Cocaine increases intracellular calcium in the interferon-gamma-primed macrophages but not in the LPS-primed-macrophages.

Calcium is required for antigen presentation. In the past study, we found that cocaine increased macrophage antigen-presenting activity. To investigate whether cocaine induces a calcium influx into macrophage cytosol, we used Fura2-AM to directly test the macrophage intracellular calcium concentration [Ca2+]i under the influence of different concentrations of cocaine after macrophages were primed by IFN-gamma or LPS. We report here that cocaine increases the IFN-gamma-primed macrophage [Ca2+]i, but it does not affect the LPS-primed macrophage [Ca2+]i. Furthermore, calcium blocker, nifedipine, blocks the effect of cocaine, suggesting that extracellular calcium enters the cytosol through the L-channel.

Stimulation of mouse macrophage antigen presentation by cocaine.

Cocaine has been demonstrated to have multiple effects on the immune system. Here, we determined the effects of cocaine on macrophage antigen presentation, using an in vitro antigen presentation assay after macrophages were treated with cocaine both in vitro and in vivo. Our results showed that in vitro treatment of macrophages with cocaine significantly enhanced macrophage's ability to present ovalbumin (OVA) and the enhancement was also demonstrated in the macrophages of cocaine-injected mice. The presentation of an OVA-derived antigenic peptide (OVA323-339), however, was not affected. In vitro cocaine treatment neither affected antigen uptake nor major histocompatibility complex (MHC) II expression and the expression of co-stimulatory molecules B7. These results suggest that cocaine may act on an early event in the antigen handling by accessory cells.

The effects of cocaine injections on mouse thymocyte population.

C57 BL mice were injected daily with either saline or varied doses of cocaine (5-50 mg/kg), and thymocyte subpopulations were analyzed 4 hr after the fifth injection. Mice injected with either 25 or 50 mg/kg of cocaine showed a decrease in the percentage of CD4+8+ cells and increase of CD4-8-, CD4+, and CD8+ cells. The absolute numbers of each subpopulation, calculated by multiplying the percentage of each subpopulation with the total cell number, revealed an extensive decline in CD4+8+, a decrease in CD8+, an increase in CD4-8-, and no change in the CD4+ subpopulation. Flow cytometric analysis of thymocytes and electrophoresis of the thymocyte DNA revealed a dosage-dependent increase in cells undergoing programed cell death with apoptosis. Culturing of thymocytes from control or drug-treated mice demonstrated an inverse relationship between cell viability and cocaine concentrations, suggesting that in vivo cocaine, or its biological products, may damage thymocytes. Incubation of normal cells with cocaine showed a dose-dependent decrease of viability with identical patterns of the alteration of cell subpopulations observed in vivo. A dose-dependent increase of apoptosis was also observed. In summary, we demonstrate a selective in vivo cocaine-induced alteration of the thymocyte subpopulations and identified programed cell death with apoptosis as the likely mechanism mediating this thymic atrophy. The comparable findings observed in vivo and in vitro support the concept that cocaine may directly affect some features of thymocyte biology, and suggest the usefulness of the in vitro system in studying cocaine effects on thymocyte biology.

Adenosine deaminase isoenzyme levels in patients with human T-cell lymphotropic virus type 1 and human immunodeficiency virus type 1 infections.

In serum, the enzyme adenosine deaminase (ADA) is known to be divided into two isoenzymes, ADA1 and ADA2, which have different molecular weights and kinetic properties. The present study investigated ADA isoenzyme levels in the sera of patients infected with retroviruses associated with adult T-cell leukemia (ATL), human T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy (HAM), and AIDS, ADA isoenzyme activities were found to be significantly (P < 0.001) higher in the sera of patients with ATL, HAM, and AIDS than in the sera of healthy controls. In the case of the ADA subtypes in the sera of patients with ATL, ADA1 activity was significantly (P < 0.001) elevated in patients with the acute and lymphoma types of ATL compared with that in patients with the chronic and smoldering types of ATL. ADA2 activity was significantly elevated in the sera of patients with the acute, lymphoma, and chronic types of ATL (P < 0.001) compared with that in patients with smoldering ATL and HTLV-1 carriers. In the case of patients with human immunodeficiency virus type 1 (HIV-1) infection, ADA1 and ADA2 activities in the sera of patients with AIDS and HIV-1 antibody-positive individuals were significantly (P < 0.001) higher than those in the sera of HIV-1 antibody-negative individuals. A significant elevation in ADA2 activity was also seen in the sera of AIDS patients (P < 0.01) compared with that in the sera of HIV-1 antibody-positive individuals. These results suggest that the magnitude of elevation of ADA isoenzyme levels in serum correlates well with the clinical conditions of the patients with these diseases. Measurement of the activities of ADA isoenzymes may therefore provide an additional parameter for distinguishing the subtypes of ATL and may prove to be useful as prognostic and therapeutic monitors in diseases associated with HTLV-1 and HIV-1 infections.

Suppression of macrophage reactive intermediates by cocaine.

