Exosomal-miRNas expression and growth factors released by mononuclear cells of CLAD patients in response to extracorporeal photopheresis.

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.

New therapeutic approaches in pediatric diseases: Mesenchymal stromal cell and mesenchymal stromal cell-derived extracellular vesicles as new drugs.

Mesenchymal Stromal Cell (MSC) clinical applications have been widely reported and their therapeutic potential has been documented in several diseases. MSCs can be isolated from several human tissues and easily expanded in vitro, they are able to differentiate in a variety of cell lineages, and they are known to interact with most immunological cells, showing immunosuppressive and tissue repair properties. Their therapeutic efficacy is closely associated with the release of bioactive molecules, namely Extracellular Vesicles (EVs), effective as their parental cells. EVs isolated from MSCs act by fusing with target cell membrane and releasing their content, showing a great potential for the treatment of injured tissues and organs, and for the modulation of the host immune system. EV-based therapies provide, as major advantages, the possibility to cross the epithelium and blood barrier and their activity is not influenced by the surrounding environment. In the present review, we deal with pre-clinical reports and clinical trials to provide data in support of MSC and EV clinical efficacy with particular focus on neonatal and pediatric diseases. Considering pre-clinical and clinical data so far available, it is likely that cell-based and cell-free therapies could become an important therapeutic approach for the treatment of several pediatric diseases.

Liposome Formulation and In Vitro Testing in Non-Physiological Conditions Addressed to Ex Vivo Kidney Perfusion.

This work focuses on formulating liposomes to be used in isolated kidney dynamic machine perfusion in hypothermic conditions as drug delivery systems to improve preservation of transplantable organs. The need mainly arises from use of kidneys from marginal donors for transplantation that are more exposed to ischemic/reperfusion injury compared to those from standard donors. Two liposome preparation techniques, thin film hydration and microfluidic techniques, are explored for formulating liposomes loaded with two model proteins, myoglobin and bovine serum albumin. The protein-loaded liposomes are characterized for their size by DLS and morphology by TEM. Protein releases from the liposomes are tested in PERF-GEN perfusion fluid, 4 °C, and compared to the in vitro protein release in PBS, 37 °C. Fluorescent liposome uptake is analyzed by fluorescent microscope in vitro on epithelial tubular renal cell cultures and ex vivo on isolated pig kidney in hypothermic perfusion conditions. The results show that microfluidics are a superior technique for obtaining reproducible spherical liposomes with suitable size below 200 nm. Protein encapsulation efficiency is affected by its molecular weight and isoelectric point. Lowering incubation temperature slows down the proteins release; the perfusion fluid significantly affects the release of proteins sensitive to ionic media (such as BSA). Liposomes are taken up by epithelial tubular renal cells in two hours' incubation time.

CD73-Adenosinergic Axis Mediates the Protective Effect of Extracellular Vesicles Derived from Mesenchymal Stromal Cells on Ischemic Renal Damage in a Rat Model of Donation after Circulatory Death.

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.

Large Area Nanohole Arrays for Sensing Fabricated by Interference Lithography.

Several fabrication techniques are recently used to produce a nanopattern for sensing, as focused ion beam milling (FIB), e-beam lithography (EBL), nanoimprinting, and soft lithography. Here, interference lithography is explored for the fabrication of large area nanohole arrays in metal films as an efficient, flexible, and scalable production method. The transmission spectra in air of the 1 cm2 substrate were evaluated to study the substrate behavior when hole-size, periodicity, and film thickness are varied, in order to elucidate the best sample for the most effective sensing performance. The efficiency of the nanohole array was tested for bulk sensing and compared with other platforms found in the literature. The sensitivity of ~1000 nm/RIU, achieved with an array periodicity in the visible range, exceeds near infrared (NIR) performances previously reported, and demonstrates that interference lithography is one of the best alternative to other expensive and time-consuming nanofabrication methods.

Coupling Single-Drop Microextraction with SERS: A Demonstration Using p-MBA on Gold Nanohole Array Substrate.

