Regulation of NKG2D-Dependent NK Cell Functions: The Yin and the Yang of Receptor Endocytosis.

Natural-killer receptor group 2, member D (NKG2D) is a well characterized natural killer (NK) cell activating receptor that recognizes several ligands poorly expressed on healthy cells but up-regulated upon stressing stimuli in the context of cancer or viral infection. Although NKG2D ligands represent danger signals that render target cells more susceptible to NK cell lysis, accumulating evidence demonstrates that persistent exposure to ligand-expressing cells causes the decrease of NKG2D surface expression leading to a functional impairment of NKG2D-dependent NK cell functions. Upon ligand binding, NKG2D is internalized from the plasma membrane and sorted to lysosomes for degradation. However, receptor endocytosis is not only a mechanism of receptor clearance from the cell surface, but is also required for the proper activation of signalling events leading to the functional program of NK cells. This review is aimed at providing a summary of current literature relevant to the molecular mechanisms leading to NKG2D down-modulation with particular emphasis given to the role of NKG2D endocytosis in both receptor degradation and signal propagation. Examples of chronic ligand-induced down-regulation of NK cell activating receptors other than NKG2D, including natural cytotoxicity receptors (NCRs), DNAX accessory molecule-1 (DNAM1) and CD16, will be also discussed

Discrete-time quantum walks: continuous limit and symmetries

The continuous limit of one dimensional discrete-time quantum walks with time- and space-dependent coefficients is investigated. A given quantum walk does not generally admit a continuous limit but some families (1-jets) of quantum walks do. All families (1-jets) admitting a continuous limit are identified. The continuous limit is described by a Dirac-like equation or, alternately, a couple of Klein-Gordon equations. Variational principles leading to these equations are also discussed, together with local invariance properties

Innate immune activating ligand SUMOylation affects tumor cell recognition by NK cells

Natural Killer cells are innate lymphocytes involved in tumor immunosurveillance. They express activating receptors able to recognize self-molecules poorly expressed on healthy cells but up-regulated upon stress conditions, including transformation. Regulation of ligand expression in tumor cells mainly relays on transcriptional mechanisms, while the involvement of ubiquitin or ubiquitin-like modifiers remains largely unexplored. Here, we focused on the SUMO pathway and demonstrated that the ligand of DNAM1 activating receptor, PVR, undergoes SUMOylation in multiple myeloma. Concurrently, we found that PVR is preferentially located in intracellular compartments in human multiple myeloma cell lines and malignant plasma cells and that inhibition of the SUMO pathway promotes its translocation to the cell surface, increasing tumor cell susceptibility to NK cell-mediated cytolysis. Our findings provide the first evidence of an innate immune activating ligand regulated by SUMOylation, and confer to this modification a novel role in impairing recognition and killing of tumor cells.Natural Killer cells are innate lymphocytes involved in tumor immunosurveillance. They express activating receptors able to recognize self-molecules poorly expressed on healthy cells but up-regulated upon stress conditions, including transformation. Regulation of ligand expression in tumor cells mainly relays on transcriptional mechanisms, while the involvement of ubiquitin or ubiquitin-like modifiers remains largely unexplored. Here, we focused on the SUMO pathway and demonstrated that the ligand of DNAM1 activating receptor, PVR, undergoes SUMOylation in multiple myeloma. Concurrently, we found that PVR is preferentially located in intracellular compartments in human multiple myeloma cell lines and malignant plasma cells and that inhibition of the SUMO pathway promotes its translocation to the cell surface, increasing tumor cell susceptibility to NK cell-mediated cytolysis. Our findings provide the first evidence of an innate immune activating ligand regulated by SUMOylation, and confer to this modification a novel role in impairing recognition and killing of tumor cells

