Development and validation of cryopreserved or freeze-dried decellularized human dermis for transplantation.

For decades, dermal tissue grafts have been used in various regenerative, reconstructive, and augmentative procedures across the body. To eliminate antigenicity and immunogenic response while still preserving the individual components and collective structural integrity of the extracellular matrix (ECM), dermis can be decellularized. Acellular dermal matrix (ADM) products like such are produced to accurately serve diverse clinical purposes. The aim of the present study is to evaluate the efficacy of a novel decellularization protocol of the human dermis, which eliminates residual human genetic material without compromising the biomechanical integrity and collagenous content of the tissue. Moreover, a freeze-drying protocol was validated. The results showed that though our decellularization protocol, human dermis can be decellularized obtaining a biocompatible matrix. The procedure is completely realized in GMP aseptic condition, avoiding tissue terminal sterilization.

Decellularized cryopreserved human pericardium: a validation study towards tissue bank practice.

Pericardial patches are currently used as reconstructive material in cardiac surgery for surgical treatment of cardiac septal defects. Autologous pericardial patches, either treated with glutaraldehyde or not, can be used as an alternative to synthetic materials or xenograft in congenital septal defects repair. The availability of an allogenic decellularized pericardium could reduce complication during and after surgery and could be a valid alternative. Decellularization of allogenic tissues aims at reducing the immunogenic reaction that might trigger inflammation and tissue calcification over time. The ideal graft for congenital heart disease repair should be biocompatible, mechanically resistant, non-immunogenic, and should have the ability to growth with the patients. The aim of the present study is the evaluation of the efficacy of a new decellularization protocol of homologous pericardium, even after cryopreservation. The technique has proven to be suitable as a tissue bank procedure and highly successful in the removal of cells and nucleic acids content, but also in the preservation of collagen and biomechanical properties of the human pericardium.

Validation of orthopedic allograft preparation process: a new application of media fill test.

Musculoskeletal allografts represent an important practice in orthopedic surgeries and the demand for them has been growing. For this reason, in order to reduce clinical risk and to more efficiently manage the increase of allograft usage and also to optimize timing of the surgeries, the thawing and washing processes with aseptic technique were centralized in the department of Hospital Pharmacy. This study describe the design and execution of an adapted Media Fill Test (MFT) to demonstrate aseptic thawing and washing of allografts. For this specific and innovative setting, to better simulate the actual processing steps, a surrogate system was developed to simulate the tendon allograft. The aseptic technique of four operators was assessed and an initial performance validation and the first revalidation were described. All MFT were completed successfully, with no observation of turbidity. The readapted MFT shown in this study can provide insight into this innovative and growing field to other health professionals who want to implement this service.

Characterization of Human Subcutaneous Adipose Tissue and Validation of the Banking Procedure for Autologous Transplantation.

Adipose tissue (AT) is composed of a heterogeneous population which comprises both progenitor and differentiated cells. This heterogeneity allows a variety of roles for the AT, including regenerative functions. In fact, autologous AT is commonly used to repair soft tissue defects, and its cryopreservation could be a useful strategy to reduce the patient discomfort caused by multiple harvesting procedures. Our work aimed to characterize the cryopreserved AT and to validate its storage for up to three years for clinical applications. AT components (stromal vascular fraction-SVF and mature adipocytes) were isolated in fresh and cryopreserved samples using enzymatic digestion, and cell viability was assessed by immunofluorescence (IF) staining. Live, apoptotic and necrotic cells were quantified using cytometry by evaluating phosphatidylserine binding to fluorescent-labeled Annexin V. A multiparametric cytometry was also used to measure adipogenic (CD34+CD90+CD31-CD45-) and endothelial (CD34+CD31+CD45-) precursors and endothelial mature cells (CD34-CD31+CD45-). The maintenance of adipogenic abilities was evaluated using in vitro differentiation of SVF cultures and fluorescent lipid staining. We demonstrated that AT that is cryopreserved for up to three years maintains its differentiation potential and cellular composition. Given our results, a clinical study was started, and two patients had successful transplants without any complications using autologous cryopreserved AT.

Human amniotic membrane: an improvement in the treatment of Medication-related osteonecrosis of the jaw (MRONJ)? A case-control study.

