Silvana Belletti

Comparison of Annexin V and Calcein-AM as Early Vital Markers of Apoptosis in Adherent Cells by Confocal Laser Microscopy

SUMMARY Although morphological criteria for apoptosis are in general reliable, no systematic comparison of the techniques employed thus far has yet been performed. In this study, using confocal laser microscopy, we compared the performance of annexin V-FITC and calcein-AM for early detection of apoptosis in living adherent cells. Experiments were carried out on two distinct cell lines, PC 12 and NIH3T3, endowed with different shape and adhesion properties. The apoptotic process was followed for a prolonged period in the same cells of a predetermined field by means of a special flow chamber. Our results show that both probes allowed the detection of apoptotic cells in either cell line. However, some cells that clearly exhibited apoptotic changes on calcein visualization were annexin-negative. In NIH3T3 cells, annexin negativity of apoptotic cells was correlated with the preservation of cell shape and adhesion properties. These findings show that, at least in PC12 and NIH3T3 cells, annexin might be less sensitive than calcein-AM for early apoptosis detection and, for NIH3T3 cells, suggest that phosphatidilserine exposure is in some way linked to changes in cell shape and/or adhesion to culture substrate.

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.

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.

PD-L1 genetic overexpression or pharmacological restoration in hematopoietic stem and progenitor cells reverses autoimmune diabetes

Restoration of a PD-L1 defect in HSPCs reverses diabetes in NOD mice and thus may represent a potential cure for T1D. Stemming attacks on the pancreas In type 1 diabetes, autoreactive CD4 T cells attack and kill pancreatic β cells, disrupting insulin production. Many approaches have been taken to inhibit this process, but few have translated into real benefit for diabetic patients. Ben Nasr et al. demonstrate that hematopoietic stem and progenitor cells from NOD mice and diabetic patients express less PD-L1, which is a T cell inhibitory molecule. Induction of PD-L1 expression on stem cells reversed diabetes in NOD mice and inhibited human autoimmune responses in vitro. Either gene therapy or pharmacological modulation of PD-L1 on stem cells could be brought into the clinic, providing a new way to interrupt the autoimmune response and help people with diabetes. Immunologically based clinical trials performed thus far have failed to cure type 1 diabetes (T1D), in part because these approaches were nonspecific. Because the disease is driven by autoreactive CD4 T cells, which destroy β cells, transplantation of hematopoietic stem and progenitor cells (HSPCs) has been recently offered as a therapy for T1D. Our transcriptomic profiling of HSPCs revealed that these cells are deficient in programmed death ligand 1 (PD-L1), an important immune checkpoint, in the T1D nonobese diabetic (NOD) mouse model. Notably, the immunoregulatory molecule PD-L1 plays a determinant role in controlling/inhibiting activated T cells and thus maintains immune tolerance. Furthermore, our genome-wide and bioinformatic analysis revealed the existence of a network of microRNAs (miRNAs) controlling PD-L1 expression, and silencing one of key altered miRNAs restored PD-L1 expression in HSPCs. We therefore sought to determine whether restoration of this defect would cure T1D as an alternative to immunosuppression. Genetically engineered or pharmacologically modulated HSPCs overexpressing PD-L1 inhibited the autoimmune response in vitro, reverted diabetes in newly hyperglycemic NOD mice in vivo, and homed to the pancreas of hyperglycemic NOD mice. The PD-L1 expression defect was confirmed in human HSPCs in T1D patients as well, and pharmacologically modulated human HSPCs also inhibited the autoimmune response in vitro. Targeting a specific immune checkpoint defect in HSPCs thus may contribute to establishing a cure for T1D.

Polydeoxyribonucleotide promotes cyclobutane pyrimidine dimer repair in UVB- exposed dermal fibroblasts

Background: DNA is the main cellular chromophore for ultraviolet B (UVB). Its absorption leads to the generation of typical photoproducts. The most frequent types (about 80%) are cyclobutane pyrimidine dimers (CPDs). Several studies have suggested that treatment with deoxyribonucleosides can protect some cell types from DNA damage. The aim of this work was to evaluate the ability of the polydeoxyribonucleotide (PDRN) to protect human dermal fibroblasts from UVB‐induced DNA damage.