Single-drop microextraction (SDME) was coupled with surface-enhanced Raman scattering (SERS) to provide sample extraction and pre-concentration for detection of analyte at low concentrations. A gold nanohole array substrate (AuNHAS), fabricated by interference lithography, was used as SERS substrate and para-mercaptobenzoic acid (p-MBA) was tested as a probe molecule, in the concentration range 10-8-10-4 mol L-1. With this approach, a limit of 10-7 mol L-1 was clearly detected. To improve the detection to lower p-MBA concentration, as 10-8 mol L-1, the SDME technique was applied. The p-MBA Raman signature was detected in two performed extractions and its new concentration was determined to be ~4.6 × 10-5 mol L-1. This work showed that coupling SDME with SERS allowed a rapid (5 min) and efficient pre-concentration (from 10-8 mol L-1 to 10-5 mol L-1), detection, and quantification of the analyte of interest, proving to be an interesting analytical tool for SERS applications.

Fecal Clostridiales distribution and short-chain fatty acids reflect bowel habits in irritable bowel syndrome.

Irritable bowel syndrome (IBS), a common functional gastrointestinal disorder, is classified according to bowel habits as IBS with constipation (IBS-C), with diarrhea (IBS-D), with alternating constipation and diarrhea (IBS-M), and unsubtyped (IBS-U). The mechanisms leading to the different IBS forms are mostly unknown. This study aims to evaluate whether specific fecal bacterial taxa and/or short-chain fatty acids (SCFAs) can be used to distinguish IBS subtypes and are relevant for explaining the clinical differences between IBS subcategories. We characterized five fecal samples collected at 4-weeks intervals from 40 IBS patients by 16S rRNA gene profiling and SCFA quantification. Finally, we investigated the potential correlations in IBS subtypes between the fecal microbial signatures and host physiological and clinical parameters. We found significant differences in the distribution of Clostridiales OTUs among IBS subtypes and reduced levels of SCFAs in IBS-C compared to IBS-U and IBS-D patients. Correlation analyses showed that the diverse representation of Clostridiales OTUs between IBS subtypes was associated with altered levels of SCFAs; furthermore, the same OTUs and SCFAs were associated with the fecal cytokine levels and stool consistency. Our results suggest that intestinal Clostridiales and SCFAs might serve as potential mechanistic biomarkers of IBS subtypes and represent therapeutic targets.

HLA-DQ genetics in children with celiac disease: a meta-analysis suggesting a two-step genetic screening procedure starting with HLA-DQ ß chains.

BackgroundSpecific HLA-DQ genes have been recognized as necessary - but not sufficient - factors for the occurrence of Celiac Disease (CD). Through a meta-analysis, evaluating the distribution of CD-related HLA genotypes in children, we aimed at providing insights for a potential widened screening strategy.MethodsAfter a systematic search on the association between class II HLA genes and CD in children, 46 publications were obtained and assessed for eligibility. A total of 13 eligible studies were submitted to data extraction and analysis (10 case-control studies and 3 cohort studies). Case-control studies collectively enrolled 740 CD patients and 943 controls.ResultsIn the population-stratified analysis, the following alleles conferred a significantly increased risk for CD: HLA-DQB1*02 (odds ratio [OR]=10.28) and HLA-DQB1*03:02 (OR=2.24). By drafting a risk gradient to develop CD according to HLA genetic background, the highest risk is confirmed to exist for DQ2/DQ2 homozygous subjects, regardless of the ethnicities (OR=5.4). Actually, the genotype DQ2/ß2 showed basically the same risk (OR=5.3). Indeed, no differences have been found in CD risk between DQ2/ß2 and DQ2/DQ2, as well as between DQ8/ß2 and DQ2/DQ8, and between ß2/DQX and DQ2/X.ConclusionThe HLA-DQB1*02:01 allele is present in more than 90% CD children. In the perspective of a widened pediatric population screening for CD, a double-step process might be suggested: HLA-DQB1*02:01 might be investigated first and, only if this result is positive, children might be candidate for a prospective serologic screening, as a second step.

Entomotoxic activity of Rhinella icterica (Spix, 1824) toad skin secretion in Nauphoeta cinerea cockroaches: An octopamine-like modulation.

