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Massimo Allegri,1,2 Dario Bugada,1–3 Manuela De Gregori,2,4 Maria A Avanzini,5 Annalisa De Silvestri,6 Anna Petroni,7 Angelo Sala,7,8 Claudia Filisetti,9–11 Antonia Icaro Cornaglia,12 Lorenzo Cobianchi13,14 1Department of Medicine and Surgery, University of Parma, Parma 2SIMPAR Group (Study in Multidisciplinary PAin Research), 3Department of Anaesthesia and ICU, ASST Papa Giovanni XXIII, Bergamo, 4Pain Therapy Service, Fondazione IRCCS Policlinico San Matteo, 5Laboratory of Transplant Immunology/Cell Factory, IRCCS Foundation Policlinico San Matteo, 6Clinical epidemiology and Biometrics Unit, Fondazione IRCCS Policlinico San Matteo, Pavia, 7Department of Pharmacological and Biomolecular Sciences, University of Milan, Milan, 8I.B.I.M., C.N.R., Palermo, 9PhD School, University of Pavia, 10Department of Pediatric Surgery, Fondazione IRCCS Policlinico San Matteo, Pavia, 11Department of Pediatric Surgery, “V. Buzzi” Children’s Hospital, Milan, 12Department of Public Health, Experimental and Forensic Medicine, University of Pavia, 13Department of Surgical, Clinical, Paediatric and Diagnostic Science, University of Pavia, 14General Surgery 1, IRCCS Fondazione Policlinico San Matteo, Pavia, Italy Abstract: Continuous wound infusion (CWI) may protect from inflammation, hyperalgesia and persistent pain. Current local anesthetics display suboptimal pharmacokinetic profile during CWI; chloroprocaine (CP) has ideal characteristics, but has never been tested for CWI. We performed an animal study to investigate the pharmacokinetic profile and anti-inflammatory effect of CP during CWI. A total of 14 piglets received an infusion catheter after pararectal laparotomy and were randomly allocated to one of three groups: 5 mL/h infusion of saline (group A), CP 1.5% (group B) and CP 0.5% (group C). Blood sampling was performed to assess absorption and systemic inflammation at 0, 3, 6, 12, 24, 48, 72, 96, 102 and 108 hours. The wound and contralateral healthy abdominal wall were sampled for histological analyses. Absorption of CP from the site of infusion, evaluated as the plasmatic concentrations of CP and its metabolite, 4-amino-2-chlorobenzoic acid (CABA), showed a peak during the first 6 hours, but both CP and its metabolite rapidly disappeared after stopping CP infusion. Local inflammation was reduced in groups B and C (CP-treated p < 0.001), in a CP dose-dependent fashion. While CP inhibited in a dose-dependent manner pig mononuclear cells (MNCs) in vitro proliferation to a polyclonal activator, no effect on systemic cytokines’ concentrations or on ex vivo monocytes’ responsiveness was observed, suggesting the lack of systemic effects, in line with the very short half-life of CP in plasma. CP showed a very good profile for use in CWI, with dose-dependent local anti-inflammatory effects, limited absorption and rapid clearance from the bloodstream upon discontinuation. No cytotoxicity or side effects were observed. CP, therefore, may represent an optimal choice for clinical CWI, adaptable to each patient’s need, and protective on wound inflammatory response (and hyperalgesia) after surgery. Keywords: continuous wound infusion, pig model, chloroprocaine, pharmacokinetics, inflammation, postoperative pain

