Adolfo Turano

Flow cytometric analysis of activation markers on stimulated T cells and their correlation with cell proliferation.

The expression of activation antigens, namely CD25, CD69, CD71, and HLA-DR on T cells from 15 healthy individuals stimulated with different mitogens and specific antigens was evaluated by immunofluorescence assay and flow cytometric analysis and compared with cell proliferation as a function of [3H]thymidine incorporation. CD69 was the earliest expressed antigen on stimulated cells, while HLA-DR was the latest. Regardless of the stimulus used, lymphocytes expressing CD25 and CD71 were always more numerous than cells expressing CD69 and HLA-DR. Variations in the proportion of CD4+ and CD8+ T cells expressing each activation marker were observed with different antigenic stimuli. The expression of each activation marker showed overall agreement with the [3H]thymidine incorporation assay in discriminating between positive and negative immune response. However, no correlation was observed between the percentage of CD25-, CD69-, CD71-, and HLA-DR-positive T cells and the amount of [3H]thymidine incorporation. Moreover, low doses of mitogens and antigens as well as short time of stimulation were sufficient to induce T cells to express activation antigens but not to proliferate. Our data show that results obtained by flow cytometry and [3H]thymidine incorporation may differ qualitatively, at least under certain conditions; this suggests that the 2 assays are complementary, and when combined, may gives a clearer understanding of events leading to efficient cell-mediated immune response.

Expression of CD28 on CD8+ and CD4+ Lymphocytes During HIV Infection

CD28 interaction with B7 molecules, expressed on the membranes of antigen‐presenting cells, costimulates cytokine production, T‐cell proliferation and generation of cytotoxic lymphocytes. The expression of CD28 markers on CD4+ and CD8+ lymphocytes was studied in a group of subjects at various stages of HIV infection. A reduction in the percentage of CD28‐bearing CD4+ and CD8+ cell subsets was observed during the asymptomatic stage of the disease. This reduction was more pronounced in AIDS than in non‐AIDS patients. At the same time, an increase in the absolute CD8+CD28− cell number (greater in stage A than in stage B and C subjects) was observed in HIV‐infected patients. The finding of an altered pattern of CD28 expression on T cells might per se explain certain early defects in the cytokine pattern and in the immune response peculiar to HIV‐infected patients.

CD11b Expression Identifies CD8+CD28+ T Lymphocytes with Phenotype and Function of Both Naive/Memory and Effector Cells

A previously unreported CD8+CD28+CD11b+ T cell subset occurs in healthy individuals and expands in patients suffering from primary viral infections. In functional terms, these cells share the features of naive/memory CD8+CD28+CD11b− and terminally differentiated effector CD8+CD28−CD11b+ subpopulations. Like CD28− cells, CD28+CD11b+ lymphocytes have the ability to produce IFN-γ, to express perforin granules in vivo, and to exert a potent cytolytic activity. Moreover, these cells can respond to chemotactic stimuli and can efficiently cross the endothelial barrier. In contrast, like their CD11b− counterpart, they still produce IL-2 and retain the ability to proliferate following mitogenic stimuli. The same CD28+CD11b+ subpopulation detected in vivo could be generated by culturing naive CD28+CD11b− cells in the presence of mitogenic stimuli following the acquisition of a CD45RO+ memory phenotype. Considering both phenotypic and functional properties, we argue that this subset may therefore constitute an intermediate phenotype in the process of CD8+ T cell differentiation and that the CD11b marker expression can distinguish between memory- and effector-type T cells in the human CD8+CD28+ T cell subset.

