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Purpose: To investigate the potential link between C-reactive protein (CRP), a known biomarker of acute and chronic inflammation, and TRAIL, a cytokine which plays a key role in the immune-surveillance against tumors.Experimental Design: Primary normal peripheral blood mononuclear cell (PBMC) and CD14+ monocytes were exposed to recombinant CRP (1–10 μmol/L). TRAIL expression was analyzed by ELISA and/or by quantitative real-time PCR (qRT-PCR). In parallel, the potential role of the transcription factor Egr-1 was investigated by analyzing its modulation in response to CRP and by transfection experiments.Results:In vitro CRP exposure induced downregulation of TRAIL expression, both at the mRNA and protein level, in unfractionated PBMC and in purified CD14+ monocytes. TRAIL downregulation was not due to a specific toxicity or to contaminating lipopolysaccharide (LPS), as shown by the lack of induction of monocyte apoptosis and by the inability of the inhibitor of LPS polymyxin B to interfere with CRP activity. Of note, CRP downregulated TRAIL expression/release in CD14+ monocytes also in response to IFN-α, the most potent inducer of TRAIL. At the molecular level, the downmodulation of TRAIL by CRP was accompanied by a significant increase of Egr-1. Consistently, Egr-1 overexpression reduced the baseline levels of TRAIL mRNA, whereas knocking down Egr-1 counteracted the ability of CRP to downregulate TRAIL.Conclusions: Our findings suggest that a chronic elevation of CRP, which occurs during systemic inflammation and often in patients with cancer, might contribute to promote cancer development and/or progression by downregulating TRAIL in immune cells. Clin Cancer Res; 19(8); 1949–59. ©2013 AACR.

Despite advances in acute graft-versus-host disease (aGVHD) prophylaxis, current pharmacological approaches fail to prevent aGVHD. The protective effect of defibrotide on GVHD incidence and GVHD-free survival has not been sufficiently studied. 91 pediatric patients included in this retrospective study were divided into two groups based on defibrotide use. We compared the incidence of aGVHD and chronic GVHD-free survival between the defibrotide and control groups. The incidence and severity of aGVHD were significantly lower in patients who received defibrotide prophylactic administration than in the control group. This improvement was observed in the liver and intestinal aGVHD. No defibrotide prophylaxis benefit was observed in the prevention of chronic GVHD. The pro-inflammatory cytokine levels were significantly higher in the control group. Our findings suggest that prophylactic administration of defibrotide in pediatric patients significantly reduces the incidence and severity of aGVHD, with a modification of cytokine pattern, both strongly coherent with the protective drug’s action. This evidence adds to pediatric retrospective studies and preclinical data suggesting a possible defibrotide role in this setting.

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.

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 lymphoblastic leukemia (ALL) represents a heterogeneous group of hematologic malignancies, and it is normally characterized by an aberrant proliferation of immature lymphoid cells. Moreover, dysregulation of multiple signaling pathways that normally regulate cellular transcription, growth, translation, and proliferation is frequently encountered in this malignancy. ALL is the most frequent tumor in childhood, and adult ALL patients still correlate with poor survival. This review focuses on modern therapies in ALL that move beyond standard chemotherapy, with a particular emphasis on immunotherapeutic approaches as new treatment strategies. Bi-specific T-cell Engagers (BiTE) antibodies, the chimeric antigen receptor (CAR)-T cells, or CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats [CRISPR]-associated nuclease 9) represent other new innovative approaches for this disease. Target and tailored therapy could make the difference in previously untreatable cases, i.e., precision and personalized medicine. Clinical trials will help to select the most efficient novel therapies in ALL management and to integrate them with existing treatments to achieve durable cures.

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.

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.

