Search

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.

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.

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.

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.

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.

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.

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.

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.

Mesenchymal stem cells (MSCs) were first isolated more than 50 years ago from the bone marrow. Currently MSCs may also be isolated from several alternative sources and they have been used in more than a hundred clinical trials worldwide to treat a wide variety of diseases. The MSCs mechanism of action is undefined and currently under investigation. For in vivo purposes MSCs must be produced in compliance with good manufacturing practices and this has stimulated research on MSCs characterization and safety. The objective of this review is to describe recent developments regarding MSCs properties, physiological effects, delivery, clinical applications and possible side effects.

Kidney donation after circulatory death (DCD) is a less than ideal option to meet organ shortages. Hypothermic machine perfusion (HMP) with Belzer solution (BS) improves the viability of DCD kidneys, although the graft clinical course remains critical. Mesenchymal stromal cells (MSC) promote tissue repair by releasing extracellular vesicles (EV). We evaluated whether delivering MSC‐/MSC‐derived EV during HMP protects rat DCD kidneys from ischaemic injury and investigated the underlying pathogenic mechanisms. Warm ischaemic isolated kidneys were cold‐perfused (4 hrs) with BS, BS supplemented with MSC or EV. Renal damage was evaluated by histology and renal gene expression by microarray analysis, RT‐PCR. Malondialdehyde, lactate, LDH, glucose and pyruvate were measured in the effluent fluid. MSC‐/EV‐treated kidneys showed significantly less global ischaemic damage. In the MSC/EV groups, there was up‐regulation of three genes encoding enzymes known to improve cell energy metabolism and three genes encoding proteins involved in ion membrane transport. In the effluent fluid, lactate, LDH, MDA and glucose were significantly lower and pyruvate higher in MSC/EV kidneys as compared with BS, suggesting the larger use of energy substrates by MSC/EV kidneys. The addition of MSC/EV to BS during HMP protects the kidney from ischaemic injury by preserving the enzymatic machinery essential for cell viability and protects the kidney from reperfusion damage.

Background: Anti-T lymphocyte globulin (ATLG) modulates the alloreactivity of T lymphocytes, reducing the risk of immunological posttransplant complications, in particular rejection and graft-versushost disease, after allogeneic hematopoietic stem cell transplantation (HSCT). We developed and validated a new enzyme-linked immunosorbent assay (ELISA) method to measure serum levels of total ATLG and evaluate the pharmacokinetics (PK) of the drug in children with b-Thalassemia, receiving allogeneic HSCT. Methods: Diluted serum samples were incubated with Goat-anti- Rabbit IgG antibody coated on a microtiter plate and then, with Goat-anti-Human IgG labeled with horseradish peroxidase. After incubation and washings, substrate solution was added and absorbance was read at 492 nm. ATLG concentrations in samples were determined by interpolation from a standard curve (range: 200-0.095 ng/mL), prepared by diluting a known amount of ATLG in phosphate-buffered saline (PBS). Low, medium, and high-quality control concentrations were 1.56, 6.25, and 25 ng/ mL, respectively. This method was developed and validated within the acceptance criteria in compliance with the Guidelines for a biological method validation: the sensitivity of the method was 0.095 ng/mL. We analyzed serum samples from 14 children with b-Thalassemia who received ATLG (Grafalon) at a dose of 10 mg/kg administered as intravenous (IV) infusion on days 25, 24, and 23 before HSCT (day 0). Blood sampling for PK evaluation was performed on days 25, 24, and 23 before and after drug infusion; and then from day 22 to +56. Results: The median total ATLG levels pre-IVand post-IV were 0 and 118 mcg/mL on day 25; 85.9 and 199.2 mcg/mL on day 24; 153 and 270.9 mcg/mL on day 23, respectively. The median PK values of CL was 0.0029 (range: 0.0028-0.0057) L$kg21$d21, Vd was 0.088 (range: 0.025-0.448) L/kg and t1/2 was 20.2 (range: 5.8- 50.2) days. Conclusions: These data suggest that given the marked interindividual variability of total ATLG disposition, the development of a validated ELISA method and the possibility to measure PK parameters in paediatric populations are essential steps to optimize drug therapeutic regimens.

Human mesenchymal stromal cells (MSCs) expanded in vitro for cell therapy approaches need to be carefully investigated for genetic stability, by employing both molecular and conventional karyotyping. Reliability of cytogenetic analysis may be hampered in some MSC samples by the difficulty of obtaining an adequate number of metaphases. In an attempt to overcome this problem, a methodology apt to evaluate the cell-cycle structure on synchronous MSCs was optimised. Results obtained in five independent experiments by comparing cell-cycle analysis of synchronous and asynchronous MSC populations evaluated at early and late culture passages documented that in synchronous MSCs, 30% of cells entered G2/M phase after about 27–28 h of culture, while in asynchronous MSCs only 8% of cells in G2/M phase could be observed at the same time point. Cytogenetic analysis on synchronous MSCs allowed us to obtain 20–25 valuable metaphases/slide, whereas only 0–4 metaphases/slide were detectable in asynchronous preparations. J. Cell. Biochem. 112: 1817–1821, 2011. © 2011 Wiley-Liss, Inc.

Significant improvement in the understanding of mesenchymal stem cell (MSC) biology has opened the way to their clinical use. However, concerns regarding the possibility that MSCs undergo malignant transformation have been raised. We investigated the susceptibility to transformation of human bone marrow (BM)–derived MSCs at different in vitro culture time points. MSCs were isolated from BM of 10 healthy donors and propagated in vitro until reaching either senescence or passage (P) 25. MSCs in the senescence phase were closely monitored for 8 to 12 weeks before interrupting the cultures. The genetic characterization of MSCs was investigated through array-comparative genomic hybridization (array-CGH), conventional karyotyping, and subtelomeric fluorescent in situ hybridization analysis both before and after prolonged culture. MSCs were tested for the expression of telomerase activity, human telomerase reverse transcriptase (hTERT) transcripts, and alternative lengthening of telomere (ALT) mechanism at different passages. A huge variability in terms of proliferative capacity and MSCs life span was noted between donors. In eight of 10 donors, MSCs displayed a progressive decrease in proliferative capacity until reaching senescence. In the remaining two MSC samples, the cultures were interrupted at P25 to pursue data analysis. Array-CGH and cytogenetic analyses showed that MSCs expanded in vitro did not show chromosomal abnormalities. Telomerase activity and hTERT transcripts were not expressed in any of the examined cultures and telomeres shortened during the culture period. ALT was not evidenced in the MSCs tested. BM-derived MSCs can be safely expanded in vitro and are not susceptible to malignant transformation, thus rendering these cells suitable for cell therapy approaches. [Cancer Res 2007;67(19):9142–9]

Alemtuzumab is a humanized (IgG,) rat monoclonal antibody to CD52 antigen and is currently used in the treatment of chronic lymphocytic leukemia (CLL) and other CD52-positive lymphoproliferative disorders. Various techniques have been developed to measure Alemtuzumab levels in human serum/plasma. The authors report on the validation of a very sensitive enzyme-linked immunosorbent assay (ELISA) to measure serum concentrations of the humanized IgG, using a rabbit polyclonal antibody specifically produced against the rat sequence of Alemtuzumab after papain digestion. The assay was successfully applied to test the serum samples of patients with B-lymphocyte CLL who received Alemtuzumab subcutaneously. This ELISA assay could be easily used to determine human serum levels of Alemtuzumab pre- and post-treatment to optimize dosing and scheduling and to study the relationship between dose and clinical response.