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In December 2019 in China, after a pneumonia outbreak of unknown etiology, a new RNA virus has been identified and called Sars-CoV-2. Sars-CoV-2 induced severe respiratory infections, with global and rapid epidemic diffusion, designated coronavirus disease 2019 (Covid-19). Sars-CoV-2 infection can lead to severe complications, such as acute respiratory distress syndrome (ARDS) with progression to pulmonary fibrosis. Recent clinical studies described that in patients with severe Covid-19, MSC infusions, promote regenerative and reparative effects with anti-inflammatory and anti-fibrotic action. MSCs do not express ACE2 and TMPRSS2, the two main human receptors for host-pathogen interaction, and are not permissive to in vitro Sars-CoV-2 infection, making them suitable for clinical application. The aim of our study was to evaluate the safety and efficacy of MSCs as cellular therapy in ARDS secondary to Sars-CoV-2 in patients undergoing mechanical ventilation, in order to prevent pulmonary fibrosis. MSCs for infusions are thawed at 2x106/ml cellular concentration. The intravenous infusion protocol consists of two doses of third party allogenic MSCs at 1x106/Kg, 15 day apart. From April 2020, six adult patients median age 65 years, median body weight 80 Kg, in mechanical ventilation for ARDS secondary to Sars-CoV-2 infection have been treated. Early or late adverse events were not recorded. Four out six patients showed a significant gas exchange improvement with extubation within seven days from the first infusion. Our results underline the safety and efficacy of MSC infusions for ARDS patients in mechanical ventilation, supporting the need of a phase I/II clinical trial.

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.

Neuroblastoma tumor-associated mesenchymal stromal cells (NB-TA-MSC) have been extensively characterized for their pro-tumorigenic properties, while their immunosuppressive potential, especially against NK cells, has not been thoroughly investigated. Herein, we study the immune-regulatory potential of six primary young and senescent NB-TA-MSC on NK cell function. Young cells display a phenotype (CD105+/CD90+/CD73+/CD29+/CD146+) typical of MSC cells and, in addition, express high levels of immunomodulatory molecules (MHC-I, PDL-1 and PDL-2 and transcriptional-co-activator WWTR1), able to hinder NK cell activity. Notably, four of them express the neuroblastoma marker GD2, the most common target for NB immunotherapy. From a functional point of view, young NB-TA-MSC, contrary to the senescent ones, are resistant to activated NK cell-mediated lysis, but this behavior is overcome using anti-CD105 antibody TRC105 that activates antibody-dependent cell-mediated cytotoxicity. In addition, proliferating NB-TA-MSC, but not the senescent ones, after six days of co-culture, inhibit proliferation, expression of activating receptors and cytolytic activity of freshly isolated NK. Inhibitors of the soluble immunosuppressive factors L-kynurenine and prostaglandin E2 efficiently counteract this latter effect. Our data highlight the presence of phenotypically heterogeneous NB-TA-MSC displaying potent immunoregulatory properties towards NK cells, whose inhibition could be mandatory to improve the antitumor efficacy of targeted immunotherapy.

Este estudo toma as Diretrizes Curriculares Nacionais para a Educação de Jovens e Adultos, de 2000, como marco regulatório que balizou, nos últimos vinte anos, a construção de Diretrizes estaduais e municipais, sob os princípios da educação humana emancipatória, atendendo às especificidades que delimita a EJA, como modalidade da educação básica. Aponta que as Diretrizes Nacionais são resultado da confluência das forças políticas que disputavam, no interior do Estado, diferentes projetos de educação. Afirma que as Diretrizes Estaduais, no Paraná, de 2006, coadunam com os princípios emancipatórios e demonstra, por meio, de pesquisa bibliográfica e da análise dos atos normativos da Secretaria de Estado da Educação e do Esporte e do Conselho Estadual de Educação, emitidos nos últimos dez anos, que a EJA se distancia das Diretrizes, para alinhar-se à Base Nacional Comum Curricular, cujo objetivo é a rápida certificação, por meio da educação a distância.

Lymphomatoid papulosis (LyP) is a rare condition in pediatrics; LyP histological type D has been reported in only 7 children. The differential diagnosis of LyP in the spectrum of lymphoid proliferation remains controversial.A 6-year-old boy presented to Emergency Department with a 3-week history of an erythematous papulo-vesicular itchy eruption over the submandibular regions, trunk and extremities. History, symptoms and laboratory tests were unremarkable. SARS-CoV-2 antigen was negative. The clinical suspicion of pityriasis lichenoides et varioliformis acuta (PLEVA) was posed, and topical steroids were introduced. One week after, he returned with an extensive painful scaly papulo-erythematous rash, with some ulcerated and necrotic lesions, and fever; therefore the child was hospitalized. Biochemical results were within reference limits, except for high level of C-reactive protein, aspartate aminotransferase, alanine transaminase and bilirubin. Due to a persistently high fever, systemic corticosteroid treatment was administered, with a good clinical response and an improvement of the skin lesions. Anti-PVB-19 Immunoglobulin M was detected. Elevated levels of IL-6, IL-10 and IFN-γ were also recorded. Five days post-admission, most of the lesions had cleared, and the child was discharged. Methotrexate was started, with a positive response. At skin biopsy a "PLEVA-like" pattern was apparent, with a dense, wedge shaped lymphoid infiltrate featuring epidermotropism and morphologically comprising pleomorphic and blastic cells. The pattern of infiltration was highlighted by immunohistochemical stains, which prove the process to feature a CD8+/CD30 + phenotype, the latter being intense on larger cells, with antigenic loss. Polymerase chain reaction for T-cell receptor gamma (TCRG) chain clonality assessment documented a monoclonal peak. A diagnosis of LyP type D was favored.The reported case encompasses most of the critical features of two separated entities-PLEVA and LyP-thus providing further support to the concept of them representing declinations within a sole spectrum of disease. Studying the role of infectious agents as trigger potential in lymphoproliferative cutaneous disorders and detecting novel markers of disease, such as cytokines, could have a crucial impact on pathogenic disease mechanisms and perspective therapies.

