Jacopo Uggeri

CD8 + T lymphocyte responses are induced during acute hepatitis C virus infection but are not sustained

Cellular immune responses are likely to play a key role in determining the clinical outcome in acute infection with hepatitis C virus (HCV), but the dynamics of such responses and their relationship to viral clearance are poorly understood. In a previous study we have shown highly activated, multispecific cytotoxic T lymphocyte responses arising early and persisting in an individual who subsequently cleared the virus. In this study the HCV‐specific CD8+ lymphocytes response has been similarly analyzed, using peptide‐HLA class I tetramers, in a further nine individuals with documented acute HCV infection, six of whom failed to clear the virus. Significant populations of virus‐specific CD8+ lymphocytes were detected at the peak of acute hepatic illness (maximally 3.5 % of CD8+ lymphocytes). Frequencies were commonly lower than those seen previously and were generally not sustained. Early HCV‐specific CD8+ lymphocytes showed an activated phenotype in all patients (CD38+ and HLA class II+), but this activation was short‐lived. Failure to sustain sufficient numbers of activated virus‐specific CD8+ lymphocytes may contribute to persistence of HCV.

Time course assessment of methylmercury effects on C6 glioma cells: submicromolar concentrations induce oxidative DNA damage and apoptosis

Organic mercury is a well‐known neurotoxicant although its mechanism of action has not been fully clarified. In addition to a direct effect on neurons, much experimental evidence supports an involvement of the glial component. We assessed methylmercury hydroxide (MeHgOH) toxicity in a glial model, C6 glioma cells, exposed in the 10−5–10−8 M range. The time course of the effects was studied by time‐lapse confocal microscopy and supplemented with biochemical data. We have monitored cell viability and proliferation rate, reactive oxygen species (ROS), mitochondrial transmembrane potential, DNA oxidation, energetic metabolism and modalities of cell death. The earliest effect was a measurable ROS generation followed by oxidative DNA damage paralleled by a partial mitochondrial depolarization. The effect on cell viability was dose dependent. TUNEL, caspase activity and real‐time morphological observation of calcein‐loaded cells showed that apoptosis was the only detectable mode of cell death within this concentration range. N‐acetyl‐cysteine (NAC) or reduced glutathione (GSH) completely prevent the apoptotic effect of MeHgOH. The lowest effective MeHgOH concentration was 10−7 M for ROS and DNA OH‐adducts generation. The effect of submicromolar concentrations of MeHgOH on C6 cells could be relevant in the developmental neurotoxicity caused by low dose, long‐term exposures, such as those of food origin. In addition, we have shown that the same concentrations are effective in the induction of DNA oxidative damage, with further potential pathogenetic implications.

Ex vivo characterization of tumor-derived melanoma antigen encoding gene-specific CD8 + cells in patients with hepatocellular carcinoma

BACKGROUND/AIMS Members of the melanoma antigen encoding gene family are expressed in tumors of different histological types but not in normal tissue. For this reason, they are attractive targets for cancer immunotherapy. METHODS In the present study, we analyzed the expression of MAGE-1 and -3 genes in the hepatocellular carcinoma (HCC) tissue as well as frequency, phenotype and function of circulating and tumor infiltrating CD8+ cells specific for HLA-A1 and -A2 restricted epitopes of MAGE-1 and -3. RESULTS Our study shows for the first time the presence of MAGE/tetramer+ CD8 cells in the tumor tissue of patients with HCC. These cells are able to recognize the MAGE-1 sequence 161-169 and the MAGE-3 sequence 271-279. In a patient with a particularly high frequency of MAGE-1 sequence 161-169-specific T cells, phenotypic and functional analysis was performed showing a phenotype of recently-primed CD8 cells (CD28+CD27+CD45RA-CCR7). CONCLUSIONS The observation of a spontaneous in vivo priming of a MAGE-specific T cell response in patients with HCC and the high frequency of MAGE antigens expression in this tumor, makes this antigen a potential candidate for a MAGE-specific immunotherapy in hepatocellular carcinoma.

Calcein-AM is a detector of intracellular oxidative activity

Calcein-acetoxymethylester (calcein-AM) is a non-fluorescent, cell permeant compound, which is converted by intracellular esterases into calcein, an anionic fluorescent form. It is used in microscopy and fluorometry and provides both morphological and functional information of viable cells. In this study we have tested the response of calcein-AM to oxidation. In cell-free fluorometric assays, H2O2 and xanthine–xanthine oxidase induced a dose-dependent emission of the AM form but had no effects on calcein. Fluorometric and confocal microscopy tests on human fibroblasts confirmed that the cell permeant AM form is the actual sensor since its removal from culture medium, and its consequent back-diffusion, made the system insensitive to oxidative stimuli. In time-lapse confocal microscopy, calcein-AM detected changes in the intracellular redox state following direct oxidation (H2O2, xanthine–xanthine oxidase) and phorbol ester treatment. Comparative tests showed that calcein-AM sensitivity to oxidation is about one order of magnitude higher than other fluorescein derivatives. The absence of leakage, due to the presence of the probe in the extracellular compartment, and its low toxicity allow to perform experiments for prolonged times following the response to the same or different stimuli repeatedly applied. We propose calcein-AM as a sensitive tool for intracellular ROS generation in living cells with useful applications for real-time imaging in confocal microscopy.

