Biomedical subjects

E Garaci

Publications and source records attributed to E Garaci.

At least 37 records · Page 2Linked to original sources

PubMed1999

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.

Animals↗

PubMed1998

Cocaine induced T cell proliferation in the rat: role of amygdala dopamine D1 receptors.

The immunomodulatory effects of local administration of cocaine into the amygdala were studied in the rat. Intra-amygdala infusion of cocaine significantly and dose-dependently increased the proliferative response of splenocytes to concanavalin A (Con A). A similar effect on the immune response was also observed in rats, microinfused into the central amygdala with the selective D1 receptor agonist SKF 38393. The increase of the proliferative response of splenocytes to Con A was inhibited by coinfusion within the central amygdala of the dopamine D1 receptor antagonist SCH 23390, together with cocaine, but not by coinfusion of the dopamine D2 receptor antagonist eticlopride. These results suggest that cocaine may produce at least some of its effects on the immune system through the activation of brain dopamine neurotransmission and that the central amygdala may represent a critical structure mediating cocaine-induced T cell proliferation.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗

PubMed1998

Detection of high nerve growth factor serum levels in AIDS-related and -unrelated Kaposi's sarcoma patients.

OBJECTIVE: Nerve growth factor (NGF) is an autocrine survival factor for memory B lymphocytes. As functional B-cell deregulation is present during HIV infection, NGF serum levels were measured in HIV-infected patients and compared with the patients' clinical features. DESIGN: Sera from 48 consecutive HIV-infected patients and matched healthy controls were retrospectively and blindly analysed. Sera from seven patients with classical Kaposi's sarcoma (KS) were subsequently included in the study. The effects of NGF on spindle-shaped cells from KS lesions (KS cells) were also investigated. METHODS: NGF titration was performed by enzyme-linked immunosorbent assay (ELISA) and human herpesvirus 8 (HHV-8) antibody testing by immunofluorescent assay (IFA). NGF receptors were assessed by Western blot analysis. Cell growth assays were performed by cell counting. RESULTS: Very high median NGF serum levels were detected in all seven patients with AIDS-related KS (2500 pg/ml) compared with HIV-infected patients without KS (40 pg/ml), as well as in all seven classical KS patients (550 pg/ml) compared with healthy controls (20 pg/ml). In HIV-infected patients, NGF serum levels were significantly related to KS (P=0.0038) by stepwise multiple regression analysis, and HHV-8 seropositivity was significantly associated with KS (P=0.045) and to NGF levels (P=0.001) by logistic regression analysis. KS cells did not produce NGF but expressed both NGF receptors and presented increased proliferation rate after exogenous NGF addition. CONCLUSIONS: These results strongly suggest that increased NGF levels, possibly related to HHV-8 infection, may be involved in KS progression.

Antibodies, Viral↗

PubMed1998

Differential effects of acute morphine administrations on polymorphonuclear cell metabolism in various mouse strains.

This paper shows that an acute morphine treatment dose-dependently alters the energetic and oxidative metabolism of polymorphonuclear leukocytes obtained from BALB/c and DBA/2 mice, while phagocytic cells from C57BL/6 were not affected. In sensitive mouse strains, i.e. BALB/c and DBA/2, morphine decreased both ATP concentration and energy charge potential. At the same time, ATP catabolic products, i.e. nucleosides (inosine+adenosine) and oxypurines (hypoxanthine+xanthine+uric acid), significantly increased, indicating an imbalance between energy production and consumption. Morphine treatment also induced malondialdehyde and superoxide anions production in leukocyte cells from sensitive mice. The opiate antagonist naloxone blocked morphine-induced modifications by the lower morphine dose. The same parameters in cells from C57BL/6 mice were not affected. These findings confirm that: i) the phagocytic cells are an important target for the in vivo effects of morphine, and ii) the genotype-dependent variation influences the immunological responsiveness to opiates.

Animals↗

PubMed1998

The conformation of peptide thymosin alpha 1 in solution and in a membrane-like environment by circular dichroism and NMR spectroscopy. A possible model for its interaction with the lymphocyte membrane.