Background: In end-stage chronic liver disease, transplantation represents the only curative option. However, the shortage of donors results in the death of many patients. To overcome this gap, it is mandatory to develop new therapeutic options. In the present study, we decellularised pig livers and reseeded them with allogeneic porcine mesenchymal stromal cells (pMSCs) to understand whether extracellular matrix (ECM) can influence and/or promote differentiation into hepatocyte-like cells (HLCs). Methods: After decellularisation with SDS, the integrity of ECM-scaffolds was examined by histological staining, immunofluorescence and scanning electron microscope. DNA quantification was used to assess decellularisation. pMSCs were plated on scaffolds by static seeding and maintained in in vitro culture for 21 days. At 3, 7, 14 and 21 days, seeded ECM scaffolds were evaluated for cellular adhesion and growth. Moreover, the expression of specific hepatic genes was performed by RT-PCR. Results: The applied decellularisation/recellularisation protocol was effective. The number of seeded pMSCs increased over the culture time points. Gene expression analysis of seeded pMSCs displayed a weak induction due to ECM towards HLCs. Conclusions: These results suggest that ECM may address pMSCs to differentiate in hepatocyte-like cells. However, only contact with liver-ECM is not enough to induce complete differentiation.

We propose a new organ-conditioning strategy based on mesenchymal stromal cell (MSCs)/extracellular vesicle (EVs) delivery during hypothermic perfusion. MSCs/EVs marker CD73 is present on renal proximal tubular cells, and it protects against renal ischemia-reperfusion injury by converting adenosine monophosphate into adenosine (ADO). In this study, after checking if CD73-silenced EVs (EVsi) would impact in vitro tubular-cell proliferation, we perfused kidneys of a rat model of donation after circulatory death, with Belzer solution (BS) alone, BS supplemented with MSCs, EVs, or EVsi. The ADO and ATP levels were measured in the effluents and tissues. Global renal ischemic damage score (GRS), and tubular cell proliferation index (IPT) were evaluated in the tissue. EVsi did not induce cell proliferation in vitro. Ex vivo kidneys perfused with BS or BS + EVsi showed the worst GRS and higher effluent ADO levels than the MSC- and EV-perfused kidneys. In the EV-perfused kidneys, the tissue and effluent ATP levels and IPT were the highest, but not if CD73 was silenced. Tissue ATP content was positively correlated with tissue ADO content and negatively correlated with effluent ADO level in all groups. In conclusion, kidney conditioning with EVs protects against ischemic damage by activating the CD73/ADO system.

Congenital anomalies may have an increased risk of noncommunicable diseases (NCDs) We performed a clinical exome analysis in an infant affected by “Vertebral, Anorectal, Cardiac, Tracheoesophageal, Genitourinary, and Limb” (VACTERL) malformation association to identify potential biomarkers that may be helpful for preventing malignancy risk or other chronic processes. Among the variants, six variants that may be linked with VACTERL were identified in the exome analysis. The variants c.501G>C on OLR1 and c.-8C>G on PSMA6 were previously associated with myocardial infarction. The variants c.1936A>G on AKAP10 and c.575A>G on PON1 are linked to defects in cardiac conduction and artery disease, respectively. Alterations in metabolism were also suggested by the variants c.860G>A on EPHX2 and c.214C>A on GHRL. In addition, three variants associated with colon cancer were discovered. Specifically, the reported variants were c.723G>A on CCND1 and c.91T>A on AURKA proto-oncogenes as well as c.827A>C in the tumor suppressor PTPRJ. A further inspection identified 15 rare variants carried by cancer genes. Specifically, these mutations are located on five tumor suppressors (SDHA, RB1CC1, PTCH1, DMBT1, BCR) and eight proto-oncogenes (MERTK, CSF1R, MYB, ROS1, PCM1, FGFR2, MYH11, BRCC3) and have an allele frequency lower than 0.01 in the Genome Aggregation Database (GnomAD). We observed that the cardiac and metabolic phenotypic traits are linked with the genotype of the patient. In addition, the risk of developing neoplasia cannot be excluded a priori. Long-term surgical issues of patients with VATER syndrome could benefit from the clinical exome sequencing of a personalized risk assessment for the appearance of further disease in pubertal timing and adult age.

Acquired congenital esophageal malformations, such as malignant esophageal cancer, require esophagectomy resulting in full thickness resection, which cannot be left untreated. The proposed approach is a polymeric full-thickness scaffold engineered with mesenchymal stem cells (MSCs) to promote and speed up the regeneration process, ensuring adequate support and esophageal tissue reconstruction and avoiding the use of autologous conduits. Copolymers poly-L-lactide-co-poly-ε-caprolactone (PLA-PCL) 70:30 and 85:15 ratio were chosen to prepare electrospun tubular scaffolds. Electrospinning apparatus equipped with two different types of tubular mandrels: cylindrical (∅ 10 mm) and asymmetrical (∅ 10 mm and ∅ 8 mm) were used. Tubular scaffolds underwent morphological, mechanical (uniaxial tensile stress) and biological (MTT and Dapi staining) characterization. Asymmetric tubular geometry resulted in the best properties and was selected for in vivo surgical implantation. Anesthetized pigs underwent full thickness circumferential resection of the mid-lower thoracic esophagus, followed by implantation of the asymmetric scaffold. Preliminary in vivo results demonstrated that detached stitch suture achieved better results in terms of animal welfare and scaffold integration; thus, it is to be preferred to continuous suture.