A comparison of Legionella pneumophila occurrence in hot water tanks and instantaneous devices in domestic, nosocomial, and community environments

The aim of this study was to compare the occurrence of L. pneumophila in hot water samples from hot water tanks and instantaneous devices. Tanks and devices were all operated by heat exchangers employed in the towns district heating system. Thirty-six out of 171 (21%) hot water samples tested positive for L. pneumophila isolation, with 14.6% belonging to serogroup 1 and 6.4% to serogroups 2–14. The proportion of L. pneumophila detected in hot water reservoirs (30%) was higher than that observed in hot water instantaneous devices (6.2%). Differences in L. pneumophila isolation reflected different temperatures registered at the faucet: ≤50°C for hot water from reservoir devices, and >60°C for hot water from instantaneous devices. These data emphasize the need to control temperature in hot water distribution devices, thus inhibiting the formation of biofilm and L. pneumophila colonization.

Phenotypic and functional characteristics of tumour-derived microvascular endothelial cells.

We recently developed a method for the isolation and purification of tumour-derived endothelium. In this study the phenotypic and functional properties of human tumour-derived microvascular endothelial cells (TdMEC) were examined. Endothelium obtained from human adrenal gland specimens (HAMEC) was used as a reference microvascular endothelial cell population. TdMEC formed a confluent monolayer with the typical morphological appearance of endothelium and were positive for endothelial markers such as Ulex-1 lectin, CD31 antigen, von Willebrand Factor and VE-cadherin. The addition of acidic Fibroblast Growth Factor (aFGF), basic FGF (bFGF) or Vascular Endothelial Growth Factor (VEGF) substantially improved proliferation of TdMEC; and kidney carcinoma derived endothelial cells were more responsive to FGFs, whereas glioblastoma derived endothelial cells greatly responded to VEGF. TdMEC expressed high levels of the VEGF receptors, KDR/flk-1 and Flt-1, as shown by northern blot analysis. TdMEC expressed the adhesion molecules ICAM-1, VCAM-1 and E-selectin that could be further increased by exposing TdMEC culture to interleukin-1. All the TdMEC expressed interleukin-8 mRNA. These findings show that TdMEC in vitro maintain several of the features described for microvasculature. Thus, TdMEC represent a useful tool to study markers for tumor vasculature.

Natural antibodies to interferon-gamma

Natural antibodies to interferon (IFN)-γ were detected in the serum of virus-infected patients and also, at a low titre, in the serum of healthy subjects. The increased titre of antibodies to IFN-γ in the sera of virus-infected patients, and its decrease with clinical resolution, indicate that these antibodies are related to viral infection and probably reflect IFN-γ production as a result of antigenic stimulationin vivo. Natural antibodies to IFN-γ were affinity purified and studied for their capability to interferein vitro with the multiple activities of the lymphokine. Data obtained show that these human anti-IFN-γ antibodies have no inhibitory effect on the antiviral and antiproliferative activity of IFN-γ and do not interfere with the binding of the lymphokine to its specific cell receptor. Instead, they can inhibit the expression of HLA-DR antigens induced by IFN-γ on U937 cells and interfere, in mixed lymphocyte culture, with the proliferation of lymphocytes and the generation of cytotoxic lymphocytes. Experiments in animal models suggest that natural antibodies to IFN-γ may have a role in the immunoregulatory process limiting the intensity and/or duration of immune response. As they can interfere only with the immunomodulating activities of IFN-γ, these antibodies might open up new therapeutic approaches to diseases with evidence of activated cell-mediated immunity.

Resistance to cadmium salts and metal absorption by different microbial species

The present study evaluates the inhibitory activity and the absorption of cadmium (Cd) salts by different microbial species, including Gram-positive (Staphylococcus aureus andS. epidermidis), Gram-negative (Pseudomonas aeruginosa, Escherichia coli, Salmonella typhimurium, andProteus mirabilis) bacteria and one yeast (Candida albicans). The metal absorption by growing cells was considered both in liquid and in solid medium. For one strain ofP. aeruginosa the presence of Cd deposits inside the cell was investigated by transmission electron microscopy. Generally, the Gram-negative species tested proved to be highly resistant to Cd ions and accumulated great amounts of Cd during growth. Two strains ofP. aeruginosa showed a high degree of resistance to Cd and were particularly efficient in removing the metal from solutions. The Gram-positive bacteria showed a heterogeneous behavior: anS. aureus strain susceptible to Cd absorbed, at low metal concentrations, higher amounts of metal than a Cd-resistant one. The metal absorption for Gram-negative species was dose dependent, while for the Cd-resistant staphylococci it reached a plateau. Our results suggest that microorganisms can represent a good model to study the interactions between heavy metals and living organisms.

