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Simple Summary P-selectin glycoprotein ligand-1 (PSGL-1), coded by the SELPLG gene, is the major ligand of selectins and plays a pivotal role in tethering, rolling and extravasation of immune cells. PSGL-1 involvement in core molecular programs, such as SYK, PLCγ2, PI3Kγ or MAPK pathways, suggests additional functions beyond the modulation of cell trafficking. Recently, several studies identified a novel mechanism responsible for PSGL-1-mediated immune suppression in the tumor microenvironment and proved a novel concept of PSGL-1 as a critical checkpoint molecule for tumor immunotherapy. The immunotherapeutic approach has gained an ever-growing interest in the treatment of several hematological malignancies, and in particular, novel targets for immunotherapy are still highly sought-after in T-cell lymphomas. Based on our results obtained through gene expression profiling and immunohistochemical analysis, PSGL-1, already suggested as a potential target in multiple myeloma humoral immunotherapy, could be considered noteworthy among the candidates. Abstract Due to the high expression of P-selectin glycoprotein ligand-1 (PSGL-1) in lymphoproliferative disorders and in multiple myeloma, it has been considered as a potential target for humoral immunotherapy, as well as an immune checkpoint inhibitor in T-cells. By investigating the expression of SELPLG in 678 T- and B-cell samples by gene expression profiling (GEP), further supported by tissue microarray and immunohistochemical analysis, we identified anaplastic large T-cell lymphoma (ALCL) as constitutively expressing SELPLG at high levels. Moreover, GEP analysis in CD30+ ALCLs highlighted a positive correlation of SELPLG with TNFRSF8 (CD30-coding gene) and T-cell receptor (TCR)-signaling genes (LCK, LAT, SYK and JUN), suggesting that the common dysregulation of TCR expression in ALCLs may be bypassed by the involvement of PSGL-1 in T-cell activation and survival. Finally, we evaluated the effects elicited by in vitro treatment with two anti-PSGL-1 antibodies (KPL-1 and TB5) on the activation of the complement system and induction of apoptosis in human ALCL cell lines. In conclusion, our data demonstrated that PSGL-1 is specifically enriched in ALCLs, altering cell motility and viability due to its involvement in CD30 and TCR signaling, and it might be considered as a promising candidate for novel immunotherapeutic approaches in ALCLs.

BackgroundT-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with a poor cure rate for relapsed/resistant patients. Due to the lack of T-cell restricted targetable antigens, effective immune-therapeutics are not presently available and the treatment of chemo-refractory T-ALL is still an unmet clinical need. To develop novel immune-therapy for T-ALL, we generated an afucosylated monoclonal antibody (mAb) (ahuUMG1) and two different bispecific T-cell engagers (BTCEs) against UMG1, a unique CD43-epitope highly and selectively expressed by T-ALL cells from pediatric and adult patients.MethodsUMG1 expression was assessed by immunohistochemistry (IHC) on a wide panel of normal tissue microarrays (TMAs), and by flow cytometry on healthy peripheral blood/bone marrow-derived cells, on 10 different T-ALL cell lines, and on 110 T-ALL primary patient-derived cells. CD43-UMG1 binding site was defined through a peptide microarray scanning. ahuUMG1 was generated by Genetic Glyco-Engineering technology from a novel humanized mAb directed against UMG1 (huUMG1). BTCEs were generated as IgG1-(scFv)2 constructs with bivalent (2+2) or monovalent (2+1) CD3ε arms. Antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP) and redirected T-cell cytotoxicity assays were analysed by flow cytometry. In vivo antitumor activity of ahUMG1 and UMG1-BTCEs was investigated in NSG mice against subcutaneous and orthotopic xenografts of human T-ALL.ResultsAmong 110 T-ALL patient-derived samples, 53 (48.1%) stained positive (24% of TI/TII, 82% of TIII and 42.8% of TIV). Importantly, no expression of UMG1-epitope was found in normal tissues/cells, excluding cortical thymocytes and a minority (2 log lower as compared with ahuUMG1, with significant mice survival advantage in different T-ALL models in vivo.ConclusionAltogether our findings, including the safe UMG1-epitope expression profile, provide a framework for the clinical development of these innovative immune-therapeutics for this still orphan disease.

In this work, we studied the degradability of PLA-based biocomposites containing Posidonia Oceanica flour at different loading levels and aspect ratios. Hydrolytic tests were carried out in neutral (pH = 7.4) and alkaline (pH = 10) environment. Time-dependent evolution of some key features, including residual mass and solution uptake, was monitored, and correlated with the changes observed in both morphology and chemical structure of the matrix. The results pointed out that biocomposites degraded much faster than neat PLA in both conditions, up to lose 70% of their initial weight after 1000 h immersion. A complex mechanism was unveiled, evidencing the crucial role of the fillers, capable of both imparting degradation to PLA during processing with enhancement of hydrophilicity and offering preferential gateways for solution penetration through filler-matrix interface by capillarity and swelling-aided polymer cracking. Based on data collected, we propose a new model allowing to predict the triggering and final extent of degradation pathways by considering starting morphological and chemical features of composites via the use of a novel yet simple indicator of chemical stability, which we called morphochemical parameter.

Review of: The Science of Writing Characters, Kira-Anne Pelican (2020)New York: Bloomsbury Academic, 200 pp.,ISBN 978-1-50135-724-4, p/bk, $26.95; h/bk, $90.00; Kindle, $24.25

Decreases in social behavior are a hallmark aspect of acute “sickness behavior” in response to infection. However, immune insults that occur during the perinatal period may have long-lasting consequences for adult social behavior by impacting the developmental organization of underlying neural circuits. Microglia, the resident immune cells of the central nervous system, are sensitive to immune stimulation and play a critical role in the developmental sculpting of neural circuits, making them likely mediators of this process. Here, we investigated the impact of a postnatal day (PND) 4 lipopolysaccharide (LPS) challenge on social behavior in adult mice. Somewhat surprisingly, neonatal LPS treatment decreased sociability in adult female, but not male mice. LPS-treated females also displayed reduced social interaction and social memory in a social discrimination task as compared to saline-treated females. Somatostatin (SST) interneurons within the anterior cingulate cortex (ACC) have recently been suggested to modulate a variety of social behaviors. Interestingly, the female-specific changes in social behavior observed here were accompanied by an increase in SST interneuron number in the ACC. Finally, these changes in social behavior and SST cell number do not appear to depend on microglial inflammatory signaling, because microglia-specific genetic knock-down of myeloid differentiation response protein 88 (MyD88; the removal of which prevents LPS from increasing proinflammatory cytokines such as TNFα and IL-1β) did not prevent these LPS-induced changes. This study provides novel evidence for enduring effects of neonatal immune activation on social behavior and SST interneurons in females, largely independent of microglial inflammatory signaling.

BackgroundWithin the bone marrow (BM), mature T cells are maintained under homeostatic conditions to facilitate proper hematopoietic development. This homeostasis depends upon a peculiar elevated frequency of regulatory T cells (Tregs) and immune regulatory activities from BM-mesenchymal stem cells (BM-MSCs). In response to BM transplantation (BMT), the conditioning regimen exposes the BM to a dramatic induction of inflammatory cytokines and causes an unbalanced T-effector (Teff) and Treg ratio. This imbalance negatively impacts hematopoiesis, particularly in regard to B-cell lymphopoiesis that requires an intact cross-talk between BM-MSCs and Tregs. The mechanisms underlying the ability of BM-MSCs to restore Treg homeostasis and proper B-cell development are currently unknown.MethodsWe studied the role of host radio-resistant cell-derived CD40 in restoring Teff/Treg homeostasis and proper B-cell development in a murine model of BMT. We characterized the host cellular source of CD40 and performed radiation chimera analyses by transplanting WT or Cd40-KO with WT BM in the presence of T-reg and co-infusing WT or - Cd40-KO BM-MSCs. Residual host and donor T cell expansion and activation (cytokine production) and also the expression of Treg fitness markers and conversion to Th17 were analyzed. The presence of Cd40+ BM-MSCs was analyzed in a human setting in correlation with the frequency of B-cell precursors in patients who underwent HSCT and variably developed acute graft-versus-host (aGVDH) disease.ResultsCD40 expression is nearly undetectable in the BM, yet a Cd40-KO recipient of WT donor chimera exhibited impaired B-cell lymphopoiesis and Treg development. Lethal irradiation promotes CD40 and OX40L expression in radio-resistant BM-MSCs through the induction of pro-inflammatory cytokines. OX40L favors Teff expansion and activation at the expense of Tregs; however, the expression of CD40 dampens OX40L expression and restores Treg homeostasis, thus facilitating proper B-cell development. Indeed, in contrast to dendritic cells in secondary lymphoid organs that require CD40 triggers to express OX40L, BM-MSCs require CD40 to inhibit OX40L expression.ConclusionsCD40+ BM-MSCs are immune regulatory elements within BM. Loss of CD40 results in uncontrolled T cell activation due to a reduced number of Tregs, and B-cell development is consequently impaired. GVHD provides an example of how a loss of CD40+ BM-MSCs and a reduction in B-cell precursors may occur in a human setting.

