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Abstract Background Lung regions excluded from mechanical insufflation are traditionally assumed to be spared from ventilation-associated lung injury. However, preliminary data showed activation of potential mechanisms of injury within these non-ventilated regions (e.g., hypoperfusion, inflammation). Methods In the present study, we hypothesized that non-ventilated lung injury (NVLI) may develop within 24 h of unilateral mechanical ventilation in previously healthy pigs, and we performed extended pathophysiological measures to profile NVLI. We included two experimental groups undergoing exclusion of the left lung from the ventilation with two different tidal volumes (15 vs 7.5 ml/kg) and a control group on bilateral ventilation. Pathophysiological alteration including lung collapse, changes in lung perfusion, lung stress and inflammation were measured. Lung injury was quantified by histological score. Results Histological injury score of the non-ventilated lung is significantly higher than normally expanded lung from control animals. The histological score showed lower intermediate values (but still higher than controls) when the tidal volume distending the ventilated lung was reduced by 50%. Main pathophysiological alterations associated with NVLI were: extensive lung collapse; very low pulmonary perfusion; high inspiratory airways pressure; and higher concentrations of acute-phase inflammatory cytokines IL-6, IL-1β and TNF-α and of Angiopoietin-2 (a marker of endothelial activation) in the broncho-alveolar lavage. Only the last two alterations were mitigated by reducing tidal volume, potentially explaining partial protection. Conclusions Non-ventilated lung injury develops within 24 h of controlled mechanical ventilation due to multiple pathophysiological alterations, which are only partially prevented by low tidal volume.

BackgroundBreast cancer during pregnancy (PrBC) is a rare condition known for its aggressive clinical behavior. The presence of tumor-infiltrating lymphocytes (TILs) has been shown to have a significant impact on the prognosis of these patients. Despite some biological characteristics of the tumor that may differ depending on the gestational age, little is known about the dynamics of the immune landscape within the tumor microenvironment (TME) in PrBC. Therefore, in this study, our objective was to gain comprehensive insights into the relationship between gestational age at breast cancer diagnosis and the composition of the TME.Methodsn = 108 PrBC were selected from our institutional registry and categorized based on the gestational age by trimester. For all cases, TILs were profiled according to the International TILs Working Group recommendations, and subtyped by CD4, CD8, and forkhead box P3 (FOXP3) immunohistochemistry. PD-L1 was tested according to the combined positive score (CPS) using the IHC 22C3 pharmDx assay, with a cutoff value of ≥10 for positivity. The statistical approach encompassed Fisher’s and Chi-squared tests, with appropriate adjustments for multiple comparisons, logistic regression models, and survival analyses based on the Kaplan–Meier method.ResultsThe proportion of patients with poorly differentiated (G3) neoplasms increased as the gestational age advanced (first trimester, n = 25, 56.8%; second trimester, n = 27, 69.2%; third trimester, n = 21, 87.5%; p = 0.03). The histologic subtypes as well as the hormone receptor (HR) and HER2 status did not show significant changes across different pregnancy trimesters. In the HR+/HER2– subtype, there was a higher proportion of tumors with high/moderate TILs in the early phases of pregnancy, similar to FOXP3 expression (TILs: first trimester, n = 10, 35.7%; second trimester, n = 2, 10.5%; third trimester, n = 0; p = 0.02; FOXP3: first trimester, n = 10, 40%; second trimester, n = 3, 15.8%; third trimester, n = 0; p = 0.03). The median follow-up for our cohort was 81 months. Patients who relapsed after a breast cancer diagnosis during the first trimester were more frequently PD-L1-negative, unlike those with no disease recurrence (n = 9, 100% vs. n = 9, 56.3%; p = 0.03; hormone therapy and n = 9, 100% vs. n = 7, 53.9%; p = 0.02; chemotherapy). No statistically significant differences were seen among the three trimesters in terms of survival outcome.ConclusionThe TME dynamics of HR+/HER2− PrBC vary based on gestational age, suggesting that immune tolerance expression during later gestational age could explain the increased aggressiveness of tumors diagnosed at that stage.

Of the about 300 gas-phase molecular species so far detected in the interstellar medium (ISM), mostly via observations of their rotational lines, around 40% contain nitrogen (N) atoms. Likewise, of the less than a dozen interstellar molecules, firmly or likely detected in the solid-state water-dominated icy matrix by means of infrared observations, two bear N. A crucial parameter that regulates whether a species is in the gas or adsorbed on the icy phase is their binding energy (BE) toward the icy grain. Therefore, an accurate quantification of the BE is of paramount importance to properly model the ISM chemistry through numerical models. However, very few BEs are available in the literature, either determined experimentally or theoretically. In the present study, we calculate the BEs of 21 among the most abundant interstellar N-bearing species. We adopted two structural water ice models, representing a crystalline and an amorphous surface, using a reliable cost-effective procedure based on the density functional theory. While on the crystalline surface model only one BE per species is obtained due to the high symmetry of the unit cell, on the amorphous model from 5 to 10 BEs are obtained, due to its richer surface morphological variety. Most of our computed BEs agree with available experimental and other computational values. Finally, we discuss how the newly computed BEs can help estimate which N-bearing species can be frozen at the water snow line and, therefore, incorporated in water-rich ice planetesimals.

IntroductionChronic lung allograft dysfunction (CLAD) is the main cause of the reduced survival of lung transplanted (LTx) patients. The possible role of immune checkpoint molecules in establishing tolerance has been scarcely investigated in the setting of lung transplantation.MethodsWe conducted a retrospective, observational pilot study on a consecutive series of transbronchial cryobiopsies (TCB) obtained from 24 patients during LTx follow-up focusing on PD-1, one of the most investigated immune checkpoint molecules.ResultsResults showed that PD-1-expressing T lymphocytes were present in all TCB with a histological diagnosis of acute rejection (AR; 9/9), but not in most (11/15) of the TCB not resulting in a diagnosis of AR (p=0.0006). Notably, the presence of PD-1-expressing T lymphocytes in TCB resulted in a 10-times higher risk of developing chronic lung allograft dysfunction (CLAD), the main cause of the reduced survival of lung transplanted patients, thus being associated with a clearly worst clinical outcome.DiscussionResults of this pilot study indicate a central role of PD-1 in the development of AR and its evolution towards CLAD and suggest that the evaluation of PD-1-expressing lymphocytes in TCB could offer a prognostic advantage in monitoring the onset of AR in patients who underwent lung transplantation.

