Extracellular nicotinamide phosphoribosyltransferase (eNAMPT) neutralization counteracts T cell immune evasion in breast cancer.

BACKGROUND: Nicotinamide phosphoribosyltransferase (NAMPT) is a key intracellular enzyme that participates in nicotinamide adenine dinucleotide (NAD) homeostasis as well as a released cytokine (eNAMPT) that is elevated in inflammatory conditions and in cancer. In patients with breast cancer, circulating eNAMPT is elevated and its plasma levels correlate with prognosis and staging. In light of this, we investigated the contribution of eNAMPT in triple negative mammary carcinoma progression by investigating the effect of its neutralization via a specific neutralizing monoclonal antibody (C269). METHODS: We used female BALB/c mice injected with 4T1 clone 5 cells and female C57BL6 injected with EO771 cells, evaluating tumoral size, spleen weight and number of metastases. We injected two times a week the anti-eNAMPT neutralizing antibody and we sacrificed the mice after 28 days. Harvested tumors were analyzed by histopathology, flow cytometry, western blot, immunohistochemistry, immunofluorescence and RNA sequencing to define tumor characteristics (isolating tumor infiltrating lymphocytes and tumoral cells) and to investigate the molecular mechanisms behind the observed phenotype. Moreover, we dissected the functional relationship between T cells and tumoral cells using three-dimensional (3D) co-cultures. RESULTS: The neutralization of eNAMPT with C269 led to decreased tumor size and reduced number of lung metastases. RNA sequencing and functional assays showed that eNAMPT controlled T-cell response via the programmed death-ligand 1/programmed cell death protein 1 (PD-L1/PD-1) axis and its neutralization led to a restoration of antitumoral immune responses. In particular, eNAMPT neutralization was able to activate CD8+IFNγ+GrzB+ T cells, reducing the immunosuppressive phenotype of T regulatory cells. CONCLUSIONS: These studies indicate for the first time eNAMPT as a novel immunotherapeutic target for triple negative breast cancer.

Feeding Next-Generation Nanomedicines to Europe: Regulatory and Quality Challenges.

New and innovative nanomedicines have been developed and marketed over the past half-century, revolutionizing the prognosis of many human diseases. Although a univocal regulatory definition is not yet available worldwide, the term "nanomedicines" generally identifies medicinal products that use nanotechnology in their design or production. Due to the intrinsic high structural complexity of these products, the scientific and regulatory communities are reflecting on how to revise the regulatory framework to provide a more appropriate benefit/risk balance to authorize them on the market, considering the impact of their peculiar physicochemical features in the evaluation of efficacy and safety patterns. Herein, a critical perspective is provided on the current open issues regarding regulatory qualification and physicochemical characterization of nanosystems considering the current European regulatory framework on nanomedicine products. Practicable paths for improving their quality assurance and predicting their fate in vivo are also argued. Strengthening the multilevel alliance among academic institutions, industrial stakeholders, and regulatory authorities seems strategic to support innovation by standard approaches (e.g., qualification, characterization, risk assessment), and to expand current knowledge, also benefiting from the new opportunities offered by artificial intelligence and digitization in predictive modelling of the impact of nanomedicine characteristics on their fate in vivo.

A Versatile and Sustainable Multicomponent Platform for the Synthesis of Protein Degraders: Proof-of-Concept Application to BRD4-Degrading PROTACs.

The use of small molecules to induce targeted protein degradation is increasingly growing in the drug discovery landscape, and protein degraders have progressed rapidly through the pipelines. Despite the advances made so far, their synthesis still represents a significant burden and new approaches are highly demanded. Herein we report an unprecedented platform that leverages the modular nature of both multicomponent reactions and degraders to enable the preparation of highly decorated PROTACs. As a proof of principle, our platform has been applied to the preparation of potential BRD4-degrading PROTACs, resulting in the discovery of a set of degraders endowed with high degradation efficiency. Compared to the existing methods, our approach offers a versatile and cost-effective means to access libraries of protein degraders and increase the chance of identifying successful candidates.

Malignant melanoma in HIV: Epidemiology, pathogenesis, and management.

