A rescue approach in refractory diffuse large B-cell lymphoma with obinutuzumab-redirected cytokine-induced killer cells: a first-in-human case report.

Not available.

Enalaprilat and losartan decrease erythroid precursors frequency in cells from patients with polycythemia vera.

OBJECTIVE: Polycythemia Vera (PV) is a myeloproliferative neoplasm characterized by the overproduction of red blood cells. First-line therapies are directed at lowering hematocrit levels. After the discovery of a mutation in the Janus kinase 2 (JAK2V617F), JAK2 inhibitors have been tested as second-line therapies. Despite these approaches, there is still the need for a major comprehension of the mechanisms involved in PV erythrocytosis and of more effective therapies. Angiotensin-converting enzyme (ACE) stimulates hematopoietic precursors proliferation and erythroid differentiation. We thus hypothesized that ACE inhibition could help in controlling erythrocytosis in PV. METHODS: We assessed the clonogenic potential by colony-forming unit (CFU) assay of mononuclear cells isolated from PV JAK2 positive or JAK2 negative patients with erythrocytosis treated with enalaprilat or losartan. RESULTS: Treatment with drugs led to a decrease of erythroid precursor frequency both in the presence and absence of JAK2 mutation, with a high extent in JAK2 positive cells and without affecting other types of precursors. No dose-dependent effect was observed. CONCLUSIONS: Our results demonstrate that ACE inhibition reduces erythroid precursor frequency, confirming the involvement of ACE in erythrocytosis despite the presence of JAK2 mutation and encouraging the hypothesis that ACE inhibitors and AT1R antagonists could help in directly managing erythrocytosis in PV.

Decellularized esophageal tubular scaffold microperforated by quantum molecular resonance technology and seeded with mesenchymal stromal cells for tissue engineering esophageal regeneration.

Current surgical options for patients requiring esophageal replacement suffer from several limitations and do not assure a satisfactory quality of life. Tissue engineering techniques for the creation of customized "self-developing" esophageal substitutes, which are obtained by seeding autologous cells on artificial or natural scaffolds, allow simplifying surgical procedures and achieving good clinical outcomes. In this context, an appealing approach is based on the exploitation of decellularized tissues as biological matrices to be colonized by the appropriate cell types to regenerate the desired organs. With specific regard to the esophagus, the presence of a thick connective texture in the decellularized scaffold hampers an adequate penetration and spatial distribution of cells. In the present work, the Quantum Molecular Resonance® (QMR) technology was used to create a regular microchannel structure inside the connective tissue of full-thickness decellularized tubular porcine esophagi to facilitate a diffuse and uniform spreading of seeded mesenchymal stromal cells within the scaffold. Esophageal samples were thoroughly characterized before and after decellularization and microperforation in terms of residual DNA content, matrix composition, structure and biomechanical features. The scaffold was seeded with mesenchymal stromal cells under dynamic conditions, to assess the ability to be repopulated before its implantation in a large animal model. At the end of the procedure, they resemble the original esophagus, preserving the characteristic multilayer composition and maintaining biomechanical properties adequate for surgery. After the sacrifice we had histological and immunohistochemical evidence of the full-thickness regeneration of the esophageal wall, resembling the native organ. These results suggest the QMR microperforated decellularized esophageal scaffold as a promising device for esophagus regeneration in patients needing esophageal substitution.

Selective cell cycle arrest in glioblastoma cell lines by quantum molecular resonance alone or in combination with temozolomide.

BACKGROUND: Glioblastoma is the most aggressive form of brain cancer, characterised by high proliferation rates and cell invasiveness. Despite advances in surgery and radio-chemotherapy, patients continue to have poor prognoses, with a survival rate of 14-15 months. Thus, new therapeutic strategies are needed. Non-ionising electromagnetic fields represent an emerging option given the potential advantages of safety, low toxicity and the possibility to be combined with other therapies. METHODS: Here, the anticancer activity of quantum molecular resonance (QMR) was investigated. For this purpose, three glioblastoma cell lines were tested, and the QMR effect was evaluated on cancer cell proliferation rate and aggressiveness. To clarify the QMR mechanism of action, the proteomic asset after stimulation was delineated. Mesenchymal stromal cells and astrocytes were used as healthy controls. RESULTS: QMR affected cancer cell proliferation, inducing a significant arrest of cell cycle progression and reducing cancer tumorigenicity. These parameters were not altered in healthy control cells. Proteomic analysis suggested that QMR acts not only on DNA replication but also on the machinery involved in the mitotic spindle assembly and chromosome segregation. Moreover, in a combined therapy assessment, QMR significantly enhanced temozolomide efficacy. CONCLUSIONS: QMR technology appears to be a promising tool for glioblastoma treatment.

