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In December 2019 in China, after a pneumonia outbreak of unknown etiology, a new RNA virus has been identified and called Sars-CoV-2. Sars-CoV-2 induced severe respiratory infections, with global and rapid epidemic diffusion, designated coronavirus disease 2019 (Covid-19). Sars-CoV-2 infection can lead to severe complications, such as acute respiratory distress syndrome (ARDS) with progression to pulmonary fibrosis. Recent clinical studies described that in patients with severe Covid-19, MSC infusions, promote regenerative and reparative effects with anti-inflammatory and anti-fibrotic action. MSCs do not express ACE2 and TMPRSS2, the two main human receptors for host-pathogen interaction, and are not permissive to in vitro Sars-CoV-2 infection, making them suitable for clinical application. The aim of our study was to evaluate the safety and efficacy of MSCs as cellular therapy in ARDS secondary to Sars-CoV-2 in patients undergoing mechanical ventilation, in order to prevent pulmonary fibrosis. MSCs for infusions are thawed at 2x106/ml cellular concentration. The intravenous infusion protocol consists of two doses of third party allogenic MSCs at 1x106/Kg, 15 day apart. From April 2020, six adult patients median age 65 years, median body weight 80 Kg, in mechanical ventilation for ARDS secondary to Sars-CoV-2 infection have been treated. Early or late adverse events were not recorded. Four out six patients showed a significant gas exchange improvement with extubation within seven days from the first infusion. Our results underline the safety and efficacy of MSC infusions for ARDS patients in mechanical ventilation, supporting the need of a phase I/II clinical trial.

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.

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.

This paper presents a study on the variation of strain rate influence with the scale effect in quasi-brittle materials. Works in the literature report an increase of dynamic resistance with strain rate. To study this relation, a numerical model that combines the Finite Element Method and the Lattice Discrete Element Method is employed. This mixed model was implemented on the commercial software Abaqus/Explicit. The samples are prepared with FEM at the extremity and LDEM for the central part, which has a reduction in the cross-section, so the rupture occurs in the middle. In the simulations, four model specimens with dimensional variations were subjected to direct tensile testing with different strain rates. The results showed that with the increase of applied strain rate, the resistances also increase and the body dimension influences this behavior. The variation of the tensile strength is found without modifications on the elemental constitutive relationship or material parameters. At last, a method that could lead to an independent relationship between the dynamic intensification and the stain rate of scale effect is also proposed.

Neurotoxicity (NT) testing for regulatory purposes is based on in vivo animal testing. There is general consensus, however, about the need for the development of alternative methodologies to allow researchers to more rapidly and cost effectively screen large numbers of chemicals for their potential to cause NT, or to investigate their mode of action. In vitro assays are considered an important source of information for making regulatory decisions, and human cell-based systems are recommended as one of the most relevant models in toxicity testing, to reduce uncertainty in the extrapolation of results from animal-based models. Human neuronal models range from various neuroblastoma cell lines to stem cell-derived systems, including those derived from mesenchymal stem/stromal cells (hMSC). hMSCs exhibit numerous advantages, including the fact that they can be obtained in high yield from healthy human adult tissues, can be cultured with a minimal laboratory setup and without genetic manipulations, are able of continuous and repeated self-renewal, are nontumorigenic, and can form large populations of stably differentiated cells representative of different tissues, including neuronal cells. hMSCs derived from human umbilical cord (hUC) in particular possess several prominent advantages, including a painless, non-invasive, and ethically acceptable collection procedure, simple and convenient preparation, and high proliferation capacity. In addition, hMSCs can be efficiently differentiated into neuron-like cells (hNLCs), which can then be used for the assessment of neuronal toxicity of potential neurotoxic compounds in humans. Here, we describe a step-by-step procedure to use hMSCs from the umbilical cord for in vitro neurotoxicity testing. First, we describe how to isolate, amplify, and store hMSCs derived from the umbilical cord. We then outline the steps to transdifferentiate these cells into hNLCs, and then use the hNLCs for neurotoxicity testing by employing multiple common cytotoxicity assays after treatment with test compounds. The approach follows the most updated guidance on using human cell-based systems. These protocols will allow investigators to implement an alternative system for obtaining primary NLCs of human origin, and support advancement in neurotoxicity research. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation and maintenance of human mesenchymal stem/stromal cells (hMSCs) obtained from the umbilical cord lining membrane Basic Protocol 2: Transdifferentiation of hMSCs into neuron-like cells (hNLCs) and basic neurotoxicity assessment.