Hematopoietic stem cells (HSCs) are regulated by signals from the bone marrow (BM) niche that tune hematopoiesis at steady state and in hematologic disorders. To understand HSC-niche interactions in altered nonmalignant homeostasis, we selected β-thalassemia, a hemoglobin disorder, as a paradigm. In this severe congenital anemia, alterations secondary to the primary hemoglobin defect have a potential impact on HSC-niche cross talk. We report that HSCs in thalassemic mice (th3) have an impaired function, caused by the interaction with an altered BM niche. The HSC self-renewal defect is rescued after cell transplantation into a normal microenvironment, thus proving the active role of the BM stroma. Consistent with the common finding of osteoporosis in patients, we found reduced bone deposition with decreased levels of parathyroid hormone (PTH), which is a key regulator of bone metabolism but also of HSC activity. In vivo activation of PTH signaling through the reestablished Jagged1 and osteopontin levels correlated with the rescue of the functional pool of th3 HSCs by correcting HSC-niche cross talk. Reduced HSC quiescence was confirmed in thalassemic patients, along with altered features of the BM stromal niche. Our findings reveal a defect in HSCs in β-thalassemia induced by an altered BM microenvironment and provide novel and relevant insight for improving transplantation and gene therapy approaches.

In this work, a lignocellulosic flour was achieved by grinding the cladodes of Opuntia Ficus Indica and then added to a poly-lactic acid (PLA) in order to prepare biocomposites by melt processing. The influence of filler content and size on the morphological, rheological, and mechanical properties of the green composites was assessed. Moreover, solvent-aided filler extraction enabled to evaluate the homogeneity of filler dispersion, as well as the effect of processing on the geometrical features of the fillers. The experimental data obtained by tensile tests proved to be remarkably higher than those predicted by Halpin–Tsai model, presumably due to the capability of the polymer to enter the empty channels of the fillers, thus dramatically increasing the interphasic region.

Amyotrophic lateral sclerosis (ALS) is one of the most common motoneuronal disease, characterized by motoneuronal loss and progressive paralysis. Despite research efforts, ALS remains a fatal disease, with a survival of 2-5 years after disease onset. Numerous gene mutations have been correlated with both sporadic (sALS) and familiar forms of the disease, but the pathophysiological mechanisms of ALS onset and progression are still largely uncertain. However, a common profile is emerging in ALS pathological features, including misfolded protein accumulation and a cross-talk between neuroinflammatory and degenerative processes. In particular, astrocytes and microglial cells have been proposed as detrimental influencers of perineuronal microenvironment, and this role may be exerted via gap junctions (GJs)- and hemichannels (HCs)-mediated communications. Herein we investigated the role of the main astroglial GJs-forming connexin, Cx43, in human ALS and the effects of focal spinal cord motoneuronal depletion onto the resident glial cells and Cx43 levels. Our data support the hypothesis that motoneuronal depletion may affect glial activity, which in turn results in reactive Cx43 expression, further promoting neuronal suffering and degeneration.

B lymphocytes are among the cell types whose effector functions are modulated by mast cells (MCs). The B/MC crosstalk emerged in several pathological settings, notably the colon of inflammatory bowel disease (IBD) patients is a privileged site in which MCs and IgA+ cells physically interact. Herein, by inducing conditional depletion of MCs in red MC and basophil (RMB) mice, we show that MCs control B cell distribution in the gut and IgA serum levels. Moreover, in dextran sulfate sodium (DSS)-treated RMB mice, the presence of MCs is fundamental for the enlargement of the IgA+ population in the bowel and the increase of systemic IgA production. Since both conventional B-2 and peritoneal-derived B cells populate the intestine and communicate with MCs in physiological conditions and during inflammation, we further explored this interplay through the use of co-cultures. We show that MCs finely regulate different aspects of splenic B cell biology while peritoneal B cells are unresponsive to the supporting effects provided by MCs. Interestingly, peritoneal B cells induce a pro-inflammatory skewing in MCs, characterized by increased ST2 and TNF-α expression. Altogether, this study uncovers the versatility of the B/MC liaison and highlights key aspects for the resolution of intestinal inflammation.

Background Intra-tumour heterogeneity in lymphoid malignancies encompasses selection of genetic events and epigenetic regulation of transcriptional programs. Clonal-related neoplastic cell populations are unsteadily subjected to immune editing and metabolic adaptations within different tissue microenvironments. How tissue-specific mesenchymal cells impact on the diversification of aggressive lymphoma clones is still unknown. Methods Combining in situ quantitative immunophenotypical analyses and RNA sequencing we investigated the intra-tumour heterogeneity and the specific mesenchymal modifications that are associated with A20 diffuse large B-cell lymphoma (DLBCL) cells seeding of different tissue microenvironments. Furthermore, we characterized features of lymphoma-associated stromatogenesis in human DLBCL samples using Digital Spatial Profiling, and established their relationship with prognostically relevant variables, such as MYC. Findings We found that the tissue microenvironment casts a relevant influence over A20 transcriptional landscape also impacting on Myc and DNA damage response programs. Extending the investigation to mice deficient for the matricellular protein SPARC, a stromal prognostic factor in human DLBCL, we demonstrated a different immune imprint on A20 cells according to stromal Sparc proficiency. Through Digital Spatial Profiling of 87 immune and stromal genes on human nodal DLBCL regions characterized by different mesenchymal composition, we demonstrate intra-lesional heterogeneity arising from diversified mesenchymal contextures and impacting on the stromal and immune milieu. Interpretation Our study provides experimental evidence that stromal microenvironment generates topological determinants of intra-tumour heterogeneity in DLBCL involving key transcriptional pathways such as Myc expression, damage response programs and immune checkpoints. Funding This study has been supported by the Italian Foundation for Cancer Research (AIRC) (grants 15999 and 22145 to C. Tripodo) and by the University of Palermo.

Extensive loss of noradrenaline-containing neurons and fibers is a nearly invariant feature of Alzheimer’s Disease (AD). However, the exact noradrenergic contribution to cognitive and histo- pathological changes in AD is still unclear. Here, this issue was addressed following selective lesioning and intrahippocampal implantation of embryonic noradrenergic progenitors in developing rats. Starting from about 3 months and up to 12 months post-surgery, animals underwent behav- ioral tests to evaluate sensory-motor, as well as spatial learning and memory, followed by post- mortem morphometric analyses. At 9 months, Control, Lesioned and Lesion1Transplant animals exhibited equally efficient sensory-motor and reference memory performance. Interestingly, working memory abilities were seen severely impaired in Lesion-only rats and fully recovered in Transplanted rats, and appeared partly lost again 2 months after ablation of the implanted neuroblasts. Morphological analyses confirmed the almost total lesion-induced noradrenergic neuronal and terminal fiber loss, the near-normal reinnervation of the hippocampus promoted by the transplants, and its complete removal by the second lesion. Notably, the noradrenergic-rich transplants normalized also the nuclear expression of the transactive response DNA-binding protein 43 (TDP- 43) in various hippocampal subregions, whose cytoplasmic (i.e., pathological) occurrence appeared dramatically increased as a result of the lesions. Thus, integrity of ascending noradrenergic inputs to the hippocampus may be required for the regulation of specific aspects of learning and memory and to prevent TDP-43 tissue pathology.

Patterns of somatic single nucleotide variants observed in human cancers vary widely between different tumor types. They depend not only on the activity of diverse mutational processes, such as exposure to ultraviolet light and the deamination of methylated cytosines, but largely also on the sequence content of different genomic regions on which these processes act. With MutViz (http://gmql.eu/mutviz/), we have presented a user-friendly web tool for the identification of mutation enrichments that offers preloaded mutations from public datasets for a variety of cancer types, well organized within an effective database architecture. Somatic mutation patterns can be visually and statistically analyzed within arbitrary sets of small, user-provided genomic regions, such as promoters or collections of transcription factor binding sites. Here, we present MutViz 2.0, a largely extended and consolidated version of the tool: we took into account the immediate (trinucleotide) sequence context of mutations, improved the representation of clinical annotation of tumor samples and devised a method for signature refitting on limited genomic regions to infer the contribution of individual mutational processes to the mutation patterns observed in these regions. We described both the features of MutViz 2.0, concentrating on the novelties, and the substantial re-engineering of the cloud-based architecture., Graphical Abstract Graphical AbstractSimplified workflow of data analysis and visualization with MutViz: Analysis of somatic mutations within user-defined genomic regions for identification and visualization of region-specific mutation characteristics.