The in vivo or in vitro effects of cocaine on the production of hydrogen peroxide (H2O2), superoxide (O2-), and nitrite (NO2-) by sodium periodate-elicited macrophages were studied. Hydrogen peroxide and superoxide productions were suppressed by both in vitro treatments and in vivo administrations of cocaine. The H2O2 and O2- production partially recovered after 150 min in vitro. However, cocaine inhibited NO2- production only in vitro, failing to suppress it through in vivo administration.

The suppression of macrophage secretion by calcium blockers and adenosine.

In an effort to examine the effects of calcium blockers and adenosine on superoxide, hydrogen peroxide, and nitrite secretions by mouse peritoneal macrophages, we have utilized the phenol red method, the superoxide dismutase-inhibitable reduction of ferricytochrome c method, and the Griess reagent method to test products after treating periodate-elicited mouse peritoneal macrophages with verapamil, nifedipine, and adenosine. The results show that after treating the macrophages with chemicals 10 minutes before adding PMA (100 ng/ml), all three chemicals inhibited superoxide (O2-) and hydrogen peroxide (H2O2) secretions dose-dependently, yet they failed to suppress macrophage reactive oxygen intermediates (ROI) after a 24 hour treatment. On the other hand, calcium blockers, verapamil and nifedipine, could reduce nitrite secretion (NO2-), while adenosine did not show the ability to inhibit NO2-. This indicates that calcium, as a secondary messenger, is important for the production of ROI and RNI. The reason behind the loss of the ability to suppress macrophage ROI production in a 24 hour treatment remains unexplored.

Adrenergic receptors regulate macrophage secretion.

Macrophages have been shown to possess both alpha 2 and beta 2-adrenergic receptors which appear to antagonize each other. The ability of these two kinds of receptors to mediate macrophage secretion was tested, using alpha- and beta-antagonists, idazoxan and propranolol. Both antagonists could inhibit hydrogen peroxide and superoxide secretion, although the timing of the inhibition is different. However, only idazoxan has the ability to reduce nitrite accumulation, while propranolol fails to affect macrophage production. In addition, sodium nitrite was able to inhibit hydrogen peroxide and superoxide secretion, and hydrogen peroxide could reduce nitrite accumulation, suggesting that a negative cross-feedback mechanism exists.

Inhibition of cytokine release by cocaine.

The influence of cocaine on cytokine release by mouse peritoneal macrophages has been investigated in vitro. LPS-stimulated TNF and IL-1 secretion were suppressed by cocaine in higher dosages. This suppression was shown to be dose-related. The synthesis of cAMP, however, was stimulated by cocaine. Though cAMP is generally considered a mediator for cytokine release, further studies are required to demonstrate whether cocaine directly affects cytokine release by macrophages or indirectly influences cytokine release through stimulating cAMP synthesis.

Effects of cannabinoids and cocaine on the mitogen-induced transformations of lymphocytes of human and mouse origins.

The effects of delta 9-tetrahydrocannabinol (d9THC), delta 6-tetrahydrocannabinol (d6THC), and cocaine on the mitogen-induced transformations of lymphocytes of human and mouse origins were examined in an in vitro system. None of the three compounds is mitogenic in nature. The effects of the two components of marijuana on the mitogen-induced transformation are biphasic. Both compounds stimulated the lymphocyte transformation at low concentrations and inhibited mitogenesis at high concentrations. Cocaine, on the other hand, neither affected mitogen-induced lymphocyte transformations nor altered d9THC's transformation effects when both compounds were added together. Human lymphocytes and mouse splenocytes appeared to respond in similar patterns.

Immunomopharmacological effects of polysaccharides from Acanthopanax senticosus on experimental animals.

Polysaccharides PES isolated from a common Oriental herb Acanthopanax senticosus were found to have a wide spectrum of immunomodulatory activities on experimental animals. The potential usefulness of these polysaccharides is suggested in the observations that PES inhibited transplanted tumor growths and ameliorated toxicities of the toxic substances in experimental animals. Of most interest is the observations that they suppressed human TB propagation in mice and guinea pigs, as evaluated by lymph node responses and OT skin tests in the guinea pig model, and the quantitation of the TB in the lungs in the mouse model.

The effects of pH and temperature on the in vitro bindings of delta-9-tetrahydrocannabinol and other cannabinoids to bovine serum albumin.

Albumin is a major carrier of drugs and fatty acids in biological fluids. These protein-drug complexes serve to solubilize, transport these compounds to sites of action, and have been associated with increased half-life for these compounds. The authors are interested in the pH and temperature effects of the binding of delta-9-tetrahydrocannabinol to albumin. Ultrafiltration techniques were used in the separation of free to bound compounds. Cannabinoids bind to bovine serum albumin rapidly. The cannabinoid binding sites are more sensitive to temperature changes (37-47 degrees C) than changes in pH with 37 degrees C and pH 7.4 resulting in optimal binding. These conditions would result in the greatest viability in the cells, while allowing for the use of a variety of compounds in in vitro studies for the administration of compounds to isolated cells and cell lines.

Effects of cocaine on the immune system of Balb/C mice.

