Microfluidic Fabricated Liposomes for Nutlin-3a Ocular Delivery as Potential Candidate for Proliferative Vitreoretinal Diseases Treatment.

Purpose: Proliferative vitreoretinal diseases (PVDs) represent a heterogeneous group of pathologies characterized by the presence of retinal proliferative membranes, in whose development retinal pigment epithelium (RPE) is deeply involved. As the only effective treatment for PVDs at present is surgery, we aimed to investigate the potential therapeutic activity of Nutlin-3a, a small non-genotoxic inhibitor of the MDM2/p53 interaction, on ARPE-19 cell line and on human RPE primary cells, as in vitro models of RPE and, more importantly, to formulate and evaluate Nutlin-3a loaded liposomes designed for ophthalmic administration. Methods: Liposomes were produced using an innovative approach by a microfluidic device under selection of different conditions. Liposome size distribution was evaluated by photon correlation spectroscopy and centrifugal field flow fractionation, while the liposome structure was studied by transmission electron microscopy and Fourier-transform infrared spectroscopy. The Nutlin-3a entrapment capacity was evaluated by ultrafiltration and HPLC. Nutlin-3a biological effectiveness as a solution or loaded in liposomes was evaluated by viability, proliferation, apoptosis and migration assays and by morphological analysis. Results: The microfluidic formulative study enabled the selection of liposomes composed of phosphatidylcholine (PC) 5.4 or 8.2 mg/mL and 10% ethanol, characterized by roundish vesicular structures with 150-250 nm mean diameters. Particularly, liposomes based on the lower PC concentration were characterized by higher stability. Nutlin-3a was effectively encapsulated in liposomes and was able to induce a significant reduction of viability and migration in RPE cell models. Conclusion: Our results lay the basis for a possible use of liposomes for the ocular delivery of Nutlin-3a.

Convolvulus pluricaulis Choisy's Extraction, Chemical Characterization and Evaluation of the Potential Effects on Glycaemic Balance in a 3T3-L1 Adipocyte Cell Model.

Convolvulus pluricaulis (CP) is a common Indian herb, largely employed in Ayurvedic medicine and known for its neuroprotective and neuroinflammatory action. Its effectiveness against several pathologic/sub-pathologic conditions is widely accepted, but it is not yet completely chemically characterized. In recent years, several researchers have pointed out the involvement of CP and other Convolvulaceae in lipidic and glucidic metabolism, particularly in the control of hyperlipidaemia and diabetic conditions. In this scenario, the aim of the study was to chemically characterize the medium polarity part of the CP whole plant and its fractions and to shed light on their biological activity in adipocyte differentiation using the 3T3-L1 cell model. Our results demonstrated that the CP extract and fractions could upregulate the adipocyte differentiation through the modulation of the nuclear receptor PPARγ (Peroxisome Proliferator-Activated Receptor γ), broadly recognized as a key regulator of adipocyte differentiation, and the glucose transporter GLUT-4, which is fundamental for cellular glucose uptake and for metabolism control. CP also showed the ability to exert an anti-inflammatory effect, downregulating cytokines such as Rantes, MCP-1, KC, eotaxin, and GM-CSF, which are deeply involved in insulin resistance and glucose intolerance. Taken together, these data suggest that CP could exert a potential beneficial effect on glycemia and could be employed as an anti-diabetic adjuvant or, in any case, a means to better control glucose homeostasis.

Palmitic Acid Induced a Long-Lasting Lipotoxic Insult in Human Retinal Pigment Epithelial Cells, which Is Partially Counteracted by TRAIL.