The MinION is a miniaturized high-throughput next generation sequencing platform of novel conception. The use of nucleic acids derived from formalin-fixed paraffin-embedded samples is highly desirable, but their adoption for molecular assays is hurdled by the high degree of fragmentation and by the chemical-induced mutations stemming from the fixation protocols. In order to investigate the suitability of MinION sequencing on formalin-fixed paraffin-embedded samples, the presence and frequency of BRAF c.1799T > A mutation was investigated in two archival tissue specimens of Hairy cell leukemia and Hairy cell leukemia Variant. Despite the poor quality of the starting DNA, BRAF mutation was successfully detected in the Hairy cell leukemia sample with around 50% of the reads obtained within 2 h of the sequencing start. Notably, the mutational burden of the Hairy cell leukemia sample as derived from nanopore sequencing proved to be comparable to a sensitive method for the detection of point mutations, namely the Digital PCR, using a validated assay. Nanopore sequencing can be adopted for targeted sequencing of genetic lesions on critical DNA samples such as those extracted from archival routine formalin-fixed paraffin-embedded samples. This result let speculating about the possibility that the nanopore sequencing could be trustably adopted for the real-time targeted sequencing of genetic lesions. Our report opens the window for the adoption of nanopore sequencing in molecular pathology for research and diagnostics.

Microbiomes are vast communities of microorganisms and viruses that populate all natural ecosystems. Viruses have been considered to be the most variable component of microbiomes, as supported by virome surveys and examples of high genomic mosaicism. However, recent evidence suggests that the human gut virome is remarkably stable compared with that of other environments. Here, we investigate the origin, evolution and epidemiology of crAssphage, a widespread human gut virus. Through a global collaboration, we obtained DNA sequences of crAssphage from more than one-third of the world's countries and showed that the phylogeography of crAssphage is locally clustered within countries, cities and individuals. We also found fully colinear crAssphage-like genomes in both Old-World and New-World primates, suggesting that the association of crAssphage with primates may be millions of years old. Finally, by exploiting a large cohort of more than 1,000 individuals, we tested whether crAssphage is associated with bacterial taxonomic groups of the gut microbiome, diverse human health parameters and a wide range of dietary factors. We identified strong correlations with different clades of bacteria that are related to Bacteroidetes and weak associations with several diet categories, but no significant association with health or disease. We conclude that crAssphage is a benign cosmopolitan virus that may have coevolved with the human lineage and is an integral part of the normal human gut virome. ispartof: NATURE MICROBIOLOGY vol:4 issue:10 pages:1727-1736 ispartof: location:England status: published

Data Availability Statement: FISH data are available on request from the corresponding authors. Hi- C data are in the references [5,7,23,24,26], and visible in the Juicebox software version 1.5.2 from the Broad Institute and Aiden Lab (https://aidenlab.org/juicebox/ accessed on 16 June 2018) referenced in [7,32]. © 2021 by the authors. FISH and Hi-C methods are largely used to investigate the three-dimensional organization of the genome in the cell nucleus, and are applied here to study the organization of genes (LMBR1, NOM1, MNX1, UBE3C, PTPRN2) localized in the human 7q36.3 band. This region contains the MNX1 gene, which is normally not expressed in human lymphocytes beyond embryonic development. However, this homeobox gene is frequently activated in leukemic cells and its expression is associated with an altered gene positioning in the leukemia cell nuclei. In this study, we used FISH on 3D-preserved nuclei to investigate the nuclear positioning of MNX1 in the leukemia-derived cell line K562. Of the five copies of the MNX1 gene present n K562, four alleles were positioned in the nuclear periphery and only one in the nuclear interior. Using the Juicebox’s Hi-C dataset, we identified five chromatin loops in the 7q36.3 band, with different extensions related to the size and orientation of the genes located here, and independent from their expression levels. We identified similar loops in 11 human and 3 mouse cell lines, showing that these loops are highly conserved in different human cell lines and during evolution. Moreover, the chromatin loop organization is well conserved also during neuronal cell differentiation, showing consistency in genomic organization of this region in development. In this report, we show that FISH and Hi-C are two different approaches that complement one another and together give complete information on the nuclear organization of specific chromosomal regions in different conditions, including cellular differentiation and genetic diseases. Research Plan PIACERI L.3 Starting Grant from Department of Biological, Geological and Environmental Sciences, University of Catania to C.F.

Variant visualization plays an important role in supporting the viral evolution analysis, extremely valuable during the COVID-19 pandemic. VirusViz is a web-based application for comparing variants of selected viral populations and their sub-populations; it is primarily focused on SARS-CoV-2 variants, although the tool also supports other viral species (SARS-CoV, MERS-CoV, Dengue, Ebola). As input, VirusViz imports results of queries extracting variants and metadata from the large database ViruSurf, which integrates information about most SARS-CoV-2 sequences publicly deposited worldwide. Moreover, VirusViz accepts sequences of new viral populations as multi-FASTA files plus corresponding metadata in CSV format; a bioinformatic pipeline builds a suitable input for VirusViz by extracting the nucleotide and amino acid variants. Pages of VirusViz provide metadata summarization, variant descriptions, and variant visualization with rich options for zooming, highlighting variants or regions of interest, and switching from nucleotides to amino acids; sequences can be grouped, groups can be comparatively analyzed. For SARS-CoV-2, we manually collect mutations with known or predicted levels of severity/virulence, as indicated in linked research articles; such critical mutations are reported when observed in sequences. The system includes light-weight project management for downloading, resuming, and merging data analysis sessions. VirusViz is freely available at http://gmql.eu/virusviz/., Graphical Abstract Graphical AbstractVirusViz accepts as input user-defined viral sequences or results from external applications; it supports data preparation by defining groups and regions of interest for producing powerful data visualization and comparisons of variants.

BackgroundIn consideration of the increasing prevalence of COVID-19 cases in several countries and the resulting demand for unbiased sequencing approaches, we performed a direct RNA sequencing experiment using critical oropharyngeal swab samples collected from Italian patients infected with SARS-CoV-2 from the Palermo region in Sicily.MethodsHere, we identified the sequences SARS-CoV-2 directly in RNA extracted from critical samples using the Oxford Nanopore MinION technology without prior cDNA retro-transcription.ResultsUsing an appropriate bioinformatics pipeline, we could identify mutations in the nucleocapisid (N) gene, which have been reported previously in studies conducted in other countries.ConclusionTo the best of our knowledge, the technique used in this study has not been used for SARS-CoV-2 detection previously owing to the difficulties in the extraction of RNA of sufficient quantity and quality from routine oropharyngeal swabs.Despite these limitations, this approach provides the advantages of true native RNA sequencing, and does not include amplification steps that could introduce systematic errors.This study can provide novel information relevant to the current strategies adopted in SARS-CoV-2 next-generation sequencing.We deposited the gene sequence in the NCBI database under the following URL:https://www.ncbi.nlm.nih.gov/nuccore/MT457389

Surfactant protein D (SP-D) is a pattern recognition molecule belonging to the Collectin (collagen-containing C-type lectin) family that has pulmonary as well as extra-pulmonary existence. In the lungs, it is a well-established opsonin that can agglutinate a range of microbes, and enhance their clearance via phagocytosis and super-oxidative burst. It can interfere with allergen–IgE interaction and suppress basophil and mast cell activation. However, it is now becoming evident that SP-D is likely to be an innate immune surveillance molecule against tumor development. SP-D has been shown to induce apoptosis in sensitized eosinophils derived from allergic patients and a leukemic cell line via p53 pathway. Recently, SP-D has been shown to suppress lung cancer progression via interference with the epidermal growth factor signaling. In addition, a truncated form of recombinant human SP-D has been reported to induce apoptosis in pancreatic adenocarcinoma via Fas-mediated pathway in a p53-independent manner. To further establish a correlation between SP-D presence/levels and normal and cancer tissues, we performed a bioinformatics analysis, using Oncomine dataset and the survival analysis platforms Kaplan–Meier plotter, to assess if SP-D can serve as a potential prognostic marker for human lung cancer, in addition to human gastric, breast, and ovarian cancers. We also analyzed immunohistochemically the presence of SP-D in normal and tumor human tissues. We conclude that (1) in the lung, gastric, and breast cancers, there is a lower expression of SP-D than normal tissues; (2) in ovarian cancer, there is a higher expression of SP-D than normal tissue; and (3) in lung cancer, the presence of SP-D could be associated with a favorable prognosis. On the contrary, at non-pulmonary sites such as gastric, breast, and ovarian cancers, the presence of SP-D could be associated with unfavorable prognosis. Correlation between the levels of SP-D and overall survival requires further investigation. Our analysis involves a large number of dataset; therefore, any trend observed is reliable. Despite apparent complexity within the results, it is evident that cancer tissues that produce less levels of SP-D compared to their normal tissue counterparts are probably less susceptible to SP-D-mediated immune surveillance mechanisms via infiltrating immune cells.