The 28-residue peptide thymosin alpha1 was studied by circular dichroism and two-dimensional NMR. Circular dichroism indicates that thymosin alpha1 in water solution does not assume a preferred conformation, while in the presence of small unilamellar vesicles of dimiristoylphosphatidylcholine and dimiristoylphosphatidic acid (10:1) and in sodium dodecyl sulphate, it assumes a partly structured conformation. Presence of zinc ions produces similar effects. In a more hydrophobic environment like a solution of a mixed solvent water-2,2,2 trifluoroethanol, it adopts a structured conformation. NMR spectra indicated that in this mixture as solvent, thymosin alpha1 has a structure characterized by two regions. A beta-turn is present between residue 5 and residue 8, while the region between residues 17 and 24 shows an alpha helix conformation. These changes of conformation in different environments may be considered structural requirements in the steps of its interaction with the lymphocyte membrane. In fact, these conformational changes may correspond to the first event of the mechanism of lymphocyte activation in the immune response modulation by thymosin alpha1.

Adjuvants, Immunologic↗

PubMed1998

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.

Animals↗

PubMed1997

Antiretroviral effect of combined zidovudine and reduced glutathione therapy in murine AIDS.

A combination of antiretroviral drugs acting at different points in the virus replication cycle was evaluated in a murine retrovirus-induced immunodeficiency model of AIDS (MAIDS). Intramuscular administration of high doses of reduced glutathione (GSH, 100 mg/mouse/day) and AZT (0.25 mg/ml in drinking water) was found to reduce lymphoadenopathy (92%), splenomegaly (80%), and hypergammaglobulinemia (90%) significantly more than AZT alone. Combined treatment resulted in a reduction in proviral DNA content of 69, 66, and 60%, respectively, in lymph nodes, spleen, and bone marrow. Furthermore, the stimulation index of B cells was also significantly higher in animals receiving GSH and AZT whereas additional responses were not observed in the T cell stimulation index and blood lymphocyte phenotype analyses. In conclusion, the administration of high doses of GSH and AZT, a new combination of antiviral drugs, seems to provide additional advantages compared to single-agent therapy.

Animals↗

PubMed1997

Loss of GSH, oxidative stress, and decrease of intracellular pH as sequential steps in viral infection.

Madin-Darby canine kidney cells infected with Sendai virus rapidly lose GSH without increase in the oxidized products. The reduced tripeptide was quantitatively recovered in the culture medium of the cells. Since the GSH loss in infected cells was not blocked by methionine, a known inhibitor of hepatocyte GSH transport, a nonspecific leakage through the plasma membrane is proposed. UV-irradiated Sendai virus gave the same results, confirming that the major loss of GSH was due to membrane perturbation upon virus fusion. Consequent to the loss of the tripeptide, an intracellular pH decrease occurred, which was due to a reversible impairment of the Na+/H+ antiporter, the main system responsible for maintaining unaltered pHi in those cells. At the end of the infection period, a rise in both pHi value and GSH content was observed, with a complete recovery in the activity of the antiporter. However, a secondary set up of oxidative stress was observed after 24 h from infection, which is the time necessary for virus budding from cells. In this case, the GSH decrease was partly due to preferential incorporation of the cysteine residue in the viral proteins and partly engaged in mixed disulfides with intracellular proteins. In conclusion, under our conditions of viral infection, oxidative stress is imposed by GSH depletion, occurring in two steps and following direct virus challenge of the cell membrane without the intervention of reactive oxygen species. These results provide a rationale for the reported, and often contradictory, mutual effects of GSH and viral infection.

Adsorption↗

PubMed1997

Intracellular GSH content and HIV replication in human macrophages.

In vitro HIV-1 infection induced a significant decrease in intracellular reduced glutathione (GSH) in human macrophages. Such a decrease was observed at the time of infection corresponding to maximum release of virus from infected cells and was not related to cell cytotoxicity. GSH los was not related to its oxidation or leakage through the cell membrane. Inhibition of intracellular GSH synthesis by buthionine sulfoximine (BSO) did not further decrease GSH levels with respect to the decrease caused by HIV alone. However, treatment of macrophages with BSO significantly increased the HIV yield in the supernatant. Exogenous GSH strongly suppressed the production of p24 gag protein as well as the virus infectivity. Previous observations with other RNA and DNA viruses consistently showed that GSH antiviral effect occurred at late stages of virus replication and was related to the selective decrease of specific glycoproteins, such as gp120, which are particularly rich in disulfide bonds.

Buthionine Sulfoximine↗

PubMed1997

Combination therapy with BRMs in cancer and infectious diseases.

In recent years many studies have stressed the importance of using biological response modifiers (BRMs) in the treatment of different conditions of immune-impairment correlated with ageing, cancer and infectious diseases. In particular, the use of different BRMs in conjunction with conventional therapies has been extensively explored. Our studies have demonstrated that treatment with Thymosin alpha-1 and low doses of IFN or IL-2 exert powerful biological effects both in vitro and in vivo. They are highly effective in restoring cytotoxic activities in immunosuppression induced by tumors and/or cytostatic drugs. In addition, when combined with specific chemotherapy, they are able to induce a dramatic inhibition of tumor growth in both experimental models and in humans. Immunotherapeutic treatment also has an application in controlling infectious diseases, especially those occurring in the immuno-compromised host. The advantage of using the combined immunotherapy treatment with antiviral drugs has been recently demonstrated by our group both in a murine experimental influenza model and in patients infected with HBV, HCV and HIV.