Cd4+ and Cd8+ Lymphocytes of Patients with Aids Synthesize Increased Amounts of Interferon-γ

Individual cells capable of interferon-gamma (IFN-gamma) synthesis are easily detected by immunofluorescence and flow cytometric analysis using an anti-IFN-gamma monoclonal antibody as specific reagent. By IFN-gamma flow cytometry assay, we demonstrated that HIV-seropositive patients, starting at the early stage of viral infection, generally have an increased percentage of lymphocytes potentially able to produce IFN-gamma, compared with healthy blood donors. IFN-gamma expression in patient lymphocytes was observed to increase with the progressive stages of HIV infection, with the highest figures occurring in stage C patients. Such increased IFN-gamma expression involved both CD4+ and CD8+ T cell subsets. Most interestingly, we found patients at the same stage of HIV infection who had similar numbers of total and CD4+ lymphocytes but highly different percentages of lymphocytes potentially capable of producing IFN-gamma.

Differential production of IFN‐γ, analyzed at the single‐cell level, by specific subsets of human NK and T cells from healthy and HIV+ subjects

BACKGROUND Interferon gamma is a cytokine that plays a central role in immunity, and is physiologically secreted by T and NK cells under appropriate stimuli during the immune response. By means of flow cytometry, we performed a single cell analysis of interferon gamma producing NK cells and their surface phenotype in normal and HIV(+) individuals that show several defects of cytokine production and cellular immunity. METHODS PBMC or purified NK cells were stimulated for 1-12 h with PMA/ionomycin in the presence of monensin, subsequently stained for surface CD56 and CD3 or CD8, and for intracytoplasmic IFN-gamma, and analysed by flow cytometry. RESULTS Our results show that CD56(+) NK cells are more efficient interferon gamma producers than T cells. Moreover, within the CD56(+) NK cell population, those that co-express low density CD8 are the best producers. Finally, we show that NK cells during HIV infection are more massively recruited to interferon gamma production than those from normal subjects. CONCLUSIONS Both in the normal and HIV(+) subjects, a higher percentage of NK cells than T cells can produce IFN-gamma although differences can be identified within the NK cells subset in terms of IFN-gamma production. The production of IFN-gamma is fully achievable in the HIV(+) subjects, which is consistent with their elevated plasmatic levels of the cytokine. The possibility that NK cells that produce interferon gamma could represent a functionally distinct population committed to the production of this cytokine, is discussed.

INCREASED LEVELS OF ANTIBODIES TO IFN-GAMMA IN HUMAN AND EXPERIMENTAL AFRICAN TRYPANOSOMIASIS

In African trypanosomiasis the occurrence of antibodies to interferon‐γ (IFN‐γ) was studied in both humans and experimental rats. Sera from patients infected with Trypanosoma brucei gambiense showed increased levels of antibodies to IFN‐γ as compared with controls from the same regions in Africa. In Sprague‐Dawley rats infected with Trypanosoma brucei brucei an early appearance of IFN‐γ‐producing spleen cells was observed, followed by an increase in levels of antibodies against IFN‐γ in the sera. Previously, IFN‐γ has been found to play a crucial role in trypanosome infections in rats by promoting proliferation of Trypanosoma brucei brucei. The appearance of antibodies to IFN‐γ in humans, as in rats, indicates that this cytokine is produced also in the human infection. Its parasitic growthstimulating and pathophysiological effects on the organism may be reduced by the antibodies.