Withdrawal from morphine reduces cell-mediated immunity against herpes simplex virus generated by natural immunization.

In a previous study, the authors have shown that herpes simplex virus type 1 (HSV-1) glycoprotein B DNA vaccine but not live vaccine (non-virulent KOS strain) failed to induce protective immunity against acute HSV-1 challenge in morphine-dependent mice. The present study reports the effect of morphine withdrawal on protective immunity induced by live HSV-1 immunization. BALB/c mice were vaccinated with KOS strain as a live vaccine. Three weeks later, they were exposed to morphine for 14 days. On day 14, withdrawal was induced by administration of normal saline instead of morphine. One day later, immune responses against HSV-1 were assessed by measuring cytotoxicity, lymphocyte proliferation and interferon-γ production. Protection against HSV-1 was assessed by measuring the mortality rate after acute HSV-1 challenge. The results showed that withdrawal from morphine reduces protective immunity against acute HSV-1 challenge. These findings raise the possibility that withdrawal from morphine may increase the susceptibility of drug addicts to infectious diseases.

Multiplexed detection of metabolites of narcotic drugs from a single latent fingermark.

An immunoassay based technique is used for the detection of psychoactive substances in the sweat deposited within fingermarks of a narcotic drug user. Magnetic particles functionalized with antimorphine and antibenzoylecgonine antibodies were used for the detection of a metabolite of heroin (morphine) and a metabolite of cocaine (benzoylecgonine), respectively. The drug metabolites were detected individually as well as simultaneously from a single fingermark. The images of the fingermarks obtained using brightfield and fluorescence microscopy were of high evidential quality with resolution to enable identification of an individual in addition to providing information on drug usage.

Determination of propoxyphene in oral fluid.

The determination of propoxyphene in oral fluid using solid-phase extraction and gas chromatography-mass spectrometry is described for the first time. The method employs collection of oral fluid with the Quantisal device, immunoassay screening of the specimen, confirmation of the positive screened samples after extraction using cation exchange/hydrophobic solid-phase extraction columns, optimized derivative formation, and gas chromatography-mass spectrometry in electron impact mode. Validated parameters including selectivity, linearity, accuracy, intra- and interday precision, extraction efficiency, and limit of quantitation were all within acceptable limits. The method was applied to authentic specimens taken from an individual prescribed propoxyphene following surgery.

Comparison of an automated and point-of-care immunoassay to GC-MS for urine oxycodone testing in the clinical laboratory.

OxyContin, a controlled-release formulation of oxycodone, is increasingly abused. Monitoring patient compliance by urine drug testing may deter illegal diversion of OxyContin. Two urine immunoassays were evaluated with a 100 ng/mL cutoff for oxycodone. The Microgenics Corporation Oxycodone DRI on the Bayer ADVIA 1650 and a point-of-care (POC) immunoassay, Monitect Oxycodone POC from Branan Medical Corporation, were compared to gas chromatography-mass spectrometry (GC-MS) with a detection limit of 50 ng/mL free oxycodone. Between-day precision for DRI yielded coefficients of variation from 3.9% to 7.0% at 75 and 125 ng/mL. Fifty-two positive and 52 negative urines were tested. The DRI had a 100% agreement with GC-MS. Two positive specimens had free oxycodone < 50 ng/mL, but oxycodone metabolites, oxymorphone and oxycodone glucuronide > 100 ng/mL, were identified by GC-MS analysis. The POC assay had two false positives and 15 indeterminate (+/-) results. Codeine or hydrocodone was present in all but one of these samples. There was no interference with DRI from morphine, codeine, hydrocodone, hydromorphone, dihydrocodeine, or 6-monoacetyl morphine. Four-hundred and ninety urine samples were subsequently tested with DRI to estimate the oxycodone-positive rate at our hospital, and 47 (9.4%) were positive. The confirmation rate with GC-MS for free oxycodone, not including metabolites, was 93%. The Microgenics DRI offers good performance for oxycodone urine testing and is a better choice for the clinical laboratory than the POC assay. Confirmation of screened positive samples requires a method that can detect total oxycodone and oxymorphone.

Humoral Dysregulation Associated with Increased Systemic Inflammation among Injection Heroin Users.

