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Background: CLAD (Chronic Lung Allograft Dysfunction) remains a serious complication following lung transplantation. Some evidence shows that portions of Extracorporeal Photopheresis (ECP)-treated patients improve/stabilize their graft function. In spite of that, data concerning molecular mechanisms are still lacking. Aims of our study were to assess whether ECP effects are mediated by Mononuclear Cells (MNCs) modulation in term of microRNAs (miRNAs) expression and growth factors release., Methods: Cells from leukapheresis of 16 CLAD patients, at time 0 and 6-months (10 cycles), were cultured for 48h ± PHA (10 ug/ml) or LPS (2 ug/ml). Expression levels of miR-146a-5p, miR-155-5p, miR-31-5p, miR181a-5p, miR-142-3p, miR-16-5p and miR-23b-5p in MNCs-exosomes were evaluated by qRT-PCR, while ELISA assessed different growth factors levels on culture supernatants., Results: Our result showed miR-142-3p down-regulation (p = 0.02) in MNCs of ECP-patients after the 10 cycles and after LPS stimulation (p = 0.005). We also find miR-146a-5p up-regulation in cells after the 10 cycles stimulated with LPS (p = 0.03). Connective tissue growth factor (CTGF) levels significantly decreased in MNCs supernatant (p = 0.04). The effect of ECP is translated into frequency changes of Dendritic Cell (DC) subpopulations and a slight increase in T regulatory cells (Treg) number and a significant decrease in CTGF release., Conclusions: ECP might affect regulatory T cell functions, since both miR-142 and miR-146a have been shown to be involved in the regulation of suppressor regulatory T cell functions and DCs. On the other side ECP, possibly by regulating macrophage activation, is able to significantly down modulate CTGF release., (© 2024. The Author(s).)

Extracorporeal photopheresis (ECP) is a cell therapy originally employed for cutaneous T cell lymphoma and later for GvHD, solid organ rejection and other immunological diseases demonstrating an excellent safety profile. Mononuclear cell (MNCs) apoptosis triggered by UV-A light irradiation in the presence of 8-methoxypsoralene has a key role in priming the cells, ultimately leading to immunomodulation. We report preliminary data about an evaluation of the new automated irradiator device LUMILIGHT (Pelham Crescent srl) for off-line ECP. Fifteen MNCs samples collected by apheresis from 15 adult patients undergoing ECP at our Center were cultured immediately after irradiation along with untreated samples and evaluated at 24, 48 and 72 h timepoints for T cell apoptosis and viability by flow cytometry with Annexin V and Propide Iodidum staining. Post irradiation Hematocrit (HCT), calculated by the device, was compared with that of the automated cell counter. Bacterial contamination was also tested. In irradiated samples after 24-48 and 72 h, the average total apoptosis was 47 %, 70 % and 82 %, respectively, showing a significant difference from untreated samples; residual viable lymphocytes at 72 h were, on average, 18 %. The greatest initiation of apoptosis occurred from 48 h of irradiation onwards. Average early apoptosis of irradiated samples decreased over time (26 %, 17 % and 10 % at 24, 48 and 72 h, respectively). HCT measured by LUMILIGHT was over-estimated, possibly due to the low pre irradiation red blood cell contamination. Bacterial tests resulted negative. Our study showed the LUMILIGHT device to be a valid instrument for MNCs irradiation with good handling and no major technical problems as well as no adverse events in the patients. Our data need to be confirmed in larger studies., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: Test the ability of Mirasol Pathogen Reduction Technology (PRT, Terumo BCT, Lakewood Co, USA) treatment with riboflavin and ultraviolet light (R + UV) in reducing SARS-CoV-2 infectivity while maintaining blood product quality., Material and Methods: SARS-CoV-2 strains were isolated and titrated to prepare cell free virus for plasma units infection. The units were then under treatment with Mirasol PRT. The infectious titers were determined before and after treatment with an in house microtitration assay on Vero E6 cells. Thirty-six plasma pool bags underwent PRT treatment., Results: In all the experiments, the measured titer following riboflavin and UV treatment was below the limit of detection of microtitration assay for all the different SARS-CoV-2 strains. Despite the high copies number detected by RT-PCR for each viral strain after treatment, viruses were completely inactivated and not able to infect VERO E6 cells., Conclusion: Riboflavin and UV light treatment effectively reduced the virus titers of human plasma to the limit of detection in tissue culture, regardless of the strain. These data suggest that pathogen reduction in blood products highlight the safety of CP therapy procedures for critically ill COVID-19 patients, while maintaining blood product quality., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The adhesion, proliferation, and migration of cells over nanomaterials is regulated by a cascade of biochemical signals that originate at the interface of a cell with a substrate and propagate through the cytoplasm to the nucleus. The topography of the substrate plays a major role in this process. Cell adhesion molecules (CAMs) have a characteristic size of some nanometers and a range of action of some tens of nanometers. Controlling details of a surface at the nanoscale-the same dimensional over which CAMs operate-offers ways to govern the behavior of cells and create organoids or tissues with heretofore unattainable precision. Here, using electrochemical procedures, we generated mesoporous silicon surfaces with different values of pore size (PS≈11 nm and PS≈21 nm), roughness (Ra≈7 nm and Ra≈13 nm), and fractal dimension (Df≈2.48 and Df≈2.15). Using electroless deposition, we deposited over these substrates thin layers of gold nanoparticles. Resulting devices feature (i) nanoscale details for the stimulation and control of cell assembly, (ii) arrays of pores for drug loading/release, (iii) layers of nanostructured gold for the enhancement of the electromagnetic signal in Raman spectroscopy (SERS). We then used these devices as cell culturing substrates. Upon loading with the anti-tumor drug PtCl (O,O'-acac)(DMSO) we examined the rate of adhesion and growth of breast cancer MCF-7 cells under the coincidental effects of surface geometry and drug release. Using confocal imaging and SERS spectroscopy we determined the relative importance of nano-topography and delivery of therapeutics on cell growth-and how an unbalance between these competing agents can accelerate the development of tumor cells.