Rhinella icterica is a poisonous toad whose toxic secretion has never been studied against entomotoxic potential. Sublethal doses of Rhinella icterica toxic secretion (RITS) were assayed in Nauphoeta cinerea cockroaches, in order to understand the physiological and behavioral parameters, over the insect central and peripheral nervous system. RITS (10 µg/g) injections, induced behavioral impairment as evidenced by a significant decrease (38 ±â€¯14%) in the distance traveled (p < .05), followed by an increase (90 ±â€¯6%) of immobile episodes (p < .001, n = 28, respectively). In cockroaches semi-isolated heart preparations, RITS (16 µg/200 µl) induced a significant irreversible dose-dependent negative chronotropism, reaching ~40% decrease in heart rate in 20 min incubation. In in vivo cockroach neuromuscular preparations, RITS (20, 50 and 100 µg/g of animal weight) induced a time-dependent inhibition of twitch tension that was complete for 20 µg/g, in 120 min recordings. RITS (10 µg/g) also induced a significant increase in the insect leg grooming activity (128 ±â€¯10%, n = 29, p < .01), but not in the antennae counterparts. The RITS increase in leg grooming activity was prevented in 90% by the pretreatment of cockroaches with phentolamine (0.1 µg/g). The electrophysiological recordings of spontaneous neural compound action potentials showed that RITS (20 µg/g) induced a significant increase in the number of events, as well as in the rise time and duration of the potentials. In conclusion, RITS showed to be entomotoxic, being the neuromuscular failure and cardiotoxic activity considered the main deleterious effects. The disturbance of the cockroaches' behavior together with the electrophysiological alterations, may unveil the presence of some toxic components present in the poison with inherent biotechnological potentials.

Gut Microbiota and Obesity.

Intestinal microbiota is composed by symbiotic innocuous bacteria and potential pathogens also called pathobionts. The human gut normally hosts roughly 1014 bacterial organisms of up to 1000 different species. The genome size of this microbial organ, collectively named microbiome, exceeds the size of the human nuclear genome by 2 orders of magnitude.

Confined water in imidazolium based ionic liquids: a supramolecular guest@host complex case.

It is well known that the macroscopic physico-chemical properties of ionic liquids (ILs) are influenced by the presence of water that strongly interferes with the supramolecular organization of these fluids. However, little is known about the function of water traces within this confined space and restricted ionic environments, i.e. between cations and anions. Using specially designed ILs namely 1,2,3-trimethyl-1H-imidazol-3-ium imidazol-1-ide (MMMI·Im) and 3-n-butyl-1,2-dimethyl-1H-imidazol-3-ium imidazol-1-ide (BMMI·Im), the structure and function of water have been determined in condensed, solution and gas phases by X-ray diffraction studies, NMR, molecular dynamics simulations (MDS) and DFT calculations. In the solid state the water molecule is trapped inside the ionic network (constituted of contact ion pairs formed by π(+)-π(-) interaction) through strong H-bonds involving the water hydrogens and the nitrogens of two imidazolate anions forming a guest@host supramolecular structure. A similar structural arrangement was corroborated by DFT calculations and MDS. The presence of a guest@host species (H2O@ILpair) is maintained to a great extent even in solution as detected by (1)H-(1)H NOESY-experiments of the ILs dissolved in solvents with low and high dielectric constants. This confined water catalyses the H/D exchange with other substrates containing acidic-H such as chloroform.

Improving the performance of gold nanohole array biosensors by controlling the optical collimation conditions.

An experimental investigation on how the bulk and surface sensitivities of gold nanohole arrays fabricated by interference lithography affect the degree of white light beam collimation is presented. The optical transmission response of nanohole arrays has been recorded by focused and collimated beam transmission spectra. The results show that both the bulk and surface sensitivities for the collimated case are much larger than for the focused case. In particular, the shape of the spectra was dependent on the degree of beam collimation. The results showed that improved sensing performance (around 3.5 times) and higher figure of merit (around 4.4 times) can be obtained by simply adjusting the incident/collection experimental conditions in transmission measurements.

The Benefits of the Mediterranean Diet on Inflamm-Aging in Childhood Obesity.

(1) Background: Numerous elements of the Mediterranean diet (MD) have antioxidant and anti-inflammatory qualities. (2) Methods: We present a narrative review of the potential benefits of the Mediterranean dietary pattern (MD) in mitigating aging-related inflammation (inflamm-aging) associated with childhood obesity. The mechanisms underlying chronic inflammation in obesity are also discussed. A total of 130 papers were included after screening abstracts and full texts. (3) Results: A complex interplay between obesity, chronic inflammation, and related comorbidities is documented. The MD emerges as a promising dietary pattern for mitigating inflammation. Studies suggest that the MD may contribute to weight control, improved lipid profiles, insulin sensitivity, and endothelial function, thereby reducing the risk of metabolic syndrome in children and adolescents with obesity. (4) Conclusions: While evidence supporting the anti-inflammatory effects of the MD in pediatric obesity is still evolving, the existing literature underscores its potential as a preventive and therapeutic strategy. However, MD adherence remains low among children and adolescents, necessitating targeted interventions to promote healthier dietary habits. Future high-quality intervention studies are necessary to elucidate the specific impact of the MD on inflammation in diverse pediatric populations with obesity and associated comorbidities.

