CAR+ extracellular vesicles predict ICANS in patients with B cell lymphomas treated with CD19-directed CAR T cells.

BACKGROUND: Predicting Immune-effector Cell Associated Neurotoxicity Syndrome (ICANS) in patients infused with Chimeric Antigen Receptor T cells (CAR-T) is still a conundrum. This complication, thought to be consequent to CAR-T cell activation, arises a few days after infusion, when circulating CAR-T cells are scarce and specific CAR-T cell-derived biomarkers are lacking. METHODS: Human CD19.CAR-T cells were generated to gain insight into CAR+ extracellular vesicle (CAR+EV) release upon target engagement. A prospective cohort of 100 B-cell lymphoma patients infused with approved CD19.CAR-T cell products (axi-cel, brexu-cel and tisa-cel) was assessed for plasma CAR+EVs as potential biomarkers of in vivo CD19.CAR-T cell activation and predictors of ICANS. Human induced pluripotent stem cells (iPSCs)-derived neural cells were used as a model for CAR+EV-induced neurotoxicity. RESULTS: In vitro, exosome-like CAR+EVs were released by CD19.CAR-T cells upon target engagement. In vivo, CAR+EVs were detectable as early as 1 hour in the plasma of patients. A concentration > 132.8 CAR+EVs/µl at hour +1 or > 224.5 CAR+EVs/µl at day +1 predicted ICANS in advance of 4 days, with a sensitivity up to 96.55% and a specificity up to 80.36%, outperforming other potential ICANS predictors. Enolase 2 (ENO2+) nanoparticles were released by iPSCs-derived neural cells upon CAR+EVs exposure and were increased in the plasma of ICANS patients. CONCLUSIONS: These results convey that plasma CAR+EVs are an immediate signal of CD19.CAR-T cell activation, are suitable predictors of neurotoxicity, and may be involved in ICANS pathogenesis. TRIAL REGISTRATION: NCT04892433, NCT05807789.

Outcome of High-Dose Chemotherapy Followed by Autologous Stem Cell Transplantation in Relapsed/Refractory Hodgkin Lymphoma after Different Numbers of Salvage Regimens.

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.

Early anakinra treatment improves cardiac outcome of multisystem inflammatory syndrome in children, regardless of disease severity.

OBJECTIVE: The main aim of this study was to define the best treatment option for multisystem inflammatory syndrome in children (MIS-C) and to analyse the role of anakinra. METHODS: This is a multicentre retrospective cohort study. Patients were treated according to the attending physician's decision. The patients were divided into four groups on the basis of the first treatment at time of admittance: (i) IVIG, (ii) IVIG and methylprednisolone (≤2 mg/kg/day), (iii) IVIG with high-dose methylprednisolone (>2 mg/kg/day) and (iv) anakinra with or without IVIG and/or methylprednisolone. Primary outcomes were defined as the presence of at least one of the following features: death, the failure of initial treatment, meaning the need for additional treatment for clinical worsening and cardiac involvement at the end of follow-up. RESULTS: Two hundred thirty-nine patients were recruited. At univariate analysis, persistent heart involvement at discharge was more frequent in those not receiving anakinra as initial treatment (3/21 vs 66/189; P = 0.047). After comparisons between the four treatment regimens, adjusting for the propensity score, we observed that early treatment with anakinra was associated with a lower probability of developing persistent heart disease at the end of follow-up (odds ratio: 0.6; 95% CI: 0.4-1.0). CONCLUSION: We report that early treatment with anakinra is safe and very effective in patients with severe MIS-C. In addition, our study suggests that early treatment with anakinra is the most favourable option for patients with a higher risk of developing a severe disease outcome.

Detection of Cleaved Stx2a in the Blood of STEC-Infected Patients.

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.

Bilateral adrenal hemorrhage: learning notes from clinical practice and literature review.

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.

Unfolding dermatologic spectrum of Behçet's disease in Italy: real-life data from the International AIDA Network Behçet's disease Registry.

