Effects of in vivo morphine treatment on antibody responses in C57BL/6 bgJ/bgJ (beige) mice

C57BL/6J bg^J/bg^J (beige) mice are less sensitive than other strains to the analgesic effects of morphine, although they have normal numbers of μ receptors. In the present study, beige mice and their normal littermates (beige⁺) were treated in vivo with morphine or the opioid antagonist, naltrexone and their primary in vitro antibody responses were assessed. Morphine treatment caused splenic atrophy and suppressed the primary in vitro antibody response in beige and beige⁺ mice. However, these effects were not blocked by naltrexone co-treatment. In these mouse strains, naltrexone decreased spleen size and antibody responses by itself, which may mask its ability to antagonize morphine. In beige mice, placebo pellet implantation suppressed the primary in vitro antibody response. Morphine did not cause a further suppression of the antibody response in beige mice compared to placebo. Because of this anomalous response to placebo treatment, the immunosuppressive effects of morphine on the antibody response/10⁷ cells can not be attributed to a specific drug effect in this strain. However, when antibody responses were expressed on a per spleen basis, the overall capacity to respond to antigenic challenge was suppressed by morphine treatment.     

Regulation of mucosal B cell immunoglobulin secretion by intestinal epithelial cell-derived cytokines

Intestinal epithelial cells (IEC) secrete a variety of cytokines and, because of their close proximity to B cells in the lamina propria, may affect local antibody production via these cytokines. However, studies have not yet addressed which and to what extent these IEC-derived cytokines may affect B cell antibody production. In this study, rat mesenteric lymph node B cells were cultured with culture supernatants from the rat IEC-6 intestinal epithelial cell line to determine their effect on immunoglobulin (Ig) secretion. Unstimulated IEC-6 cells were found to secrete sufficient levels of IL-6 to enhance IgA, IgG and IgM secretion by unstimulated B cells. However, culture of lipopolysaccharide (LPS)-stimulated B cells with the unstimulated IEC-6 supernatant resulted in an enhancement of IgA secretion while IgM secretion was significantly suppressed. Depletion of the IEC-6 supernatant using cytokine specific antibodies revealed that both interleukin 6 (IL-6) and transforming growth factor beta (TGF-beta) were responsible for the enhanced IgA secretion while TGF-beta suppressed IgM secretion. More importantly, culture supernatants from LPS stimulated IEC-6 cells contained enhanced levels of IL-6 which enhanced both IgG and IgA production and partially overcame the suppressive effect of TGF-beta on IgM secretion. These results suggest that intestinal epithelial cells may secrete IL-6 and TGF-beta to regulate local B cell antibody secretion and their effect may be highly dependent upon the activation state of the epithelial cells.     

Role of p38 mitogen-activated protein kinase phosphorylation and Fas-Fas ligand interaction in morphine-induced macrophage apoptosis

In this study, we evaluated the molecular mechanisms involved in morphine-induced macrophage apoptosis. Both morphine and TGF-beta promoted P38 mitogen-activated protein kinase (MAPK) phosphorylation, and this phosphorylation was inhibited by SB 202190 as well as by SB 203580. Anti-TGF-beta Ab as well as naltrexone (an opiate receptor antagonist) inhibited morphine-induced macrophage P38 MAPK phosphorylation. Anti-TGF-beta Ab also attenuated morphine-induced p53 as well as inducible NO synthase expression; in contrast, N(G)-nitro-L-arginine methyl ester, an inhibitor of NO synthase, inhibited morphine-induced P38 MAPK phosphorylation and Bax expression. Morphine also enhanced the expression of both Fas and Fas ligand (FasL), whereas anti-FasL Ab prevented morphine-induced macrophage apoptosis. Moreover, naltrexone inhibited morphine-induced FasL expression. In addition, macrophages either deficient in FasL or lacking p53 showed resistance to the effect of morphine. Inhibitors of both caspase-8 and caspase-9 partially prevented the apoptotic effect of morphine on macrophages. In addition, caspase-3 inhibitor prevented morphine-induced macrophage apoptosis. These findings suggest that morphine-induced macrophage apoptosis proceeds through opiate receptors via P38 MAPK phosphorylation. Both TGF-beta and inducible NO synthase play an important role in morphine-induced downstream signaling, which seems to activate proteins involved in both extrinsic (Fas and FasL) and intrinsic (p53 and Bax) cell death pathways.     

