Andrea Fattorossi

FLOW CYTOMETRIC ANALYSIS OF THE EARLY PHASES OF APOPTOSIS BY CELLULAR AND NUCLEAR TECHNIQUES

The early events occurring during apoptosis at the plasma membrane, chromatin, and mitochondrial levels were investigated in freshly isolated irradiated human lymphocytes, growth factor-deprived cultured human lymphocytes, and dexamethasone (DEX)-treated murine thymocytes. In intact, unfixed cells, evaluation of the light scatter properties and of DNA stainability with ethidium bromide (EB) allowed a cell subset suggestive for initial apoptosis to be identified. The apoptotic nature of these cells was confirmed by cell sorting in irradiated human lymphocyte model. EB could not be substituted for by propidium iodide, indicating that the nature of DNA probe used is of major importance for detecting initial apoptotic changes. Because mitochondria are thought to represent a primary target during apoptosis, we measured the uptake of mitochondria transmembrane potential sensitive (Rhodamine 123) and nonsensitive (10-nonyl-acridine-orange) probes concomitantly with EB uptake. Cells starting apoptosis had an enhanced incorporation of both mitochondria dyes, which in combination with EB identified several cell subsets. This suggests that complex alterations in mitochondrial structure and functioning occur in the early stages of apoptosis. To investigate phenomena occurring at the chromatin level in similar phases of apoptosis, irradiated human lymphocytes and DEX-treated murine thymocytes were disrupted and DNA stainability assessed in nuclear suspensions. A transient increase in DNA stainability, i.e., the appearance of distinct hyperdiploid peaks in the human model and a generalised upward shift of the G0/1 peak in the murine model, was observed in the early phases of apoptosis concomitantly with specific alterations in light scattering properties. These findings suggest that chromatin texture is altered in early apoptosis and affects DNA stainability.

Circulating and thymic CD4+ CD25+ T regulatory cells in myasthenia gravis: effect of immunosuppressive treatment

Accumulating evidence indicates an immunosuppressive role of the thymus‐derived CD4+ T‐cell population constitutively expressing high level of CD25, T regulatory (Treg) cells, in autoimmune diseases. Here we show that the number of Treg cells in the blood is significantly lower in untreated myasthenia gravis patients than in age‐matched healthy subjects, whereas it is normal or elevated in patients on immunosuppressive therapy (prednisone frequently associated with azathioprine). Therapeutic thymectomy (Tx) for either the thymoma or non‐neoplastic thymic alterations that are often associated with myasthenia gravis provided unique material for studying intrathymic Treg cells and correlating them with their peripheral counterparts. We observed that Tx prevents the increase of Treg cells in the circulation that follows immunosuppressive therapy (particularly evident if the thymus is not neoplastic), indicating that the thymus contributes to Treg‐cell normalization. However, thymic Treg cells are not modulated by immunosuppressive therapy and even in thymectomized patients Treg‐cell numbers in the blood eventually recover. The present findings suggest that a deficiency in Treg cells favours the development of myasthenia gravis and that their normalization is an important clinical benefit of immunosuppressive therapy. Treg normalization appears to be largely thymus independent and possibly reflects the reported capacity of corticosteroids to promote Treg‐cell development.

Protective actions of l-carnitine and acetyl-l-carnitine on the neurotoxicity evoked by mitochondrial uncoupling or inhibitors

The mechanism for the pathological increase in cell death in various disease states e.g. HIV immunodefficiency or even ageing or Alzheimers disease, occurs by complex and as yet undefined mechanism(s) related to immunological, virological or biochemical disturbances (i.e. energy depletion, oxidative stress, increased protein degradation). We have studied mitochondrial uncoupling or inhibitor toxicity on neurones at the cellular level and at the mitochondrial level using rhodamine (Rh123) and 10-nonylacridine orange (NAO) fluorescence with confocal microscopy. Blockade of the mitochondrial chain complexes at various points was studied. The possible protective effects of the compound L-carnitine, which plays a central role in mitochondrial function, was tested in this form of neurotoxicity. It appears that L-carnitine and its acetylated form, acetyl-L-carnitine, can attenuate the cell damage, as assessed by lactate dehydrogenase (LDH) release, evoked by the uncoupler, p-(trifluoromethoxy)phenylhdyrazone (FCCP), or by the inhibitors, 3-nitropropionic acid (3-NPA) or rotenone. Further, the FCCP-induced inhibition of Rh123 uptake was antagonized by the preincubation of cells with L-carnitine. Since such neurotoxic mechanisms may be operating in the various pathological forms of myotoxicity and neurotoxicity, these observations suggest potential for a therapeutic approach.

Interference with cell cycle progression and induction of apoptosis by dideoxynucleoside analogs.

