Host-cell dependent role of phosphorylated keratin 8 during influenza A/NWS/33 virus (H1N1) infection in mammalian cells.

In this study, we investigated the involvement of keratin 8 during human influenza A/NWS/33 virus (H1N1) infection in semi-permissive rhesus monkey-kidney (LLC-MK2) and permissive human type II alveolar epithelial (A549) cells. In A549 cells, keratin 8 showed major expression and phosphorylation levels. Influenza A/NWS/33 virus was able to subvert keratin 8 structural organization at late stages of infection in both cell models, promoting keratin 8 phosphorylation in A549 cells at early phases of infection. Accordingly, partial colocalizations of the viral nucleoprotein with keratin 8 and its phosphorylated form were assessed by confocal microscopy at early stages of infection in A549 cells. The employment of chemical activators of phosphorylation resulted in structural changes as well as increased phosphorylation of keratin 8 in both cell models, favoring the influenza A/NWS/33 virus's replicative efficiency in A549 but not in LLC-MK2 cells. In A549 and human larynx epidermoid carcinoma (HEp-2) cells inoculated with respiratory secretions from pediatric patients positive for, respectively, influenza A virus or respiratory syncytial virus, the keratin 8 phosphorylation level had increased only in the case of influenza A virus infection. The results obtained suggest that in A549 cells the influenza virus is able to induce keratin 8 phosphorylation thereby enhancing its replicative efficiency.

3D-printed chitosan scaffolds modified with D-(+) raffinose and enriched with type IV collagen to improve epithelial cell colonization.

Tissue regeneration often requires the use of biocompatible resorbable scaffolds to support the ingrowth of cells from neighboring tissues into a localized tissue defect. Such scaffolds must possess surface molecular cues that stimulate cells to populate the device, the first necessary condition for the formation of a healthy tissue. Chitosan is a natural polymer that has long been tested in biomedical applications because of its high biocompatibility, which can be further increased by modifying its formulation, e.g. adding D-(+) raffinose. We used this formulation in an ad hoc designed 3D printer to create regularly ordered scaffolds, which we then enriched with type IV collagen, an isoform of collagen that is exclusively found in basement membranes. Human epithelial A549 cells were then seeded on control scaffolds or on scaffolds coated with collagen, which was precipitated, or on scaffolds first collagenized and then exposed to either UVB or UVC radiation. Observations by the transmission light microscope, confocal microscope after staining with calcein-AM/propidium iodide, and by environmental scanning electron microscope revealed that collagen-enriched UV-treated scaffolds promoted the attachment of a higher number of cells, which covered a more extensive area of the scaffold, as also confirmed by alamar blue viability assay. Together these data confirm that coating 3D-printed scaffolds made of D-(+) raffinose-modified chitosan with type IV collagen and exposing them to UV light sensibly increases the cell compatibility of scaffolds, making them a better candidate to serve as a tool for the regeneration of epithelia.

Dissection of the Structural Features of a Fungicidal Antibody-Derived Peptide.

The synthetic peptide T11F (TCRVDHRGLTF), derived from the constant region of human IgM antibodies, proved to exert a significant activity in vitro against yeast strains, including multidrug resistant isolates. Alanine substitution of positively charged residues led to a decrease in candidacidal activity. A more dramatic reduction in activity resulted from cysteine replacement. Here, we investigated the conformational properties of T11F and its alanine-substituted derivatives by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. Peptide interaction with Candida albicans cells was studied by confocal and scanning electron microscopy. T11F and most of its derivatives exhibited CD spectra with a negative band around 200 nm and a weaker positive band around 218 nm suggesting, together with NMR coupling constants, the presence of a polyproline II (PPII) helix, a conformational motif involved in a number of biological functions. Analysis of CD spectra revealed a critical role for phenylalanine in preserving the PPII helix. In fact, only the F11A derivative presented a random coil conformation. Interestingly, the loss of secondary structure influenced the rate of killing, which turned out to be significantly reduced. Overall, the obtained results suggest that the PPII conformation contributes in characterising the cell penetrating and fungicidal properties of the investigated peptides.

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

Design and Synthesis of New Cell Penetrating Peptides: Diffusion and Distribution Inside the Cornea.

