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Biomedical subjects

E Cozzi

Publications and source records attributed to E Cozzi.

At least 55 records · Page 3Linked to original sources
PubMed1995

Chlorpromazine inhibits tumour necrosis factor synthesis and cytotoxicity in vitro.

Chlorpromazine (CPZ) has been previously shown to protect against endotoxin [lipopolysaccharide (LPS)] lethality and inhibit the release of tumour necrosis factor in vivo. We investigated at the cellular level whether this was due to direct inhibition of tumour necrosis factor-alpha (TNF-alpha) synthesis, using LPS-stimulated THP-1 human monocytic leukemia cells. We also studied the effect of CPZ on human TNF-alpha action by assessing TNF-alpha cytotoxicity on mouse fibrosarcoma L929 cells. CPZ (1-100 microM) inhibited TNF-alpha production in THP-1 cells in a dose dependent manner by a maximum of 80%. This effect was comparable to that of two well-known inhibitory drugs, dexamethasone and cyclicAMP. Inhibition was also evident at the mRNA level. On the other hand CPZ (10-25 microM) also inhibited TNF-alpha activity: in fact it reduced the cytotoxicity of TNF-alpha on L929 cells (EC50 was increased four times) and could provide protection even as a post-treatment. CPZ inhibited TNF-induced apoptosis in L929 cells, as detected by analysis of nuclear morphology. However, since we showed that apoptosis was very limited, and was not the main mode of cell death in our conditions, this could not explain the overall protection. Since CPZ did not interfere with either the oligomerization state of TNF-alpha or its receptor binding, our data suggest that it reduced cytotoxicity by inhibiting some steps in the TNF-alpha signalling pathways.

Apoptosis↗
PubMed1994

Tumor necrosis factor-driven formation of disulfide-linked receptor aggregates.

We have characterized, by ligand blotting, solubilized tumor necrosis factor receptors (TNFR) from K562 cells. Preparations that had been partially purified by gel filtration chromatography yielded two prominent bands of M(r) 60,000 and 75,000 corresponding to the two known TNFR (types I and II, respectively). In addition to these, types I and II TNFR-related species of M(r) > 100,000 were detected after purification by tumor necrosis factor (TNF)-affinity chromatography, suggesting that TNF had driven receptor aggregation during this step. To test this hypothesis ligand blots were performed on receptor preparations that had been partially purified by gel filtration chromatography and incubated with TNF before electrophoretic separation. Indeed, type II TNFR aggregates, but not type I TNFR aggregates, were generated at optimal TNF concentrations. Formation of type II TNFR aggregates in this last experimental setting and of both type I and type II TNFR aggregates during affinity purification could be prevented if an alkylating agent (N-ethylmaleimide) was added during the TNFR-TNF incubation step. Similar results were obtained when intact K562 cells were incubated with TNF and then analyzed for receptor aggregation; type II TNF receptor aggregates were generated at TNF concentrations ranging from 10(-9) to 10(-10) M and their formation was prevented in the presence of N-ethylmaleimide.

Blotting, Western↗
PubMed1993

Suramin induces deoligomerization of human tumor necrosis factor alpha.

Suramin inhibits the biological activity of human tumor necrosis factor alpha (TNF) through a direct action on the ligand rather than on its receptors (Grazioli, L., Alzani, R., Ciomei, M., Mariani, M., Restivo, A., Cozzi, E., and Marcucci, F. (1992) Int. J. Immunopharmacol. 14, 637-642). In order to clarify the mechanism whereby suramin leads to inhibition of TNF, we investigated the possibility that suramin might modify the quaternary structure of TNF which is biologically active as a trimer. For this purpose we used a new assay (double streptavidin sandwich assay) designed for the rapid detection of oligomer-monomer conversion of proteins. Taking advantage of this assay we observed, upon incubation with suramin, dissociation of TNF. Suramin-induced dissociation of TNF was confirmed by gel filtration chromatography. Under conditions of partial dissociation, two molecular species were separated. One of higher molecular weight, corresponding to trimeric TNF, was biologically active, whereas the other, corresponding to monomeric TNF, was inactive. These results are at variance with others recently reported, where suramin has been shown to induce microaggregation of several polypeptides (Middaugh, C. R., Mach, H., Burke, C. J., Volkin, D. B., Dabora, J. M., Tsai, P. K., Bruner, M. W., Ryan, J. A., and Marfia, K. E. (1992) Biochemistry 31, 9016-9024). This suggests that suramin inhibits the bioactivity of different protein molecules through opposite effects on their quaternary structure. The present results are, to our knowledge, the first demonstration of a drug inhibiting a target molecule through dissociation of its quaternary structure.