The in vitro effect of single or combined doses of zidovudine (AZT) and dideoxycytidine (ddC) on PHA-activated human peripheral blood mononuclear cells (PBMC) proliferative response and lymphoblastoid T cell line CEM cell growth was evaluated. Clinically relevant amounts (0.1, 1 and 10 microM) of AZT, ddC and AZT/ddC combination (10 + 10 microM) inhibited 3H TdR uptake in both cell models in a dose-dependent manner. The inhibitory effect on cell growth was confirmed by counting the amount of viable CEM cells recovered after 24, 48 and 72 h exposure to the drugs. On equimolar basis, ddC was considerably more efficient than AZT although the latter potentiates the activity of the former Flow cytometric analysis of PBMC and CEM cells exposed to the dideoxynucleosides revealed a decrease in the rate of DNA synthesis (rate of passage through the S phase of the cell cycle) and a reduced number of cell generations, the latter assessed by measuring the halving of the fluorescent probe 5-6-carboxyfluorescein diacetate succinimidyl ester by flow cytometry. The analysis of CEM cells recovered after exposure to ddC or AZT/ddC combination (10 + 10 microM), showed that in addition to perturbing cell cycle progression, ddC, and most efficiently the AZT/ddC combination, induced cell death by apoptosis. The latter was manifested as enhanced side scatter and decreased, sub-G1, DNA content by flow cytometry, and as DNA breakdown in nucleosomal fragments by gel electrophoresis. Present findings indicate that clinically relevant concentrations of dideoxynucleosides reduce cell growth by hampering DNA replication and inducing apoptosis.

Okadaic acid induces changes in the organization of F-actin in intestinal cells

Okadaic acid, a polyether fatty acid associated with diarrhetic seafood poisoning, is capable of inhibiting protein phosphatases 1 and 2A which are considered among the major protein phosphatases in the cytosol of mammalian cells. One of the substrates for these phosphatases has been reported to be the cytoskeleton. In this paper, we focused on the effects of okadaic acid in intestinal cells, the more physiological target for this toxin. By fluorescence and scanning electron microscopy, we evidenced a dose- and time-dependent effect on F-actin which preceded any detectable change of tubulin and vimentin network. By a flow cytometric approach, we observed that plasma membrane permeability and transmembrane potential, two indicators of early cell damage or activation, respectively, remained unaffected in intoxicated cells. The present data strongly support the theory that actin is one of the main cytosolic targets for the phosphatases inhibited by okadaic acid in intestinal cells.

Clinical and immunological response to typhoid vaccination with parenteral or oral vaccines in two groups of 30 recruits

The clinical and immunological responses to typhoid vaccination with parenteral and oral vaccines in two groups of 30 adult male subjects were studied. Specific anti-Salmonella typhi cell-mediated immunity and total or specific anti-lipopolysaccharide faecal immunoglobulin (Ig) A titres in vaccinated subjects were monitored. Cellular antibacterial activity was significantly increased only in orally vaccinated subjects. Serum arming activity and inhibition experiments suggested an IgA-dependent cellular cytotoxicity in those orally vaccinated. In these subjects, a total and anti-lipopolysaccharide faecal IgA increase was observed lasting up to 8 months after completion of the vaccination schedule. In parenteral vaccinated subjects, an early onset transitory increase of IgM rheumatoid factor was observed. Oral vaccine was well tolerated and free of side effects, whereas 65% of parenterally vaccinated subjects reported side effects such as fever, headache, malaise and local tenderness in the injection site.

Potentiation of human polymorphonuclear leukocyte activation by atrial natriuretic peptide. inhibitory effect of carnitine congeners

Polymorphonuclear leukocytes (PMN), atrial natriuretic peptide (ANP) and leukotriene B4 (LTB4) reportedly play a major role in ischemia/reperfusion states of coronary artery disease. We sought to determine whether ANP and LTB4 cooperate in inducing PMN activation with consequent modulation of membrane molecules required for adherence to endothelium and myocardial cells, namely CD11b and L-selectin and the release of toxic oxygen radicals. ANP (from 10−16 to 10−8 M), LTB4 (from 10−10 to 1−6 M) and combinations of the two were incubated with normal PMN at 37°C for 15 minutes. Membrane molecules modulation was measured by flow cytometry using specific monoclonal antibodies. Hydrogen peroxide production, an indicator of the capacity of PMN to release toxic oxygen species was quantified by flow cytometry using the peroxide-sensitive fluorescent probe dichlorofluorescein diacetate. ANP, uneffective when used alone, dose-dependently potentiated the PMN response to LTB4 (10−9 M) as evidenced by an up-regulation of CD11b expression and peroxide production, and a down-regulation of L-selectin expression. These effects were prevented dose-dependently by the protein kinase C (PKC) inhibitor staurosporine (from 10 to 160μM). Two carnitine congeners, palmytoylcamitine (tested from 125 pg to 2μg/ml) that also possesses an established ability to antagonise PKC and L-carnitine (tested from 12 to 200 ng/ml) were also effective. These data indicate that ANP potentiates LTB4 in inducing PMN mobilization and activation with a possible consequent detrimental effect on cardiac tissue and evisages the usefulness of PMN metabolism modulators.