The inflammatory response plays a central role in the complications of congenital pulmonary airway malformations (CPAM) and severe coronavirus disease 2019 (COVID-19). The aim of this study was to evaluate the transcriptional changes induced by SARS-CoV-2 exposure in pediatric MSCs derived from pediatric lung (MSCs-lung) and CPAM tissues (MSCs-CPAM) in order to elucidate potential pathways involved in SARS-CoV-2 infection in a condition of exacerbated inflammatory response. MSCs-lung and MSCs-CPAM do not express angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TRMPSS2). SARS-CoV-2 appears to be unable to replicate in MSCs-CPAM and MSCs-lung. MSCs-lung and MSCs-CPAM maintained the expression of stemness markers MSCs-lung show an inflammatory response (IL6, IL1B, CXCL8, and CXCL10), and the activation of Notch3 non-canonical pathway, this route appears silent in MSCs-CPAM, and cytokine genes expression is reduced. Decreased value of p21 in MSCs-lung suggested no cell cycle block, and cells did not undergo apoptosis. MSCs-lung appears to increase genes associated with immunomodulatory function but could contribute to inflammation, while MSCs-CPAM keeps stable or reduce the immunomodulatory receptors expression, but they also reduce their cytokines expression. These data indicated that, independently from their perilesional or cystic origin, the MSCs populations already present in a patient affected with CPAM are not permissive for SARS-CoV-2 entry, and they will not spread the disease in case of infection. Moreover, these MSCs will not undergo apoptosis when they come in contact with SARS-CoV-2, on the contrary, they maintain their staminality profile.

Immunoglobulin light-chain amyloidosis (AL) is caused by misfolded light chains produced by a small B cell clone. Mesenchymal stromal cells (MSCs) have been reported to affect plasma cell behavior. We aimed to characterize bone marrow (BM)-MSCs from AL patients, considering functional aspects, such as proliferation, differentiation, and immunomodulatory capacities. MSCs were in vitro expanded from the BM of 57 AL patients and 14 healthy donors (HDs). MSC surface markers were analyzed by flow cytometry, osteogenic and adipogenic differentiation capacities were in vitro evaluated, and co-culture experiments were performed in order to investigate MSC immunomodulatory properties towards the ALMC-2 cell line and HD peripheral blood mononuclear cells (PBMCs). AL-MSCs were comparable to HD-MSCs for morphology, immune-phenotype, and differentiation capacities. AL-MSCs showed a reduced proliferation rate, entering senescence at earlier passages than HD-MSCs. The AL-MSC modulatory effect on the plasma-cell line or circulating plasma cells was comparable to that of HD-MSCs. To our knowledge, this is the first study providing a comprehensive characterization of AL-MSCs. It remains to be defined if the observed abnormalities are the consequence of or are involved in the disease pathogenesis. BM microenvironment components in AL may represent the targets for the prevention/treatment of the disease in personalized therapies.

The explanation for cancer recurrence still remains to be fully elucidated. Moreover, tumor dormancy, which is a process whereby cells enter reversible G0 cell cycle arrest, appears to be a critical step in this phenomenon. We evaluated the cell cycle proliferation pattern in pediatric tumor-derived mesenchymal stromal cells (MSCs), in order to provide a better understanding of the complex mechanisms underlying cancer dormancy. Specimens were obtained from 14 pediatric patients diagnosed with solid tumors and submitted to surgery. Morphology, phenotype, differentiation, immunological capacity, and proliferative growth of tumor MSCs were studied. Flow cytometric analysis was performed to evaluate the cell percentage of each cell cycle phase. Healthy donor bone marrow-derived mesenchymal stromal cells (BM-MSCs) were employed as controls. It was noted that the DNA profile of proliferating BM-MSC was different from that of tumor MSCs. All BM-MSCs expressed the typical DNA profile of proliferating cells, while in all tumor MSC samples, ≥70% of the cells were detected in the G0/G1 phase. In particular, seven tumor MSC samples displayed intermediate cell cycle behavior, and the other seven tumor MSC samples exhibited a slow cell cycle. The increased number of tumor MSCs in the G0–G1 phase compared with BM-MSCs supports a role for quiescent MSCs in tumor dormancy regulation. Understanding the mechanisms that promote dormant cell cycle arrest is essential in identifying predictive markers of recurrence and to promote a dedicated surgical planning.

Background Mesenchymal stromal cell (MSC)-mediated therapeutic effects have been observed in the treatment of lung diseases. For the first time, this treatment was used as rescue therapy in a pediatric patient with a life-threatening respiratory syndrome associated with the filamin A (FLNA) gene mutation. Methods A child with a new pathogenic variant of the FLNA gene c.7391_7403del (p.Val2464AlafsTer5), at the age of 18 months, due to serious and irreversible chronic respiratory failure, was treated with repeated intravenous infusions of allogeneic bone marrow (BM)-MSCs. The child's respiratory condition was monitored. Immunologic studies before each MSC treatment were performed. Results No acute adverse events related to the MSC infusions were observed. After the second infusion, the child's respiratory condition progressively improved, with reduced necessity for mechanical ventilation support. A thorax computed tomography (CT) scan showed bilateral recovery of the basal parenchyma, anatomical-functional alignment and aerial penetration improvement. After the first MSC administration, an increase in Th17 and FoxP3+ T percentages in the peripheral blood was observed. After the second MSC infusion, a significant rise in the Treg/Th17 ratio was noted, as well as an increased percentage of CD20+ /CD19+ B lymphocytes and augmented PHA-induced proliferation. Discussion MSC infusions are a promising therapeutic modality for patients in respiratory failure, as observed in this pediatric patient with an FLNA mutation. MSCs may have an immunomodulatory effect and thus mitigate lung injury; although in this case, MSC antimicrobial effects may have synergistically impacted the clinical improvements. Further investigations are planned to establish the safety and efficacy of this treatment option for interstitial lung diseases in children.

This work was aimed at defining the suitable test for evaluating Fe3O4 NPs cytotoxicity after short-term exposure in human mesenchymal stem cells (hMSCs) using different viability tests, na...