BACKGROUND: Injection drug use is a growing major public health concern. Injection drug users (IDUs) have a higher incidence of co-morbidities including HIV, Hepatitis, and other infections. An effective humoral response is critical for optimal homeostasis and protection from infection; however, the impact of injection heroin use on humoral immunity is poorly understood. We hypothesized that IDUs have altered B cell and antibody profiles. METHODS AND FINDINGS: A comprehensive systems biology-based cross-sectional assessment of 130 peripheral blood B cell flow cytometry- and plasma- based features was performed on HIV-/Hepatitis C-, active heroin IDUs who participated in a syringe exchange program (n = 19) and healthy control subjects (n = 19). The IDU group had substantial polydrug use, with 89% reporting cocaine injection within the preceding month. IDUs exhibited a significant, 2-fold increase in total B cells compared to healthy subjects, which was associated with increased activated B cell subsets. Although plasma total IgG titers were similar between groups, IDUs had significantly higher IgG3 and IgG4, suggestive of chronic B cell activation. Total IgM was also increased in IDUs, as well as HIV Envelope-specific IgM, suggestive of increased HIV exposure. IDUs exhibited numerous features suggestive of systemic inflammation, including significantly increased plasma sCD40L, TNF-α, TGF-α, IL-8, and ceramide metabolites. Machine learning multivariate analysis distilled a set of 10 features that classified samples based on group with absolute accuracy. CONCLUSIONS: These results demonstrate broad alterations in the steady-state humoral profile of IDUs that are associated with increased systemic inflammation. Such dysregulation may impact the ability of IDUs to generate optimal responses to vaccination and infection, or lead to increased risk for inflammation-related co-morbidities, and should be considered when developing immune-based interventions for this growing population.

Evaluation of the DRI oxycodone immunoassay for the detection of oxycodone in urine.

We evaluated the performance of the DRI Oxycodone (DRI-Oxy) enzyme immunoassay for the detection of oxycodone and its primary metabolite, oxymorphone, in urine, by testing 1523 consecutive urine specimens collected from pain management patients. All 1523 specimens were tested with the DRI-Oxy assay at a cut-off of 100 ng/mL and then analyzed by gas chromatography-mass spectrometry (GC-MS) for opiates, including oxycodone and oxymorphone. Approximately 29% (435) of the 1523 specimens yielded positive results by the DRI-Oxy assay. Of these 435 specimens, GC-MS confirmed the presence of oxycodone and/or oxymorphone at >100 ng/mL in 433 specimens, an agreement of 99.5%. In addition to oxycodone and/or oxymorphone, 189 of the 433 positive specimens contained other opiates including codeine, hydrocodone, hydromorphone, and morphine. These other opiates were also present in 54% (590/1084) of the oxycodone negative specimens. The DRI-Oxy assay demonstrated no cross-reactivity, yielding negative results, with specimens containing concentrations of codeine, >75,000 ng/mL; hydrocodone, >75,000 ng/mL; hydromorphone, >12,000 ng/mL; and morphine, >163,000 ng/mL. From the presented study, the sensitivity of the DRI-Oxy was 0.991 and the selectivity 0.998. The DRI-Oxy assay provided a highly reliable method for the detection of oxycodone and/or oxymorphone in urine specimens.

The detection of oxycodone in meconium specimens.

A procedure for the determination of oxycodone in meconium using direct ELISA microplate technology followed by electron impact gas chromatography-mass spectrometry (GC-MS) is described for the first time. The abuse of oxycodone (OxyContin) has been widely discussed in mainstream media, and it has been described as a cheap form of heroin. Oxycodone has been reported as having a high degree of abuse and potential complications in neonates from maternal drug use. Using a standard enzyme multiplied immunoassay screening technology, the cross-reactivity of oxycodone to the morphine antibody is only 5-6%. A positive screening value would require a high concentration of drug to be present, so a protocol for the detection of oxycodone in meconium using a direct ELISA microplate immunoassay followed by GC-MS was developed. The assay is now routinely used in our laboratory.

Opioid mediated effects on the immune system: sympathetic nervous system involvement.

Opioids have been hypothesized to suppress parameters of immune function by acting within the central nervous system to increase the activity of the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system. Production of catecholamines and adrenocorticoids have been demonstrated to be responsible for many of the observed immunomodulatory effects which occur following opioid administration. In general, the sympathetic nervous system has been shown to play a role in regulating lymphocyte proliferation and natural killer cell activity as well as several other parameters of immune function. Here, we will focus primarily on the role of the sympathetic nervous system in modulating opioid induced immunosuppression. The role of the hypothalamic-pituitary adrenal axis is reviewed elsewhere in this issue.

Opiate binding sites in the cellular immune system: expression and regulation.

