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Objective: Steroidogenic factor-1 (SF1) neurons of the ventromedial hypothalamus play key roles in the regulation of food intake, body weight and glucose metabolism. The bile acid receptor Takeda G protein-coupled receptor 5 (TGR5) is expressed in the hypothalamus, where it determines some of the actions of bile acids on food intake and body weight through still poorly defined neuronal mechanisms. Here, we examined the role of TGR5 in SF1 neurons in the regulation of energy balance and glucose metabolism. Methods: We used a genetic approach combined with metabolic phenotyping and molecular analyses to establish the effect of TGR5 deletion in SF1 neurons on meal pattern, body weight, body composition, energy expenditure and use of energy substrates as well as on possible changes in glucose handling and insulin sensitivity. Results: Our findings reveal that TGR5 in SF1 neurons does not play a major role in the regulation of food intake or body weight under standard chow, but it is involved in the adaptive feeding response to the acute exposure to cold or to a hypercaloric, high-fat diet, without changes in energy expenditure. Notably, TGR5 in SF1 neurons hinder glucose metabolism, since deletion of the receptor improves whole-body glucose uptake through heightened insulin signaling in the hypothalamus and in the brown adipose tissue. Conclusions: TGR5 in SF1 neurons favours satiety by differently modifying the meal pattern in response to specific metabolic cues. These studies also reveal a novel key function for TGR5 in SF1 neurons in the regulation of whole-body insulin sensitivity, providing new insight into the role played by neuronal TGR5 in the regulation of metabolism.

Pembrolizumab (an anti-PD1 antibody) alone or combined with chemotherapy represented the standard of care for advanced non-oncogene addicted non-small cell lung cancer (NSCLC) patients. These therapies induced early modifications of the immune response impacting the clinical outcome. Identifying early changes in the immune system was critical to directing the therapeutic choice and improving the clinical outcome. In this study, we aim to analyze the activating and inhibiting immune cells of NSCLC patients before and during therapy to identify patients who will benefit from immunotherapies. Forty-eight NSCLC patients were analyzed before (T0) and after the first cycle of immunotherapy (T1), evaluating several activating (CD137+and PD1+), proliferating (Ki67+) and immunosuppressing immune subsets (Tregs: total, active, resting, and non-suppressive; MDSCs: PMN(Lox1+)-MDSC and M-MDSCs) by cytofluorimetry. Concurrently, 14 soluble immune checkpoints were analyzed by Luminex assay. Immunotherapy significantly increased the levels of Ki67+(total and CD8+) T cells, PMN(Lox1+)-MDSCs, non-suppressive Tregs (nsTregs), and soluble PD1 from T0 to T1 in the entire NSCLC population, while decreased active Tregs. These changes were partially attributed to responding patients who showed an increase of Ki67+ and CD8+T cells and nsTregs at T1. CD137+(total, CD8+, and CD4+) T cells and soluble LAG3 were predictor factors at T0 and T1. A low ratio of Tregs/CD137+ T cells and high levels of Ki67+CD137+ T cells positively correlated with response to therapy at T0 and T1, respectively. Results highlighted that immunotherapy improved the immunological fitness of those patients who benefited from immunotherapy, changing the immunological balance towards immune activation.

Abstract The hypothalamus contains a remarkable diversity of neurons that orchestrate behavioural and metabolic outputs in a highly plastic manner. Neuronal diversity is key to enabling hypothalamic functions and, according to the neuroscience dogma, it is predetermined during embryonic life. Here, by combining lineage tracing of hypothalamic pro-opiomelanocortin (Pomc) neurons with single-cell profiling approaches in adult male mice, we uncovered subpopulations of ‘Ghost’ neurons endowed with atypical molecular and functional identity. Compared to ‘classical’ Pomc neurons, Ghost neurons exhibit negligible Pomc expression and are ‘invisible’ to available neuroanatomical approaches and promoter-based reporter mice for studying Pomc biology. Ghost neuron numbers augment in diet-induced obese mice, independent of neurogenesis or cell death, but weight loss can reverse this shift. Our work challenges the notion of fixed, developmentally programmed neuronal identities in the mature hypothalamus and highlight the ability of specialised neurons to reversibly adapt their functional identity to adult-onset obesogenic stimuli.

Abstract Background Immune checkpoint inhibitors (ICIs), administered alone or combined with chemotherapy, are the standard of care in advanced non-oncogene addicted Non-Small Cell Lung Cancer (NSCLC). Despite these treatments' success, most long-term survival benefit is restricted to approximately 20% of patients, highlighting the need to identify novel biomarkers to optimize treatment strategies. In several solid tumors, immune soluble factors, the activatory CD137+ Tcells, and the immunosuppressive cell subsets Tregs and MDSCs (PMN(Lox1+)-MDSC and M-MDSCs) correlated with responses to ICIs and clinical outcomes thus becoming appealing predictive and prognostic factors. This study investigated the role of distinct CD137+ Tcell subsets, Tregs, MDSCs, and immune-soluble factors in NSCLC patients as possible biomarkers. Methods The levels of T cells, MDSCs and soluble factors were evaluated in 89 metastatic NSCLC patients who underwent ICIs as first- or second-line treatment. T cell analysis was performed by cytoflurimetry evaluating Tregs and different CD137+ Tcell subsets also combined with CD3+, CD8+, PD1+, and Ki67+ markers. Circulating cytokines and immune checkpoints were also evaluated by Luminex analysis. All these parameters were correlated with several clinical factors (age, sex, smoking status, PS and TPS), response to therapy, PFS , and OS . The analyses were conducted in the overall population and in patients treated with ICIs as first-line (naïve patients). Results In both groups of patients, high levels of circulating CD137+ and CD137+PD1+ T cells (total, CD4 and CD8) and the soluble factor LAG3 positively correlated with response to therapy. In naïve patients, PMN(Lox1+)-MDSCs negatively correlated with clinical response, and a high percentage of Tregs was associated with favorable survival. Moreover, the balance between Treg/CD137+ Tcells or PMN(Lox1+)-MDSC/CD137+ Tcells was higher in non-responding patients and was associated with poor survival. CD137+ Tcells and Tregs resulted as two positive independent prognostic factors. Conclusion High levels of CD137+, CD137+PD1+ Tcells and sLAG3 could predict the response to ICIs in NSCLC patients independently by previous therapy. Combining the evaluation of CD137+ Tcells and Tregs also as Treg/CD137+ T cells ratio it is possible to identify naive patients with longer survival.