Purpose To examine the ultrastructure of the natural plane of separation in grafted corneas and evaluate the outcomes of stromal peeling. Design Interventional case series. Methods In this multicenter study, stromal peeling was attempted in 96 consecutive eyes with unsatisfactory vision following penetrating keratoplasty (PK) for keratoconus (n = 79), herpetic keratitis (n = 11), and granular dystrophy (n = 6). Stromal exchange was performed by (1) 9 mm partial-thickness trephination; (2) creation of a corneal flap across the PK wound; (3) opening of the stromal component of the PK wound until a smooth, translucent natural plane was identified; (4) severing the attachment of the PK scar; (5) stromal peeling along the identified plane; and (6) suturing of donor lamella. Grafted corneas from cases that mandated conversion to PK were processed for transmission electron microscopy. Results The natural plane of separation was identified in all cases. Stromal exchange was successfully completed in 84 cases (87.5%). Snellen visual acuity ≥20/40 and ≥20/25 was reached in 93% and 72% of cases at 3 years (n = 49) and 86% and 62% at 4 years (n = 21) postoperatively. Mean endothelial cell loss at 1 year was 6.6% ± 9.5%. Stromal peeling occurred along a plane lined with a continuous layer of keratocytes separating pre-Descemet membrane (DM) stroma, DM, and endothelium from the anterior stroma. Pre-DM stroma was made of poorly organized lamellae containing widely spaced, randomly arranged collagen fibrils. Conclusions Ultrastructural alterations in the stromal microarchitecture of grafted corneas provide evidence of a natural plane of separation identified intraoperatively. Stromal peeling can be successfully performed in post-PK eyes with various stromal pathology.

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.

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. 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., Graphical abstract Image 1, Highlights • The vitamin D deficiency represents a risk factor for cardiovascular disease. • The vitamin D deficiency has been correlated to atheromatosis progression. • A model of vitamin D deficiency was fundamental to evaluate the role of the vitamin D the onset/progression of atheromatosis. • The vitamin D deficiency was clearly involved in an early formation of atherosclerotic lesions. • The vitamins play a potential role in the pharmacological treatments of cardiovascular disorders.

microRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at the posttranscriptional level, inducing the degradation of the target mRNA or translational repression. MiRNAs are involved in the control of a multiplicity of biological processes, and their absence or altered expression has been associated with a variety of human diseases, including cancer. Recently, extracellular miRNAs (ECmiRNAs) have been described as mediators of intercellular communication in multiple contexts, including tumor microenvironment. Cancer cells cooperate with stromal cells and elements of the extracellular matrix (ECM) to establish a comfortable niche to grow, to evade the immune system, and to expand. Within the tumor microenvironment, cells release ECmiRNAs and other factors in order to influence and hijack the physiological processes of surrounding cells, fostering tumor progression. Here, we discuss the role of miRNAs in the pathogenesis of multicomplex diseases, such as Alzheimer’s disease, obesity, and cancer, focusing on the contribution of both intracellular miRNAs, and of released ECmiRNAs in the establishment and development of cancer niche. We also review growing evidence suggesting the use of miRNAs as novel targets or potential tools for therapeutic applications.

Objective: To determine whether maternal serum concentrations of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a cytokine with anti-inflammatory activity, also involved in cardiovascular morbidity, differ between women with early preeclampsia (

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.

Purpose To identify risk factors predictive of postoperative double anterior chamber formation after deep anterior lamellar keratoplasty (DALK). Design Retrospective institutional cohort study. Methods The study group included all consecutive eyes undergoing primary DALK between May 2015 and October 2018 at Villa Igea private hospital (Forli, Italy). The indications for surgery were categorized as (1) keratoconus without scarring; (2) keratoconus with scarring; (3) non-keratoconus without scarring; and (4) non-keratoconus with scarring. Multivariate binary logistic regression analysis was performed, introducing, as independent variables, those that reached a significance level of less than .05 in univariate analysis. The main outcome measure was whether or not postoperative double anterior chamber (AC) occurred. Results A total of 591 eyes of 591 patients were included. The main indication for DALK was keratoconus (67.2%, n = 397), and pneumatic dissection was achieved in 72.9% (n = 431) of patients. Postoperative double AC was observed in 8.1% (n = 48) of cases. Age, intraoperative central DM perforation, type 2 bubble formation, and presence of scar in keratoconic and nonkeratoconic corneas were all associated with an increased risk of postoperative double AC formation in the univariate analysis. Manual dissection was not associated with double AC formation. The factors that remained significant in multivariate analysis were keratoconus with scarring (odds ratio [OR] = 3.56, P = .02), non-keratoconus with scarring (OR = 5.09, P = .002), intraoperative central perforation (OR = 6.09, P = .03), and type 2 bubble formation (OR = 14.17, P Conclusions Scarred corneas of both normal and abnormal shape are independent risk factors for double AC formation following DALK, along with intraoperative perforation and the occurrence of a type 2 bubble.