Blockade of morphine supersensitivity by an antisense oligodeoxynucleotide targeting the delta opioid receptor (DOR-1)

An antisense oligodeoxynucleotide (ODN) targeting 20 bases of the coding sequence of the cloned delta opioid receptor (DOR-1), a mismatched ODN (different from the antisense ODN at 4 bases) or saline was administered to 3 groups of CD-1 mice implanted with naltrexone pellets (7.5 mg) for 7 days. Morphine supersensitivity (i.e., increased potency as defined by decreased morphine ED₅₀ values) was observed 24 h after pellet removal (day 8) in mice treated with saline or mismatch ODN, but not in antisense ODN treated mice. Antisense ODN alone had no effect on basal nociceptive thresholds or morphine analgesia but reduced the analgesic potency of the delta₂ opioid agonist [D-Ala²]deltorphin II. These data suggest that the delta₂ opioid receptor system participates in the adaptive changes contributing to increased morphine potency following chronic naltrexone treatment.     

Reinstatement of precipitated narcotic withdrawal hypothermia in the rat

Withdrawal hypothermia can be induced in rats by injection of naltrexone 72 h after subcutaneous implantation of a morphine pellet. At 45 days after implantation the same dose of naltrexone is without effect on body temperature and the animals are normally sensitive to the hypothermic effect of acute morphine administration. This acute administration of morphine re-sensitizes the animals to naltrexone so that administration of the antagonist again causes withdrawal hypothermia. These results are consistent with the view that narcotic dependence can be reinstated in previously dependent, but not naive, animals by acute administration of the narcotic.     

Maternally Administered Sustained-Release Naltrexone in Rats Affects Offspring Neurochemistry and Behaviour in Adulthood

Naltrexone is not recommended during pregnancy. However, sustained-release naltrexone implant use in humans has resulted in cases of inadvertent foetal exposure. Here, we used clinically relevant dosing to examine the effects of maternally administered sustained-release naltrexone on the rat brain by examining offspring at birth and in adulthood. Maternal treatment (naltrexone or placebo implant) started before conception and ceased during gestation, birth or weaning. Morphometry was assessed in offspring at birth and adulthood. Adult offspring were evaluated for differences in locomotor behaviour (basal and morphine-induced, 10 mg/kg, s.c.) and opioid neurochemistry, propensity to self-administer morphine and cue-induced drug-seeking after abstinence. Blood analysis confirmed offspring exposure to naltrexone during gestation, birth and weaning. Naltrexone exposure increased litter size and reduced offspring birth-weight but did not alter brain morphometry. Compared to placebo, basal motor activity of naltrexone-exposed adult offspring was lower, yet they showed enhanced development of psychomotor sensitization to morphine. Developmental naltrexone exposure was associated with resistance to morphine-induced down-regulation of striatal preproenkephalin mRNA expression in adulthood. Adult offspring also exhibited greater operant responding for morphine and, in addition, cue-induced drug-seeking was enhanced. Collectively, these data show pronounced effects of developmental naltrexone exposure, some of which persist into adulthood, highlighting the need for follow up of humans that were exposed to naltrexone in utero.     

Inhibition of calcium mobilization is an early event in opiate-induced immunosuppression

Morphine administered as a subcutaneous implant inhibits the initial increase in cytoplasmic free-calcium [Ca2+]i induced by mitogens in mouse splenocytes. This effect was not reproduced by incubation of splenocytes with morphine (10(-8)-10(-4) M). Analysis of splenocyte subpopulations demonstrates that this effect was manifest in both B and T cells. However, within T cell subpopulations, CD4+ but not CD8+ cells were affected. Adrenalectomy abolished this effect of morphine in CD4+ T but not CD4-, CD8- spleen cells (most likely Thy 1.2- B cells). Moreover, simultaneous administration of the opiate antagonist naltrexone blocked the effect of morphine in CD4-, CD8- spleen cells, but not in CD4+ T cells. These data indicate that the effects of morphine on mitogen-stimulated increase in [Ca2+]i may be mediated through distinct glucocorticoid-dependent and -independent mechanisms. The morphine-induced inhibition of an increase in [Ca2+]i in immune cells reported here may be an early event mediating opiate-induced immunosuppression.     

Effects of morphine and beta-endorphin on basal and elevated plasma levels of alpha-MSH and vasopressin.

Morphine induced an increase of plasma α-MSH levels and a decrease of AVP levels after peripheral or intracerebroventricular administration. This increase of α-MSH levels and decrease of AVP levels after morphine treatment was observed in non-stimulated animals as well as in rats in which the hormone levels were elevated by water deprivation or by administration of hypertonic saline. These latter effects of morphine on plasma levels of α-MSH and AVP could be blocked by simultaneous administration of naltrexone.β-Endorphin also increased plasma α-MSH levels and lowered plasma AVP levels. From these effects only the increase of the plasma α-MSH level and not the decrease of plasma AVP could be blocked by naltrexone. Moreover PLG treatment was ineffective with respect to the endorphin-induced decrease in plasma AVP, but it partly blocked the increase of plasma α-MSH when this tripeptide was given in combination with β-endorphin.     

