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.

A new highly specific buprenorphine immunoassay for monitoring buprenorphine compliance and abuse.

Urine buprenorphine screening is utilized to assess buprenorphine compliance and to detect illicit use. Robust screening assays should be specific for buprenorphine without cross-reactivity with other opioids, which are frequently present in patients treated for opioid addiction and chronic pain. We evaluated the new Lin-Zhi urine buprenorphine enzyme immunoassay (EIA) as a potentially more specific alternative to the Microgenics cloned enzyme donor immunoassay (CEDIA) by using 149 urines originating from patients treated for chronic pain and opioid addiction. The EIA methodology offered specific detection of buprenorphine use (100%) (106/106) and provided superior overall agreement with liquid chromatography-tandem mass spectrometry, 95% (142/149) and 91% (135/149) using 5 ng/mL (EIA[5]) and 10 ng/mL (EIA[10]) cutoffs, respectively, compared to CEDIA, 79% (117/149). CEDIA generated 27 false positives, most of which were observed in patients positive for other opioids, providing an overall specificity of 75% (79/106). CEDIA also demonstrated interference from structurally unrelated drugs, chloroquine and hydroxychloroquine. CEDIA and EIA[5] yielded similar sensitivities, both detecting 96% (22/23) of positive samples from patients prescribed buprenorphine, and 88% (38/43) and 81% (35/43), respectively, of all positive samples (illicit and prescribed users). The EIA methodology provides highly specific and sensitive detection of buprenorphine use, without the potential for opioid cross-reactivity.

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.

Immunomodulatory effects of opioids.

OBJECTIVE: To review the immunomodulatory effects of opioids. DATA SOURCES: Original research publications and review articles using the PubMed search engine with the following keywords--opioids, morphine, immuomodulation, and immunosuppression. VETERINARY AND HUMAN DATA SYNTHESIS: Opioids have been shown to modulate the immune system in animal models by affecting both the acquired and innate arms of the immune system. Natural killer cell activity, T-cell proliferation, antibody production, phagocytic cell function, and cytokine production have all been shown to be affected by opioids. Many of these effects are reversed by opioid antagonists. Opioids have also been shown to induce sepsis in laboratory animals. Opioid administration alters immune parameters in healthy humans at analgesic doses and may increase the risk of infection in some patient populations. CONCLUSIONS: While opioids remain the most powerful and widely used analgesics available, their negative effects on the immune system are well established in the laboratory setting. Thoughtful consideration should be given to the use of certain opioids in critically ill patients, especially those with pre-existing immunocompromise.

Immunoglobulin E antibodies to rocuronium: a new diagnostic tool.

BACKGROUND: Diagnosis of allergy from neuromuscular blocking agents is not always straightforward. The objectives of the current study were to investigate the value of quantification of immunoglobulin E (IgE) by ImmunoCAP (Phadia AB, Uppsala, Sweden) in the diagnosis of rocuronium allergy and to study whether IgE inhibition tests can predict clinical cross-reactivity between neuromuscular blocking agents. METHODS: Twenty-five rocuronium-allergic patients and 30 control individuals exposed to rocuronium during uneventful anesthesia were included. Thirty-two sera (total IgE > 1,500 kU/l) were analyzed for potential interference of elevated total IgE titers. Results were compared with quantification of IgE for suxamethonium, morphine, and pholcodine. Cross-reactivity between drugs was assessed by IgE inhibition and skin tests. RESULTS: Sensitivity of IgE for rocuronium, suxamethonium, morphine, and pholcodine was 68, 60, 88, and 86%, respectively. Specificity was 100% for suxamethonium, morphine, and pholcodine IgE and 93% for rocuronium IgE. ROC analysis between patients and control individuals changed the threshold to 0.13 kUa/l for rocuronium, 0.11 kUa/l for suxamethonium, 0.36 kUa/l for morphine, and 0.43 kUa/l for pholcodine. Corresponding sensitivity was 92, 72, 88, and 86%, respectively. Specificity was unaltered. Interference of elevated total IgE with quantification of IgE was demonstrated by the analysis in sera with a total IgE greater than 1,500 kU/l. IgE inhibition did not predict clinical relevant cross-reactivity. CONCLUSIONS: The rocuronium ImmunoCAP constitutes a reliable technique to diagnose rocuronium allergy, provided an assay-specific decision threshold is applied. IgE assays based on compounds bearing ammonium epitopes are confirmed to represent reliable tools to diagnose rocuronium allergy. High total IgE titers were observed to affect specificity of the assays.