Inkjet printing is considered a key technology in the fabrication of organic light-emitting diodes (OLEDs), but achieving a fully inkjet-printable OLED structure is still a challenge. Here, we propose the fabrication of OLEDs with an inkjet-printed multilayer structure (i.e. anode/hole injection layer (HIL)/electron blocking layer (EBL)/emitting layer (EML)) by properly developing new HIL and EBL inks to achieve uniform and homogeneous printed thin films. In the fabricated multilayer device, the dissolution process of the EBL, which occurs during the printing of the EML, creates a blurred interface, resulting in device performance that achieves maximum current efficiencies of 20 cd/A and 7 cd/A with ITO and printed polymeric anode, respectively. With the aim of simplifying the structure of the device and mimicking the formation of such a blurred interface, another printed multilayer structure (i.e. anode/printed HIL/printed EBL:EML) was proposed, achieving maximum current efficiencies of 13 and 6 cd/A with ITO and polymeric anode, respectively. Such results represent a compromise between simplifying the fabrication process and achieving good electro-optical properties and thus represent a further step towards the fabrication of a fully inkjet-printed ITO-free OLED.

Abstract Background Fibroblast growth factor receptor (FGFR) gene family alterations are found in several cancers, indicating their importance as potential therapeutic targets. The FGFR-tyrosine kinase inhibitor (TKI) pemigatinib has been introduced in the treatment of advanced cholangiocarcinoma and more recently for relapsed or refractory myeloid/lymphoid neoplasms with FGFR2 and FGFR1 rearrangements, respectively. Several clinical trials are currently investigating the possible combination of pemigatinib with immunotherapy. In this study, we analyzed the biological and molecular effects of pemigatinib on different cancer cell models (lung, bladder, and gastric), which are currently objective of clinical trial investigations. Methods NCI-H1581 lung, KATO III gastric and RT-112 bladder cancer cell lines were evaluated for FGFR expression by qRT-PCR and Western blot. Cell lines were treated with Pem and then characterized for cell proliferation, apoptosis, production of intracellular reactive oxygen species (ROS), and induction of senescence. The expression of microRNAs with tumor suppressor functions was analyzed by qRT-PCR, while modulation of the proteins coded by their target genes was evaluated by Western blot and mRNA. Descriptive statistics was used to analyze the various data and student’s t test to compare the analysis of two groups. Results Pemigatinib exposure triggered distinct signaling pathways and reduced the proliferative ability of all cancer cells, inducing G1 phase cell cycle arrest and strong intracellular stress resulting in ROS production, senescence and apoptosis. Pemigatinib treatment also caused the upregulation of microRNAs (miR-133b, miR-139, miR-186, miR-195) with tumor suppressor functions, along with the downregulation of validated protein targets with oncogenic roles (c-Myc, c-MET, CDK6, EGFR). Conclusions These results contribute to clarifying the biological effects and molecular mechanisms mediated by the anti-FGFR TKI pemigatinib in distinct tumor settings and support its exploitation for combined therapies.

Abstract The fabrication of optoelectronic devices using low‐cost inkjet printing techniques is a topic of great interest to the scientific and industrial community and represents a step toward the full deployment of solution‐processable organic light emitting diodes (OLEDs), particularly for commercial lighting and signaling applications. Herein, the inkjet printing of tBuCzDBA (9,10‐bis(4‐(3,6‐di‐tert‐butyl‐9H‐carbazol‐9‐yl)−2,6‐dimethylphenyl)−9,10‐diboraanthracene) is reported, a high‐performing thermally activated delayed fluorescence (TADF) emitter for OLEDs. Optimizing the surface tension values of the ink formulations and the associated wetting behavior are crucial parameters for achieving a uniform and homogeneous printed thin film. In particular, it is observed that using a proper mixture of solvents with different surface tensions, it is possible to generate Marangoni flows inside the drop, which triggers a very fast drying process, ensuring optimized morphological and optical properties in the inkjet printed tBuCzDBA‐based film. OLEDs exploiting this film as an emissive layer are then fabricated, achieving a maximum luminance of 32 000 cd m−2, a current efficiency of 27.5 cd A−1, and an external quantum efficiencyof 10%. To the best of the knowledge, this is the highest efficiency reported to date for self‐hosted TADF inkjet‐printed OLEDs.

The exposure to sublethally high temperature reduces reproductive performance in diverse organisms. Although this effect has been particularly emphasized for males or male reproductive functioning, it remains largely unknown whether the effect of heat on fertility is sex-specific. Here we examined the impact of sublethally high temperature on male and female functions in a simultaneously hermaphroditic snail species, Lymnaea stagnalis. Examining hermaphrodites is useful to evaluate the sex-specific impacts of heat exposure, since they possess male and female functions within a single individual, sharing genetic and environmental factors. Moreover, previously developed sex allocation theory allows us to compare the differential performance of sex functions. In this study, we exposed snails to 20°C (control), 24°C and 28°C for 14 days and assessed their egg and sperm production, sperm transfer, mating behaviour and growth. Both types of gamete production were significantly reduced by higher temperature, leading to an overall reduction of reproductive investment. By quantifying sex allocation, we furthermore revealed that the heat-stressed snails reduced the relative investment in their male function. This study illustrates that examining simultaneous hermaphrodites can provide significant insights for the impact of heat, and the proximate mechanism, on reproduction in diverse organisms.