The Hedgehog (Hh) pathway is essential for embryonic development and tissue homeostasis. Aberrant Hh signaling may occur in a wide range of human cancers, such as medulloblastoma, the most common brain malignancy in childhood. Here, we identify endoplasmic reticulum aminopeptidase 1 (ERAP1), a key regulator of innate and adaptive antitumor immune responses, as a previously unknown player in the Hh signaling pathway. We demonstrate that ERAP1 binds the deubiquitylase enzyme USP47, displaces the USP47-associated βTrCP, the substrate-receptor subunit of the SCFβTrCP ubiquitin ligase, and promotes βTrCP degradation. These events result in the modulation of Gli transcription factors, the final effectors of the Hh pathway, and the enhancement of Hh activity. Remarkably, genetic or pharmacological inhibition of ERAP1 suppresses Hh-dependent tumor growth in vitro and in vivo. Our findings unveil an unexpected role for ERAP1 in cancer and indicate ERAP1 as a promising therapeutic target for Hh-driven tumors., ERAP1 is an endoplasmic reticulum aminopeptidase that trims MHC Class-I peptides for antigen presentation. Here, the authors show that ERAP1 enhances Hedgehog signalling by sequestering USP47 from βTrCP and promoting tumorigenesis through βTrCP degradation and increased Gli protein stability.

Allogeneic bone marrow transplantation remains the only therapeutic option for a wide range of hematological malignancies despite the risk of possible adverse, immune-related events, such as infection and acute graft-versus-host disease (aGVHD). aGVHD is characterized by T-cell activation, defective B-cell development and osteoblastic niche destruction in bone marrow (BM) among other issues. Transplant conditioning regimens cause excessive inflammatory cytokines production and impaired regulatory T-cell control of aberrant T-cell activation. Here, we show that mesenchymal cells (MSCs) upregulated CD40 upon irradiation at the expense of mesenchymal markers, and that CD40 endows MSC of regulatory function on Treg homeostasis and fitness. Transplantation of wild type hematopoietic cells into a CD40-null recipient reduces Treg numbers allowing persistent T-cell activation and pro-inflammatory cytokines production causing, impaired B-lymphopoiesis. These evidences find correlation in aGVHD patients showing the loss of CD40+ BM-MSCs along with reduction in cells of the B-lineage. Modeling aGVHD in mice we show that the elimination of CD40+ BM-MSCs relies on their higher expression of MHC-I molecules. Indeed, aGVHD mice compared to MHC-matched controls showed the loss of MHC-I + radio-resistant host BM-MSCs. Our data point to CD40+ MHC-I+BM-MSCs as a key regulator of BM tolerogenic niches.Key pointsCD40 regulates BM immunological tolerance following total body irradiation (TBI) and transplantation (BMT).Loss of CD40+MHC-IhighBM-MSCs is associated to BM manifestation of aGVHD in human and murine model.

Wastewater is a rich source of microbial life and contains bacteria, viruses, and other microbes found in human waste as well as environmental runoff sources. As part of an effort to characterize the New York City wastewater metagenome, we profiled the viral community of sewage samples across all five boroughs of NYC and found that local sampling sites have unique sets of viruses. We focused on bacteriophages, or viruses of bacteria, to understand how they may influence the microbial ecology of this system. We identified several new clusters of phages and successfully associated them with bacterial hosts, providing insight into virus-host interactions in urban wastewater. This study provides a first look into the viral communities present across the wastewater system in NYC and points to their functional importance in this environment., Bacteriophages are abundant members of all microbiomes studied to date, influencing microbial communities through interactions with their bacterial hosts. Despite their functional importance and ubiquity, phages have been underexplored in urban environments compared to their bacterial counterparts. We profiled the viral communities in New York City (NYC) wastewater using metagenomic data collected in November 2014 from 14 wastewater treatment plants. We show that phages accounted for the largest viral component of the sewage samples and that specific virus communities were associated with local environmental conditions within boroughs. The vast majority of the virus sequences had no homology matches in public databases, forming an average of 1,700 unique virus clusters (putative genera). These new clusters contribute to elucidating the overwhelming proportion of data that frequently goes unidentified in viral metagenomic studies. We assigned potential hosts to these phages, which appear to infect a wide range of bacterial genera, often outside their presumed host. We determined that infection networks form a modular-nested pattern, indicating that phages include a range of host specificities, from generalists to specialists, with most interactions organized into distinct groups. We identified genes in viral contigs involved in carbon and sulfur cycling, suggesting functional importance of viruses in circulating pathways and gene functions in the wastewater environment. In addition, we identified virophage genes as well as a nearly complete novel virophage genome. These findings provide an understanding of phage abundance and diversity in NYC wastewater, previously uncharacterized, and further examine geographic patterns of phage-host association in urban environments. IMPORTANCE Wastewater is a rich source of microbial life and contains bacteria, viruses, and other microbes found in human waste as well as environmental runoff sources. As part of an effort to characterize the New York City wastewater metagenome, we profiled the viral community of sewage samples across all five boroughs of NYC and found that local sampling sites have unique sets of viruses. We focused on bacteriophages, or viruses of bacteria, to understand how they may influence the microbial ecology of this system. We identified several new clusters of phages and successfully associated them with bacterial hosts, providing insight into virus-host interactions in urban wastewater. This study provides a first look into the viral communities present across the wastewater system in NYC and points to their functional importance in this environment.

Intra-tumor heterogeneity in lymphoid malignancies is articulated around several fundamentals, encompassing selection of genetic subclonal events and epigenetic regulation of transcriptional programs. Clonally-related neoplastic cell populations are unsteadily subjected to immune editing and metabolic adaptations within different tissue microenvironments. How tissue-intrinsic mesenchymal determinants impact on the diversification of aggressive lymphomas is still unknown. In this study we adopted the established A20 line-based model of Diffuse Large B-cell Lymphoma (DLBCL), to investigate the intra-tumor heterogeneity associated with the infiltration of different tissue microenvironments and the specific mesenchymal modifications that characterize stromal adaptation to lymphoma seeding. Combining in situ quantitative immunophenotypical analyses and RNA sequencing, we found that the tissue microenvironment casts a relevant influence over A20 transcriptional landscape also impacting on Myc and DNA damage response programs. Extending the investigation to mice deficient for the matricellular protein Sparc, a stromal determinant endowed with a strong prognostic significance in human DLBCL, we demonstrated a different immune imprint on A20 cells related to stromal Sparc proficiency. The study provides the first evidence of human DLBCL intra-lesional heterogeneity arising from diversified mesenchymal contextures and impacting on MYC expression, advancing the challenge for resolving intra-tumor heterogeneity probing stromal/immune interfaces.KEY POINTSDiversified stromal adaptations of infiltrated tissues shape DLBCL intra-tumor heterogeneity regulating transcriptional/phenotypic featuresStromal Sparc, which endorses prognostic significance in DLBCL, tunes the immune pressure exerted on lymphoma cells by the microenvironment

Decreases in social behavior are a hallmark aspect of acute “sickness behavior” in response to infection. However, immune insults that occur during the perinatal period may have long-lasting consequences for adult social behavior by impacting the developmental organization of underlying neural circuits. Microglia, the resident immune cells of the central nervous system, are sensitive to immune stimulation and play a critical role in the developmental sculpting of neural circuits, making them likely mediators of this process. Here, we investigated the impact of a postnatal day (PND) 4 lipopolysaccharide (LPS) challenge on social behavior in adult mice. Somewhat surprisingly, neonatal LPS treatment decreased sociability in adult female, but not male mice. LPS-treated females also displayed reduced social interaction and social memory in a social discrimination task as compared to saline-treated females. Somatostatin (SST) interneurons within the anterior cingulate cortex (ACC) have recently been suggested to modulate a variety of social behaviors. Interestingly, the female-specific changes in social behavior observed here were accompanied by an increase in SST interneuron number in the ACC. Finally, these changes in social behavior and SST cell number do not appear to depend on microglial inflammatory signaling, because microglia-specific genetic knock-down of myeloid differentiation response protein 88 (MyD88; the removal of which prevents LPS from increasing proinflammatory cytokines such as TNFα and IL-1β) did not prevent these LPS-induced changes. This study provides novel evidence for enduring effects of neonatal immune activation on social behavior and SST interneurons in females, independent of microglial inflammatory signaling.

