In vitro induction of neoantigen-specific T cells in myelodysplastic syndrome,a disease with low mutational burden

Therapies that utilize immune checkpoint inhibition work by leveraging mutation-derived neoantigens and have shown greater clinical efficacy in tumors with higher mutational burden. Whether tumors with a low mutational burden are susceptible to neoantigen-targeted therapy has not been fully addressed. To examine the feasibility of neoantigen-specific adoptive T-cell therapy, the authors studied the T-cell response against somatic variants in five patients with myelodysplastic syndrome (MDS), a malignancy with a very low tumor mutational burden. DNA and RNA from tumor (CD34⁺) and normal (CD3⁺) cells isolated from the patients’ blood were sequenced to predict patient-specific MDS neopeptides. Neopeptides representing the somatic variants were used to induce and expand autologous T cells ex vivo, and these were systematically tested in killing assays to determine the proportion of neopeptides yielding neoantigen-specific T cells. The authors identified a total of 32 somatic variants (four to eight per patient) and found that 21 (66%) induced a peptide-specific T-cell response and 19 (59%) induced a T-cell response capable of killing autologous tumor cells. Of the 32 somatic variants, 11 (34%) induced a CD4⁺ response and 11 (34%) induced a CD8⁺ response that killed the tumor. These results indicate that in vitro induction of neoantigen-specific T cells is feasible for tumors with very low mutational burden and that this approach warrants investigation as a therapeutic option for such patients.     

Extremely low frequency electromagnetic field exposure promotes differentiation of pituitary corticotrope-derived AtT20 D16V cells

The pituitary corticotrope-derived AtT20 D16V cell line responds to nerve growth factor (NGF) by extending neurite-like processes and differentiating into neurosecretory-like cells. The aim of this work is the study of the effect of extremely low frequency electromagnetic fields (ELF-EMF) at a frequency of 50 Hz on these differentiation activities. To establish whether exposure to the field could influence the molecular biology of the cells, they were exposed to a magnetic flux density of 2 milli-Tesla (mT). Intracellular calcium ([Ca2+]i) and intracellular pH (pHi) were monitored in single exposed AtT20 D16V cells using fluorophores Indo-1 and SNARF for [Ca2+]i and pHi, respectively. Single-cell fluorescence microscopy showed a statistically significant increase in [Ca2+]i followed by a drop in pHi in exposed cells. Both scanning electron microscopy (SEM) and transmission microscopy of exposed AtT20 D16V cells show morphological changes in plasma membrane compared to non-exposed cells; this modification was accompanied by a rearrangement in actin filament distribution and the emergence of properties typical of peptidergic neuronal cells-the appearance of secretory-like granules in the cytosol and the increase of synaptophysin in synaptic vesicles, changes typical of neurosecretory-like cells. Using a monoclonal antibody toward the neurofilament protein NF-200 gave additional evidence that exposed cells were in an early stage of differentiation compared to control. Pre-treatment with 0.3 microM nifedipine, which specifically blocks L-type Ca2+ channels, prevented NF-200 expression in AtT20 D16V exposed cells. The above findings demonstrate that exposure to 50 Hz ELF-EMF is responsible for the premature differentiation in AtT20 D 16 V cells.     

Mechanism of Folding and Activation of Subtilisin Kexin Isozyme-1 (SKI-1)/Site-1 Protease (S1P)

