Extensive Characterization of Platelet Gel Releasate From Cord Blood in Regenerative Medicine.

Platelet gel derived from peripheral blood is widely applied in many clinical fields of surgery as biomaterial containing growth factors with high proliferative properties. In 2010, we studied and patented a platelet gel derived from cord blood. In this study, due to the crucial role of the factors released by the platelet gel, we first extended the characterization of its releasate. Using a wide proteomic array and splitting the two components of the releasate, that is, platelets and plasma, we have been able to study their growth factor content. Interestingly, we discovered high levels of hormones and molecules able to support tissue growth in the cord blood platelet gel releasate and, in addition, higher concentrations of several angiogenic factors if compared with the peripheral blood counterpart. On the contrary, the latter was much richer in inflammatory factors. The second aim of our work was to study the effects on cell culture, immunophenotype, and function of mesenchymal stem cells exposed to these two platelet gel releasates as substitute for the animal serum. Since our findings nicely show that the use of the peripheral versus the cord blood platelet gel releasate can differently influence the mesenchymal stem cell commitment, we can suggest that in addition to its peculiar angiogenic properties cord blood platelet gel releasate shows excellent proliferative properties as cell culture supplement

A Chemically Defined Medium-Based Strategy to Efficiently Generate Clinically Relevant Cord Blood Mesenchymal Stromal Colonies.

During the last decade it has been demonstrated that mesenchymal progenitors are present and can be isolated also from cord blood (CB). Recently, we managed to set up a standard protocol allowing the isolation of mesenchymal stromal cells (MSCs) with high proliferative potential and multiple differentiation capabilities, whereas the generation rate of MSC-initiating colonies could still be further improved. Herein, we strikingly succeeded in defining some simple and basic culture conditions based on the use of a chemically defined medium that increased the colony isolation efficiency up to almost 80% of processed CB units. Importantly, this result was achieved irrespective of CB unit white blood cell content and time elapsed from delivery, two limiting parameters involved with processing CB units. Thus, this high efficiency is guaranteed without strict selection of the starting material. In addition, since we are profoundly concerned about how different culture conditions can influence cell behavior, we devoted part of this study to in-depth characterization of the established CB-MSC populations to confirm their stemness features in this novel isolation and culture system. Therefore, an extended study of their immunophenotype, including classical pericytic markers, and a detailed molecular analysis addressing telomere length and also stemness-related microRNA contribution were performed. In summary, we propose a straightforward, extremely efficient, and reliable approach to isolate and expand thoroughly characterized CB-MSCs, even when poor-quality CB units are the only available source, or there is no space for an isolation to fail

Functional relevance of circRNA aberrant expression in pediatric acute leukemia with KMT2A::AFF1 fusion

: Circular RNAs (circRNAs) are emerging molecular players in leukemogenesis and promising therapeutic targets. In KMT2A::AFF1 (MLL::AF4)-rearranged leukemia, an aggressive disease compared with other pediatric B-cell precursor (BCP) acute lymphoblastic leukemia (ALL), data about circRNAs are limited. Here, we disclose the circRNA landscape of infant patients with KMT2A::AFF1 translocated BCP-ALL showing dysregulated, mostly ectopically expressed, circRNAs in leukemia cells. Most of these circRNAs, apart from circHIPK3 and circZNF609, previously associated with oncogenic behavior in ALL, are still uncharacterized. An in vitro loss-of-function screening identified an oncogenic role of circFKBP5, circKLHL2, circNR3C1, and circPAN3 in KMT2A::AFF1 ALL, whose silencing affected cell proliferation and apoptosis. Further study in an extended cohort disclosed a significantly correlated expression of these oncogenic circRNAs and their putative involvement in common regulatory networks. Moreover, it showed that circAFF1 upregulation occurs in a subset of cases with HOXA KMT2A::AFF1 ALL. Collectively, functional analyses and patient data reveal oncogenic circRNA upregulation as a relevant mechanism that sustains the malignant cell phenotype in KMT2A::AFF1 ALL

Extracellular ATP is increased by release of ATP-loaded microparticles triggered by nutrient deprivation

Rationale: Caloric restriction improves the efficacy of anti-cancer therapy. This effect is largely dependent on the increase of the extracellular ATP concentration in the tumor microenvironment (TME). Pathways for ATP release triggered by nutrient deprivation are largely unknown. Methods: The extracellular ATP (eATP) concentration was in vivo measured in the tumor microenvironment of B16F10-inoculated C57Bl/6 mice with the pmeLuc probe. Alternatively, the pmeLuc-TG-mouse was used. Caloric restriction was in vivo induced with hydroxycitrate (HC). B16F10 melanoma cells or CT26 colon carcinoma cells were in vitro exposed to serum starvation to mimic nutrient deprivation. Energy metabolism was monitored by Seahorse. Microparticle release was measured by ultracentrifugation and by Nanosight. Results: Nutrient deprivation increases eATP release despite the dramatic inhibition of intracellular energy synthesis. Under these conditions oxidative phosphorylation was dramatically impaired, mitochondria fragmented and glycolysis and lactic acid release were enhanced. Nutrient deprivation stimulated a P2X7-dependent release of ATP-loaded, mitochondria-containing, microparticles as well as of naked mitochondria. Conclusions: Nutrient deprivation promotes a striking accumulation of eATP paralleled by a large release of ATP-laden microparticles and of naked mitochondria. This is likely to be a main mechanism driving the accumulation of eATP into the TME