The direct actions of opiates on the mammalian immune system depend on the existence of ligand binding sites either on the surface of the affected cell or in the interior of the cell. With the cloning of various opiate receptors from neuronal tissue, numerous researchers have screened leukocyte cDNA libraries for the expression of these receptors with some positive results. However, the pattern of expression of neuronal opiate receptors in the cellular immune system does not completely explain the biological action of opiates there. Several possibilities could account for this non-congruence including differential expression of the receptors as determined by such factors as cell population or prior history of the cells; the existence of sequence modified versions of the neuronal receptors such that the amplification methods miss their presence; or the opiates act by a different, non-receptor mechanism in the cellular immune system.

The opioid-cytokine connection.

Opioids (exogenous opiates and endogenous opioid peptides) have a diversity of effects on the immune system. Although numerous studies have shown that opioid-induced immunosuppression can be mediated indirectly via the central nervous system (CNS) or through direct interactions with immunocytes, the precise cellular mechanisms underlying the immunomodulatory effects of opioids are largely unknown. In recent years, investigations from several laboratories have indicated that opioids can operate as cytokines, the principal communication signals of the immune system. All of the major properties of cytokines are shared by opioids, i.e., production by immune cells with paracrine, autocrine, and endocrine sites of action, functional redundancy, pleiotropy and effects that are both dose- and time-dependent. Studies of the effects of opioids on peripheral blood mononuclear cells (PBMC) or brain cells cocultured with HIV-infected cells suggest that some of the immunoregulatory actions of opioids are mediated by ultrahigh affinity receptors on PBMC and glial cells. Because the CNS is populated predominantly by astroglia and microglia which have properties of immune cells, it is possible that certain of the CNS effects of opioids involve cytokine-like interactions with glial cells. Although there is mounting evidence supporting the concept that opioids are members of the cytokine family, the relative contribution of the opioids to immunoregulation remains unclear. The importance of opiate addiction in the AIDS epidemic means that gaining a better understanding of the mechanisms of opioid-induced immunomodulation is of more than academic interest.

Opioid modulation of immune responses: effects on phagocyte and lymphoid cell populations.

The literature describing effects of morphine on cells of the immune system points to the clear conclusion that morphine given in vivo suppresses a variety of immune responses that involve the major cell types in the immune system, including natural killer (NK) cells, T cells, B cells, macrophages and polymorphonuclear leukocytes (PMNs). Depression of NK cell activity has been reported in humans, monkeys and rodents. Similarly, responses of T cells are depressed by morphine, as assessed by inhibition of induction of delayed-type hypersensitivity reactions and cytotoxic T-cell activity, modulation of T-cell antigen expression, and depression of responses to T-cell mitogens. Effects on T cells have been reported in humans, monkeys and rodents. Effects of morphine on B-cell activity have mainly been tested in rodents using assays of antibody formation, which also require macrophages and T cells, preventing a conclusion as to the cell type being affected. Consistent effects on phagocytic cell function have been reported in rodents given morphine. In contrast, studies on immunomodulatory effects of morphine added to cells of the immune system in vitro have shown robust effects on some of these cell types, but not others. There is a rich literature demonstrating downregulation of phagocytic cell function by morphine, particularly for human peripheral blood mononuclear cells (PBMCs) and PMNs. Phagocytosis, chemotactic responses, interleukin production, and generation of activated oxygen intermediates and arachidonic acid products have all been reported to be inhibited. On the contrary, the literature does not support direct effects of morphine on NK cell function, is inconclusive concerning effects on B cells, and provides limited evidence for effects on T cells. The divergence between the in vivo and in vitro data suggests that effects on some cells in the immune system observed after in vivo morphine are probably not direct, but mediated. In aggregate, the literature supports the existence of an in vivo neural-immune circuit through which morphine acts to depress the function of all cells of the immune system. Further, there is strong evidence that morphine can directly depress the function of macrophages and PMNs, and modulate expression of one type of T-cell surface marker. There is, however, little evidence for direct effects of morphine on NK cells and B cells. A further complication emerges from reports of immunopotentiation of immune function in in vitro assays using endogenous opioids. The possibility of different receptors for endogenous and exogenous opioids or of interactions among the activated opioid receptors may account for these opposing effects.

Distinct domains of IFNalpha mediate immune and analgesic effects respectively.