Non-apoptotic programmed cell death induced by a copper(II) complex in human fibrosarcoma cells

A0, a Cu(II) thioxotriazole complex, produces severe cytotoxic effects on HT1080 human fibrosarcoma cells with a potency comparable to that exhibited by cisplatin. A0 induced a characteristic series of changes, hallmarked by the formation of eosin- and Sudan Black-B-negative vacuoles. No evidence of nuclear fragmentation or caspase-3 activation was detected in cells treated with A0 which, rather, inhibited cisplatin-stimulated caspase-3 activity. Membrane functional integrity, assessed with calcein and propidium iodide, was spared until the late stages of the death process induced by the copper complex. Vacuoles were negative to the autophagy marker monodansylcadaverine and their formation was not blocked by 3-methyladenine, an inhibitor of autophagic processes. Negativity to the extracellular marker pyranine excluded vacuole derivation from the extracellular fluid. Ultrastructural analysis indicated that A0 caused the appearance of many electronlight cytoplasmic vesicles, possibly related to the endoplasmic reticulum, which progressively enlarge and coalesce to form large vacuolar structures that eventually fill the cytoplasm. It is concluded that A0 triggers a non-apoptotic, type 3B programmed cell death (Clarke in Anat Embryol (Berl) 181:195–213, 1990), characterized by an extensive cytoplasmic vacuolization. This peculiar cytotoxicity pattern may render the employment of A0 to be of particular interest in apoptosis-resistant cell models.

Inhibition of Glutamine Synthetase Triggers Apoptosis in Asparaginase-Resistant Cells

The resistance to L-asparaginase (ASNase) has been associated to the overexpression of asparagine synthetase (AS), although the role played by other metabolic adaptations has not been yet defined. Both in ASNase-sensitive Jensen rat sarcoma cells and in ARJ cells, their ASNase-resistant counterparts endowed with a five-fold increased AS activity, ASNase treatment rapidly depletes intracellular asparagine. Under these conditions, cell glutamine is also severely reduced and the activity of glutamine synthetase (GS) is very low. After 24h of treatment, while sensitive cells have undergone massive apoptosis, ARJ cells exhibit a marked increase in GS activity, associated with overexpression of GS protein but not of GS mRNA, and a partial restoration of glutamine and asparagine. However, when ARJ cells are treated with both ASNase and L-methionine-sulfoximine (MSO), an inhibitor of GS, no restoration of cell amino acids occurs and the cell population undergoes a typical apoptosis. No toxicity is observed upon MSO treatment in the absence of ASNase. The effects of MSO are not referable to depletion of cell glutathione or inhibition of AS. These findings indicate that, in the presence of ASNase, the inhibition of GS triggers apoptosis. GS may thus constitute a target for the suppression of ASNase-resistant phenotypes.

Immunopathogenesis of hepatitis C virus infection

ESOLUTION of acute hepatitis C is generally associR ated with detection of neutralizing antibodies, a high frequency of circulating HCV-specific T cells with a predominant production of Thl cytokines. In contrast, T cell responses in the peripheral blood are usually undetectable in the acute phase of disease and are oriented towards a predominant production of Th2 cytokines when infection becomes chronic. Therefore, different strengths and quality of T cell responses at the early stages of infection may influence the evolution of hepatitis C but the primary causes of these different behaviors are still undefined. A good proportion of patients with long-lasting chronic HCV infection display detectable levels of peripheral blood cytotoxic T lymphocyte (CTL) responses and produce neutralizing anti-envelope antibodies. Moreover, the frequency of intrahepatic HCVspecific CTL seems to be high in these patients, the CTL response is multispecific and the liver environment is characterized by a predominant production of Thl cytokines. Therefore, at this stage of infection the virus appears to have acquired the capacity to escape immune surveillance and to persist in the face of an active antiviral immune response. If these observations derived from in vitro studies actually reflect the strength and the quality of the immune responses in vivo, inhibition of viral replication rather than modulation of the immune response should represent the main objective of anti-HCV therapies once a chronic

Human osteoblast behavior on as‐synthesized SiO4 and B‐CO3 co‐substituted apatite

The functional behavior of synthetic apatite, commonly used as fillers or scaffolds, depends on physical and chemical parameters, which vary in response to chemical substitutions and to thermal treatments. The effect of silicon co-substituting with carbonate ions in the apatite lattice on the properties of the as-synthesized powder and finally on human osteoblast in vitro behavior was investigated. Dose-response curves of Si-free and Si-substituted carbonated apatites (namely CHA and SiCHA-1 and SiCHA-2 with 0.88 and 0.55 wt % of Si, respectively) showed that SiCHA-1 had toxic effect, whereas CHA and SiCHA-2, at worst, hindered osteoblast proliferation, but no toxicity occurred. Subsequent experiments compared the effects of CHA and SiCHA-2 used at the doses of 0.3 and 1 mg/mL. After 7 days of treatment, both the powders stimulated cell proliferation and protein content and inhibited alkaline phosphatase activity. However, SiCHA-2 slightly stimulated osteoblast differentiation, as shown by higher calcium deposition, compared with CHA. The cell behaviors were linked to the peculiar powder characteristics. The as-synthesized powder represents the most critical system in terms of reactivity toward cells and can inform on the limits for positively exploiting the characteristics of SiCHA powders in making bone fillers or scaffolds, using no thermal treatments. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.

