A microbially produced AhR ligand promotes a Tph1-driven tolerogenic program in multiple sclerosis.

Multiple sclerosis is a debilitating autoimmune disease, characterized by chronic inflammation of the central nervous system. While the significance of the gut microbiome on multiple sclerosis pathogenesis is established, the underlining mechanisms are unknown. We found that serum levels of the microbial postbiotic tryptophan metabolite indole-3-carboxaldehyde (3-IAld) inversely correlated with disease duration in multiple sclerosis patients. Much like the host-derived tryptophan derivative L-Kynurenine, 3-IAld would bind and activate the Aryl hydrocarbon Receptor (AhR), which, in turn, controls endogenous tryptophan catabolic pathways. As a result, in peripheral lymph nodes, microbial 3-IAld, affected mast-cell tryptophan metabolism, forcing mast cells to produce serotonin via Tph1. We thus propose a protective role for AhR-mast-cell activation driven by the microbiome, whereby natural metabolites or postbiotics will have a physiological role in immune homeostasis and may act as therapeutic targets in autoimmune diseases.

Biologics, theranostics, and personalized medicine in drug delivery systems.

The progress in human disease treatment can be greatly advanced through the implementation of nanomedicine. This approach involves targeted and cell-specific therapy, controlled drug release, personalized dosage forms, wearable drug delivery, and companion diagnostics. By integrating cutting-edge technologies with drug delivery systems, greater precision can be achieved at the tissue and cellular levels through the use of stimuli-responsive nanoparticles, and the development of electrochemical sensor systems. This precision targeting - by virtue of nanotechnology - allows for therapy to be directed specifically to affected tissues while greatly reducing side effects on healthy tissues. As such, nanomedicine has the potential to transform the treatment of conditions such as cancer, genetic diseases, and chronic illnesses by facilitating precise and cell-specific drug delivery. Additionally, personalized dosage forms and wearable devices offer the ability to tailor treatment to the unique needs of each patient, thereby increasing therapeutic effectiveness and compliance. Companion diagnostics further enable efficient monitoring of treatment response, enabling customized adjustments to the treatment plan. The question of whether all the potential therapeutic approaches outlined here are viable alternatives to current treatments is also discussed. In general, the application of nanotechnology in the field of biomedicine may provide a strong alternative to existing treatments for several reasons. In this review, we aim to present evidence that, although in early stages, fully merging advanced technology with innovative drug delivery shows promise for successful implementation across various disease areas, including cancer and genetic or chronic diseases.

Engineering carrier nanoparticles with biomimetic moieties for improved intracellular targeted delivery of mRNA therapeutics and vaccines.

Biological membrane-engineered lipid nanoparticles (LNP) have shown enormous potential as vehicles for drug delivery due to their outstanding biomimetic properties. To make these nanoparticles more adaptable to complex biological systems, several methods and cellular sources have been adopted to introduce biomembrane-derived moieties onto LNP and provide the latter with more functions while preserving their intrinsic nature. In this review, we focus on LNP decoration with specific regard to mRNA therapeutics and vaccines. The bio-engineering approach exploits a variety of biomembranes for functionalization, such as those derived from red blood cells, white blood cells, cancer cells, platelets, exosomes, and others. Biomembrane engineering could greatly enhance efficiency in targeted drug delivery, treatment, and diagnosis of cancer, inflammation, immunological diseases, and a variety of pathologic conditions. These membrane-modification techniques are expected to advance biomembrane-derived LNP into wider applications in the future.

A Stable Micellar Formulation of RAD001 for Intracerebroventricular Delivery and the Treatment of Alzheimer's Disease and Other Neurological Disorders.

A large body of evidence, replicated in many mouse models of Alzheimer's disease (AD), supports the therapeutic efficacy of the oral mammalian target of rapamycin inhibitors (mTOR-Is). Our preliminary data show that intracerebroventricular (ICV) administration of everolimus (RAD001) soon after clinical onset greatly diminished cognitive impairment and the intracellular beta amyloid and neurofibrillary tangle load. However, RAD001 shows >90% degradation after 7 days in solution at body temperature, thus hampering the development of proper therapeutic regimens for patients. To overcome such a drawback, we developed a stable, liquid formulation of mTOR-Is by loading RAD001 into distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) micelles using the thin layer evaporation method. The formulation showed efficient encapsulation of RAD001 and a homogeneous colloidal size and stabilised RAD001, with over 95% of activity preserved after 14 days at 37 °C with a total decay only occurring after 98 days. RAD001-loaded DSPE-PEG2000 micelles were unchanged when stored at 4 and 25 °C over the time period investigated. The obtained formulation may represent a suitable platform for expedited clinical translation and effective therapeutic regimens in AD and other neurological diseases.