Antitumor strategies based on positive modulation of the immune system currently represent therapeutic options with prominent acceptance for cancer patients’ treatment due to its selectivity and higher tolerance compared to chemotherapy. Racotumomab is an anti-idiotype (anti-Id) monoclonal antibody (mAb) directed to NeuGc-containing gangliosides such as NeuGcGM3, a widely reported tumor-specific neoantigen in many human cancers. Racotumomab has been approved in Latin American countries as an active immunotherapy for advanced non-small cell lung cancer (NSCLC) treatment. In this work, we evaluated the induction of Ab-dependent cell-mediated cytotoxicity (ADCC) in NSCLC patients included in a phase III clinical trial, in response to vaccination with racotumomab. The development of anti-NeuGcGM3 antibodies (Abs) in serum samples of immunized patients was first evaluated using the NeuGcGM3-expressing X63 cells, showing that racotumomab vaccination developed antigen-specific Abs that are able to recognize NeuGcGM3 expressed in tumor cell membranes. ADCC response against NeuGcGM3-expressing X63 (target) was observed in racotumomab-treated- but not in control group patients. When target cells were depleted of gangliosides by treatment with a glucosylceramide synthase inhibitor, we observed a significant reduction of the ADCC activity developed by sera from racotumomab-vaccinated patients, suggesting a target-specific response. Our data demonstrate that anti-NeuGcGM3 Abs induced by racotumomab vaccination are able to mediate an antigen-specific ADCC response against tumor cells in NSCLC patients. Fil: Segatori, Valeria Inés. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentina Fil: Cuello, Héctor Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentina Fil: Gulino, Cynthia Antonella. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentina Fil: Albertó, Marina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentina Fil: Venier, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; Argentina Fil: Guthmann, Marcelo D.. Elea Laboratories; Argentina Fil: Demarco, Ignacio A.. Elea Laboratories; Argentina Fil: Alonso, Daniel Fernando. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Gabri, Mariano Rolando. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

SummaryThe extracellular matrix (ECM) contributes to the biological and clinical heterogeneity of breast cancer, and different prognostic groups can be identified according to specific ECM signatures. In high-grade, but not low-grade, tumors, an ECM signature characterized by high SPARC expression (ECM3) identifies tumors with increased epithelial-to-mesenchymal transition (EMT), reduced treatment response, and poor prognosis. To better understand how this ECM3 signature is contributing to tumorigenesis, we expressed SPARC in isogenic cell lines and found that SPARC overexpression in tumor cells reduces their growth rate and induces EMT. SPARC expression also results in the formation of a highly immunosuppressive microenvironment, composed by infiltrating T regulatory cells, mast cells, and myeloid-derived suppressor cells (MDSCs). The ability of SPARC to induce EMT depended on the localization and suppressive function of myeloid cells, and inhibition of the suppressive function MDSCs by administration of aminobisphosphonates could revert EMT, rendering SPARC-overexpressing tumor cells sensitive to Doxil. We conclude that that SPARC is regulating the interplay between MDSCs and the ECM to drive the induction of EMT in tumor cells.

Background & Aim Homozygosity for a common non-coding rs4374383 G>A polymorphism in MERTK (myeloid-epithelial-reproductive tyrosine kinase) has been associated with the protection against fibrosis progression in chronic hepatitis C. The main study objective was to assess whether MERTK AA genotype influences liver fibrosis, and secondarily MERTK expression in patients with non-alcoholic fatty liver disease (NAFLD). We also investigated whether MERTK is expressed in human hepatic stellate cells (HSC) and in murine models of fibrogenesis. Methods We considered 533 consecutive patients who underwent liver biopsy for suspected non-alcoholic steatohepatitis (NASH) without severe obesity from two Italian cohorts. As controls, we evaluated 158 patients with normal liver enzymes and without metabolic disturbances. MERTK rs4374383 genotype was assessed by 5′-nuclease assays. MERTK expression was analysed in mouse models of fibrosis, and the effect of the MERTK ligand GAS6 were investigated in human HSC. Results Clinically significant fibrosis (stage F2-F4) was observed in 19% of patients with MERTK AA compared to 30% in those with MERTK GG/GA (OR 0.43, CI 0.21–0.88, p =0.02; adjusted for centre, and genetic, clinical-metabolic and histological variables). The protective rs4374383 AA genotype was associated with lower MERTK hepatic expression. MERTK was overexpressed in the liver of NAFLD patients with F2-F4 fibrosis and in in vivo models of fibrogenesis. Furthermore, exposure of cultured human HSC to the MERTK ligand GAS6, increased cell migration and induced procollagen expression. These effects were counteracted by inhibition of MERTK activity, which also resulted in apoptotic death of HSC. Conclusions The rs4374383 AA genotype, associated with lower intrahepatic expression of MERTK , is protective against F2-F4 fibrosis in patients with NAFLD. The mechanism may involve modulation of HSC activation.

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignantCD51 B lymphocytes in blood, bone marrow, and lymphoid organs. CD1d-restricted invariant natural killer T (iNKT) cells are innate-like T lymphocytes strongly implicated in tumor surveillance.Weinvestigated the impact of iNKT cells in the natural history of the disease in the Em-Tcl1 (Tcl1) CLL mouse model and 68 CLL patients.Wefound that Tcl1-CLL cells express CD1d and that iNKT cells critically delay disease onset but become functionally impaired upon disease progression. In patients, disease progression correlates with high CD1d expression on CLL cells and impaired iNKT cells. Conversely, disease stability correlates with negative or low CD1d expression on CLL cells and normal iNKT cells, suggesting indirect leukemia control. iNKT cells indeed hinder CLL survival in vitro by restraining CD1d-expressing nurse-like cells, a relevant proleukemia macrophage population. Multivariable analysis identified iNKT cell frequency as an independent predictor of disease progression. Together, these results support the contribution of iNKT cells to CLL immune surveillance and highlight iNKT cell frequency as a prognostic marker for disease progression. © 2017 by The American Society of Hematology.

Two-dimensional (2D) cell cultures have been extensively used to investigate stem cell biology, but new insights show that the 2D model may not properly represent the potential of the tissue of origin. Conversely, three-dimensional cultures exhibit protein expression patterns and intercellular junctions that are more representative of their in vivo condition. Multiclonal cells that grow in suspension are defined as "spheroids," and we have previously demonstrated that spheroids from adipose-derived stem cells (S-ASCs) displayed enhanced regenerative capability. With the current study, we further characterized S-ASCs to further understand the molecular mechanisms underlying their stemness properties. Recent studies have shown that microRNAs (miRNAs) are involved in many cellular mechanisms, including stemness maintenance and proliferation, and adipose stem cell differentiation. Most studies have been conducted to identify a specific miRNA profile on adherent adipose stem cells, although little is still known about S-ASCs. In this study, we investigate for the first time the miRNA expression pattern in S-ASCs compared to that of ASCs, demonstrating that cell lines cultured in suspension show a typical miRNA expression profile that is closer to the one reported in induced pluripotent stem cells. Moreover, we have analyzed miRNAs that are specifically involved in two distinct moments of each differentiation, namely early and late stages of osteogenic, adipogenic, and chondrogenic lineages during long-term in vitro culture. The data reported in the current study suggest that S-ASCs have superior stemness features than the ASCs and they represent the true upstream stem cell fraction present in adipose tissue, relegating their adherent counterparts.