Background: Human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, hold enormous promise for many biomedical applications, such as regenerative medicine, drug testing, and disease modeling. Although induced pluripotent stem cells resemble embryonic stem cells both morphologically and functionally, the extent to which these cell lines are truly equivalent, from a molecular point of view, remains controversial., Methods: Principal component analysis and K-means cluster analysis of collected Raman spectroscopy data were used for a comparative study of the biochemical fingerprint of human induced pluripotent stem cells and human embryonic stem cells. The Raman spectra analysis results were further validated by conventional biological assays., Results: Raman spectra analysis revealed that the major difference between human embryonic stem cells and induced pluripotent stem cells is due to the nucleic acid content, as shown by the strong positive peaks at 785, 1098, 1334, 1371, 1484, and 1575 cm -1 , which is enriched in human induced pluripotent stem cells., Conclusions: Here, we report a nonbiological approach to discriminate human induced pluripotent stem cells from their native embryonic stem cell counterparts.

Background: Circulating tumor cells (CTCs) represent one of the most interesting target in improving diagnosis, prognosis and treatment. Herein we evaluate the possibility of using an emo-cytometric approach on the evaluation of the heterogeneous population of CTCs to improve personalized metastatic risk assessment. We benchmarked ex vivo behavior of distinct subsets of circulating colon tumor cells with correspondent clinical behavior of patients from which we isolated CTCs., Methods: Isolation and CTC expansion were performed by a gradient protocol. In vitro characterization was determined by flow cytometry, immunofluorescence, western blotting and proteomic profiling. Cell sorter was performed with immunomagnetic beads. Confocal microscopy was used to evaluate tissue sections. Kaplan Mayer curves was cared for through Medcalc program., Results: We collected heterogeneous CTCs, derived from the whole blood of seven patients affected by colon cancer, expressing CD133(pos)CD45(neg) (5 ± 1) and (2 ± 1) and CK20(pos)CD45(neg) of (29 ± 3) (11 ± 1) cells/ml in Dukes D and A stage respectively. Proliferation rate of 57 ± 16 %, expression for CXCR4(pos) of 18 ± 7 % and detectable levels of IL-6, IL-8 and SDF-1 cytokines in conditioned culture medium characterized short-time expanded-CTCs (eCTCs). ECTCs organized in tumor sphere were CD45(neg)CD133(pos) while in adhesion were CXCR4(pos)CK20(pos). These two subsets were separately injected in mice. The first group of xenografts developed superficial lesions within 2 weeks. In the second group, in absence of growing tumour, the survival of injected eCTCs was monitored through SDF-1 serum levels detection. The detection of human cancer cells expressing CK20, in mice tissues sections, suggested a different biological behaviour of injected eCTC-subsets: tumorigenic for the first and disseminating for the second. The benchmarking of the experimental data with the clinical course highlights that patients with prevalence of circulating cancer stem cells (CD45(neg)CD133(pos)) have a lower overall survival. Conversely, patients with prevalence of circulating differentiated cells (CXCR4(pos)CK20(pos)) have a low disease-free survival., Conclusion: On the basis of the heterogeneous composition and despite the low number of CTCs, it was possible to distinguish two subgroups of CTCs, suggesting a different clinical outcome. CTC-subsets detailing is useful to better define the metastatic-risk personalized score thus improving disease management and reducing patient care cost.