The aim of this article is to report the results obtained by the use of HAM in surgical wound healing and the reduction of relapse in patients affected by Medication-related osteonecrosis of the jaw (MRONJ).The study involved patients with the diagnosis of MRONJ, surgically treated between October 2016 and April 2019, in a case-control setting. Enrolled patients were randomly divided into 2 groups. One group will be treated with resective surgery and with the insertion of HAM patch (Group A), while the second group had been treated exclusively with resective surgery (Group B).The patients underwent MRONJ surgical treatment with the placement of amniotic membrane patches at the wound site. Data regarding the long-term complications/functions were evaluated at 3, 6, 12, and 24 months after surgery. Pain measurements were performed before the intervention (T0), 7(T1) and 30(T2) days after surgery. 49 patients were included in the study. 2 patients of GROUP A after 30 days since they were surgically treated showed persistent bone exposure. 5 patients of group B demonstrated a lack of healing of the surgical wound with the persistence of bone exposed to 30 days after surgery. Statistical analysis ruled out any difference in OUTCOME (relapse) between GROUP A and B (p = 0.23). However, the Fisher test highlighted a significant difference between the use of HAM and only surgical treatment in pain at rest (p = 0.032). The use of amniotic membrane implement the patient's quality of life and reduce pain perception. has a learning curve that is fast enough to justify its routine use.

Impact of Sars-CoV-2 pandemic on the Veneto Region multitissue bank activity.

Covid pandemic affected donation activities worldwide, especially for living donation due to the lack of elective surgery. Moreover, the number of heart-beating and non-heart beating donors has recorded a decrease. Fondazione Banca dei Tessuti di Treviso (FBTV) is a non-profit healthcare organisation, located in Veneto Region, tasked with procurement, processing, preserving, validating and distributing human tissue for clinical use. During Covid-19 outbreak, operations in FBTV have never stopped and a great effort was required to maintain a standard trend of activity. The aim of this study was to describe the impact of Sars-CoV-2 on the activity of a multitissue bank in Italy. Moreover, we investigated the presence of the virus in tissues retrieved from two Sars-CoV-2 positive cadaver donors. Our survey demonstrated that the transplantation network of Veneto Region has positively reacted to the pandemic scenario, thanks to the effort of all personnel involved. Statistical analyses underlined that most of the activities of the tissue bank were unaffected during the Sars-CoV-2 pandemic.

Contamination profile in allografts retrieved from multitissue donors: longitudinal analysis.

Microbiological contamination of retrieved tissues has become an issue of key importance and is a critical aspect of allograft safety, especially in the case of multi-tissue donations, which frequently become contaminated during retrieval and handling. We analysed contamination in 11,129 tissues with a longitudinal contamination profile for each individual tissue. Specifically, 10,035 musculoskeletal tissues and 1094 cardiovascular tissues were retrieved from a total of 763 multi-tissue donors, of whom 105 heart-beating organ donors and 658 deceased tissue donors. Of the 1955 tissues found to be contaminated after the first decontamination step, 1401 tissues (72%) were contaminated by the same species as the one(s) isolated at retrieval (Time1) and 554 (28%) by different species. Among the 113 tissues testing positive after the 2nd decontamination (Time3), 36 tissues (32%) were contaminated by the same species detected at Timel while the contaminating species differed from Time1 in 77 tissues (68%). The higher the number of contaminating species per tissue the higher the percentage of tissues in which contamination changed over time compared to Time1. The analysis revealed a 28% incidence of new species in tissues already testing positive after retrieval and of 3.5% of tissues becoming positive after admission to the tissue bank. Of these, coagulase-negative Staphylococcus accounted for over 70% of new contaminations.

Stability analysis of the antibiotic cocktail used by Treviso Tissue Bank Foundation for tissues decontamination.

Although careful donor selection reduces tissue contamination, close microbiological control of harvested allografts remains a key task of tissue banks. To guarantee the safety of human tissues for allograft transplantation, a decontamination regimen must be adopted which, as recommended by European guidelines, is active against the majority of microorganisms isolated in tissues. Antibiotic decontamination methods differ from one tissue bank to another in terms of antimicrobial agents, temperature and length of exposure. After identifying the most effective antibiotics against the bacterial strains most commonly isolated in allografts, Treviso Tissue Bank Foundation demonstrated the efficacy of an antibiotic cocktail for tissue decontamination containing Gentamicin, Vancomycin and Meropenem. The aim of this study was to analyse the degradation kinetics of the three antibiotics according to preparation method and use. The results show that only Meropenem is unstable at + 4 °C, while Gentamicin and Vancomycin are valid for over 10 days. We thus established to add Meropenem before the start of the tissue decontamination phase.