Abstract Exposure to extreme temperatures can negatively affect animal reproduction, by disrupting the ability of individuals to produce any offspring (fertility), or the number of offspring produced by fertile individuals (fecundity). This has important ecological consequences, because reproduction is the ultimate measure of population fitness: a reduction in reproductive output lowers the population growth rate and increases the extinction risk. Despite this importance, there have been no large‐scale summaries of the evidence for effect of temperature on reproduction. We provide a systematic map of studies testing the relationship between temperature and animal reproduction. We systematically searched for published studies that statistically test for a direct link between temperature and animal reproduction, in terms of fertility, fecundity or indirect measures of reproductive potential (gamete and gonad traits). Overall, we collated a large and rich evidence base, with 1654 papers that met our inclusion criteria, encompassing 1191 species. The map revealed several important research gaps. Insects made up almost half of the dataset, but reptiles and amphibians were uncommon, as were non‐arthropod invertebrates. Fecundity was the most common reproductive trait examined, and relatively few studies measured fertility. It was uncommon for experimental studies to test exposure of different life stages, exposure to short‐term heat or cold shock, exposure to temperature fluctuations, or to independently assess male and female effects. Studies were most often published in journals focusing on entomology and pest control, ecology and evolution, aquaculture and fisheries science, and marine biology. Finally, while individuals were sampled from every continent, there was a strong sampling bias towards mid‐latitudes in the Northern Hemisphere, such that the tropics and polar regions are less well sampled. This map reveals a rich literature of studies testing the relationship between temperature and animal reproduction, but also uncovers substantial missing treatment of taxa, traits, and thermal regimes. This database will provide a valuable resource for future quantitative meta‐analyses, and direct future studies aiming to fill identified gaps.

Abstract The endocannabinoid system (ECS) regulates energy metabolism, has been implicated in the pathogenesis of metabolic diseases and exerts its actions mainly through the type 1 cannabinoid receptor (CB1). Likewise, autophagy is involved in several cellular processes. It is required for the normal development of muscle mass and metabolism, and its deregulation is associated with diseases. It is known that the CB1 regulates signaling pathways that control autophagy, however, it is currently unknown whether the ECS could regulate autophagy in the skeletal muscle of obese mice. This study aimed to investigate the role of the CB1 in regulating autophagy in skeletal muscle. We found concomitant deregulation in the ECS and autophagy markers in high-fat diet-induced obesity. In obese CB1-KO mice, the autophagy-associated protein LC3 II does not accumulate when mTOR and AMPK phosphorylation levels do not change. Acute inhibition of the CB1 with JD-5037 decreased LC3 II protein accumulation and autophagic flux. Our results suggest that the CB1 regulates autophagy in the tibialis anterior skeletal muscle in both lean and obese mice.

Microfluidics offers a highly controlled and reproducible route to synthesize lipid vesicles. In recent years, several microfluidic approaches have been introduced for this purpose, but double emulsions, such as Water-in-Oil-in-Water (W/O/W) droplets, are preferable to produce giant vesicles that are able to maximize material encapsulation. Flow focusing (FF) is a technique used to generate double emulsion droplets with high monodispersity, a controllable size, and good robustness. Many researchers use polydimethylsiloxane as a substrate material to fabricate microdroplet generators, but it has some limitations due to its hydrophobicity, incompatibility with organic solvents, and the molecular adsorption on the microchannel walls. Thus, specific surface modification and functionalization steps, which are uncomfortable to perform in closed microchannels, are required to overcome these shortcomings. Here, we propose glass as a material to produce a chip with a six-inlet junction geometry. The peculiar geometry and the glass physicochemical properties allow for W/O/W droplet formation without introducing microchannel wall functionalization and using a variety of reagents and organic solvents. The robust glass chip can be easily cleaned and used repeatedly, bringing advantages in terms of cost and reproducibility in emulsion preparation.

Membrane-based sensors (MePSs) exhibit remarkable precision and sensitivity in detecting pressure changes. MePSs are commonly used to monitor catalytic reactions in solution, generating gas products crucial for signal amplification in bioassays. They also allow for catalyst quantification by indirectly measuring the pressure generated by the gaseous products. This is particularly interesting for detecting enzymes in biofluids associated with disease onset. To enhance the performance of a MePS, various structural factors influence membrane flexibility and response time, ultimately dictating the device’s pressure sensitivity. In this study, we fabricated MePSs using polydimethylsiloxane (PDMS) and investigated how structural modifications affect the Young’s modulus (E) and residual stress (σ0) of the membranes. These modifications have a direct impact on the sensors’ sensitivity to pressure variations, observed as a function of the volume of the chamber (Σ) or of the mechanical properties of the membrane itself (S). MePSs exhibiting the highest sensitivities were then employed to detect catalyst quantities inducing the dismutation of hydrogen peroxide, producing dioxygen as a gaseous product. As a result, a catalase enzyme was successfully detected using these optimized MePSs, achieving a remarkable sensitivity of (22.7 ± 1.2) µm/nM and a limit of detection (LoD) of 396 pM.

BackgroundThe immune profile of each patient could be considered as a portrait of the fitness of his/her own immune system. The predictive role of the immune profile in immune-related toxicities (irAEs) development and tumour response to treatment was investigated.MethodsA prospective, multicenter study evaluating, through a multiplex assay, the soluble immune profile at the baseline of 53 patients with advanced cancer, treated with immunotherapy as single agent was performed. Four connectivity heat maps and networks were obtained by calculating the Spearman correlation coefficients for each group: responder patients who developed cumulative toxicity (R-T), responders who did not develop cumulative toxicity (R-NT), non-responders who developed cumulative toxicity (NR-T), non-responders who did not develop cumulative toxicity (NR-NT).ResultsA statistically significant up-regulation of IL-17A, sCTLA4, sCD80, I-CAM-1, sP-Selectin and sEselectin in NR-T was detected. A clear loss of connectivity of most of the soluble immune checkpoints and cytokines characterized the immune profile of patients with toxicity, while an inversion of the correlation for ICAM-1 and sP-selectin was observed in NR-T. Four connectivity networks were built for each group. The highest number of connections characterized the NR-T.ConclusionsA connectivity network of immune dysregulation was defined for each subgroup of patients, regardless of tumor type. In patients with the worst prognosis (NR-T) the peculiar connectivity model could facilitate their early and timely identification, as well as the design of a personalized treatment approach to improve outcomes or prevent irAEs.