The immune modulatory effects of umbilical cord-derived mesenchymal stromal cells in severe COVID-19 pneumonia.

Coronavirus disease 2019 (COVID-19) may result in a life-threatening condition due to a hyperactive immune reaction to severe acute respiratory syndrome-coronavirus-2 infection, for which no effective treatment is available. Based on the potent immunomodulatory properties of mesenchymal stromal cells (MSCs), a growing number of trials are ongoing. This prompted us to carry out a thorough immunological study in a patient treated with umbilical cord-derived MSCs and admitted to the Intensive Care Unit for COVID-19-related pneumonia. The exploratory analyses were assessed on both peripheral blood and bronchoalveolar fluid lavage samples at baseline and after cellular infusion by means of single-cell RNA sequencing, flow cytometry, ELISA, and functional assays. Remarkably, a normalization of circulating T lymphocytes count paralleled by a reduction of inflammatory myeloid cells, and a decrease in serum levels of pro-inflammatory cytokines, mostly of interleukin-6 and tumor necrosis factor-α, were observed. In addition, a drop of plasma levels of those chemokines essential for neutrophil recruitment became evident that paralleled the decrease of lung-infiltrating inflammatory neutrophils. Finally, circulating monocytes and low-density gradient neutrophils acquired immunosuppressive function. This scenario was accompanied by an amelioration of respiratory, renal, inflammatory, and pro-thrombotic indexes. Our results provide the first immunological data possibly related to the use of umbilical cord-derived MSCs in severe COVID-19 context.

Logistics of an advanced therapy medicinal product during COVID-19 pandemic in Italy: successful delivery of mesenchymal stromal cells in dry ice.

BACKGROUND: During the coronavirus disease-2019 (COVID-19) pandemic, Italian hospitals faced the most daunting challenges of their recent history, and only essential therapeutic interventions were feasible. From March to April 2020, the Laboratory of Advanced Cellular Therapies (Vicenza, Italy) received requests to treat a patient with severe COVID-19 and a patient with acute graft-versus-host disease with umbilical cord-derived mesenchymal stromal cells (UC-MSCs). Access to clinics was restricted due to the risk of contagion. Transport of UC-MSCs in liquid nitrogen was unmanageable, leaving shipment in dry ice as the only option. METHODS: We assessed effects of the transition from liquid nitrogen to dry ice on cell viability; apoptosis; phenotype; proliferation; immunomodulation; and clonogenesis; and validated dry ice-based transport of UC-MSCs to clinics. RESULTS: Our results showed no differences in cell functionality related to the two storage conditions, and demonstrated the preservation of immunomodulatory and clonogenic potentials in dry ice. UC-MSCs were successfully delivered to points-of-care, enabling favourable clinical outcomes. CONCLUSIONS: This experience underscores the flexibility of a public cell factory in its adaptation of the logistics of an advanced therapy medicinal product during a public health crisis. Alternative supply chains should be evaluated for other cell products to guarantee delivery during catastrophes.

Successful muscle regeneration by a homologous microperforated scaffold seeded with autologous mesenchymal stromal cells in a porcine esophageal substitution model.