Animals↗
PubMed1993

Increased cytokine production in mononuclear cells of healthy elderly people.

The production of cytokines during aging, except interleukin (IL)-2, has been neglected in humans. We measured the in vitro production of IL-6, tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma and IL-1 beta by peripheral mononuclear cells from selected healthy young (mean age 26.8 years) and aged (mean age 80.2 years) subjects. Significant increases of IL-6, TNF-alpha and IL-1 beta levels were found in mitogen-stimulated cultures from aged donors, occurring at 24 to 72 h after stimulation. No significant differences were observed for IFN-gamma production. Proliferative capability of cells stimulated with PHA was not impaired in aged subjects. Since the amounts of all cytokines studied were similar in unstimulated cultures from young and aged subjects, and also serum levels of TNF-alpha did not differ, these data indicate that the cellular machinery for the production of these cytokines is well preserved in aging, and also that cells from old people are able to up-regulate their production in response to appropriate stimuli. The increases in cytokine synthesis were not dependent on changes in the number of monocytes, nor were they related to the significant rise of CD45RO+, and the concomitant decrease of CD45RA+, occurring in both CD4+ and CD8+ lymphocytes from aged subjects. The increased production of pro-inflammatory cytokines by stimulated mononuclear cells of healthy aged subjects may be relevant to several aspects of age-associated pathological events, including atherosclerosis, osteoporosis, fibrosis and dementia.

Adult↗
PubMed1993

Humoral and cellular immune response to influenza virus vaccination in aged humans.

Aging is characterized by an increased susceptibility to infectious diseases; influenza virus infection, which is easily managed by an intact immune system, represents a life-threatening disease in aged subjects. We studied 18 healthy aged subjects (> 65 years of age), vaccinated yearly with conventional anti-influenza vaccine, and 9 healthy young volunteers (mean age 26 years), without previous anti-influenza vaccination, who were vaccinated with the conventional trivalent 1990 anti-influenza preparation. Six out of the 18 aged individuals received a second boost of the same vaccine about 4 months later. In all subjects, we analyzed the humoral response to type A and B influenza viruses and the influenza type A virus-specific CTL generation. Among the elderly population with a single vaccination, 6 and 5 subjects seroconverted against type A and type B influenza virus respectively. Young subjects seroconverted in 5 cases against type A, and in 5 cases against type B influenza virus. Seroconversion took place after the second vaccination in only one subject, and the antibody production was type A specific. Influenza type A virus-specific CTL activity was significantly lower in aged subjects, compared with the values observed in the young volunteers (p = 0.017). The second vaccination partially restored this immunological impairment. These data clearly demonstrate that the elderly do not have the same ability as younger subjects to mount an antibody response, and generate influenza type A virus-specific CTL after conventional anti-influenza vaccination. Moreover, a double anti-influenza vaccination generates CTL activity levels comparable to young subjects, although it does not seem to substantially modify the antibody production.

Adult↗
PubMed1993

Expression, in Escherichia coli, of a soluble human tumor necrosis factor receptor.

We have expressed in Escherichia coli a soluble, truncated form of the human 55 kDa Tumor Necrosis Factor (TNF) receptor. For this purpose a plasmid was constructed which contains the extracellular domain of the 55 kDa TNF receptor fused to the coding sequence of the IgG binding domains of protein A from Staphylococcus aureus. The fusion product (TNFR-PA) obtained in E. coli is a soluble protein which bound human TNF alpha (huTNF alpha) with high affinity. In ligand-blotting experiments huTNF alpha bound to a single 52 kDa protein, a molecular mass corresponding to that expected for the monomeric fusion product. In gel filtration experiments binding activity was recovered from fractions that eluted at a volume corresponding to 140-150 kDa. TNFR-PA neutralized huTNF alpha in an in vitro cytotoxicity assay.

Chromatography, Gel↗
PubMed1993

Quantitative and qualitative analysis of anti-tetanus toxoid antibody response in the elderly. Humoral immune response enhancement by thymostimulin.