Analytic and Dynamic Secretory Profile of Patient-Derived Cytokine-Induced Killer Cells.

Adoptive immunotherapy with Cytokine Induced Killer (CIK) cells has shown antitumor activity against several kinds of cancers in preclinical models and clinical trials. CIK cells are a subset of ex vivo expanded T lymphocytes with T-NK phenotype and MHC-unrestricted antitumor activity. Literature provides scanty information on cytokines, chemokines and growth factors secreted by CIK cells. Therefore, we investigated the secretory profile of CIK cells generated from tumor patients. The secretome analysis was performed at specific time points (day 1, day 14 and day 21) of CIK cells expansion. Mature CIK cells (day 21) produce a great variety of interleukins and secreted proteins that can be divided into 3 groups based on their secretion quantity: high (IL-13, RANTES, MIP-1α and 1ß), medium (IL-1Ra, IL-5, IL-8, IL-10, IL-17, IP-10, INF-γ, VEGF and GMCSF) and low (IL-1ß, IL-4, IL-6, IL-7, IL-9, IL-12, IL-15, Eotaxin, PDGF-bb, FGF basic, G-CSF and MCP-1) secreted. Moreover, comparing PBMC (day 1) and mature CIK cells (day 14 and 21) secretome, we observed that IL-5, IL-10, IL-13, GM-CSF, VEGF resulted greatly up-regulated, while IL-1ß, IL-6, IL-8, IL-15, IL-17, eotaxin, MCP-1, and RANTES were down-regulated. We also performed a gene expression profile analysis of patient-derived CIK cells showing that mRNA for the different cytokines and secreted proteins were modulated during PBMC to CIK differentiation. We highlighted previously unknown secretory properties and provided for the first time a comprehensive molecular characterization of CIK cells. Our findings provide rationale to explore the functional implications and possible therapeutic modulation of CIK secretome.

An Aγ-globin G->A gene polymorphism associated with ß039 thalassemia globin gene and high fetal hemoglobin production.

BACKGROUND: Increase of the expression of γ-globin gene and high production of fetal hemoglobin (HbF) in ß-thalassemia patients is widely accepted as associated with a milder or even asymptomatic disease. The search for HbF-associated polymorphisms (such as the XmnI, BCL11A and MYB polymorphisms) has recently gained great attention, in order to stratify ß-thalassemia patients with respect to expectancy of the first transfusion, need for annual intake of blood, response to HbF inducers (the most studied of which is hydroxyurea). METHODS: Aγ-globin gene sequencing was performed on genomic DNA isolated from a total of 75 ß-thalassemia patients, including 31 ß039/ß039, 33 ß039/ß+IVSI-110, 9 ß+IVSI-110/ß+IVSI-110, one ß0IVSI-1/ß+IVSI-6 and one ß039/ß+IVSI-6. RESULTS: The results show that the rs368698783 polymorphism is present in ß-thalassemia patients in the 5'UTR sequence (+25) of the Aγ-globin gene, known to affect the LYAR (human homologue of mouse Ly-1 antibody reactive clone) binding site 5'-GGTTAT-3'. This Aγ(+25 G->A) polymorphism is associated with the Gγ-globin-XmnI polymorphism and both are linked with the ß039-globin gene, but not with the ß+IVSI-110-globin gene. In agreement with the expectation that this mutation alters the LYAR binding activity, we found that the Aγ(+25 G->A) and Gγ-globin-XmnI polymorphisms are associated with high HbF in erythroid precursor cells isolated from ß039/ß039 thalassemia patients. CONCLUSIONS: As a potential explanation of our findings, we hypothesize that in ß-thalassemia the Gγ-globin-XmnI/Aγ-globin-(G->A) genotype is frequently under genetic linkage with ß0-thalassemia mutations, but not with the ß+-thalassemia mutation here studied (i.e. ß+IVSI-110) and that this genetic combination has been selected within the population of ß0-thalassemia patients, due to functional association with high HbF. Here we describe the characterization of the rs368698783 (+25 G->A) polymorphism of the Aγ-globin gene associated in ß039 thalassemia patients with high HbF in erythroid precursor cells.