Interferon-alpha (IFNalpha) is not only an immunoregulatory factor, but is also an analgesic molecule. The analgesic effect of IFNalpha was mediated by mu opioid receptor. After the 129th Tyr residue of human IFNalpha was mutated to Ser, the antiviral activity almost disappeared, but there still remained a strong analgesic activity that could be blocked by naloxone. These results indicate that there exist distinct domains in the IFNalpha molecule, which mediate immune and analgesic effects respectively, and suggest that there are different receptor mechanisms inducing immune and analgesic effects of IFNalpha. However, although the antiviral activity of IFNalpha decreased to 34.1% of wild type IFNalpha after the 122nd Tyr residue was changed to Ser, the analgesic activity of this mutant was lost completely. There were significant cross reactivities between INFalpha and anti-opioid sera. These studies show strong structural and functional similarities between INFalpha and opioid peptides, and inferred that the analgesic domain locates around the 122nd Tyr residue of IFNalpha molecule in tertiary structure.

Initial characterization and autoradiographic localization of a novel sigma/opioid binding site in immune tissues.

High concentrations of novel, haloperidol- and DTG-inaccessible (+)-[3H]-3-PPP binding sites were found in human peripheral blood leukocytes rat spleen and splenocytes, but not in rat brain. Splenic sites were localized in a course punctate pattern in the marginal zones and red pulp. The pharmacology of the splenic sites was: (-)-SKF 10,047 > or = naltrexone = (-)-pentazocine > (+)-pentazocine = (-)-3-PPP = (+)-SKF 10,047 > or = (+)-3-PPP > or = dextrorphan > dextromethorphan > PCP > clorgyline. DTG, haloperidol, TCP, (-)-deprenyl and SKF 525-A did not complete. Binding activity was destroyed by heating and phospholipase C, but not by proteases or glycosidases. These sites may be involved in immunomodulation by opiate and sigma receptor agonists.

Chemistry of drug allergenicity.

Of the very large number and variety of drugs used in medicine, those that are frequently implicated in immediate allergic reactions are relatively small in number and include neuromuscular blocking drugs used in anaesthesia, beta-lactam antibiotics, some other antibacterial agents including broad-spectrum antibiotics and quinolones and, less often, some narcotics. Structure-activity and immunochemical investigations have been most numerous and detailed for neuromuscular blocking drugs and beta-lactams, particularly penicillins. For the former group of drugs, morphine is proving to be a useful agent for the in-vitro detection of clinically relevant neuromuscular blocking drug-- as well as morphine- and fentanyl-reactive IgE antibodies. The employment of so-called 'major' and 'minor' determinants for a range of different penicillins and cephalosporins has revealed previously unsuspected heterogeneity in patient recognition responses, and has reinforced findings on the allergenic importance of side-chain groups. Many reports have been published on anaphylaxis to chlorhexidine, and progress in identifying allergenic determinants is reviewed together with the still inadequately understood subject of IgE antibody recognition of quinolone antibacterial agents.

Bidirectional heterologous desensitization of opioid and chemokine receptors.

Opioids are known to suppress a number of elements of the immune response, including antimicrobial resistance, antibody production, and delayed-type hypersensitivity. Phagocytic cells may be particularly susceptible to opioid administration, since reduced production of the cytokines IL-1, IL-6 and TNF-alpha, monocyte-mediated phagocytosis, and both neutrophil and monocyte chemotaxis have all been well established. Earlier studies have shown that both mu- and delta-opioid agonists induce a chemotactic response in monocytes and neutrophils. In addition, mu- and delta-opioid administration inhibited the chemotactic response of these cell populations to a number of chemokines through a process of heterologous desensitization. We report here that mu-, delta-, and kappa-opioid agonists also induce a chemotactic response in T lymphocytes. Using the human T-cell line Jurkat, we have confirmed previous observations that pre-incubation with met-enkephalin (MetEnk), an endogenous opioid agonist, prevents the subsequent chemotactic response to the chemokine RANTES. On the other hand, treatment with MetEnk does not alter the response to the chemokine SDF-1 alpha. Moreover, we found that pre-treatment with RANTES prevented a subsequent response of monocytes to the mu-opioid agonist DAMGO. These results suggest that activation of members of the opioid and chemokine receptor families leads to downregulation of each other's leukocyte migratory activities.

Long-term methadone treatment: effect on CD4+ lymphocyte counts and HIV-1 plasma RNA level in patients with HIV infection.