Polymeric membranes are useful tools for water filtration processes, with their performance strongly dependent on the presence of hydrophilic dopants. In this study, polyaniline (PANI)-capped aluminosilicate (halloysite) nanotubes (HNTs) are dispersed into polyether sulfone (PES), with concentrations ranging from 0.5 to 1.5 wt%, to modify the properties of the PES membrane. Both undoped and HNT-doped PES membranes are investigated in terms of wettability (static and time-dependent contact angle), permeance, mechanical resistance, and morphology (using scanning electron microscopy (SEM)). The higher water permeance observed for the PES membranes incorporating PANI-capped HNTs is, finally, assessed and discussed vis-à-vis the real distribution of HNTs. Indeed, the imaging and characterization in terms of composition, spatial arrangement, and counting of HNTs embedded within the polymeric matrix are demonstrated using non-destructive Micro Particle Induced X-ray Emission (µ-PIXE) and Scanning Transmission Ion Microscopy (STIM) techniques. This approach not only exhibits the unique ability to detect/highlight the distribution of HNTs incorporated throughout the whole thickness of polymer membranes and provide volumetric morphological information consistent with SEM imaging, but also overcomes the limits of the most common analytical techniques exploiting electron probes. These aspects are comprehensively discussed in terms of practical analysis advantages.

Primary cutaneous CD30+ lymphoproliferative disorders include primary cutaneous anaplastic large cell lymphoma (pcALCL) and lymphomatoid papulosis. The prognosis of the disease is usually excellent but, in a minority of cases, it presents with extracutaneous involvement and aggressive behavior. The case we present—relapsed after surgical excision, immunosuppressive therapy, and conventional chemotherapy—is the first one treated with Autologous Stem Cell transplant followed by Brentuximab Vedotin consolidation, a scheme already used for high risk Hodgkin Lymphoma.

Objective. The role of T cells in the pathogenesis of systemic lupus erythematosus (SLE) has recently gained attention. Costimulatory molecules are membrane proteins strictly associated with T-cell receptor (TCR), acting by activating or inhibiting T cells and antigen-presenting cells (APC) through direct and reverse signaling, thus becoming responsible for the development of effector T cells or regulatory T cells. The primary objective of the present case–control study was to evaluate the cell membrane expression of CD137 on T cells and the serum concentration of CD137 (sCD137) in a SLE cohort. Materials. We enrolled SLE patients and sex/age-matched healthy subjects (HS). Disease activity was assessed by SLEDAI-2K. By application of flow cytometry, we evaluated the expression of CD137 on CD4+ and CD8+ lymphocytes. ELISA test was performed to evaluate serum levels of sCD137. Results. Twenty-one SLE patients (M/F 1/20; median age 48 years (IQR 17); median disease duration 144 months (IQR 204)) were evaluated. SLE patients showed %CD3+CD137+ cells significantly higher compared to HS (median 5.32 (IQR 6.11) versus 3.3 (IQR 1.8), p=0.001). In SLE patients, %CD4+CD137+ cells positively correlated with SLEDAI-2K (p=0.0082, r = 0.58, CI (0.15–0.82); indeed, %CD4+CD137+ cells were significantly lower in SLE patients with a remission status compared to those not reaching this condition (median 1.07 (IQR 0.91) versus 1.58 (IQR 2.42), p=0.013). Accordingly, sCD137 levels were significantly lower in remission status (31.30 pg/mL (IQR 102.2 versus median 122.8 pg/mL (IQR 536); p=0.03) and correlated with %CD4+CD137+ cells (p=0.012, r = 0.60, CI (0.15–0.84)). Conclusion. Our results suggest a possible involvement of CD137-CD137L axis in SLE pathogenesis, as demonstrated by higher expression of CD137 on CD4+ cells in SLE compared with HS. Furthermore, the positive correlation between SLEDAI-2K and membrane CD137 expression on CD4+ cells, as well as soluble CD137, indicates a possible use as biomarkers for disease activity.

BackgroundDespite the efficacy of immunotherapy, only a small percentage of patients achieves a long-term benefit in terms of overall survival. The aim of this study was to define an immune profile predicting the response to immune checkpoint inhibitors (ICIs).MethodsPatients with advanced solid tumors, who underwent ICI treatment were enrolled in this prospective study. Blood samples were collected at the baseline. Thirteen soluble immune checkpoints, 3 soluble adhesion molecules, 5 chemokines and 11 cytokines were analyzed. The results were associated with oncological outcomes.ResultsRegardless of tumor type, patients with values of sTIM3, IFNα, IFNγ, IL1β, IL1α, IL12p70, MIP1β, IL13, sCD28, sGITR, sPDL1, IL10 and TNFα below the median had longer overall survival (p

Metastatic uveal melanoma (UM) is a poor prognosis malignancy. Immunotherapy is commonly employed, despite the low activity, considering the lack of other effective systemic treatments. In this study, the prognostic and predictive role of soluble immune checkpoints and inflammatory cytokines/chemokines in 22 metastatic UM patients was evaluated. Baseline levels of these molecules were assessed, as well as their changes during anti-PD-1 therapy. The correlation between soluble immune checkpoints/cytokines/chemokines and survival was analyzed. A comparison between circulating immune profile of metastatic cutaneous melanoma (CM), for which immunotherapy is a mainstay of treatment, and UM during anti-PD-1 therapy was also performed. Three immune molecules resulted significantly higher in metastatic UM patients with survival 30 months. We also observed an increase of sCD137, sCD28, sPD-1, sPD-L2 sLAG3, sCD80 and sTim3 during anti-PD-1 treatment, as well as IDO activity, IP-10 and CCL2. Several of these molecules were significantly higher in UM compared to CM patients during anti-PD-1 therapy. The analysis of circulating immune molecules allows to identify patients with poor prognosis despite immunotherapy and patients with long survival treated with an anti-PD-1 agent. The different serum concentration of these molecules during anti-PD-1 therapy between UM and CM reflects the different efficacy of immune checkpoint inhibitors.