Morphine modulates gammadelta lymphocytes cytolytic activity following BCG vaccination.

Chronic opioid administration modulates lymphocytes' functional capabilities increasing susceptibility to infectious diseases. Bacille-Calmette-Guérin (BCG) vaccination initiates a non-specific and specific cell-mediated immunity orchestrated by T lymphocytes including gammadelta T lymphocytes. gammadelta T lymphocytes increase in natural killer and antigen-directed cytolytic response following BCG vaccination. The objective of this study was to determine morphine effects on gammadelta T lymphocytes' cytolytic activity. Pigs were chronically administered morphine and subsequently vaccinated with Mycobacterium bovis BCG. By administering morphine prior to BCG vaccination, natural killer response was significantly suppressed (p=.034). Furthermore, innate cytolytic response against M. bovis-infected monocytes (p=.002) as well as antigen specific cytolytic functions (p=.04) were significantly altered due to morphine administration. It was concluded that administering morphine prior to BCG vaccination significantly altered gammadelta T lymphocyte cytolytic responses.

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.

Multiple opioid receptors on immune cells modulate intracellular signaling.

In 1979, Joseph Wybran reported his insights into the existence of different opioid receptor subtypes on T-cells. He observed that morphine and methionine enkephalin had different effects on human T-cell rosetting to sheep red blood cells. Since that time, a wide array of laboratories have shown that opiate alkyloids and opioid peptides exert pleiotropic effects on immune cell function. These compounds are immunomodulators, modifying immune responses to extracellular stimuli such as mitogens, antigens, and antibodies that cross-link the T-cell receptor. It has been demonstrated that cells involved in host defense and immunity express mRNA transcripts encoding the various opioid receptors originally described in neuronal tissues. Molecular imaging approaches have demonstrated the regulated expression of both delta and kappa opioid receptors, predominantly on T-cells. Moreover, atypical opiate and opioid binding sites are present on these cells. This review will consider the evidence for both classical and atypical opioid receptors and their effects on signaling within immune cells; our emphasis is the T-cell and its delta opioid receptor.

Immunomodulatory effect of morphine: therapeutic implications.

The immunosuppressive as well as modulatory effects of morphine have been known in clinical medicine for > 100 years. Recent developments in molecular immunology, including experiments in mu (mu) opioid receptor knockout mice has led to a better understanding of central and peripheral mechanisms involved in this process. Though there is a large volume of literature documenting adverse effects of immunosupression following the use of morphine, several reports confirm its potential usefulness as an immunomodulator. In vitro and in vivo animal experiments have demonstrated wide-spectrum effects of morphine, including anti-inflammatory, antifibrotic, antitumour, cardioprotective and renoprotective. Immunomodulation is an important field in modern medicine with rapid advancement in recent years. Though a final statement regarding the clinical relevance of morphine-induced immunomodulation cannot be made at this juncture, nevertheless, it is worthwhile to review current developments. It may encourage further clinical studies to elucidate the influence of morphine treatment on immune regulation in different specialties of medicine.

Anaphylaxis during anesthesia: results of a 12-year survey at a French pediatric center.