Despite the growing interest in nanoparticles (NPs), their toxicity has not yet been defined and the development of new strategies and predictive models are required. Human stem cells (SCs) offer a promising and innovative cell-based model. Among SCs, mesenchymal SCs (MSCs) derived from cord lining membrane (CL) may represent a new species-specific tool for establishing efficient platforms for primary screening and toxicity/safety testing of NPs. Superparamagnetic iron oxide NPs, including magnetite (Fe3 O4 NPs), have aroused great public health and scientific concerns despite their extensive uses. In this study, CL-MSCs were characterized and applied for in vitro toxicity screening of Fe3 O4 NPs. Cytotoxicity, internalization/uptake, differentiation and proliferative capacity were evaluated after exposure to different Fe3 O4 NP concentrations. Data were compared with those obtained from bone marrow (BM)-MSCs. We observed, at early passages (P3), that: (1) cytotoxicity occurred at 10 μg/mL in CL-MSCs and 100 μg/mL in BM-MSCs (no differences in toxicity, between CL- and BM-MSCs, were observed at higher dosage, 100-300 μg/mL); (2) cell density decrease and monolayer features loss were affected at ≥50 μg/mL in CL-MSCs only; and (3) NP uptake was concentration-dependent in both MSCs. After 100 μg/mL Fe3 O4 NP exposures, the capacity of proliferation was decreased (P5-P9) in CL-MSCs without morphology alteration. Moreover, a progressive decrease of intracellular Fe3 O4 NPs was observed over culture time. Antigen surface expression and multilineage differentiation were not influenced. These findings suggest that CL-MSCs could be used as a reliable cell-based model for Fe3 O4 NP toxicity screening evaluation and support the use of this approach for improving the confidence degree on the safety of NPs to predict health outcomes.

Aim: To validate the use of ultrafiltration (UF) as an alternative applicable industrial method to replace ultracentrifugation (UC) in the purification of mesenchymal stromal cell (MSC)-secretome. Materials & methods: Pharmaceutical formulations containing secretome and/or extracellular vesicles were extracted from adipose-MSCs and bone marrow-MSCs by combining UF or UC with lyophilization. Results & conclusion: UF led to higher protein, lipid, cytokine and exosomes yields compared with UC. The isolation procedure and cell source influenced immunomodulatory activity, which was in vitro evaluated by inhibition of phytohemagglutinin-activated peripheral blood mononuclear cell proliferation, and by modulation of IL-10, IFN-γ and IL-6. A secretome dosage was identified to obtain the same immunomodulatory activity of MSCs, paving the way for cell-free therapy.

BACKGROUND. The human bone marrow (BM) niche contains a population of mesenchymal stromal cells (MSCs) that provide physical support and regulate hematopoietic stem cell (HSC) homeostasis. β-Thalassemia (BT) is a hereditary disorder characterized by altered hemoglobin beta-chain synthesis amenable to allogeneic HSC transplantation and HSC gene therapy. Iron overload (IO) is a common complication in BT patients affecting several organs. However, data on the BM stromal compartment are scarce. METHODS. MSCs were isolated and characterized from BM aspirates of healthy donors (HDs) and BT patients. The state of IO was assessed and correlated with the presence of primitive MSCs in vitro and in vivo. Hematopoietic supportive capacity of MSCs was evaluated by transwell migration assay and 2D coculture of MSCs with human CD34+ HSCs. In vivo, the ability of MSCs to facilitate HSC engraftment was tested in a xenogenic transplant model, whereas the capacity to sustain human hematopoiesis was evaluated in humanized ossicle models. RESULTS. We report that, despite iron chelation, BT BM contains high levels of iron and ferritin, indicative of iron accumulation in the BM niche. We found a pauperization of the most primitive MSC pool caused by increased ROS production in vitro which impaired MSC stemness properties. We confirmed a reduced frequency of primitive MSCs in vivo in BT patients. We also discovered a weakened antioxidative response and diminished expression of BM niche–associated genes in BT-MSCs. This caused a functional impairment in MSC hematopoietic supportive capacity in vitro and in cotransplantation models. In addition, BT-MSCs failed to form a proper BM niche in humanized ossicle models. CONCLUSION. Our results suggest an impairment in the mesenchymal compartment of BT BM niche and highlight the need for novel strategies to target the niche to reduce IO and oxidative stress before transplantation. FUNDING. This work was supported by the SR-TIGET Core grant from Fondazione Telethon and by Ricerca Corrente.

Different mechanisms were proposed as responsible for COVID-19 neurological symptoms but a clear one has not been established yet. In this work we aimed to study SARS-CoV-2 capacity to infect pediatric human cortical neuronal HCN-2 cells, studying the changes in the transcriptomic profile by next generation sequencing. SARS-CoV-2 was able to replicate in HCN-2 cells, that did not express ACE2, confirmed also with Western blot, and TMPRSS2. Looking for pattern recognition receptor expression, we found the deregulation of scavenger receptors, such as SR-B1, and the downregulation of genes encoding for Nod-like receptors. On the other hand, TLR1, TLR4 and TLR6 encoding for Toll-like receptors (TLRs) were upregulated. We also found the upregulation of genes encoding for ERK, JNK, NF-κB and Caspase 8 in our transcriptomic analysis. Regarding the expression of known receptors for viral RNA, only RIG-1 showed an increased expression, downstream RIG-1, the genes encoding for TRAF3, IKKε and IRF3 were downregulated. We also found the upregulation of genes encoding for chemokines and accordingly we found an increase in cytokine/chemokine levels in the medium. According to our results, it is possible to speculate that additionally to ACE2 and TMPRSS2, also other receptors may interact with SARS-CoV-2 proteins and mediate its entry or pathogenesis in pediatric cortical neurons infected with SARS-CoV-2. In particular, TLRs signaling could be crucial for the neurological involvement related to SARS-CoV-2 infection.

