In vivo effect of an immunostimulating bacterial lysate on human B lymphocytes.

The aim of the present study is to investigate in humans the mechanism by which the oral vaccine Polyvalent Mechanical Bacterial Lysate (PMBL) can rapidly mobilize specific immune response and evaluate the efficacy of its immunostimulating activity in preventing recurrent infections of the upper respiratory tract (URTIs) in a group of patients with a medical history of URTI recurrence. Patients received, by sublingual route, PBML, an immunostimulating lysate obtained by mechanical lysis of the most common bacteria responsible for upper respiratory tract infections. The treatment was administered for 10 consecutive days/month for 3 consecutive months. After the end of the treatment period the patients were followed up for an additional 3 months. The frequency of IgM memory B cells and the expression of the activation marker CD25 in peripheral blood lymphocytes were measured using the flow cytometric method before the start and at days 30 and 90 of the treatment cycle. To correlate clinical results to immunological parameters, the patients were monitored at different time-points during the treatment and at the end of follow-up period. The results showed that PMBL exerts a therapeutic and preventing effect in acute and recurrent infections of the upper respiratory tract and that this effect correlated with the activation and enhancement of both IgM memory B lymphocytes (CD24+/CD27+ cells) and IL2 receptor-expressing lymphocytes (CD25+ cells) involved either in humoral or cellular immunity.

Effect of resveratrol on proliferation and telomerase activity of human colon cancer cells in vitro.

A number of studies performed in our laboratory and elsewhere, showed that resveratrol is able to prevent carcinogenesis and to impair tumor growth and progression. In order to provide additional information on the pleiotropic effects of resveratrol on malignant cells, the present study was performed to test the in vitro influence of the compound on the growth and TLMA of HT-29 and WiDr human colon cancer cell lines. The results confirmed that resveratrol has a direct, dose dependent, inhibitory effect on cell proliferation in both lines. In addition, for the first time, relatively high concentrations of this compound were found to be able to substantially down-regulate telomerase activity. These preliminary results further support the potential role of resveratrol in chemoprevention/chemotherapy of human colon tumor cells and provide the rational basis for novel strategies in cancer control.

Effect of morphine on cell-mediated immune responses of human lymphocytes against allogeneic malignant cells.

Opioid drugs, including morphine, are largely used as pain control in cancer patients at different stages of neoplastic growth and progression. Therefore, the possible influence of these drugs on host immunity appears to be of considerable interest. We have examined in vitro the effect of morphine on the generation of human cytotoxic T lymphocytes (CTL) against HTLV-I induced T-cell leukemia cells (MT-2 line). The results show that the drug, at graded concentrations (from 3 pg/ml to 32 microg/ml), that include those detectable in treated patients, enhances CTL activity whereas natural killer cell activity was unaffected. The enhancing effect is particularly evident when morphine was present at the onset of lymphocyte/MT-2 co-culture. On the contrary, the drug was ineffective when added on the last day of co-culture, thus indicating that morphine operates during the generation phase of CTL, but not on mature CTL. Flow cytometric analysis of intracellular cytokine expression showed that morphine increases the percentage of interferon gamma-producing CD8+ T cells in co-culture assay. Collectively, these results suggest that in our experimental model morphine enhances CTL responses by directly affecting the induction phase of T-dependent cell-mediated immunity.

In vitro effects of an immunostimulating bacterial lysate on human lymphocyte function.