BACKGROUND: Following adverse reactions to anesthesia, tests are carried out to determine the mechanism of the reaction and to identify the agent responsible. No specific data are available in France concerning such skin tests in children. METHODS: Between 1989 and 2001, we assessed hypersensitivity reactions to general anesthesia in 68 children. Thirty underwent more than one operation, for congenital malformations. Immunoglobulin (Ig)E-mediated anaphylaxis was diagnosed on skin tests combined with the clinical history. RESULTS: Grade I, II and III reactions were observed in 20, 27 and 21 children, respectively. IgE-mediated anaphylaxis was diagnosed in 51 children: 31 (60.8%) for neuromuscular blocking agents (NMBA), 14 (27%) for latex, seven (14%) for colloids, five (9%) for opioids and six (12%) for hypnotics. Vecuronium was the NMBA causing the largest number of reactions. Cross reactivity to NMBA available in France was observed in 23 of 30 children (76%), particularly for vecuronium and atracurium or pancuronium. The estimated frequency of IgE mediated anaphylactic reactions was one in 2100 operations. Based on our results, 25 children subsequently received a different anesthetic with no adverse reaction. CONCLUSIONS: As in adults, NMBA, then latex were responsible for most anaphylactic reactions during anesthesia. Our results confirm that skin tests with anesthetic agents are feasible and safe in children and improve the safety of subsequent anesthetic procedures.

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.

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.

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.

Validation of a microtiter plate ELISA for screening of postmortem blood for opiates and benzodiazepines.

The object of this study was to evaluate the suitability of the Neogen Corp. microtiter plate enzyme-linked immunoassays (ELISA) for opiates and benzodiazepines for screening of postmortem blood. Ninety postmortem whole blood specimens were obtained from drug-involved deaths which had been screened and confirmed positive for opiates and/or benzodiazepines. Forty negative specimens were obtained from non-opiate-involved deaths. Specimens were tested using the Neogen Opiates Group and Neogen Benzodiazepines Group microtiter plate ELISA assays. No matrix effects were found for whole blood in these assays and a dilution of 1:5 was chosen to facilitate pipetting and to bring the IC50 of the microtiter plate ELISA assay within the range of opiates and benzodiazepines encountered in medical examiner specimens. True positive, true negatives, false positives, and false negatives were determined and graphed for the ELISA results against gas chromatography-mass spectrometry (GC-MS), gas chromatography-nitrogen-phosphorus detection and case histories. From these graphs and the ROC curves, the optimal cut-off for the Neogen Opiates Group ELISA was found to be between 20 and 50 ng/mL morphine equivalents and the optimum cut-off for the Neogen Benzodiazepines Group ELISA was between 20 and 50 ng/mL temazepam equivalents. The Neogen Opiates Group ELISA had a sensitivity of 95.2% +/- 2.7% and a specificity of 92.2% +/- 3.4% versus GC-MS at a cut-off of 20-ng/mL cut-off and a sensitivity of 88.8% +/- 3.9% and specificity of 96.8% +/- 2.1% versus GC-MS at a 50-ng/mL morphine equivalents cut-off. The Neogen Benzodizepines Group ELISA had a sensitivity of 100% +/- 1.3% and a specificity of 94.6% +/- 2.9% versus GC-MS (20-ng/mL temazepam equivalents cut-off) and a sensitivity of 95.8% +/- 2.5% and specificity of 98.2% +/- 1.8% versus GC-MS at a 50-ng/mL cut-off.

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.

Laboratory analysis of remotely collected oral fluid specimens for opiates by immunoassay.

The performance characteristics of a method for detecting opiates (morphine, codeine, heroin, and 6-acetylmorphine [6-AM]) in oral fluid specimens were examined and compared with methods for urine specimens. The oral fluid was easily obtained using a simple device that collects between 1 and 1.5 mL of fluid for laboratory analysis. Simultaneously collected specimens from 60 known opiate abusers from a drug-treatment center were first tested using an immunoassay cutoff of 10 ng/mL in oral fluids and 2,000 ng/mL in urine. Using a second aliquot, opiate confirmation in urine was performed by gas chromatography-mass spectrometry (GC-MS) and in oral fluids by GC-MS-MS. The combined immunoassay and GC-MS-MS procedures were completed with less than 250 pL of oral fluid. Opiates identified in oral fluid specimens from heroin users included morphine, codeine, heroin, and 6-AM. The immunoassay was tested for precision, stability, and the effects of potential cross-reactants. The results yielded 93.6% agreement between oral fluid and urine, suggesting that oral fluid may be a reliable matrix for opiate detection.