Thiophilic interaction chromatography of prostate-specific antigen.

Prostate-specific antigen (PSA) protein and complexes of PSA with alpha1-antichymotrypsin (PSA-ACT) or alpha2-macroglobulin (PSA-A2M) prepared in vitro, have strong affinity for different thiophilic gels (T-gel). Free PSA, and these PSA complexes can be isolated due to their affinity for T-gels. The average recovery of PSA from several of the T-gels, based upon ELISA measurements, was 84 to 94%. The data suggest that T-gel affinity can be explored for the purification of free and complexed PSA from various biologic fluids.

Mild hyperthermia modulates biological activities of interferons.

A significant enhancement of antiviral activity of human IFN-alpha, -beta and -gamma and murine IFN-gamma is observed when cells are treated with mild hyperthermia (39 degrees C) during antiviral assays. Treatment of primary human fibroblast cells with mild hyperthermia for 4 and 24 hours prior to interferon antiviral assays (pre-assay hyperthermia) further enhances interferon antiviral activity. An enhancement of interferon induced enzyme, 2,5-oligoadenylate synthetase, is also observed in cells treated with interferon and mild hyperthermia. This increase in enzyme activity is, in part, responsible for the observed increase in interferon antiviral activity with hyperthermia. Besides antiviral activity, mild hyperthermia also increases interferon antiproliferative activity on different tumour cells beyond its effect at normal physiological temperatures. On the other hand, mild hyperthermia decreases human interferon production in both human and murine cells when challenged with a viral or non-viral inducer. Also, mild hyperthermia suppresses interferon-mediated enhancement of natural killer (NK) cell activity in human and murine cells. The findings demonstrate that, although mild hyperthermia has suppressive effects upon interferon production and NK cell activity, it significantly increases both antiviral and antiproliferative activities of all three human interferons. These observations have direct bearing upon clinical utilization of exogenously administered interferons to late stage cancer patients who for the most part have a weaker immune system. In these patients, the antiviral and antiproliferative efficacies of administered interferon can be enhanced by combining interferon and hyperthermia.

Cocaine differentially modulates chemokine production by mononuclear cells from normal donors and human immunodeficiency virus type 1-infected patients.

Earlier studies have supported a significant role for cocaine in the susceptibility to and the progression of human immunodeficiency virus type 1 (HIV-1) infection. Recently, several unique HIV-1 entry coreceptors (e.g., CCR5 and CCR3) and a trio of HIV-1-specific suppressor chemokines, namely, RANTES (regulated-upon-activation T expressed and secreted), macrophage inflammatory protein 1alpha (MIP-1alpha) and MIP-1beta, were identified. Although cocaine has been linked to the immunopathogenesis of HIV-1 infection, the corresponding cellular and molecular mechanism(s) have not been well defined. We hypothesize that cocaine mediates these pathologic effects through the downregulation of HIV-1-suppressing chemokines and/or upregulating HIV-1 entry coreceptors in HIV-1-infected subjects, resulting in disease progression to AIDS. Our results show that cocaine selectively downregulates endogenous MIP-1beta secretion by normal peripheral blood mononuclear cells (PBMC), while cocaine did not affect the MIP-1beta production by PBMC from AIDS patients. Cocaine also selectively suppresses lipopolysaccharide-induced MIP-1beta production by PBMC from HIV-infected patients. Further, cocaine significantly downregulates endogenous MIP-1beta gene expression, while it upregulates HIV-1 entry coreceptor CCR5 by normal PBMC. These studies suggests a role for cocaine as a cofactor in the pathogenesis of HIV infection and support the premise that cocaine increases susceptibility to and progression of HIV-1 infection by inhibiting the synthesis of HIV-1 protective chemokines and/or upregulating the HIV-1 entry coreceptor, CCR5.

Mechanism(S) of interferon inhibitory activity in blood from patients with AIDS and patients with lupus erythematosus with vasculitis.