Abstract Background Phosphatase and tensin homolog (PTEN) loss is associated with tumorigenesis, tumor progression, and therapy resistance in breast cancer. However, the clinical value of PTEN as a biomarker in these patients is controversial. We sought to determine whether the benefit of traditional biomarkers testing is improved by the analysis of PTEN status for the identification of high-risk breast cancer. Methods A cohort of 608 patients with breast cancer was included in this study. Based on the expression on the neoplastic cells compared to the normal internal controls by immunohistochemistry (IHC), cases were classified as PTEN-low (PTEN-L) or PTEN-retained (PTEN-WT). The former constituted the study group, while the latter the control group. Analysis of gene expression was performed on publicly available genomic data and included 4265 patients from the METABRIC and MSK cohorts retrieved from cBioPortal. The Shapiro-Wilk test was used to analyze the normal distributions of continuous variables. Relationships between PTEN status and the clinicopathologic and molecular features of the patient population were assessed using Fisher’s exact test or Chi-squared/Wilcoxon rank-sum test. Survival curves were built according to the Kaplan-Meier method. Results Alteration in PTEN status was significantly different at protein and gene levels, where the reduced protein expression was observed in 280/608 cases (46.1%) from our group, while genetic aberrations in only 315/4265 (7.4%) cases of the METABRIC and MSK cohorts. PTEN-L tumors were significantly enriched for hormone receptors (HR) and HER2 negativity (n = 48, 17.1%) compared to PTEN-WT tumors (n = 22, 6.7%; p = 0.0008). Lack of HR with or without HER2 overexpression/amplification was significantly associated with worse overall survival (OS) in PTEN-L but not in PTEN-WT breast cancers (p

The neurofibromatosis-1 gene (NF1) was initially characterized because its germline mutation is responsible for an inherited syndromic disease predisposing tumor development, in particular neurofibromas but also various malignancies. Recently, large-scale tumor sequencing efforts have demonstrated NF1 as one of the most frequently mutated genes in human cancer, being mutated in approximately 5–10% of all tumors, especially in malignant peripheral nerve sheath tumors and different skin tumors. NF1 acts as a tumor suppressor gene that encodes neurofibromin, a large protein that controls neoplastic transformation through several molecular mechanisms. On the other hand, neurofibromin loss due to NF1 biallelic inactivation induces tumorigenic hyperactivation of Ras and mTOR signaling pathways. Moreover, neurofibromin controls actin cytoskeleton structure and the metaphase–anaphase transition. Consequently, neurofibromin deficiency favors cell mobility and proliferation as well as chromosomal instability and aneuploidy, respectively. Growing evidence supports the role of oxidative stress in NF1-related tumorigenesis. Neurofibromin loss induces oxidative stress both directly and through Ras and mTOR signaling activation. Notably, innovative therapeutic approaches explore drug combinations that further increase reactive oxygen species to boost the oxidative unbalance of NF1-altered cancer cells. In our paper, we review NF1-related tumors and their pathogenesis, highlighting the twofold contribution of oxidative stress, both tumorigenic and therapeutic.

Background: Breast cancer with osteoclast-like stromal giant cells (OSGC) is an exceedingly rare morphological pattern of invasive breast carcinoma. The tumor immune microenvironment (TIME) of these tumors is populated by OSGC, which resemble osteoclasts and show a histiocytic-like immunophenotype. Their role in breast cancer is unknown. The osteoclast maturation in the bone is regulated by the expression of cytokines that are also present in the TIME of tumors and in breast cancer tumor-associated macrophages (TAMs). TAMs-mediated anti-tumor immune pathways are regulated by miRNAs akin to osteoclast homeostasis. Here, we sought to characterize the different cellular compartments of breast cancers with OSGC and investigate the similarities of OSGC with tumor and TIME in terms of morphology, protein, and miRNA expression, specifically emphasizing on monocytic signatures.Methods and Results: Six breast cancers with OSGC were included. Tumor-infiltrating lymphocytes (TILs) and TAMs were separately quantified. The different cellular populations (i.e., normal epithelium, cancer cells, and OSGC) were isolated from tissue sections by laser-assisted microdissection. After RNA purification, 752 miRNAs were analyzed using a TaqMan Advanced miRNA Low-Density Array for all samples. Differentially expressed miRNAs were identified by computing the fold change (log2Ratio) using the Kolmogorov-Smirnov test and p values were corrected for multiple comparisons using the false discovery rate (FDR) approach. As a similarity analysis among samples, we used the Pearson test. The association between pairs of variables was investigated using Fisher exact test. Classical and non-classical monocyte miRNA signatures were finally applied. All OSGC displayed CD68 expression, TILs (range, 45–85%) and high TAMs (range, 35–75%). Regarding the global miRNAs profile, OSGC was more similar to cancer cells than to non-neoplastic ones. Shared deregulation of miR-143-3p, miR-195-5p, miR-181a-5p, and miR-181b-5p was observed between OSGC and cancer cells. The monocyte-associated miR-29a-3p and miR-21-3p were dysregulated in OSGCs compared with non-neoplastic or breast cancer tissues.Conclusion: Breast cancers with OSGC have an activated TIME. Shared epigenetic events occur during the ontogenesis of breast cancer cells and OSGC but the innumophenotype and miRNA profiles of the different cellular compartmens suggest that OSGC likely belong to the spectrum of M2 TAMs.

TIGIT (T cell immunoreceptor with Ig and ITIM domains) is a co-inhibitory receptor expressed on various immune cells, including T cells, NK cells, and dendritic cells. TIGIT interacts with different ligands, such as CD155 and CD112, which are highly expressed on cancer cells, leading to the suppression of immune responses. Recent studies have highlighted the importance of TIGIT in regulating immune cell function in the tumor microenvironment and its role as a potential therapeutic target, especially in the field of lung cancer. However, the role of TIGIT in cancer development and progression remains controversial, particularly regarding the relevance of its expression both in the tumor microenvironment and on tumor cells, with prognostic and predictive implications that remain to date essentially undisclosed. Here, we provide a review of the recent advances in TIGIT-blockade in lung cancer, and also insights on TIGIT relevance as an immunohistochemical biomarker and its possible theranostic implications.