Hyperglycaemia and increased circulating saturated fatty acids are key metabolic features of type 2 diabetes mellitus (T2DM) that contribute to diabetic retinopathy pathogenesis. Contrarily, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has been shown to improve or prevent T2DM. This study aimed at investigating the effect of TRAIL in an in vitro model of human retinal pigment epithelium: the ARPE-19 cell line, treated with palmitic acid (PA) in the presence of high glucose concentration. PA caused a drop in cellular metabolic activity and cell viability as well as an increase in apoptosis rates, which were paralleled by an upregulation of reactive oxygen species (ROS) generation as well as mitochondrial fragmentation. Despite ARPE-19 cells expressing TRAIL-R2 at the cell surface, TRAIL failed to counteract the cytotoxic effects of PA. However, when TRAIL was used alongside PA and then removed or used alone following PA challenge, it partially attenuated PA-induced lipotoxicity. This effect of TRAIL appeared to rely upon the modulation of inflammation and ROS production. Thus, TRAIL exerted a trophic effect on ARPE-19 cells, which became evident only when the lipotoxic insult was removed. Nevertheless, whether recombinant TRAIL might have a therapeutic potential for the treatment of diabetic retinopathy requires further investigation.

Prenylation Defects and Oxidative Stress Trigger the Main Consequences of Neuroinflammation Linked to Mevalonate Pathway Deregulation.

The cholesterol biosynthesis represents a crucial metabolic pathway for cellular homeostasis. The end products of this pathway are sterols, such as cholesterol, which are essential components of cell membranes, precursors of steroid hormones, bile acids, and other molecules such as ubiquinone. Furthermore, some intermediates of this metabolic system perform biological activity in specific cellular compartments, such as isoprenoid molecules that can modulate different signal proteins through the prenylation process. The defects of prenylation represent one of the main causes that promote the activation of inflammation. In particular, this mechanism, in association with oxidative stress, induces a dysfunction of the mitochondrial activity. The purpose of this review is to describe the pleiotropic role of prenylation in neuroinflammation and to highlight the consequence of the defects of prenylation.

Acute Neurological Involvement after Donor Lymphocyte Infusion for Post-Transplant Viral Infection: The Same Pattern of Novel Cancer Immunotherapy-Related CNS Toxicity?

Early post-transplant is the critical phase for the success of hematopoietic stem cell transplantation (HSCT). New viral infections and the reactivations associated with complete ablation of the recipient's T-cell immunity and inefficient reconstitution of the donor-derived system represent the main risks of HSCT. To date, the pharmacological treatments for post-HSCT viral infection-related complications have many limitations. Adoptive cell therapy (ACT) represents a new pharmacological strategy, allowing us to reconstitute the immune response to infectious agents in the post-HSC period. To demonstrate the potential advantage of this novel immunotherapy strategy, we report three cases of pediatric patients and the respective central nervous system complications after donor lymphocyte infusion.

New Applications of JAK/STAT Inhibitors in Pediatrics: Current Use of Ruxolitinib.

Janus kinases (JAK) are a family of tyrosine kinases (JAK1, JAK2, JAK3, and TYK2) that transduce cytokine-mediated signals through the JAK-STAT metabolic pathway. These kinases act by regulating the transcription of specific genes capable of inducing biological responses in several immune cell subsets. Inhibition of Janus kinases interferes with the JAK-STAT signaling pathway. Besides being used in the treatment of cancer and inflammatory diseases, in recent years, they have also been used to treat inflammatory conditions, such as graft-versus-host disease (GVHD) and cytokine release syndrome as complications of allogeneic hematopoietic stem cell transplantation and cell therapy. Recently, the FDA approved the use of ruxolitinib, a JAK1/2 inhibitor, in the treatment of acute steroid-refractory GVHD (SR-aGVHD), highlighting the role of JAK inhibition in this immune deregulation. Ruxolitinib was initially used to treat myelofibrosis and true polycythemia in a high-dose treatment and caused hematological toxicity. Since a lower dosage often could not be effective, the use of ruxolitinib was suspended. Subsequently, ruxolitinib was evaluated in adult patients with SR-aGVHD and was found to achieve a rapid and effective response. In addition, its early low-dose use in pediatric patients affected by GVHD has proved effective, safe, and reasonably preventive. The review aims to describe the potential properties of ruxolitinib to identify new therapeutic strategies.