Dual species sphingosine-1-phosphate lyase inhibitors to combine antifungal and anti-inflammatory activities in cystic fibrosis: a feasibility study.

Cystic fibrosis (CF) is an autosomal recessive disorder characterized by respiratory failure due to a vicious cycle of defective Cystic Fibrosis Transmembrane conductance Regulator (CFTR) function, chronic inflammation and recurrent bacterial and fungal infections. Although the recent introduction of CFTR correctors/potentiators has revolutionized the clinical management of CF patients, resurgence of inflammation and persistence of pathogens still posit a major concern and should be targeted contextually. On the background of a network-based selectivity that allows to target the same enzyme in the host and microbes with different outcomes, we focused on sphingosine-1-phosphate (S1P) lyase (SPL) of the sphingolipid metabolism as a potential candidate to uniquely induce anti-inflammatory and antifungal activities in CF. As a feasibility study, herein we show that interfering with S1P metabolism improved the immune response in a murine model of CF with aspergillosis while preventing germination of Aspergillus fumigatus conidia. In addition, in an early drug discovery process, we purified human and A. fumigatus SPL, characterized their biochemical and structural properties, and performed an in silico screening to identify potential dual species SPL inhibitors. We identified two hits behaving as competitive inhibitors of pathogen and host SPL, thus paving the way for hit-to-lead and translational studies for the development of drug candidates capable of restraining fungal growth and increasing antifungal resistance.

Evaluation of ß-Lactamase Enzyme Activity in Outer Membrane Vesicles (OMVs) Isolated from Extended Spectrum ß-Lactamase (ESBL) Salmonella Infantis Strains.

Outer membrane vesicles (OMVs) are nanoparticles released by Gram-negative bacteria, which contain different cargo molecules and mediate several biological processes. Recent studies have shown that OMVs are involved in antibiotic-resistance (AR) mechanisms by including ß-lactamase enzymes in their lumen. Since no studies have as yet been conducted on Salmonella enterica subs. enterica serovar Infantis' OMVs, the aim of the work was to collect OMVs from five S. Infantis ß-lactam resistant strains isolated from a broiler meat production chain and to investigate whether ß-lactamase enzymes are included in OMVs during their biogenesis. OMVs were isolated by means of ultrafiltration and a Nitrocefin assay quantified the presence of ß-lactamase enzymes in the OMVs. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) were used to identify the OMVs. The results showed that all strains release spherical OMVs, ranging from 60 to 230 nm. The Nitrocefin assay highlighted the presence of ß-lactamase enzymes within the OMVs. This suggests that ß-lactamase enzymes also get packaged into OMVs from bacterial periplasm during OMV biogenesis. An investigation into the possible role played by OMVs in AR mechanisms would open the door for an opportunity to develop new, therapeutic strategies.

Nickel oxide nanoparticles exposure as a risk factor for male infertility: "In vitro" effects on porcine pre-pubertal Sertoli cells.