Carmelo Gigliuto,1 Manuela De Gregori,2 Valentina Malafoglia,3 William Raffaeli,3 Christian Compagnone,4 Livia Visai,5,6 Paola Petrini,7 Maria Antonietta Avanzini,9 Carolina Muscoli,8 Jacopo Viganò,11 Francesco Calabrese,11 Tommaso Dominioni,11 Massimo Allegri,2,10 Lorenzo Cobianchi111Anaesthesia and Intensive Care, University of Pavia, Pavia, 2Pain Therapy Service, Fondazione IRCCS Policlinico San Matteo, Pavia, 3ISAL Foundation, Institute for Research on Pain, Torre Pedrera, Rimini, 4Department of Anaesthesia, Intensive Care and Pain Therapy, Azienda Ospedaliera Universitaria Parma, University of Parma, Parma, 5Department of Molecular Medicine, Center for Tissue Engineering (CIT), INSTM UdR of Pavia, University of Pavia, Pavia, 6Department of Occupational Medicine, Ergonomy and Disability, Laboratory of Nanotechnology, Salvatore Maugeri Foundation, IRCCS, Veruno, 7Dipartimento di Chimica, Materiali e Ingegneria Chimica 'G Natta' and Unità di Ricerca Consorzio INSTM, Politecnico di Milano, Milan, 8Department of Health Science, University Magna Grecia of Catanzaro and Centro del Farmaco, IRCCS San Raffaele Pisana, Roma, 9Laboratory of Transplant Immunology/Cell Factory, Fondazione IRCCS Policlinico "San Matteo", Pavia, 10Department of Clinical, Surgical, Diagnostic and Paediatric Sciences, University of Pavia, Pavia, 11University of Pavia, Department of Surgical, Clinical, Paediatric and Diagnostic Science, General Surgery 1, IRCCS Fondazione Policlinico San Matteo, Pavia, ItalyAbstract: In the last two decades, animal models have become important tools in understanding and treating pain, and in predicting analgesic efficacy. Although rodent models retain a dominant role in the study of pain mechanisms, large animal models may predict human biology and pharmacology in certain pain conditions more accurately. Taking into consideration the anatomical and physiological characteristics common to man and pigs (median body size, digestive apparatus, number, size, distribution and communication of vessels in dermal skin, epidermal–dermal junctions, the immunoreactivity of peptide nerve fibers, distribution of nociceptive and non-nociceptive fiber classes, and changes in axonal excitability), swines seem to provide the most suitable animal model for pain assessment. Locomotor function, clinical signs, and measurements (respiratory rate, heart rate, blood pressure, temperature, electromyography), behavior (bright/quiet, alert, responsive, depressed, unresponsive), plasma concentration of substance P and cortisol, vocalization, lameness, and axon reflex vasodilatation by laser Doppler imaging have been used to assess pain, but none of these evaluations have proved entirely satisfactory. It is necessary to identify new methods for evaluating pain in large animals (particularly pigs), because of their similarities to humans. This could lead to improved assessment of pain and improved analgesic treatment for both humans and laboratory animals.Keywords: pain assessment, experimental model, translational research

The idea that landscape fulfils many different functions and provides multiple benefits for human society is gaining strength. This new vision of landscape changes both, the interests and the needs of the persons involved and the use and management of the landscape itself. The objective of this paper is to study landscape conservation in a particular mountainous area of Italy, which requires an institutional, non-generic approach. Using the contingent valuation method, we investigated whether there is a relationship between experts’ and casual observers’ evaluations of ten different scenarios in the Leno Valleys. Results show that direct knowledge of the site characteristics increases the awareness of the importance of the site, significantly impacting on conservation choices and strategies.

Abstract Aortitis may be due to infectious and non-infectious causes. We observed aortitis, associated with glaucoma, thyroiditis, pericarditis, pleural effusion and neuropathy in a 63-years old woman. Despite antibiotic therapy, inflammatory signs persisted and resolved only after initiation of glucocorticoid therapy. Increasing aortic ectasia necessitated resection of the ascending aorta and implantation of a Vascutek 30 mm prosthesis. Histologically a granulomatous aortitis was diagnosed. Since all other possible causes were excluded, an immunological mechanism of the aortitis is suspected and possible triggering factors are discussed.