Transient Receptor Potential Ankyrin 1 Channels Modulate Inflammatory Response in Respiratory Cells from Patients with Cystic Fibrosis.

Pseudomonas aeruginosa colonization, prominent inflammation with massive expression of the neutrophil chemokine IL-8, and luminal infiltrates of neutrophils are hallmarks of chronic lung disease in patients with cystic fibrosis (CF). The nociceptive transient receptor potential ankyrin (TRPA) 1 calcium channels have been recently found to be involved in nonneurogenic inflammation. Here, we investigate the role of TRPA1 in CF respiratory inflammatory models in vitro. Expression of TRPA1 was evaluated in CF lung tissue sections and cells by immunohistochemistry and immunofluorescence. Epithelial cell lines (A549, IB3-1, CuFi-1, CFBE41o-) and primary cells from patients with CF were used to: (1) check TRPA1 function modulation, by Fura-2 calcium imaging; (2) down-modulate TRPA1 function and expression, by pharmacological inhibitors (HC-030031 and A-967079) and small interfering RNA silencing; and (3) assess the effect of TRPA1 down-modulation on expression and release of cytokines upon exposure to proinflammatory challenges, by quantitative RT-PCR and 27-protein Bioplex assay. TRPA1 channels are expressed in the CF pseudostratified columnar epithelium facing the bronchial lumina exposed to bacteria, where IL-8 is coexpressed. Inhibition of TRPA1 expression results in a relevant reduction of release of several cytokines, including IL-8 and the proinflammatory cytokines IL-1ß and TNF-α, in CF primary bronchial epithelial cells exposed to P. aeruginosa and to the supernatant of mucopurulent material derived from the chronically infected airways of patients with CF. In conclusion, TRPA1 channels are involved in regulating the extent of airway inflammation driven by CF bronchial epithelial cells.

Cytokines profile and peripheral blood mononuclear cells morphology in Rett and autistic patients.

A potential role for immune dysfunction in autism spectrum disorders (ASD) has been well established. However, immunological features of Rett syndrome (RTT), a genetic neurodevelopmental disorder closely related to autism, have not been well addressed yet. By using multiplex Luminex technology, a panel of 27 cytokines and chemokines was evaluated in serum from 10 RTT patients with confirmed diagnosis of MECP2 mutation (typical RTT), 12 children affected by classic autistic disorder and 8 control subjects. The cytokine/chemokine gene expression was assessed by real time PCR on mRNA of isolated peripheral blood mononuclear cells (PBMCs). Moreover, ultrastructural analysis of PBMCs was performed using transmission electron microscopy (TEM). Significantly higher serum levels of interleukin-8 (IL-8), IL-9, IL-13 were detected in RTT compared to control subjects, and IL-15 shows a trend toward the upregulation in RTT. In addition, IL-1ß and VEGF were the only down-regulated cytokines in autistic patients with respect to RTT. No difference in cytokine/chemokine profile between autistic and control groups was detected. These data were also confirmed by ELISA real time PCR. At the ultrastructural level, the most severe morphological abnormalities were observed in mitochondria of both RTT and autistic PBMCs. In conclusion, our study shows a deregulated cytokine/chemokine profile together with morphologically altered immune cells in RTT. Such abnormalities were not quite as evident in autistic subjects. These findings indicate a possible role of immune dysfunction in RTT making the clinical features of this pathology related also to the immunology aspects, suggesting, therefore, novel possible therapeutic interventions for this disorder.

Incorporation of Naked Peptide Nucleic Acids into Liposomes Leads to Fast and Efficient Delivery.

The delivery of peptide nucleic acids (PNAs) to cells is a very challenging task. We report here that a liposomal formulation composed of egg PC/cholesterol/DSPE-PEG2000 can be loaded, according to different encapsulation techniques, with PNA or fluorescent PNA oligomers. PNA loaded liposomes efficiently and quickly promote the uptake of a PNA targeting the microRNA miR-210 in human erythroleukemic K562 cells. By using this innovative delivery system for PNA, down-regulation of miR-210 is achieved at a low PNA concentration.