We have earlier reported that patients suffering from acquired immuno-deficiency syndrome (AIDS), systemic lupus erythematosus (SLE) with vasculitis, Wegner granulomatosis and certain types of late stage cancer have interferon inhibitory activity in their serum. The purpose of this study was to identify the factor(s) involved in this interferon inhibitory activity. Twenty patients with advanced AIDS, twenty patients with SLE and vasculitis and twenty normal healthy controls between ages 25-40 years were studied. In contrast to normal, healthy controls, significant interferon inhibitory activity was found in AIDS and SLE patients. This appears to be largely due to: (a) increased soluble circulating interferon alpha/beta receptors, (b) increased prostaglandin E2 levels which inhibits interferon and (c) a interferon inhibitory protein. Further understaging of the nature of interferon inhibitory activity in the patient's sera and development of anti-interferon inhibitory agents would greatly enhance interferons potential as a treatment modality.

Immunoregulatory effects of morphine on human lymphocytes.

It is now well established that parenteral drug abuse is a significant risk factor for contracting human immunodeficiency virus type 1 (HIV-1) infection and subsequently developing AIDS. Earlier studies have shown that morphine can modulate various immune responses and therefore support the premise that morphine is a cofactor in susceptibility to and progression of HIV infection. Dysregulation of interferon (IFN) production, nonspecific apoptosis of T cells, and the immune response to soluble HIV gene products have been associated with potential mechanisms of pathogenesis in HIV disease. The present study was undertaken to examine the immunomodulatory role of morphine on HIV protein-induced lymphocyte proliferative responses, Sendai and Newcastle disease virus-induced alpha IFN (IFN-alpha) and IFN-beta production by lymphocytes and fibroblast cells, respectively, and induction of apoptosis of normal lymphocytes in vitro. Our results demonstrate that HIV protein-induced human lymphocyte proliferative responses were significantly inhibited by morphine in a dose-dependent manner. Furthermore, morphine significantly inhibited both IFN-alpha and IFN-beta production by normal lymphocytes and fibroblasts but induced apoptosis of normal lymphocytes. Inhibition of IFN-alpha production by morphine could be reversed by the opiate receptor antagonist naloxone. This suggests that the immunomodulatory effects of morphine are mediated through the opioid receptor. These studies support a role of morphine as a cofactor in the pathogenesis of HIV infection and describe some of the possible pathologic mechanisms which underlie the immunoregulatory effects of morphine.

Differential effects of human immunodeficiency virus type 1 envelope protein gp120 on interferon production by mononuclear cells from adults and neonates.

While considerable progress in examining the course of human immunodeficiency virus (HIV) infection in adults has been made, a better understanding of the natural history of perinatal HIV infection remains to be obtained. Dysregulation of the production and functions of various cytokines, especially the interferons (IFNs), during HIV infections has been reported. Using an in vitro model system, we examined the effects of the HIV type 1 envelope protein, gp120 (10, 50, and 100 ng/ml), on gamma IFN (IFN-gamma) and IFN-alpha production by lymphocytes from neonates and adults and also examined the potential regulatory effects of gp120 on phorbol 12-myristate acetate (PMA)- and Sendai virus-induced IFN-gamma and IFN-alpha production by lymphocytes. PMA at a concentration of 50 ng/ml plus 50 ng of calcium ionophore A23187 per ml was used to induce IFN-gamma, while 150 hemagglutinating units of Sendai virus was used to induce IFN-alpha production. The antiviral activity of both IFN-alpha and IFN-gamma in leukocyte culture supernatants was assayed on BG-9 cells by a dye uptake technique using vesicular stomatitis virus as a challenge virus. Placental cord blood leukocyte (CBL) samples from healthy, term infants and adult peripheral blood leukocytes (APBL) produced no IFN in response to gp120. However, CBL produced significantly decreased levels of IFN-gamma compared with APBL in response to PMA plus ionophore. gp120 significantly suppressed both Sendai virus-induced IFN-alpha and PMA-induced IFN-gamma production by both CBL and APBL in a dose-dependent manner. However, gp120-induced suppression of IFN-alpha and IFN-gamma was significantly greater with CBL than with APBL. Treatment of CBL and APBL with gp120 did not induce any phenotypic alteration of the CD45 RO+ subset. Increased suppression of IFN-alpha and IFN-gamma production by gp120 in neonates may partially explain their apparent increased susceptibility to the clinical progression of HIV infections compared with that of adults.