Endothelin receptor antagonists restore morphine analgesia in morphine tolerant rats

Several neurotransmitter mechanisms have been proposed to play a role in the development of morphine tolerance. The present study provides evidence for the first time that endothelin (ET) antagonists can restore morphine analgesia in morphine tolerant rats. Tolerance to morphine was induced by subcutaneous implantation of six morphine pellets during a 7-day period. The degree of tolerance to morphine was measured by determining analgesic response (tail-flick latency) and hyperthermic response to morphine sulfate (8 mg/kg, subcutaneously (s.c.)) in placebo and morphine pellet implanted rats. The maximal tail-flick latency in morphine pellet-vehicle treated rats (7.54 s) was significantly lower (P<0.05) when compared to placebo pellet-vehicle treated rats (10s), indicating that tolerance developed to the analgesic effect of morphine. In separate sets of experiments, ET antagonists, BQ123 (10 microg, intracerebroventricularly (i.c.v.)) and BMS182874 (50 microg, i.c.v.) were administered in placebo and morphine tolerant rats. BQ123 was injected twice daily for 7 days and once on day 8. BMS182874 was administered only on day 8. Morphine (8 mg/kg, s.c.) was administered 30min after BQ123 or BMS182874 administration. It was found that both BQ123 and BMS182874 potentiated morphine analgesia in placebo and morphine tolerant rats. BQ123 potentiated tail-flick latency by 30.0% in placebo tolerant rats and 94.5% in morphine tolerant rats compared to respective controls. BMS182874 potentiated tail-flick latency by 30.2% in placebo tolerant rats and 66.7% in morphine tolerant rats. Morphine-induced hyperthermic effect was also potentiated by BQ123 and BMS182874. The duration of analgesic action was also prolonged by BQ123 and BMS182874. The effect of BMS182874 was less as compared to BQ123. BQ123 and BMS182874 are selective ET(A) receptor antagonists. Therefore, it is concluded that ET(A) receptor antagonists restore morphine analgesia in morphine tolerant rats.     

Development of a mucosal complex vaccine against oral Salmonella infection in mice

We examined the immunogenicity of a Salmonella enterica complex vaccine (CV), consisting of flagellin and polysome purified from serotype Typhimurium LT2. CV plus cholera toxin (CT), in three oral doses given at 7-day intervals, conferred complete protection on C57BL/6 mice against lethal oral infection with a wild-type strain. It elicited mucosal IgA > IgG2a > IgG1 and systemic IgG2a > IgG1 > IgA antibodies to flagellin and polysome, and delayed footpad response (DFR) to both antigens. In Peyer's patches (PPs) and lamina propria (LP), IgA was produced under a Th1-dominant environment; CD4+T cells from produced interleukin (IL)-2, interferon (IFN)-gamma, and IL-10 by stimulation with salmonella extract. On the same protocol, flagellin plus CT induced flagellin-specific mucosal and systemic IgA and IgG1 antibodies, CD4+T cells producing IL-10 and IFN-gamma in PPs and LP, and only minimal levels of flagellin-specific DFR. Polysome plus CT induced polysome-specific mucosal and systemic IgG2a in addition to IgG1 and IgA antibodies, CD4+T cells producing IFN-gamma and IL-2 in PPs and LP, and polysome-specific DFR. These two vaccines, however, conferred at most 50-60% survival rates. Our results suggest that polysomes in CV provide effective adjuvant activity for the induction of both mucosal and systemic Th1-biased responses toward flagellin.     

Transforming growth factor-beta 1 (TGF-beta 1) is a bifunctional immune regulator for mucosal IgA responses

Porcine transforming growth factor-beta 1 (TGF-beta 1) exerts a unique bifunctional immunoregulatory effect on IgA responses in murine mesenteric and Peyer's patches lymphocyte cultures. TGF-beta 1 is a potent costimulator of LPS-induced IgA responses. The enhancement was observed at TGF-beta 1 at 0.1 to 10 ng/ml in both early and late phases of IgA responses from Day 5 to Day 14 in cultures. On the contrary, TGF-beta 1 exerts a profound immunosuppressive effect on IL-5-induced IgA synthesis. TGF-beta 1 is a natural cytokine produced by intestinal epithelial cells and may account for the polyclonal production and secretion of IgA at the mucosal surface and ensure the integrity of the primary host defense by mucosa-associated lymphoid tissues (MALT).     