Regulation of IL-8 gene expression in gliomas by microRNA miR-93.

BACKGROUND: Different strategies have been proposed to target neoangiogenesis in gliomas, besides those targeting Vascular Endothelial Growth Factor (VEGF). The chemokine Interleukin-8 (IL-8) has been shown to possess both tumorigenic and proangiogenic properties. Although different pathways of induction of IL-8 gene expression have been already elucidated, few data are available on its post-transcriptional regulation in gliomas. METHODS: Here we investigated the role of the microRNA miR-93 on the expression levels of IL-8 and other pro-inflammatory genes by RT-qPCR and Bio-Plex analysis. We used different disease model systems, including clinical samples from glioma patients and two glioma cell lines, U251 and T98G. RESULTS: IL-8 and VEGF transcripts are highly expressed in low and high grade gliomas in respect to reference healthy brain; miR-93 expression is also increased and inversely correlated with transcription of IL-8 and VEGF genes. Computational analysis showed the presence of miR-93 consensus sequences in the 3'UTR region of both VEGF and IL-8 mRNAs, predicting possible interaction with miR-93 and suggesting a potential regulatory role of this microRNA. In vitro transfection with pre-miR-93 and antagomiR-93 inversely modulated VEGF and IL-8 gene expression and protein release when the glioma cell line U251 was considered. Similar data were obtained on IL-8 gene regulation in the other glioma cell line analyzed, T98G. The effect of pre-miR-93 and antagomiR-93 in U251 cells has been extended to the secretion of a panel of cytokines, chemokines and growth factors, which consolidated the concept of a role of miR-93 in IL-8 and VEGF gene expression and evidenced a potential regulatory role also for MCP-1 and PDGF (also involved in angiogenesis). CONCLUSION: In conclusion, our results suggest an increasing role of miR-93 in regulating the level of expression of several genes involved in the angiogenesis of gliomas.

Expression of microRNA-93 and Interleukin-8 during Pseudomonas aeruginosa-mediated induction of proinflammatory responses.

In this study we analyzed the microRNA profile of cystic fibrosis (CF) bronchial epithelial IB3-1 cells infected with Pseudomonas aeruginosa by microarray and quantitative RT-PCR, demonstrating that microRNA 93 (miR-93), which is highly expressed in basal conditions, decreases during infection in parallel with increased expression of the IL-8 gene. The down-regulation of miR-93 after P. aeruginosa infection was confirmed in other bronchial cell lines derived from subjects with and without CF, namely CuFi-1 and NuLi-1 cells. Sequence analysis shows that the 3'-UTR region of IL-8 mRNA is a potential target of miR-93 and that the consensus sequence is highly conserved throughout molecular evolution. The possible involvement of miR-93 in IL-8 gene regulation was validated using three luciferase vectors, including one carrying the complete 3'-UTR region of the IL-8 mRNA and one carrying the same region with a mutated miR-93 site. Up-modulation of IL-8 after P. aeruginosa infection was counteracted in IB3-1, CuFi-1, and NuLi-1 cells by pre-miR-93 transfection. In addition, IL-8 was up-regulated in uninfected cells treated with antagomiR-93. Our results support the concept of a possible link between microRNA expression and IL-8 induction in bronchial epithelial cells infected with P. aeruginosa. Specifically, the data presented here indicate that, in addition to NF-κB-dependent up-regulation of IL-8 gene transcription, IL-8 protein expression is posttranscriptionally regulated by interactions of the IL-8 mRNA with the inhibitory miR-93.

Uptake by human glioma cell lines and biological effects of a peptide-nucleic acids targeting miR-221.