Segregation of normal and pathological human red blood cells, lymphocytes and fibroblasts by immobilized metal-ion affinity partitioning.

Immobilized metal-ion affinity partitioning (IMAP) is shown to be useful as a preliminary screening test and for the separation of different cell populations based upon recognition of the differences in the proteins on cell surfaces. The feasibility of using IMAP to segregate a spectrum of normal human cells (red blood cells, lymphocytes and fibroblasts) from their counterpart pathological cells has been demonstrated. A clear segregation between normal and sickle-cell anemia red blood cells (RBC), or malaria (Plasmodium vivax) infected RBCs was obtained. Further, the partition differences were found to depend on the nature and the concentrations of metal used. Cells from breast cancer and those from the lung adenocarcinoma showed differences in their partition pattern as compared to normal fibroblasts when PEG-IDA-M(II) was added to the phase system. Maximum differences between the three cell populations were observed in the presence of 10% PEG-IDA-Ni(II). Normal lymphocytes and Burkitt's lymphoma cells (Raji and Namalwa cell lines) were shown to partition differently in the presence of PEG-IDA-M(II) in the phase system. Normal lymphocytes could be distinguished from the Burkitt's lymphoma cell lines in all three phases (top, interface and bottom), in the presence of 10% PEG-IDA-Ni(II) in the system. These differences in the partition behavior could mainly be attributed to the density, surface exposure and micro-environment of histidine residues of cell membrane-associated proteins. These data, along with those obtained for normal and pathological human cells show that IMAP could be a simple and versatile tool for the segregation and study of cells.

Selective inhibition by alcohol and cortisol of natural killer cell activity of lymphocytes from cord blood.

1. The immunosuppressive effects of drugs such as alcohol or hormones such as cortisol may be age-related. To test this hypothesis, the authors investigated the in vitro effects of ethanol (EtOH) and cortisol on Natural Killer (NK) cell activity of lymphocytes from normal cord blood in comparison with that of lymphocytes from normal adult peripheral blood. 2. K562, an erythroleukemia cell line, was used as a target in a 4 hr 51Cr release assay. 3. Ethanol at 0.3% (V/V) and cortisol at 0.05, 0.1 and 0.2 microgram/ml concentrations, added directly to a mixture of effector and target cells significantly suppressed the NK activity of cord blood lymphocytes in a dose dependent fashion, whereas similar concentrations of either EtOH or cortisol did not manifest significant immunoregulatory effects on NK cell activity of normal adult lymphocytes. 4. Pre-treatment of the target with either EtOH or cortisol for 4 hours did not affect cytotoxicity. Inhibition of cytotoxicity was also not due to direct toxicity of effector cells because lymphocytes treated with either EtOH or cortisol showed normal 51Cr release and their viability was comparable to that of untreated control cells. 5. This suggests a selective inhibitory effect of EtOH and cortisol on NK activity of neonatal lymphocytes that may be of clinical significance.

Pentoxifylline and meclofenamic acid treatment reduces clinical manifestations in a murine model of AIDS.