Although the detection of methylated cell free DNA represents one of the most promising approaches for relapse risk assessment in cancer patients, the low concentration of cell-free circulating DNA constitutes the biggest obstacle in the development of DNA methylation-based biomarkers from blood. This paper describes a method for the measurement of genomic methylation content directly on circulating tumor cells (CTC), which could be used to deceive the aforementioned problem. Since CTC are disease related blood-based biomarkers, they result essential to monitor tumor's stadiation, therapy, and early relapsing lesions. Within surface's bio-functionalization and cell's isolation procedure standardization, the presented approach reveals a singular ability to detect high 5-methylcytosine CTC-subset content in the whole CTC compound, by choosing folic acid (FA) as transducer molecule. Sensitivity and specificity, calculated for FA functionalized surface (FA-surface), result respectively on about 83% and 60%. FA-surface, allowing the detection and characterization of early metastatic dissemination, provides a unique advance in the comprehension of tumors progression and dissemination confirming the presence of CTC and its association with high risk of relapse. This functionalized surface identifying and quantifying high 5-methylcytosine CTC-subset content into the patient's blood lead significant progress in cancer risk assessment, also providing a novel therapeutic strategy., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Activation of the Ras-Raf-extracellular signal-regulated kinase (ERK) pathway causes not only proliferation and suppression of apoptosis but also the antioncogenic response of senescence. How these contrasting effects are reconciled to achieve cell transformation and cancer formation is poorly understood. In a system of two-step carcinogenesis (dedifferentiated PC EIA, transformed PC EIA-polyoma-middle T [PC EIA + Py] and PC EIA-v-raf [PC EIA + raf] cells], v-raf cooperated with EIA by virtue of a strong prosurvival effect, not elicited by Py-middle T, evident toward serum-deprivation-and H(2)O(2)-induced apoptosis. Apoptosis was detected by DNA fragmentation and annexin V staining. The prosurvival function of v-raf was, in part, mitogen-activated protein kinase/ERK kinase (MEK)-dependent, as shown by pharmacological MEK inhibition. The MEK-dependent antiapoptotic effect of v-raf was exerted despite a lower level of P-ERK1/2 in EIA + raf cells with respect to EIA + Py/EIA cells, which was dependent on a high tyrosine phosphatase activity, as shown by orthovanadate blockade. An ERK1/2 tyrosine phosphatase was likely involved. The high tyrosine phosphatase activity was instrumental to the complete suppression of senescence, detected by β-galactosidase activity, because tyrosine phosphatase blockade induced senescence in EIA + raf but not in EIA + Py cells. High tyrosine phosphatase activity and evasion from senescence were confirmed in an anaplastic thyroid cancer cell line. Therefore, besides EIA, EIA + raf cells suppress senescence through a new mechanism, namely, phosphatase-mediated P-ERK1/2 inhibition, but, paradoxically, retain the oncogenic effects of the Raf-ERK pathway. We propose that the survival effect of Raf is not a function of absolute P-ERK1/2 levels at a given time but is rather dynamically dependent on greater variations after an apoptotic stimulus.

Transformed stem cells have been isolated from some human cancers. We report that, unlike other brain cancers, the lethal glioblastoma multiforme contains neural precursors endowed with all of the critical features expected from neural stem cells. Similar, yet not identical, to their normal neural stem cell counterpart, these precursors emerge as unipotent (astroglial) in vivo and multipotent (neuronal-astroglial-oligodendroglial) in culture. More importantly, these cells can act as tumor-founding cells down to the clonal level and can establish tumors that closely resemble the main histologic, cytologic, and architectural features of the human disease, even when challenged through serial transplantation. Thus, cells possessing all of the characteristics expected from tumor neural stem cells seem to be involved in the growth and recurrence of adult human glioblastomas multiforme.