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Abstract We investigated the relationship between the degree of alveolarization and ultrasound-assessed lung aeration in a validated preterm rabbit model of experimental bronchopulmonary dysplasia (BPD). Lung ultrasound findings were heterogeneously abnormal and consisted of zones with interstitial, interstitial-alveolar or consolidated patterns. The median radial alveolar count was 10.1 [8.4–11.5], 7.8 [6.1–9] and 7.3 [1.8–10.1] in rabbits with interstitial, interstitial-alveolar or consolidated ultrasound pattern, respectively (overall p = 0.036). Alveolar count and lung ultrasound score were significantly correlated (ρ = − 0.044 (95%CI: − 1; − 0.143), p = 0.009; τ-b = − 0.362 (95%CI: − 0.6; − 0.1), p = 0.017).

IntroductionDespite the advances in perinatal care, bronchopulmonary dysplasia (BPD) continues to be a highly prevalent chronic lung disease that affects newborns, especially affecting premature newborns. There is no specific cure for BPD, and treatments aimed at reducing the risk of developing BPD focus mainly on lung-protective ventilation strategies, surfactant therapy, and/or corticosteroid administration. Our objective was to evaluate whether systemic postnatal administration of a new glucocorticoid, ciclesonide, can attenuate the alteration of lung structure and pulmonary hypertension in a rat model of chorioamnionitis-induced BPD, with minimal adverse effects on the developing brain.MethodsEndotoxin (ETX) or saline was administered to pregnant rats by intra-amniotic (i.a.) injection on day 20 of pregnancy, and pups were delivered by cesarean section on day 22. Ciclesonide (0.5 mg/kg) was administered postnatally for five consecutive days to pups previously exposed to i.a. ETX. On postnatal day 14, we assessed lung function (compliance), lung structure (radial alveolar count, mean linear intercept, pulmonary vessel density), pulmonary hypertension, and brain histology (edema, inflammation, apoptosis, hemorrhage, and infarction).ResultOn postnatal day 14, the effects of i.a. ETX administration were evident in neonatal rats not receiving treatment; these animals showed impaired lung compliance, disrupted lung structure, and developing pulmonary hypertension compared to those receiving i.a. saline. Postnatal administration of ciclesonide for 5 days was associated with significantly better outcomes in terms of lung compliance, alveolarization, lung vascular growth, and pulmonary hypertension, without affecting the brain histological parameters evaluated.ConclusionPostnatal ciclesonide administration preserved lung function and structure and prevented pulmonary hypertension in a BPD model induced by antenatal i.a. ETX administration, without causing any adverse effects on brain development. These findings suggest that the new glucocorticoid, ciclesonide, may provide a novel strategy for the prevention of BPD; however, more long-term studies are required.

Abstract Background Premature birth, perinatal inflammation, and life-saving therapies such as postnatal oxygen and mechanical ventilation are strongly associated with the development of bronchopulmonary dysplasia (BPD); these risk factors, alone or combined, cause lung inflammation and alter programmed molecular patterns of normal lung development. The current knowledge on the molecular regulation of lung development mainly derives from mechanistic studies conducted in newborn rodents exposed to postnatal hyperoxia, which have been proven useful but have some limitations. Methods Here, we used the rabbit model of BPD as a cost-effective alternative model that mirrors human lung development and, in addition, enables investigating the impact of premature birth per se on the pathophysiology of BPD without further perinatal insults (e.g., hyperoxia, LPS-induced inflammation). First, we characterized the rabbit’s normal lung development along the distinct stages (i.e., pseudoglandular, canalicular, saccular, and alveolar phases) using histological, transcriptomic and proteomic analyses. Then, the impact of premature birth was investigated, comparing the sequential transcriptomic profiles of preterm rabbits obtained at different time intervals during their first week of postnatal life with those from age-matched term pups. Results Histological findings showed stage-specific morphological features of the developing rabbit’s lung and validated the selected time intervals for the transcriptomic profiling. Cell cycle and embryo development, oxidative phosphorylation, and WNT signaling, among others, showed high gene expression in the pseudoglandular phase. Autophagy, epithelial morphogenesis, response to transforming growth factor β, angiogenesis, epithelium/endothelial cells development, and epithelium/endothelial cells migration pathways appeared upregulated from the 28th day of gestation (early saccular phase), which represents the starting point of the premature rabbit model. Premature birth caused a significant dysregulation of the inflammatory response. TNF-responsive, NF-κB regulated genes were significantly upregulated at premature delivery and triggered downstream inflammatory pathways such as leukocyte activation and cytokine signaling, which persisted upregulated during the first week of life. Preterm birth also dysregulated relevant pathways for normal lung development, such as blood vessel morphogenesis and epithelial-mesenchymal transition. Conclusion These findings establish the 28-day gestation premature rabbit as a suitable model for mechanistic and pharmacological studies in the context of BPD.

Adrenal hemorrhage is a rare, but important, diagnosis to recognize, in particular when there is involvement of both adrenal glands. Bilateral adrenal hemorrhage can in fact lead to adrenal insufficiency, with dramatic consequences if not promptly recognized and treated. It is normally caused by systemic conditions that lead to the vasoconstriction and thrombosis of the adrenal vein. Oftentimes, the clinical diagnosis of this condition can be very challenging, as its signs and symptoms are generalized and nonspecific (abdominal pain, nausea, and fatigue). Here, we present the cases of two patients admitted to the Emergency Department in 2016 and 2022 with acute abdominal pain, having recently undergone surgery and subsequently prescribed low-molecular-weight heparin. In both cases, laboratory results revealed neutrophilic leukocytosis and an unexplained anemia. Due to the persistence of abdominal pain despite medication, a CT scan was performed, showing an enlargement of both adrenal glands suggestive of bilateral adrenal hemorrhage. Adrenal function was tested that correlated with a diagnosis of adrenal insufficiency, and both patients were promptly treated with parenteral hydrocortisone as a result. On 5 years’ follow-up from the acute event, the second patient’s adrenal function had returned to normal, and he has not needed further adrenal replacement therapy; the first patient however demonstrated persistence of adrenal failure requiring replacement therapy. In this paper, through our experience and a literature analysis, we will aim to outline some clues to identify patients at potential risk of bilateral adrenal hemorrhage.

The introduction of novel drugs (PD-1 inhibitors and/or brentuximab vedotin) into salvage regimens has improved the response rate and the outcome of patients with relapsed/refractory Hodgkin lymphoma. However, the impact of new drugs on the outcome has not been adequately investigated so far. We retrospectively analyzed 42 consecutive patients treated at our institution with high-dose chemotherapy/autologous stem cell transplantation after either one standard chemotherapy represented by BEGEV (n = 28) or >1 salvage therapy (ST) comprising novel drugs (n = 14). With a median follow-up of 24 months, the 2-year cumulative incidence of relapse was similar between the two cohorts: 26% for 1 ST and 18% for >1 ST (p = 0.822). Consistently, overall survival and progression-free survival did not differ among the two groups: 3-year overall survival was 91% and 89% (p = 0.731), respectively, and 3-year progression-free survival was 74% and 83% (p = 0.822) for only one and more than one salvage regimens, respectively. Of note, the post-transplant side effects and engraftment rates were similar between the 1 ST and >1 ST cohorts. In conclusion, consolidation with high-dose chemotherapy/autologous stem cell transplantation is a safe and curative option, even for patients achieving disease response after more than one rescue line of therapy.