Objectives. Taking into account that genetic predisposition, marked by human leukocyte antigen (HLA) class I and II genes, augments the probability of developing an autoimmune disorder after a triggering vaccination, as largely debated, we investigated the frequency of autoantibody production after recombinant hepatitis B vaccine (rHBv) in 6-year-old children immunized at birth to evaluate an association between autoimmune disorders and hepatitis B virus vaccination.Methods. We investigated the presence of autoantibodies in 210 6-year-old children who were immunized at birth with rHBv: 200 showed anti-hepatitis B surface antigen concentrations = >/=10 mUI/mL at seroconversion (responders), and 10 were nonresponders. Data were compared with those obtained in 109 unvaccinated children. All participants were screened for the presence of antinuclear antibodies (ANAs), anti-DNA, antimitochondrial, anti-liver/kidney microsomal, antireticulin, anti-smooth muscle (SMA), and antiribosomal antibodies. All participants were also screened for the presence of antithyroid antibodies, such as antithyroglobulin and antiperoxidase, and for antibodies found in type 1 diabetes, such as tyrosine phosphatase (IA-2A) and glutamic acid decarboxylase (GADA). HLA typing was extended to all 10 nonresponders.Results. Autoantibodies were found in 16 of the 200 responders: ANAs were found in 12 (6%), smooth muscle antibodies were found in 4 (2.0%), and antireticulin antibodies and endomysial antibodies were found in 1 girl with ANAs. Antithyroid antibodies, IA-2A, and GADA were not present in any of the participants. No significant difference was found in the frequency of autoantibodies between vaccinated and control children. Three of the 10 nonresponder children were SMA-positive (30% vs 2% of responders); they also carried the supratype HLA-C4AQ0,DRB1*0301,DQB1*02. A family history for autoimmune disorders was present in 3 (18%; 95% confidence interval [CI]: 4.0%-45.6%) of the 16 responder infants with autoantibodies, in 15 (8.4%; 95% CI: 4.6%-13.1%) of responder children without autoantibodies, and in 1 (10%) of the 10 nonsreponder children.Conclusions. From our data, vaccination with rHBv given during the neonatal period does not seem to increase autoantibody production in a 6-year-old children. Autoantibodies, referred to as natural autoantibodies, can be found in healthy participants, but their significance is unclear. These autoantibodies often cross-react with bacteria or tumor antigens, suggesting their importance in innate immunity. It has been demonstrated in an animal model that self-antigen can promote B-cell accumulation, and that a significant proportion of natural autoantibodies is the product of this self-antigen- dependent process. Consequently, it has been speculated that self-antigens play a positive role in recruiting B cells as a part of innate immunity, but this process carries a potential risk for unregulated growth.Spreading of the immune response is a common theme in organ-specific and systemis autoimmune diseases, and this could be initiated by exogenous agents, in genetically susceptible hosts, owing to molecular mimicry of natural antigen. Moreover, 3 (18%) of the 16 children who had autoantibodies had a family history of autoimmume diseases. Thus, it is apparent that susceptibility to autoimmunity is determined by genetic factors rather than by vaccine challenge. Among all the children considered, only 1 girl (0.5%) developed celiac disease, reflecting the prevalence described in the literature. GADA and IA-2A were not found in our children; this observation is in agreement with data showing that type 1 diabetes risk may not be altered by vaccinations administered during childhood. On the contrary, a high frequency (30%) of autoantibodies, in particular SMA, was observed in the nonresponder children. The 3 SMA-positive children carried the HLA-C4Q0,DRB1*0301,DQB1*02 haplotype, a well-known predisposing factor for autoimmune disorders. On the other hand, the presence of autoantibodies to smooth muscle is known to be common in hepatitis B infection, and, it has been shown that cross-reactive immunity targeting homologous self-protein may partly account for autoantibody production. Although hepatitis B vaccination given during the neonatal period does not increase autoantibody production in 6-year-old immunized children, we deem useful a more prolonged follow-up for these nonresponder children carrying certain HLA haplotypes (such as C4AQ0,DRB1*0301,DQB1*02), particularly because most autoimmune diseases do not develop until later in life. [Abstract for this article also available on page 159-6 of printed version. Full article available at http://www.pediatrics.org/cgi/content/full/110/1/e4] [ABSTRACT FROM AUTHOR]

Bone marrow-mesenchymal stromal cells (BM-MSCs) are key components of the BM niche, where they regulate hematopoietic stem progenitor cell (HSPC) homeostasis by direct contact and secreting soluble factors. BM-MSCs also protect the BM niche from excessive inflammation by releasing anti-inflammatory factors and modulating immune cell activity. Thanks to these properties, BM-MSCs were successfully employed in pre-clinical HSPC transplantation models, increasing the rate of HSPC engraftment, accelerating the hematological reconstitution, and reducing the risk of graft failure. However, their clinical use requires extensive in vitro expansion, potentially altering their biological and functional properties. In this work, we analyzed the transcriptomic profile of human BM-MSCs sorted as CD45 - , CD105 + , CD73 + , and CD90 + cells from the BM aspirates of heathy-donors and corresponding ex-vivo expanded BM-MSCs. We found the expression of immune and inflammatory genes downregulated upon cell culture and selected the transcription factor EGR1 to restore the MSC properties. We overexpressed EGR1 in BM-MSCs and performed in vitro tests to study the functional properties of EGR1-overexpressing BM-MSCs. We concluded that EGR1 increased the MSC response to inflammatory stimuli and immune cell control and potentiated the MSC hematopoietic supportive activity in co-culture assay, suggesting that the EGR1-based reprogramming may improve the BM-MSC clinical use., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier B.V.)