Simple Summary The most common cause of late allograft failure is chronic active antibody-mediated rejection (ABMR), but no effective therapy is available. Different immunosuppressive drugs in combination with procedures that remove serum antibodies have been used and the results have not shown to improve graft and patient outcome, but only an increased risk of adverse events. Extracorporeal pho-topheresis (ECP) is leukapheresis-based immunomodulatory therapy not associated with adverse effect, in which lymphocytes treat-ed with 8-methoxypsoralen (8-MOP) are irradiated with ultraviolet-A (UVA) ex vivo and re-infused into the patient. In this study we investigated therapeutic long-term effect of ECP in patients with biopsy proved chronic ABMR. Abstract Objective: Chronic renal antibody-mediated rejection (ABMR) is a common cause of allograft failure, but an effective therapy is not available. Extracorporeal photopheresis (ECP) has been proven successful in chronic lung and heart rejection, and graft versus host disease. The aim of this study was to evaluate the effectiveness of ECP in chronic ABMR patients. Patients and Methods: We investigated ECP treatment in 14 patients with biopsy-proven chronic ABMR and stage 2–3 chronic renal failure. The primary aim was to e valuate the eGFR lowering after 1 year of ECP therapy. The ECP responders (R) showed eGFR reduction greater than 20% vs the basal levels. We also evaluated the effectiveness of ECP on proteinuria, anti-HLA antibodies (HLAab), interleukin 6 (IL-6) serum levels, and CD3, CD4, CD8, CD19, NK, Treg and T helper 17 (Th17) circulating cells. Results: Three patients dropped out of the study. The R patients were eight (72.7%) out of the 11 remaining patients. Because ECP was not associated with any adverse reaction, the R patients continued such treatment for up to 3 years, showing a persisting eGFR stabilization. Twenty four hour proteinuria did not increase in the R patients over the follow-up when compared to the non-responder patients (NR). In the R patients, the HLAab levels were reduced and completely cleared in six out of eight patients when compared with the NR patients. The NR HLAab levels also increased after the discontinuation of the ECP. The ECP in the R patients showed a decrease in CD3, CD4, CD8, CD19, and NK circulating cells. The ECP treatment in the R patients also induced Tregs and Th17 cell increases, and a decrease of the IL-6 serum levels. Conclusions: ECP abates the HLAab titer and renal failure progression in patients with chronic renal ABMR, modulating the immune cellular and humoral responses.

In the last few decades, obesity has increased dramatically in pediatric patients. Obesity is a chronic disease correlated with systemic inflammation, characterized by the presence of CD4 and CD8 T cell infiltration and modified immune response, which contributes to the development of obesity related diseases and metabolic disorders, including impaired glucose metabolism. In particular, Treg and Th17 cells are dynamically balanced under healthy conditions, but imbalance occurs in inflammatory and pathological states, such as obesity. Some studies demonstrated that peripheral Treg and Th17 cells exhibit increased imbalance with worsening of glucose metabolic dysfunction, already in children with obesity. In this review, we considered the role of adipose tissue immunomodulation and the potential role played by Treg/T17 imbalance on the impaired glucose metabolism in pediatric obesity. In the patient care, immune monitoring could play an important role to define preventive strategies of pediatric metabolic disease treatments.

Neste trabalho são analisadas as motivações que auxiliaram a efetivação do convenio que cedeu professores a lecionar para crianças num hospital infantil em Curitiba - PR entre os anos de 1988 a 2002, e assim identificar o papel da educação para as crianças curitibanas num espaço totalmente diferenciado e paradoxal ao escolar, mas com objetivo educacional, o universo hospitalar. Além disto, a tese busca descrever o processo de efetivação dos atendimentos educacionais, analisar os objetivos e o modelo de educação para as crianças curitibanas internadas num hospital infantil e como esta prática de educação dentro de um hospital se desdobra. O ofício do Historiador dá elementos para tratar a temática pelas bordas, pois a pesquisa trata de algo que se encontra dentro de um hospital, sendo que a análise que se propõe com esse trabalho não é com o enaltecimento, mas sim a análise e produção de inteligibilidade, apontando as dificuldades do projeto. Ao falar do passado como diferença, a história mostra seu presente no qual é escrita, mostrando uma ausência no passado e um limite no interior do presente do qual é possível ultrapassar. Considera-se que o atendimento educacional hospitalar é relevante para a educação, pois com sua existência, crianças e jovens que estão impossibilitados de frequentar as escolas em decorrência de internamento hospitalar ou tratamento de saúde são beneficiadas e têm seus direitos garantidos, afinal, com as aulas durante seus internamentos, os alunos não interrompem seu processo de escolarização, o que é um direito previsto principalmente no Cap., IV, Artigos 53 e 54 do Estatuto da Criança e do Adolescente (BRASIL, 1990). Além disso, o direito à educação foi aceito internacionalmente a partir da Declaração dos Direitos da Criança feito pelas Nações Unidas em 1959 (com exceções da África do Sul) e significou igualdade de acesso para todos. A educação em hospitais vem procurando cumprir este direito, garantido em lei, para que a igualdade realmente se efetive. O recorte temporal é balizado pela realização do primeiro convênio entre a Secretaria de Estado da Educação para cessão de professores, que realizariam atendimentos educacionais às crianças internadas em um hospital infantil de Curitiba, no ano de 1988, ligado ao setor da assistência social, até o ano de 2002, quando é transferido o convênio para o setor de educação e cultura criado no hospital em questão. Com a cessão de professoras que iniciariam o atendimento educacional às crianças hospitalizadas, surgem alguns questionamentos, que serão respondidos com esse trabalho. Dentre eles destaco quais eram os espaços, tempos e práticas de educação que se desdobravam dentro do hospital? Neste sentido, as primeiras professoras do hospital destacaram que é possível identificar, por conta da especificidade do trabalho, necessidades das crianças ou até mesmo possibilidade de efetivação das atividades por conta do estado físico em virtude da doença, que elas precisaram se adaptar a uma educação diferenciada que acabava se distanciando da escola regular e do ensino tradicional, afinal a questão do tempo e espaço escolar era totalmente diferenciadas. This paper will analyze the motivations that helped to implement the agreement that reallocated teachers to teach children in a children\'s hospital in Curitiba - PR between 1988 and 2002, thus identifying the importance of education for the children in Curitiba in a totally different and paradoxical to the school, but for educational purposes, the hospital universe. In addition, to understand the process of effecting educational care, to analyze the objectives and the model of education for the children in Curitiba, hospitalized in a children\'s hospital and to investigate how this practice of education within a hospital unfolds. The office of the Historian gives elements to deal with the issue by the edges, since the research deals with something that is found inside a hospital, and the analysis that is proposed with this work is not with the exaltation, but the analysis and production of intelligibility, pointing out the difficulties of the project. In speaking of the past as difference, history shows its present in which it is written, showing an absence in the past and a limit within the present from which it is possible to overcome. It is considered that the hospital pedagogical service is relevant for education, because with its existence, children and young people who are unable to attend schools due to hospital admission or health treatment are benefited and their rights are guaranteed, after all, with the classes during their hospitalizations, students do not interrupt their schooling process, which is a right foreseen mainly in Cap., IV, Articles 53 and 54 of the Statute of the Child and Adolescent (BRASIL, 1990). In addition, the right to education was accepted internationally on the basis of the United Nations Declaration of the Rights of the Child in 1959 (with the exceptions of South Africa) and meant equal access for all, education in hospitals has been seeking to fulfill this right, guaranteed by law, so that equality really takes effect. The temporal cut-off is marked by the first agreement between the State Secretariat of Education for the release of teachers, which would provide educational assistance to children admitted to a children\'s hospital in Curitiba in 1988, linked to the social assistance sector, until the year of 2002, when the agreement for the education and culture sector of the hospital in question is created. With the release of teachers who would initiate educational services to hospitalized children, some questions arise, which will be answered with this work: What were the spaces, times and practices of education that unfolded within the hospital? In this sense, the first teachers of the hospital emphasized that it is possible to identify, due to the specificity of the work, children\'s needs or even the possibility of carrying out the activities due to the physical state due to the disease, that they needed to adapt to an education differentiated from the regular school and traditional education, after all the question of time and school space was totally different.