Synthesis and Biological Investigation of Bile Acid-Paclitaxel Hybrids.

Chenodeoxycholic acid and ursodeoxycholic acid (CDCA and UDCA, respectively) have been conjugated with paclitaxel (PTX) anticancer drugs through a high-yield condensation reaction. Bile acid-PTX hybrids (BA-PTX) have been investigated for their pro-apoptotic activity towards a selection of cancer cell lines as well as healthy fibroblast cells. Chenodeoxycholic-PTX hybrid (CDC-PTX) displayed cytotoxicity and cytoselectivity similar to PTX, whereas ursodeoxycholic-PTX hybrid (UDC-PTX) displayed some anticancer activity only towards HCT116 colon carcinoma cells. Pacific Blue (PB) conjugated derivatives of CDC-PTX and UDC-PTX (CDC-PTX-PB and UDC-PTX-PB, respectively) were also prepared via a multistep synthesis for evaluating their ability to enter tumor cells. CDC-PTX-PB and UDC-PTX-PB flow cytometry clearly showed that both CDCA and UDCA conjugation to PTX improved its incoming into HCT116 cells, allowing the derivatives to enter the cells up to 99.9%, respect to 35% in the case of PTX. Mean fluorescence intensity analysis of cell populations treated with CDC-PTX-PB and UDC-PTX-PB also suggested that CDC-PTX-PB could have a greater ability to pass the plasmatic membrane than UDC-PTX-PB. Both hybrids showed significant lower toxicity with respect to PTX on the NIH-3T3 cell line.

Mevalonate Kinase Deficiency and Squalene Synthase Inhibitor (TAK-475): The Balance to Extinguish the Inflammation.

Mevalonate Kinase Deficiency (MKD) is a rare inborn disease belonging to the family of periodic fever syndromes. The MKD phenotype is characterized by systemic inflammation involving multiple organs, including the nervous system. Current anti-inflammatory approaches to MKD are only partially effective and do not act specifically on neural inflammation. According to the new emerging pharmacology trends, the repositioning of drugs from the indication for which they were originally intended to another one can make mechanistic-based medications easily available to treat rare diseases. According to this perspective, the squalene synthase inhibitor Lapaquistat (TAK-475), originally developed as a cholesterol-lowering drug, might find a new indication in MKD, by modulating the mevalonate cholesterol pathway, increasing the availability of anti-inflammatory isoprenoid intermediates. Using an in vitro model for MKD, we mimicked the blockade of the cholesterol pathway and evaluated the potential anti-inflammatory effect of Lapaquistat. The results obtained showed anti-inflammatory effects of Lapaquistat in association with a low blockade of the metabolic pathway, while this effect did not remain with a tighter blockade. On these bases, Lapaquistat could be configured as an effective treatment for MKD's mild forms, in which the residual enzymatic activity is only reduced and not almost completely absent as in the severe forms.

Autoinflammatory Diseases and Cytokine Storms-Imbalances of Innate and Adaptative Immunity.

Innate and adaptive immune responses have a well-known link and represent the distinctive origins of several diseases, many of which may be the consequence of the loss of balance between these two responses. Indeed, autoinflammation and autoimmunity represent the two extremes of a continuous spectrum of pathologic conditions with numerous overlaps in different pathologies. A common characteristic of these dysregulations is represented by hyperinflammation, which is an exaggerated response of the immune system, especially involving white blood cells, macrophages, and inflammasome activation with the hyperproduction of cytokines in response to various triggering stimuli. Moreover, hyperinflammation is of great interest, as it is one of the main manifestations of COVID-19 infection, and the cytokine storm and its most important components are the targets of the pharmacological treatments used to combat COVID-19 damage. In this context, the purpose of our review is to provide a focus on the pathogenesis of autoinflammation and, in particular, of hyperinflammation in order to generate insights for the identification of new therapeutic targets and strategies.