Lately, nickel oxide nanoparticles (NiO NPs) have been employed in different industrial and biomedical fields. Several studies have reported that NiO NPs may affect the development of reproductive organs inducing oxidative stress and, resulting in male infertility. We investigated the in vitro effects of NiO NPs on porcine pre-pubertal Sertoli cells (SCs) which undergone acute (24 h) and chronic (from 1 up to 3 weeks) exposure at two subtoxic doses of NiO NPs of 1 µg/ml and 5 µg/ml. After NiO NPs exposure we performed the following analysis: (a) SCs morphological analysis (Light Microscopy); (b) ROS production and oxidative DNA damage, gene expression of antioxidant enzymes (c) SCs functionality (AMH, inhibin B Real-time PCR analysis and ELISA test); (d) apoptosis (WB analysis); (e) pro-inflammatory cytokines (Real-time PCR analysis), and (f) MAPK kinase signaling pathway (WB analysis). We found that the SCs exposed to both subtoxic doses of NiO NPs didn't sustain substantial morphological changes. NiO NPs exposure, at each concentration, reported a marked increase of intracellular ROS at the third week of treatment and DNA damage at all exposure times. We demonstrated, un up-regulation of SOD and HO-1 gene expression, at both concentrations tested. The both subtoxic doses of NiO NPs detected a down-regulation of AMH and inhibin B gene expression and secreted proteins. Only the 5 µg/ml dose induced the activation of caspase-3 at the third week. At the two subtoxic doses of NiO NPs a clear pro-inflammatory response was resulted in an up-regulation of TNF-α and IL-6 in terms of mRNA. Finally, an increased phosphorylation ratio of p-ERK1/2, p-38 and p-AKT was observed up to the third week, at both concentrations. Our results show the negative impact of subtoxic doses NiO NPs chronic exposure on porcine SCs functionality and viability.

Boosting Immunity and Management against Wheat Fusarium Diseases by a Sustainable, Circular Nanostructured Delivery Platform.

Fusarium head blight (FHB) and Fusarium crown rot (FCR) are managed by the application of imidazole fungicides, which will be strictly limited by 2030, as stated by the European Green Deal. Here, a novel and eco-sustainable nanostructured particle formulation (NPF) is presented by following the principles of the circular economy. Cellulose nanocrystals (CNC) and resistant starch were obtained from the bran of a high amylose (HA) bread wheat and employed as carrier and excipient, while chitosan and gallic acid were functionalized as antifungal and elicitor active principles. The NPF inhibited conidia germination and mycelium growth, and mechanically interacted with conidia. The NPF optimally reduced FHB and FCR symptoms in susceptible bread wheat genotypes while being biocompatible on plants. The expression level of 21 genes involved in the induction of innate immunity was investigated in Sumai3 (FHB resistant) Cadenza (susceptible) and Cadenza SBEIIa (a mutant characterized by high-amylose starch content) and most of them were up-regulated in Cadenza SBEIIa spikes treated with the NPF, indicating that this genotype may possess an interesting genomic background particularly responsive to elicitor-like molecules. Quantification of fungal biomass revealed that the NPF controlled FHB spread, while Cadenza SBEIIa was resistant to FCR fungal spread. The present research work highlights that the NPF is a powerful weapon for FHB sustainable management, while the genome of Cadenza SBEIIa should be investigated deeply as particularly responsive to elicitor-like molecules and resistant to FCR fungal spread.

Extracellular Vesicles in Aging: An Emerging Hallmark?

Extracellular vesicles (EVs) are membrane-enclosed particles secreted by cells and circulating in body fluids. Initially considered as a tool to dispose of unnecessary material, they are now considered an additional method to transmit cell signals. Aging is characterized by a progressive impairment of the physiological functions of tissues and organs. The causes of aging are complex and interconnected, but there is consensus that genomic instability, telomere erosion, epigenetic alteration, and defective proteostasis are primary hallmarks of the aging process. Recent studies have provided evidence that many of these primary stresses are associated with an increased release of EVs in cell models, able to spread senescence signals in the recipient cell. Additional investigations on the role of EVs during aging also demonstrated the great potential of EVs for the modulation of age-related phenotypes and for pro-rejuvenation therapies, potentially beneficial for many diseases associated with aging. Here we reviewed the current literature on EV secretion in senescent cell models and in old vs. young individual body fluids, as well as recent studies addressing the potential of EVs from different sources as an anti-aging tool. Although this is a recent field, the robust consensus on the altered EV release in aging suggests that altered EV secretion could be considered an emerging hallmark of aging.

Turning Microbial AhR Agonists into Therapeutic Agents via Drug Delivery Systems.