Traditionally, lymphovascular invasion (LVI) has represented one of the foremost pathological features of malignancy and has been associated with a worse prognosis in different cancers, including breast carcinoma. According to the most updated reporting protocols, the assessment of LVI is required in the pathology report of breast cancer surgical specimens. Importantly, strict histological criteria should be followed for LVI assessment, which nevertheless is encumbered by inconsistency in interpretation among pathologists, leading to significant interobserver variability and scarce reproducibility. Current guidelines for breast cancer indicate biological factors as the main determinants of oncological and radiation therapy, together with TNM staging and age. In clinical practice, the widespread use of genomic assays as a decision-making tool for hormone receptor-positive, HER2-negative breast cancer and the subsequent availability of a reliable prognostic predictor have likely scaled back interest in LVI’s predictive value. However, in selected cases, the presence of LVI impacts adjuvant therapy. This review summarizes current knowledge on LVI in breast cancer with regard to definition, histopathological assessment, its biological understanding, clinicopathological association, and therapeutic implications.

Long non-coding RNAs (lncRNAs) are an important class of epigenetic regulators involved in both physiological processes and cancer development. Preliminary evidence suggested that lncRNAs could act as accurate prognostic and diagnostic biomarkers. Parathyroid cancer is a rare endocrine neoplasia, whose management represents a clinical challenge due to the lack of accurate molecular biomarkers. Our previous findings showed that human parathyroid tumors are characterized by a different lncRNAs signature, suggesting heterogeneity through the different histotypes. Particularly, we found that the lncRNA BC200/BCYRN1 could represent a candidate biomarker for parathyroid carcinomas (PCas). Here we aimed to extend our preliminary data evaluating whether BC200 could be an accurate non-invasive biomarker of PCas to support the clinical management of patients affected by parathyroid tumors at diagnosis, prognosis and follow-up. To provide a non-invasive point-of-care for parathyroid carcinoma diagnosis and follow-up, we analyzed BC200 expression in patients’ serum through digital PCR. Our results show that BC200 counts are higher in serum from patients harboring PCa (n=4) compared to patients with parathyroid adenoma (PAd; n=27). Further, in PAd patients circulating BC200 levels are positively correlated with serum total calcium. Then, we found that BC200 is overexpressed in metastatic PCas (n=4) compared to non-metastatic ones (n=9). Finally, the lncRNA expression in PCa patients’ serum drops are reduced after parathyroidectomy, suggesting its possible use in the post-operative setting for patients follow-up. Overall, these findings extend the knowledge on BC200 in parathyroid tumors, supporting its role as a useful biomarker for management of PCa.

BackgroundUnilateral ligation of the pulmonary artery (UPAL) induces bilateral lung injury in pigs undergoing controlled mechanical ventilation. Possible mechanisms include redistribution of ventilation toward the non-ligated lung and hypoperfusion of the ligated lung. The addition of 5% CO2 to the inspiratory gas (FiCO2) prevents the injury, but it is not clear whether lung protection is a direct effect of CO2 inhalation or it is mediated by plasmatic hypercapnia. This study aims to compare the effects and mechanisms of FiCO2vs. hypercapnia induced by low tidal volume ventilation or instrumental dead space.MethodsHealthy pigs underwent left UPAL and were allocated for 48 h to the following: Volume-controlled ventilation (VCV) with VT 10 ml/kg (injury, n = 6); VCV plus 5% FiCO2 (FiCO2, n = 7); VCV with VT 6 ml/kg (low VT, n = 6); VCV plus additional circuit dead space (instrumental VD, n = 6). Histological score, regional compliance, wet-to-dry ratio, and inflammatory infiltrate were assessed to evaluate lung injury at the end of the study. To investigate the mechanisms of protection, we quantified the redistribution of ventilation to the non-ligated lung, as the ratio between the percentage of tidal volume to the right and to the left lung (VTRIGHT/LEFT), and the hypoperfusion of the ligated lung as the percentage of blood flow reaching the left lung (PerfusionLEFT).ResultsIn the left ligated lung, injury was prevented only in the FiCO2 group, as indicated by lower histological score, higher regional compliance, lower wet-to-dry ratio and lower density of inflammatory cells compared to other groups. For the right lung, the histological score was lower both in the FiCO2 and in the low VT groups, but the other measures of injury showed lower intensity only in the FiCO2 group. VTRIGHT/LEFT was lower and PerfusionLEFT was higher in the FiCO2 group compared to other groups.ConclusionIn a model of UPAL, inhaled CO2 but not hypercapnia grants bilateral lung protection. Mechanisms of protection include reduced overdistension of the non-ligated and increased perfusion of the ligated lung.

Abstract Background SARS-coronavirus-2 enters host cells through binding of the Spike protein to ACE2 receptor and subsequent S priming by the TMPRSS2 protease. We aim to assess differences in both ACE2 and TMPRSS2 expression in normal tissues from oral cavity, pharynx, larynx and lung tissues as well as neoplastic tissues from the same areas. Methods The study has been conducted using the TCGA and the Regina Elena Institute databases and validated by experimental model in HNSCC cells. We also included data from one COVID19 patient who went under surgery for HNSCC. Results TMPRSS2 expression in HNSCC was significantly reduced compared to the normal tissues. It was more evident in women than in men, in TP53 mutated versus wild TP53 tumors, in HPV negative patients compared to HPV positive counterparts. Functionally, we modeled the multivariate effect of TP53, HPV, and other inherent variables on TMPRSS2. All variables had a statistically significant independent effect on TMPRSS2. In particular, in tumor tissues, HPV negative, TP53 mutated status and elevated TP53-dependent Myc-target genes were associated with low TMPRSS2 expression. The further analysis of both TCGA and our institutional HNSCC datasets identified a signature anti-correlated to TMPRSS2. As proof-of-principle we also validated the anti-correlation between microRNAs and TMPRSS2 expression in a SARS-CoV-2 positive HNSCC patient tissues Finally, we did not find TMPRSS2 promoter methylation. Conclusions Collectively, these findings suggest that tumoral tissues, herein exemplified by HNSCC and lung cancers might be more resistant to SARS-CoV-2 infection due to reduced expression of TMPRSS2. These observations may help to better assess the frailty of SARS-CoV-2 positive cancer patients.