Objective To investigate the biological effects of a novel endovenous scaffold in a porcine model. Methods Petalo is a compliant venous scaffold implanted into the internal jugular veins of 12 healthy pigs. The pigs were sacrificed at one, two, three, and six months, respectively. Microscopic investigations were performed at two blinded laboratories. Results Neo-intima formation progressively covering up the stent metallic bars was observed. The inflammatory response of the venous wall showed a peak after three months by the implant, followed by marked reduction after six months. The device induced a significant ( p Conclusions The implant of Petalo compliant venous scaffold in the venous wall of this porcine model is characterized by neointima formation and by an inflammatory reaction which tends to decrease after six months. Our data point against the induction of smooth muscle cells proliferation and migration as confirmed by electronic transmission microscopy analyses.

// Rebecca Voltan 1, * , Erika Rimondi 2, * , Elisabetta Melloni 1 , Gian Matteo Rigolin 3 , Fabio Casciano 1 , Maria Vittoria Arcidiacono 1 , Claudio Celeghini 2 , Antonio Cuneo 3 , Giorgio Zauli 1 , Paola Secchiero 1 1 Department of Morphology, Surgery and Experimental Medicine and LTTA Centre, University of Ferrara, Ferrara, Italy 2 Department of Life Sciences, University of Trieste, Trieste, Italy 3 Department of Medical Sciences, Section of Hematology, University of Ferrara, Ferrara, Italy * These authors have contributed equally to this work Correspondence to: Paola Secchiero, email: paola.secchiero@unife.it Keywords: B leukemic cells, Ibrutinib, MDM-2 inhibitors, apoptosis, combination therapy Received: July 20, 2016 Accepted: September 09, 2016 Published: September 20, 2016 ABSTRACT Objective: The aim of this study was to investigate the anti-leukemic activity of the Bruton tyrosine kinase inhibitor Ibrutinib in combination with the small molecule MDM-2 inhibitor Nutlin-3 in preclinical models. Methods: The potential efficacy of the Ibrutinib/Nutlin-3 combination was evaluated in vitro in a panel of B leukemic cell lines (EHEB, JVM-2, JVM-3, MEC-1, MEC-2) and in primary B-chronic lymphocytic leukemia (B-CLL) patient samples, by assessing cell viability, cell cycle profile, apoptosis and intracellular pathway modulations. Validation of the combination therapy was assessed in a B leukemic xenograft mouse model. Results: Ibrutinib exhibited variable anti-leukemic activity in vitro and the combination with Nutlin-3 synergistically enhanced the induction of apoptosis independently from the p53 status. Indeed, the Ibrutinib/Nutlin-3 combination was effective in promoting cytotoxicity also in primary B-CLL samples carrying 17p13 deletion and/or TP53 mutations, already in therapy with Ibrutinib. Molecular analyses performed on both B-leukemic cell lines as well as on primary B-CLL samples, while confirming the switch-off of the MAPK and PI3K pro-survival pathways by Ibrutinib, indicated that the synergism of action with Nutlin-3 was independent by p53 pathway and was accompanied by the activation of the DNA damage cascade signaling through the phosphorylation of the histone protein H2A.X. This observation was confirmed also in the JVM-2 B leukemic xenograft mouse model. Conclusions: Taken together, our data emphasize that the Ibrutinib/Nutlin-3 combination merits to be further evaluated as a therapeutic option for B-CLL.

Compliance is a characteristic of every deformable system. Compliance is very clear concept in physics and mechanics but in clinics, perhaps, is not the same. However, in veins compliance fits perfectly with the function of drainage of the venous system. Volumetric increase (dV) of the content is correlated with pressure increase (dP) inside the vein according to the equation C’= dV/dP. In humans 75% of the blood is located in the venous system, primarily because the molecular components of a vein media layer is significantly more compliant to that of arteries. This property is fundamental to understanding the change in blood volume in response to a change in posture. Measurements of venous compliance in clinical practice can be done by the means of ultrasound, as well as with the plethysmography. Ultrasound methods assimilate the cross sectional area to the volume of the vein, because it reflects the blood content. Changes in cross sectional area can be reliably measured in response to a change in posture, while pressure can be derived from the hydrostatic pressure changes. Venous compliance is of paramount importance also in pulsatile veins such as the inferior or superior vena cava and the jugular veins, where high resolution ultrasound may accurately derive the cross sectional area. Clinical implications of the mechanical properties of the venous wall are extensively discussed, including the need of dedicated venous stenting, which takes into account venous compliance as the main parameter of the venous function. In addition, venous compliance is the interpretative key for a better understanding of plethysmography curves, as well as of varicose veins and of their return to normal cross sectional area following ambulatory venous pressure reduction.