Bacterial Infections↗

PubMed1997

Red blood cells mediated delivery of 9-(2-phosphonylmethoxyethyl)adenine to primary macrophages: efficiency metabolism and activity against human immunodeficiency virus or herpes simplex virus.

Red blood cells (RBC) may act as selective carriers of drugs to macrophages, an important reservoir of viruses such as human immunodeficiency virus (HIV) and herpes simplex virus type 1 (HSV-1). We therefore assessed the incorporation of 9-(2-phosphonylmethoxyethyl)adenine (PMEA), a potent inhibitor of HIV and HSV-1) into RBC, its delivery to macrophages and its activity against HIV or HSV-1. Loading of PMEA in artificially aged opsonized RBC affords significant levels of intracellular PMEA. RBC metabolize PMEA to its active congener PMEA-diphosphate, although with low efficiency. Exposure of macrophages to RBC-encapsulated PMEA inhibits the replication of both HIV and HSV-1 (about 90% inhibition at the highest RBC:macrophages ratios) even if RBC were removed before virus challenge. By contrast, the antiviral activity of free PMEA removed before virus challenge was irrelevant at concentrations up to 150-fold higher than the 50% effective concentration (EC50). Finally, the antiviral effect of RBC-encapsulated PMEA correlates with PMEA levels in macrophages about 500-fold higher than those achieved by free PMEA (at concentrations 10-fold higher than the EC50). The efficacy of RBC-mediated delivery to macrophages of PMEA (and perhaps of compounds with shorter intracellular half-lives) warrants further studies in infectious diseases involving phagocytizing cells as main targets of the pathogen.

Adenine↗

PubMed1997

Interleukin 4 stimulates infection and temporary growth of human neonatal lymphocytes exposed in vitro to human T-lymphotropic virus type I, but fails to substitute for interleukin 2 in the immortalization of infected cultures.

It has been shown that interleukin 4 (IL-4) stimulates the proliferation of cells from patients affected by adult T-cell leukaemia, the haematological malignancy aetiologically associated with human T-lymphotropic virus type I (HTLV-I). In the present study, human neonatal lymphocytes were exposed to HTLV-I in vitro in the presence of IL-4. The results showed that: (i) cultures exposed to HTLV-I in the presence of either IL-4 or IL-2 bound IL-4; (ii) IL-4 did not substitute for IL-2 as a growth factor in cell lines previously infected and maintained in IL-2; (iii) cultures exposed to HTLV-I and maintained in IL-4 or IL-2 became infected; and (iv) IL-4 sustained the growth of HTLV-I-infected cultures for a maximum of 14 weeks. Moreover, HTLV-I-infected cultures grown in IL-4 showed upregulation of the IL-4 message and lower expression of HLA-DR and CD25 when compared with counterpart cultures maintained in IL-2. These results suggest that continuous growth of T-lymphocytes induced in vitro by HTLV-I infection, at least temporarily, requires signals specifically provided by IL-2 and not by IL-4.

Cell Division↗

PubMed1996

Cocaine increases Sendai virus replication in cultured epithelial cells: critical role of the intracellular redox status.

Cocaine was found to increase parainfluenza-1 Sendai virus (SV) replication in Madin Darby canine kidney (MDCK) cells. Its effect was maximal when it was added before SV infection, while practically no effect was observed when cocaine was added at the time of or after infection. Enhanced SV replication was associated with increased viral protein expression. Cocaine also greatly reduced the intracellular level of glutathione (GSH), namely the most abundant cell thiol with antioxidant functions, recently proposed as an important factor influencing viral infection. Support for this view was provided in the present study by the reversal of cocaine-induced enhancement of SV replication when the intracellular content of GSH was restored by addition of exogenous GSH.

Animals↗

PubMed1996

Glutathione inhibits HIV replication by acting at late stages of the virus life cycle.