The proprotein convertase subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P) is implicated in lipid homeostasis, the unfolded protein response, and lysosome biogenesis. The protease is further hijacked by highly pathogenic emerging viruses for the processing of their envelope glycoproteins. Zymogen activation of SKI-1/S1P requires removal of an N-terminal prodomain, by a multistep process, generating the mature enzyme. Here, we uncover a modular structure of the human SKI-1/S1P prodomain and define its function in folding and activation. We provide evidence that the N-terminal AB fragment of the prodomain represents an autonomous structural and functional unit that is necessary and sufficient for folding and partial activation. In contrast, the C-terminal BC fragment lacks a defined structure but is crucial for autoprocessing and full catalytic activity. Phylogenetic analysis revealed that the sequence of the AB domain is highly conserved, whereas the BC fragment shows considerable variation and seems even absent in some species. Notably, SKI-1/S1P of arthropods, like the fruit fly Drosophila melanogaster, contains a shorter prodomain comprised of full-length AB and truncated BC regions. Swapping the prodomain fragments between fly and human resulted in a fully mature and active SKI-1/S1P chimera. Our study suggests that primordial SKI-1/S1P likely contained a simpler prodomain consisting of the highly conserved AB fragment that represents an independent folding unit. The BC region appears as a later evolutionary acquisition, possibly allowing more subtle fine-tuning of the maturation process.     

A Novel Topology of Proline-rich Transmembrane Protein 2 (PRRT2): HINTS FOR AN INTRACELLULAR FUNCTION AT THE SYNAPSE*

Proline-rich transmembrane protein 2 (PRRT2) has been identified as the single causative gene for a group of paroxysmal syndromes of infancy, including epilepsy, paroxysmal movement disorders, and migraine. On the basis of topology predictions, PRRT2 has been assigned to the recently characterized family of Dispanins, whose members share the two-transmembrane domain topology with a large N terminus and short C terminus oriented toward the outside of the cell. Because PRRT2 plays a role at the synapse, it is important to confirm the exact orientation of its N and C termini with respect to the plasma membrane to get clues regarding its possible function. Using a combination of different experimental approaches, including live immunolabeling, immunogold electron microscopy, surface biotinylation and computational modeling, we demonstrate a novel topology for this protein. PRRT2 is a type II transmembrane protein in which only the second hydrophobic segment spans the plasma membrane, whereas the first one is associated with the internal surface of the membrane and forms a helix-loop-helix structure without crossing it. Most importantly, the large proline-rich N-terminal domain is not exposed to the extracellular space but is localized intracellularly, and only the short C terminus is extracellular (N_cyt/C_exo topology). Accordingly, we show that PRRT2 interacts with the Src homology 3 domain-bearing protein Intersectin 1, an intracellular protein involved in synaptic vesicle cycling. These findings will contribute to the clarification of the role of PRRT2 at the synapse and the understanding of pathogenic mechanisms on the basis of PRRT2-related neurological disorders.     

Hydroxytyrosol glucuronides protect renal tubular epithelial cells against H(2)O(2) induced oxidative damage

Hydroxytyrosol (2-(3′,4′-dihydroxyphenyl)ethanol; HT), the most active ortho-diphenolic compound, present either in free or esterified form in extravirgin olive oil, is extensively metabolized in vivo mainly to O-methylated, O-sulfated and glucuronide metabolites. We investigated the capacity of three glucuronide metabolites of HT, 3′-O-β-d-glucuronide and 4′-O-β-d-glucuronide derivatives and 2-(3′,4′-dihydroxyphenyl)ethanol-1-O-β-d-glucuronide, in comparison with the parent compound, to inhibit H₂O₂ induced oxidative damage and cell death in LLC-PK1 cells, a porcine kidney epithelial cell line. H₂O₂ treatment exerted a toxic effect inducing cell death, interacting selectively within the pro-death extracellular-signal relate kinase (ERK 1/2) and the pro-survival Akt/PKB signaling pathways. It also produced direct oxidative damage initiating the membrane lipid peroxidation process. None of the tested glucuronides exhibited any protection against the loss in renal cell viability. They also failed to prevent the changes in the phosphorylation states of ERK and Akt, probably reflecting their inability to enter the cells, while HT was highly effective. Notably, pretreatment with glucuronides exerted a protective effect at the highest concentration tested against membrane oxidative damage, comparable to that of HT: the formation of malondialdehyde, fatty acid hydroperoxides and 7-ketocholesterol was significantly inhibited.     