MLBL is an oral immunostimulating vaccine consisting of bacterial standardized lysates obtained by mechanical lysis of different strains of Gram-positive and Gram-negative bacteria that can cause acute and chronic infections of the respiratory tract. Previous studies suggested a stimulating effect of MLBL both on humoral and cellular immune responses. In the present study, the in vitro effects of MLBL on human lymphocyte effector functions and its mechanisms of action were evaluated. The results show that the most remarkable effects of MLBL on the immune system are: i) activation of the IL-2 receptor (IL-2Ralpha) on different lymphocyte subsets (B, CD4+ T and CD8+ T cells) involved both in humoral and cellular immune responses; ii) induction of cytokine synthesis (IL-2, IL-10, IL-12, IFNgamma) in the immune competent cells that induce and regulate immune responses; iii) generation of CD4+ and CD8+ effector T cells. Overall, these results suggest that the therapeutic effect of MLBL on acute and recurrent infections of the respiratory tract is related to its ability to activate the responses of different subsets of immune competent cells both for humoral and cellular immunity. Moreover, these effects can be induced either by direct immune cell activation or through the generation and activation of immune effector cells.

Effects of resveratrol on human immune cell function.

Resveratrol (3,5,4'-trihydroxystilbene), a polyphenol found in grapes and grape products such as red wine, has been reported to exhibit a wide range of biological and pharmacological activities both in vitro and in vivo. Because many of the biological activities of resveratrol, like the inhibition of cyclooxygenase, induction of CD95 signaling-dependent apoptosis, effects on cell division cycle and modulation of NF-kB activation, suggest a possible effect on the immune system, we evaluated the in vitro effects of resveratrol in three immune response models: i) development of cytokine-producing CD4+ and CD8+ T cells induced by stimulation of peripheral blood mononuclear cells (PBMC) with anti-CD3/anti-CD28; ii) specific antigen-induced generation of cytotoxic T lymphocytes; iii) natural killer (NK) activity of PBMC. The results showed that in vitro exposure to resveratrol produces a biphasic effect on the anti-CD3/anti-CD28-induced development of both IFN-gamma- IL2- and IL4-producing CD8+ and CD4+ T cells, with stimulation at low resveratrol concentrations and suppression at high concentrations. Similarly, the compound was found to induce a significant enhancement at low concentrations and suppression at high concentrations of both CTL and NK cell cytotoxic activity. On the whole, the results of the study indicate that resveratrol modulates several human immune cell functions and suggest that this activity may be related to its effects on cytokine production by both CD4+ and CD8+ T cells.

Effects of cocaine administration to influenza virus-immunized mice on cytokine profiles of individual splenic CD4+ and CD8+ T cells.

We have analysed the effects of cocaine, administered to mice during the in vivo differentiation of effector T cells stimulated by antigen (influenza virus) recognition, on the frequency of IL-2-, IL-4- and interferon-gamma (IFN-gamma)-expressing CD4+ and CD8+ T cells. Each animal was injected intraperitoneally with 10 mg/kg of cocaine 6, 24, 48 and 72 h after immunization with A/PR8 influenza virus (PR8). This enabled the determination of the pharmacological effects of cocaine on T cells during the initial step of the immune response, which is characterized by the production of large amounts of immunoregulatory cytokines. The distribution of IL-2-, IL-4- and IFN-gamma-producing CD4+ and CD8+ T cells was assayed on unseparated PR8-immune spleen cells, obtained from mice treated with cocaine or vehicle, and restimulated in vitro with UV-inactivated PR8 virus. The frequency of T cells singly or co-expressing the above three cytokines was determined at single-cell level by simultaneous flow cytometric analysis of intracellular cytokines and surface antigen expression. In parallel, the levels of IL-2, IL-4 and IFN-gamma in the culture supernatants were quantified by ELISA. The results showed that cocaine, administered during the in vivo virus-induced differentiation of T cells, caused an increase of both the frequencies of CD8+ T cells singly and co-expressing IL-2 and IFN-gamma and the levels of these cytokines in virus-restimulated spleen cell culture supernatants, compared with those of untreated controls. In contrast, no effect was found on IL-4-positive CD8+ T cells and on IL-2-, IFN-gamma- and IL-4-positive CD4+ T cells. Our findings suggest that the immunomodulatory effects of cocaine may be due to the up-regulation of the production of IL-2 and IFN-gamma by CD8+ T cells with a type 0 cytokine profile.