Effects of acute and chronic morphine and narcotic antagonists on brain energy metabolism

Morphine sulphate (4 mg/kg to 32 mg/kg) produced a dose-dependent decrease in brain malate as antinociception increased. Decreased brain malate persisted 72 hours after implantation of morphine pellets by which time mice had become tolerant to antinociception. This finding suggests that malate decrease, unlike changes of other metabolites in other studies, might not be simply a result of general metabolic changes. Malate change as well as antinociception was prevented by prior injection of naloxone (3.0 mg/kg) or naltrexone (0.6 mg/kg) in acute experiments. Malate decrease in pelleted mice was no longer present if withdrawal was produced by naloxone or naltrexone in mice implanted with morphine pellets for 72 hours. Brain P-creatine was elevated in all mice implanted with morphine pellets even after withdrawal, thus, apparently, representing a more generalized effect than malate change.     

Systematic and intestinal antibody-secreting cell responses and correlates of protective immunity to human rotavirus in a gnotobiotic pig model of disease.

Neonatal gnotobiotic pigs orally inoculated with virulent (intestinal-suspension) Wa strain human rotavirus (which mimics human natural infection) developed diarrhea, and most pigs which recovered (87% protection rate) were immune to disease upon homologous virulent virus challenge at postinoculation day (PID) 21. Pigs inoculated with cell culture-attenuated Wa rotavirus (which mimics live oral vaccines) developed subclinical infections and seroconverted but were only partially protected against challenge (33% protection rate). Isotype-specific antibody-secreting cells (ASC were enumerated at selected PID in intestinal (duodenal and ileal lamina propria and mesenteric lymph node [MLN]) and systemic (spleen and blood) lymphoid tissues by using enzyme-linked immunospot assays. At challenge (PID 21), the numbers of virus-specific immunoglobulin A (IgA) ASC, but not IgG ASC, in intestines and blood were significantly greater in virulent-Wa rotavirus-inoculated pigs than in attenuated-Wa rotavirus-inoculated pigs and were correlated (correlation coefficients: for duodenum and ileum, 0.9; for MLN, 0.8; for blood, 0.6) with the degree of protection induced. After challenge, the numbers of IgA and IgG virus-specific ASC and serum-neutralizing antibodies increased significantly in the attenuated-Wa rotavirus-inoculated pigs but not in the virulent-Wa rotavirus-inoculated pigs (except in the spleen and except for IgA ASC in the duodenum). The transient appearance of IgA ASC in the blood mirrored the IgA ASC responses in the gut, albeit at a lower level, suggesting that IgA ASC in the blood of humans could serve as an indicator for IgA ASC responses in the intestine after rotavirus infection. To our knowledge, this is the first report to study and identify intestinal IgA ASC as a correlate of protective active immunity in an animal model of human-rotavirus-induced disease.     

Morphine decreases the voltage sensitivity of the slow sodium channels]

Morphine was shown to decrease in a dose-dependent manner the effective charge transfer in tetrodotoxin-resistant (TTXr) sodium channel activation system in short-term cultured dorsal root ganglion cells. Morphine seems to interact with opioid receptors because of total block of the binding by naloxone and naltrexone. Neither activating, nor inhibiting G-protein agents exerted any effect on this process. The morphine signal was blocked by extracellular application of 2 x 10(-4) M ouabain. The findings suggest existence of sodium signalling pathway involving receptors, Na+, K(+)-ATPase and the TTXr sodium channels.     

Effects of morphine dependence,withdrawal and tolerance on prolactin and growth hormone secretion in the rat

The effects of morphine dependence and withdrawal on prolactin (Prl) and growth hormone (GH) secretion were examined in the rat. Morphine dependence, induced by morphine pellet implantation, had no effect on nonstress concentrations of plasma Prl or GH, but it potentiated the response of Prl secretion to the stress associated with blood collection + injection of saline. Naloxone-induced withdrawal had no demonstrable effect on the changes in Prl and GH secretion produced by stress. In addition, signs of tolerance to both the Prl- and GH-stimulating effects of morphine injection were observed in morphine-dependent rats.     