Developing therapeutics for inflammatory diseases is challenging due to physiological mucosal barriers, systemic side effects, and the local microbiota. In the search for novel methods to overcome some of these problems, drug delivery systems that improve tissue-targeted drug delivery and modulate the microbiota are highly desirable. Microbial metabolites are known to regulate immune responses, an observation that has resulted in important conceptual advances in areas such as metabolite pharmacology and metabolite therapeutics. Indeed, the doctrine of "one molecule, one target, one disease" that has dominated the pharmaceutical industry in the 20th century is being replaced by developing therapeutics which simultaneously manipulate multiple targets through novel formulation approaches, including the multitarget-directed ligands. Thus, metabolites may not only represent biomarkers for disease development, but also, being causally linked to human diseases, an unexploited source of therapeutics. We have shown the successful exploitation of this approach: by deciphering how signaling molecules, such as the microbial metabolite, indole-3-aldehyde, and the repurposed drug anakinra, interact with the aryl hydrocarbon receptor may pave the way for novel therapeutics in inflammatory human diseases, for the realization of which drug delivery platforms are instrumental.

Sustainable Protocols for Cellulose Nanocrystals Synthesis from Tomato Waste and Their Antimicrobial Properties against Pseudomonas syringae pv. tomato.

Nanotechnology is rapidly gaining ground in crop protection, with the growing quest for sustainable nanopesticides and nanocarriers for plant pathogen management. Among them, cellulose nanocrystals (CNC) are emerging as innovative agrofood-waste-derived antimicrobial materials. In this work, new chemical and enzymatic CNC extraction methods from tomato harvest residues were evaluated. The obtained nanomaterials were characterized and tested for their antimicrobial properties on Pseudomonas syringae pv. tomato (Pto), the causal agent of bacterial speck disease on tomato. Both protocols were efficient. The enzymatic extraction method was greener, producing purer CNC at slightly lower yield. The obtained CNC, although they weakly inhibited cell growth and did not promote reactive oxygen species (ROS) formation, provoked bacterial aggregation and the inhibition of biofilm production and swimming motility. Both protocols produced CNC with similar morpho-chemical features, as well as promising antimicrobial activity against plant bacterial pathogens, suggesting their potential role in sustainable crop protection strategies. The new protocols could be a valuable alternative to conventional methods.

Biodrug Delivery Systems: Do mRNA Lipid Nanoparticles Come of Age?

As an appealing alternative to treat and prevent diseases ranging from cancer to COVID-19, mRNA has demonstrated significant clinical effects. Nanotechnology facilitates the successful implementation of the systemic delivery of mRNA for safe human consumption. In this manuscript, we provide an overview of current mRNA therapeutic applications and discuss key biological barriers to delivery and recent advances in the development of nonviral systems. The relevant challenges that LNPs face in achieving cost-effective and widespread clinical implementation when delivering mRNA are likewise discussed.

Pulmonary drug delivery technology enables anakinra repurposing in cystic fibrosis.

Inflammation is a key pathological driver in cystic fibrosis (CF). Current therapies are ineffective in treating and preventing the escalation of inflammatory events often exacerbated by superimposed infection. In this work, we propose a novel treatment based on the pulmonary administration of anakinra, a non-glycosylated recombinant form of IL-1Ra. An inhalable dry powder of anakinra was successfully developed to meet the specific needs of lung drug delivery. The new formulation was investigated in vitro for aerodynamic performances and activity and in vivo for its pharmacological profile, including the pharmacokinetics, treatment schedule, antimicrobial and anti-inflammatory activity and systemic toxicity. The protein was structurally preserved inside the formulation and retained its pharmacological activity in vitro immediately after preparation and over time when stored at ambient conditions. Anakinra when delivered to the lungs showed an improved and extended therapeutic efficacy in CF models in vivo as well as higher potency compared to systemic delivery. Peripheral side effects were significantly reduced and correlated with lower serum levels compared to systemic treatment. These findings provide proof-of-concept demonstration for anakinra repurposing in CF through the pulmonary route.

Development of a new indole derivative dry powder for inhalation for the treatment of biofilm-associated lung infections.

The aim of this work was to produce an inhalable dry powder formulation of a new anti-biofilm compound (SC38). For this purpose, chitosan was used as a polymeric carrier and l-leucine as a dispersibility enhancer. SC38 was entrapped by spray-drying into previously optimized chitosan microparticles. The final formulation was fully characterized in vitro in terms of particle morphology, particle size and distribution, flowability, aerodynamic properties, anti-biofilm activity and effects on lung cell viability. The SC38-loaded chitosan microparticles exhibited favorable aerodynamic properties with emitted and respirable fractions higher than 80 % and 45 % respectively. The optimized formulation successfully inhibited biofilm formation at microparticle concentrations starting from 20 µg/mL for methicillin-sensitive and 100 µg/mL for methicillin-resistant Staphylococcus aureus and showed a relatively safe profile in lung cells after 72 h exposure. Future in vivo tolerability and efficacy studies are needed to unravel the potential of this novel formulation for the treatment of difficult-to-treat biofilm-mediated lung infections.