Determination of cytokine co-expression in individual splenic CD4+ and CD8+ T cells from influenza virus-immune mice.

We have studied the patterns of interleukin-2 (IL-2), IL-4 and interferon-gamma (IFN-gamma) co-expression displayed by individual splenic CD4+ and CD8+ T cells in response to influenza virus immunization. Unseparated spleen cells obtained from mice intraperitoneally (i.p.) injected with A/PR8 (H1N1) influenza virus (PR8) were cultured for 24 hr in the presence of ultraviolet-inactivated PR8. As controls, cultures of both naive spleen cells stimulated with PR8 or of immune cells lacking the inactivated virus were used. The frequencies of CD4+ and CD8+ T cells expressing IL-2, IL-4 and IFN-gamma were determined by three-colour flow cytometric analysis of fixed and saponin-permeabilized cells fluorescent-stained for either CD4 or CD8 surface molecules and for one of the following combinations of two intracellular cytokines: IL-2/IL-4, IL-2/IFN-gamma and IL-4/IFN-gamma. The results showed that immunization with influenza virus induces in both CD4+ and CD8+ T cells a heterogeneity of cytokine response patterns that do not follow the type 1/type 2 polarized response model, but with substantial differences between the two populations. In fact, the analysis of the phenotypes of virus-immune CD8+ T cells revealed similar significant proportions of cells either expressing any one of the three cytokines or co-expressing combinations of them (i.e. IL-4/IL-2, IL-4/IFN-gamma and IL-2/IFN-gamma), whereas immune CD4+ T cells were seen to express almost exclusively a single cytokine per cell. The observed patterns of cytokine production suggest that influenza virus immunization induces the expression of a type 0 cytokine pattern at both population and single cell levels in CD8+ T cells and exclusively at the population level in CD4+ T cells.

Chronic morphine alters calbindin D-28k immunostaining patterns in mouse forebrain.

The influence of chronic morphine treatment on the brain of adult mouse has been studied. Female Swiss mice were daily administered saline or morphine (30 or 60 mg/kg body weight) for a period comprising 7 days before mating, during gestation and until 21 days post-partum. Their brains were then perfusion-fixed and examined for histology and calbindin D-28k protein-immunoreactivity. Histological observations revealed no significant changes in the various brain regions; whereas a reduced number of calbindin-positive cells was encountered in the cingulate and parietal cortices and the lateral septal regions of morphine-treated brains compared with those of controls. The alteration in the expression-patterns of this neuroprotective calcium-binding protein in specific regions of the adult brain might be one of the mechanisms by which the addictive drugs modify the functional aspects of the CNS.

Splenic CD4+ and CD8+ T cells from influenza immune mice concurrently produce in vitro IL2, IL4, and IFN-gamma.

The cytokine responses exerted by virus-primed spleen T cells upon in vitro restimulation were studied. Spleen cells obtained from mice injected intraperitoneally with A/PR8 (H1N1) influenza virus (PR8) were restimulated in vitro with UV-inactivated PR8 virus. The percentage of both CD4+ and CD8+ T cells producing IL2, IL4, or IFN-gamma was assayed at the single cell level by flow cytometric analysis of intracytoplasmic cytokine content. In parallel, the levels of the different cytokines in spleen cell culture supernatants were quantitated by enzyme linked immunosorbent assay. The results showed that in vitro virus restimulation of immune spleen cells induced the concurrent increase, in both CD4+ and CD8+ T cells, of the frequency of IL2-, IFN-gamma-, and IL4-producing cells. The frequency of IFN-gamma-producing T cells was found to be significantly higher in CD8+ T cells. Significant levels of the three cytokines were also detected in the culture supernatants. These data suggest that both CD4+ and CD8+ T cells play an important role in cytokine response to virus infection and that the synthesis and secretion of antiviral and regulatory cytokines is not mutually exclusive either between or within the two T cell subsets. The results of the experiments also indicated that the virus restimulation did not induce a dominant type 1 or type 2 cytokine response.