The reactivity of interstellar carbon atoms (C) on water-dominated ices is one of the possible ways to form interstellar complex organic molecules (iCOMs). In this work, we report a quantum chemical study of the coupling reaction of C ( ^3 P) with an icy water molecule, alongside possible subsequent reactions with the most abundant closed-shell frozen species (NH _3 , CO, CO _2 , and H _2 ), atoms (H, N, and O), and molecular radicals (OH, NH _2 , and CH _3 ). We found that C reacts spontaneously with the water molecule, resulting in the formation of ^3 C–OH _2 , a highly reactive species due to its triplet electronic state. While reactions with the closed-shell species do not show any reactivity, reactions with N and O form CN and CO, respectively, the latter ending up in methanol upon subsequent hydrogenation. The reactions with OH, CH _3 , and NH _2 form methanediol, ethanol, and methanimine, respectively, upon subsequent hydrogenation. We also propose an explanation for methane formation observed in experiments through additions of H to C in the presence of ices. The astrochemical implications of this work are: (i) atomic C on water ice is locked into ^3 C–OH _2 , making difficult the reactivity of bare C atoms on icy surfaces, contrary to what is assumed in current astrochemical models; and (ii) the extraordinary reactivity of ^3 C–OH _2 provides new routes toward the formation of iCOMs in a nonenergetic way, in particular ethanol, the mother of other iCOMs once it is in the gas phase.

The carbon ( ^3 P) atom is a reactive species that, according to laboratory experiments and theoretical calculations, condensates with interstellar ice components. This fact is of uttermost importance for the chemistry in the interstellar medium (ISM) because the condensation reaction is barrierless, and the subsequent species formed are still reactive given their open-shell character. Carbon condensation on CO-rich ices forms the C=C=O ( ^3 Σ ^− ) species, which can be easily hydrogenated twice to form ketene (H _2 CCO). Ketene is very reactive in terrestrial conditions, usually found as an intermediate that is difficult to isolate in chemical synthesis laboratories. These characteristics suggest that ketene can be a good candidate to form interstellar complex organic molecules via a two-step process, i.e., its activation followed by a radical–radical coupling. In this work, reactions between ketene and atomic H and the OH and NH _2 radicals on a CO-rich ice model have been explored by means of quantum chemical calculations complemented by kinetic calculations to evaluate if they are favorable in the ISM. Results indicate that the addition of H to ketene (helped by tunneling) to form the acetyl radical (CH _3 CO) is the most preferred path as the reactions with OH and NH _2 possess activation energies (≥9 kJ mol ^−1 ) hard to surmount in the ISM conditions unless external processes provide energy to the system. Thus, acetaldehyde (CH _3 CHO) and, probably, ethanol (CH _3 CH _2 OH) formation via further hydrogenations, are the possible unique operating synthetic routes. Moreover, from the computed, relatively large binding energies of OH and NH _2 on CO ice, slow diffusion is expected, hampering possible radical–radical couplings with CH _3 CO. The astrophysical implications of these findings are discussed considering the incoming James Webb Space Telescope observations.

In the coldest (10–20 K) regions of the interstellar medium, the icy surfaces of interstellar grains serve as solid-state supports for chemical reactions. Among their plausible roles, that of third body is advocated, in which the reaction energies of surface reactions dissipate throughout the grain, stabilizing the product. This energy dissipation process is poorly understood at the atomic scale, although it can have a high impact on astrochemistry. Here we study, by means of quantum mechanical simulations, the formation of NH _3 via successive H-additions to atomic N on water ice surfaces, paying special attention to the third-body role. We first characterize the hydrogenation reactions and the possible competitive processes (i.e., H-abstractions), in which the H-additions are more favorable than the H-abstractions. Subsequently, we study the fate of the hydrogenation reaction energies by means of ab initio molecular dynamics simulations. Results show that around 58%–90% of the released energy is quickly absorbed by the ice surface, inducing a temporary increase of the ice temperature. Different energy dissipation mechanisms are distinguished. One mechanism, more general, is based on the coupling of the highly excited vibrational modes of the newly formed species and the libration modes of the icy water molecules. A second mechanism, exclusive during the NH _3 formation, is based on the formation of a transient H _3 O ^+ /NH _2 ^− ion pair, which significantly accelerates the energy transfer to the surface. Finally, the astrophysical implications of our findings relative to the interstellar synthesis of NH _3 and its chemical desorption into the gas are discussed.

Despite their rarity, thymic epithelial tumors (TETs) have attracted much interest over the years, leading to an impressive number of histological and staging classifications. At present, TETs are divided by the WHO classification into four main subtypes: type A, type AB, and type B thymomas (subdivided into B1, B2, and B3), and thymic carcinomas, going from the more indolent to the most aggressive ones. Among many debated staging proposals, the TNM and the Masaoka–Koga staging systems have been widely accepted and used in routine practice. The four-tiered histological classification is symmetrically mirrored by the molecular subgrouping of TETs, which identifies an A-like and an AB-like cluster, with frequent GTF2I and HRAS mutations; an intermediate B-like cluster, with a T-cell signaling profile; and a carcinoma-like cluster comprising thymic carcinomas with frequent CDKN2A and TP53 alterations and a high tumor molecular burden. Molecular investigations have opened the way to tailored therapies, such as tyrosine kinase inhibitors targeting KIT, mTOR, and VEGFR, and immune-checkpoints that have been adopted as second-line systemic treatments. In this review, we discuss the crucial events that led to the current understanding of TETs, while disclosing the next steps in this intriguing field.

Abstract Background Worldwide approximately 2.6 million are stillborn, mostly occurring in developing countries. In the great part these deaths are inexplicable. The evenness and standardisation of the diagnostic criteria are prerequisites to understand their pathogenesis. The core goal of this article is to propose new evidence based investigative post-mortem guidelines that should be adopted in all the Institutions especially when a fetal death, after a routine autopsy procedure, is diagnosed as “unexplained”. The proposed protocol is mainly focused on the anatomopathological examination of the autonomic nervous system and in particular of the brainstem where the main centers that control vital functions are located. Methods Updated investigative guidelines for the examination of unexplained stillbirths, prevalently focused on the histological examination of the brainstem, where the main centers that are involved in monitoring the vital functions are located, are here presented. A section of this protocol concerns the Immunohistochemical evaluation of specific functional markers such as the neuronal nuclear antigen, nicotinic acetylcholine receptors, serotonin, orexin, apoptosis and gliosis. The important role of risk factors, having regard in particular to maternal smoking and air pollution is also contemplated in these guidelines. Results Specific morphological and/or functional alterations of vital brainstem structures have been found with high incidence in over 100 cases of unexplained fetal death sent to the “Lino Rossi Research Center” of the Milan University according to the Italian law. These alterations were rarely detected in a group of control cases. Conclusions We hope this protocol can be adopted in all the Institutions notably for the examination of unexplained fetal deaths, in order to make uniform investigations. This will lead to identify a plausible explanation of the pathogenetic mechanism behind the unexplained fetal deaths and to design preventive strategies to decrease the incidence of these very distressing events for both parents and clinicians. Trial registration not applicable for this study.