In the last decade, the secreting activity of mesenchymal stem/stromal cells (MSCs) has been widely investigated, due to its possible therapeutic role. In fact, MSCs release extracellular vesicles (EVs) containing relevant biomolecules such as mRNAs, microRNAs, bioactive lipids, and signaling receptors, able to restore physiological conditions where regenerative or anti-inflammatory actions are needed. An actual advantage would come from the therapeutic use of EVs with respect to MSCs, avoiding the possible immune rejection, the lung entrapment, improving the safety, and allowing the crossing of biological barriers. A number of concerns still have to be solved regarding the mechanisms determining the beneficial effect of MSC-EVs, the possible alteration of their properties as a consequence of the isolation/purification methods, and/or the best approach for a large-scale production for clinical use. Most of the preclinical studies have been successful, reporting for MSC-EVs a protecting role in acute kidney injury following ischemia reperfusion, a potent anti-inflammatory and anti-fibrotic effects by reducing disease associated inflammation and fibrosis in lung and liver, and the modulation of both innate and adaptive immune responses in graft versus host disease (GVHD) as well as autoimmune diseases. However, the translation of MSC-EVs to the clinical stage is still at the initial phase. Herein, we discuss the therapeutic potential of an acellular product such as MSC derived EVs (MSC-EVs) in acute and chronic pathologies.

Background and Aims Adenosine (Ado), the main substrate of ATP, is a potent endogenous anti-inflammatory nucleoside. Hypoxia induces ATP depletion, AMP extracellular increase that is phosphohydrolyzed to ADO by the enzyme ecto-5′-nucleotidase (CD73). Constitutive CD73 expression is higher in deep cortex outer medulla that is the most vulnerable region to ischemic injury. Notably CD73 is also expressed on Mesenchymal Stromal Cell (MSC) and is essential phenotypic marker. Previously in a rat model of Donation after Circulatory Death (DCD), we demonstrated that the perfusion of ischemic kidney with MSC or MSC derived Extracellular Vesicles (EV) protects tissue up regulating mitochondrial energetic metabolism genes. The aim of this study was to investigate the role of CD73/Ado system in MSC/EV protection from ischemia. Method Fisher rats were used as kidney donors and Lewis rats as MSC donors. MSC missing CD73 were produced by electroporation in the presence of specific siRNA to block CD73 expression (siMSC). EV were isolated from supernatants of MSC and siMSC (siEV) medium through differential ultracentrifugations. Size distribution and enumeration analysis were performed using NanoSight NS300. EV phenotypic characterization was performed in flow cytometer using CD45, CD49e, CD63, CD9, CD81 and CD73 monoclonal antibodies. DCD model was obtained by renal artery clamping for 20 minutes. This warm ischemia time represents the “no touch period” imposed by the Italian law to death declaration. After nephrectomy, kidneys were perfused in hypothermia (4°C) with Belzer Solution (BS), or BS supplemented with 3x106 MSC (MSC) or BS supplemented with 28,5x109 EV/siEV (EV/siEV). The effluent fluid (EF) was collected at the beginning (T0), every hours (T1, T2, T3) and at the end of the perfusion (T4). Ado and ATP determinations in EF and tissues were performed by HPLC and ELISA, respectively. Results EF Ado was significantly higher in MSC vs BS from T1 and in EV vs BS from T0 to T4 (p Conclusion CD73 expressed on MSC /EV impacts on cell energy metabolism pathway and ATP generation. This is the first evidence that MSC/EV act through CD73/Ado system to prevent ischaemic damage.

To date, relatively few studies have been specifically designed to identify the proteome profile in pulmonary pediatric disease. The aim of this study was to survey proteins present in bronchoalveo...