Exposure to reversine affects the chondrocyte morphology and phenotype in vitro

Articular chondrocytes derived from osteoarthritic tissues (OA HAC) show a severely reduced chondrogenic commitment. This impairment undermines their use for tissue-engineered cartilage repair, which relies on cell proliferation and growth to meet therapeutic needs, but also on efficient cell plasticity to recover the chondrogenic phenotype. Reversine (Rev), a 2,6-disubstituted purine inhibitor of spindle-assembly checkpoints, was described to convert differentiated mesenchymal cells to their undifferentiated precursors. We hypothesized that Rev exposure could divert OA HAC to more plastic cells, re-boosting their subsequent commitment. HAC were enzymatically released from OA cartilage specimens, expanded for 2 weeks and treated with 5 \u3bcm Rev in dimethylsulphoxide (DMSO) or with DMSO alone for 6 days. Cell growth was assessed using the AlamarBlueTM assay. Cytoskeletal structure, endoproliferation and caspase-3-immunopositivity were assayed by epifluorescence microscopy. The OA HAC chondrogenic performance was evaluated by quantitative reverse transcription-polymerase chain reaction (RT-PCR) for glyceraldehyde-3-phosphate dehydrogenase, Sox9, Aggrecan (Agg), type II collagen (Col2), Ki67, cyclinD1, transforming growth factor-\u3b21 (TGF-\u3b21), -2 and -3, interleukin-1\u3b2 (IL-1\u3b2) and -6 , SMAD3 and -7, and vascular endothelial growth factor. Rev-treated OA HAC recovered polygonal morphology and reduced Ki67 expression and proliferation. Cell-cycle impairment accounted for altered cytoskeletal organization, endoproliferation and apoptosis, whereas a compensatory mechanism sustained the increased cyclinD1 transcript levels. Sox9, Agg and TGFs were overexpressed, but not Col2. IL transcripts were massively downregulated. These events were dose-related and transient. Overall, in spite of a higher Rev-induced transcriptional activity for extracellular matrix components and in spite of a Rev-treated cell phenotype closer to that of the three-dimensional native articular chondrocyte, Rev effects seem unleashed from a full regained chondrogenic potential

Clinical use of a 180-day implantable glucose sensor improves glycated haemoglobin and time in range in patients with type 1 diabetes

Aims: This real-world study evaluated the changes in glycated haemoglobin (HbA1c) and continuous glucose monitoring (CGM) metrics associated with use of the implantable 180-day Eversense CGM System (Eversense) in patients with type 1 diabetes. Materials and methods: This was a prospective, multicentre, observational study among adult participants aged ≥18 years with type 1 diabetes across seven diabetes-care centres in Italy who had Eversense inserted for the first time. HbA1c was measured at baseline and at 180 days. Changes in time in range [TIR (glucose 70–180 mg/dL)], time above range [TAR (glucose >180 mg/dL)], time below range [TBR (glucose <70 mg/dL)] and glycaemic variability were also assessed. Data were also analysed by previous CGM use and by mode of insulin delivery. Results: One-hundred patients were enrolled (mean age 36 ± 12 years, mean baseline HbA1c 7.4 ± 0.92% [57 ± 10 mmol/mol]). Fifty-six per cent of patients were users of the continuous subcutaneous insulin infusion pump and 45% were previous users of CGM. HbA1c significantly decreased in patients after 180 days of sensor wear (−0.43% ± 0.69%, 5 ± 8 mmol/mol, P < 0.0001). As expected, CGM-naïve patients achieved the greatest reduction in HbA1c (−0.74% ± 0.48%, 8 ± 5 mmol/mol). TIR significantly increased and TAR and mean daily sensor glucose significantly decreased while TBR did not change after 180 days of sensor wear. Conclusions: Real-world clinical use of the Eversense CGM System for 180 days was associated with significant improvements in HbA1c and CGM metrics among adults with type 1 diabetes. The study is registered on clinicaltrials.gov (NCT04160156)

Samhd1 phosphorylation and cytoplasmic relocalization after human cytomegalovirus infection limits its antiviral activity

SAMHD1 is a host restriction factor that functions to restrict both retroviruses and DNA viruses, based on its nuclear deoxynucleotide triphosphate (dNTP) hydrolase activity that limits availability of intracellular dNTP pools. In the present study, we demonstrate that SAMHD1 expression was increased following human cytomegalovirus (HCMV) infection, with only a modest effect on infectious virus production. SAMHD1 was rapidly phosphorylated at residue T592 after infection by cellular cyclin-dependent kinases, especially Cdk2, and by the viral kinase pUL97, resulting in a significant fraction of phosho-SAMHD1 being relocalized to the cytoplasm of infected fibroblasts, in association with viral particles and dense bodies. Thus, our findings indicate that HCMV-dependent SAMHD1 cytoplasmic delocalization and inactivation may represent a potential novel mechanism of HCMV evasion from host antiviral restriction activities

Lipid Raft-Dependent FcεRI Ubiquitination Regulates Receptor Endocytosis through the Action of Ubiquitin Binding Adaptors