In the modern age, the idea of property tends to become simpler and more defined than in the past. Likewise, the notion of public assets begins to become more definite. As a consequence, a new perception emerges also in relation with goods of artistic value and their protection. A conflict arises between public needs and the prerogatives of the owner. This paper focuses on the legislative provisions adopted by the Italian States between the Sixteenth and Eighteenth centuries and wonders about the purposes and reasons which move political authorities.

The mucosal-associated lymphoid tissues of the upper respiratory tract, including adenoids and palatine tonsils, are considered as the first line of defense against respiratory infections, being important effector organs in both mucosal-type and systemic-type adaptive immunity. They are strategically located for mediating both local and regional immune functions, as they are exposed to antigens from both the inhaled air (allergens and pathogens) and the alimentary tract. Adenoids play a major role in the early and effective immune responses against viral and bacterial upper airway infections, as well as in the development of allergic reactions to respiratory allergens, being influenced by several environmental antigens and pollutants, such as tobacco smoke. In addition, recent studies have focused on new immune-modulating strategies for adenoidal cells as a preventive and therapeutic approach for chronic upper airways inflammation.:Herein, we aimed to summarize what is known about the cellular and molecular mechanisms regulating adenoidal immune responses in the context of inflammation and allergy, with particular reference to scientific literature published within the last five years.

In the last decade, the secreting activity of mesenchymal stem/stromal cells (MSCs) has been widely investigated, due to its possible therapeutic role. In fact, MSCs release extracellular vesicles (EVs) containing relevant biomolecules such as mRNAs, microRNAs, bioactive lipids, and signaling receptors, able to restore physiological conditions where regenerative or anti-inflammatory actions are needed. An actual advantage would come from the therapeutic use of EVs with respect to MSCs, avoiding the possible immune rejection, the lung entrapment, improving the safety, and allowing the crossing of biological barriers. A number of concerns still have to be solved regarding the mechanisms determining the beneficial effect of MSC-EVs, the possible alteration of their properties as a consequence of the isolation/purification methods, and/or the best approach for a large-scale production for clinical use. Most of the preclinical studies have been successful, reporting for MSC-EVs a protecting role in acute kidney injury following ischemia reperfusion, a potent anti-inflammatory and anti-fibrotic effects by reducing disease associated inflammation and fibrosis in lung and liver, and the modulation of both innate and adaptive immune responses in graft versus host disease (GVHD) as well as autoimmune diseases. However, the translation of MSC-EVs to the clinical stage is still at the initial phase. Herein, we discuss the therapeutic potential of an acellular product such as MSC derived EVs (MSC-EVs) in acute and chronic pathologies.

Respiratory infections, mainly in children, are a demanding challenge for physicians. Commonly, a relative immune-defect sustains their recurrence. At present, there is no standardized treatment for their prevention acting on the immune system. Citomix is a low-dose multicomponent medication largely used in this issue. The current study evaluated its ex vivo effect on adenoidal mononuclear cells recovered from children operated for adenoid hypertrophy. B cell phenotype, and IFN-γ, IL-6, IL-10, IgG, IgA, IgM in culture supernatants were evaluated. Citomix was able to significantly increase the expression of B memory cells, IFN-γ, IL-6, IgA and IgM, and significantly decrease IL-10 and IgG. The current outcomes could be consistent with a strategy deputed to improve the early immune response to pathogens. In conclusion, the present ex vivo study suggests that Citomix might be a promising medication in preventing and early treating respiratory infections.

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.

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/β2 showed basically the same risk (OR=5.3). Indeed, no differences have been found in CD risk between DQ2/β2 and DQ2/DQ2, as well as between DQ8/β2 and DQ2/DQ8, and between β2/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.

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.