We aimed to evaluate the effects of intermittent pneumatic compression (IPC) in patients at low mobility with leg edema.A pilot, two-arm, randomized controlled clinical trial was performed. Fifty patients (age, 58.4 ± 9 years; male, 14), randomly allocated to a group (IPC) undergoing 1 month (n = 29) of an in-home cycle of IPC and to a control (C) group (n = 21), were studied. Leg edema was evaluated by measuring subcutaneous thickness (high-resolution ultrasound) and circumferences (metric tape), both assessed at different levels of the lower limbs, and volume (water plethysmography). Ankle range of motion (ROM, goniometer), quality of life (QoL) by the 36-Item Short Form Health Survey, and a pool of plasma inflammatory markers were also evaluated.Edema significantly decreased in the IPC group (for all outcome measures, P .0001), whereas it significantly increased in the C group (P .0001). Ankle ROM was significantly enhanced in the IPC group (dorsiflexion, P .0001; plantar flexion, P = .002) and remained stable in the C group. QoL showed an improvement in the IPC group, particularly significant for the general health subscale (P = .004), whereas no changes were highlighted in the C group. The two groups exhibited different trends and variations for some plasma inflammatory markers, mainly for granulocyte colony-stimulating factor.In a sample of patients at reduced mobility with leg edema, IPC treatment was effective in reducing the edema, improving the ankle ROM, and determining a positive impact on QoL together with a slight modulation of some plasma inflammatory markers.

Background: Chronic kidney disease (CKD) patients experience a high risk of cardiovascular disease (CV); however, the factors involved in CV-related morbidity and mortality in these patients have not been fully defined. Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) is a cytokine, which exhibits pleiotropic activities on endothelial, vascular smooth muscle and inflammatory cells, with relevant effects on atheromatous plaque formation. On this basis, the present study aims to investigate the role of TRAIL in atheromatosis progression in CKD patients. Methods: Circulating TRAIL levels were measured in 378 CKD patients belonging to the Spanish National Observatory of Atherosclerosis in Nephrology (NEFRONA) study. All patients were free of previous CV events. Carotid and femoral B-mode ultrasound was performed to detect the presence of plaque at baseline and after 24 months of follow-up. Results: The lowest levels of TRAIL at baseline were significantly (p

Photorefractive keratectomy (PRK) represents a therapeutic option to remodel corneal stroma and to compensate refractive errors, which involves inflammatory and/or regenerative processes. In this context, the modulation of cytokines/chemokines in the conjunctival sac fluid and their role in the maintenance of the corneal microenvironment during the healing process upon refractive procedures has not been deeply investigated. In this study, serial samples of conjunctival sac fluid of patients (n=25) undergoing PRK were harvested before and at different time points after surgery. The levels of 29 cytokines/chemokines/growth factors involved in inflammatory/immune processes were measured with a multiplex array system. The results have firstly highlighted the different pattern of cytokine expression between the microenvironment at the anterior surface of the eye and the systemic circulation. More importantly, the kinetic of modulation of cytokines/chemokines at the conjunctival level following PRK revealed that while the majority of cytokines/chemokines showed a significant decrease, MCP-1 emerged in light of its pronounced and significant increase soon after PRK and during the follow-up. This methodological approach has highlighted the role of MCP-1 in the healing process following PRK and has shown a potential for the identification of expression/modulation of soluble factors for biomarker profiling in ocular surface diseases.