BACKGROUND: Since the esophagus has no redundancy, congenital and acquired esophageal diseases often require esophageal substitution, with complicated surgery and intestinal or gastric transposition. Peri-and-post-operative complications are frequent, with major problems related to the food transit and reflux. During the last years tissue engineering products became an interesting therapeutic alternative for esophageal replacement, since they could mimic the organ structure and potentially help to restore the native functions and physiology. The use of acellular matrices pre-seeded with cells showed promising results for esophageal replacement approaches, but cell homing and adhesion to the scaffold remain an important issue and were investigated. METHODS: A porcine esophageal substitute constituted of a decellularized scaffold seeded with autologous bone marrow-derived mesenchymal stromal cells (BM-MSCs) was developed. In order to improve cell seeding and distribution throughout the scaffolds, they were micro-perforated by Quantum Molecular Resonance (QMR) technology (Telea Electronic Engineering). RESULTS: The treatment created a microporous network and cells were able to colonize both outer and inner layers of the scaffolds. Non seeded (NSS) and BM-MSCs seeded scaffolds (SS) were implanted on the thoracic esophagus of 4 and 8 pigs respectively, substituting only the muscle layer in a mucosal sparing technique. After 3 months from surgery, we observed an esophageal substenosis in 2/4 NSS pigs and in 6/8 SS pigs and a non-practicable stricture in 1/4 NSS pigs and 2/8 SS pigs. All the animals exhibited a normal weight increase, except one case in the SS group. Actin and desmin staining of the post-implant scaffolds evidenced the regeneration of a muscular layer from one anastomosis to another in the SS group but not in the NSS one. CONCLUSIONS: A muscle esophageal substitute starting from a porcine scaffold was developed and it was fully repopulated by BM-MSCs after seeding. The substitute was able to recapitulate in shape and function the original esophageal muscle layer.

Synthesis of 2,6-Diamino-Substituted Purine Derivatives and Evaluation of Cell Cycle Arrest in Breast and Colorectal Cancer Cells.

Reversine is a potent antitumor 2,6-diamino-substituted purine acting as an Aurora kinases inhibitor and interfering with cancer cell cycle progression. In this study we describe three reversine-related molecules, designed by docking calculation, that present structural modifications in the diamino units at positions 2 and 6. We investigated the conformations of the most stable prototropic tautomers of one of these molecules, the N6-cyclohexyl-N6-methyl-N2-phenyl-7H-purine-2,6-diamine (3), by Density Functional Theory (DFT) calculation in the gas phase, water and chloroform, the last solvent considered to give insights into the detection of broad signals in NMR analysis. In all cases the HN(9) tautomer resulted more stable than the HN(7) form, but the most stable conformations changed in different solvents. Molecules 1⁻3 were evaluated on MCF-7 breast and HCT116 colorectal cancer cell lines showing that, while being less cytotoxic than reversine, they still caused cell cycle arrest in G2/M phase and polyploidy. Unlike reversine, which produced a pronounced cell cycle arrest in G2/M phase in all the cell lines used, similar concentrations of 1⁻3 were effective only in cells where p53 was deleted or down-regulated. Therefore, our findings support a potential selective role of these structurally simplified, reversine-related molecules in p53-defective cancer cells.

Impaired Neuronal Differentiation of Neural Stem Cells Lacking the Engrailed-2 Gene.

The Engrailed-2 (En2) gene codes for a homeobox-containing transcription factor, involved in midbrain-hindbrain embryonic development. In postnatal brain, En2 is expressed in the ventral mesencephalon, cerebellum, hippocampus and neocortex. Two single-nucleotide polymorphisms (SNPs) that are associated to autism spectrum disorders (ASD) have been identified in the human EN2 gene. Accordingly, mice lacking the En2 homeodomain (En2hd/hd, referred to as En2-/-) show molecular, anatomical and behavioral "ASD-like" features. Among these, we previously showed a partial loss of GABAergic interneurons in the En2-/- postnatal hippocampus and neocortex, accompanied by a marked decrease of brain-derived neurotrophic factor (BDNF) signaling, a crucial determinant of GABAergic differentiation. In order to better investigate the role of En2 in GABAergic interneuron differentiation, we generated and subsequently differentiated neural stem cells (NSCs) from basal ganglia and neocortex of En2+/+ and En2-/- mouse embryos. Wild-type NSCs from both basal ganglia and neocortex express En2, while mutant ones do not, as expected. As compared to En2+/+ NSCs, En2-/- NSCs derived from basal ganglia show impaired GABAergic differentiation accompanied by a reduced expression of the BDNF receptor trkB. Conversely, En2-/- NSCs derived from the neocortex expressed high levels of trkB and readily differentiated into neurons, as En2+/+ NSCs. Our results suggest that En2 contributes to GABAergic neuron differentiation from basal ganglia NSCs through a trkB-dependent BDNF signaling, thus providing a possible explanation for the reduced number of GABAergic interneurons detected in the En2-/- postnatal forebrain.