In the present work, we report the synthesis, Raman characterization and comparison of the Raman spectra of the wood sawdust ash (WSA) and graphene produced from this WSA at different temperatures and burning times. For this purpose, the wood sawdust (WS) was burnt at different temperatures (300, 400 and 500 °C) at a constant time of 60 min, and different times (40, 60 and 120 min.) for a constant temperature of 400 °C. For graphene production, WSA was activated with potassium hydroxide (KOH) at different activation temperatures (700, 750, 800 and 850 °C) at a constant time of 60 min, and different burning times (30, 60 and 90 min) for the constant temperature of 850 °C. Comparing the results, it was found that 400 °C and 60 min are the optimum parameters to obtain the most efficient WSA for graphene production. On the other hand, 850 °C and 60 min are the optimum parameters to activate the WSA with KOH for graphene synthetization. Disorder analysis through the ratio between the integrated intensities of the D=DS + DA, G = GS + GA, DA and GA bands (ID/IG, IDA/IGA), showed that the WSA produced at 400 °C/60 min and graphene obtained at 850 °C/60 min have lower disorder, in respect to other temperatures and burning times. These results show that graphene production from wood-based industry residue is one of the most environmentally friendly solutions of transforming negative-valued wastes to valuable product in an easy, fast and economical way.

Plasmonic biosensors, particularly arrays of nanoholes on thin gold films, have been widely explored in recent years as possible platforms for fast medical diagnostic. In this work, we present a screening method for leukemia cancer markers that uses a plasmonic biosensor based on nanohole arrays fabricated on plastic substrates. The low-cost, scalable, and reproducible nanohole array structures were fabricated by UV nanoimprinting technique. The relative concentration of human immunoglobulin kappa and lambda light chains in blood serum was employed as a screening method. The kappa/lambda concentration ratio was used to determine an unbalance in the immunoglobulin production due to leukemia. The platform was tested using serum samples from patients with known leukemia diagnoses. The results indicated that this inexpensive and flexible plasmonic platform is a promising tool for routine screening in clinical settings.

We report the interaction between 5.0 wt.% and 20 wt.% Yb3+ doped phosphate glasses substrates with graphene single layer (1L) and bilayer (2L) by Raman spectroscopy. This interaction is studied in terms of shifting of the G, 2D and 2D’ bands of graphene, which is higher in 1L than in 2L graphene on the 20% Yb3+ substrate, in respect to the 5.0 wt.% substrate. Furthermore, changes in the full width at half maximum of the G, 2D and 2D’ bands, together with the I2D/IG ratio, for doped and undoped substrates, reveal this interaction. Additionally, when a single layer graphene is found on the surface of both substrates, some Raman peaks on the less doped sample are absent, whereas they are present on the more doped sample, indicating that the presence of Yb3+ ions increases the interaction between the substrate and the single layer graphene. The reported results give insights about graphene interaction with doped rare earth substrates.

The interaction and solvent influence on two different imidazolium based ion pairs have been investigated by molecular dynamics simulations and Nuclear Magnetic Resonance. The cations 1,2,3,4,5-pentamethyl imidazolium and 1,3,4,5-tetramethyl imidazolium were considered with the imidazolate anion to evaluate the influence of the acidic hydrogen at the position 2 of the imidazolium ring on the ion pair formation in different solvents. The selected solvents are chloroform, dichloromethane, acetone, dimethylsulfoxide, and water, covering a broad range of polarity and permittivity. The binding free energy of the ion pair was computed by umbrella sampling. We observed that, with the increase of the dielectric constant, the ion pairs become more transient being separated in water. The free energies of binding corroborate ion pair stabilization by the hydrogen bond at carbon 2 of the imidazolium cations. In dichloromethane, we obtained weaker bound ion pairs than in acetone due to intercalation of dichloromethane into the ion pair. Thus, the ion pair stability is not only a consequence of the solvent's dielectric constant, but also due to local structural details.

The aim of the present study is to evaluate the effects of 60-day artichoke leaf extract (ALE) supplementation (250mg, twice daily) on cytokines levels, natural killer cell (NK) response, and lipo-metabolic profile (HDL, LDL, and total-cholesterol, triglycerides (TG), ApoB, ApoA, lipid accumulation product (LAP), glucose, insulin, and homeostasis model assessment of insulin resistance (HOMA-IR)) in twenty adults (9/11 males/females, age=49.10 ± 13.74 years, and BMI=33.12 ± 5.14 kg/m2) with low HDL-C and mild hypercholesterolemia. Hierarchical generalized linear model, adjusted for sex, BMI, and age, has been used to evaluate pre-post treatment changes. A significant increase for HDL-C (β=0.14, p=0.0008) and MCP-1 (β=144.77, p=0.004) and a significant decrease for ApoB/ApoA (β=-0.07, p=0.03), total-C/HDL-C ratio (β=-0.58, pβ=0.43, p=0.04), medium (β=0.40, pβ=0.42, p=0.001) have been found. These results support the benefits of ALE supplementation on metabolic profile.