C57/BL/6 mice infected with LP-BM5 MuLV virus developed an AIDS-like disease (MAIDS) with splenomegaly, leukopenia, thrombocytopenia, anemia, decreased numbers of helper/inducer and suppressor/cytotoxic T-cells and decreased production of interferon alpha. We have shown previously that HIV-associated Kaposi's sarcoma tissue contains high levels of prostaglandin E2 (PgE2), and this inhibits interferon synthesis through a cAMP-dependent second-messenger process. In this study we treated groups of MAIDS-infected mice with combinations of pentoxifylline, an agent which increases cAMP and inhibits phosphodiesterases, and sodium meclofenamic acid, a PgE2 inhibitor. Treated mice showed: 1) significantly higher total leukocyte and platelet counts, 2) higher total L3T4+ (helper/inducer) and Lyt-2+ (suppressor-cytotoxic) T-cell population. Pathologic examination also showed significantly less hepatosplenomegaly and lymphadenopathy in animals treated with pentoxifylline and meclofenamic acid. Partly, PgE2-induced suppression of interferon alpha production may mediate expression of retrovirus infection in this murine model of AIDS.

The alpha-interferon system in patients undergoing renal transplantation and treated with cyclosporine and prednisone.

The interferon system was investigated in 65 normal control patients and ten patients with chronic renal disease, approximately a half year after renal transplantation and treatment with prednisone and cyclosporine. In previous studies, comparable doses of prednisone had no effect on the interferon system. It was assumed that the changes observed were primarily the result of cyclosporine therapy. In contrast with normal persons, low levels of interferon-alpha (IFN-alpha) were found in the circulation of patients. It was thought that this may be related to low grade infections in immunosuppressed persons. In patients, the IFN-alpha synthesizing capacity of leukocytes was significantly decreased. IFN-alpha inhibitor level was slightly increased in two patients. Inhibition of IFN-alpha (IFN-alpha) synthesizing capacity may be part of the immunosuppression mechanism of action of cyclosporine in patients undergoing transplant.

A discriminant function analysis of various interferon parameters among alcoholics and heavy smokers.

A three-group design of alcoholics, heavy smokers and controls was used to investigate the status of the interferon system, including natural killer cell activity. Group differences were indicated via discriminant function analysis which correctly classified 86% of subjects. Test-retest relations were investigated for 14 subjects following a 30-day alcoholic inpatient program. Whereas significant immune suppression was indicated at the time of detoxification, recovery was evident at the 30-day follow-up. Results are discussed in terms of the significance of alcohol abuse on immune system functioning with consequences for susceptibility to viruses, bacteria and medical illnesses.

Effect of alcohol on spleen cells and their functions in C57BL/6 mice.

Spleen cells from C57BL/6 mice maintained on alcohol containing liquid diet for two weeks were evaluated for different immune functions. On an average, 22% fewer spleen cells were recovered from alcohol-fed mice when compared to cells from control animals. In alcohol-fed mice, the relative frequency of B cells increased, whereas total T cells including CD4+ cells decreased significantly. Alcoholic mice, when challenged with poly(rI) poly(rC), produced significantly less interferon than control mice. In vitro production of interferon alpha and gamma by the spleen cells of alcoholic mice was reduced by 67-90%. No significant differences were seen in the level of natural killer cell activity in spleen cells of control and alcoholic mice. These results suggest that chronic alcohol intake can result in not only changes in the number of immune cells, but more importantly affect their biological functions such as their ability to produce interferons.

Effect of an interferon inhibitor on the antiproliferative signal of interferon-alpha.

We have described a nonantibody type inhibitor of interferons (IFN) in the blood of patients with AIDS, advanced neoplastic disorders, and lupus erythematosus in earlier reports (1,2). In the present study we show that the semipurified inhibitor blocks the antiproliferative signal of IFN-alpha in Daudi cells and the membrane potential shifting ability of IFN-alpha is modulated by the interferon inhibitor preparation (IFI).

Effects of meclofenamate on natural killer cell activity.