MRE11 is a component of the MRE11/RAD50/NBS1 (MRN) complex, whose activity is essential to control faithful DNA replication and to prevent accumulation of deleterious DNA double-strand breaks. In humans, hypomorphic mutations in these genes lead to DNA damage response (DDR)-defective and cancer-prone syndromes. Moreover, MRN complex dysfunction dramatically affects the nervous system, where MRE11 is required to restrain MYCN-dependent replication stress, during the rapid expansion of progenitor cells. MYCN activation, often due to genetic amplification, represents the driving oncogenic event for a number of human tumors, conferring bad prognosis and predicting very poor responses even to the most aggressive therapeutic protocols. This is prototypically exemplified by neuroblastoma, where MYCN amplification occurs in about 25% of the cases. Intriguingly, MRE11 is highly expressed and predicts bad prognosis in MYCN-amplified neuroblastoma. Due to the lack of direct means to target MYCN, we explored the possibility to trigger intolerable levels of replication stress-dependent DNA damage, by inhibiting MRE11 in MYCN-amplified preclinical models. Indeed, either MRE11 knockdown or its pharmacological inhibitor mirin induce accumulation of replication stress and DNA damage biomarkers in MYCN-amplified cells. The consequent DDR recruits p53 and promotes a p53-dependent cell death, as indicated by p53 loss- and gain-of-function experiments. Encapsulation of mirin in nanoparticles allowed its use on MYCN-amplified neuroblastoma xenografts in vivo, which resulted in a sharp impairment of tumor growth, associated with DDR activation, p53 accumulation, and cell death. Therefore, we propose that MRE11 inhibition might be an effective strategy to treat MYCN-amplified and p53 wild-type neuroblastoma, and suggest that targeting replication stress with appropriate tools should be further exploited to tackle MYCN-driven tumors.

Although the progression of chronic lymphocytic leukemia (CLL) requires the cooperation of the microenvironment, the exact cellular and molecular mechanisms involved are still unclear. We investigated the interleukin (IL)-23 receptor (IL-23R)/IL-23 axis and found that circulating cells from early-stage CLL patients with shorter time-to-treatment, but not of those with a more benign course, expressed a defective form of the IL-23R complex lacking the IL-12Rβ1 chain. However, cells from both patient groups expressed the complete IL-23R complex in tissue infiltrates and could be induced to express the IL-12Rβ1 chain when cocultured with activated T cells or CD40L+ cells. CLL cells activated in vitro in this context produced IL-23, a finding that, together with the presence of IL-23 in CLL lymphoid tissues, suggests the existence of an autocrine/paracrine loop inducing CLL cell proliferation. Interference with the IL-23R/IL-23 axis using an anti-IL-23p19 antibody proved effective in controlling disease onset and expansion in xenografted mice, suggesting potential therapeutic strategies.

It is known that exists a relationship between listening to music and food intake. Hypothalamus appears to integrate the orexigenic properties of a novel peptide, ghrelin (Ghre) that induces food intake through neuropeptide Y (NPY). Ghre stimulates appetite by acting on the ventral hypothalamus, which controls food intake. Ghre is secreted from the stomach and circulates in the bloodstream under fasting conditions, sending a hunger signal from the periphery to the Central Nervous System. The aim of this study was to evaluate, in the rat, the effects of different musical frequencies (432 and 440 Hz) on the Ghre and NPY expression in the hypothalamic neurons through immunohistochemistry; in addition, we investigated on the different production of Ghre in the serum through Western blot assay (Wb), in relation to the body weight of animals. Ghre-immunopositive cells were counted, showing a significant increase in music-treated compared to the control (CTR) group. Similarly, music-treated rats showed increased levels of Ghre in the serum compared to CTR animals. Finally, the body weight of animals was affected by music. In particular, music exposure was able to stimulate the body weight increase and, interestingly, the increase was higher when animals were exposed to music at 440 Hz. Together, the results strongly support the hypothesis that different musical frequencies could affect food intake by modulating the hypothalamic Ghre expression and its release.

High-risk and MYCN-amplified neuroblastomas are among the most aggressive pediatric tumors. Despite intense multimodality therapies, about 50% of these patients succumb to their disease, making the search for effective therapies an absolute priority. Due to the important functions of poly (ADP-ribose) polymerases, PARP inhibitors have entered the clinical settings for cancer treatment and are being exploited in a variety of preclinical studies and clinical trials. PARP inhibitors based combination schemes have also been tested in neuroblastoma preclinical models with encouraging results. However, the expression of PARP enzymes in human neuroblastoma and the biological consequences of their inhibition remained largely unexplored. Here, we show that high PARP1 and PARP2 expression is significantly associated with high-risk neuroblastoma cases and poor survival, highlighting its previously unrecognized prognostic value for human neuroblastoma. In vitro, PARP1 and 2 are abundant in MYCN amplified and MYCN-overexpressing cells. In this context, PARP inhibitors with high 'PARP trapping' potency, such as olaparib or talazoparib, yield DNA damage and cell death preceded by intense signs of replication stress. Notwithstanding the activation of a CHK1-CDC25A replication stress response, PARP-inhibited MYCN amplified and overexpressing cells fail to sustain a prolonged checkpoint and progress through mitosis in the presence of damaged DNA, eventually undergoing mitotic catastrophe. CHK1-targeted inhibition of the replication stress checkpoint exacerbated this phenotype. These data highlight a novel route for cell death induction by PARP inhibitors and support their introduction, together with CHK1 inhibitors, in therapeutic approaches for neuroblastomas with high MYC(N) activity.Oncogene advance online publication, 10 April 2017; doi:10.1038/onc.2017.40.

Seminomas are the most frequent kind of testicular germ cell tumors (TGCTs), accounting for 50% of tumor diagnosis in young men, whereas nonseminomas account for 40% and mixed forms for 10% of cases. It is currently supposed that TGCTs evolve from a pre-invasive stage of carcinoma in situ (CIS). Octamer-binding transcription factor 4 (OCT4) is essential for self-renewal of stem cells. It is considered as a major regulator of cell pluripotency. Prior studies have shown that seminoma expresses OCT4. Transcription factor Kruppel-like factor 4 (KLF4) has moreover associated with embryonic stem cell maintenance. Finally, we previously demonstrated the expression of PTTG1 in CIS and seminomas. In this pilot study, we compared the combined expression of PTTG1 with KLF4 and OCT4 in seminoma, in order to validate our hypotesis that PTTG1 mark a specific population of stem cells in neoplastic tissue, strictly related with tumor. Formalin-fixed and paraffin-embedded testicular tissues by 5 patients who underwent an orchidectomy for seminoma have been collected and immunofluorescence analysis was performed using antibody rabbit monoclonal PTTG-1 and mouse monoclonal OCT4 or mouse monoclonal KLF4 antibody. In seminoma we observed that tumor cells strongly express OCT-4 in all seminomas and in the intratubular areas of seminoma. Expression of KLF-4 was observed in many tumor cells (Fig. 1B). PTTG1 marks some specific OCT4- and KLF4-positive tumor cells, mainly localized at the periphery of the neoplasm. In the intertubular infiltration areas nests of cells expressing both OCT4/KLF4 and PTTG1 have been observed. This is the first identification of a cell population in seminoma characterized for being OCT4, KLF4 and PTTG1 positive cells in seminoma, associated with cancer invasiveness. Further investigation is needed to elucidate if a functional abrogation of PTTG1 might be used in order to offer new therapeutic approaches in the clinical workout of seminoma.

Purpose: The testis-sparing surgery (TSS) is surgical technique accepted for small testicular masses (STMs). Frozen section examination (FSE) is an essential assessment at the time of TSS. The aim of this study is to measure the maximum distance of the foci of ITGCN from STMs. Methods: In our hospital between June 2010 and October 2017 a total of 68 patients with STM underwent a TSS. All the testis specimens were totally embedded and processed via the whole-mount method and a diagnosis of germ cell tumor with GCNIS were made. The distance between STMs and GCNIS were calculated by two pathologists directly on the slides considering for the third dimension the number of the paraffin blocks in which the foci of GCNIS were found. Results: The STMs were classic seminoma in 62 out 68 cases, embryonal carcinoma in 4 cases, while in 2 case a diagnose of mixed germ cell tumor were made. The size of the STMs was between 0.5 and 2 cm and the foci of GCNIS were observed in seminiferous tubules very closed to SMTs or as skip lesions in the surrounding testicular parenchyma, dispersed in normal testis. In 48 out of 68 cases (70.5%) foci of GCNIS were at the distance from SMTS of 1.5 cm or below and in 60 out of 68 cases (88%) at the distance of 2 cm or below The distance of GCNIS from the STMs was not related to the histological subtype of the germ cell tumor, while there is a linear correlation between size of the STMs and the distance of foci of GCNIS (p = 0.0105; r = 0.9167). Conclusion: Our data showed that foci of ITGCN were not observed beyond 2.5 cm from the STM. In particular we demonstrated that exist a linear correlation between size of STMs and distance of the foci of GCNIS from STMs (p = 0.0105; r = 0.9167). In conclusion mapping the tissue around the tumor not randomly but in targeted areas could reduce the false negative biopsies of the testis with GCNIS, increasing the radicality of the TSS procedure.