Summary: Background: Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) are innovative small target molecules that, in combination with endocrine therapy, have recently been employed in the treatment of patients with HR+/HER2− metastatic breast cancer (mBC). In this prospective study, we investigate the impact of CDK4/6i on the immune profile of patients with HR+/HER2− mBC. Methods: Immune cell subsets were analysed using flow cytometry of peripheral blood mononuclear cells (PBMCs) isolated from patients with HR+/HER2− mBC, both before and during treatment. Regulatory T cells (Tregs) were identified using the markers CD4, CD25, CTLA4, CD45RA, and intracellular FOXP3. Monocytic and polymorphonuclear myeloid-derived suppressor cells (M-MDSCs and PMN-MDSCs) and other immune populations were analysed using CD45, CD14, CD66b, CD11c, HLA-DR, CD3, CD8, CD28, CD137, PD1, CD45RA, CCR7, and Ki67. Findings: The percentage of circulating Tregs and M/PMN-MDSCs was significantly downregulated from baseline during CDK4/6i-treatment (p

Abstract Background Despite the efficacy of immune checkpoint inhibitors (ICIs) only the 20–30% of treated patients present long term benefits. The metabolic changes occurring in the gut microbiota metabolome are herein proposed as a factor potentially influencing the response to immunotherapy. Methods The metabolomic profiling of gut microbiota was characterized in 11 patients affected by non-small cell lung cancer (NSCLC) treated with nivolumab in second-line treatment with anti-PD-1 nivolumab. The metabolomics analyses were performed by GC–MS/SPME and 1H-NMR in order to detect volatile and non-volatile metabolites. Metabolomic data were processed by statistical profiling and chemometric analyses. Results Four out of 11 patients (36%) presented early progression, while the remaining 7 out of 11 (64%) presented disease progression after 12 months. 2-Pentanone (ketone) and tridecane (alkane) were significantly associated with early progression, and on the contrary short chain fatty acids (SCFAs) (i.e., propionate, butyrate), lysine and nicotinic acid were significantly associated with long-term beneficial effects. Conclusions Our preliminary data suggest a significant role of gut microbiota metabolic pathways in affecting response to immunotherapy. The metabolic approach could be a promising strategy to contribute to the personalized management of cancer patients by the identification of microbiota-linked “indicators” of early progressor and long responder patients.

Highly conductive PEDOT:PSS is one of the most promising materials for indium tin oxide (ITO) substitution in printed electronics. Here, we report the development and optimisation of two PEDOT:PSS ink formulations for the fabrication of inkjet-printed transparent conductive layers. Starting from aqueous commercial solutions, co-solvents and a non-ionic surfactant were employed to modify the surface tension, improve the wetting capability of the ink, and obtain uniform and homogeneous thin films. In particular, the quantities of ethanol and surfactant were systematically adjusted to determine the optimal conditions for inkjet printing. The results demonstrate that a surface tension value between 28 and 40 mN/m and approximately 40 vol.% of a low-boiling-point co-solvent are fundamental to ensure the proper wetting of the glass substrate and a quick-drying process that confers uniformity to the printed thin film. The printed PEDOT:PSS thin films show good morphological, optical, and electrical properties that are similar to those observed for the corresponding spin-coated layers. The organic light-emitting diodes (OLEDs) fabricated with the inkjet-printed PEDOT:PSS electrodes showed a maximum quantum efficiency of 5.5% and a maximum current efficiency of 15 cd/A, which is comparable to spin-coated reference devices. Our study demonstrates the great potential of polymeric electrodes for the fabrication of high-efficiency printed OLED devices that are compatible with flexible and stretchable substrates.

Introduction Gastric Diffuse large B‐cell lymphoma (DLBCL) is the most common extra-nodal site of lymphoma’s involvement (30%-40% of all extranodal lymphomas and 55%-65% of all gastrointestinal lymphomas). However, gastric localizations are also sometimes found in systemic DLBCL. Gastric complications such as bleeding, perforation and stenosis under chemotherapy are well documented. Methods We retrospectively analyzed 15 patients with newly diagnosed DLBCL with gastrointestinal involvement. Endoscopies were performed in these patients before and after treatment. Treatment consisted in cyclophosphamide low-dose pre-phase chemotherapy before conventional-dose chemotherapy. Results Endoscopy at staging detected ulcers in 12 patients (80%). After low-dose pre-phase chemotherapy GI ulcers healed in 91.6% of cases (1 ulcer detected). After the whole treatment (Low-dose pre-phase + chemotherapy) 9 patients (60%) achieved complete response, 4 patients (26.6%) partial response, 2 (13,3%) patients presented disease progression. The most frequent adverse event was neutropenia (73.3%); the most frequent non hematological adverse event was transaminases elevation (20%). Conclusion Cyclophosphamide low-dose pre-phase chemotherapy resulted a safe and effective way to prevent adverse events in systemic DLBCL with gastrointestinal involvement.

Using preproghrelin-deficient mice (Ghrl-/-), we previously observed that preproghrelin modulates pulsatile growth hormone (GH) secretion in post-pubertal male mice. However, the role of ghrelin and its derived peptides in the regulation of growth parameters or feeding in females is unknown. We measured pulsatile GH secretion, growth, metabolic parameters and feeding behavior in adult Ghrl-/- and Ghrl+/+ male and female mice. We also assessed GH release from pituitary explants and hypothalamic growth hormone-releasing hormone (GHRH) expression and immunoreactivity. Body weight and body fat mass, linear growth, spontaneous food intake and food intake following a 48-h fast, GH pituitary contents and GH release from pituitary explants ex vivo, fasting glucose and glucose tolerance were not different among adult Ghrl-/- and Ghrl+/+ male or female mice. In vivo, pulsatile GH secretion was decreased, while approximate entropy, that quantified orderliness of secretion, was increased in adult Ghrl-/- females only, defining more irregular GH pattern. The number of neurons immunoreactive for GHRH visualized in the hypothalamic arcuate nucleus was increased in adult Ghrl-/- females, as compared to Ghrl+/+ females, whereas the expression of GHRH was not different amongst groups. Thus, these results point to sex-specific effects of preproghrelin gene deletion on pulsatile GH secretion, but not feeding, growth or metabolic parameters, in adult mice.