(1) Background: Numerous elements of the Mediterranean diet (MD) have antioxidant and anti-inflammatory qualities. (2) Methods: We present a narrative review of the potential benefits of the Mediterranean dietary pattern (MD) in mitigating aging-related inflammation (inflamm-aging) associated with childhood obesity. The mechanisms underlying chronic inflammation in obesity are also discussed. A total of 130 papers were included after screening abstracts and full texts. (3) Results: A complex interplay between obesity, chronic inflammation, and related comorbidities is documented. The MD emerges as a promising dietary pattern for mitigating inflammation. Studies suggest that the MD may contribute to weight control, improved lipid profiles, insulin sensitivity, and endothelial function, thereby reducing the risk of metabolic syndrome in children and adolescents with obesity. (4) Conclusions: While evidence supporting the anti-inflammatory effects of the MD in pediatric obesity is still evolving, the existing literature underscores its potential as a preventive and therapeutic strategy. However, MD adherence remains low among children and adolescents, necessitating targeted interventions to promote healthier dietary habits. Future high-quality intervention studies are necessary to elucidate the specific impact of the MD on inflammation in diverse pediatric populations with obesity and associated comorbidities.

Background: CLAD (Chronic Lung Allograft Dysfunction) remains a serious complication following lung transplantation. Some evidence shows that portions of Extracorporeal Photopheresis (ECP)-treated patients improve/stabilize their graft function. In spite of that, data concerning molecular mechanisms are still lacking. Aims of our study were to assess whether ECP effects are mediated by Mononuclear Cells (MNCs) modulation in term of microRNAs (miRNAs) expression and growth factors release., Methods: Cells from leukapheresis of 16 CLAD patients, at time 0 and 6-months (10 cycles), were cultured for 48h ± PHA (10 ug/ml) or LPS (2 ug/ml). Expression levels of miR-146a-5p, miR-155-5p, miR-31-5p, miR181a-5p, miR-142-3p, miR-16-5p and miR-23b-5p in MNCs-exosomes were evaluated by qRT-PCR, while ELISA assessed different growth factors levels on culture supernatants., Results: Our result showed miR-142-3p down-regulation (p = 0.02) in MNCs of ECP-patients after the 10 cycles and after LPS stimulation (p = 0.005). We also find miR-146a-5p up-regulation in cells after the 10 cycles stimulated with LPS (p = 0.03). Connective tissue growth factor (CTGF) levels significantly decreased in MNCs supernatant (p = 0.04). The effect of ECP is translated into frequency changes of Dendritic Cell (DC) subpopulations and a slight increase in T regulatory cells (Treg) number and a significant decrease in CTGF release., Conclusions: ECP might affect regulatory T cell functions, since both miR-142 and miR-146a have been shown to be involved in the regulation of suppressor regulatory T cell functions and DCs. On the other side ECP, possibly by regulating macrophage activation, is able to significantly down modulate CTGF release., (© 2024. The Author(s).)

We previously published that in patients with infantile hemangioma (IH) at the onset (T0) colony forming unit-fibroblasts (CFU-Fs) are present in in vitro cultures from PB. Herein, we characterize these CFU-Fs and investigate their potential role in IH pathogenesis, before and after propranolol therapy. The CFU-F phenotype (by flow cytometry), their differentiation capacity and ability to support angiogenesis (by in vitro cultures) and their gene expression (by RT-PCR) were evaluated. We found that CFU-Fs are actual circulating MSCs (cMSCs). In patients at T0, cMSCs had reduced adipogenic potential, supported the formation of tube-like structures in vitro and showed either inflammatory (IL1β and ESM1 ) or angiogenic ( F3 ) gene expression higher than that of cMSCs from CTRLs. In patients receiving one-year propranolol therapy, the cMSC differentiation in adipocytes improved, while their support in in vitro tube-like formation was lost; no difference was found between patient and CTRL cMSC gene expressions. In conclusion, in patients with IH at T0 the cMSC reduced adipogenic potential, their support in angiogenic activity and the inflammatory/angiogenic gene expression may fuel the tumor growth. One-year propranolol therapy modifies this picture, suggesting cMSCs as one of the drug targets.