The effect of meclofenamate on natural killer cell activity was studied in a four hour 51Cr release assay. Meclofenamate, a nonsteroidal anti-inflammatory drug, had no effect on natural killer cell activity when added to the mixture of target (K562) and effector (peripheral blood mononuclear) cells either at the beginning of the reaction or when effector cells were pretreated with meclofenamate for 12-24 hours. However, a significant increase (p less than 0.0005) in the natural killer cell activity was seen when target cells were pretreated with meclofenamate for 12-24 hours prior to their mixing with effector cells. This increase in natural killer cell activity was observed consistently when target cells were taken from different donors.

Interferon inhibitor in the blood of patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) patients at advanced stages of the disease have an interferon inhibitor in the blood circulation. This inhibitor can block antiviral activity of all three types of human interferons and can significantly reduce the synthesis of interferon alpha by the treated lymphocytes obtained from normal healthy individuals. Available evidence suggests that inhibitor activity is neither because of the antibody to interferon nor due to high level of protease-like activity in the plasma. The inhibitor has also been shown to be effective in eliminating the interferon-mediated enhancement of natural killer cell activity. Interferon inhibitory activity was not detected in any of the sera taken from normal healthy individuals. Identification and characterization of interferon inhibitor has direct bearing upon effective utilization of interferons in the clinic.

On the mechanism of action of interferons: interaction with nonsteroidal anti-inflammatory agents, pentoxifylline (Trental) and cGMP inducers.

In earlier studies, we showed that when interferon alpha (IFN alpha) binds to cell receptors, it alters membrane potentials. In IFN-sensitive Daudi cells derived from Burkitt's lymphoma patients, IFNs produce dose-dependent hyperpolarization, decreased plasma membrane viscosity, modulation of the microfilament system, and altered synthesis of cAMP, cGMP and prostaglandins. No such changes were seen in IFN alpha-resistant Daudi cells. In this study, we found that indomethacin, pentoxifylline, or the cGMP inducer sodium azide (NaN3) had no significant effect on the IFN alpha induced membrane potential changes, or on membrane viscosity changes as measured by flow cytometry and electron spin resonance spectrometry. In tissue culture, indomethacin did not alter the anti-proliferative effect of IFN alpha on Daudi cells. Indomethacin may influence IFN synthesis, but not it's antiproliferative actions. Relatively high doses of pentoxifylline slightly inhibited proliferation of Daudi cells and synergized with IFN alpha. Measurement of early biophysical changes induced by IFNs may represent a new screening method to rapidly explore certain types of IFN alpha potentiating agents.

The interferon system in carcinoma of the cervix. Effect of radiation and chemotherapy.

Thirteen patients with advanced carcinoma of the cervix were studied for parameters of the interferon system compared with 40 age-matched and sex-matched controls. All patients had measurable serum interferon levels; controls did not. All patients had non-antibody-type interferon-inhibitory activity, and controls had none. Interferon-synthesizing potential was higher in controls than in patients. After successful radiation and chemotherapy, these parameters normalized in the patients. No change was seen in one patient who did not respond to therapy.

Effect of calmodulin antagonists on the interferon system: induction and action of interferons.

Synthesis of interferon (IFN) in response to Sendai virus and the development of the resulting antiviral state were studied in human (BG-9) and murine (L-929) fibroblast cell cultures in the presence of the calmodulin antagonists, trifluoperazine (TFP) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). Compared to control cultures, 16-fold and 8-fold more IFN was formed in human and murine cells, respectively, when 10 microM TFP was present in the medium for 24 h prior to IFN induction with Sendai virus. W-7 did not affect IFN production in human cells, but enhanced it in L-929 cells by 4- to 8-fold. TFP inhibited the antiviral state induced by homologous IFN in the two cell systems, and at 20 microM, there was a 3,000-fold increase in vesicular stomatitis virus (VSV) yield. It also reduced the maintenance of the antiviral state in human cells. In contrast, W-7 had no effect on the development of the antiviral state in either of the two cell systems. Thus, calcium-calmodulin dependent cellular processes are involved in both induction of IFN and its action. The several patterns response to TFP and W-7 may reflect different ligand-binding sites on calmodulin.