Typical hemolytic uremic syndrome (HUS) is mainly caused by Shiga toxin-producing Escherichia coli (STEC) releasing Shiga toxin 2 (Stx2). Two different structures of this AB5 toxin have been described: uncleaved, with intact B and A chains, and cleaved, with intact B and a nicked A chain consisting of two fragments, A1 and A2, connected by a disulfide bond. Despite having the same toxic effect on sensitive cells, the two forms differ in their binding properties for circulating cells, serum components and complement factors, thus contributing to the pathogenesis of HUS differently. The outcome of STEC infections and the development of HUS could be influenced by the relative amounts of uncleaved or cleaved Stx2 circulating in patients’ blood. Cleaved Stx2 was identified and quantified for the first time in four out of eight STEC-infected patients’ sera by a method based on the inhibition of cell-free translation. Cleaved Stx2 was present in the sera of patients with toxins bound to neutrophils and in two out of three patients developing HUS, suggesting its involvement in HUS pathogenesis, although in association with other bacterial or host factors.

Abstract Background Treatment with PARP inhibitors (PARPi) is primarily effective against high-grade serous ovarian cancers (HGSOC) with BRCA1/2 mutations or other deficiencies in homologous recombination (HR) repair mechanisms. However, resistance to PARPi frequently develops, mostly as a result of BRCA1/2 reversion mutations. The tumour suppressor CCDC6 is involved in HR repair by regulating the PP4c phosphatase activity on γH2AX. In this work, we reported that in ovarian cancer cells, a physical or functional loss of CCDC6 results synthetic lethal with the PARP-inhibitors drugs, by affecting the HR repair. We also unravelled a role for CCDC6 as predictive marker of PARPi sensitivity in ovarian cancer, and the impact of CCDC6 downregulation in overcoming PARPi resistance in these tumours. Methods A panel of HGSOC cell lines (either BRCA-wild type or mutant) were treated with PARPi after CCDC6 was attenuated by silencing or by inhibiting USP7, a CCDC6-deubiquitinating enzyme, and the effects on cell survival were assessed. At the cellular and molecular levels, the processes underlying the CCDC6-dependent modification of drugs’ sensitivity were examined. Patient-derived xenografts (PDXs) were immunostained for CCDC6, and the expression of the protein was analysed statistically after digital or visual means. Results HGSOC cells acquired PARPi sensitivity after CCDC6 depletion. Notably, CCDC6 downregulation restored the PARPi sensitivity in newly generated or spontaneously resistant cells containing either wild type- or mutant-BRCA2. When in an un-phosphorylated state, the CCDC6 residue threonine 427 is crucial for effective CCDC6-PP4 complex formation and PP4 sequestration, which maintains high γH2AX levels and effective HR. Remarkably, the PP4-dependent control of HR repair is influenced by the CCDC6 constitutively phosphorylated mutant T427D or by the CCDC6 loss, favouring PARPi sensitivity. As a result, the PP4 regulatory component PP4R3α showed to be essential for both the activity of the PP4 complex and the CCDC6 dependent PARPi sensitivity. It's interesting to note that immunohistochemistry revealed an intense CCDC6 protein staining in olaparib-resistant HGSOC cells and PDXs. Conclusions Our findings suggest that the physical loss or the functional impairment of CCDC6 enhances the PP4c complex activity, which causes BRCAness and PARPi sensitivity in HGSOC cells. Moreover, CCDC6 downregulation might overcome PARPi resistance in HGSOCs, thus supporting the potential of targeting CCDC6 by USP7 inhibitors to tackle PARPi resistance.

IntroductionExtracellular vesicles (EVs) and particles (EPs) represent reliable biomarkers for disease detection. Their role in the inflammatory microenvironment of severe COVID-19 patients is not well determined. Here, we characterized the immunophenotype, the lipidomic cargo and the functional activity of circulating EPs from severe COVID-19 patients (Co-19-EPs) and healthy controls (HC-EPs) correlating the data with the clinical parameters including the partial pressure of oxygen to fraction of inspired oxygen ratio (PaO2/FiO2) and the sequential organ failure assessment (SOFA) score.MethodsPeripheral blood (PB) was collected from COVID-19 patients (n=10) and HC (n=10). EPs were purified from platelet-poor plasma by size exclusion chromatography (SEC) and ultrafiltration. Plasma cytokines and EPs were characterized by multiplex bead-based assay. Quantitative lipidomic profiling of EPs was performed by liquid chromatography/mass spectrometry combined with quadrupole time-of-flight (LC/MS Q-TOF). Innate lymphoid cells (ILC) were characterized by flow cytometry after co-cultures with HC-EPs or Co-19-EPs.ResultsWe observed that EPs from severe COVID-19 patients: 1) display an altered surface signature as assessed by multiplex protein analysis; 2) are characterized by distinct lipidomic profiling; 3) show correlations between lipidomic profiling and disease aggressiveness scores; 4) fail to dampen type 2 innate lymphoid cells (ILC2) cytokine secretion. As a consequence, ILC2 from severe COVID-19 patients show a more activated phenotype due to the presence of Co-19-EPs.DiscussionIn summary, these data highlight that abnormal circulating EPs promote ILC2-driven inflammatory signals in severe COVID-19 patients and support further exploration to unravel the role of EPs (and EVs) in COVID-19 pathogenesis.

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a rare entity whose neoplastic cells retain a B-cell phenotype with expression of CD20. Radiotherapy is recommended for favorable stage IA disease while for other stages guidelines suggest therapeutic strategies similar to those used for classic HL. The role of rituximab, although quite widespread, is not completely elucidated. We retrospectively analyzed baseline characteristics of 308 consecutive patients with NLPHL diagnosed in 19 Italian centers from 2000 to 2018. With a median follow-up of 8.4 years (interquartile range: 4.5–12.4) for treated patients, median overall survival (OS) was not reached and estimated 5-year OS was 97.8% and 5-year progression-free survival (PFS) was 84.5%. Five-year cumulative incidence of histological transformation was 1.4%, 95% confidence interval (CI), 0.5%-3.8%. After adjusting for lymphocyte count, splenic involvement, bulky disease and B symptoms (fever, drenching night sweats, unintentional loss >10% of body weight within the preceding 6 months), patients with stage II or more showed superior PFS with immunochemotherapy in comparison to chemotherapy alone (hazard ratio = 0.4, 95% CI, 0.2-0.8; P = 0.015). Our data suggest an advantage of the use of rituximab combined with chemotherapy ± radiotherapy in the treatment of stage II–III–IV NLPHL.