Role of the EGFR ligand/receptor system in the secretion of angiogenic factors in mesenchymal stem cells

Increasing evidence suggests that bone marrow-derived mesenchymal stem cells (MSCs) are recruited into the stroma of developing tumors where they contribute to cancer progression. MSCs produce different growth factors that sustain tumor-associated neo-angiogenesis. Since the majority of carcinomas secrete ligands of the epidermal growth factor receptor (EGFR), we assessed the role of EGFR signaling in regulating the release of angiogenic factors in MSCs. Treatment of human primary MSCs and of the human osteoblastic cell line hFOB with transforming growth factor α (TGF-α), one of the main ligands of the EGFR, significantly induced activation of this receptor and of different intracellular signaling proteins, including the PI3K/AKT and the MEK/MAPK pathways. TGF-α induced a significant increase in the levels of secretion of vascular endothelial growth factor in both MSCs and hFOB. Conditioned medium from TGF-α treated MSCs showed an higher in vivo angiogenic effect as compared with medium from untreated cells. Treatment of MSCs with TGF-α also produced a significant increase in the secretion of other angiogenic growth factors such as angiopoietin-2, granulocyte-colony stimulating factor, hepatocyte growth factor, interleukin (IL)-6, IL-8, and platelet-derived growth factor-BB. Using selective MEK and PI3K inhibitors, we found that both MEK/MAPK and the PI3K/AKT signaling pathways mediate the ability of TGF-α to induce secretion of angiogenic factors in MSCs. Finally, stimulation with TGF-α increased the ability of MSCs to induce migration of MCF-7 breast cancer cells. These data suggest that EGFR signaling regulates the ability of MSCs to sustain cancer progression through the release of growth factors that promote neo-angiogenesis and tumor cell migration.     

ENPP1 affects insulin action and secretion: evidences from in vitro studies

The aim of this study was to deeper investigate the mechanisms through which ENPP1, a negative modulator of insulin receptor (IR) activation, plays a role on insulin signaling, insulin secretion and eventually glucose metabolism. ENPP1 cDNA (carrying either K121 or Q121 variant) was transfected in HepG2 liver-, L6 skeletal muscle- and INS1E beta-cells. Insulin-induced IR-autophosphorylation (HepG2, L6, INS1E), Akt-Ser(473), ERK1/2-Thr(202)/Tyr(204) and GSK3-beta Ser(9) phosphorylation (HepG2, L6), PEPCK mRNA levels (HepG2) and 2-deoxy-D-glucose uptake (L6) was studied. GLUT 4 mRNA (L6), insulin secretion and caspase-3 activation (INS1E) were also investigated. Insulin-induced IR-autophosphorylation was decreased in HepG2-K, L6-K, INS1E-K (20%, 52% and 11% reduction vs. untransfected cells) and twice as much in HepG2-Q, L6-Q, INS1E-Q (44%, 92% and 30%). Similar data were obtained with Akt-Ser(473), ERK1/2-Thr(202)/Tyr(204) and GSK3-beta Ser(9) in HepG2 and L6. Insulin-induced reduction of PEPCK mRNA was progressively lower in untransfected, HepG2-K and HepG2-Q cells (65%, 54%, 23%). Insulin-induced glucose uptake in untransfected L6 (60% increase over basal), was totally abolished in L6-K and L6-Q cells. GLUT 4 mRNA was slightly reduced in L6-K and twice as much in L6-Q (13% and 25% reduction vs. untransfected cells). Glucose-induced insulin secretion was 60% reduced in INS1E-K and almost abolished in INS1E-Q. Serum deficiency activated caspase-3 by two, three and four folds in untransfected INS1E, INS1E-K and INS1E-Q. Glyburide-induced insulin secretion was reduced by 50% in isolated human islets from homozygous QQ donors as compared to those from KK and KQ individuals. Our data clearly indicate that ENPP1, especially when the Q121 variant is operating, affects insulin signaling and glucose metabolism in skeletal muscle- and liver-cells and both function and survival of insulin secreting beta-cells, thus representing a strong pathogenic factor predisposing to insulin resistance, defective insulin secretion and glucose metabolism abnormalities.