We investigated the effect of glutathione on the replication of human immunodeficiency virus (HIV) in chronically infected macrophages, a known reservoir of the virus in the body. We found that exogenous GSH strongly suppresses the production of p24gag protein as well as the virus infectivity. This is related to a dramatic decrease in both budding and release of virus particles from chronically infected cells (either macrophages or lymphocytes), together with a selective decrease in the expression of gp120, the major envelope glycoprotein, rich in intrachain disulfide bonds and thus potentially sensitive to the effect of a reducing agent such as GSH. Overall data suggest that GSH can interfere with late stages of virus replication. This would be in agreement with data obtained in cells exposed to herpesvirus type 1 (a DNA virus) or to Sendai (an RNA virus), showing that the suppression of virus replication by GSH is related to the selective inhibition of envelope glycoproteins. These results suggest a potential role of GSH in combination with other antivirals in the treatment of virus-related diseases.

CD4-Positive T-Lymphocytes↗

PubMed1996

Inhibition of murine AIDS by reduced glutathione.

The imbalance of the redox state in cells and body fluids in HIV-1-infected patients may result in progression of the disease as well as in immunologic disfuctions. In this report, we have evaluated whether the direct administration of high doses of reduced glutathione (GSH) exerts any antiviral activity and/or improves immune functions in a murine immunodeficiency animal model. Intramuscular administration of 50 or 100 mg GSH/mouse for five consecutive days weekly to LP-BM5-infected mice did not show local or systemic signs of acute toxicity. During the first 3 weeks from infection, a period in which clinical signs of disease were not yet detectable, GSH significantly reduced the viral load in lymph nodes and spleen as evaluated by a PCR semiquantitative assay of the proviral DNA content. At 10 weeks a GSH concentration-dependent reduction of splenomegaly, lymphadenopathy and hypergammaglobulinemia was evident in all treated mice. Evaluation of proviral DNA content showed that GSH was effective in inhibiting LP-BM5 infectivity in lymph nodes, spleen, and bone marrow at 100 mg/day, while it was less effective when administered at 50 mg/day. At 10 weeks some animals receiving the highest GSH dose died, thus only the mice receiving 50 mg GSH were followed up to 15 weeks without signs of toxicity. In this case, almost not significant differences among infected untreated or treated animals were observed. Thus, GSH is effective in reducing the proviral DNA load in the first period of infection. These data and the failure of sulfhydril supplementation to further counteract the progression of disease after 10 weeks of infection suggest that combinations of GSH and other antiviral agents may be useful for improving current antiviral therapies.

Acquired Immunodeficiency Syndrome↗

PubMed1996

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.

Animals↗

PubMed1996

Nerve growth factor is an autocrine survival factor for memory B lymphocytes.

Production of nerve growth factor (NGF) was assessed in cultures of human T and B lymphocytes and macrophages. NGF was constitutively produced by B cells only, which also expressed surface p140trk-A and p75NGFR molecules and hence efficiently bound and internalized the cytokine. Neutralization of endogenous NGF caused disappearance of Bcl-2 protein and apoptotic death of resting lymphocytes bearing surface IgG or IgA, a population comprising memory cells, while surface IgM/IgD "virgin" B lymphocytes were not affected. In vivo administration of neutralizing anti-NGF antibodies caused strong reduction in the titer of specific IgG in mice immunized with tetanus toxoid, nitrophenol, or arsonate and reduced numbers of surface IgG or IgA B lymphocytes. Thus, NGF is an autocrine survival factor for memory B lymphocytes.

Animals↗

PubMed1996

Effect of combined alpha IFN and prostaglandin A1 treatment on vesicular stomatitis virus replication and heat shock protein synthesis in epithelial cells.

The antiviral activity of prostaglandin A (PGA) and interferons (IFNs) has been widely described. In the present report, we investigated the effect of combined alpha IFN and PGA1 treatment on vesicular stomatitis virus (VSV) replication and on heat shock protein (HSP) induction in monkey epithelia cells. In uninfected cells, PGA1 caused a dose-dependent induction of HSP70, HSP90 and HSP110, while alpha IFN did not affect HSP synthesis. Alpha-IFN suppressed VSV replication dose-dependently, even when cells were treated after virus infection. VSV protein synthesis was not affected by alpha IFN, indicating a block at the level of virus assembly or maturation. PGA1 caused a dose-dependent inhibition of VSV replication, and suppressed VSV protein synthesis at concentrations which induced the synthesis of high levels of HSP70. The combined treatment with low doses of alpha IFN or PGA1, which only moderately inhibited VSV replication when administered separately, was found to suppress VSV production by more than 95%, and resulted in a 3-fold increase of HSP70 synthesis as compared to PGA1 alone. These results demonstrate a co-operative effect of PGA1 and alpha IFN against VSV infection and suggest that alpha IFN can potentiate the cellular response to HSP induction in virus-infected cells.

Animals↗