Overcoming of Microenvironment Protection on Primary Chronic Lymphocytic Leukemia Cells after Treatment with BTK and MDM2 Pharmacological Inhibitors.

In B-chronic lymphocytic leukemia (B-CLL), the interaction between leukemic cells and the microenvironment promotes tumor cell survival. The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib is one of the first-in-class molecules for the treatment of B-CLL patients; however, the emerging mechanisms of resistance to ibrutinib call for new therapeutic strategies. The purpose of the current study was to investigate the ability of ibrutinib plus the MDM2-inhibitor nutlin-3 to counteract the tumor microenvironment protective effect. We observed that primary B-CLL cells cultivated in microenvironment mimicking conditions were protected from apoptosis by the up-regulation of c-MYC and of p53. In the same setting, combined treatments with ibrutinib plus nutlin-3 led to significantly higher levels of apoptosis compared to the single treatments, counteracting the c-MYC up-regulation. Moreover, the combination induced high p53 levels and a significant dissipation of the mitochondrial membrane potential, together with BAX cleavage in the more active p18 form and phospho-BAD down-regulation, that are key components of the mitochondrial apoptotic pathway, enhancing the apoptosis level. Our findings propose a new therapeutic strategy to overcome the tumor microenvironment protection involved in B-CLL resistance to drugs, with possible clinical implications also for other hematologic and solid tumors for which ibrutinib is considered a therapeutic option.

MitoQ Is Able to Modulate Apoptosis and Inflammation.

Mitoquinone (MitoQ) is a mitochondrial reactive oxygen species scavenger that is characterized by high bioavailability. Prior studies have demonstrated its neuroprotective potential. Indeed, the release of reactive oxygen species due to damage to mitochondrial components plays a pivotal role in the pathogenesis of several neurodegenerative diseases. The present study aimed to examine the impact of the inflammation platform activation on the neuronal cell line (DAOY) treated with specific inflammatory stimuli and whether MitoQ addition can modulate these deregulations. DAOY cells were pre-treated with MitoQ and then stimulated by a blockade of the cholesterol pathway, also called mevalonate pathway, using a statin, mimicking cholesterol deregulation, a common parameter present in some neurodegenerative and autoinflammatory diseases. To verify the role played by MitoQ, we examined the expression of genes involved in the inflammation mechanism and the mitochondrial activity at different time points. In this experimental design, MitoQ showed a protective effect against the blockade of the mevalonate pathway in a short period (12 h) but did not persist for a long time (24 and 48 h). The results obtained highlight the anti-inflammatory properties of MitoQ and open the question about its application as an effective adjuvant for the treatment of the autoinflammatory disease characterized by a cholesterol deregulation pathway that involves mitochondrial homeostasis.

Baseline and overtime variations of soluble adhesion molecule plasma concentrations are associated with mobility recovery after rehabilitation in multiple sclerosis patients.

Rehabilitative exercise outcomes and plasma concentrations of soluble adhesion molecules (sEndoglin, sE-Selectin, sL-Selectin, sICAM-1, sNCAM, sNCAM-1, sVCAM-1, sPECAM-1, sVAP-1) were evaluated in 60 severely disabled progressive multiple sclerosis (MS) patients at 4-time points. Changes of sE-Selectin, sL-Selectin, and sPECAM-1 concentrations were observed over time, and their variations were significantly correlated with rehabilitative outcome variations. Baseline sVAP-1 concentrations were able to predict functional mobility recovery. Our data suggest that the evaluation of adhesion molecules in plasma provides useful information to interpret rehabilitative exercise processes and to identify potential predictors of the rehabilitation-induced changes in mobility outcomes in MS patients.

Role of vitamin D in the pathogenesis of atheromatosis.