An in vitro model for the assessment of manganese neurotoxicity

PC12 (undifferentiated and differentiated) and C6 cells have been used to investigate kinetics, morphological and functional endpoints following exposure to MnCl(2) and manganic transferrin (Mn-Tf). [Mn](i) in undifferentiated (non-differentiated cells) exposed to both free (MnCl(2)) and bound Mn (Mn-Tf), was three- to fivefold lower as compared to differentiated (differentiated) PC12 cells and higher by one order of magnitude as compared to glial C6 cells. Exposure to both MnCl(2) and Mn-Tf was followed by time- and dose-dependent morphological changes characteristic of apoptosis, which was never observed in Mn-exposed C6 glial cells. Results from cell viability assays were consistent with apoptotic response rates quantified by cell count. Threshold concentrations for undifferentiated and differentiated PC12 cells were 10(-6) and 10(-5)m, respectively. Thus, despite their greater ability to accumulate Mn, differentiated PC12 cells are less sensitive to Mn-induced apoptosis. This model might be relevant to neuronal degeneration induced by Mn occurring in the developing brain and possibly in clinical manganism. Such critical doses at the cellular level seem to be consistent with Mn levels (5x10(-6)m) recorded in the basal ganglia of monkeys chronically exposed to Mn and developing clinical signs of manganism.

Isolation and Characterization of Circulating Tumor Cells in Squamous Cell Carcinoma of the Lung Using a Non-EpCAM-Based Capture Method.

Introduction The exclusion of circulating tumor cells (CTCs) that have lost epithelial antigens during the epithelial-to-mesenchymal transition (EMT) process by using Epithelial Cell Adhesion Molecule (EpCAM) based capture methods is still a matter of debate. In this study, cells obtained after depletion procedure from blood samples of squamous cell lung cancer (SQCLC) patients were identified based on morphology and characterized with the combination of FISH assessment and immunophenotypic profile. Materials and Methods Five mL blood samples, collected from 55 advanced SQCLC patients, were analyzed by a non-EpCAM-based capture method. After depletion of leukocytes and erythroid cells, the negative fraction was characterized by both FISH using a fibroblast growth factor receptor 1 (FGFR1) probe and by immunocytochemistry. Thirty healthy donors were also tested. Results Based on morphology (nuclear dimension ≥10 μm, shape and hypercromatic aspect) suspicious circulating cells clearly distinguishable from contaminant leukocytes were observed in 49/55 (89%) SQCLC patients. Thirty-four of the 44 (77%) samples evaluable for FGFR1 FISH showed ≥ 6 FGFR1 gene copy number on average per cell. Vimentin expression involved 43% (18/42) of pooled circulating SQCLC cells, whereas only 29% (14/48) were EpCAM positive. Confocal microscopy confirmed the localization of FGFR1 probe in suspicious circulating cells. Suspicious circulating elements were also observed in healthy donors and did not show any epithelial associated antigens. A significantly lower number of suspicious circulating cells in healthy donors compared to SQCLC patients was found. Conclusions Among the heterogeneous cell population isolated by depletion procedure, the coexistence of cells with epithelial and/or mesenchymal phenotype suggests that EMT may participate to transendothelial invasion and migration of tumor cells in advanced SQCLC. The finding of cells with neither EpCAM or EMT phenotype, retrieved after non-EpCAM-based systems, underlines the presence of suspicious elements in the blood of both SQCLC patients and healthy donors. Further phenotyping and molecular analyses are necessary to fully characterize these circulating elements.