Messa a punto di una metodica molecolare per l'identificazione di specie in ceppi di Pseudomonas spp. isolata da matrici alimentari

La tassonomica batterica del genere Pseudomonas è a tutt'oggi ancora in evoluzione e si modifica parallelamente l'introduzione di nuovi metodi che sfruttano i criteri più appropiati per la delineazione di genere e specie

Autologous mesenchymal stem cell therapy for progressive supranuclear palsy: translation into a phase I controlled, randomized clinical study

Background: Progressive Supranuclear Palsy (PSP) is a sporadic and progressive neurodegenerative disease which belongs to the family of tauopathies and involves both cortical and subcortical structures. No effective therapy is to date available. Methods/design: Autologous bone marrow (BM) mesenchymal stem cells (MSC) from patients affected by different type of parkinsonisms have shown their ability to improve the dopaminergic function in preclinical and clinical models. It is also possible to isolate and expand MSC from the BM of PSP patients with the same proliferation rate and immuphenotypic profile as MSC from healthy donors. BM MSC can be efficiently delivered to the affected brain regions of PSP patients where they can exert their beneficial effects through different mechanisms including the secretion of neurotrophic factors. Here we propose a randomized, placebo-controlled, double-blind phase I clinical trial in patients affected by PSP with MSC delivered via intra-arterial injection. Discussion: To our knowledge, this is the first clinical trial to be applied in a no-option parkinsonism that aims to test the safety and to exploit the properties of autologous mesenchymal stem cells in reducing disease progression. The study has been designed to test the safety of this " first-in-man" approach and to preliminarily explore its efficacy by excluding the placebo effect. Trial registration: NCT0182412

Screening of PRKAR1A and PDE4D in a Large Italian Series of Patients Clinically Diagnosed with Albright Hereditary Osteodystrophy and/or Pseudohypoparathyroidism

The cyclic adenosine monophosphate (cAMP) intracellular signaling pathway mediates the physiological effects of several hormones and neurotransmitters, acting by the activation of G-protein coupled receptors (GPCRs) and several downstream intracellular effectors, including the heterotrimeric stimulatory G-protein (Gs), the cAMP-dependent protein kinase A (PKA), and cAMP-specific phosphodiesterases (PDEs). Defective G-protein-mediated signaling has been associated with an increasing number of disorders, including Albright hereditary osteodistrophy (AHO) and pseudohypoparathyroidism (PHP), a heterogeneous group of rare genetic metabolic disorders resulting from molecular defects at the GNAS locus. Moreover, mutations in PRKAR1A and PDE4D genes have been recently detected in patients with acrodysostosis (ACRDYS), showing a skeletal and endocrinological phenotype partially overlapping with AHO/PHP. Despite the high detection rate of molecular defects by currently available molecular approaches, about 30% of AHO/PHP patients still lack a molecular diagnosis, hence the need to screen patients negative for GNAS epi/genetic defects also for chromosomal regions and genes associated with diseases that undergo differential diagnosis with PHP. According to the growing knowledge on Gsα-cAMP signaling-linked disorders, we investigated our series of patients (n = 81) with a clinical diagnosis of PHP/AHO but negative for GNAS anomalies for the presence of novel genetic variants at PRKAR1A and PDE4D genes. Our work allowed the detection of 8 novel missense variants affecting genes so far associated with ACRDYS in 9 patients. Our data further confirm the molecular and clinical overlap among these disorders. We present the data collected from a large series of patients and a brief review of the literature in order to compare our findings with already published data; to look for PRKAR1A/PDE4D mutation spectrum, recurrent mutations, and mutation hot spots; and to identify specific clinical features associated with ACRDYS that deserve surveillance during follow-up

Application of LCA modelling in integrated waste management

Life cycle assessment (LCA) has been used in waste management for the last two decades and hundreds of journal papers have been published. The use of LCA in waste management has provided a much improved holistic view of waste management including waste flows and potential environmental impacts. Although much knowledge has been obtained from LCA studies, there is still a need to use LCA models in integrated waste management. This paper describes six areas where LCA is expected to play a role in waste management in the future: 1) understanding an existing waste management system; 2) improving existing waste management systems; 3) comparing alternative technologies/ technology performance; 4) technology development/prospective technologies; 5) policy development/strategic development; and 6) reporting. Illustrative examples are provided for each application area