Phospholipid fatty acid remodeling and carbonylated protein increase in extracellular vesicles released by airway epithelial cells exposed to cigarette smoke extract.

Cigarette smoke (CS) represents one of the most relevant environmental risk factors for several chronic pathologies. Tissue damage caused by CS exposure is mediated, at least in part, by oxidative stress induced by its toxic and pro-oxidant components. Evidence demonstrates that extracellular vesicles (EVs) released by various cell types exposed to CS extract (CSE) are characterized by altered biochemical cargo and gained pathological properties. In the present study, we evaluated the content of oxidized proteins and phospholipid fatty acid profiles of EVs released by human bronchial epithelial BEAS-2B cells treated with CSE. This specific molecular characterization has hitherto not been performed. After confirmation that CSE reduces viability of BEAS-2B cells and elevates intracellular ROS levels, in a dose-dependent manner, we demonstrated that 24 h exposure at 1% CSE, a concentration that only slight modifies cell viability but increases ROS levels, was able to increase carbonylated protein levels in cells and released EVs. The release of oxidatively modified proteins via EVs might represent a mechanism used by cells to remove toxic proteins in order to avoid their intracellular overloading. Moreover, 1% CSE induced only few changes in the fatty acid asset in BEAS-2B cell membrane phospholipids, whereas several rearrangements were observed in EVs released by CSE-treated cells. The impact of changes in acyl chain composition of CSE-EVs accounted for the increased saturation levels of phospholipids, a membrane parameter that might influence EV stability, uptake and, at least in part, EV-mediated biological effects. The present in vitro study adds new information concerning the biochemical composition of CSE-related EVs, useful to predict their biological effects on target cells. Furthermore, the information regarding the presence of oxidized proteins and the specific membrane features of CSE-related EVs can be useful to define the utilization of circulating EVs as marker for diagnosing of CS-induced lung damage and/or CS-related diseases.

Aryl Hydrocarbon Receptor Agonism Antagonizes the Hypoxia-driven Inflammation in Cystic Fibrosis.

Hypoxia contributes to the exaggerated yet ineffective airway inflammation that fails to oppose infections in cystic fibrosis (CF). However, the potential for impairment of essential immune functions by HIF-1α (hypoxia-inducible factor 1α) inhibition demands a better comprehension of downstream hypoxia-dependent pathways that are amenable for manipulation. We assessed here whether hypoxia may interfere with the activity of AhR (aryl hydrocarbon receptor), a versatile environmental sensor highly expressed in the lungs, where it plays a homeostatic role. We used murine models of Aspergillus fumigatus infection in vivo and human cells in vitro to define the functional role of AhR in CF, evaluate the impact of hypoxia on AhR expression and activity, and assess whether AhR agonism may antagonize hypoxia-driven inflammation. We demonstrated that there is an important interferential cross-talk between the AhR and HIF-1α signaling pathways in murine and human CF, in that HIF-1α induction squelched the normal AhR response through an impaired formation of the AhR:ARNT (aryl hydrocarbon receptor nuclear translocator)/HIF-1ß heterodimer. However, functional studies and analysis of the AhR genetic variability in patients with CF proved that AhR agonism could prevent hypoxia-driven inflammation, restore immune homeostasis, and improve lung function. This study emphasizes the contribution of environmental factors, such as infections, in CF disease progression and suggests the exploitation of hypoxia:xenobiotic receptor cross-talk for antiinflammatory therapy in CF.

Development and Pharmacokinetics of a Novel Acetylsalicylic Acid Dry Powder for Pulmonary Administration.