The role of cell penetrating peptides (CPPs) has been challenged in recent years for drug delivery to ocular tissues for the targeting of both anterior and posterior segments. The enhancement of trans-corneal transport for anterior segment targeting is a very important issue possibly leading to important outcomes on efficacy and to the opportunity of topical administration of molecules with unfavorable penetration properties. The aim of the present work was the design and synthesis of new CPPs, deriving from the structure of PEP-1 peptide. Synthesized peptides were labeled with 5-carboxyfluorescein (5-FAM), and their diffusion behavior and distribution inside the cornea were evaluated by a validated ex vivo model and a confocal microscopy approach. Newly synthesized peptides showed similar corneal permeation profiles as PEP-1 (Papp = 0.75 ± 0.56 × 10-6 cm/s), about 2.6-fold higher than 5-FAM (Papp = 0.29 ± 0.08 × 10-6 cm/s) despite the higher molecular weight. Confocal microscopy experiments highlighted the tendency of PEP-1 and its derived peptides to localize in the intercellular space and/or in the plasma membrane. Noteworthy, using penetratin as positive control, a higher trans-corneal permeation (Papp = 6.18 ± 1.46 × 10-6 cm/s) was evidenced together with a diffusion by intracellular route and a different accumulation between wings and basal epithelial cells, probably depending on the stage of cell development. Finally, PEP-1 and pep-7 proved to be safe and well tolerated when tested on human conjuctival cell line.

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.

Synergistic activity of letrozole and sorafenib on breast cancer cells.

Estrogens induce breast tumor cell proliferation by directly regulating gene expression via the estrogen receptor (ER) transcriptional activity and by affecting growth factor signaling pathways such as mitogen-activated protein kinase (MAPK) and AKT/mammalian target of rapamycin Complex1 (mTORC1) cascades. In this study we demonstrated the preclinical therapeutic efficacy of combining the aromatase inhibitor letrozole with the multi-kinase inhibitor sorafenib in aromatase-expressing breast cancer cell lines. Treatment with letrozole reduced testosterone-driven cell proliferation, by inhibiting the synthesis of estrogens. Sorafenib inhibited cell proliferation in a concentration-dependent manner; this effect was not dependent on sorafenib-mediated inhibition of Raf1, but involved the down-regulation of mTORC1 and its targets p70S6K and 4E-binding protein 1 (4E-BP1). At concentrations of 5-10 µM the growth-inhibitory effect of sorafenib was associated with the induction of apoptosis, as indicated by release of cytochrome c and Apoptosis-Inducing Factor into the cytosol, activation of caspase-9 and caspase-7, and PARP-1 cleavage. Combination of letrozole and sorafenib produced a synergistic inhibition of cell proliferation associated with an enhanced accumulation of cells in the G(0)/G(1) phase of the cell cycle and with a down-regulation of the cell cycle regulatory proteins c-myc, cyclin D1, and phospho-Rb. In addition, longer experiments (12 weeks) demonstrated that sorafenib may be effective in preventing the acquisition of resistance towards letrozole. Together, these results indicate that combination of letrozole and sorafenib might constitute a promising approach to the treatment of hormone-dependent breast cancer.

Dose-dependent effects of platelet gel releasate on activities of human osteoblasts.

BACKGROUND: Platelet-rich plasma is used in oral and maxillofacial surgery; however, its real efficacy is debated. Also, the in vitro effects on bone-specific functions are contradictory. Understanding the mechanisms of action of platelet-derived factors could be the basis for their proper use in clinical applications. METHODS: The functional parameters of osteoblasts (proliferation, alkaline phosphatase, collagen synthesis, and calcium deposition) were analyzed in vitro for 14 days in the presence of different concentrations (100%, 33%, and 11%) of platelet gel releasate (PGR). RESULTS: Concentrations of 100% PGR and 33% PGR stimulated cells to proliferate more than 10% fetal calf serum. The effect on cell proliferation was dose dependent, and the addition of dexamethasone (dex) and beta-glycerophosphate (beta-GP) reduced the proliferative effects. Alkaline phosphatase activity was stimulated by 33% PGR and 11% PGR after 7 days and was induced further by dex and beta-GP. Also, collagen synthesis, measured on day 11, was stimulated by 33% PGR and 11% PGR. Calcium deposition, evaluated after 7 and 14 days, was greatest in cells treated with PGR supplemented with dex and GP. The mineralization process increased with time; on day 14, calcium aggregates were observed in all cultures treated with PGR (100%, 33%, and 11%). CONCLUSIONS: PGR stimulated osteoblast proliferation in a dose dependent manner and, when used at 33% and 11%, induced maximum levels of alkaline phosphatase and collagen synthesis. Moreover, in the presence of dex and beta-GP, PGR stimulated the end maturative status of cells as expressed by the deposition of calcium nodules.

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. METHODS: Human dermal fibroblasts were irradiated with 600 mJ/cm(2) of UVB radiation. Cells were analyzed at increasing time points from irradiation to study the recovery from UVB-induced DNA photodamage. Damage repair was subsequently assessed by immunocytochemical analysis of CPDs levels and by measurement of p53 protein expression. RESULTS: The extracellular addition of 100 microg/ml PDRN immediately after irradiation caused a strong activation of p53 protein in the first 24 h. This signal was accompanied by an increase in CPDs repair rates at early time points of recovery. CONCLUSIONS: The addition of PDRN to the culture medium supports CPDs repair probably providing a faster supply of precursors for the deoxyribonucleotide triphosphates pool necessary to UVB-damaged cells. This condition could promote the action of the salvage pathway, thereby accelerating DNA repair, but other inducible responses linked to increased p53 expression could be involved.

