Ubiquitin-dependent endocytosis of NKG2D-DAP10 receptor complexes activates signaling and functions in human NK cells

Cytotoxic lymphocytes share the presence of the activating receptor NK receptor group 2, member D (NKG2D) and the signaling-competent adaptor DNAX-activating protein 10 (DAP10), which together play an important role in antitumor immune surveillance. Ligand stimulation induces the internalization of NKG2D-DAP10 complexes and their delivery to lysosomes for degradation. In experiments with human NK cells and cell lines, we found that the ligand-induced endocytosis of NKG2D-DAP10 depended on the ubiquitylation of DAP10, which was also required for degradation of the internalized complexes. Moreover, through combined biochemical and microscopic analyses, we showed that ubiquitin-dependent receptor endocytosis was required for the activation of extracellular signal-regulated kinase (ERK) and NK cell functions, such as the secretion of cytotoxic granules and the inflammatory cytokine interferon-γ. These results suggest that NKG2D-DAP10 endocytosis represents a means to decrease cell surface receptor abundance, as well as to control signaling outcome in cytotoxic lymphocytes

Early instrumental predictors of long term neurodevelopmental impairment in newborns with perinatal asphyxia treated with therapeutic hypothermia

Background. Hypoxic-ischemic encephalopathy (HIE) is a leading cause of disability in full-term newborns. Long-term consequences of HIE, even when treated by hypothermia, are not easily predictable. Aims. To assess the potential role of electroencephalography and neuroimaging parameters as early predictors of neurodevelopmental outcome in HIE newborns treated with hypothermia. Methods. We retrospectively evaluated 13 HIE patients treated with hypothermia in January 2012-September 2014. We reviewed their amplitude-integrated electroencephalography (a-EEG) at 6, 12 and 24 hours (h), cranial ultrasonography (US) at 12, 72 h and >7 days of life (DOL) and brain magnetic resonance (MRI) performed at 7-28 DOL, according to validated scores. aEEG, US and MRI patterns were correlated to neurodevelopmental outcome at 18- 24 months, considered as negative if one of the following was present: Mental Development Index (MDI)<85, motor, visual or hearing impairment. Results. Te severity of a-EEG, US and MRI alterations at each time point was not diferent according to the outcome. MDI was negatively correlated with aEEG score at 12h (R= -0.571, p=0.04) and with US score at 72h (R= -0.630, p=0.02). A positive correlation was found between aEEG score at 6h and US score at >7DOL (R=0.690, p=0.013). US alterations of the cortical matter at 72h were directly correlated with a-EEG score at 12h (R = 0.606, p=0.028) and 24h (R=0.605, p=0.029). Conclusions. Early instrumental evaluations, in particular aEEG and US, seem to predict neurodevelopmental outcome at 18-24 months in HIE newborns treated with hypothermia

The IMiDs targets IKZF-1/3 and IRF4 as novel negative regulators of NK cell-activating ligands expression in multiple myeloma

Immunomodulatory drugs (IMiDs) have potent anti-tumor activities in multiple myeloma (MM) and are able to enhance the cytotoxic function of natural killer (NK) cells, important effectors of the immune response against MM. Here, we show that these drugs can enhance the expression of the NKG2D and DNAM-1 activating receptor ligands MICA and PVR/CD155 in human MM cell lines and primary malignant plasma cells. Depletion of cereblon (CRBN) by shRNA interference strongly impaired upregulation of these ligands and, more interestingly, IMiDs/CRBN-mediated downregulation of the transcription factors Ikaros (IKZF1), Aiolos (IKZF3) and IRF4 was critical for these regulatory mechanisms. Indeed, shRNA knockdown of IKZF1 or IKZF3 expression was both necessary and sufficient for the upregulation of MICA and PVR/CD155 expression, suggesting that these transcription factors can repress these genes; accordingly, the direct interaction and the negative role of IKZF1 and IKZF3 proteins on MICA and PVR/CD155 promoters were demonstrated. Finally, MICA expression was enhanced in IRF4-silenced cells, indicating a specific suppressive role of this transcription factor on MICA gene expression in MM cells. Taken together, these findings describe novel molecular pathways involved in the regulation of MICA and PVR/CD155 gene expression and identify the transcription factors IKZF-1/IKZF-3 and IRF4 as repressors of these genes in MM cells

A Multicentre Database for Normative Brainstem Auditory Evoked Potentials (BAEPs) in Children: Methodology for Data Collection and Evaluation