Stress affects the immune system and intestinal microbiota composition and can lead to imbalance between pro- and anti-inflammatory cytokines or to uncontrolled production of cytokines. The effect of emotional stress on secretory IgA levels also indicates that stress decreases mucosal integrity. Our aim was to evaluate whether a probiotic product (Lactoflorene® Plus) can prevent alterations in the immune response associated with self-reported stress and microbiota composition. Healthy adult volunteers who self-reported psychological stress were enrolled and randomised into a placebo and a probiotic group. Salivary stress markers (α-amylase, cortisol, chromogranin A) and immunological parameters (sIgA, NK cell activity, IL-8, IL-10, TNF-α) in feces and the composition of intestinal microbiota were evaluated. Administration of the product did not exert a direct effect on the salivary stress markers or NK cell activity but did reduce abdominal pain and increase faecal IgA and IL-10 levels. The probiotic product induced a moderate increase in Bifidobacterium and Lactobacillus spp., as expected, and in Faecalibacterium spp., and decreased the size of the Dialister spp. and Escherichia and Shigella populations. Administration of the product helped protect the mucosal barrier by supporting the number of short-chain fatty acid producers and decreasing the load of potentially harmful bacteria, thus reducing intestinal inflammation and abdominal discomfort. ClinicalTrials.gov: NCT03234452. Keywords: BB-12®, LA-5®, Immune response, Stress, Abdominal pain, IgA

In this work, an optical-diffraction biosensor consisted of gold nanohole arrays was fabricated by interference lithography and lift-off process. The transmitted first diffraction order intensity of different streptavidin concentrations was analysed using a 458 nm wavelength and a photodetector connected to an oscilloscope. The biosensor limit of detection (LOD) was found to be 122 ng.mL−1. The possibility of real time measurements and the high sensibility achieved demonstrate the great potential of the device.

The current scientific research is continuously aiming at identifying new therapeutic targets with the purpose of modifying the immune response to allergens. The evolution in immunological methods has led to the identification of immunoglobulin E (IgE) as both a diagnostic biomarker and potential therapeutic target in allergic diseases, such as allergic rhinitis. Allergen immunotherapy has been used for more than 100 years to treat allergic diseases and it is today considered the only disease-modifying treatment capable of inducing a long-lasting immunological and clinical tolerance toward the causal allergen. During the past 20 years, major advances have been made in understanding the molecular and cellular mechanisms of allergen tolerance in humans. Moreover, there has been considerable progress in allergen extract modifications and additions to standard extracts. The recognition that IgE plays a pivotal role in basic regulatory mechanisms of allergic inflammation has recently stimulated research into the therapeutic potential of directly targeting this antibody. Omalizumab, the most advanced humanized anti-IgE monoclonal antibody, is currently approved for the treatment of uncontrolled allergic asthma and chronic spontaneous urticaria. Interesting results also arise from studies in which omalizumab was administered in patients with allergic rhinitis. The aim of this review is to provide an update on current findings on immunological and clinical effects of allergen immunotherapy and anti-IgE therapy, which have been shown to have synergistic modes of action for the treatment of allergic rhinitis.

The adenoids are exposed to a wide number and variety of microbes, environmental pollutants, and food antigens. Atopy and passive smoke may significantly affect immune responses, mainly in children. The aim of the present study was to investigate whether passive exposure to tobacco smoke and/or atopy could affect immunoglobulin production by adenoidal lymphocytes in a cohort of children presenting with adenoid hypertrophy. A total of 277 children (151 males and 126 females; median age 5.5 years), with adenoidal hypertrophy requiring adenoidectomy and or adeno-tonsillectomy, were consecutively enrolled in the study. Adenoid mononuclear cells were in vitro stimulated with LPS or CpG. When considering both the presence of smoke exposure and atopy, we observed that the CpG-induced decrease in IgA and IgM production was significantly associated with this combination of risk factors. In the T-independent immunoglobulin production assay we found a positive association between the two risk factors and IgA and IgM production. In particular, the presence of both risk factors, showed a significant increase in IgA and IgM production after stimulation. In conclusion, this is the first study that investigated the in vitro adenoidal B cell response after different stimuli in children, also evaluating possible exposure to passive smoke and/or an atopic condition.