// Paola Secchiero 1, * , Rebecca Voltan 1, * , Erika Rimondi 1 , Elisabetta Melloni 1 , Emmanouil Athanasakis 2 , Veronica Tisato 1 , Stefania Gallo 1 , Gian Matteo Rigolin 3 and Giorgio Zauli 1 1 Department of Morphology, Surgery and Experimental Medicine and LTTA Centre, University of Ferrara, Ferrara, Italy 2 Institute for Maternal and Child Health, IRCCS “Burlo Garofolo”, Trieste, Italy 3 Department of Medical Sciences, Section of Hematology, University of Ferrara, Ferrara, Italy * These authors have contributed equally to this work Correspondence to: Giorgio Zauli, email: giorgio.zauli@unife.it Paola Secchiero, email: paola.secchiero@unife.it Keywords: B-leukemic cells, Ibrutinib, γ-secretase inhibitors, NOTCH1, combination therapy Received: May 05, 2017 Accepted: June 20, 2017 Published: July 22, 2017 ABSTRACT Ibrutinib blocks B-cell receptor signaling and interferes with leukemic cell-to-microenvironment interactions. Ibrutinib plays a key role in the management of B-CLL and is recommended for first line treatment of high-risk CLL patients with 17p deletion. Therefore, elucidating the factors governing sensitivity/resistance to Ibrutinib represents a relevant issue. For this purpose, in 3 B-CLL patient samples harboring functional TP53 mutations, the frequency of the mutated clones was monitored during in vivo Ibrutinib therapy, revealing a progressive decline of the frequency of TP53 mut clones during 12 months of treatment. In parallel, the anti-leukemic activity of Ibrutinib was assessed in vitro on B-CLL patient cell cultures in combination with γ-secretase inhibitors (GSI). In the in vitro assays, the combination of Ibrutinib+GSI exhibited enhanced cytotoxicity on B-CLL cells also in the presence of stroma and it was coupled to the down-regulation of the stroma-activated NOTCH1 and c-MYC pathways. Moreover, the combined treatment was effective in reducing CXCR4 expression and functions. Therefore, the ability of GSI to enhance the Ibrutinib anti-leukemic activity in B-CLL cells, by down-regulating the NOTCH1 and c-MYC pathways, warrants further experimentation for its potential therapeutic applications.

Large vein endothelium plays important roles in clinical diseases such as chronic venous disease (CVD) and thrombosis; thus to characterize CVD vein endothelial cells (VEC) has a strategic role in identifying specific therapeutic targets. On these bases we evaluated the effect of the natural anti-inflammatory compoundsα-Lipoic acid and Ginkgoselect phytosome on cytokines/chemokines released by CVD patient-derived VEC. For this purpose, we characterized the levels of a panel of cytokines/chemokines (n=31) in CVD patients’ plasma compared to healthy controls and their release by VEC purified from the same patients, in unstimulated and TNF-αstimulated conditions. Among the cytokines/chemokines released by VEC, which recapitulated the systemic profile (IL-8, TNF-α, GM-CSF, INF-α2, G-CSF, MIP-1β, VEGF, EGF, Eotaxin, MCP-1, CXCL10, PDGF, and RANTES), we identified those targeted byex vivotreatment withα-Lipoic acid and/or Ginkgoselect phytosome (GM-CSF, G-CSF, CXCL10, PDGF, and RANTES). Finally, by investigating the intracellular pathways involved in promoting the VEC release of cytokines/chemokines, which are targeted by natural anti-inflammatory compounds, we documented thatα-Lipoic acid significantly counteracted TNF-α-induced NF-κB and p38/MAPK activation while the effects ofGinkgo bilobaappeared to be predominantly mediated by Akt. Ourdataprovide new insights into the molecular mechanisms of CVD pathogenesis, highlighting new potential therapeutic targets.

The effect of the multi-kinase inhibitor Sorafenib was investigated in an in vitro model of human osteoclastogenesis, represented by peripheral blood mononuclear cells (PBMCs) induced to differentiate into osteoclast-like cells in presence of receptor activator of nuclear factor kappa B ligand (RANKL) plus macrophage-colony stimulating factor (M-CSF). Sorafenib significantly inhibited osteoclastic formation at clinically achievable concentrations (1-3 μM) and promoted autophagia with minimal induction of apoptosis. At the molecular levels, the M-CSF + RANKL combination increased the expression level of the Bcl-2 family member Mcl-1 protein, which is known to play a key role in the control of both cell survival and autophagia. The simultaneous treatment with Sorafenib significantly down-regulated endogenous Mcl-1 expression. Conversely, over-expression of Mcl-1 in primary human macrophages significantly counteracted the anti-osteoclastic activity of Sorafenib, strongly suggesting that Mcl-1 down-regulation played a major role in mediating the inhibitory activity of Sorafenib in cells of the osteoclastic lineage.