In order to explore the humoral primary and secondary response to tetanus toxoid (TT), and to define the possible immunopotentiating effect of the thymic hormone thymostimulin, we studied 13 elderly people, selected according to the Senieur Eurage protocol, vaccinated against TT, an antigen never encountered before. Six of them were treated with thymostimulin before and during the immunization protocol. Specific anti-TT antibody level measurement and spectrotypic analysis were performed on the sera collected from the subjects at different times over the immunization protocol. In addition, spontaneous in vitro production of anti-TT antibodies as well as cutaneous delayed hypersensitivity reactions were also studied. Only one patient showed a detectable humoral immune response after the first immunization. After the booster, four of six thymostimulin-treated individuals, compared with only two of seven controls, showed in vivo anti-TT humoral response; at the same time, spontaneous anti-TT production was detected in peripheral blood mononuclear cells from five of six thymostimulin-treated individuals but only three of seven untreated controls. These differences were highly significant (p < 0.0001). In addition, only in thymostimulin-treated subjects were the levels of serum anti-TT antibodies 14, 21 and 28 days after the booster significantly (p < 0.05) higher than the baseline values. The spectrotypic analysis of anti-TT antibodies performed by isoelectric focusing and reverse blotting showed total agreement with the results from enzyme-linked immunosorbent assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Adjuvants, Immunologic↗
PubMed1993

Early phenotypic activation of circulating helper memory T cells in scleroderma: correlation with disease activity.

OBJECTIVES: The differential expression of several accessory/activation molecules (CD26, CD29, CD45RA, CD25, MLR4, HLA-DR) on peripheral blood CD4+ and CD8+ T lymphocytes in patients with scleroderma was compared with that in controls and patients with other connective systemic diseases to look for evidence of the involvement of T cells in the disease process of scleroderma. METHODS: The two colour expression of surface molecules by circulating T cells was analysed with a panel of monoclonal antibodies and flow cytometry in 17 patients with scleroderma, 10 patients with systemic lupus erythematosus, and five patients with rheumatoid arthritis, and the results compared with those for 10 normal controls. The two colour T CD4+ phenotype was further compared between patients with active and quiescent disease in these patients with scleroderma. The coexpression of surface molecules by CD4+ T cells was also analysed by three colour flow cytometry in eight patients with scleroderma. RESULTS: Patients with scleroderma showed increased CD4+CD26+ and CD4+CD25+ percentages and absolute numbers and decreased CD8+CD29+ percentages compared with controls. Moreover, a significant correlation between the higher CD4+CD26+ T cell percentage and absolute cell numbers with disease activity was observed. Most of the CD4+ peripheral blood T cells from patients with scleroderma showed the CD26+CD45RA- phenotype by three colour flow cytometry analysis. CONCLUSIONS: The distinctive pattern of early helper memory T cell activation in these patients with rapidly evolving scleroderma supports the role of a T cell mediated mechanism in the progression of scleroderma.

Adult↗
PubMed1992

Detection of mycoplasma contamination through modulation (stimulation or inhibition) of thymidine incorporation by unstimulated mouse spleen cells.

In the present report we describe a rapid and sensitive assay for mycoplasma detection in cell cultures. The assay is based on the ability of contaminated culture supernatants to modulate [3H]TdR incorporation by unstimulated mouse splenocytes. Several mycoplasma species (Mycoplasma orale, culturable and non-culturable strains of Mycoplasma hyorhinis) inhibited [3H]TdR incorporation and permitted the detection of some contaminated cell cultures that would otherwise have escaped detection in assays measuring [3H]TdR incorporation by mitogen-stimulated splenocytes. On the other hand, several other mycoplasma species (Mycoplasma arginini, Mycoplasma hominis) strongly enhanced [3H]TdR incorporation by unstimulated splenocytes. This enhancement was optimally detectable on day 2 after initiation of the cultures. The sensitivity of the assay was determined for a mycoplasma species (culturable M. hyorhinis) that inhibited as well as for one (M. arginini) that enhanced [3H]TdR incorporation. In both cases, the sensitivity was such that 1-3 x 10(2) mycoplasma colony-forming units (CFU) could be detected.

Animals↗
PubMed1992

Inhibitory effect of suramin on receptor binding and cytotoxic activity of tumor necrosis factor alpha.

The effect of suramin on the binding of human Tumor Necrosis Factor alpha (huTNF alpha) to specific cell-surface receptors as well as on its cytotoxic activity in vitro was investigated. Suramin inhibited both activities in a dose-dependent manner. Experiments designed to discriminate if suramin exerted its inhibitory activity on the ligand or on the receptor showed that the ligand (huTNF alpha) was the most likely target for suramin in this system. These results may explain, in part, the immunosuppressive activities of suramin that have been observed in vivo and suggest that suramin could be useful in those disease states in which hyperproduction of huTNF alpha has been shown to play a pathogenic role.

Cell Survival↗