BACKGROUND AND AIMS: Cardiovascular disease is the main cause of death worldwide, but the collective efforts to prevent this pathological condition are directed exclusively to individuals at higher risk due to hypercholesterolemia, hypertension, obesity, diabetes. Recently, vitamin D deficiency was identified as a risk factor for cardiovascular disease in healthy people, as it predisposes to different vascular dysfunctions that can result in plaque development and fragility. In this scenario, the fundamental aim of the study was to reproduce a disease model inducing vitamin D deficiency and atheromatosis in ApoE-/- mice and then to evaluate the impact of this vitamin D status on the onset/progression of atheromatosis, focusing on plaque formation and instability. METHODS AND RESULTS: In our murine disease model, vitamin D deficiency was achieved by 3 weeks of vitamin D deficient diet along with intraperitoneal paricalcitol injections, while atheromatosis by western-type diet administration. Under these experimental conditions, vitamin D deficient mice developed more unstable atheromatous plaques with reduced or absent fibrotic cap. Since calcium and phosphorus metabolism and also cholesterol and triglycerides systemic concentration were not affected by vitamin D level, our results highlighted the role of vitamin D deficiency in the formation/instability of atheromatous plaque and, although further studies are needed, suggested a possible intervention with vitamin D to prevent or delay the atheromatous disease. CONCLUSIONS: The data obtained open the question about the potential role of the vitamins in the pharmacological treatments of cardiovascular disorders as coadjutant of the primary drugs used for these pathologies.

Eosinophils and Purinergic Signaling in Health and Disease.

Eosinophils are major effector cells against parasites, fungi, bacteria, and viruses. However, these cells also take part in local and systemic inflammation, which are central to eczema, atopy, rhinitis, asthma, and autoimmune diseases. A role for eosinophils has been also shown in vascular thrombotic disorders and in cancer. Many, if not all, above-mentioned conditions involve the release of intracellular nucleotides (ATP, ADP, UTP, etc.) and nucleosides (adenosine) in the extracellular environment. Simultaneously, eosinophils further release ATP, which in autocrine and paracrine manners, stimulates P2 receptors. Purinergic signaling in eosinophils mediates a variety of responses including CD11b induction, ROI production, release of granule contents and enzymes, as well as cytokines. Exposure to extracellular ATP also modulates the expression of endothelial adhesion molecules, thereby favoring eosinophil extravasation and accumulation. In addition, eosinophils express the immunosuppressive adenosine P1 receptors, which regulate degranulation and migration. However, pro-inflammatory responses induced by extracellular ATP predominate. Due to their important role in innate immunity and tissue damage, pharmacological targeting of nucleotide- and nucleoside-mediated signaling in eosinophils could represent a novel approach to alleviate eosinophilic acute and chronic inflammatory diseases. These innovative approaches might also have salutary effects, particularly in host defense against parasites and in cancer.

Colorectal Cancer Study with Nanostructured Sensors: Tumor Marker Screening of Patient Biopsies.

Despite the great progress in screening techniques and medical treatments, colorectal cancer remains one of the most widespread cancers in both sexes, with a high death rate. In this work, the volatile compounds released from human colon cancer tissues were detected by a set of four different chemoresistive sensors, made with a nanostructured powder of metal-oxide materials, inserted into an innovative patented device. The sensor responses to the exhalation of a primary cancer sample and of a healthy sample (both of the same weight, collected during colorectal surgery from the intestine of the same patient) were statistically analyzed. The sensors gave reversible, reproducible, and fast responses for at least one year of continuous use, making them quite superior in respect to the existing diagnostic methods. Preliminary results obtained using principal component analysis of the sensor responses to samples removed from 13 patients indicate that the nanostructured sensors employed in this study were able to distinguish between healthy and tumor tissue samples with coherent responses (the discrimination power of the most sensitive sensor was about 17%), highlighting a strong potential for clinical practice.

Crosstalk Between Adipokines and Paraoxonase 1: A New Potential Axis Linking Oxidative Stress and Inflammation.