In vitro effects of cocaine on cytokine secretion induced in murine splenic CD4+ T cells by antigen-specific stimulation.

The in vitro effects of cocaine on antigen-specific-induced cytokine production by murine splenocytes was evaluated both by quantitation by ELISA of the cytokines in culture supernatants and by flow cytometric analysis of the frequency of the cytokine-producing CD4+ T cells. Spleen cells from mice immunized with ovalbumin (OVA) were restimulated with OVA in the presence or absence of cocaine for different periods of time and then evaluated for production of cytokines. Exposure to cocaine was found to reduce the levels in culture supernatants of IL2 and IFN-gamma, whereas IL4 and IL5 levels were not changed. Flow cytometric analysis showed that cocaine increased the frequency of IL2- but not of IL4-producing CD4+ T cells. Kinetics studies indicated that the in vitro antigen-specific-induced production of IL2 is faster than that of IL4 and that cocaine did not affect the production kinetics of either cytokine. Collectively, the results suggest that in vitro cocaine acts by interfering with the secretion rather than with the synthesis of cytokines and that the drug exerts different effects on cytokines with different production kinetics.

Differential effects of short-term or prolonged cocaine exposure on peripheral blood cells in mice.

The effects of cocaine on the number of murine peripheral blood cells and on phenotypic expression of lymphocyte subsets were examined using different regimens of drug exposure. Cocaine administered to mice for 7 consecutive days at 1 and 10 mg/kg/day reduced the absolute number of circulating white blood cells and the relative number of cells stained as lymphocytes, polymorphonuclear cells and monocytes. Cocaine did not modify the relative proportion between lymphocytes, polymorphonuclear cells and monocytes. Parallely, there were no changes in the percentage of circulating CD4+ and CD8+ lymphocytes, while the number of natural killer cells increased. In sharp contrast, we didn't observe any significant change in peripheral blood cells after 30 days of consecutive drug administrations.

Effect of synthetic thymic hormones on the cocaine-induced inhibition of the primary immune response in mice.

The effects of thymosin alpha 1 (T alpha 1) and thymopentin (TP5) on the cocaine-induced impairment of the primary antibody response to sheep red blood cells (SRBC) was studied. The administration of cocaine from day -4 to the day of immunization (day 0) induced a significant impairment of the response to the T-dependent antigen SRBC, as evaluated on day 5 post-immunization by the Splenocyte-Induced SRBC Hemolysis (SIH) assay. The analysis of the responses to immunogen elicited from each single mouse indicated that, under the experimental conditions used, cocaine acted by exerting more an "all or nothing" effect rather than by modulating the strength of the immune response. Both T alpha 1 and TP5, injected into mice during cocaine administration and for 4 days after, induced a significant recovery of the response to SRBC. Our experiments did not show any great differences in the overall efficacy of the two drugs, although they showed quite a different dose-response effect. The results of the present investigation demonstrated the capability of TP5 and T alpha 1 to reverse the cocaine-induced impairment of the response to SRBC and suggested that the effect of the two peptides may be related to their immunomodulating activities on T-cell functions.

Thymic hormone modulation of CD38 (T10) antigen on human cord blood lymphocytes.

We studied the in vitro effect of three different thymic factors on the expression of CD38 (T10) antigen on cord T-lymphoid cell surface. The results showed that cord mononuclear cell populations contain variable percentages of CD38+ cells. The CD38 molecule was expressed on cord T and B lymphocyte and monocyte surfaces. Incubation with thymic agents induced a significant increases in the CD38+ cell percentage only in the samples with low CD38 antigen expression, and this modulation was mainly attributable to the T-cell subset. The effect seems to be specific and not correlated with the known high spontaneous DNA synthesis rate of cord mononuclear cells.