The most recent approaches to the initial treatment of respiratory distress syndrome (RDS)- involve non-invasive ventilation (NIV) and less-invasive surfactant (SF) administration (LISA). Combining these techniques has been proven a useful treatment option for SF-deficient neonates. The objective of this study was to explore the impact on the brain (using cerebral near infrared spectroscopy, NIRS) of different LISA methods during NIV, using nasal intermittent positive pressure ventilation (NIPPV) for treating neonatal RDS. For this, we used five groups of spontaneously breathing newborn piglets (n = 6/group) with bronchoalveolar lavage (BAL)-induced respiratory distress which received NIPPV only (controls), poractant-alfa using the INSURE-like method (bolus delivery) followed by NIPPV, or poractant-alfa using one of three LISA devices, 1) a nasogastric tube (NT), 2) a vascular catheter (VC) or 3) the LISAcath® catheter. We assessed pulmonary, hemodynamic and cerebral effects, and performed histological analysis of lung and brain tissue. Following BALs, the piglets developed severe RDS (pH70 mmHg, PaO2

Graft versus host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (HSCT). Rabbit anti-T lymphocyte globulin (ATLG) in addition to calcineurin inhibitors and antimetabolites is a suitable strategy to prevent GVHD in several transplant settings. Randomized studies already demonstrated its efficacy in terms of GVHD prevention, although the effect on relapse remains the major concern for a wider use. Tailoring of ATLG dose on host characteristics is expected to minimize its side effects (immunological reconstitution, relapse, and infections). Here, day -6 to day +15 pharmacokinetics of active ATLG serum level was first assayed in an explorative cohort of 23 patients by testing the ability of the polyclonal serum to bind antigens on human leukocytes. Significantly lower levels of serum active ATLG were found in the patients who developed GVHD (ATLG_AUCCD45: 241.52 ± 152.16 vs. 766.63 +/- 283.52 (μg*day)/ml, p = 1.46e-5). Consistent results were obtained when the ATLG binding capacity was assessed on CD3+ and CD3+/CD4+ T lymphocytes (ATLG_AUCCD3: 335.83 ± 208.15 vs. 903.54 ± 378.78 (μg*day)/ml, p = 1.92e-4; ATLG_AUCCD4: 317.75 ± 170.70 vs. 910.54 ± 353.35 (μg*day)/ml, p = 3.78e-5. Concomitantly, at pre-infusion time points, increased concentrations of CD69+ extracellular vesicles (EVs) were found in patients who developed GVHD (mean fold 9.01 ± 1.33; p = 2.12e-5). Consistent results were obtained in a validation cohort of 12 additional ATLG-treated HSCT patients. Serum CD69+ EVs were mainly represented in the nano (i.e. 100 nm in diameter) EV compartment and expressed the leukocyte marker CD45, the EV markers CD9 and CD63, and CD103, a marker of tissue-resident memory T cells. The latter are expected to set up a host pro-inflammatory cell compartment that can survive in the recipient for years after conditioning regimen and contribute to GVHD pathogenesis. In summary, high levels of CD69+ EVs are significantly correlated with an increased risk of GVHD, and they may be proposed as a tool to tailor ATLG dose for personalized GVHD prevention.

Abstract Background Overweight and obese patients with solid tumors receiving anti-programmed cell death-1 (PD-1)/PD-ligand-1(PD-L1) immune checkpoint inhibitors exhibit improved survival and higher risk of immune-related adverse events (irAEs) than those with a normal body mass index (BMI). In classic Hodgkin lymphoma (cHL), the impact of BMI on survival and immune-related toxicity is unknown. We evaluated for the first time associations of BMI with survival and irAEs in patients with relapsed/refractory (RR)-cHL undergoing PD-1 blockade. Methods Data from a multicenter study on 133 patients treated with the anti-PD1 antibody nivolumab (July 2015–December 2016) were retrieved from a prospective database. Progression-free (PFS), overall survival (OS), incidence and severity of irAEs according to BMI categories were estimated by Kaplan–Meier method, landmark-analyses and Cox regressions. Results Patients, mostly males (63%, n = 84) with a median age of 35 years (range, 15–82), advanced stage (75%), B symptoms (63%), bulky disease (24%), a median of 4 previous treatments (range, 1–9), received a median of 18 nivolumab doses (range, 1–57). No statistically significant differences across BMI subgroups emerged as to PFS, with 1-year rates of 67.1% for both normal weight (n = 66; 49.6%) and overweight (n = 31; 23.3%) patients. Underweight (n = 12; 9%) and obese (n = 24; 18%) patients had a 1-year PFS of 54.5% and 49%, respectively. In survival analyses, BMI either as a continuous (P = 0.5) or categorical (P for trend = 0.63) variable failed to associate with PFS. Response rates and time-to-response did not cluster in any BMI subset. No BMI-related differences in OS emerged across normal, overweight and obese patients but underweight patients had the worst survival. Occurrence of irAEs of whatever severity did not statistically associate with BMI. Conclusions In patients with RR-cHL receiving nivolumab, no statistically significant differences emerged in response rates, PFS and OS across BMI categories of normal weight, overweight and obese. Overweight/obese patients did not display an increased risk of irAEs. The exquisite sensitivity to anti-PD-1 antibodies, the unique cytokine milieu and effector pathways triggered by nivolumab in cHL, may represent biologic ‘equalizers’ counteracting the immunoregulatory effects of adiposity. Differently from solid tumors, BMI is not associated with treatment efficacy and immune-related toxicity and does not represent a predictive tool for PD-1-targeted immunotherapies in cHL.

ObjectiveThe present manuscript aims to describe an international, electronic-based, user-friendly and interoperable patient registry for monogenic autoinflammatory diseases (mAIDs), developed in the contest of the Autoinflammatory Diseases Alliance (AIDA) Network.MethodsThis is an electronic platform, based on the Research Electronic Data Capture (REDCap) tool, used for real-world data collection of demographics, clinical, laboratory, instrumental and socioeconomic data of mAIDs patients. The instrument has flexibility, may change over time based on new scientific acquisitions, and communicate potentially with other similar registries; security, data quality and data governance are corner stones of the platform.ResultsAIDA project will share knowledge and expertise on mAIDs. Since its start, 118 centers from 24 countries and 4 continents have joined the AIDA project. Fifty-nine centers have already obtained the approval from their local Ethics Committees. Currently, the platform counts 337 users (122 Principal Investigators, 210 Site Investigators, 2 Lead Investigators, and 3 data managers). The Registry collects baseline and follow-up data using 3,748 fields organized into 21 instruments, which include demographics, patient history, symptoms, trigger/risk factors, therapies, and healthcare information for mAIDs patients.ConclusionsThe AIDA mAIDs Registry, acts both as a research tool for future collaborative real-life studies on mAIDs and as a service to connect all the figures called to participate. On this basis, the registry is expected to play a pivotal role in generating new scientific evidence on this group of rare diseases, substantially improving the management of patients, and optimizing the impact on the healthcare system. NCT 05200715 available at https://clinicaltrials.gov.

Small molecules that can modulate or stabilize cell–cell interactions are valuable tools for investigating the impact of collective cell behavior on various biological processes such as development/morphogenesis, tissue regeneration and cancer progression. Recently, we showed that budesonide, a glucocorticoid widely used as an anti-asthmatic drug, is a potent regulator of stem cell pluripotency. Here we tested the effect of different budesonide derivatives and identified CHD-030498 as a more effective analogue of budesonide. CHD-030498 was able to prevent stem cell pluripotency exit in different cell-based models, including embryonic stem-to-mesenchymal transition, spontaneous differentiation and 3D gastruloid development, and at lower doses compared to budesonide.