Behçet's disease (BD) is a heterogeneous multifactorial autoinflammatory disease characterized by a plethora of clinical manifestations. Cutaneous lesions are considered hallmarks of the disease. However, their evolution over time and a thorough description are scarcely reported in non-endemic regions. The aim of this study was to detail BD skin manifestations and their evolution over time in Italy, as well as the dermatological prognostic impact of specific cutaneous features in long-standing disease. Data were collected in a double fashion, both retrospectively and prospectively, from the AutoInflammatory Disease Alliance (AIDA) international registry dedicated to BD, between January 2022 and December 2022. A total of 458 Italian patients were included. When assessing skin manifestations course, the constant or sporadic presence or absence of cutaneous involvement between onset and follow-up was considered. Oral ulcers (OU) (88.4%) and genital ulcers (GU) (52.6%), followed by skin involvement (53.7%) represented the most common presenting mucocutaneous manifestations at disease onset. Up to the time of enrolment into the AIDA registry, 411 (93.8%) patients had suffered from OU and 252 (57.9%) from GU; pseudofolliculitis (PF) accounted for the most common skin manifestation (170 patients, 37.1%), followed by erythema nodosum (EN) (102 patients, 22.3%), skin ulcers (9 patients, 2%) and pyoderma gangrenosum (4 patients, 0.9%). A prospective follow-up visit was reported in 261/458 patients; 24/148 (16.2%) subjects with skin involvement as early as BD onset maintained cutaneous lesions for the entire period of observation, while 120 (44.1%) patients suffered from sporadic skin involvement. Conversely, 94/113 (83.2%) with no skin involvement at disease onset did not develop skin lesions thereafter. At follow-up visits, cutaneous involvement was observed in 52 (20%) patients, with a statistically significant association between PF and constant skin involvement (p = 0.031). BD in Italy is characterized by a wide spectrum of clinical presentations and skin manifestations in line with what is described in endemic countries. Patients with skin disease at the onset are likely to present persistent cutaneous involvement thereafter; mucocutaneous lesions observed at the onset, especially PF, could represent a warning sign for future persistent skin involvement requiring closer dermatological care.

The Impact of Nitroglycerin on the Evaluation of Coronary Stenosis in Coronary-CT: Preliminary Study in 131 Patients.

BACKGROUND: The sublingual administration of short-acting nitroglycerin (NTG) before coronary computed tomography (CCT) improves the visualization of coronary arteries, causing vasodilatation. The aim of this study was to evaluate whether and how nitroglycerin can influence the concordance between radiologists and cardiologists in the evaluation of vessel stenosis measured in CCT by the former and during the following coronarography by the latter. METHODS: We conducted a retrospective analysis of 131 patients who underwent CCT for cardiac symptoms in 2022, followed by coronarography performed six months later because of significant stenosis revealed by the CCT. First, the patients were divided into two groups: an NTG group who received sublingual nitroglycerin before CCT and a non-NTG group who did not because of contraindications. Second, 254 stenoses were measured by two radiologists after CCT and by two interventional cardiologists during the next coronarography; moreover, stenoses were classified on the basis of their location and plaque pattern (calcific, mixed and lipidic). Third, the strength of agreement was evaluated between the two radiologists, between the two cardiologists and finally between the radiologists and cardiologists in order to evaluate whether and how the interdisciplinary discrepancy in stenosis evaluation could change with or without the use of nitroglycerin before CCT and in relation to the different plaque pattern. RESULTS: In the NTG group, the use of nitroglycerine reduced the agreement between radiologists and cardiologists in calcific stenosis but did not change the concordance in the case of mixed or lipidic plaques on the same vessels. CONCLUSIONS: The use of sublingual nitroglycerin before CCT may lead to a radiological overestimation of calcific stenosis.

Relation of the work ability index to fitness for work in healthcare and public employees in a region of Northeastern Italy.

PURPOSE: Work ability indicates an individual's capacity to match job demands according to his/her physical and mental conditions and work circumstances. Occupational physicians should take into consideration the global health status of a worker in order to correctly assess if he/she is fit for the job. The aim of this study was to verify the association between fitness for work evaluation and Work Ability Index scores, as well as individual factors (age, gender, and anthropometric characteristics) and work-related variables (job type, years of working duration). METHODS: A cross-sectional study was conducted within the occupational health surveillance of health and public employers in the Friuli-Venezia Giulia region (2018-2022). The participants voluntarily agreed to answer the standard Work Ability Index questionnaire. Data were investigated by univariable as well as multivariable regression analysis. RESULTS: The Work Ability Index of the workers included in the study (N = 6893) resulted negatively associated with age, female sex, and body mass index. It was averagely lower in nurses and assistive personnel, and the highest in medical doctors and public employers. The fitness for work assessments was also statistically related to WAI scores. The results obtained from the univariable and the multivariable analysis were consistent. CONCLUSIONS: The Work Ability Index is an efficient tool to measure an individual's capability to sustain job demands, and can be taken into account to produce a correct fitness for work evaluation and consequently preserve workers' health status.