Chronic lung allograft dysfunction (CLAD) is the main cause of poor survival and low quality of life of lung transplanted patients. Several studies have addressed the role of dendritic cells, macrophages, T cells, donor specific as well as anti-HLA antibodies, and interleukins in CLAD, but the expression and function of immune checkpoint molecules has not yet been analyzed, especially in the two CLAD subtypes: BOS (bronchiolitis obliterans syndrome) and RAS (restrictive allograft syndrome). To shed light on this topic, we conducted an observational study on eight consecutive grafts explanted from patients who received lung re-transplantation for CLAD. The expression of a panel of immune molecules (PD1/CD279, PDL1/CD274, CTLA4/CD152, CD4, CD8, hFoxp3, TIGIT, TOX, B-Cell-Specific Activator Protein) was analyzed by immunohistochemistry in these grafts and in six control lungs. Results showed that RAS compared to BOS grafts were characterized by 1) the inversion of the CD4/CD8 ratio; 2) a higher percentage of T lymphocytes expressing the PD-1, PD-L1, and CTLA4 checkpoint molecules; and 3) a significant reduction of exhausted PD-1-expressing T lymphocytes (PD-1pos/TOXpos) and of exhausted Treg (PD-1pos/FOXP3pos) T lymphocytes. Results herein, although being based on a limited number of cases, suggest a role for checkpoint molecules in the development of graft rejection and offer a possible immunological explanation for the worst prognosis of RAS. Our data, which will need to be validated in ampler cohorts of patients, raise the possibility that the evaluation of immune checkpoints during follow-up offers a prognostic advantage in monitoring the onset of rejection, and suggest that the use of compounds that modulate the function of checkpoint molecules could be evaluated in the management of chronic rejection in LTx patients.

Background: Mitochondrial functions are exploited in cancer and provide a validated therapeutic target. However, how this process is regulated has remained mostly elusive and the identification of new pathways that control mitochondrial integrity in cancer is an urgent priority. Methods: We studied clinically-annotated patient series of primary and metastatic prostate cancer, representative cases of multiple myeloma (MM) and publicly available genetic databases. Gene regulation studies involved chromatin immunoprecipitation, PCR amplification and Western blotting of conditional Myc-expressing cell lines. Transient or stable gene silencing was used to quantify mitochondrial functions in bioenergetics, outer membrane permeability, Ca2+ homeostasis, redox balance and cell death. Tumorigenicity was assessed by cell proliferation, colony formation and xenograft tumour growth. Findings: We identified Mitochondrial Fission Factor (MFF) as a novel transcriptional target of oncogenic Myc overexpressed in primary and metastatic cancer, compared to normal tissues. Biochemically, MFF isoforms, MFF1 and MFF2 associate with the Voltage-Dependent Anion Channel-1 (VDAC1) at the mitochondrial outer membrane, in vivo. Disruption of this complex by MFF silencing induces general collapse of mitochondrial functions with increased outer membrane permeability, loss of inner membrane potential, Ca2+ unbalance, bioenergetics defects and activation of cell death pathways. In turn, this inhibits tumour cell proliferation, suppresses colony formation and reduces xenograft tumour growth in mice. Interpretation: An MFF-VDAC1 complex is a novel regulator of mitochondrial integrity and actionable therapeutic target in cancer. Keywords: Mitochondria, MFF, Cell death, Tumour metabolism, VDAC1, Cancer therapy

Summary Febrile infection–related epilepsy syndrome (FIRES) is a severe epileptic encephalopathy with presumed inflammatory origin and lacking effective treatments. Anakinra is the human recombinant interleukin 1 receptor antagonist clinically used in autoinflammatory or autoimmune conditions. We report a case of FIRES for which the spatial and temporal match between electroencephalography (EEG) and magnetic resonance imaging (MRI) focal alterations provides support for the detrimental synergic interplay between seizures and inflammation that may evolve to permanent focal lesions and progressive brain atrophy in weeks to months. Brain biopsy showed aspects of chronic neuroinflammation with scarce parenchymal lymphocytes. We report the novel evidence that anakinra reduces the relapse of highly recurrent refractory seizures at 1.5 years after FIRES onset. Our evidence, together with previously reported therapeutic effects of anakinra administered since the first days of disease onset, support the hypothesis that interleukin 1β and inflammation‐related factors play a crucial role in seizure recurrence in both the acute and chronic stages of the disease.

Background: The V-ATPase proton pump controls acidification of intra and extra-cellular milieu in both physiological and pathological conditions. We previously showed that some V-ATPase subunits are enriched in glioma stem cells and in patients with poor survival. In this study, we investigated how expression of a GBM-like V-ATPase pump influences the non-neoplastic brain microenvironment. Methods: Large oncosome (LO) vesicles were isolated from primary glioblastoma (GBM) neurospheres, or from patient sera, and co-cultured with primary neoplastic or non-neoplastic brain cells. LO transcript and protein contents were analyzed by qPCR, immunoblotting and immunogold staining. Activation of pathways in recipient cells was determined at gene and protein expression levels. V-ATPase activity was impaired by Bafilomycin A1 or gene silencing. Findings: GBM neurospheres influence their non-neoplastic microenvironment by delivering the V-ATPase subunit V1G1 and the homeobox genes HOXA7, HOXA10, and POU3F2 to recipient cells via LO. LOs reprogram recipient cells to proliferate, grow as spheres and to migrate. Moreover, LOs are particularly abundant in the circulation of GBM patients with short survival time. Finally, impairment of V-ATPase reduces LOs activity. Interpretation: We identified a novel mechanism adopted by glioma stem cells to promote disease progression via LO-mediated reprogramming of their microenvironment. Our data provide preliminary evidence for future development of LO-based liquid biopsies and suggest a novel potential strategy to contrast glioma progression. Fund: This work was supported by Fondazione Cariplo (2014-1148 to VV) and by the Italian Minister of Health-Ricerca Corrente program 2017 (to SF). Keywords: V-ATPase, Homeobox genes, Glioma stem cells, Large oncosome