Background: Multisystem Inflammatory Syndrome in Children (MIS-C) is a severe complication of SARS-CoV-2 infection caused by hyperactivation of the immune system. Methods: this is a retrospective analysis of clinical data, biochemical parameters, and immune cell subsets in 40 MIS-C patients from hospital admission to outpatient long-term follow-up. Results: MIS-C patients had elevated inflammatory markers, associated with T- and NK-cell lymphopenia, a profound depletion of dendritic cells, and altered monocyte phenotype at disease onset, while the subacute phase of the disease was characterized by a significant increase in T- and B-cell counts and a rapid decline in activated T cells and terminally differentiated B cells. Most of the immunological parameters returned to values close to the normal range during the remission phase (20–60 days after hospital admission). Nevertheless, we observed a significantly reduced ratio between recently generated and more differentiated CD8+ T- and B-cell subsets, which partially settled at longer-term follow-up determinations. Conclusions: The characterization of lymphocyte distribution in different phases of MIS-C may help to understand the course of diseases that are associated with dysregulated immune responses and to calibrate prompt and targeted treatments.

In recent decades, cardiac computed tomography (CT) has emerged as a powerful non-invasive tool for risk stratification, as well as the detection and characterization of coronary artery disease (CAD), which remains the main cause of morbidity and mortality in the world. Advances in technology have favored the increasing use of cardiac CT by allowing better performance with lower radiation doses. Coronary artery calcium, as assessed by non-contrast CT, is considered to be the best marker of subclinical atherosclerosis, and its use is recommended for the refinement of risk assessment in low-to-intermediate risk individuals. In addition, coronary CT angiography (CCTA) has become a gate-keeper to invasive coronary angiography (ICA) and revascularization in patients with acute chest pain by allowing the assessment not only of the extent of lumen stenosis, but also of its hemodynamic significance if combined with the measurement of fractional flow reserve or perfusion imaging. Moreover, CCTA provides a unique incremental value over functional testing and ICA by imaging the vessel wall, thus allowing the assessment of plaque burden, composition, and instability features, in addition to perivascular adipose tissue attenuation, which is a marker of vascular inflammation. There exists the potential to identify the non-obstructive lesions at high risk of progression to plaque rupture by combining all of these measures.

Background: Human subcutaneous dirofilariasis caused by Dirofilaria repens is a vector-borne zoonotic disease mostly transmitted from dogs to humans through a mosquito’s blood meal. Heartwarms replication is amplified by the climate change, the increase of the range of suitable vectors, the facilitation of pet travel and the high rate of undiagnosed dirofilariasis in dogs. Observation: We describe a case of a young Romanian woman, resident in Rome for 18 years, that came to our attention for the appearance for five months of a subcutaneous nodule in the left arm. The patient reported that she first felt an insect bite, after which she noticed the onset of an erythematous and itchy wheal in the same skin area, turned into a subcutaneous nodule within a few weeks. The ultrasound examination showed a hypoechoic subcutaneous formation of 1,2 cm in diameter, containing a ribbon-like structure made up of hyperechoic parallel double lines, reminiscent of a warm. Based on this suspicion, we opted for the surgical radicalization of the lesion. The histological examination confirmed the radiological hypothesis of a warm-like foreign body morphologically compatible with Dirofilaria repens. Key message: Our experience shows how a clinical nonspecific skin nodular lesion may conceal an unexpected and unsettling diagnosis of subcutaneous Dirofilaria repens.

ObjectiveAim of this paper is to illustrate the methodology, design, and development of the AutoInflammatory Disease Alliance (AIDA) International Registry dedicated to patients with the Periodic Fever, Aphthous stomatitis, Pharyngitis, and cervical Adenitis (PFAPA) syndrome.MethodsThis is a physician-driven, non-population- and electronic-based registry proposed to gather real-world demographics, clinical, laboratory, instrumental and socioeconomic data from PFAPA patients. Data recruitment is realized through the on-line Research Electronic Data Capture (REDCap) tool. This registry is thought to collect standardized information for clinical research leading to solid real-life evidence. The international scope and the flexibility of the registry will facilitate the realization of cutting-edge study projects through the constant updating of variables and the possible merging and transfer of data between current and future PFAPA registries.ResultsA total of 112 centers have already been involved from 23 countries and 4 continents starting from August 24th, 2021, to April 6th, 2022. In total 56/112 have already obtained the formal approval from their local Ethics Committees. The platform counts 321 users (113 principal investigators, 203 site investigators, two lead investigators, and three data managers). The registry collects retrospective and prospective data using 3,856 fields organized into 25 instruments, including PFAPA patient's demographics, medical histories, symptoms, triggers/risk factors, therapies, and impact on the healthcare systems.ConclusionsThe development of the AIDA International Registry for PFAPA patients will enable the on-line collection of standardized data prompting real-life studies through the connection of worldwide groups of physicians and researchers. This project can be found on https://clinicaltrials.gov NCT 05200715.

ObjectiveThe aim of this paper is to present the AutoInflammatory Disease Alliance (AIDA) international Registry dedicated to Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic (VEXAS) syndrome, describing its design, construction, and modalities of dissemination.MethodsThis Registry is a clinical, physician-driven, population- and electronic-based instrument designed for the retrospective and prospective collection of real-life data. Data gathering is based on the Research Electronic Data Capture (REDCap) tool and is intended to obtain real-world evidence for daily patients' management. The Registry may potentially communicate with other on-line tools dedicated to VEXAS syndrome, thus enhancing international collaboration and data sharing for research purposes. The Registry is practical enough to be easily modified to meet future needs regarding VEXAS syndrome.ResultsTo date (April 22nd, 2022), 113 Centers from 23 Countries in 4 continents have been involved; 324 users (114 Principal Investigators, 205 Site Investigators, 2 Lead Investigators, and 3 data managers) are currently able to access the registry for data entry (or data sharing) and collection. The Registry includes 4,952 fields organized into 18 instruments designed to fully describe patient's details about demographics, clinical manifestations, symptoms, histologic details about skin and bone marrow biopsies and aspirate, laboratory features, complications, comorbidities, therapies, and healthcare access.ConclusionThis international Registry for patients with VEXAS syndrome will allow the achievement of a comprehensive knowledge about this new disease, with the final goal to obtain real-world evidence for daily clinical practice, especially in relation to the comprehension of this disease about the natural history and the possible therapeutic approaches. This Project can be found on https://clinicaltrials.gov NCT05200715.