Cocaine was given to Balb/C mice by intramuscular injection to assess the effects of the drug on their immune system. An injection of 5 mg/kg of cocaine 24 hr before assay suppressed phagocytic activity of peritoneal macrophages and decreased the numbers of thymocytes and white blood cells in a dose-dependent manner. The suppression appeared to be reversible. Tumor-implanted mice received 10 consecutive days of injections of smaller doses of cocaine (0.05, 0.25, or 5 mg/kg), also resulting in a dose-dependent suppression of phagocytosis 24 hr after the last injection. Cocaine decreased the number of plaque-forming cells when 5 mg/kg of cocaine was injected on the day of immunization but no inhibition was detected if the drug was given later. The size of tumors appeared to be increased in mice injected with cocaine for 10 consecutive days in comparison to the control mice. The effects were concentration dependent. Our study showed that cocaine had general suppressive effects on the mouse immune system.

Cocaine or delta 9-tetrahydrocannabinol does not affect cellular cytotoxicity in vitro.

Cocaine or delta 9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana, has no effect on in vitro cytotoxicity mediated by natural killer (NK) cells and cytotoxic T-lymphocytes (CTL) at concentrations similar to those observed in vivo.

Confirmation of drugs of abuse in urine drug screens by direct exposure probe mass spectrometry.

A direct exposure probe (DEP) mass spectrometric method was developed to confirm the presence of cocaine (C), phencyclidine (P), benzoyl ecognine (BE) and 11-norcarboxy tetrahydrocannabinol (11-nor-c-THC) in previously screened urine specimens. Essentially, a urine sample is lyophilized overnight and reconstituted in 30 microliters of a 3:1 mixture of ethyl acetate:methanol. One microliter is placed on a rhenium filament and left to air dry. The specimen was analyzed by negative ion chemical ionization DEP using ammonia as reagent gas at 0.20 Torr with an ion source temperature of 100 degrees C. An electronics setting of 1700 V (EM), 0.30 mA filament current, and 100 eV with scanning at m/z 100-650 (1 sec/SCAN) resulted in simple spectra with easily identifiable molecular ions for C, BE, P and 11-nor-c-THC. The sensitivity of the assay was 1 ng for the drugs of abuse. The method was validated by analyzing 50 urine samples that have been previously screened and confirmed for drugs at the University of Illinois Toxicology Laboratory. The results showed that the DEP method confirmed 87%, 71%, 100%, and 85% of the BE, C, P and 11-nor-c-THC. In summary, a rapid and sensitive DEP method for the confirmation of drugs of abuse in urine has been developed which can serve as a useful adjunct to gas chromatographic/mass spectrometric confirmation.

Variable response to intravenous gamma-globulin therapy in a patient with autoimmune neutropenia, thrombocytopenia, and pemphigoid.

Intravenous high-dose gamma-globulin has been shown to be useful in some patients with immune thrombocytopenia or neutropenia. In the case we have reported, this regimen elevated the platelet but not the neutrophil count. This observation emphasizes the heterogeneity in patients with immune cytopenia, and shows that treatment outcome is likely to be influenced by a number of pathogenetic variables.

Acute-phase decrease of T lymphocyte subsets in rhinovirus infection.

Populations of peripheral blood leukocytes were enumerated in 15 volunteers challenged by intranasal inoculation with rhinovirus serotype 25. The results demonstrated a significant decrease in total lymphocyte count among infected persons on the third day after challenge with the virus (P less than .01). The change in lymphocyte count was associated with a significant decrease in total T cells, as determined by monoclonal antibodies (both T11+ and T3+, P less than .02), but not in B cells (B7+). Among the subsets of T cells, T4+ (T helper/inducer) and T8+ (T suppressor/cytotoxic) lymphocytes both declined in number, but only the change in the T4+ subset was significant. For each of the lymphocyte populations that decreased significantly (T3+, T11+, and T4+) there was a strong correlation with increased severity of symptoms. Persons who had the greatest decrease in total lymphocyte count also shed virus most frequently. The number of nonlymphocyte leukocytes increased with the severity of the symptoms. These data show that T lymphocytes (particularly the T4+ population) are related to both the progression of infection and the symptoms of the rhinovirus common cold.

Increased double marker lymphocytes in systemic lupus erythematosus and lymphoproliferative disorders.

In this study we were able to confirm the presence of double marker lymphocytes (D cells) in human peripheral blood from normal subjects in addition to patients with SLE and malignant lymphoma. Quantitation of D cells and Null lymphocytes (N cells) in peripheral blood was not feasible via conventional rosette methods. The present combination method, using two indicators, neuraminidase treated sheep red blood cells (nSRBC) and Immunobeads, allows for simultaneous demonstration of T, B, N and D lymphocytes in a simple reproducible manner. The mean percentage of the D cell counts with this method was 1.8% of total lymphocytes from normal donors, 7.0% in patients with SLE, and 29.2% in patients with malignant lymphoma. The simultaneous quantitation of T, B and N cells in these blood samples was compared to the results of conventional methods, i.e. E rosettes for T cells and EAC rosettes for B cells, which gave similar results. This combination method for simultaneous enumeration of human peripheral lymphocyte subpopulations may offer significant advances in the study and diagnosis of various clinical entities.