Noggin 1 overexpression in retinal progenitors affects bipolar cell generation.

Waves of Bone Morphogenetic Proteins (BMPs) and their antagonists are present during initial eye development, but their possible roles in retinogenesis are still unknown. We have recently shown that noggin 1, a BMP antagonist, renders pluripotent cells able to differentiate into retinal precursors, and might be involved in the maintenance of retinal structures in the adult vertebrate eye. Here, we report that noggin 1, differently from noggin 2 and noggin 4, is expressed during all phases of Xenopus laevis retinal development. Gain-of-function experiments by electroporation in the optic vesicle show that overexpression of noggin 1 significantly decreases the number of bipolar cells in the inner nuclear layer of the retina, without significantly affecting the generation of the other retinal cell types. Our data suggest that BMP signaling could be involved in the differentiation of retinal progenitors into specific retinal subtypes during late phases of vertebrate retinal development.

Microbiological quality of water from the rivers of Curitiba, Paraná State, Brazil, and the susceptibility to antimicrobial drugs and pathogenicity of Escherichia coli.

Water safety is determined by several markers, and Escherichia coli is one of the most important indicators of water quality. The objective of this study was to evaluate the microbiological parameters in environmental samples of fresh water from rivers of Curitiba and its metropolitan area in Paraná State, Brazil. In addition, we evaluated the pathogenicity and susceptibility to antimicrobial drugs in E. coli. These evaluations were performed by quantitative and qualitative methods employing selective media for isolating thermotolerant coliforms and biochemical tests for identifying E. coli. Pathogenic strains of E. coli were detected by PCR multiplex using specific primers. From the water samples, 494 thermotolerant coliforms were obtained, of which 96 (19.43%) isolates were characterized as E. coli. Three isolates were identified as enteroaggregative E. coli, one as enterotoxigenic E. coli, one as enteropathogenic E. coli, and two carried the Eae virulence gene. E. coli susceptibility to commonly employed antimicrobial drugs was analyzed by the disc diffusion method. The results showed 49 (51.04%) isolates resistant to all the drugs assayed, 16 (16.67%) with an intermediate resistance to all drugs, and 31 (32.29%) intermediately or fully resistant to one or more drugs tested. The highest rate of resistance was observed for tetracycline 30 µg, streptomycin 10 µg, and ceftazidime 30 µg. Detection of E. coli is associated with water contamination by fecal material from humans and warm-blooded animals. The occurrence of resistant strains can be the result of the indiscriminate use of antimicrobial drugs and poor sanitation in the areas assayed.

Noggin-Mediated Retinal Induction Reveals a Novel Interplay Between Bone Morphogenetic Protein Inhibition, Transforming Growth Factor ß, and Sonic Hedgehog Signaling.

It has long been known that the depletion of bone morphogenetic protein (BMP) is one of the key factors necessary for the development of anterior neuroectodermal structures. However, the precise molecular mechanisms that underlie forebrain regionalization are still not completely understood. Here, we show that Noggin1 is involved in the regionalization of anterior neural structures in a dose-dependent manner. Low doses of Noggin1 expand prosencephalic territories, while higher doses specify diencephalic and retinal regions at the expense of telencephalic areas. A similar dose-dependent mechanism determines the ability of Noggin1 to convert pluripotent cells in prosencephalic or diencephalic/retinal precursors, as shown by transplant experiments and molecular analyses. At a molecular level, the strong inhibition of BMP signaling exerted by high doses of Noggin1 reinforces the Nodal/transforming growth factor (TGF)ß signaling pathway, leading to activation of Gli1 and Gli2 and subsequent activation of Sonic Hedgehog (SHH) signaling. We propose a new role for Noggin1 in determining specific anterior neural structures by the modulation of TGFß and SHH signaling.

Noggin Expression in the Adult Retina Suggests a Conserved Role during Vertebrate Evolution.