Splenic hematopoiesis is a major feature in the course of myelofibrosis (MF). In fact, the spleen of patients with MF contains malignant hematopoietic stem cells retaining a complete differentiation program, suggesting both a pivotal role of the spleen in maintaining the disease and a tight regulation of hematopoiesis by the splenic microenvironment, in particular by mesenchymal stromal cells (MSCs). Little is known about splenic MSCs (Sp-MSCs), both in normal and in pathological context. In this work, we have in vitro expanded and characterized Sp-MSCs from 25 patients with MF and 13 healthy subjects (HS). They shared similar phenotype, growth kinetics, and differentiation capacity. However, MF Sp-MSCs expressed significant lower levels of nestin, and favored megakaryocyte (Mk) differentiation in vitro at a larger extent than their normal counterpart. Moreover, they showed a significant upregulation of matrix metalloprotease 2 (MMP2) and fibronectin 1 (FN1) genes both at mRNA expression and at protein level, and, finally, developed genetic abnormalities which were never detected in HS-derived Sp-MSCs. Our data point toward the existence of a defective splenic niche in patients with MF that could be responsible of some pathological features of the disease, including the increased trafficking of CD34+ cells and the expansion of the megakaryocytic lineage.

Mesenchymal stromal cells (MSCs) have been proposed as a potential therapy to treat congenital and acquired lung diseases. Due to their tissue-regenerative, anti-fibrotic, and immunomodulatory properties, MSCs combined with other therapy or alone could be considered as a new approach for repair and regeneration of the lung during disease progression and/or after post- surgical injury. Children interstitial lung disease (chILD) represent highly heterogeneous rare respiratory diseases, with a wild range of age of onset and disease expression. The chILD is characterized by inflammatory and fibrotic changes of the pulmonary parenchyma, leading to gas exchange impairment and chronic respiratory failure associated with high morbidity and mortality. The therapeutic strategy is mainly based on the use of corticosteroids, hydroxychloroquine, azithromycin, and supportive care; however, the efficacy is variable, and their long-term use is associated with severe toxicity. The role of MSCs as treatment has been proposed in clinical and pre-clinical studies. In this narrative review, we report on the currently available on MSCs treatment as therapeutical strategy in chILD. The progress into the therapy of respiratory disease in children is mandatory to ameliorate the prognosis and to prevent the progression in adult age. Cell therapy may be a future therapy from both a pediatric and pediatric surgeon’s point of view.

Introduction The association between congenital pulmonary airway malformations (CPAM) and malignancy is reported in the literature. Interactions between the tumor, immune, and mesenchymal stromal/stem cells (MSCs) have been recognized as crucial for understanding tumorigenesis. We characterized MSCs isolated from CPAM lesions in order to define potential malignancy risks. Methods CPAM II pulmonary tissue was used for MSC expansion; a “healthy” lung section from the same child was used as a comparator. Morphology, immunophenotype, differentiation and immunological capacity, proliferative growth, gene signature telomerase activity, and in vivo tumorigenicity in nude mice were evaluated. Results MSCs were successfully isolated and propagated from CPAM tissue. CPAM-MSCs presented the typical MSC morphology and phenotype, while exhibiting high proliferative capacity, reaching confluence at a median time of 5 days as well as differentiation capabilities. CPAM-MSCs at early passages were not neoplastic and chromosomally normal, even though unbalanced chromosomal rearrangements were noted by molecular karyotype. Conclusions CPAM-MSCs exhibited specific features similar to tumor derived MSCs. Whilst there was no evidence of malignant transformation in the cystic tissue, our results provide evidence that this abnormal tissue has malignant potential. MSCs are considered important players in the tumor microenvironment and they have been closely linked to regulation of tumor survival, growth, and progression. Thus, early lesion resection also in asymptomatic patients might be indicated to exclude that the microenvironment may be potentially permissive to cancer development.

Allogeneic liver transplantation is still deemed the gold standard solution for end-stage organ failure; however, donor organ shortages have led to extended waiting lists for organ transplants. In order to overcome the lack of donors, the development of new therapeutic options is mandatory. In the last several years, organ bioengineering has been extensively explored to provide transplantable tissues or whole organs with the final goal of creating a three-dimensional growth microenvironment mimicking the native structure. It has been frequently reported that an extracellular matrix-based scaffold offers a structural support and important biological molecules that could help cellular proliferation during the recellularization process. The aim of the present review is to underline the recent developments in cell-on-scaffold technology for liver bioengineering, taking into account: (1) biological and synthetic scaffolds; (2) animal and human tissue decellularization; (3) scaffold recellularization; (4) 3D bioprinting; and (5) organoid technology. Future possible clinical applications in regenerative medicine for liver tissue engineering and for drug testing were underlined and dissected.

Highlights • Seventeen EBMT centers participated to a questionnaire on MSC manufacturing. • 88% of centers manufacture MSC from bone marrow and only 2 centers from umbilical cord. • Human platelet lysate has replaced bovine serum as culture medium supplement. • Release criteria extensively differ among centers. • The results highlight the need to harmonize MSC manufacturing., The immunosuppressive properties of mesenchymal stromal cells (MSC) have been successfully tested to control clinical severe graft-versus host disease and improve survival. However, clinical studies have not yet provided conclusive evidence of their efficacy largely because of lack of patients’ stratification criteria. The heterogeneity of MSC preparations is also a major contributing factor, as manufacturing of therapeutic MSC is performed according to different protocols among different centers. Understanding the variability of the manufacturing protocol would allow a better comparison of the results obtained in the clinical setting among different centers. In order to acquire information on MSC manufacturing we sent a questionnaire to the European Society for Blood and Marrow Transplantation centers registered as producing MSC. Data from 17 centers were obtained and analyzed by means of a 2-phase questionnaire specifically focused on product manufacturing. Gathered information included MSC tissue sources, MSC donor matching, medium additives for ex vivo expansion, and data on MSC product specification for clinical release. The majority of centers manufactured MSC from bone marrow (88%), whilst only 2 centers produced MSC from umbilical cord blood or cord tissue. One of the major changes in the manufacturing process has been the replacement of fetal bovine serum with human platelet lysate as medium supplement. 59% of centers used only third-party MSC, whilst only 1 center manufactured exclusively autologous MSC. The large majority of these facilities (71%) administered MSC exclusively from frozen batches. Aside from variations in the culture method, we found large heterogeneity also regarding product specification, particularly in the markers used for phenotypical characterization and their threshold of expression, use of potency assays to test MSC functionality, and karyotyping. The initial data collected from this survey highlight the variability in MSC manufacturing as clinical products and the need for harmonization. Until more informative potency assays become available, a more homogeneous approach to cell production may at least reduce variability in clinical trials and improve interpretation of results.