Aspirin is an historic blockbuster product, and it has been proposed in a wide range of formulas. Due to exacerbation risks, the pulmonary route has been seldom considered as an alternative to conventional treatments. Only recently, owing to overt advantages, inhalable acetylsalicylic acid dry powders (ASA DPI) began to be considered as an option. In this work, we developed a novel highly performing inhalable ASA DPI using a nano spray-drying technique and leucine as an excipient and evaluated its pharmacokinetics compared with oral administration. The formulation obtained showed remarkable respirability and quality features. Serum and lung ASA DPI profiles showed faster presentation in blood and higher retention compared with oral administration. The dry powder was superior to the DPI suspension. The relative bioavailability in serum and lungs claimed superiority of ASA DPI over oral administration, notwithstanding a fourfold lower pulmonary dose. The obtained ASA DPI formulation shows promising features for the treatment of inflammatory and infectious lung pathologies.

Pharmaceutically Active Microbial AhR Agonists as Innovative Biodrugs in Inflammation.

Alterations of the microbiome occur in inflammatory and autoimmune diseases, a finding consistent with the role of the microbiome in the maintenance of the immune system homeostasis. In this regard, L-tryptophan (Trp) metabolites, of both host and microbial origin, act as important regulators of host-microbial symbiosis by acting as aryl hydrocarbon receptor (AhR) ligands. The intestinal and respiratory barriers are very sensitive to AhR activity, suggesting that AhR modulation could be a therapeutic option to maintain the integrity of the epithelial barrier, which has substantial implications for health even beyond the mucosal site. A number of studies have highlighted the capacity of AhR to respond to indoles and indolyl metabolites, thus positioning AhR as a candidate indole receptor. However, the context-and ligand-dependent activity of AhR requires one to resort to suitable biopharmaceutical formulations to enable site-specific drug delivery in order to achieve therapeutic effectiveness, decrease unwanted toxicities and prevent off-target effects. In this review, we highlight the dual activity of the microbial metabolite indole-3-aldehyde at the host-microbe interface and its ability to orchestrate host pathophysiology and microbial symbiosis and discuss how its proper clinical development may turn into a valuable therapeutic strategy in local and distant inflammatory diseases.

Optimizing therapeutic outcomes of immune checkpoint blockade by a microbial tryptophan metabolite.

BACKGROUND: Despite the great success, the therapeutic benefits of immune checkpoint inhibitors (ICIs) in cancer immunotherapy are limited by either various resistance mechanisms or ICI-associated toxic effects including gastrointestinal toxicity. Thus, novel therapeutic strategies that provide manageable side effects to existing ICIs would enhance and expand their therapeutic efficacy and application. Due to its proven role in cancer development and immune regulation, gut microbiome has gained increasing expectation as a potential armamentarium to optimize immunotherapy with ICI. However, much has to be learned to fully harness gut microbiome for clinical applicability. Here we have assessed whether microbial metabolites working at the interface between microbes and the host immune system may optimize ICI therapy. METHODS: To this purpose, we have tested indole-3-carboxaldehyde (3-IAld), a microbial tryptophan catabolite known to contribute to epithelial barrier function and immune homeostasis in the gut via the aryl hydrocarbon receptor (AhR), in different murine models of ICI-induced colitis. Epithelial barrier integrity, inflammation and changes in gut microbiome composition and function were analyzed. AhR, indoleamine 2,3-dioxygenase 1, interleukin (IL)-10 and IL-22 knockout mice were used to investigate the mechanism of 3-IAld activity. The function of the microbiome changes induced by 3-IAld was evaluated on fecal microbiome transplantation (FMT). Finally, murine tumor models were used to assess the effect of 3-IAld treatment on the antitumor activity of ICI. RESULTS: On administration to mice with ICI-induced colitis, 3-IAld protected mice from intestinal damage via a dual action on both the host and the microbes. Indeed, paralleling the activation of the host AhR/IL-22-dependent pathway, 3-IAld also affected the composition and function of the microbiota such that FMT from 3-IAld-treated mice protected against ICI-induced colitis with the contribution of butyrate-producing bacteria. Importantly, while preventing intestinal damage, 3-IAld did not impair the antitumor activity of ICI. CONCLUSIONS: This study provides a proof-of-concept demonstration that moving past bacterial phylogeny and focusing on bacterial metabolome may lead to a new class of discrete molecules, and that working at the interface between microbes and the host immune system may optimize ICI therapy.