Vertebrates share common mechanisms in the control of development and in the maintenance of neural and retinal function. The secreted factor Noggin, a BMP inhibitor, plays a crucial role in neural induction during embryonic development. Moreover, we have shown its involvement in retinal differentiation of pluripotent cells. Here we show Noggin expression in the adult retina in three vertebrate species. Four Noggin genes are present in zebrafish (Danio rerio; ZbNog1, 2, 3, 5), three in frog (Xenopus laevis; XenNog1, 2 and 4), and one in mouse (Mus musculus; mNog). Quantitative RT-PCR experiments show the presence of ZbNog3 and ZbNog5 mRNAs, but not ZbNog1 and ZbNog2, in the adult zebrafish retina. All three genes are expressed in the frog retina, and mNog in the mouse. Immunohistochemistry data show that Noggin proteins are predominantly localized in the Golgi apparatus of photoreceptors and in the fibers of the outer plexiform layer. Lower expression levels are also found in inner plexiform layer fibers, in ganglion cells, in the ciliary marginal zone, and in retinal pigmented epithelium. Our results show that Noggin has a specific cellular and sub-cellular expression in the adult vertebrate retina, which is conserved during evolution. In addition to its established role during embryonic development, we postulate that Noggin also exerts a functional role in the adult retina.

Neural differentiation of pluripotent cells in 3D alginate-based cultures.

Biomaterial-supported culture methods, allowing for directed three-dimensional differentiation of stem cells are an alternative to canonical two-dimensional cell cultures. In this paper, we evaluate the suitability of alginate for three-dimensional cultures to enhance differentiation of mouse embryonic stem cells (mESCs) towards neural lineages. We tested whether encapsulation of mESCs within alginate beads could support and/or enhance neural differentiation with respect to two-dimensional cultures. We encapsulated cells in beads of alginate with or without modification by fibronectin (Fn) or hyaluronic acid (HA). Gene expression analysis showed that cells grown in alginate and alginate-HA present increased differentiation toward neural lineages with respect to the two-dimensional control and to Fn group. Immunocytochemistry analyses confirmed these results, further showing terminal differentiation of neurons as seen by the expression of synaptic markers and markers of different neuronal subtypes. Our data show that alginate, alone or modified, is a suitable biomaterial to promote in vitro differentiation of pluripotent cells toward neural fates.

Occurrence of sulphate reducing bacteria (SRB) associated with biocorrosion on metallic surfaces in a hydroelectric power station in Ibirama (SC) - Brazil

The aim of this study was evaluate, two methods for the detection and identification of sulphate reducing bacteria (SRB): ML medium and PCR with specific primers for SRB groups. SRB were detected through the selective medium only on carbon steel, which showed corrosion. Employing specific PCR primer, SBR were detected from all the metallic components assayed, even those that did not present visible corrosion spots, such stainless steel and copper alloys. Despite the presence or absence of corrosion at the later stages effectively by using the selective medium,, the initial stages of the corrosion could only be detected by the amplification of total DNA with SRB specific primers. The early detection of SRB could be employed for preventing the damages on metal surfaces before the installation of corrosion processes. Strategies for reducing the time spent on SRB isolation and identification could be auxiliary tools for controlling the corrosion of materials.

Potential of Fourier transform infrared spectroscopy (FT-IR) to differentiate environmental Aspergillus fungi species A. niger, A. ochraceus, and A. westerdijkiae using two different methodologies.

We assessed the ability of Fourier transform infrared spectroscopy (FT-IR) to differentiate three important and morphologically similar Aspergillus species: A. ochraceus and A. westerdijkiae, and A. niger. Fungi were processed by two methods, powdered mycelia and conidiospore-saline solution, and then recorded in a spectrometer. Second derivatives with nine points of smoothing were applied as spectra data pretreatment. Partial least squares regression was used for the species comparison models and a prediction test was used to evaluate the models. The powdered-mycelia methodology correctly identified 100% of the prediction test set to discriminate A. niger from A. ochraceus and A. westerdijkiae; in addition, it had a 86.6% success rate in discriminating A. ochraceus and A. westerdijkiae. This is the first time a study assessed the ability of FT-IR to differentiate A. niger, A. ochraceus, and A. westerdijkiae, and we believe this technique is very promising for classifying and distinguish fungi isolates.