Bioprinting offers new opportunities to obtain reliable 3D in vitro models of the liver for testing new drugs and studying pathophysiological mechanisms, thanks to its main feature in controlling the spatial deposition of cell-laden hydrogels. In this context, decellularized extracellular matrix (dECM)-based hydrogels have caught more and more attention over the last years because of their characteristic to closely mimic the tissue-specific microenvironment from a biological point of view. In this work, we describe a new concept of designing dECM-based hydrogels; in particular, we set up an alternative and more practical protocol to develop a hepatic lyophilized dECM (lyo-dECM) powder as an 'off-the-shelf' and free soluble product to be incorporated as a biomimetic component in the design of 3D-printable hybrid hydrogels. To this aim, the powder was first characterized in terms of cytocompatibility on human and porcine mesenchymal stem cells (MSCs), and the optimal powder concentration (i.e. 3.75 mg ml -1 ) to use in the hydrogel formulation was identified. Moreover, its non-immunogenicity and capacity to reactivate the elastase enzyme potency was proved. Afterward, as a proof-of-concept, the powder was added to a sodium alginate/gelatin blend, and the so-defined multi-component hydrogel was studied from a rheological point of view, demonstrating that adding the lyo-dECM powder at the selected concentration did not alter the viscoelastic properties of the original material. Then, a printing assessment was performed with the support of computational simulations, which were useful to define a priori the hydrogel printing parameters as window of printability and its post-printing mechanical collapse. Finally, the proposed multi-component hydrogel was bioprinted with cells inside, and its post-printing cell viability for up to 7 d was successfully demonstrated., (Creative Commons Attribution license.)

Mosquito-borne West Nile virus (WNV) infection is benign in most individuals but can cause encephalitis in <1% of infected individuals. We show that ∼35% of patients hospitalized for WNV disease (WNVD) in six independent cohorts from the EU and USA carry auto-Abs neutralizing IFN-α and/or -ω. The prevalence of these antibodies is highest in patients with encephalitis (∼40%), and that in individuals with silent WNV infection is as low as that in the general population. The odds ratios for WNVD in individuals with these auto-Abs relative to those without them in the general population range from 19.0 (95% CI 15.0-24.0, P value <10-15) for auto-Abs neutralizing only 100 pg/ml IFN-α and/or IFN-ω to 127.4 (CI 87.1-186.4, P value <10-15) for auto-Abs neutralizing both IFN-α and IFN-ω at a concentration of 10 ng/ml. These antibodies block the protective effect of IFN-α in Vero cells infected with WNV in vitro. Auto-Abs neutralizing IFN-α and/or IFN-ω underlie ∼40% of cases of WNV encephalitis., (© 2023 Gervais et al.)