Polyphenon E®, a standardized green tea extract, induces endoplasmic reticulum stress, leading to death of immortalized PNT1a cells by anoikis and tumorigenic PC3 by necroptosis

Increasing doses of Polyphenon E®, a standardized green tea extract, were given to PNT1a and PC3 prostate epithelial cells mimicking initial and advanced stages of prostate cancer (PCa), respectively. Cell death occurred in both cell lines, with PNT1a being more sensitive [half-maximal inhibitory concentration (IC50) = 35 μg/ml] than PC3 (IC50 = 145 μg/ml) to Polyphenon E®. Cell cycle arrest occurred at G0/G1 checkpoint for PNT1a, and G2/M for PC3 cells. Endoplasmic reticulum stress (ERS) and unfolded protein response (UPR) occurred in both cell lines, with each exhibiting different timing in response to Polyphenon E®. Autophagy was transiently activated in PNT1a cells within 12 h after treatment as a survival response to overcome ERS; then activation of caspases and cleavage of poly (ADP ribose) polymerase 1 occurred, committing cells to anoikis death. Polyphenon E® induced severe ERS in PC3 cells, causing a dramatic enlargement of the ER; persistent activation of UPR produced strong upregulation of GADD153/CHOP, a key protein of ERS-mediated cell death. Thereafter, GADD153/CHOP activated Puma, a BH3-only protein, committing cells to necroptosis, a programmed caspase-independent mechanism of cell death. Our results provide a foundation for the identification of novel targets and strategies aimed at sensitizing apoptosis-resistant cells to alternative death pathways.

Adhesion of human osteoblasts to titanium: A morpho-functional analysis with confocal microscopy

Properties of surface affect the interactions between the implant and osteoblasts and direct the clinical osteointegrative outcome. The aim of this in vitro study was to describe the adhesion of living human osteoblasts to titanium disks with differently prepared surfaces: sand blasted with ZrO(2) particles and acid-etched (Soft-SLA, S-SLA) or with Al(2)O(3) particles and acid-etched (Hard-SLA, H-SLA), smooth surface (SS). Confocal microscopy was exploited to follow cell morpho-functional features either on living cells (cell shape with calcein-acethoxymethylester and mitochondria with tetramethylrhodamine methyl ester) or on fixed cells (immunocytochemistry of beta1-integrin and of actin) 6h or 24h after seeding. The underlying surface was visualized simultaneously on the same field. No cytotoxic effect was detected at any time and on any surface. At 6h after seeding, osteoblasts showed either a rounded or polygonal shape on both rough surfaces. Several features suggested that adhesion was faster with a higher level of organization on S-SLA than on H-SLA. Indeed osteoblasts grown on S-SLA were wider and with more extended protrusions than those on H-SLA. Active mitochondria on S-SLA occupied perinuclear areas and cellular prolongations, whereas on H-SLA they were mainly focused around nucleus. Organization of integrin beta1-subunit and actin, confirmed different kinetics of cell adhesion. At 6h integrin beta1-subunit was distributed along the periphery on the cell-biomaterial focal complexes in cells grown on S-SLA, whereas it was unevenly dispersed in membrane of cells cultured on H-SLA. Stress actin fibers were well defined in cells cultured on S-SLA, whereas they were scarcely evident on H-SLA. Osteoblasts seeded on smooth surface for 6h had morpho-functional features typical of adhesion, with some elements characterized by an elongated shape with an evident main longitudinal axis. At 24h osteoblasts were spread-out onto all surfaces. Nonetheless, different morphologies were shown in response to the different surfaces tested: polygonal cells prevailed on SLA surfaces, whereas almost all the cells on SS were long with two principal prolongations. At 24h number of cells adhered to the three kind of surfaces was similar, but during the following three days, cells seeded on S-SLA and on SS proliferated to a greater extent than those cultured on H-SLA. Analysis of morpho-functional parameters performed in living cells, and in particular the study of mitochondria organization, proved to be a valuable tool to follow cell-biomaterial adhesion. A higher level of spreading occurring in osteoblasts grown on S-SLA and SS at early times accounted for a faster subsequent cell proliferation. Nonetheless, these comparable activities were exerted by cells showing polygonal or elongated shapes when grown respectively on S-SLA or on SS. The former is typical of osteogenic cells, whereas the latter resumes a fibroblast-like morphology, that would result in an ineffective in vivo osteointegrative process.