Cabozantinib (XL-184) is a multitarget tyrosine kinase inhibitor (TKI) targeting receptor tyrosine kinases (RTKs) involved in oncogenesis and angiogenesis. It is currently the standard therapy for medullary thyroid cancer (MTC), metastatic renal cell carcinoma (mRCC), and hepatocellular carcinoma (HCC). Combination of Cabozantinib with immunotherapy is now a standard treatment in metastatic renal cancer, and its efficacy is being tested in ongoing clinical trial in prostate cancer patients. Here, we report that Cabozantinib may exert an immunostimulatory role by inducing immunogenic stress of prostate cancer cells and directly modulating dendritic cells (DCs). Cabozantinib treatment arrested the cell cycle and triggered immunogenic cell death (ICD) in prostate cancer cells in vitro. Cabozantinib had a direct effect on DCs by the down-modulation of β-catenin and change in migratory and costimulatory phenotype of the DCs. These results may suggest possible immunomodulatory effects induced by Cabozantinib that could be exploited to optimize patient-tailored immunotherapeutic treatments.

Carbon dioxide (CO2)-laser processing of glasses is a versatile maskless writing technique to engrave micro-structures with flexible control on shape and size.In this study, we present the fabrication of hundreds of microns quartz micro-channels and micro-holes by pulsed CO2-laser ablation with a focus on the great potential of the technique in microfluidics and biomedical applications. After discussing the impact of the laser processing parameters on the design process, we illustrate specific applications. First, we demonstrate the use of a serpentine microfluidic reactor prepared by combining CO2-laser ablation and post-ablation wet etching to remove surface features stemming from laser-texturing that are undesirable for channel sealing. Then, cyclic olefin copolymer micro-pillars are fabricated using laser-processed micro-holes as molds with high detail replication. The hundreds of microns conical and square pyramidal shaped pillars are used as templates to drive 3D cell assembly. Human Umbilical Vein Endothelial Cells are found to assemble in a compact and wrapping way around the micro-pillars forming a tight junction network. These applications are interesting for both Lab-on-a-Chip and Organ-on-a-Chip devices.

ObjectiveThe monoclonal antibodies anti-programmed death protein-1 (anti–PD-1) nivolumab and pembrolizumab are the first immune checkpoint inhibitors (ICIs) approved for treatment of recurrent/metastatic head and neck carcinoma R/M HNSCC in first line and in platinum refractory disease. This network meta-analysis aims to investigate the efficacy of anti–PD-1- vs anti–PD-L1-based therapy in R/M HNSCC cancer patients through a systematic review of the literature to provide support for evidence-based treatment decisions. In particular, the effectiveness of ICIs for R/M HNSCC is analyzed according to the different mechanisms of action of the check-points inhibitory drugs in different subgroups of patients.MethodsWe did a systematic literature review and network meta-analysis (NMA) of randomized controlled trials (RCTs) in PubMed, ClinicalTrials.gov, Embase, Medline, the Cochrane Central Register of Controlled Trials, Web of Science. Our search identified a total of five randomized controlled trials: Keynote 040, Keynote 048, Eagle, Condor, Checkmate 141. These trials included 3001 patients. Treatment was sub-categorized into PD-L1–based, PD-1–based, and standard chemotherapy. Treatments were indirectly compared with anti–PD-L1-based therapy.ResultsThe network meta-analysis demonstrated no significant differences in OS between different subgroups except for the metastatic patients in which anti–PD-1-based therapy was associated with significantly less risk of death. Furthermore, anti–PD-1-based therapy appeared to be effective in smoker patients and in human papilloma–negative (HPV) patients. Conversely, anti–PD-L1-based therapy seems to be better efficient in female patients, in locally recurrent setting and in HPV positive patients.ConclusionThis is the first NMA study that aimed to indirectly compare anti–PD-1- and anti–PD-L1-based therapy in HNSCC patients. The results of our NMA could help define a profile of patient responder or resistant to specific classes of immune drugs and can be used to guide/design future studies in the novel scenario of precision immune-oncology.

Abstract Background The advent of immune checkpoint inhibitors (ICIs) has considerably expanded the armamentarium against non-small cell lung cancer (NSCLC) contributing to reshaping treatment paradigms in the advanced disease setting. While promising tissue- and plasma-based biomarkers are under investigation, no reliable predictive factor is currently available to aid in treatment selection. Methods Patients with stage IIIB–IV NSCLC receiving nivolumab at Sant’Andrea Hospital and Regina Elena National Cancer Institute from June 2016 to July 2017 were enrolled onto this study. Major clinicopathological parameters were retrieved and correlated with patients’ survival outcomes in order to assess their prognostic value and build a useful tool to assist in the decision making process. Results A total of 102 patients were included in this study. The median age was 69 years (range 44–85 years), 69 (68%) were male and 52% had ECOG PS 0. Loco-regional/distant lymph nodes were the most commonly involved site of metastasis (71%), followed by lung parenchyma (67%) and bone (26%). Overall survival (OS) in the whole patients’ population was 83.6%, 63.2% and 46.9% at 3, 6 and 12 months, respectively; while progression-free survival (PFS) was 66.5%, 44.4% and 26.4% at 3, 6 and 12 months, respectively. At univariate analysis, age ≥ 69 years (P = 0.057), ECOG PS (P