Breast cancer during pregnancy (PrBC) is a rare tumor with only a little information on its immune landscape. Here, we sought to characterize the cellular composition of the tumor microenvironment (TME) of PrBC and identify its differences from early-onset breast cancer (EOBC) in non-pregnant women. A total of 83 PrBC and 89 EOBC were selected from our Institutional registry and subjected to tumor-infiltrating lymphocytes (TILs) profiling and immunohistochemistry for CD4, CD8, forkhead box P3 (FOXP3), and programmed death-ligand 1 (PD-L1) (clone 22C3). A significantly lower frequency of hormone receptor (HR)-positive tumors was observed in PrBC. The prevalence of low/null PD-L1 and CD8+TILs was higher in PrBC than in the controls, specifically in HR+/HER2– breast cancers. PrBC had a significantly higher risk of relapse and disease-related death, compared to EOBC. The presence of TILs and each TIL subpopulation were significantly associated with disease relapse. Moreover, the death rate was higher in PrBC with CD8+ TILs. The TME of PrBC is characterized by specific patterns of TIL subpopulations with significant biological and prognostic roles. Routine assessment of TILs and TILs subtyping in these patients would be a valid addition to the pathology report that might help identify clinically relevant subsets of women with PrBC.

Anomalous microwave emission (AME) is detected in many astrophysical environments as a foreground feature typically peaking between 20–30 GHz and extending over a 10–60 GHz range. One of the leading candidates for the source of AME is small spinning dust grains. Such grains should be very small (approx. ≤1 nm diameter) in order for the rotational emission to fall within the observed frequency range. In addition, these nanosized grains should possess a significant dipole moment to account for the observed emissivities. These constraints have been shown to be compatible with spinning bare nanosilicate clusters, assuming that ∼1% of the total Si mass budget is held in these ultrasmall grains. Silicate dust can be hydroxylated by processing in the interstellar medium and is generally known to provide seeds for molecular water ice nucleation in denser regions. Herein, we use quantum chemical calculations to investigate how the dipole moment of Mg-rich pyroxenic (MgSiO3) nanoclusters is affected by both accretion of molecular water and dissociative hydration. Our work thus provides an indication of how the formation of water ice mantles is likely to affect the capacity of nanosilicates to generate AME.

COVID-19, a recently emerged disease caused by SARS-CoV-2 infection, can present with different degrees of severity and a large variety of signs and symptoms. The oral manifestations of COVID-19 often involve the tongue, with loss of taste being one of the most common symptoms of the disease. This study aimed to detect SARS-CoV-2 RNA and assess possible morphological and immunopathological alterations in the lingual tissue of patients who died with a history of SARS-CoV-2 infection. Sixteen cadavers from 8 SARS-CoV-2 positive (COVID-19+) and 8 negative (COVID-19−) subjects provided 16 tongues, that were biopsied. Samples underwent molecular analysis through Real-Time RT-PCR for the detection of SARS-CoV-2 RNA. Lingual papillae were harvested and processed for histological analysis and for immunohistochemical evaluation for ACE2, IFN-γ and factor VIII. Real-Time RT-PCR revealed the presence of SARS-CoV-2 RNA in filiform, foliate, and circumvallate papillae in 6 out of 8 COVID-19+ subjects while all COVID-19− samples resulted negative. Histology showed a severe inflammation of COVID-19+ papillae with destruction of the taste buds. ACE2 and IFN-γ resulted downregulated in COVID-19+ and no differences were evidenced for factor VIII between the two groups. The virus was detectable in most COVID-19+ tongues. An inflammatory damage to the lingual papillae, putatively mediated by ACE2 and IFN-γ in tongues from COVID-19+ cadavers, was observed. Further investigations are needed to confirm these findings and deepen the association between taste disorders and inflammation in SARS-CoV-2 infection.

The purpose of this study was to research possible developmental alterations of the substantia nigra (SN) in sudden infant death syndrome (SIDS), a syndrome frequently attributed to arousal failure from sleep. Brain stems of 46 victims of sudden infant death, aged from 1 to about 7 months (4 to 30 postnatal weeks), were investigated. Twenty-six of these cases were diagnosed as SIDS, due to the lack of any pathological finding, while the remaining 20 cases in which the cause of death was determined at autopsy served as controls. Maternal smoking was reported in 77% of SIDS and 10% of controls. Histopathological examination of the SN was done on 5-µm-thick sections of caudal midbrain stained with both hematoxylin-eosin and Klüver-Barrera. Densitometry, immunohistochemistry and histochemistry were applied to highlight the neuronal concentration, the tyrosine hydroxylase (TH) expression, and the presence of neuromelanin (NM) in this structure. Hypoplasia of the pars compacta portion of the SN was observed in 69% of SIDS but never in controls; TH expression was significantly higher in controls than in SIDS; and NM was observed only in 4 infants of the control group but not in SIDS. A significant correlation was found between SIDS, hypoplasia/low neuronal density, low TH expression in the pars compacta, and maternal smoking. Because the SN pars compacta, being the major dopamine brain center, controls many functions, including the sleep-arousal phase, its alterations, especially concurrently with smoking exposure, may contribute to explain the pathogenesis of SIDS that occur in the great part of cases at awakening from sleep.