Despite the relevant research efforts, the causes of amyotrophic lateral sclerosis (ALS) are still unknown and no effective cure is available. Many authors suggest that ALS is a multi-system disease caused by a network failure instead of a cell-autonomous pathology restricted to motoneurons. Although motoneuronal loss is the critical hallmark of ALS given their specific vulnerability, other cell populations, including muscle and glial cells, are involved in disease onset and progression, but unraveling their specific role and crosstalk requires further investigation. In particular, little is known about the plastic changes of the degenerating motor system. These spontaneous compensatory processes are unable to halt the disease progression, but their elucidation and possible use as a therapeutic target represents an important aim of ALS research. Genetic animal models of disease represent useful tools to validate proven hypotheses or to test potential therapies, and the conception of novel hypotheses about ALS causes or the study of pathogenic mechanisms may be advantaged by the use of relatively simple in vivo models recapitulating specific aspects of the disease, thus avoiding the inclusion of too many confounding factors in an experimental setting. Here, we used a neurotoxic model of spinal motoneuron depletion induced by injection of cholera toxin-B saporin in the gastrocnemius muscle to investigate the possible occurrence of compensatory changes in both the muscle and spinal cord. The results showed that, following the lesion, the skeletal muscle became atrophic and displayed electromyographic activity similar to that observed in ALS patients. Moreover, the changes in muscle fiber morphology were different from that observed in ALS models, thus suggesting that some muscular effects of disease may be primary effects instead of being simply caused by denervation. Notably, we found plastic changes in the surviving motoneurons that can produce a functional restoration probably similar to the compensatory changes occurring in disease. These changes could be at least partially driven by glutamatergic signaling, and astrocytes contacting the surviving motoneurons may support this process.

Sonic hedgehog (Shh) signaling is a key pathway within the central nervous system (CNS), during both development and adulthood, and its activation via the 7-transmembrane protein Smoothened (Smo) may promote neuroprotection and restoration during neurodegenerative disorders. Shh signaling may also be activated by selected glucocorticoids such as clobetasol, fluocinonide and fluticasone, which therefore act as Smo agonists and hold potential utility for regenerative medicine. However, despite its potential role in neurodegenerative diseases, the impact of Smo-modulation induced by these glucocorticoids on adult neural stem cells (NSCs) and the underlying signaling mechanisms are not yet fully elucidated. The aim of the present study was to evaluate the effects of Smo agonists (i.e., purmorphamine) and antagonists (i.e., cyclopamine) as well as of glucocorticoids (i.e., clobetasol, fluocinonide and fluticasone) on NSCs in terms of proliferation and clonal expansion. Purmorphamine treatment significantly increased NSC proliferation and clonal expansion via GLI-Kruppel family member 1 (Gli1) nuclear translocation and such effects were prevented by cyclopamine co-treatment. Clobetasol treatment exhibited an equivalent pharmacological effect. Moreover, cellular thermal shift assay suggested that clobetasol induces the canonical Smo-dependent activation of Shh signaling, as confirmed by Gli1 nuclear translocation and also by cyclopamine co-treatment, which abolished these effects. Finally, fluocinonide and fluticasone as well as control glucocorticoids (i.e., prednisone, corticosterone and dexamethasone) showed no significant effects on NSCs proliferation and clonal expansion. In conclusion, our data suggest that Shh may represent a druggable target system to drive neuroprotection and promote restorative therapies.

Plastic changes have been reported in the SOD1-G93A mouse model of amyotrophic lateral sclerosis, a disorder characterized by progressive motoneuronal loss; however, whether these changes related with the onset and development of motor impairments is still unclear. Here, the functional and anatomical changes taking place in SOD1-G93A mice and their time course were investigated during ongoing motoneuronal degeneration. Starting from about 4 postnatal weeks, SOD1-G93A and wild-type (WT) mice were evaluated in the rotarod test, to be sacrificed at about 12-13 or 19 weeks of age, and their lumbar spinal cords were processed for histo- and immunohistochemistry. Compared to age-matched WT controls, 12 weeks-old SOD1-G93A mice exhibited relatively mild or no motor impairments in the rotarod test, in spite of a dramatic (≈60%, as estimated by stereology) loss of choline acetyl-transferase (ChAT)-immunoreactive motoneurons which remained virtually unchanged in SOD1-G93A mice surviving up to 19 weeks. Notably, the functional sparing in SOD1-G93A mice at 12 weeks was paralleled by a marked ≈50% increase in motoneuron volume and a near-normal density of acetylcholinesterase-positive process arborization, which was significantly increased when analyzed as ratio to the decreased number of ChAT-positive motoneurons. By contrast, at 19 weeks, when motor deficits had become dramatically evident, both measures were found reverted to about 50-60% of control values. Thus, at specific stages during the progression of the disease, robust compensatory events take place in surviving motoneurons of SOD1-G93A mice, which sustain motor performance, and whose full understanding may highlight a valuable therapeutic opportunity window.

The MRE11/RAD50/NBS1 (MRN) complex is a major sensor of DNA double strand breaks, whose role in controlling faithful DNA replication and preventing replication stress is also emerging. Inactivation of the MRN complex invariably leads to developmental and/or degenerative neuronal defects, the pathogenesis of which still remains poorly understood. In particular, NBS1 gene mutations are associated with microcephaly and strongly impaired cerebellar development, both in humans and in the mouse model. These phenotypes strikingly overlap those induced by inactivation of MYCN, an essential promoter of the expansion of neuronal stem and progenitor cells, suggesting that MYCN and the MRN complex might be connected on a unique pathway essential for the safe expansion of neuronal cells. Here, we show that MYCN transcriptionally controls the expression of each component of the MRN complex. By genetic and pharmacological inhibition of the MRN complex in a MYCN overexpression model and in the more physiological context of the Hedgehog-dependent expansion of primary cerebellar granule progenitor cells, we also show that the MRN complex is required for MYCN-dependent proliferation. Indeed, its inhibition resulted in DNA damage, activation of a DNA damage response, and cell death in a MYCN- and replication-dependent manner. Our data indicate the MRN complex is essential to restrain MYCN-induced replication stress during neural cell proliferation and support the hypothesis that replication-born DNA damage is responsible for the neuronal defects associated with MRN dysfunctions.

In recent years, microRNAs (miRNAs) have received increasing attention for their important role in tumor initiation and progression. MiRNAs are a class of endogenous small non-coding RNAs that negatively regulate the expression of several oncogenes or tumor suppressor genes. MiR-19a, a component of the oncogenic miR-17-92 cluster, has been reported to be highly expressed only in anaplastic thyroid cancer, the most undifferentiated, aggressive and lethal form of thyroid neoplasia. In this work, we evaluated the putative contribution of miR-19a in de-differentiation and aggressiveness of thyroid tumors. To this aim, we induced miR-19a expression in the well-differentiated follicular thyroid cancer cell line and evaluated proliferation, apoptosis and gene expression profile of cancer cells. Our results showed that miR-19a overexpression stimulates cell proliferation and alters the expression profile of genes related to thyroid cell differentiation and aggressiveness. These findings not only suggest that miR-19a has a possible involvement in de-differentiation and malignancy, but also that it could represent an important prognostic indicator and a good therapeutic target for the most aggressive thyroid cancer.

Thyroid carcinoma is generally associated with good prognosis, but no effective treatments are currently available for aggressive forms not cured by standard therapy. To find novel therapeutic targets for this tumor type, we had previously performed a siRNA-based functional screening to identify genes essential for sustaining the oncogenic phenotype of thyroid tumor cells, but not required to the same extent for the viability of normal cells (non-oncogene addiction paradigm). Among those, we found the coatomer protein complex ζ1 (COPZ1) gene, which is involved in intracellular traffic, autophagy and lipid homeostasis. In this paper, we investigated the mechanisms through which COPZ1 depletion leads to thyroid tumor cell death. We showed that siRNA-mediated COPZ1 depletion causes abortive autophagy, endoplasmic reticulum stress, unfolded protein response and apoptosis. Interestingly, we observed that mouse tumor xenografts, locally treated with siRNA targeting COPZ1, showed a significant reduction of tumor growth. On the whole, we demonstrated for the first time the crucial role of COPZ1 in the viability of thyroid tumor cells, suggesting that it may be considered an attractive target for novel therapeutic approaches for thyroid cancer.