Introduction: Only a minority of patients with platinum refractory head and neck squamous cell carcinoma (PR/HNSCC) gain some lasting benefit from immunotherapy. Methods: The combined role of the comprehensive genomic (through the FoundationOne Cdx test) and immune profiles of 10 PR/HNSCC patients treated with the anti-PD-1 nivolumab was evaluated. The immune profiles were studied both at baseline and at the second cycle of immunotherapy, weighing 20 circulating cytokines/chemokines, adhesion molecules, and 14 soluble immune checkpoints dosed through a multiplex assay. A connectivity map was obtained by calculating the Spearman correlation between the expression profiles of circulating molecules. Results: Early progression occurred in five patients, each of them showing TP53 alteration and three of them showing a mutation/loss/amplification of genes involved in the cyclin-dependent kinase pathway. In addition, ERB2 amplification (1 patient), BRCA1 mutation (1 patient), and NOTCH1 genes alteration (3 patients) occurred. Five patients achieved either stable disease or partial response. Four of them carried mutations in PI3K/AKT/PTEN pathways. In the only two patients, with a long response to immunotherapy, the tumor mutational burden (TMB) was high. Moreover, a distinct signature, in terms of network connectivity of the circulating soluble molecules, characterizing responder and non-responder patients, was evidenced. Moreover, a strong negative and statistically significant (p-value ≤ 0.05) correlation with alive status was evidenced for sE-selectin at T1. Conclusions: Our results highlighted the complexity and heterogeneity of HNSCCs, even though it was in a small cohort. Molecular and immune approaches, combined in a single profile, could represent a promising strategy, in the context of precision immunotherapy.

Background: ICIs have strongly improved the outcome of NSCLC patients. However, primary and secondary resistance occur during treatment in most of the patients, with several of them developing fast progressions. Autoantibodies can be related with a dysfunctional immune system, although their association with immune-based anti-cancer therapies has never been investigated. Moreover, so far no reliable predictive factor is currently available to aid in treatment selection. CD137+T-cells are largely known to be the anti-tumor activated effector cells, but they have never been associated with the response to immunotherapies. Methods: Forty-two patients with metastatic NSCLC receiving anti-PD-1 ICIs at Sant'Andrea Hospital and Policlinico Umberto I, from June 2016 to September 2018 were enrolled. Circulating levels of IgM-Rheumatoid Factor were evaluated at baseline and correlated with patients clinical response following the anti-PD-1 treatment. IgM-RF interaction and effect on T-cells in vivo and in vitro were investigated. Findings: IgM-RF in NSCLC patient sera strongly predicted the development of early progression to ICIs. Also, a significant reduction of progression-free survival rate in anti-PD-1 treated patients could be identified when patients were stratified based on IgM-RF positivity and titers. IgM-RF bound preferentially circulating naïve and central memory T-cells and a significant reduction of CD137+ anti-tumor T effector cells was found in IgM-RF positive patients. In addition, a higher percentage of CD137+T-cells in peripheral blood of NSCLC patients at baseline resulted as a strong independent prognostic factor for a better outcome in terms of PFS and OS after the anti-PD-1 treatment. Furthermore, T-cells exposed to IgM-RF showed a robust defect in their migratory ability in response to CCL19 chemokine. Interpretation: In this study we showed that serum IgM-RF can be regarded as predictive factor for the development of early progression and prognostic factor of a reduced progression-free survival and overall-survival in anti-PD-1 treated NSCLC patients. The ability of IgM-RF to bind naïve and central memory T-cells and impair their migration could make account for the reduction of the tumor-reactive CD137+ T-cells population that may cause a non-effectiveness of these T-cells targeting drugs. Fundings: AIRC, MIUR and Sapienza University of Rome.

Background: Clinical trials showed that only a subset of patients benefits from immunotherapy, suggesting the need to identify new predictive biomarker of resistance. Indoleamine-2,3-dioxygenase (IDO) has been proposed as a mechanism of resistance to anti-PD-1 treatment, and serum kynurenine/tryptophan (kyn/trp) ratio represents a possible marker of IDO activity.Methods: Metastatic non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), and head and neck squamous cell carcinoma (HNSCC) treated with nivolumab as second-line treatment were included in this prospective study. Baseline serum kyn and trp levels were measured by high-performance liquid chromatography to define the kyn/trp ratio. The χ2-test and t-test were applied to compare frequencies and mean values of kyn/trp ratio between subgroups with distinct clinical/pathological features, respectively. Median baseline kyn/trp ratio was defined and used as cutoff in order to stratify the patients. The association between kyn/trp ratio, clinical/pathological characteristics, response, progression-free survival (PFS), and overall survival (OS) was analyzed.Results: Fifty-five patients were included. Mean baseline serum kyn/trp ratio was significantly lower in female than in male patients (0.048 vs. 0.059, respectively, p = 0.044) and in patients with lung metastasis than in others (0.053 vs. 0.080, respectively, p = 0.017). Mean baseline serum kyn/trp ratio was significantly higher in early progressor patients with both squamous and non-squamous NSCLC (p = 0.003) and with a squamous histology cancer (19 squamous NSCLC and 14 HNSCC, p = 0.029). The median value of kyn/trp ratio was 0.06 in the overall population. With the use of median value as cutoff, patients with kyn/trp ratio > 0.06 had a higher risk to develop an early progression (within 3 months) to nivolumab with a trend toward significance (p = 0.064 at multivariate analysis). Patients presenting a baseline kyn/trp ratio ≤0.06 showed a longer PFS [median 8 vs. 3 months; hazard ratio (HR): 0.49; 95% confidence interval (CI) 0.24–1.02; p = 0.058] and a significantly better OS than did those with a kyn/trp ratio > 0.06 (median 16 vs. 4 months; HR: 0.39; 95% CI 0.19–0.82; p = 0.013).Conclusion: Serum kyn/trp ratio could have both prognostic and predictive values in patients with solid tumor treated with immunotherapy, probably reflecting a primary immune-resistant mechanism regardless of the primary tumor histology. Its relative weight is significantly related to gender, site of metastasis, NSCLC, and squamous histology, although these suggestive data need to be confirmed in larger studies.

The paper presents the work done during the two editions of the Summer School that took place in Lampedusa in 2017 and 2018, organized by the Italian Association of Sociology – Environment and Territory Section. The aim of the paper is twofold. Firstly, it aims to illustrate the results of the data analysis and field observation carried out by the participants of the School. They consist of a multi-faceted description of the Island, with special attention to its different populations (residents, city users and especially migrants), and of some resulting project proposals. Secondly, the essay aims to promote a practice based on study and training, which is typical of the sociological analysis of territory, and that can provide useful insight on the studied places and to the people of Lampedusa, within a broader vision of justice and social sustainability.