Abstract Background Identification of predictive molecular alterations in lung adenocarcinoma is essential for accurate therapeutic decisions. Although several molecular approaches are available, a number of issues, including tumor heterogeneity, frequent material scarcity, and the large number of loci to be investigated, must be taken into account in selecting the most appropriate technique. MALDI-TOF mass spectrometry (MS), which allows multiplexed genotyping, has been adopted in routine diagnostics as a sensitive, reliable, fast, and cost-effective method. Our aim was to test the reliability of this approach in detecting targetable mutations in non-small cell lung cancer (NSCLC). In addition, we also analyzed low-quality samples, such as cytologic specimens, that often, are the unique source of starting material in lung cancer cases, to test the sensitivity of the system. Methods We designed a MS–based assay for testing 158 mutations in the EGFR, KRAS, BRAF, ALK, PIK3CA, ERBB2, DDR2, AKT, and MEK1 genes and applied it to 92 NSCLC specimens and 13 liquid biopsies from another subset of NSCLC patients. We also tested the sensitivity of the method to distinguish low represented mutations using serial dilutions of mutated DNA. Results Our panel is able to detect the most common NSCLC mutations and the frequency of the mutations observed in our cohort was comparable to literature data. The assay identifies mutated alleles at frequencies of 2.5–10%. In addition, we found that the amount of DNA template was irrelevant to efficiently uncover mutated alleles present at high frequency. However, when using less than 10 ng of DNA, the assay can detect mutations present in at least 10% of the alleles. Finally, using MS and a commercial kit for RT-PCR we tested liquid biopsy from 13 patients with identified mutations in cancers and detected the mutations in 4 (MS) and in 5 samples (RT-PCR). Conclusions MS is a powerful method for the routine predictive tests of lung cancer also using low quality and scant tissues. Finally, after appropriate validation and improvement, MS could represent a promising and cost-effective strategy for monitoring the presence and percentage of the mutations also in non-invasive sampling.

A black-spotted duodenal mucosa was observed during endoscopy of a man with several comorbidities including hypertension and end-stage kidney disease. Histopathological examination revealed pigment-laden macrophages in the lamina propria of the duodenal villi, which was consistent with duodenal pseudomelanosis.

Duodenal pseudomelanosis (also known as pseudomelanosis duodeni) is a rare endoscopic incidental finding defined by a pigmentation limited to the apex of the intestinal villi, which requires histological confirmation. While its exact pathogenesis is still poorly understood, it appears free from clinical consequences. This condition is believed to be associated with oral iron intake, antihypertensive drugs containing a sulfur moiety (i.e., hydralazine, furosemide), and several chronic diseases (i.e., hypertension, end-stage renal disease, diabetes). However, the exact prevalence of these treatments and comorbidities among patients with duodenal pseudomelanosis is not clearly defined. Several case reports and case series about duodenal pseudomelanosis have been published in recent years. In this review, we aimed to clearly define its endoscopic and microscopic presentation; its epidemiology, associated comorbidities, and drugs; the most useful special histochemical techniques used to classify the nature of the pigmentation; and the most relevant differential diagnoses. In addition, by considering our findings, we also formulated a number of hypotheses about its pathogenesis.

Tumors of the parathyroid glands are the second most common endocrine neoplasia. Epigenetic studies revealed an embryonic signature involved in parathyroid tumorigenesis. Here, we investigated the expression of the stem core genes SOX2, POU5F1/OCT4, and NANOG. Rare cells within normal parathyroid glands expressed POU5F1/OCT4 and NANOG, while SOX2 was undetectable. Nuclear SOX2 expression was detectable in 18% of parathyroid adenomas (PAds, n = 34) involving 5–30% of cells, while OCT4 and NANOG were expressed at the nuclear level in a more consistent subset of PAds involving 15–40% of cells. Most parathyroid carcinomas expressed the core stem genes. SOX2-expressing cells co-expressed parathormone (PTH). In PAds-derived primary cultures, silencing of the tumor suppressor gene MEN1 induced the expression of SOX2, likely through a MEN1/HAR1B/SOX2 axis, while calcium-sensing receptor activation increased SOX2 mRNA levels through YAP1 activation. In addition, inducing nuclear β-catenin accumulation in PAds-derived primary cultures by short-term incubation with lithium chloride (LiCl), SOX2 and POU5F1/OCT4 expression levels increased, while NANOG transcripts were reduced, and LiCl long-term incubation induced an opposite pattern of gene expression. In conclusion, detection of the core stem genes in parathyroid tumors supports their embryogenic signature, which is modulated by crucial genes involved in parathyroid tumorigenesis.

Mitochondria have a controversial role in cancer. Here, the authors demonstrate the reprogramming of a neuronal network of mitochondrial trafficking in tumor cells, and identify Syntaphilin as a key protein that suppresses organelle dynamics thereby blocking chemotaxis and metastasis in mice.

We report a case of a baby, who, after pregnancy complicated by maternal Addison's disease and Hashimoto's thyroiditis and natural delivery, unexpectedly presented a cardiorespiratory collapse and died 1 hour after birth without responding to prolonged neonatal resuscitation maneuvers. The cause of death was reliably established by carrying out a forensic postmortem examination. More specifically, the histological examination of the lungs showed the presence of abundant endoalveolar and endobronchial cornea scales caused by absorption of amniotic fluid. The neuropathological examination of the brainstem highlighted severe hypodevelopment of the retrotrapezoid/parafacial respiratory group, which is a complex of neurons located in the caudal pons that is involved in respiratory rhythm coordination, especially expiration, in conditions of enhanced respiratory drive, as well as in chemoreception. This neuropathological finding shed new light on the mechanisms underlying the massive amniotic fluid aspiration which led to this early death.

We herein characterize the immunopathological features of two Italian COVID-19 patients who underwent bilateral lung transplantation (bLTx). Removed lungs underwent histopathological evaluation. Gene expression profiling (GEP) for immune-related signatures was performed on lung specimens and SARS-CoV-2-stimulated peripheral blood mononuclear cells (PBMCs). Cytokine levels were measured on lungs, bronchoalveolar lavage fluids and in culture supernatants. Pathological assessment showed extensive lung damage with the pattern of proliferative to fibrotic phases, with diffuse alveolar damage mimicking usual interstitial pneumonia (UIP). Lungs’ GEP revealed overexpression of pathogen recognition receptors, effector cytokines and chemokines, immune activation receptors and of the inflammasome components. Multiplex cytokine analysis confirmed a proinflammatory state, with high levels of monocyte/macrophage chemotactic and activating factors and of IL-6 and TNF-α. A similar profile was observed in SARS-CoV-2-stimulated PBMCs collected 7 days after transplant. The pattern of tissue damage observed in the lungs suggests that this may represent the output of protracted disease, resembling a diffuse UIP-like picture. The molecular immune profiling supports the paradigm of a persistent proinflammatory state and sustained humoral immunity, conditions that are maintained despite the iatrogenic immunosuppression.