Background: A few studies assessed the clinical and immunological features of selective IgM deficiency (SIgMD), especially in the pediatric age. We aimed to characterize the clinical and immunological phenotypes of a cohort of pediatric patients with SIgMD according to the different diagnostic criteria available., Methods: In this multicenter study, we evaluated pediatric SIgMD patients diagnosed at the Pediatric Clinic in Pavia, Italy, or through the Italian Primary Immunodeficiency NETwork (IPINET) and monitored changes in their diagnosis over a time frame that ranges from several months to several years., Results: Forty-eight patients with SIgMD were included (mean serum IgM: 33 mg/dL). The most common clinical manifestations were recurrent infections (67%) and allergies (48%). Subgroup analysis according to SIgMD definition criteria of the European Society for Immunodeficiencies (ESID) showed no significant difference in clinical manifestations, also considering the group with additional immunological abnormalities. Sixteen patients had long-term follow-up, during which 87% preserved their SIgMD diagnosis, while two patients showed a reduction in IgA in addition to low IgM., Conclusions: Our data suggest that the identification of a reduction in serum IgM in children should lead to a complete immunological work-up to obtain a comprehensive clinical and immunological characterization of the patient. The follow-up of these patients is fundamental to define the disease evolution and appropriate management., (© 2023 The Authors. Pediatric Allergy and Immunology published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Mesenchymal Stromal Cell (MSC) clinical applications have been widely reported and their therapeutic potential has been documented in several diseases. MSCs can be isolated from several human tissues and easily expanded in vitro, they are able to differentiate in a variety of cell lineages, and they are known to interact with most immunological cells, showing immunosuppressive and tissue repair properties. Their therapeutic efficacy is closely associated with the release of bioactive molecules, namely Extracellular Vesicles (EVs), effective as their parental cells. EVs isolated from MSCs act by fusing with target cell membrane and releasing their content, showing a great potential for the treatment of injured tissues and organs, and for the modulation of the host immune system. EV-based therapies provide, as major advantages, the possibility to cross the epithelium and blood barrier and their activity is not influenced by the surrounding environment. In the present review, we deal with pre-clinical reports and clinical trials to provide data in support of MSC and EV clinical efficacy with particular focus on neonatal and pediatric diseases. Considering pre-clinical and clinical data so far available, it is likely that cell-based and cell-free therapies could become an important therapeutic approach for the treatment of several pediatric diseases., Competing Interests: Declaration of Competing Interest We declare that the manuscript has not been published previously in print or electronic format and is not under consideration by another journal. All the authors have reviewed the manuscript, agree with its contents, and consent to its submission. No honorarium, grant or other form of payment, was given to anyone to produce this manuscript. Conflicts of interest: none to be declare., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

A subset of severe COVID19 patients develop pulmonary fibrosis, but the pathophysiology of this complication is still unclear. We previously described the possibility to isolate lung mesenchymal cells (LMC) by culturing broncho-alveolar lavage (BAL) cells from patients with pulmonary fibrosis or chronic lung allograft dysfunction. Aim of this study was to investigate the possibility to isolate and characterize LMC from BAL of patients that, two months after discharge for severe COVID19, show CT signs of post-COVID19 fibrosis (Post-COVID) and in some cases has been considered transplant indication. Results were compared with those from BAL of patients with collagen tissue disease-associated interstitial fibrosis (CTD-ILD). BAL fluid levels of TGFβ, VEGF, TIMP2, RANTES, IL6, IL8, and PAI1 were assessed. LMC were cultured and expanded, phenotyped by flow cytometry, and tested for osteogenic and adipogenic differentiation. Finally, we tested immunomodulatory and proliferative capabilities, collagen I production + /- TGF-beta stimulation. BAL cytokine and growth factor levels were comparable in the two groups. Efficiency of isolation from BAL was 100% in post-COVID compared to 63% in CTD-ILD. LMC from post-COVID were positive for CD105, CD73, CD90, and negative for CD45, CD34, CD19 and HLA-DR as in CTD-ILD samples. Post-COVID LMC displayed higher collagen production with respect to CTD-ILD LMC. Immunomodulatory capacity towards lymphocytes was very low, while Post-COVID LMC significantly upregulated pro-inflammatory cytokine production by healthy PBMCs. Our preliminary data suggest that LMC from post-COVID19 fibrosis patients share several features with CTD-ILD ones but might have a higher response to fibrogenic signals and pro-inflammatory profile., Competing Interests: Declaration of Competing Interest None declare., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

In the next-generation sequencing era, RT-qPCR is still widely employed to quantify levels of nucleic acids of interest due to its popularity, versatility, and limited costs. The measurement of transcriptional levels through RT-qPCR critically depends on reference genes used for normalization. Here, we devised a strategy to select appropriate reference genes for a specific clinical/experimental setting based on publicly available transcriptomic datasets and a pipeline for RT-qPCR assay design and validation. As a proof-of-principle, we applied this strategy to identify and validate reference genes for transcriptional studies of bone-marrow plasma cells from patients with AL amyloidosis. We performed a systematic review of published literature to compile a list of 163 candidate reference genes for RT-qPCR experiments employing human samples. Next, we interrogated the Gene Expression Omnibus to assess expression levels of these genes in published transcriptomic studies on bone-marrow plasma cells from patients with different plasma cell dyscrasias and identified the most stably expressed genes as candidate normalizing genes. Experimental validation on bone-marrow plasma cells showed the superiority of candidate reference genes identified through this strategy over commonly employed "housekeeping" genes. The strategy presented here may apply to other clinical and experimental settings for which publicly available transcriptomic datasets are available.