In vitro effects of Mg/Sr–substituted hydroxyapatite on osteoblast activity

Stoichiometric hydroxyapatite (HA: Ca10(PO4)6(OH)2) has been used as a model for the apatite of human bone tissue for many years. Besides calcium, phosphate and carbonate, mineral bone contains many inorganic compounds such as sodium, fluoride, chloride, magnesium, strontium, zinc, copper and iron. This work investigates in vitro effects of magnesium and strontium substitutions in the apatite lattice on the behaviour of human osteoblasts. We compared powders of Mg and Sr substituted apatites (MgSrHA) with those of stoichiometric HA. Powders were prepared as-synthesized and not sintered, which is the most critical condition in terms of reactivity towards cells. Human osteoblasts were seeded in 24 well plates at the density of 15,000 cells/cm2 in DMEM supplemented with 250 μM ascorbic acid and 10% FCS. Treatments with powders were applied 24 hours after seeding. Dose-response (range 0.1-10 mg/ml) curves of HA and MgSrHA powders were performed with MTT assay after 3 days of treatment. At 10 mg/ml, viability level of HA-treated osteoblasts was lower than that of MgSrHAtreated cells (respectively 35% and 50% of untreated cells). At 3 mg/ml viability values were comparable and proliferation was slightly hindered (80% of untreated). No sign of cell death, evaluated with calcein-propidium iodide assay on living cells, was detectable at any of the tested doses. MgSrHA and HA did not affect cell proliferation (evaluated by cell counting and cell protein content) after 3 or 7 days of treatment with 0.3 and 1 mg/ml. At these dosages, alkaline phosphatase (ALP) activity was not affected after 3 days, but was slightly lower than that of untreated cells at 0.3 mg/ml after 7 days. Calcium deposition was measured with red alizarin after 7, 14 and 21 days of treatment with 0.3 or 1 mg/ml of powders in the presence of 100 nM dexamethasone and 10 mM β-glicero phosphate. Calcium deposition increased in a time-dependent way and was highly stimulated in cells treated with both powders. These preliminary results suggest that MgSrHA has no toxicity effects and seems to have a better biocompatibility than stoichiometric HA. Further investigations are needed to elucidate the cell-biomaterial interactions.

Mammalian Diaphanous-related formin-1 restricts early phases of influenza A/NWS/33 virus (H1N1) infection in LLC-MK2 cells by affecting cytoskeleton dynamics

Viruses depend on cellular machinery to efficiently replicate. The host cytoskeleton is one of the first cellular systems hijacked by viruses in order to ensure their intracellular transport and promote the development of infection. Our previous results demonstrated that stable microfilaments and microtubules interfered with human influenza A/NWS/33 virus (H1N1) infection in semi-permissive LLC-MK2 cells. Although formins play a key role in cytoskeletal remodelling, few studies addressed a possible role of these proteins in development of viral infection. Here, we have demonstrated that mammalian Diaphanous-related formin-1 (mDia1) is involved in the control of cytoskeleton dynamics during human influenza A virus infection. First, by employing cytoskeleton-perturbing drugs, we evidenced a cross-talk occurring between microtubules and microfilaments that also has implications on the intracellular localization of mDia1. In influenza A/NWS/33 virus-infected LLC-MK2 cells, mDia1 showed a highly dynamic intracellular localization and partially co-localized with actin and tubulin. A depletion of mDia1 by RNA-mediated RNA interference was found to improve the outcome of influenza A/NWS/33 virus infection and to increase the dynamics of microfilament and microtubule networks in LLC-MK2 cells. Consistent with these findings, observations made in epithelial respiratory cells from paediatric patients with acute respiratory disease assessed that the expression of mDia1 is stimulated by influenza A virus but not by respiratory syncytial virus. Taken together, the obtained results suggest that mDia1 restricts the initiation of influenza A/NWS/33 virus infection in LLC-MK2 cells by counteracting cytoskeletal dynamics.

Novel Activity of a Synthetic Decapeptide Against Toxoplasma gondii Tachyzoites

The killer peptide KP is a synthetic decapeptide derived from the sequence of the variable region of a recombinant yeast killer toxin-like microbicidal single-chain antibody. KP proved to exert significant activities against diverse microbial and viral pathogens through different mechanisms of action, but little is known of its effect on apicomplexan protozoa. The aim of the present study was to evaluate the in vitro activity of KP against Toxoplasma gondii, a globally widespread protozoan parasite of great medical interest. The effect of KP treatment and its potential mechanism of action on T. gondii were evaluated by various methods, including light microscopy, quantitative PCR, flow cytometry, confocal microscopy, and transmission electron microscopy. In the presence of KP, the number of T. gondii tachyzoites able to invade Vero cells and the parasite intracellular proliferation were significantly reduced. Morphological observation and analysis of apoptotic markers suggested that KP is able to trigger an apoptosis-like cell death in T. gondii. Overall, our results indicate that KP could be a promising candidate for the development of new anti-Toxoplasma drugs with a novel mechanism of action.