Polo-like kinase 1 (PLK1) is the principle member of the well conserved serine/threonine kinase family. PLK1 has a key role in the progression of mitosis and recent evidence suggest its important involvement in regulating the G2/M checkpoint, in DNA damage and replication stress response, and in cell death pathways. PLK1 expression is tightly spatially and temporally regulated to ensure its nuclear activation at the late S-phase, until the peak of expression at the G2/M-phase. Recently, new roles of PLK1 have been reported in literature on its implication in the regulation of inflammation and immunological responses. All these biological processes are altered in tumors and, considering that PLK1 is often found overexpressed in several tumor types, its targeting has emerged as a promising anti-cancer therapeutic strategy. In this review, we will summarize the evidence suggesting the role of PLK1 in response to DNA damage, including DNA repair, cell cycle progression, epithelial to mesenchymal transition, cell death pathways and cancer-related immunity. An update of PLK1 inhibitors currently investigated in preclinical and clinical studies, in monotherapy and in combination with existing chemotherapeutic drugs and targeted therapies will be discussed.

ObjectiveThis paper points out the design, development and deployment of the AutoInflammatory Disease Alliance (AIDA) International Registry dedicated to pediatric and adult patients affected by Undifferentiated Systemic AutoInflammatory Diseases (USAIDs).MethodsThis is an electronic registry employed for real-world data collection about demographics, clinical, laboratory, instrumental and socioeconomic data of USAIDs patients. Data recruitment, based on the Research Electronic Data Capture (REDCap) tool, is designed to obtain standardized information for real-life research. The instrument is endowed with flexibility, and it could change over time according to the scientific acquisitions and potentially communicate with other similar tools; this platform ensures security, data quality and data governance.ResultsThe focus of the AIDA project is connecting physicians and researchers from all over the world to shed a new light on heterogeneous rare diseases. Since its birth, 110 centers from 23 countries and 4 continents have joined the AIDA project. Fifty-four centers have already obtained the approval from their local Ethics Committees. Currently, the platform counts 290 users (111 Principal Investigators, 179 Site Investigators, 2 Lead Investigators, and 2 data managers). The Registry is collecting baseline and follow-up data using 3,769 fields organized into 23 instruments, which include demographics, history, symptoms, trigger/risk factors, therapies, and healthcare information access for USAIDs patients.ConclusionsThe development of the AIDA International Registry for USAIDs patients will facilitate the online collection of real standardized data, connecting a worldwide group of researchers: the Registry constitutes an international multicentre observational groundwork aimed at increasing the patient cohort of USAIDs in order to improve our knowledge of this peculiar cluster of autoinflammatory diseases. NCT 05200715 available at https://clinicaltrials.gov/.

Background: Ovarian carcinoma is extremely sensitive to (platinum-based) chemotherapy; however, most patients will relapse with platinum-resistant disease, badly affecting their prognosis. Effective therapies for relapsing resistant tumors are urgently needed. Methods: We used patient-derived xenografts (PDXs) of ovarian carcinoma resistant to cisplatin (DDP) to test in vivo the combination of paclitaxel (15 mg/kg i.v. once a week for 3 weeks) and onvansertib, a plk1 inhibitor, (50 mg/kg orally 4 days a week for 3 weeks). The PDX models were subcutaneously (s.c.) or orthotopically transplanted in nude mice and antitumor efficacy was evaluated as tumor growth inhibition and survival advantages of the combination over untreated and single agent treatment. Results: The combination of onvansertib and paclitaxel was very well tolerated with weight loss no greater than 15% in the combination group compared with the control group. In the orthotopically transplanted PDXs, single onvansertib and paclitaxel treatments prolonged survival; however, the combined treatment was much more active, with median survival from three- to six-fold times that of untreated mice. Findings were similar with the s.c. transplanted PDX, though there was greater heterogeneity in tumor response. Ex vivo tumors treated with the combination showed greater induction of γH2AX, marker of apoptosis and DNA damage, and pSer10H3, a marker of mitotic block. Conclusion: The efficacy of onvansertib and paclitaxel combination in these preclinical ovarian cancer models supports the clinical translatability of this combination as an effective therapeutic approach for platinum-resistant high-grade ovarian carcinoma.

Abstract Background An ascending aortic pseudoaneurysm is a severe and rare complication following cardiothoracic surgery. This case report demonstrates its possible misinterpretation and the consequent importance of multidisciplinary evaluation. Case presentation We present a case of an 18-year-old Caucasian man with Marfan syndrome who developed an ascending aortic pseudoaneurysm about 1 year after undergoing cardiac surgery with the Bentall procedure. Computed tomographic examination of the thoracic aorta and positron emission tomography–computed tomography initially suggested a lymphomatous pathology. However, these imaging results were in contrast to the transesophageal echocardiogram and the laboratory data that showed negative results for hematological pathology. A second computed tomographic scan redirected the diagnosis toward a pseudoaneurysm. Conclusion This case demonstrates the utility of close communication and interdisciplinary consultation between cardiovascular radiologists and the cardiac surgery team, which are mandatory in order to maximize their diagnostic skills in identifying postoperative complications.

Abstract Background According to published data, radiomics features differ between lesions of refractory/relapsing HL patients from those of long-term responders. However, several methodological aspects have not been elucidated yet. Purpose The study aimed at setting up a methodological framework in radiomics applications in Hodgkin’s lymphoma (HL), especially at (a) developing a novel feature selection approach, (b) evaluating radiomic intra-patient lesions’ similarity, and (c) classifying relapsing refractory (R/R) vs non-(R/R) patients. Methods We retrospectively included 85 patients (male:female = 52:33; median age 35 years, range 19–74). LIFEx ( www.lifexsoft.org ) was used for [18F]FDG-PET/CT segmentation and feature extraction. Features were a-priori selected if they were highly correlated or uncorrelated to the volume. Principal component analysis-transformed features were used to build the fingerprints that were tested to assess lesions’ similarity, using the silhouette. For intra-patient similarity analysis, we used patients having multiple lesions only. To classify patients as non-R/R and R/R, the fingerprint considering one single lesion (fingerprint_One) and all lesions (fingerprint_All) was tested using Random Undersampling Boosting of Tree Ensemble (RUBTE). Results HL fingerprints included up to 15 features. Intra-patient lesion similarity analysis resulted in mean/median silhouette values below 0.5 (low similarity especially in the non-R/R group). In the test set, the fingerprint_One classification accuracy was 62% (78% sensitivity and 53% specificity); the classification by RUBTE using fingerprint_All resulted in 82% accuracy (70% sensitivity and 88% specificity). Conclusions Lesion similarity analysis was developed, and it allowed to demonstrate that HL lesions were not homogeneous within patients in terms of radiomics signature. Therefore, a random target lesion selection should not be adopted for radiomics applications. Moreover, the classifier to predict R/R vs non-R/R performed the best when all the lesions were used.

Abstract: The complete remission (CR) rate achieved with induction chemotherapy prior to autologous stem cell transplantation (ASCT) represents the strongest prognostic factor in relapsed/refractory (R/R) classical Hodgkin lymphoma (cHL). By inducing a CR rate of 75%, the bendamustine, gemcitabine, vinorelbine (BEGEV) regimen represents an optimal chemotherapy regimen prior to ASCT. Presented here are the 5-year results of BEGEV followed by ASCT in R/R cHL. With a median follow-up of 5 years, progression-free survival (PFS) and overall survival (OS) for the whole series (n = 59) were 59% and 78%, respectively. ASCT was performed in 43 of 49 responding patients (73% by intention to treat [ITT]; 88% by response to BEGEV) and resulted in 33 with continuous CR (56% by ITT; 77% of transplanted patients), 7 with disease relapse, and 3 with nonrelapse mortality. For patients who received transplants, the 5-year PFS and OS were 77% and 91%, respectively, with no significant difference between relapsed and refractory patients. No patient experienced secondary leukemia or myelodysplasia. In summary, the long-term efficacy data, the benefits for both relapsed and refractory patients, and the excellent safety profile provide a strong rationale for further development of the BEGEV regimen. This trial was registered at EudraCT as #2010-022169-91 and at www.clinicaltrials.gov as #NCT01884441.