Circulating Mesenchymal Stromal Cells in Patients with Infantile Hemangioma: Evaluation of Their Functional Capacity and Gene Expression Profile.

We previously published that in patients with infantile hemangioma (IH) at the onset (T0) colony forming unit-fibroblasts (CFU-Fs) are present in in vitro cultures from PB. Herein, we characterize these CFU-Fs and investigate their potential role in IH pathogenesis, before and after propranolol therapy. The CFU-F phenotype (by flow cytometry), their differentiation capacity and ability to support angiogenesis (by in vitro cultures) and their gene expression (by RT-PCR) were evaluated. We found that CFU-Fs are actual circulating MSCs (cMSCs). In patients at T0, cMSCs had reduced adipogenic potential, supported the formation of tube-like structures in vitro and showed either inflammatory (IL1ß and ESM1) or angiogenic (F3) gene expression higher than that of cMSCs from CTRLs. In patients receiving one-year propranolol therapy, the cMSC differentiation in adipocytes improved, while their support in in vitro tube-like formation was lost; no difference was found between patient and CTRL cMSC gene expressions. In conclusion, in patients with IH at T0 the cMSC reduced adipogenic potential, their support in angiogenic activity and the inflammatory/angiogenic gene expression may fuel the tumor growth. One-year propranolol therapy modifies this picture, suggesting cMSCs as one of the drug targets.

Periodic metallic nanostructures as plasmonic chemical sensors.

Periodic plasmonic nanostructures are being widely studied, optimized, and developed to produce a new generation of low-cost and efficient chemical sensors and biosensors. The extensive variety of nanostructures, interrogation approaches, and setups makes a direct comparison of the reported performance from different sensing platforms a challenging exercise. In this feature Article, the most common parameters used for the evaluation of plasmonic nanostructures will be reviewed, with particular focus on the advances in periodic plasmonic nanostructures. Recent progress in the fabrication methods that allow for the high-volume production of periodic plasmonic sensors at low cost will be described, together with an assessment of the state of the art in terms of periodic structures employed for chemical sensing.

Multifunctional 3D magnetic carbon aerogel for adsorption separation and highly sensitive SERS detection of malachite green.

This work presents a novel strategy for the synthesis of a recyclable aerogel and its multifunctional application as effective adsorption material for organic pollutants and as a high-quality SERS substrate for on-site detection measurement. Silver nanoparticles (Ag NPs) were uniformly dispersed and adsorbed on the surface of an Fe3C-loaded carbon aerogel, resulting in the formation of a three-dimensional Ag-Fe3C-MCA (magnetic carbon aerogel) composite. The substrate preparation led to Ag-Fe3C-MCA with a mesoporous structure for high adsorption capacity, together with magnetic properties for easy separation capability. The Ag-Fe3C-MCA composite demonstrated an efficient removal ability for malachite green (MG), with an adsorption capacity of 296.7 mg g-1. Moreover, Ag-Fe3C-MCA composite provided ultrasensitive surface-enhanced Raman scattering detection for MG molecules, obtaining a limit of detection (LOD) of 3 × 10-10 M. Aquaculture water samples with spiked MG concentrations were used to simulate practical scenarios. The Ag-Fe3C-MCA presented has a significant potential for the removal of hazardous residues in wastewater, together with an efficient and sensitive method of quantification, all on the same substrate.

Lung mesenchymal cells from patients with COVID-19 driven lung fibrosis: Several features with CTD-ILD derived cells but with higher response to fibrogenic signals and might be more pro-inflammatory.

