 2001). Analysis of blood levels of morphine showed that no drug was detected 48 h post pump implantation in mice receiving a dose of 1 mg/kg/day, a time and dose at which the immune response is suppressed. At doses of morphine of 10 or 25 mg/kg/day administered by pump, the pellet still resulted in 10 times the blood level of morphine compared to the pump. These differences in blood levels may account for lower numbers of culturable bacteria obtained from mice receiving a morphine pump in comparison to mice receiving a morphine pellet. Comparison of the effects of morphine with those of exogenous opioid receptor agonists selective for kappa and delta1 receptors demonstrated that neither U50,488H nor DPDPE potentiated Salmonella infection. Deltorphin II, the delta2 agonist, resulted in colonization of the spleen in all mice which received pumps dispensing the drug. The median numbers of organisms were approximately a log lower in mice given deltorphin pumps compared with mice given morphine pellets, and significantly greater than that of mice given morphine pumps. It is generally accepted that Salmonella reach the spleen by first trafficking through the Peyer's Patches and mesenteric lymph nodes as described above. It is not clear why the mice which received deltrophin had Salmonella in the spleen, but only two animals were colonized in the Peyer's Patches or mesenteric lymph nodes. It is possible that the delta opioid receptor agonist opens another pathway by which Salmonella can move to the periphery from the gut, either via movement between epithelial cells or by penetration of epithelial cells and escape via their basolateral surface. Recent evidence supports a model in which dendritic cells can extend pseudopods between tight junctions of epithelial cells and ingest bacteria in the gut lumen (Rescigno et al., 2001; Neiss et al., 2005).

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Morphine and other opioids with primary effects at the mu opioid receptor are known to slow intestinal propulsion (Kromer, 1988). Constipation is a major side effect of these compounds when used for relief of pain (Mancini and Bruera, 1998). Therefore, a correlation was sought between gut stasis and potentiation of oral Salmonella infection. Gastrointestinal transit was measured using the charcoal meal test. The results show that only the morphine pellet (compared to the placebo pellet), and the 25 mg/kg/day morphine pump (compared to the saline pump) significantly inhibited gut transit of the charcoal meal. Neither morphine at other doses given by pump, nor U50,488H or deltrophin II at any of the doses used significantly inhibited gut transit. Thus, alteration in transit time does not explain the effect of deltorphin II on spleen colonization by Salmonella at the 0.1 mg dose, perhaps lending support to a different mechanism of action by which deltorphin affects Salmonella infection, as compared with morphine. Delta opioid receptors have been demonstrated in the mouse small intestine (De Luca and Coupar, 1996), yet an effect of the delta opioid receptor agonist was not observed on gut transit. While kappa receptors have been demonstrated in the rodent gastrointestinal tract, they are not generally associated with marked inhibition of gut transit (Burks et al., 1988). Our results are consonant with a report showing that morphine did not alter gut transit in mu opioid receptor knockout mice, and that kappa and delta opioid receptor agonists had no effect in the absence of the mu receptor (Roy et al., 1998b). There may, however, be species variation in control of gastrointestinal functions by opioid receptors, as delta receptors predominate in the porcine enteric nervous system (Townsend and Brown, 2002), and effects of delta ligands on circular smooth muscle contraction have been reported in the pig (Brown et al., 1998). The current studies raise a number of questions. First is the issue of why the gastrointestinal response to morphine in the pumps is minimal at doses that are highly immunosuppressive. Second is the mechanism by which deltorphin enhances movement of Salmonella out of the gastrointestinal tract without colonizing the Peyer's Patches or the mesenteric lymph nodes. Future experiments will be needed to elucidate the mechanisms underlying these observations. Acknowledgements This work was supported by National Institute on Drug Abuse Grants DA06650, DA14223, and DA13429. References Arakawa, K., Akami, T., Okamoto, M., Akioka, K., Nakai, I., Oka, T., Nagase, H., 1993. Immunosuppression by delta opioid receptor antagonist. Transplant. Proc. 36, 738–740. Bayer, B.M., Daussin, S., Hernandez, M., Irvin, L., 1990. Morphine inhibition of lymphocyte activity is mediated by an opioid dependent mechanism. Neuropharmacology 29, 369–374. Brown, D.R., Poonyachoti, S., Osinski, M.A., Kowalski, T.R., Pampusch, M.S., Elde, R.P., Murtaugh, M.P., 1998. Delta-opioid receptor mRNA expression and immunohistochemical localization in porcine ileum. Dig. Dis. Sci. 43, 1402–1410.

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