The aim of this work is to propose a keratinocytes (KC) culture method for clinical practice with irradiated adipose-derived mesenchymal stromal cells (ASCs) as human feeder layer, avoiding murine immortalized fibroblasts, commonly request for producing skin substitutes. ASCs were isolated, expanded, irradiated, and co-cultured with autologous or allogeneic KC. All experiments were performed using murine fibroblasts as control. Cell counts, flow cytometric analysis and ELISA were carried out, in order to define cell yield, viability and cytokine secretion. Results indicate that the optimal X-ray dose for ASCs is 120 Gy and the optimal seeding density is 625 cells/cm2; moreover, flow cytometric analysis shows that the percentage of feeder layer cells reaches values lower than 1%, within 8 days of co-culture. KC reach confluence in 6.9 days on ASCs substrate and, after confluence, the number of live cells increases again in a multilayered structure. Moreover, results show higher levels of interleukin (IL)-1a in co-culture with ASCs compared with 3T3, while no differences were observed for IL-6 and IL-8. Therefore, human ASCs enable to obtain effectively in vitro expanded KC and represent a viable alternative to murine fibroblasts for the production of clinical use skin substitutes.

Aim: We evaluated the effects of the intradermal injection of extracellular vesicles (EVs) derived from adipose stem cells (ASC-EVs) and bone marrow cells (BM-EVs) in an experimental cutaneous wound repair model. Methods: Mesenchymal stem cells (MSCs) were in vitro expanded from adipose (ASC) or BM tissues (BM-MSC) of rabbits. EVs were separated from the supernatants of confluent ASC and BM-MSCs. Two skin wounds were induced in each animal and treated with MSC or EV injections. Histological examination was performed postinoculation. Results: EV-treated wounds exhibited a better restoration compared with the counterpart MSC treatment. ASC-EV-treated wounds were significantly better than BM-EVs (p = 0.036). Conclusion: EV topical inoculation provides restored architecture during cutaneous wound healing and represents a promising solution for regenerative medicine in children.

Background It has been proposed that mesenchymal stromal cells (MSCs) promote tumor progression by interacting with tumor cells and other stroma cells in the complex network of the tumor microenvironment. We characterized MSCs isolated and expanded from tumor tissues of pediatric patients diagnosed with neuroblastomas (NB-MSCs) to define interactions with the tumor microenvironment. Methods Specimens were obtained from 7 pediatric patients diagnosed with neuroblastoma (NB). Morphology, immunophenotype, differentiation capacity, proliferative growth, expression of stemness and neural differentiation markers were evaluated. Moreover, the ability of cells to modulate the immune response, i.e. inhibition of phytohemagglutinin (PHA) activated peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cytotoxic function, was examined. Gene expression profiles, known to be related to tumor cell stemness, Wnt pathway activation, epithelial-mesenchymal transition (EMT) and tumor metastasis were also evaluated. Healthy donor bone marrow-derived MSCs (BM-MSC) were employed as controls. Results NB-MSCs presented the typical MSC morphology and phenotype. They showed a proliferative capacity superimposable to BM-MSCs. Stemness marker expression (Sox2, Nanog, Oct3/4) was comparable to BM-MSCs. NB-MSC in vitro osteogenic and chondrogenic differentiation was similar to BM-MSCs, but NB-MSCs lacked adipogenic differentiation capacity. NB-MSCs reached senescence phases at a median passage of P7 (range, P5-P13). NB-MSCs exhibited greater immunosuppressive capacity on activated T lymphocytes at a 1:2 (MSC: PBMC) ratio compared with BM-MSCs (p = 0.018). NK cytotoxic activity was not influenced by co-culture, either with BM-MSCs or NB-MSCs. Flow-cytometry cell cycle analysis showed that NB-MSCs had an increased number of cells in the G0-G1 phase compared to BM-MSCs. Transcriptomic profiling results indicated that NB-MSCs were enriched with EMT genes compared to BM-MSCs. Conclusions We characterized the biological features, the immunomodulatory capacity and the gene expression profile of NB-MSCs. The NB-MSC gene expression profile and their functional properties suggest a potential role in promoting tumor escape, invasiveness and metastatic traits of NB cancer cells. A better understanding of the complex mechanisms underlying the interactions between NB cells and NB-derived MSCs should shed new light on potential novel therapeutic approaches. Electronic supplementary material The online version of this article (10.1186/s12885-018-5082-2) contains supplementary material, which is available to authorized users.

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: 5mL/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 108hours. 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 6hours, 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

Background:The association between oxidative stress (OS) and metabolic syndrome (MetS) has been reported in adults. We analyzed the relation between circulating oxidized low-density lipoproteins (Ox-LDL) and MetS in pediatric ages in order to define whether plasma Ox-LDL levels are correlated to obesity and whether oxidative damage, using serum Ox-LDL levels as a proxy, are associated with MetS.Methods:We enrolled 178 children (11.8±2.6 years). On the basis of a body mass index (BMI) threshold, the subjects were classified as: normal weight BMI 97th percentile. Patients were classified as having MetS if they met three or more of the following criteria for age and sex: BMI >97th percentile, triglyceride levels >95th percentile, high-density lipoprotein (HDL) cholesterol level 95th percentile and impaired glucose tolerance.Results:Obese children showed increased MetS prevalence (p=0.001) and higher Ox-LDL levels compared to normal- and overweight subjects (pConclusions:Oxidative damage was correlated with a hypertriglyceridemic waist phenotype and can be a precocious marker of MetS and cardiometabolic risk in obese children.