Nicotinic acetylcholine receptors (nAChRs) are cationic channels of the neuronal cell membrane, differentially expressed in the central nervous system which, when activated by endogenous acetylcholine or exogenous nicotine, are able to enhance cholinergic transmission. The aim of this study was to investigate in human perinatal age the immunohistochemical expression of the α7-nAChR subtype, given its involvement in neuronal differentiation and its significant vulnerability to the toxic effects of nicotine. Thirty fetuses (with a gestational age between 25 and 40 weeks) and 35 infants (1–6 months old), suddenly died of known (controls) and unknown causes (unexplained deaths), with smoking and nonsmoking mothers, were included in this study. A negative or low immunoexpression of α7-nAChRs, indicative of their inactivation, was observed in the granular layers of the cerebellar cortex in 66% of the sudden unexplained perinatal deaths and 11% of the controls. A high correlation was also observed between these findings and maternal smoking. Apart from the well-known adverse effects of nicotine exposure during pregnancy, it may also cause significant alterations in cerebellar cholinergic transmission in areas of the brain involved in vital functions. These events may give us insights into the pathogenetic mechanisms leading to sudden unexplained fetal and infant death.

We explain the state of the art of the immunohistochemical markers of response in rectal cancers treated with neoadjuvant medical therapies and its implication with prognosis. Neoadjuvant chemoradiotherapy is widely used to improve the outcome of patients with locally advanced rectal cancer, and the evaluation of the effects of medical therapy is to date based on histomorphological examination by applying four grading systems of response to therapy (tumor regression grade (TRG)). The need to identify immunohistochemical markers that could ensure a better assessment of response and possibly provide additional prognostic information has emerged. We identified p53, p27kip1, Ki67, matrix metalloprotease-9, survivin, Ki67 proliferative index, CD133, COX2, CD44v6, thymidylate synthase, thymidine phosphorylase, and dihydropyrimidine dehydrogenase as the most common markers studied in literature to date, and we explained their prognostic potential and their implications in the evaluation of the response to preoperative therapies in rectal cancers.

Most men diagnosed with prostate cancer will have an indolent and curable disease, whereas approximately 15% of these patients will rapidly progress to a castrate-resistant and metastatic stage with high morbidity and mortality. Therefore, the identification of molecular signature(s) that detect men at risk of progressing disease remains a pressing and still unmet need for these patients. Here, we used an integrated discovery platform combining prostate cancer cell lines, a Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model and clinically-annotated human tissue samples to identify loss of expression of microRNA-34b as consistently associated with prostate cancer relapse. Mechanistically, this was associated with epigenetics silencing of the MIR34B/C locus and increased DNA copy number loss, selectively in androgen-dependent prostate cancer. In turn, loss of miR-34b resulted in downstream deregulation and overexpression of the "stemness" marker, Sox2. These findings identify loss of miR-34b as a robust biomarker for prostate cancer progression in androgen-sensitive tumors, and anticipate a potential role of progenitor/stem cell signaling in this stage of disease.

BACKGROUND:Non Small Cell Lung Cancer is a highly heterogeneous tumor. Histologic intratumor heterogeneity could be 'major', characterized by a single tumor showing two different histologic types, and 'minor', due to at least 2 different growth patterns in the same tumor. Therefore, a morphological heterogeneity could reflect an intratumor molecular heterogeneity. To date, few data are reported in literature about molecular features of the mixed adenocarcinoma. The aim of our study was to assess EGFR-mutations and ALK-rearrangements in different intratumor subtypes and/or growth patterns in a series of mixed adenocarcinomas and adenosquamous carcinomas. METHODS:590 Non Small Cell Lung Carcinomas tumor samples were revised in order to select mixed adenocarcinomas with available tumor components. Finally, only 105 mixed adenocarcinomas and 17 adenosquamous carcinomas were included in the study for further analyses. Two TMAs were built selecting the different intratumor histotypes. ALK-rearrangements were detected through FISH and IHC, and EGFR-mutations were detected through IHC and confirmed by RT-PCR. RESULTS:10/122 cases were ALK-rearranged and 7 from those 10 showing an intratumor heterogeneity of the rearrangements. 12/122 cases were EGFR-mutated, uniformly expressing the EGFR-mutated protein in all histologic components. CONCLUSION:Our data suggests that EGFR-mutations is generally homogeneously expressed. On the contrary, ALK-rearrangement showed an intratumor heterogeneity in both mixed adenocarcinomas and adenosquamous carcinomas. The intratumor heterogeneity of ALK-rearrangements could lead to a possible impact on the therapeutic responses and the disease outcomes.

Lung cancer encompasses a constellation of malignancies with no validated prognostic markers. p16Ink4A expression has been reported in different subtypes of lung cancers; however, its prognostic value is controversial. Here, we sought to investigate the clinical significance of p16Ink4A immunoexpression according to specific staining patterns and its operational implications. A total of 502 tumors, including 277 adenocarcinomas, 84 squamous cell carcinomas, 22 large cell carcinomas, 47 typical carcinoids, 12 atypical carcinoids, 28 large cell neuroendocrine carcinomas, and 32 small cell carcinomas were reviewed and subjected to immunohistochemical analysis for p16Ink4A and Ki67. The spectrum of p16Ink4A expression was annotated for each case as negative, sporadic, focal, or diffuse. Expression at immunohistochemical level showed intra-tumor homogeneity, regardless tumor histotype. Enrichments in cells expressing p16Ink4A were observed from lower- to higher-grade neuroendocrine malignancies, whereas a decrease was seen in poorly and undifferentiated non-neuroendocrine carcinomas. Tumor proliferation indices were higher in neuroendocrine tumors expressing p16Ink4A while non-neuroendocrine malignancies immunoreactive for p16Ink4A showed a decrease in Ki67-positive cells. Quantitative statistical analyses including each histotype and the p16Ink4A status confirmed the independent prognostic role of p16Ink4A expression, being a high-risk indicator in neuroendocrine tumors and a marker of good prognosis in non-neuroendocrine lung malignancies. In this study, we provide circumstantial evidence to suggest that the routinary assessment of p16Ink4A expression using a three-tiered scoring algorithm, even in a small biopsy, may constitute a reliable, reproducible, and cost-effective substrate for a more accurate risk stratification of each individual patient.