Sphingosine 1-phosphate regulates the egress of IgA plasmablasts from Peyer's patches for intestinal IgA responses

It is well established that Peyer's patches (PPs) are sites for the differentiation of IgA plasma cell precursors, but molecular and cellular mechanisms in their trafficking remain to be elucidated. In this study, we show that alterations in type 1 sphingosine 1-phosphate (S1P) receptor expression during B cell differentiation in the PPs control the emigration of IgA plasma cell precursors. Type 1 S1P receptor expression decreased during the differentiation of IgM(+)B220(+) B cells to IgA(+)B220(+) B cells, but recovered on IgA(+)B220(-) plasmablasts for their emigration from the PPs. Thus, IgA(+)B220(-) plasmablasts migrated in response to S1P in vitro. Additionally, IgA(+) plasmablasts selectively accumulated in lymphatic regions of PPs when S1P-mediated signaling was disrupted by FTY720 treatment. This accumulation of IgA(+) plasmablasts in the PPs led to their reduction in the intestinal lamina propria and simultaneous impairment of Ag-specific intestinal IgA production against orally administered Ag. These findings suggest that S1P regulates the retention and emigration of PP B cells and plays key roles in the induction of intestinal IgA production.     

Induction of IgA-specific suppressor activity in human T-lymphocyte cultures

Cell-free supernatants of human circulating T-lymphocyte cultures incubated with secretory IgA (S-IgA) specifically suppressed both spontaneous IgA synthesis by B lymphocytes isolated from allergic individuals and pokeweek mitogen-induced IgA secretion by peripheral blood mononuclear cells. Cell-free supernatants of T-cell cultures incubated with IgE had no effect on IgA, IgG, or IgM synthesis. Hence, it is concluded that upon incubation with S-IgA, but not with another Ig class, T lymphocytes release IgA-specific suppressor factors.     

Regional brain glucose utilization during and following chronic naltrexone administration: preliminary observations in rat brain

Experiments were conducted to determine some of the metabolic correlates of tonic opioid activity in the central nervous system under conditions previously examined for changes in monoamine levels. The glucose metabolic rates in seven brain regions were determined by autoradiographic visualization of 14C-deoxyglucose incorporation in female rats after 8 days of chronic exposure to naltrexone pellets and 10 days after pellet removal. Autoradiographs were analyzed on a region-by-region basis to correspond to areas previously dissected and analyzed for changes in monoamine content under similar experimental conditions. Chronic administration of naltrexone resulted in a significant decrease in the metabolic activity of neurons in the striatum. Other brain areas examined under this condition were not significantly affected. Ten days following pellet removal, 14C-deoxy-glucose incorporation was indistinguishable from that determined in placebo treated rats in all brain regions examined. These results indicate that tonic opioid input is an important determinant of metabolic activity in the striatum. In addition, these results indicate that conditions previously shown to alter regional content of monoamines do not necessarily produce concomitant changes in regional glucose utilization.     

Partial IgA-deficiency with increased Th2-type cytokines in TGF-beta 1 knockout mice.

Though it has been shown that TGF-beta 1 directs B cells to switch to IgA in vitro, no studies have assessed TGF-beta 1 effects on mucosal vs systemic immunity in vivo. When the B cell functions of TGF-beta 1 gene-disrupted (TGF-beta 1-/-) mice were analyzed, significantly decreased IgA levels and increased IgG and IgM levels in serum and external secretions were observed. Further, analysis of Ab forming cells (AFC) isolated from both mucosal and systemic lymphoid tissue showed elevated IgM, IgG, and IgE, with decreased IgA AFC. A lack of IgA-committed B cells was seen in TGF-beta 1-/- mice, especially in the gastrointestinal (GI) tract. Splenic T cells triggered via the TCR expressed elevated Th2-type cytokines and, consistent with this observation, a 31-fold increase in serum IgE was seen in TGF-beta 1-/- mice. Thus, uncontrolled B cell responses, which include elevated IgE levels, a lack of antiinflammatory IgA, and an excess of complement-binding IgG and IgM Abs, will promote inflammation at mucosal surfaces in TGF-beta 1-/- mice and likely contribute to pulmonary and GI tract lesions, ultimately leading to the early death of these mice.     

Enhancement of pentobarbital effect by continuous administration of morphine in the mouse

These studies demonstrated that continuous morphine treatment from implantation of a 75 mg morphine pellet for 3 days potentiated pentobarbital narcosis and enhanced pentobarbital hypothermia. In the morphine implant mice, sleeping time after two different doses of pentobarbital was greater than 2.5 × the sleeping time in placebo pellet implant animals and also greater than sleeping time in animals treated acutely with morphine prior to pentobarbital. Moreover, in the morphine implant mice both the degree and duration of pentobarbital induced hypothermia were enhanced. The above findings were due to slower rate of metabolism of pentobarbital as evidenced by inhibition of hepatic N-demethylation, and higher levels of brain and serum pentobarbital in the morphine implant mice compared to both placebo and acute morphine mice.