Purpose: Mesenchymal stem cells (MSCs) represent a promising source for stem cell therapies in numerous diseases, including pediatric respiratory system diseases. Characterized by low immunogenicity, high anti-inflammatory, and immunoregulatory features, MSCs demonstrated an excellent therapeutic profile in numerous in vitro and preclinical models. MSCs reside in a specialized physiologic microenvironment, characterized by a unique combination of biophysical, biochemical, and cellular properties. The exploitation of the 3D micro-scaffold Nichoid, which simulates the native niche, enhanced the anti-inflammatory potential of stem cells through mechanical stimulation only, overcoming the limitation of biochemical and xenogenic growth factors application., Materials and Methods: In this work, we expanded pediatric bone marrow MSCs (BM-MSCs) inside the Nichoid and performed a complete cellular characterization with different approaches including viability assays, immunofluorescence analyses, RNA sequencing, and gene expression analysis., Results: We demonstrated that BM-MSCs inside the scaffold remain in a stem cell quiescent state mimicking the condition of the in vivo environment. Moreover, the gene expression profile of these cells shows a significant up-regulation of genes involved in immune response when compared with the flat control., Conclusion: The significant changes in the expression profile of anti-inflammatory genes could potentiate the therapeutic effect of BM-MSCs, encouraging the possible clinical translation for the treatment of pediatric congenital and acquired pulmonary disorders, including post-COVID lung manifestations., Lay Summary: Regenerative medicine is the research field integrating medicine, biology, and biomedical engineering. In this context, stem cells, which are a fundamental cell source able to regenerate tissues and restore damage in the body, are the key component for a regenerative therapeutic approach. When expanded outside the body, stem cells tend to differentiate spontaneously and lose regenerative potential due to external stimuli. For this reason, we exploit the scaffold named Nichoid, which mimics the in vivo cell niche architecture. In this scaffold, mesenchymal stem cells "feel at home" due to the three-dimensional mechanical stimuli, and our findings could be considered as an innovative culture system for the in vitro expansion of stem cells for clinical translation., Future Perspective: The increasing demand of safe and effective cell therapies projects our findings toward the possibility of improving cell therapies based on the use of BM-MSCs, particularly for their clinical translation in lung diseases., Competing Interests: Competing InterestsMTR is a co-founder of the university spin-off company MOAB Srl and holds shares. The other authors declare no competing financial interests., (© The Author(s) 2023.)

Mesenchymal stromal cells (MSCs) have been proposed as a new therapeutic strategy to treat congenital and acquired respiratory system diseases. We describe a case report of an 18-month-old male patient with progressive chronic respiratory failure, associated with mutations of the surfactant protein C gene (SFTPC) due to c.289G > T variant p.Gly97Ser (rs927644577) and c.176A > G variant (p.His59Arg), submitted to repeated intravenous infusions of allogeneic bone marrow (BM) MSCs. The clinical condition of the patient was monitored. Immunologic studies before and during MSC treatment were performed. No adverse events related to the MSC infusions were recorded. Throughout the MSC treatment period, the patient showed a growth recovery. Starting the second infusion, the patient experienced an improvement in his respiratory condition, with progressive adaptation to mechanical ventilation. After the third infusion, five hours/die of spontaneous breathing was shown, and after infusion IV, spontaneous ventilation for 24/24 h was recorded. A gradual decrease of lymphocytes and cell subpopulations was observed. No variations in the in vitro T cell response to PHA were determined by MSC treatment as well as for the in vitro B cell response. A decrease in IFN-γ, TNF-α, and IL-10 levels was also detected. Even though we cannot exclude an improvement of pulmonary function due to the physiological maturation, the well-known action of MSCs in the repair of lung tissue, together with the sequence of events observed in our patient, may support the therapeutic role of MSCs in this clinical condition. However, further investigations are necessary to confirm the result and long-term follow-up will be mandatory to confirm the benefits on the pulmonary condition.