MicroRNAs are a family of small noncoding RNAs regulating gene expression by sequence-selective mRNA targeting, leading to a translational repression or mRNA degradation. The oncomiR miR-221 is highly expressed in human gliomas, as confirmed in this study in samples of low and high grade gliomas, as well in the cell lines U251, U373 and T98G. In order to alter the biological functions of miR-221, a peptide nucleic acid targeting miR-221 (R8-PNA-a221) was produced, bearing a oligoarginine peptide (R8) to facilitate uptake by glioma cells. The effects of R8-PNA-a221 were analyzed in U251, U373 and T98G glioma cells and found to strongly inhibit miR-221. In addition, the effects of R8-PNA-a221 on p27(Kip1) (a target of miR-221) were analyzed in U251 and T98G cells by RT-qPCR and by Western blotting. No change of p27(Kip1) mRNA content occurs in U251 cells in the presence of PNA-a221 (lacking the R8 peptide), whereas significant increase of p27(Kip1) mRNA was observed with the R8-PNA-a221. These data were confirmed by Western blot assay. A clear increment of p27(Kip1) protein expression in the samples treated with R8-PNA-a221 was detected. In addition, R8-PNA-a221 was found able to increase TIMP3 expression (another target of miR-221) in T98G cells. These results suggest that PNAs against oncomiRNA miR-221 might be proposed for experimental treatment of human gliomas.

Antibacterial and anti-inflammatory activity of a temporin B peptide analogue on an in vitro model of cystic fibrosis.

Natural peptides with antimicrobial properties are deeply investigated as tools to fight bacteria resistant to common antibiotics. Small peptides, as those belonging to the temporin family, are very attractive because their activity can easily be tuned after small modification to their primary sequence. Structure-activity studies previously reported by us allowed the identification of one peptide, analogue of temporin B, TB_KKG6A, showing, unlike temporin B, antimicrobial activity against both Gram-positive and Gram-negative bacteria. In this paper, we investigated the antimicrobial and anti-inflammatory activity of the peptide TB_KKG6A against Pseudomonas aeruginosa. Interestingly, we found that the peptide exhibits antimicrobial activity at low concentrations, being able to downregulate the pro-inflammatory chemokines and cytokines interleukin (IL)-8, IL-1ß, IL-6 and tumor necrosis factor-α produced downstream infected human bronchial epithelial cells. Experiments were carried out also with temporin B, which was found to show pro-inflammatory activity. Details on the interaction between TB_KKG6A and the P. aeruginosa LPS were obtained by circular dichroism and fluorescence studies.

Redox Mechanisms Underlying the Cytostatic Effects of Boric Acid on Cancer Cells-An Issue Still Open.

Boric acid (BA) is the dominant form of boron in plasma, playing a role in different physiological mechanisms such as cell replication. Toxic effects have been reported, both for high doses of boron and its deficiency. Contrasting results were, however, reported about the cytotoxicity of pharmacological BA concentrations on cancer cells. The aim of this review is to briefly summarize the main findings in the field ranging from the proposed mechanisms of BA uptake and actions to its effects on cancer cells.

A retrospective hospital benefit and cost analysis of the management of human tissues for orthopaedic allografts.

OBJECTIVES: The transplantation of human tissues is a greatly expanding field of medicine with unquestionable benefits that raise questions about safety, quality and ethics. Since 1 October 2019, the Fondazione Banca dei Tessuti del Veneto (FBTV) stopped sending thawed and ready to be transplanted cadaveric human tissues to hospitals. A retrospective analysis of the period 2016-2019 found a significant number of unused tissues. For this reason, the hospital pharmacy has developed a new centralised service characterised by thawing and washing human tissues for orthopaedic allografts. This study aims to analyse the hospital cost and benefit derived from this new service. METHODS: Aggregate data relating to tissue flows were obtained retrospectively for the period 2016-2022 through the hospital data warehouse. All tissues arriving from FBTV for each year were analysed, dividing them according to the outcome (if used or wasted). The percentage of wasted tissues as well as the economic loss due to wasted allografts were analysed per year and trimester. RESULTS: We identified 2484 allografts requested for the period 2016-2022. In the last 3 years of the analysis, characterised by the new tissue management of the pharmacy department, we found a statistically significant reduction in wasted tissues (p<0.0001) from 16.33% (216/1323) with a cost to the hospital of 176 866€ during the period 2016-2019 to 6.72% (78/1161) with a cost to the hospital of 79 423€ during the period 2020-2022. CONCLUSION: This study shows how the centralised processing of human tissues in the hospital pharmacy makes the procedure safer and more efficient, demonstrating how the synergy between different hospital departments, high professional skills and ethics can lead to a clinical advantage for patients and a better economic impact for the hospital.