Probing chromatin structure in the early phases of apoptosis

Abstract. A typical flow cytometric marker of apoptosis is the appearance of a hypodiploid peak. This phenomenon is related to the chromatin fragmentation and loss that occurs during the late stages of the process. We describe herein the changes occurring at the chromatin level in purified nuclei preparations obtained from human peripheral blood mononuclear cells in a time‐course study, including the simultaneous evaluation of nuclear proteins and DNA stainability, light‐scattering properties, and spectrophotometric determination of the protein content. An augmentation of fluoroscein isothiocyanate (FITC) stainability was noticed as early as 1 h after irradiation. As this phenomenon is not correlated to changes in actual protein content, one can conclude that modifications of basic protein accessibility occur from the early phases of the apoptotic process. Also DNA stainability augmented with time, generating the transient appearance of a hyperdiploid peak that preceded the appearance of the hypodiploid peak typical of the late stages of the process, and that shared with the latter the same light‐scattering properties. Chromatin status was further explored by staining apoptotic nuclei using DNA probes with peculiar molecular weight. Propidium iodide (PI) and ethidium bromide (EB), but not the much bulkier 7‐aminoactinomycin D (7‐AAD), identified the nuclei with a transient increase in DNA stainability confirming that an increased dye accessibility to binding sites was responsible for the phenomenon. Remarkably, all dyes identified the same proportion of hypodiploid nuclei when an apoptotic nucleus shed its fragmented chromatin. Control experiments included differential interference contrast and fluorescence microscopy that showed the purity of nuclei preparations and the typical morphological apoptotic features. Finally, the simultaneous evaluation of DNA by PI and nuclear proteins by FITC in a time course study allowed a thorough assessment of changes occurring at the chromatin level in the diverse stages of apoptosis. It is suggested that proteolysis precedes endonucleolysis and probably renders it easier the final endonucleolytic step leading to DNA fragmentation and loss.

Lack of evidence for a superantigen in lymphocytes from HIV-discordant monozygotic twins

Objective:An HIV-associated superantigen (SAg) has been hypothesized. Here we test whether an SAg is functionally detectable in peripheral blood mononuclear cells (PBMC) from monozygotic twins discordant for HIV infection. Design and methods:The vβ selective T-cell depletion found in minor lymphocyte stimulation (Mls)-positive mice is caused by an SAg encoded by the mouse mammary tumour virus. Mis is a locus whose gene product stimulates a mixed lymphocyte reaction (MLR) in mice strains identical at the major histocompatibility complex locus. If an SAg is present in PBMC and/or sorted CD4+ cells from one HIV-infected monozygotic twin, it would stimulate PBMC from the corresponding healthy monozygotic twin in an MLR. In addition, if an SAg causes vβ-selective T-cell depletion in AIDS patients, a differential proliferation to a panel of staphylococcal enterotoxins (SE) of T lymphocytes from healthy and HIV-infected monozygotic twins should become measurable. Results:No positive MLR or significant differences in the SE-driven proliferation between the healthy and the HIV-infected twins were observed. Conclusions:Our results suggest that PBMC from the two HIV-infected twins do not express a functionally detectable SAg.

Celecoxib up-regulates the expression of the ζ, chain of T cell receptor complex in tumor-infiltrating lymphocytes in human cervical cancer

Purpose: We evaluated the effects of celecoxib treatment on tumor-infiltrating lymphocyte (TIL) subsets [CD3+, CD4+,CD8+, CD25+, and T cell receptor (TCR)-ζ–expressing cells] and tryptase-positive mast cells in cervical tumors. Circulating levels of cytokines [interleukin (IL)-1β, IL-10, tumor necrosis factor-α, IL-6, and IL-12] and angiogenesis-modulating factors (vascular endothelial growth factor and endostatin) have also been analyzed. Experimental Design: Cervical tumor biopsies and blood samples were obtained at the time of diagnosis and after 10 days of celecoxib treatment (400 mg b.i.d., at 8:00 a.m. and 8:00 p.m.) in 27 cases. Immunohistochemistry and ELISA assays were used to assess the expression of biological factors in tumor tissue and circulating levels of cytokines and angiogenic molecules. Results: We showed a statistically significant increase in the percentage of TIL expressing the TCR-ζ chain after celecoxib treatment: indeed, in cases exposed to celecoxib, the percentage of TCR-ζ+ cells ranged from 5.0 to 50.0 (median, 22.5) with respect to baseline expression (range, 3.0-50.0; median, 10.0; P = 0.0016). There was no significant treatment-related difference in the percentage of CD3+, CD4+, CD8+, and CD25+ TIL as well as in tryptase-positive cells. IL-12 levels were significantly reduced in posttreatment samples with respect to baseline levels (P = 0.002). We also found a reduction in the circulating levels of vascular endothelial growth factor, and a statistically significant increase of serum endostatin levels (P = 0.035). Conclusions: We reported the first evidence in humans that celecoxib restores ζ expression by TIL in primary cervical tumors, suggesting that a positive modulation of immune function may serve as an additional mechanism supporting the antitumor effect of this class of drugs.