Abstract Background Immune checkpoint inhibitors have revolutionized the treatment paradigm of highly lethal malignancies like advanced non-small cell lung cancer (NSCLC), demonstrating long-term tumour control and extended patient survival. Unfortunately, only 25–30% of patients experience a durable benefit, while the vast majority demonstrate primary or acquired resistance. Recently, indoleamine 2,3-dioxygenase (IDO) activity has been proposed as a possible mechanism of resistance to anti-PD-1 treatment leading to an immunosuppressive microenvironment. Methods Pre-treatment serum concentrations of tryptophan (trp) and kynurenine (kyn) were measured by high-performance liquid chromatography tandem mass spectrometry in NSCLC patients treated with second-line nivolumab. The IDO activity was expressed with kyn/trp ratio. The associations between kyn/trp ratio and early progression, performance status (PS), age, sex, brain metastases, pleural effusion, progression free survival (PFS) and overall survival (OS) were analyzed using Spearman test and Mann–Whitney test. Results Twenty-six NSCLC patients were included in our study; 14 of them (54%) presented early progression (

Background. Personalised medicine in oncology needs standardised immunological assays. Flow cytometry (FCM) methods represent an essential tool for immunomonitoring, and their harmonisation is crucial to obtain comparable data in multicentre clinical trials. The objective of this study was to design a harmonisation workflow able to address the most effective issues contributing to intra- and interoperator variabilities in a multicentre project. Methods. The Italian National Institute of Health (Istituto Superiore di Sanità, ISS) managed a multiparametric flow cytometric panel harmonisation among thirteen operators belonging to five clinical and research centres of Lazio region (Italy). The panel was based on a backbone mixture of dried antibodies (anti-CD3, anti-CD4, anti-CD8, anti-CD45RA, and anti-CCR7) to detect naïve/memory T cells, recognised as potential prognostic/predictive immunological biomarkers in cancer immunotherapies. The coordinating centre distributed frozen peripheral blood mononuclear cells (PBMCs) and fresh whole blood (WB) samples from healthy donors, reagents, and Standard Operating Procedures (SOPs) to participants who performed experiments by their own equipment, in order to mimic a real-life scenario. Operators returned raw and locally analysed data to ISS for central analysis and statistical elaboration. Results. Harmonised and reproducible results were obtained by sharing experimental set-up and procedures along with centralising data analysis, leading to a reduction of cross-centre variability for naïve/memory subset frequencies particularly in the whole blood setting. Conclusion. Our experimental and analytical working process proved to be suitable for the harmonisation of FCM assays in a multicentre setting, where high-quality data are required to evaluate potential immunological markers, which may contribute to select better therapeutic options.

Background: although bronchoalveolar lavage (BAL) measurements of galactomannan antigen (GM) seems to be more sensitive than serum testing to detect invasive pulmonary aspergillosis (IPA), a consensus on the most appropriate diagnostic threshold of the BAL GM test is still unclear. Moreover, there is uncertainty as to whether BAL is a safe procedure in patients with hematological malignancies (HM) and thrombocytopenia (TC). Objectives: based on this background, 102 adult patients with HM and associated thrombocytopenia were retrospectively analyzed with the twin aims of 1) determining whether BAL is a safe and feasible procedure; and, 2) identifying the most appropriate threshold for GM positivity in the diagnosis of IPA. Patients/Methods: each BAL was considered as one case/patient. One hundred twelve BALs were carried out in 102 HM patients: at the time of the BAL, the median platelets count (PLTs) in all patients was 47x109/L (1-476) and 31 patients (27%) had PLTs< 20x109/L. Results: complications from the BAL were infrequent (3.5%) and mild. No bleeding was reported. The BAL GM cut off of >0.8 was associated with the best diagnostic accuracy (sensitivity 72.97% and specificity 80%). Antifungal treatment of patients with BAL GM >0.8resulted in a clinical-radiological improvement in 35/41patients (85%). Conclusions: BAL was a safe procedure also in thrombocytopenic patients, permitting an IPA diagnosis not otherwise identifiable using EORTC/MSG criteria. Our data suggest that a BAL GM value of>0.8 represents the most useful cut-off in terms of sensibility and specificity. Further prospective studies on a larger number of patients are needed to confirm these results.

Tumor cells release extracellular microvesicles (MVs) in the microenvironment to deliver biological signals to neighboring cells as well as to cells in distant tissues. Tumor-derived MVs appear to play contradictory role promoting both immunosuppression and tumor growth and both evoking tumor specific immune response. Recent evidences indicate that tumor-derived MVs can positively impact Dendritic Cells (DCs) immunogenicity by reprogramming DC antigen processing machinery and intracellular signaling pathways, thus promoting anti-tumor response. DCs are considered pivot cells of the immune system due to their exclusive ability to coordinate the innate and acquired immune responses, cross-present exogenous antigens, and prime naïve T cells. DCs are required for the induction and maintenance of long-lasting anti-tumor immunity and their exploitation has been extensively investigated for the design of anti-tumor vaccines. However, the clinical grade culture conditions that are required to generate DCs for therapeutic use can strongly affect their functions. Here, we investigated the immunomodulatory impact of MVs carrying the MUC1 tumor glycoantigen (MVsMUC1) as immunogen formulation on clinical grade DCs grown in X-VIVO 15 (X-DCs). Results indicated that X-DCs displayed reduced performance of the antigen processing machinery in term of diminished phagocytosis and acidification of the phagosomal compartment suggesting an altered immunogenicity of clinical grade DCs. Pulsing DCs with MVsMUC1 restored phagosomal alkalinization, triggering ROS increase. This was not observed when a soluble MUC1 protein was employed (rMUC1). Concurrently, MVsMUC1 internalization by X-DCs allowed MUC1 cross-processing. Most importantly, MVsMUC1 pulsed DCs activated IFNγ response mediated by MUC1 specific CD8+ T cells. These results strongly support the employment of tumor-derived MVs as immunogen platforms for the implementation of DC-based vaccines.