Morphine modulates NF kappa B activation in macrophages.

Chronic use of morphine affects the immune system and predisposes an individual to opportunistic infections. Macrophages play an important role in conferring a first line of defense against invading pathogens. Understanding the mechanisms by which morphine affects the functioning of macrophages would have significant therapeutic benefit in treatment against infections such as HIV and AIDS related syndromes. Two of the major cytokines secreted by activated macrophages are Interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha). Our studies show that morphine differentially modulates lipopolysaccharide (LPS) induced expression of IL-6 and TNF-alpha. Nanomolar concentrations of morphine synergize with LPS and augment the secretion of both IL-6 and TNF-alpha. However, at micromolar concentrations morphine inhibits LPS induced synthesis of IL-6 and TNF-alpha. Expression of both these cytokine genes is dependent on the activation of a transcription factor, NF kappa B. Interestingly, morphine treatment also modulated the activation of NF kappa B by LPS. Pretreatment with a low dose of morphine (nanomolar) resulted in an increase in NF kappa B activation. In contrast pretreatment with a high dose of morphine (micromolar) led to a significant decrease in NF kappa B activation. Furthermore unlike the augmentation which was naloxone reversible, the inhibition of NF kappa B by morphine was not reversed by naloxone, suggesting the involvement of a nonclassical opioid receptor.

Morphine inhibits transcriptional activation of IL-2 in mouse thymocytes.

Chronic treatment of mice with morphine affects the proliferation, differentiation, and function of immune cells. In the present study, we investigated the mechanism by which morphine inhibits phytohemagglutinin (PHA)/interleukin-1 (IL-1)-induced thymocyte proliferation. When compared to control cultures, morphine-treated thymocytes showed decreased steady-state levels of bioactive IL-2 and IL-2 mRNA. The reduced IL-2 concentration and reduced transcript levels correlated well with a decreased rate of synthesis of IL-2 mRNA as determined by nuclear runoff assays. Subsequent studies showed that morphine treatment affected transcriptional control elements of the IL-2 promoter by inhibiting the synthesis of a specific trans-activating nuclear factor, c-Fos. c-Fos mRNA levels measured by semiquantitative RT-PCR were significantly decreased in thymocytes following treatment with morphine and activation with PHA and IL-1. Under identical conditions, c-Jun mRNA levels were not altered. Electrophoretic mobility shift studies with the AP-1 consensus oligonucleotide showed significantly decreased levels of AP-1-protein complex formation in nuclear extracts prepared from morphine-treated cells. These studies demonstrate for the first time that opioid alkaloids such as morphine can impair mitogen-lymphokine-activated thymocyte proliferation by interfering with transcriptional activation of the IL-2 gene.

The possible role of a central nervous system dopaminergic mechanism in hepatic c-fos protein expression following peritoneal sepsis.

OBJECTIVE: To investigate the hypothesis that a central dopaminergic mechanism may regulate hepatic c-fos and c-jun gene expression following peritoneal sepsis. METHODS: First, dopamine or vehicle was instilled into a stereotaxically placed intracerebral-ventricular (ICV) cannula with or without D1 (SCH 23390) or D2 (haloperidol) antagonist pretreatment in a rat model, and the effect on hepatic c-fos or c-jun protein expression was investigated. Second, we investigated the effect of haloperidol and vehicle treatment following cecal ligation and puncture (CLP)-induced sepsis with respect to hepatic c-fos protein expression, c-jun protein expression, and survival. RESULTS: Intracerebral-ventricular dopamine treatment increased hepatic c-fos immunoreactive protein but had no effect on hepatic c-jun immunoreactive protein expression. Pretreatment with SCH 23390 inhibited ICV dopamine treatment-induced hepatic c-fos immunoreactive protein expression. Haloperidol pretreatment synergized with ICV dopamine treatment to overexpress hepatic c-fos protein. Haloperidol treatment significantly increased CLP-induced hepatic c-fos and c-jun protein expression and improved survival following CLP. CONCLUSIONS: Hepatic c-fos protein expression may be regulated, in part, by a central nervous system-mediated dopaminergic D1 receptor mechanism. Treatment with the D2 receptor antagonist, haloperidol, increases sepsis-induced hepatic c-fos and c-jun protein expression and improves survival following peritoneal contamination.

Detection of influenza virus by centrifugal inoculation of MDCK cells and staining with monoclonal antibodies.

Two methods for detection of influenza virus in 451 clinical respiratory specimens were compared: (i) 24-well-plate centrifugation with Madin-Darby canine kidney (MDCK) cells and staining with monoclonal antibody pools to influenza viruses A and B (Centers for Disease Control, Atlanta, Ga.) in an indirect immunofluorescence assay after incubation for 40 h, and (ii) conventional tissue cell culture with primary monkey cells and hemadsorption. For 100 of these specimens, direct examination of smears by the direct fluorescence assay with monoclonal antibodies (Boots Cell Tech/API Analytab Products, Plainview, N.Y.) was also performed. Influenza A virus was recovered from 28 specimens by tissue cell culture after incubation for an average of 4.75 days (range, 2 to 14 days). Influenza B virus was recovered from 35 specimens by tissue culture after incubation for an average of 5.4 days (range, 3 to 14 days). By the centrifugation assay, 23 specimens were positive for influenza A virus and 30 were positive for influenza B virus. All specimens positive by the centrifugation assay were also positive by conventional tissue cell culture. The sensitivities of the centrifugation assay were 82% for detection of influenza A virus and 86% for influenza B virus (84% overall); the specificity of the assay was 100%. Of the 100 specimens studied by direct examination, 15 were positive for influenza virus by both conventional culture and centrifugation assay; however, the direct-smear results for these 15 specimens were negative in 13 cases and inconclusive in 2. The centrifugation assay is a rapid and specific method for detection of influenza A and B viruses in clinical specimens, and it can serve as a valuable and cost-efficient adjunct to conventional culture methods.