The best characterized role for ubiquitination of membrane receptors is to negatively regulate signaling by targeting receptors for lysosomal degradation. The high affinity receptor for IgE (FcepsilonRI) expressed on mast cells and basophils is rapidly ubiquitinated upon antigen stimulation. However, the nature and the role of this covalent modification are still largelly unknown. Here, we show that FcepsilonRI subunits are preferentially ubiquitinated at multiple sites upon stimulation, and provide evidence for a role of ubiquitin as an internalization signal: under conditions of impaired receptor ubiquitination a decrease of receptor entry is observed by FACS analysis and fluorescence microscopy. We also used biochemical approaches combined with fluorescence microscopy, to demonstrate that receptor endocytosis requires the integrity of specific membrane domains, namely lipid rafts. Additionally, by RNA interference we demonstrate the involvement of ubiquitin-binding endocytic adaptors in FcepsilonRI internalization and sorting. Notably, the triple depletion of Eps15, Eps15R and Epsin1 negatively affects the early steps of Ag-induced receptor endocytosis, whereas Hrs depletion retains ubiquitinated receptors into early endosomes and partially prevents their sorting into lysosomes for degradation. Our results are compatible with a scenario in which the accumulation of engaged receptor subunits into lipid rafts is required for receptor ubiquitination, a prerequisite for efficient receptor internalization, sorting and delivery to a lysosomal compartment

Biochemical and genetic analysis of butyrylcholinesterase (BChE) in a family, due to prolonged neuromuscular blockade after the use of succinylcholine

Butyrylcholinesterase (BChE) is a plasma enzyme that catalyzes the hydrolysis of choline esters, including the muscle-relaxant succinylcholine and mivacurium. Patients who present sustained neuromuscular blockade after using succinylcholine usually carry BChE variants with reduced enzyme activity or an acquired BChE deficiency. We report here the molecular basis of the BCHE gene underlying the slow catabolism of succinylcholine in a patient who underwent endoscopic nasal surgery. We measured the enzyme activity of BChE and extracted genomic DNA in order to study the promoter region and all exons of the BCHE gene of the patient, her parents and siblings. PCR products were sequenced and compared with reference sequences from GenBank. We detected that the patient and one of her brothers have two homozygous mutations: nt1615 GCA > ACA (Ala539Thr), responsible for the K variant, and nt209 GAT > GGT (Asp70Gly), which produces the atypical variant A. Her parents and two of her brothers were found to be heterozygous for the AK allele, and another brother is homozygous for the normal allele. Sequence analysis of exon 1 including 5′UTR showed that the proband and her brother are homozygous for –116GG. The AK/AK genotype is considered the most frequent in hereditary hypocholinesterasemia (44%). This work demonstrates the importance of defining the phenotype and genotype of the BCHE gene in patients who are subjected to neuromuscular block by succinylcholine, because of the risk of prolonged neuromuscular paralysis

Molecular investigation of coexistent chronic myeloid leukaemia and peripheral T-cell lymphoma-a case report

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm underlain by the formation of BCR-ABL1-an aberrant tyrosine kinase-in the leukaemic blasts. Long-term survival rates in CML prior to the advent of tyrosine kinase inhibitors (TKIs) were dismal, albeit the incidence of secondary malignancies was higher than that of age-matched population. Current figures confirm the safety of TKIs with conflicting data concerning the increased risk of secondary tumours. We postulate that care has to be taken when distinguishing between coexisting, secondary-to-treatment and second in sequence, but independent tumourigenic events, in order to achieve an unbiased picture of the adverse effects of novel treatments. To illustrate this point, we present a case of a patient in which CML and peripheral T-cell lymphoma (PTCL) coexisted, although the clinical presentation of the latter followed the achievement of major molecular response of CML to TKIs

Emerging therapies in pheochromocytoma and paraganglioma: Immune checkpoint inhibitors in the starting blocks

Pheochromocytoma and paraganglioma are neuroendocrine neoplasms, originating in the adrenal medulla and in parasympathetic and sympathetic autonomic nervous system ganglia, respec-tively. They usually present as localized tumours curable with surgery. However, these tumours may exhibit heterogeneous clinical course, ranging from no/minimal progression to aggressive (progres-sive/metastatic) behavior. For this setting of patients, current therapies are unsatisfactory. Immune checkpoint inhibitors have shown outstanding results for several types of solid cancers. We therefore aimed to summarize and discuss available data on efficacy and safety of current FDA-approved immune checkpoint inhibitors in patients with pheochromocytoma and paraganglioma. After an extensive search, we found 15 useful data sources (four full-published articles, four supplements of scientific journals, seven ongoing registered clinical trials). The data we detected, even with the limit of the small number of patients treated, make a great expectation on the therapeutic use of immune checkpoint inhibitors. Besides, the newly detected predictors of response will (hopefully) be of great helps in selecting the subset of patients that might benefit the most from this class of drugs. Finally, new trials are in the starting blocks, and they are expected to shed in the next future new light on a therapy, which is considered a milestone in oncology