Budesonide Analogues Preserve Stem Cell Pluripotency and Delay 3D Gastruloid Development.

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.

Nebivolol versus placebo in patients undergoing anthracyclines (CONTROL Trial): rationale and study design.

AIMS: Anthracyclines are the chemotherapeutic agents most frequently associated with cardiotoxicity, while remaining widely used. Different neurohormonal blockers have been tested as a primary prevention strategy to prevent or attenuate the onset of cardiotoxicity, with mixed results. However, prior studies were often limited by a nonblinded design and an assessment of cardiac function based only on echocardiographic imaging. Moreover, on the basis of an improved mechanistic understanding of anthracycline cardiotoxicity mechanisms, new therapeutic strategies have been proposed. Among cardioprotective drugs, nebivolol might be able to prevent the cardiotoxic effects of anthracyclines, through its protective properties towards the myocardium, endothelium, and cardiac mitochondria. This study aims to evaluate the cardioprotective effects of the beta blocker nebivolol in a prospective, placebo-controlled, superiority randomized trial in patients with breast cancer or diffuse large B cell lymphoma (DLBCL) who have a normal cardiac function and will receive anthracyclines as part of their first-line chemotherapy programme. METHODS: The CONTROL trial is a randomized, placebo-controlled, double-blinded, superiority trial. Patients with breast cancer or a DLBCL, with a normal cardiac function as assessed by echocardiography, scheduled for treatment with anthracyclines as part of their first-line chemotherapy programme will be randomized 1 : 1 to nebivolol 5 mg once daily (o.d.) or placebo. Patients will be examined with cardiological assessment, echocardiography and cardiac biomarkers at baseline, 1 month, 6 months and 12 months. A cardiac magnetic resonance (CMR) assessment will be performed at baseline and at 12 months. The primary end point is defined as left ventricular ejection fraction reduction assessed by CMR at 12 months of follow-up. CONCLUSION: The CONTROL trial is designed to provide evidence to assess the cardioprotective role of nebivolol in patients undergoing chemotherapy with anthracyclines. CLINICAL TRIAL REGISTRATION: The study is registered in the EudraCT registry (number: 2017-004618-24) and in the ClinicalTrials.gov registry (identifier: NCT05728632).

Non-Contrast and Contrast-Enhanced Cardiac Computed Tomography Imaging in the Diagnostic and Prognostic Evaluation of Coronary Artery Disease.

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.

Longitudinal Characterization of Immune Response in a Cohort of Children Hospitalized with Multisystem Inflammatory Syndrome.

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.

Circulating extracellular particles from severe COVID-19 patients show altered profiling and innate lymphoid cell-modulating ability.

Introduction: Extracellular 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. Methods: Peripheral 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. Results: We 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. Discussion: In 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.

Structural and haemodynamic evaluation of less invasive surfactant administration during nasal intermittent positive pressure ventilation in surfactant-deficient newborn piglets.

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 (pH<7.2, PaCO2>70 mmHg, PaO2<70 mmHg, dynamic compliance<0.5 ml/cmH2O/kg at FiO2 = 1). Poractant-alfa administration using different LISA techniques during NIPPV was well tolerated and efficacious in newborn piglets. In our study, although all groups showed normal physiological ranges of total lung injury score and biochemical lung analysis, VC and LISAcath® catheters were associated with better values of lung compliance and lower values of lung damage than NIPPV, NT or INSURE-like methods. Moreover, neither of the SF administration methods used (LISA or INSURE-like) had a significant impact on the histological neonatal brain injury score. Of note, the LISAcath® has been recently withdrawn from the market.

Role of Rituximab Addition to First-line Chemotherapy Regimens in Nodular Lymphocyte-predominant Hodgkin Lymphoma: A Study by Fondazione Italiana Linfomi.

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.

Time-resolved transcriptomic profiling of the developing rabbit's lungs: impact of premature birth and implications for modelling bronchopulmonary dysplasia.

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 ß, 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.

Beyond the Interface: Improved Pulmonary Surfactant-Assisted Drug Delivery through Surface-Associated Structures.

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.

Efficacy of synthetic surfactant (CHF5633) bolus and/or lavage in meconium-induced lung injury in ventilated newborn rabbits.