Methylmercury cytotoxicity in PC12 cells is mediated by primary glutathione depletion independent of excess reactive oxygen species generation.

Low doses, chronic exposure to mercurial organic compounds is a worldwide health concern and could be pathogenetically relevant as co-factor in several neurodegenerative diseases. In this in vitro study we wanted to further improve our knowledge on the mechanisms of toxicity of methylmercury hydroxide (MeHgOH) in the unprimed PC12 cell line. Cell viability, mitochondrial function, redox state, and cell morphology were recorded at different time points to sequence the events leading to cell death. The lowest cytotoxic concentration and EC50 were 0.3 and 1.3 microM, respectively. 5 microM MeHgOH was fatal for 80% of the cell population after 24 h; within 1 h it caused glutathione (GSH) depletion and a partial dissipation of Deltapsim. At this concentration, reactive oxygen species (ROS) generation was only slight and delayed. After 6h more than 50% of ATP was available and caspase 3 was active. Time-lapse confocal microscopy showed that only a fraction of the cells completed apoptosis while others turned toward necrosis (necrapoptosis). Pre-incubation with N-acetylcysteine (NAC) and GSH but not Cyclosporin A rescued over 80% of the cells. These results provide experimental evidence that, in this cell model, MeHgOH triggers cell death via a primary depletion of GSH but in the absence of ROS overproduction.

Polydeoxyribonucleotide (PDRN) promotes human osteoblast proliferation: a new proposal for bone tissue repair.

Several researchers have recently shed new light upon the importance of extracellular nucleotides and nucleosides to stimulate cells growth. PDRN, a mixture of deoxyribonucleotides polymers of different lengths, has recently demonstrated to stimulate "in vitro" fibroblast proliferation and collagen production, probably stimulating the purinergic receptor system. In this work we evaluated the effects of PDRN on human cultured osteoblasts, focusing our attention on cell proliferation and alkaline phosphatase activity. PDRN at a concentration of 100 microg/ml induce an increase in osteoblasts growth after 6 days as compared to control (+21%). The addition of DMPX 50 microM and suramine (P2 inhibitor) 10 microM give different results: suramine has no significant effect, while DPMX reduce, even if partially, the PDRN induced cell growth. The alkaline phosphatase activity shows a gradual enhancement starting from day 0 to day 10, even if PDRN treated cells, examined at day 6, present a sensibly lower phosphatase activity when compared to controls. Our data demonstrate that PDRN acts as an osteoblast growth stimulator. Its action is partially due to a stimulation of the purinergic system mediated by A2 purinoreceptors, however we can not exclude the involvement of other mechanism like salvage pathway.

[In vitro models for the evaluation of the neurotoxicity of methylmercury. Current state of knowledge]. / Modelli in vitro per la valutazione della neurotossicità del metilmercurio. Stato attuale delle conoscenze.

BACKGROUND: In adults, MeHg poisoning is characterized by damage to discrete anatomical areas of the brain (visual cortex, loss of neurons from the granule layer of the cerebellum). However, the immature central nervous system (CNS), which is extremely sensitive to MeHg neurotoxicity, shows a diffuse and widespread damage disorganization of cerebral cortex cytoarchitecture, disappearance of granule cells with narrowing of the molecular layer. While adverse effects have been unequivocally demonstrated in poisoning incidents in humans (visual abnormalities, sensory impairment of the extremities, cerebellar ataxia, hearing loss, muscle weakness, tremor and mental deterioration), the implications of lower level exposures, such as those occurring in fish-eating populations, are still controversial. The high affinity of MeHg for thiol groups makes proteins and peptides bearing cysteines the predominant targets for structural and functional modification by MeHg in all subcellular compartments. METHODS: The identification of MeHg cellular and sub-cellular targets in the CNS is complicated by the fact that it is difficult to observe the outcomes directly in vivo. In neurobiology, in vitro cell culture techniques have been successfully developed and employed to address specific questions of cell biology and nervous system functioning and provide a means to systematically study the complexity of cellular functions of the CNS elements. Moreover, they provide a convenient experimental tool for testing possible functions or postulates in vivo that otherwise might not be conducted. RESULTS: Several mechanisms have been proposed as being implicated in the neurotoxic effects of MeHg. Examples of MeHg molecular effects which may be relevant to risk assessment are presented, including cell death mode, effects on microtubules, calcium signalling, oxidative stress, effects on neurotransmitter systems. CONCLUSIONS: Molecular and cellular approaches permit exploration of early biological responses to chemical or physical agents and definition of the role of these early effects in altered cellular structure and function.

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.