JMJD6 is known to localize in the nucleus, exerting histone arginine demethylase and lysyl hydroxylase activities. A novel localization of JMJD6 in the extracellular matrix, resulting from its secretion as a soluble protein, was unveiled by a new anti-JMJD6 mAb called P4E11, which was developed to identify new targets in the stroma. Recombinant JMJD6 binds with collagen type I (Coll-I), and distinct JMJD6 peptides interfere with collagen fibrillogenesis, collagen-fibronectin interaction, and adhesion of human tumor cells to the collagen substrate. P4E11 and collagen binding to JMJD6 are mutually exclusive because the amino acid sequences of JMJD6 necessary for the interaction with Coll-I are part of the conformational epitope recognized by P4E11. In mice injected with mouse 4T1 breast carcinoma cells, treatment with P4E11 reduced fibrosis at the primary tumor and prevented lung metastases. Reduction of fibrosis has also been documented in human breast and ovarian tumors (MDA-MB-231 and IGROV1, respectively) xenotransplanted into immunodeficient mice treated with P4E11. In summary, this study uncovers a new localization and function for JMJD6 that is most likely independent from its canonical enzymatic activities, and demonstrates that JMJD6 can functionally interact with Coll-I. P4E11 mAb, inhibiting JMJD6/Coll-I interaction, represents a new opportunity to target fibrotic and tumor diseases.-Miotti, S., Gulino, A., Ferri, R., Parenza, M., Chronowska, A., Lecis, D., Sangaletti, S., Tagliabue, E., Tripodo, C., Colombo, M. P. Antibody-mediated blockade of JMJD6 interaction with collagen I exerts antifibrotic and antimetastatic activities.

Systemic immune stimulation has been associated with increased risk of myeloid malignancies, but the pathogenic link is unknown. We demonstrate in animal models that experimental systemic immune activation alters the bone marrow stromal microenvironment, disarranging extracellular matrix (ECM) microarchitecture, with downregulation of secreted protein acidic and rich in cysteine (SPARC) and collagen-I and induction of complement activation. These changes were accompanied by a decrease in Treg frequency and by an increase in activated effector T cells. Under these conditions, hematopoietic precursors harboring nucleophosmin-1 (NPM1) mutation generated myeloid cells unfit for normal hematopoiesis but prone to immunogenic death, leading to neutrophil extracellular trap (NET) formation. NET fostered the progression of the indolent NPM1-driven myeloproliferation toward an exacerbated and proliferative dysplastic phenotype. Enrichment in NET structures was found in the bone marrow of patients with autoimmune disorders and in NPM1-mutated acute myelogenous leukemia (AML) patients. Genes involved in NET formation in the animal model were used to design a NET-related inflammatory gene signature for human myeloid malignancies. This signature identified two AML subsets with different genetic complexity and different enrichment in NPM1 mutation and predicted the response to immunomodulatory drugs. Our results indicate that stromal/ECM changes and priming of bone marrow NETosis by systemic inflammatory conditions can complement genetic and epigenetic events towards the development and progression of myeloid malignancy. Cancer Res; 77(13); 3685–99. ©2017 AACR.

Endometrial cancer is now the most common gynecologic malignancy. We investigate on new scientific evidences in endometrial cancer, particularly underlined updates in advanced endometrial cancer. Early stage endometrial cancer is the most frequent presentation; however, advanced endometrial cancer that occurs in 3-13 % of cases has bad prognosis. There are two types of endometrial cancer different in molecular pattern, therapeutic strategy and prognosis. Type I endometrial cancers develop in an environment of unopposed estrogen and often arise out of endometrial hyperplasia, characterized by mutations in the PTEN gene, K-ras, and microsatellite instability inception. Type II cancer is not an estrogen-related cancer, occurs predominantly in postmenopausal women, shows typical mutations in p53 and HER2/neu and has a poor prognosis. Preoperative characterization of the type's disease is an essential step for a right diagnosis and treatment. All patients should undergo to surgical staging, except those who are inoperable, according to FIGO recommendation. Surgical debulking, neoadjuvant chemotherapy and interval debulking can be strategy options.

SummaryDevelopmental Hedgehog signaling controls proliferation of cerebellar granule cell precursors (GCPs), and its aberrant activation is a leading cause of medulloblastoma. We show here that Hedgehog promotes polyamine biosynthesis in GCPs by engaging a non-canonical axis leading to the translation of ornithine decarboxylase (ODC). This process is governed by AMPK, which phosphorylates threonine 173 of the zinc finger protein CNBP in response to Hedgehog activation. Phosphorylated CNBP increases its association with Sufu, followed by CNBP stabilization, ODC translation, and polyamine biosynthesis. Notably, CNBP, ODC, and polyamines are elevated in Hedgehog-dependent medulloblastoma, and genetic or pharmacological inhibition of this axis efficiently blocks Hedgehog-dependent proliferation of medulloblastoma cells in vitro and in vivo. Together, these data illustrate an auxiliary mechanism of metabolic control by a morphogenic pathway with relevant implications in development and cancer.

Olfactory Ensheathing Cells (OECs) are glial cells able to secrete different neurotrophic growth factors and thus promote axonal growth, also acting as a mechanical support. In the olfactory system, during development, they drive the non-myelinated axons of the Olfactory Receptor Neurons (ORNs) towards the Olfactory Bulb (OB). Ghrelin (Ghre), a gut-brain peptide hormone, and its receptor (GHS-R 1a) are expressed in different parts of the central nervous system. In the last few years, this peptide has stimulated particular interest as results show it to be a neuroprotective factor with antioxidant, anti-inflammatory and anti-apoptotic properties. Our previous studies showed that OB mitral cells express Ghre, thus being able to play an important role in regulating food behavior in response to odors. In this study, we investigated the presence of Ghre and GHS-R 1a in primary mouse OECs. The expression of both Ghre and its receptor was assessed by an immunocytochemical technique, Western Blot and Polymerase Chain Reaction (PCR) analysis. Our results demonstrated that OECs are able to express both Ghre and GHS-R 1a and that these proteins are detectable after extensive passages in vitro; in addition, PCR analysis further confirmed these data. Therefore, we can hypothesize that Ghre and GHS-R 1a interact with a reinforcement function, in the peripheral olfactory circuit, providing a neurotrophic support to the synaptic interaction between ORNs and mitral cells.

In the past, manipulation of the cholinergic system was seen as the most likely therapeutic for neurodegeneration-based cognitive decline in Alzheimer's disease (AD) (Whitehouse et al., 1982). However, targeting the noradrenergic system also seems a promising strategy, since more recent studies revealed that in post-mortem tissue from patients with AD and other neurodegenerative disorders there is a robust correlation between cognitive decline and loss of neurons from the Locus coeruleus (LC), a system with diffuse noradrenaline (NA) innervation in the central nervous system (CNS). Therefore, the hypothesis has been considered that increasing NA signaling in the CNS will prevent, or at least halt the progression of neurodegeneration and cognitive decline. A hallmark of the age- and neurodegeneration-related cognitive decline is reduced neurogenesis. We here discuss noradrenergic dysfunction in AD-related cognitive decline in humans and its potential involvement in AD pathology and disease progression. We also focus on animal models to allow the validation of the noradrenergic hypothesis of AD, including those based upon the immunotoxin-mediated ablation of LC based on saporin, a protein synthesis interfering agent, which offers selective and graded demise of LC neurons, Finally, we address how astrocytes, an abundant and functionally heterogeneous cell type of neuroglia maintaining homeostasis, may participate in the regulation of neurogenesis, a new strategy for preventing LC neuron loss.

Neuroblastoma (NB) is the most common pediatric malignancy diagnosed before the first birthday in which MYCN oncogene amplification is associated with poor prognosis. Although aberrant glycosylation is an important actor in cell biology, little is known about its role in pediatric cancers such as NB. In this work we characterized the glycophenotype and the enzyme expression involved in glycans biosynthesis in five established human NB cell lines and in patient-derived primary tumors with different MYCN status. Our results show a high expression of Lewis glycan family both in MYCN-amplified cell lines and patient samples. Additionally, we report that MYCN-amplified cells overexpressed Core 2-initiating glycosyltransferase C2GNT1 in association with specific ST3Gals and FUTs, and showed increased binding to E- and P- selectins. Silencing of C2GNT1 expression in NB cells diminished expression of Lewis glycans, decreased the E- and P-selectin binding, and reduced cell adhesion, migration and proliferation in vitro. Treatment of MYCN-non-amplified cells with Trichostatin A (TSA), an histone deacetylase inhibitor, increased the expression of Lewis glycans and the enzymes involved in their biosynthesis. Our results demonstrate that MYCN-amplified NB cells overexpress Lewis family glycans, which belong to the Core 2 O-glycans group. Their expression plays a key role in the malignant behaviour of the NB cells and it is modulated by epigenetic mechanisms.