BACKGROUND: The pathogenesis of neonatal meconium aspiration syndrome (MAS) involves meconium-induced lung inflammation and surfactant inactivation. Bronchoalveolar lavage (BAL) with diluted surfactant facilitates the removal of meconium. CHF5633, one of the most promising synthetic surfactants, is effective in neonatal respiratory distress syndrome. Here we investigated its efficacy via BAL in an experimental MAS model. METHODS: Experimental MAS was induced at birth in near-term newborn rabbits by intratracheal instillation of reconstituted human meconium. First, undiluted CHF5633 was compared with a porcine-derived surfactant (Poractant alfa) via intratracheal bolus (200 mg/kg). Second, the efficacy of BAL with diluted CHF5633 (5 mg/mL, 20 ml/kg) alone, or followed by undiluted boluses (100 or 300 mg/kg), was investigated. RESULTS: Meconium instillation caused severe lung injury, reduced endogenous surfactant pool, and poor survival. CHF5633 had similar benefits in improving survival and alleviating lung injury as Poractant alfa. CHF5633 BAL plus higher boluses exerted better effects than BAL or bolus alone in lung injury alleviation by reversing phospholipid pools and mitigating proinflammatory cytokine mRNA expression, without fluid retention and function deterioration. CONCLUSIONS: CHF5633 improved survival and alleviated meconium-induced lung injury, the same as Poractant alfa. CHF5633 BAL plus boluses was the optimal modality, which warrants further clinical investigation. IMPACT: To explore the efficacy of a synthetic surfactant, CHF5633, in neonatal lung protection comparing with Poractant alfa in a near-term newborn rabbit model with meconium-induced lung injury. Similar effects on improving survival and alleviating lung injury were found between CHF5633 and Poractant alfa. Optimal therapeutic effects were identified from the diluted CHF5633 bronchoalveolar lavage followed by its undiluted bolus instillation compared to the lavage or bolus alone regimens. Animals with CHF5633 lavage plus bolus regimen exerted neither substantial lung fluid retention nor lung mechanics deterioration but a trend of higher pulmonary surfactant-associated phospholipid pools.

Preterm rabbit-derived Precision Cut Lung Slices as alternative model of bronchopulmonary dysplasia in preclinical study: a morphological fine-tuning approach.

Bronchopulmonary dysplasia (BPD) is the most common complication of preterm delivery, with significant morbidity and mortality in a neonatal intensive care setting. Research in this field aims to identify the mechanisms of late lung development with possible therapeutic targets and the improvement of medical management. Rabbits represent a suitable lab preclinical tool for mimicking the clinical BPD phenotype. Rabbits are born at term in the alveolar phase as occurs in large animals and humans and in addition, they can be delivered prematurely in contrast to mice and rats. Continuous exposure to high oxygen concentration (95% O2) for 7 days induces functional and morphological lung changes in preterm rabbits that resemble those observed in BPD-affected babies. The preclinical research pays great attention to optimize the experimental procedures, reduce the number of animals used in experiments and, where possible, replace animal models with alternative assays, following the principle of the 3 Rs (Replace, Reduce and Refine). The use of in vitro assays based on the ex vivo culture of Precision Cut Lung Slices (PCLS) goes in this direction, representing a good compromise between controlled and flexible in vitro models and the more physiologically relevant in vivo ones. This work aims to set up morphological analyses to be applied in preclinical tests using preterm rabbits derived PCLS, cultured up to 7 days in different oxygen conditions, as a model. After a preliminary optimization of both lung preparation and histological processing methods of the lung slices of 300 µm, the morphological analysis was conducted evaluating a series of histomorphometric parameters derived from those widely used to follow the phases of lung development and its alterations in vivo. Our histomorphometric results demonstrated that the greatest differences from pseudo-normoxia and hyperoxia exposed samples at day 0, used as starting points to compare changes due to treatments and time, are detectable after 4 days of in vitro culture, representing the most suitable time point for analysis in preclinical screening. The combination of parameters suitable for evaluating PCLS morphology in vitro resulted to be Tissue Density and Septal Thickness. Shape Factor and Roughness, evaluated to highlight the increasing complexity of the airspaces, due to the formation of septal crests, gave useful information, however, without significant differences up to day 4. Other parameters like Mean Linear Intercept and Septal Density did not allow to highlight significant differences between different oxygen conditions and time points. Instead, Radial Alveolar Count, could not be applied to PCLS, due to the tissue changes following agar infusion and culture conditions.