The influence of physiological and methodological factors on recordings of brainstem auditory evoked potentials (BAEPs) is greater in children than in adults. To collect and evaluate BAEP data in normal children, and measure intra- and inter-laboratory variability. Seven hundred and fifty unselected BAEP recordings were collected and evaluated from children ranging from neonates to 14-year-olds by eight laboratories in Italy. In newborns, three laboratories showed satisfactory concordance; wave I was more broadly distributed than wave V and IPL I-V. The evaluation of pooled BAEP data from the older children showed that laboratories with age-matched data gave overlapping results; those with unmatched-age data differed significantly. The sound intensities of the laboratories did not significantly affect absolute BAEP latencies or IPLs. Females had shorter latencies than males; the difference was not significant. A single exponential regression model was an adequate but not the best predictor of normal data. The pooled data were consistent with the physiological maturation of the brainstem acoustic pathway. The BAEPs was reliably normalised using the natural logarithm of age. The differences between Centres were related to sample size, measurement accuracy, and inclusion and selection criteria. The creation of multicentre common database from an unmatched data collection is feasible and reliable enough for clinical diagnosis and multicentre clinical research

Assessing minipig compact jawbone quality at the microscale

Preclinical studies often require animal models for in vivo experiments. Particularly in dental research, pig species are extensively used due to their anatomical similarity to humans. However, there is a considerable knowledge gap on the multiscale morphological and mechanical properties of the miniature pigs’ jawbones, which is crucial for implant studies and a direct comparison to human tissue. In the present work, we demonstrate a multimodal framework to assess the jawbone quantity and quality for a minipig animal model that could be further extended to humans. Three minipig genotypes, commonly used in dental research, were examined: Yucatan, G ̈ottingen, and Sinclair. Three animals per genotype were tested. Cortical bone samples were extracted from the premolar region of the mandible, opposite to the teeth growth. Global morphological, compositional, and mechanical properties were assessed using micro-computed tomography (micro-CT) together with Raman spectroscopy and nano- indentation measurements, averaged over the sample area. Local mineral-mechanical relationships were investigated with the site-matched Raman spectroscopy and micropillar compression tests. For this, a novel femtosecond laser ablation protocol was developed, allowing high-throughput micropillar fabrication and testing without exposure to high vacuum. At the global averaged sample level, bone relative mineralization demonstrated a significant difference between the genotypes, which was not observed from the complementary micro-CT measurements. Moreover, bone hardness measured by nanoindentation showed a positive trend with the relative mineralization. For all genotypes, significant differences between the relative mineralization and elastic properties were more pronounced within the osteonal regions of cortical bone. Site-matched micropillar compression and Raman spectroscopy highlighted the differences between the genotypes’ yield stress and mineral to matrix ratios. The methods used at the global level (averaged over sample area) could be potentially correlated to the medical tools used to assess jawbone toughness and morphology in clinics. On the other hand, the local analysis methods can be applied to quantify compressive bone mechanical properties and their relationship to bone mineralization

Genotoxic stress modulates the release of exosomes from multiple myeloma cells capable of activating NK cell cytokine production: role of HSP70/TLR2/NF-kB axis

Exosomes are a class of nanovesicles formed and released through the late endosomal compartment and represent an important mode of intercellular communication. The ability of anticancer chemotherapy to enhance the immunogenic potential of malignant cells mainly relies on the establishment of the immunogenic cell death (ICD) and the release of damage-associated molecular patterns (DAMPs). Here, we investigated whether genotoxic stress could promote the release of exosomes from multiple myeloma (MM) cells and studied the immunomodulatory properties they exert on NK cells, a major component of the antitumor immune response playing a key role in the immunosurveillance of MM. Our findings show that melphalan, a genotoxic agent used in MM therapy, significantly induces an increased exosome release from MM cells. MM cell-derived exosomes are capable of stimulating IFNg production, but not the cytotoxic activity of NK cells through a mechanism based on the activation of NF-kB pathway in a TLR2/ HSP70-dependent manner. Interestingly, HSP70 positive exosomes are primarily found in the bone marrow (BM) of MM patients suggesting that they might have a crucial immunomodulatory action in the tumor microenvironment. We also provide evidence that the CD56high NK cell subset is more responsive to exosome-induced IFNg production mediated by TLR2 engagement. All together, these findings suggest a novel mechanism of synergism between chemotherapy and antitumor innate immune responses based on the drug-promotion of nanovesicles exposing DAMPs for innate receptors