The objective was to examine the effect of methadone on CD4+ lymphocyte counts and viral load and to expect to document the safety of methadone maintenance in patients with human immune deficiency syndrome. This is a retrospective chart analysis comparing the trends in CD4+ count and viral load in two populations of 21 human immunodeficiency virus (HIV) infected patients, one on methadone maintenance and a methadone non-using group. Each methadone user was matched with a control methadone non-user that had a similar CD4+ at the beginning of the study. For the CD4+ count we compared the slope of regression for each couple of patients. In 15 patients we also collected the viral load, which was measured at 4-6 monthly intervals. The mean length of follow-up was 811 days for the methadone group and 797 days in the control group. There was no statistical difference in the treatment received by the two groups of patients during the study. The slope of regression of CD4+ count showed a significantly steeper decline in the methadone-using patients compared with the methadone non-users (r= 0.487; p< 0.05). The evolution of the HIV-1 RNA levels was the same during the follow-up of mean 186 months in a few of the patients in each of the two groups. Long-term methadone use was associated with a significantly faster decrease of CD4+ count in HIV-1 affected patients compared with methadone non-users. HIV-1 RNA data were found in too few patients to enable any conclusions about the development of viral load in the two groups.

Interferon: a candidate as the endogenous substance preventing tolerance and dependence to brain opioids.

It has been suggested that protein synthesis and immunological factors play a role in the development of tolerance and dependence to opiates. Interferon (IFN) is known to influence protein synthesis and the immune system. Our and other data suggest that IFN may be an agent that prevents the development of tolerance and the physical dependence to endogenous opioids.

The morphine-binding site on human activated T-cells is not related to the mu opioid receptor.

Mitogen activation of human T-lymphocytes induces a morphine-binding site. Morphine binding is displaceable by beta-endorphin (1--31) and (--)-naloxone but not DAMGO. This site is not stereoselective for (--)-morphine. T-lymphocytes, expressing this binding site, were assayed by reverse-transcription polymerase chain reaction (RT-PCR) for expression of hMOR-1 mRNA. Several primer sets were used and each assay compared with cells known to express human or mouse MOR-1 mRNA. Neither hMOR-1 nor any homologous receptor was detected in human T-lymphocytes. Therefore, the morphine-binding site on mitogen-activated T-lymphocytes is unlikely to be closely related to hMOR-1.

Opioids, opioid receptors, and the immune response.

It is now clear that opioid receptors participate in the function of the cells of the immune system, and evidence suggests that opioids modulate both innate and acquired immune responses. We review literature here which establishes that mu-, kappa-, and delta-opioid compounds alter resistance to a variety of infectious agents, including the Human Immunodeficiency Virus (HIV). The nature of the immunomodulatory activity of the opioids has been the subject of a great deal of research over the last ten years. There is increasing evidence that effects of opioids on the immune response are mediated at several levels. Modulation of the inflammatory response appears to be a target of these compounds, including effects on phagocytic activity, as well as the response of cells to various chemoattractant molecules. Moreover, findings from several laboratories have demonstrated the impact of opioid treatment on antibody responses, and the molecular basis for this effect is likely due, at least in part, to the modulation of both cytokine and cytokine receptor expression. Future research should provide a clearer understanding of the cellular and molecular targets of opioid action within the immune system.

Immunomodulation by cocaine and ketamine in postnatal rats.

The abuse of cocaine (COC) in combination with ketamine (KET) among pregnant women was shown to be high. Transplacental exposure is not the only route by which a newborn may be exposed to these agents, but they can also distribute into breast milk. Chronic COC exposure is associated with immunological modulation in human and animal models. The effect of sub-chronic exposure to COC and KET alone and in combination on the developing immune system was assessed in neonatal male Sprague-Dawley (SD) rats. To simulate the route of exposure during lactation, newborn male rats were treated orally with saline, COC alone (20 mg/kg), KET alone (50 mg/kg), or KET (50 mg/kg) followed 15 min later by COC (20 mg/kg) from days 1 to 21 of life. Pups were sacrificed 30 min following the last treatment. Total circulating leukocyte and lymphocyte counts were decreased with relative neutrophilia, while spleen/body weight ratio and IgM antibody response to sheep red blood cells (SRBCs) were increased in animals treated with COC. Moreover, treatment with COC alone increased serum interleukin 10 (IL-10) concentration; however, it did not affect serum interferon gamma (IFN-gamma) concentration. On the other hand, KET treatment did not produce any significant change of any of these parameters. However, when co-administered with COC, the immunomodulatory effects of COC were prevented. COC caused a significant increase in serum corticosterone concentration that KET effectively prevented. Lack of significant change of plasma and tissue concentrations of norcocaine (NC) suggested no role for COC metabolism in COC-induced immunomodulation. However, the results of this study indicate that COC-induced immunomodulatory reactions and their prevention by KET most likely occurred through neuroendocrinal mechanisms.