Pulmonary surfactant (PS) has been proposed as an efficient drug delivery vehicle for inhaled therapies. Its ability to adsorb and spread interfacially and transport different drugs associated with it has been studied mainly by different surface balance designs, typically interconnecting various compartments by interfacial paper bridges, mimicking in vitro the respiratory air–liquid interface. It has been demonstrated that only a monomolecular surface layer of PS/drug is able to cross this bridge. However, surfactant films are typically organized as multi-layered structures associated with the interface. The aim of this work was to explore the contribution of surface-associated structures to the spreading of PS and the transport of drugs. We have designed a novel vehiculization balance in which donor and recipient compartments are connected by a whole three-dimensional layer of liquid and not only by an interfacial bridge. By combining different surfactant formulations and liposomes with a fluorescent lipid dye and a model hydrophobic drug, budesonide (BUD), we observed that the use of the bridge significantly reduced the transfer of lipids and drug through the air–liquid interface in comparison to what can be spread through a fully open interfacial liquid layer. We conclude that three-dimensional structures connected to the surfactant interfacial film can provide an important additional contribution to interfacial delivery, as they are able to transport significant amounts of lipids and drugs during surfactant spreading.

Abstract Recently, treatment of immune‐mediated thrombotic thrombocytopenic purpura (ITTP) has changed with the advent of caplacizumab in clinical practice. The International Working Group (IWG) has recently integrated the ADAMTS‐13 activity/autoantibody monitoring in consensus outcome definitions. We report three ITTP cases during the coronavirus disease 2019 pandemic, that received a systematic evaluation of ADAMTS‐13 activity and autoantibodies. We describe how the introduction of caplacizumab and ADAMTS‐13 monitoring could change the management of ITTP patients and discuss whether therapeutic choices should be based on the clinical response alone. ADAMTS‐13 activity/antibodies were assessed every 5 days. Responses were evaluated according to updated IWG outcome definitions. These kinetics, rather than clinical remission, guided the therapy, allowing early and safe caplacizumab discontinuation and sensible administration of rituximab. Caplacizumab was cautiously discontinued after achieving ADAMTS‐13 complete remission. These cases illustrate that prospective ADAMTS‐13 evaluation and use of updated IWG definitions may improve real‐life patients’ management in the caplacizumab era.

Polycythemia Vera (PV) is a myeloproliferative neoplasm with increased risk of thrombosis and progression to myelofibrosis. Chronic inflammation is commonly observed in myeloproliferative neoplasms including PV. The inflammatory network includes the extracellular vesicles (EVs), which play a role in cell-cell communication. Recent evidence points to circulating microbial components/microbes as potential players in hemopoiesis regulation. To address the role of EVs in PV, here we investigated phenotype and microbial DNA cargo of circulating EVs through multidimensional analysis. Peripheral blood and feces were collected from PV patients (n=38) and healthy donors (n=30). Circulating megakaryocyte (MK)- and platelet (PLT)-derived EVs were analyzed by flow cytometry. After microbial DNA extraction from feces and isolated EVs, the 16S rDNA V3-V4 region was sequenced. We found that the proportion of circulating MK-derived EVs was significantly decreased in PV patients as compared with the healthy donors. By contrast, the proportion of the PLT-derived EVs was increased. Interestingly, PV was also associated with a microbial DNA signature of the isolated EVs with higher diversity and distinct microbial composition than the healthy counterparts. Of note, increased proportion of isolated lipopolysaccharide-associated EVs has been demonstrated in PV patients. Conversely, the gut microbiome profile failed to identify a distinct layout between PV patients and healthy donors. In conclusion, PV is associated with circulating EVs harbouring abnormal phenotype and dysbiosis signature with a potential role in the (inflammatory) pathogenesis of the disease.

After 15 years from its advent in the clinical field, coronary computed tomography (CCTA) is now widely considered as the best first-step test in patients with low-to-moderate pre-test probability of coronary artery disease. Technological innovation was of pivotal importance for the extensive clinical and scientific interest in CCTA. Recently, the advent of last generation wide-coverage CT scans paved the way for new clinical applications of this technique beyond coronary arteries anatomy evaluation. More precisely, both biventricular volume and systolic function quantification and myocardial fibrosis identification appeared to be feasible with last generation CT. In the present review we would focus on potential applications of cardiac computed tomography (CCT), beyond CCTA, for a comprehensive assessment patients with newly diagnosed cardiomyopathy, from technical requirements to novel clinical applications.

Background: Assessments of airways inflammation in patients with chronic obstructive pulmonary disease (COPD) require semi-invasive procedures and specialized sample processing know-how. In this study we aimed to set up and validate a novel non-invasive processing-free method for RNA sequencing (RNAseq) of spontaneous sputum samples collected from COPD patients.Methods: Spontaneous sputum samples were collected and stabilized, with or without selection of plugs and with or without the use of a stabilizer specifically formulated for downstream diagnostic testing (PrimeStore® Molecular Transport Medium). After 8 days storage at ambient temperature RNA was isolated according to an optimized RNAzol® method. An average percentage of fragments longer than 200 nucleotides (DV200) >30% and an individual yield >50 ng were required for progression of samples to sequencing. Finally, to assess if the transcriptome generated would reflect a true endotype of COPD inflammation, the outcome of single-sample gene-set enrichment analysis (ssGSEA) was validated using an independent set of processed induced sputum samples. Results: RNA extracted from spontaneous sputum using a stabilizer showed an average DV200 higher than 30%. 70% of the samples had a yield >50 ng and were submitted to downstream analysis. There was a straightforward correlation in terms of gene expression between samples handled with or without separation of plugs. This was also confirmed by principal component analysis and ssGSEA. The top ten enriched pathways resulting from spontaneous sputum ssGSEA were associated to features of COPD, namely, inflammation, immune responses and oxidative stress; up to 70% of these were in common within the top ten enriched pathways resulting from induced sputum ssGSEA.Conclusion: This analysis confirmed that the typical COPD endotype was represented within spontaneous sputum and supported the current method as a non-invasive processing-free procedure to assess the level of sputum cell inflammation in COPD patients by RNAseq analysis.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease, including one-third of cases overexpressing MYC and BCL2 proteins (double expressor lymphoma, DEL) and 5-10% of patients with chromosomal rearrangements of MYC, BCL2 and/or BCL-6 (double/triple-hit lymphomas, DH/TH). TP53 mutations are detected in 20- 25% of DEL. We report the efficacy of dose-adjusted EPOCH and rituximab (DA-EPOCH-R) in a series of 122 consecutive patients, including DEL (n=81, 66%), DEL-MYC (n=9, 7%), DEL-BCL2 (n=13, 11%), or high-grade lymphomas (DH/TH) (n=19, 16%). Central nervous system (CNS) prophylaxis included intravenous methotrexate (n=66), intrathecal chemotherapy (IT) (n=40) or no prophylaxis (n=16). Sixty-seven patients (55%) had highintermediate or high International Prognostic Index (IPI) and 30 (25%) had high CNS-IPI. The 2-year progression-free survival (PFS) and overall survival (OS) for the entire study population were 74% and 84%, respectively. There was a trend for inferior OS for DH/TH (2-year OS: 66%, P=0.058) as compared to all the others. The outcome was significantly better for the IPI 0-2 versus IPI 3-5 (OS: 98% vs. 72%, P=0.002). DA-EPOCH-R did not overcome the negative prognostic value of TP53 mutations: 2-year OS of 62% versus 88% (P=0.036) were observed for mutated as compared to wild-type cases, respectively. Systemic CNS prophylaxis conferred a better 2-year OS (94%) as compared to IT or no prophylaxis (76% and 65%, respectively; P=0.008). DA-EPOCH-R treatment resulted in a favorable outcome in patients with DEL and DEL with single rearrangement, whereas those with multiple genetic alterations such as DEL-DH/TH and TP53 mutated cases still have an inferior outcome.