A subset of severe COVID19 patients develop pulmonary fibrosis, but the pathophysiology of this complication is still unclear. We previously described the possibility to isolate lung mesenchymal cells (LMC) by culturing broncho-alveolar lavage (BAL) cells from patients with pulmonary fibrosis or chronic lung allograft dysfunction. Aim of this study was to investigate the possibility to isolate and characterize LMC from BAL of patients that, two months after discharge for severe COVID19, show CT signs of post-COVID19 fibrosis (Post-COVID) and in some cases has been considered transplant indication. Results were compared with those from BAL of patients with collagen tissue disease-associated interstitial fibrosis (CTD-ILD). BAL fluid levels of TGFß, VEGF, TIMP2, RANTES, IL6, IL8, and PAI1 were assessed. LMC were cultured and expanded, phenotyped by flow cytometry, and tested for osteogenic and adipogenic differentiation. Finally, we tested immunomodulatory and proliferative capabilities, collagen I production + /- TGF-beta stimulation. BAL cytokine and growth factor levels were comparable in the two groups. Efficiency of isolation from BAL was 100% in post-COVID compared to 63% in CTD-ILD. LMC from post-COVID were positive for CD105, CD73, CD90, and negative for CD45, CD34, CD19 and HLA-DR as in CTD-ILD samples. Post-COVID LMC displayed higher collagen production with respect to CTD-ILD LMC. Immunomodulatory capacity towards lymphocytes was very low, while Post-COVID LMC significantly upregulated pro-inflammatory cytokine production by healthy PBMCs. Our preliminary data suggest that LMC from post-COVID19 fibrosis patients share several features with CTD-ILD ones but might have a higher response to fibrogenic signals and pro-inflammatory profile.

Allogeneic Mesenchymal Stromal Cells as a Global Pediatric Prospective Approach in the Treatment of Respiratory Failure Associated with Surfactant Protein C Dysfunction.

Mesenchymal stromal cells (MSCs) have been proposed as a new therapeutic strategy to treat congenital and acquired respiratory system diseases. We describe a case report of an 18-month-old male patient with progressive chronic respiratory failure, associated with mutations of the surfactant protein C gene (SFTPC) due to c.289G > T variant p.Gly97Ser (rs927644577) and c.176A > G variant (p.His59Arg), submitted to repeated intravenous infusions of allogeneic bone marrow (BM) MSCs. The clinical condition of the patient was monitored. Immunologic studies before and during MSC treatment were performed. No adverse events related to the MSC infusions were recorded. Throughout the MSC treatment period, the patient showed a growth recovery. Starting the second infusion, the patient experienced an improvement in his respiratory condition, with progressive adaptation to mechanical ventilation. After the third infusion, five hours/die of spontaneous breathing was shown, and after infusion IV, spontaneous ventilation for 24/24 h was recorded. A gradual decrease of lymphocytes and cell subpopulations was observed. No variations in the in vitro T cell response to PHA were determined by MSC treatment as well as for the in vitro B cell response. A decrease in IFN-γ, TNF-α, and IL-10 levels was also detected. Even though we cannot exclude an improvement of pulmonary function due to the physiological maturation, the well-known action of MSCs in the repair of lung tissue, together with the sequence of events observed in our patient, may support the therapeutic role of MSCs in this clinical condition. However, further investigations are necessary to confirm the result and long-term follow-up will be mandatory to confirm the benefits on the pulmonary condition.

Autoantibodies neutralizing type I IFNs underlie West Nile virus encephalitis in ∼40% of patients.

Mosquito-borne West Nile virus (WNV) infection is benign in most individuals but can cause encephalitis in <1% of infected individuals. We show that ∼35% of patients hospitalized for WNV disease (WNVD) in six independent cohorts from the EU and USA carry auto-Abs neutralizing IFN-α and/or -ω. The prevalence of these antibodies is highest in patients with encephalitis (∼40%), and that in individuals with silent WNV infection is as low as that in the general population. The odds ratios for WNVD in individuals with these auto-Abs relative to those without them in the general population range from 19.0 (95% CI 15.0-24.0, P value <10-15) for auto-Abs neutralizing only 100 pg/ml IFN-α and/or IFN-ω to 127.4 (CI 87.1-186.4, P value <10-15) for auto-Abs neutralizing both IFN-α and IFN-ω at a concentration of 10 ng/ml. These antibodies block the protective effect of IFN-α in Vero cells infected with WNV in vitro. Auto-Abs neutralizing IFN-α and/or IFN-ω underlie ∼40% of cases of WNV encephalitis.