Effect of thymostimulin in models of cell-mediated and humoral autoreactivity and on T-dependent suppression.

To explore the therapeutic potential of the thymic hormone preparation thymostimulin (TS) in animal models of cell-mediated and humoral autoimmunity, its effects were investigated on experimental allergic encephalomyelitis in guinea pigs and on anti-erythrocytic autoantibody production in C57B1/6 mice. In both autoimmunity models, TS produced significant therapeutic effects in terms of proportion of diseased animals, disease severity and/or disease duration; however, both the TS dose and the time of treatment start relative to the disease-inducing stimulus critically influenced results. TS effects on the generation and expression of suppressive activity induced in C57B1/6 mice by a supraoptimal immunization with 10(10) SRBC were also examined. TS given after 10(10) SRBC did not influence the level of suppression, and the activity of effectors of suppression was not modified by this agent. Conversely, using a treatment protocol analogous to that effective in reducing murine autoantibody production, TS administration prior to 10(10) SRBC was associated with a significant increase in the subsequent generation of T-dependent, antigen-specific suppressive activity. These findings suggest that effects of TS on the development of suppressor cells may be involved in the activity of this agent in animal models of autoaggression.

Combined effects of cyclophosphamide and thymostimulin treatment on the in vitro natural killer and candidacidal activity of murine spleen cells.

Two different natural cell-mediated cytotoxic reactions (lysis of YAC-1 and killing of Candida albicans) in vitro were studied in mice undergoing treatment with cyclophosphamide (Cy), thymostimulin (TP-1) or a combination of both. Enhancing effects followed the combination regime in the microbial natural cell-mediated cytotoxicity assay, whereas in the NK assay the effect of TP-1 appeared to be antagonistic to that of Cy. The possible mechanisms involved are discussed.

Impaired T-cell functions in aged guinea-pigs restored by thymostimulin (TS).

The age-related changes of different T-cell activities in guinea pigs and the effect of Thymostimulin (TS), a thymus extract, on the immunocompetence of these cells was studied. Mitogen-induced proliferation of peripheral blood lymphocytes was increased by TS in vitro. The intraperitoneal administration of TS (5 mg/kg) to aged animals restored the helper function of T lymphocytes and enhanced the reactivity to mitogens of both peripheral blood lymphocytes and spleen lymphocytes. The data obtained suggest that as in other species, there is an age-associated decline of immunological response, in guinea pigs too, probably due to a deficiency of thymic hormone(s) and that TS could correct this deficiency.

Enhancement of the immunocompetence in neonatal guinea pigs by a thymic factor (TS).

The immunocompetence of newborn guinea pigs was evaluated in comparison with that of adults. The capacity of spleen cells from neonatal guinea pigs to produce antibodies to SRBC was found to be reduced, while no differences were observed in the capacity of those cells to respond to mitogenic stimulation or to form E rosettes with rabbit red blood cells. The in vivo treatment of newborn animals with a thymic factor (TS) induced an increase in the number of antibody-producing cells and an increase in the response to mitogenic stimulation but no variation in the number of rosette-forming cells. The effect of TS on both responses to SRBC and to mitogenic stimulation, was dose dependent.

Circulating platelet aggregates: a chronic platelet activation in patients with transient ischaemic attacks.

Platelet hyperaggregability was tested in 62 TIA patients by means of the formalin fixing principle of Wu and Hoak. Compared to age-matched controls, the platelet aggregates ratio (PAR) was reduced from 0.99 +/- 0.04 SD to 0.807 +/- 0.14 SD. This means a highly significant increase of circulating platelet aggregates (CPA) in untreated TIA patients, regardless of the interval sinche the last attack (from 2 days to 1 year). PAR was especially reduced in patients with abnormal angiography (expressing pronounced cerebral or diffuse ATS) and it was possibly affected by previous antiplatelet treatment with ASA.