The rate of complete remission (CR) with the anti-PD1 immune checkpoint inhibitors (ICI) nivolumab (N) and pembrolizumab (P) in patients with relapsed/refractory (R/R) classical Hodgkin lymphoma (cHL) is low (20-30%), and the majority of patients eventually relapse. One strategy to improve their outcome is to combine ICI with radiotherapy (ICI-RT), taking advantage of a supposed synergistic effect. We retrospectively collected data of 12 adult patients with R/R cHL treated with ICI-RT delivered during or within 8 weeks from the start or after the end of ICI. Median age at ICI-RT was 37 years, 50% had previously received an autologous stem cell transplantation (SCT) and 92% brentuximab vedotin. RT was given concurrently, before or after ICI in 4, 1 and 7 patients. Median RT dose was 30Gy, for a median duration of 22 days. Median number of ICI administrations was 15. Overall response and CR rate were 100% and 58%. Nine patients received subsequent SCT consolidation (7 allogeneic and 2 autologous). After a median follow-up of 18 months, 92% of patients were in CR. No major concerns about safety were reported. ICI-RT combination appears to be a feasible and highly active bridge treatment to transplant consolidation.

Abstract Background The amount of surfactant deposited in the lungs and its overall pulmonary distribution determine the therapeutic outcome of surfactant replacement therapy. Most of the currently available methods to determine the intrapulmonary distribution of surfactant are time-consuming and require surfactant labelling. Our aim was to assess the potential of Mass Spectrometry Imaging (MSI) as a label-free technique to qualitatively and quantitatively evaluate the distribution of surfactant to the premature lamb. Methods Twelve preterm lambs (gestational age 126-127d, term ~150d) were allocated in two experimental groups. Seven lambs were treated with an intratracheal bolus of the synthetic surfactant CHF5633 (200 mg/kg) and 5 lambs were managed with mechanical ventilation for 120 min, as controls. The right lung lobes of all lambs were gradually frozen while inflated to 20 cmH2O pressure for lung cryo-sections for MSI analysis. The intensity signals of SP-C analog and SP-B analog, the two synthetic peptides contained in the CHF5633 surfactant, were used to locate, map and quantify the intrapulmonary exogenous surfactant. Results Surfactant treatment was associated with a significant improvement of the mean arterial oxygenation and lung compliance (p

Abstract Background Respiratory Distress Syndrome (RDS) is a prematurity-related breathing disorder caused by a quantitative deficiency of pulmonary surfactant. Surfactant replacement therapy is effective for RDS newborns, although treatment failure has been reported. The aim of this study is to trace exogenous surfactant by 13C variation and estimate the amount reaching the lungs at different doses of the drug. Methods Forty-four surfactant-depleted rabbits were obtained by serial bronchoalveolar lavages (BALs), that were merged into a pool (BAL pool) for each animal. Rabbits were in nasal continuous positive airway pressure and treated with 0, 25, 50, 100 or 200 mg/kg of poractant alfa by InSurE. After 90 min, rabbits were depleted again and a new pool (BAL end experiment) was collected. Disaturated-phosphatidylcholine (DSPC) was measured by gas chromatography. DSPC-Palmitic acid (PA) 13C/12C was analyzed by isotope ratio mass spectrometry. One-way non-parametric ANOVA and post-hoc Dunn’s multiple comparison were used to assess differences among experimental groups. Results Based on DSPC-PA 13C/12C in BAL pool and BAL end experiment, the estimated amount of exogenous surfactant ranged from 61 to 87% in dose-dependent way (p

Vaccination coverage against COVID-19 among health care workers (HCWs) of the University Health Agency Giuliano-Isontina (ASUGI) of Trieste (North-eastern Italy) by 1 January 2022 was 90.4% with at least one vaccine dose, 84.9% with at least 2 doses, and 75.1% with 3 doses, 98.2% with Comirnaty (Pfizer BioNtech, New York, NY, USA) versus 1.8% with Spikevax (Moderna, Cambridge, MA, USA). From 1 October 2020 to 7 February 2022, 1652 SARS-CoV-2 infections were notified in HCWs of ASUGI Trieste. Although the overall risk of SARS-CoV-2 contagion increased over time, the rate of occupational infections progressively declined, from 42.5% during the second COVID-19 wave to 15.6% in the fifth. Between 1 January–7 February 2022 (a period dominated by the Omicron variant), albeit no COVID-19-associated hospitalizations were recorded in HCWs of ASUGI Trieste, 669 SARS-CoV-2 infections were counted against 367 cases observed from 1 October to 31 December 2020, the 3 months preceding the implementation of the vaccination campaign against COVID-19. Job tasks and health care settings turned out to be the most significant risk factors for SARS-CoV-2 infection. However, the effect of workplace prevailed over job task on the biological risk, with greater rates of SARS-CoV-2 infections observed among HCWs operating in areas with higher levels of circulation of the virus, particularly COVID-19 dedicated units.

High-flow nasal cannula (HFNC) is a non-invasive respiratory support (NRS) modality to treat premature infants with respiratory distress syndrome (RDS). The delivery of nebulized surfactant during NRS would represent a truly non-invasive method of surfactant administration and could reduce NRS failure rates. However, the delivery efficiency of nebulized surfactant during HFNC has not been evaluated in vitro or in animal models of respiratory distress. We, therefore, performed first a benchmark study to compare the surfactant lung dose delivered by commercially available neonatal nasal cannulas (NCs) and HFNC circuits commonly used in neonatal intensive care units. Then, the pulmonary effect of nebulized surfactant delivered via HFNC was investigated in spontaneously breathing rabbits with induced respiratory distress. The benchmark study revealed the surfactant lung dose to be relatively low for both types of NCs tested (Westmed NCs 0.5 ± 0.45%; Fisher & Paykel NCs 1.8 ± 1.9% of a nominal dose of 200 mg/kg of Poractant alfa). The modest lung doses achieved in the benchmark study are compatible with the lack of the effect of nebulized surfactant in vivo (400 mg/kg), where arterial oxygenation and lung mechanics did not improve and were significantly worse than the intratracheal instillation of surfactant. The results from the present study indicate a relatively low lung surfactant dose and negligible effect on pulmonary function in terms of arterial oxygenation and lung mechanics. This negligible effect can, for the greater part, be explained by the high impaction of aerosol particles in the ventilation circuit and upper airways due to the high air flows used during HFNC.