Paraoxonase 1 (PON1) is a high-density lipoprotein (HDL)-associated protein that endows its carrier with (lipo-)lactonase-dependent antioxidative features. Low levels of PON1 activity have been observed in association with obesity, a major risk factor for cardiovascular disease (CVD). Considering the well-recognized atheroprotective role of PON1, exogenous/endogenous factors that might modulate its levels/activity are raising great interest. Since adipokines represent a molecular link between obesity and CVD, we here explored the possible impact of these substances on PON1 activity/expression. The levels of interleukin (IL)-6, IL-8, tumor necrosis factor alpha, monocyte chemoattractant protein-1, hepatocyte growth factor, resistin, leptin, and adiponectin were measured along with arylesterase, paraoxonase, and lactonase activities of PON1 in 107 postmenopausal women. Moreover, the direct effect of resistin on PON1 expression was evaluated in vitro. Multivariate analysis revealed that only resistin was significantly and inversely correlated with PON1-lactonase activities (r = -0.346, p < 0.001) regardless of confounding factors such as age or HDL-cholesterol. It is worth noting that no statistical link was found between adipokine and arylesterase or paraoxonase, the two promiscuous activities of PON1. Notably, resistin down-regulated PON1 expression occurred in hepatocellular carcinoma cultures. Our study suggests that resistin might be a negative modulator of PON1 expression and anti-oxidative activity.

TRAIL, OPG, and TWEAK in kidney disease: biomarkers or therapeutic targets?

Ligands and receptors of the tumor necrosis factor (TNF) superfamily regulate immune responses and homeostatic functions with potential diagnostic and therapeutic implications. Kidney disease represents a global public health problem, whose prevalence is rising worldwide, due to the aging of the population and the increasing prevalence of diabetes, hypertension, obesity, and immune disorders. In addition, chronic kidney disease is an independent risk factor for the development of cardiovascular disease, which further increases kidney-related morbidity and mortality. Recently, it has been shown that some TNF superfamily members are actively implicated in renal pathophysiology. These members include TNF-related apoptosis-inducing ligand (TRAIL), its decoy receptor osteoprotegerin (OPG), and TNF-like weaker inducer of apoptosis (TWEAK). All of them have shown the ability to activate crucial pathways involved in kidney disease development and progression (e.g. canonical and non-canonical pathways of the transcription factor nuclear factor-kappa B), as well as the ability to regulate cell proliferation, differentiation, apoptosis, necrosis, inflammation, angiogenesis, and fibrosis with double-edged effects depending on the type and stage of kidney injury. Here we will review the actions of TRAIL, OPG, and TWEAK on diabetic and non-diabetic kidney disease, in order to provide insights into their full clinical potential as biomarkers and/or therapeutic options against kidney disease.

Targeting mTOR in Acute Lymphoblastic Leukemia.

Acute Lymphoblastic Leukemia (ALL) is an aggressive hematologic disorder and constitutes approximately 25% of cancer diagnoses among children and teenagers. Pediatric patients have a favourable prognosis, with 5-years overall survival rates near 90%, while adult ALL still correlates with poorer survival. However, during the past few decades, the therapeutic outcome of adult ALL was significantly ameliorated, mainly due to intensive pediatric-based protocols of chemotherapy. Mammalian (or mechanistic) target of rapamycin (mTOR) is a conserved serine/threonine kinase belonging to the phosphatidylinositol 3-kinase (PI3K)-related kinase family (PIKK) and resides in two distinct signalling complexes named mTORC1, involved in mRNA translation and protein synthesis and mTORC2 that controls cell survival and migration. Moreover, both complexes are remarkably involved in metabolism regulation. Growing evidence reports that mTOR dysregulation is related to metastatic potential, cell proliferation and angiogenesis and given that PI3K/Akt/mTOR network activation is often associated with poor prognosis and chemoresistance in ALL, there is a constant need to discover novel inhibitors for ALL treatment. Here, the current knowledge of mTOR signalling and the development of anti-mTOR compounds are documented, reporting the most relevant results from both preclinical and clinical studies in ALL that have contributed significantly into their efficacy or failure.