Myelofibrosis (MF) is characterized by chronic inflammation and hyper-activation of the JAK-STAT pathway. Infections are one of the main causes of morbidity/mortality. Therapy with Ruxolitinib (RUX), a JAK1/2 inhibitor, may further increase the infectious risk. Monocytes are critical players in inflammation/immunity through cytokine production and release of bioactive extracellular vesicles. However, the functional behavior of MF monocytes, particularly during RUX therapy, is still unclear. In this study, we found that monocytes from JAK2V617F-mutated MF patients show an altered expression of chemokine (CCR2, CXCR3, CCR5) and cytokine (TNF-α-R, IL10-R, IL1β-R, IL6-R) receptors. Furthermore, their ability to produce and secrete free and extracellular vesicles-linked cytokines (IL1β, TNF-α, IL6, IL10) under lipopolysaccharides (LPS) stimulation is severely impaired. Interestingly, monocytes from RUX-treated patients show normal level of chemokine, IL10, IL1β, and IL6 receptors together with a restored ability to produce intracellular and to secrete extracellular vesicles-linked cytokines after LPS stimulation. Conversely, RUX therapy does not normalize TNF-R1/2 receptors expression and the LPS-driven secretion of free pro/anti-inflammatory cytokines. Accordingly, upon LPS stimulation, in vitro RUX treatment of monocytes from MF patients increases their secretion of extracellular vesicles-linked cytokines but inhibits the secretion of free pro/anti-inflammatory cytokines. In conclusion, we demonstrated that in MF the infection-driven response of circulating monocytes is defective. Importantly, RUX promotes their infection-driven cytokine production suggesting that infections following RUX therapy may not be due to monocyte failure. These findings contribute to better interpreting the immune vulnerability of MF and to envisaging strategies to improve the infection-driven immune response.

It is unknown if the lung deposition of surfactant administered via a catheter placed through a laryngeal mask airway (LMA) is equivalent to that obtained by bolus instillation through an endotracheal tube. We compare the lung deposition of surfactant delivered via two types of LMA with the standard technique of endotracheal instillation. 25 newborn piglets on continuous positive airway pressure support (CPAP) were randomized into three groups: 1—LMA-camera (integrated camera and catheter channel; catheter tip below vocal cords), 2—LMA-standard (no camera, no channel; catheter tip above the glottis), 3—InSurE (Intubation, Surfactant administration, Extubation; catheter tip below end of endotracheal tube). All animals received 100 mg·kg−1 of poractant alfa mixed with 99mTechnetium-nanocolloid. Surfactant deposition was measured by gamma scintigraphy as a percentage of the administered dose. The median (range) total lung surfactant deposition was 68% (10–85), 41% (5–88), and 88% (67–92) in LMA-camera, LMA-standard, and InSurE, respectively, which was higher (p < 0.05) in the latter. The deposition in the stomach and nasopharynx was higher with the LMA-standard. The surfactant deposition via an LMA was lower than that obtained with InSurE. Although not statistically significant, introducing the catheter below the vocal cords under visual control with an integrated camera improved surfactant LMA delivery by 65%.

Merkel cell carcinoma (MCC) is a rare and extremely aggressive neuroendocrine carcinoma of the skin, with increasing incidence worldwide. This review intends to propose a comprehensive evaluation of MCC epidemiology, clinical features, pathogenetic mechanisms, diagnosis, and therapies. A section is dedicated to immunological aspects and another to the involvement of angiogenesis and angiogenic growth factors in MCC progression, proposing novel diagnostic and therapeutic approaches. Advanced MCC tumors have been treated with immune checkpoint inhibitors with effective results. Therefore, the state of art of this immunotherapy is also examined, reporting on the most recent clinical trials in the field. We conclude by underlining the achievements in the understanding of MCC pathology and indicating the present needs for effective diagnosis and therapeutic management of the disease.

Abstract Background The existence of cancer stem cells (CSCs) within a tumor bulk has been demonstrated for many solid tumors including epithelial ovarian carcinoma (EOC). CSCs have been associated to tumor invasion, metastasis and development of chemoresistant recurrences. In this context, we aim to characterize EOC CSCs from the molecular point of view in order to identify potential biomarkers associated with chemoresistance. Methods We isolated a population of cells with stem-like characteristics (OVA-BS4 spheroids) from a primary human EOC cell line under selective conditions. OVA-BS4 spheroids were characterized for drug response by cytotoxicity assays and their molecular profile was investigated by microarray and RT-qPCR. Finally, we performed a gene expression study in a cohort of 74 high-grade serous EOC (HGSOC) patients by RT-qPCR. Results Spheroids exhibited properties of self-renewal and a pronounced expression of well-known stem cell genes. Moreover, they demonstrated greater resistance towards several anticancer drugs compared to parent cell line, consistent with their higher ABCG2 gene expression. From microarray studies MAL (T-cell differentiation protein) emerged as the most up-regulated gene in spheroids, compared to parent cell line. In HGSOC patients, MAL was significantly overexpressed in platinum-resistant compared to platinum-sensitive patients and resulted as an independent prognostic marker of survival. Conclusions This investigation provides an important contribution to the identification of molecular markers of ovarian CSCs and chemoresistance. Successful translation of molecular findings would lead to a better comprehension of the mechanisms triggering chemoresistant recurrences, to the individuation of novel therapeutic targets and to the personalization of treatment regimens.

Abstract Advanced ovarian cancer is very responsive to first line platinum therapy, however almost invariably it relapses with a resistant disease. We have reported that patient derived ovarian xenografts (PDXs), independently from the degree of the initial response to cisplatin (DDP), show a significantly lower response to a second DDP cycle. We here report the effect of new combination regimens containing a MEK inhibitor (MEK), bevacizumab (BEV) and paclitaxel (PTX) as second line therapy in platinum-relapsing PDXs. We selected three DDP-relapsing PDX models based on the presence of activation of the RAS/RAF/MEK/ERK axis, mutated p53, lack of PTEN expression and activation of the PI3K pathway. In all the selected xenograft models, the antitumor efficacy of the doublets can be summarized as PTX/BEV > BEV/MEK > PTX/MEK and the antitumor activity of the triple combination was higher than any double combination. All the different combinations were well tolerated. The present data corroborate the activity of bevacizumab in combination with chemotherapy for the treatment of relapsing ovarian tumors and suggest that the addition of another targeted agents (MEK inhibitor) can further increase the antitumor activity without any increase in toxicity. PDX models represent a useful model to test second line therapy after failure of DDP first line.