People affected by immunodeficiency, and especially those infected by HIV, are burdened by a higher risk of developing malignancies. It has been estimated that the incidence of melanoma in HIV-infected people is 2.6-fold higher than in uninfected ones. In this group of patients, melanoma shows a more aggressive phenotype and poorer survival rates compared to HIV-negative people. Standard guidelines of diagnosis and care do not exist yet. Studies suggest high index of suspicion and a low threshold for biopsy in HIV-positive patients regardless of their CD4+ count and the use of standard surgical margins for re-excision procedures. In case of diagnosis of melanoma in HIV-positive patients, a thorough search for metastatic disease is recommended because of the more aggressive course of this cancer in HIV-positive patients. Moreover, to rapidly find out any recurrence or metastatic disease after treatment, these patients need a close follow-up, every 3 months, for the first 2 years and at least twice yearly thereafter. Although surgery remains the main therapeutic option, application of immune checkpoint-based immunotherapy is being studied and seems to be promising. The aim of this review is to present the current knowledge and future options for melanoma diagnosis and treatment in people living with HIV.

Outbreak of Brucella melitensis infection in Eastern Sicily: risk factors, clinical characteristics and complication rate.

Brucellosis is one of the most common zoonoses in the world, especially in Southern Italy, where many cases are still recorded every year. 128 cases of brucellosis were observed in Messina (Sicily) in 2016, representing a tenfold increase in the number of cases of brucellosis expected. The aim of this multicenter retrospective study was to analyze clinical and microbiological aspects of a brucellosis outbreak in the province of Messina in 2016, the incidence of its complications and the treatment combinations applied. The principal transmission route was through the ingestion of unpasteurized fresh cheese. The mean latency period between the onset of the symptoms and diagnosis was 35.58±42.75 days. A late diagnosis increases the risk of developing complications. Drug-resistant strains of B. melitensis to Trimethoprim/ Sulfamethoxazole and Ciprofloxacin were found in blood cultures of 58.4% patients. Brucellosis is still present in Sicily. A diagnostic delay predisposes to complications requiring prolonged therapies. The finding of Brucella melitensis strains resistant to the most widespread treatments is worrisome and needs further investigation. Moreover, the use of alternative combination antibiotic therapy is recommended.

Circulating angiopoietin-like protein 2 levels are associated with decreased renal function in HIV+ subjects on cART: A potential marker of kidney disease.

Chronic kidney disease (CKD) is an important cause of morbidity and mortality in HIV-infected individuals, even in the antiretroviral therapy (ART) era. Inflammatory cytokines and adipokines have been suggested to play a role in the development of CKD. The aim of the present study was to examine the circulating levels of a novel proinflammatory cytokine, angiopoietin-like protein 2 (ANGPTL2), in a cohort of 72 HIV-positive subjects on ART. HIV-positive patients were on cART for at least one year. Urine and blood samples were collected. Various factors were analyzed including body mass index (BMI), smoking, and presence/treatment for comorbidities such as diabetes. The estimated glomerular filtration rate was calculated by using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation. Plasma samples obtained were stored and used to measure sCD14 and ANGPTL2 levels. Data were presented as mean (± standard deviation) or median (interquartile range) for continuous variables. Categorical variables were expressed as number (%). Variables were compared using Student's t-test, Mann-Whitney test, or χ2 test. The results showed an independent negative association between plasma ANGPTL2 and CKD-EPI values. Further prospective studies on larger cohorts are needed to evaluate the pathogenetic role of ANGPTL2 as well as its use as a diagnostic marker of renal dysfunction.

Investigational drugs in HIV: Pros and cons of entry and fusion inhibitors (Review).

Despite the profound changes and improvements reached in the field of HIV treatment, tolerability and adherence to highly active antiretroviral therapy remains a challenge. Furthermore, multi-experienced patients could take advantage of drugs with different mechanisms of action to combat the spread of resistance to actual therapy. For these reasons identification of new HIV drugs is crucial. Among all the molecules that at present are under investigation, entry and fusion inhibitors pose an interesting class owing to their peculiar characteristics, including prevention of entry of the virus into the human cells. In this study, we reviewed articles, clinical trials, and conference communications about all the drugs under investigation belonging to the class of entry and fusion inhibitors that are at least in phase I clinical trials.

HIV-associated psoriasis: Epidemiology, pathogenesis, and management.

People living with HIV (PLWH) are affected by a higher incidence skin disorders, which are often associated with high morbidity and mortality. In particular, psoriasis affects PLWH severely and for a longer time than the general population. Human immunodeficiency virus (HIV) infection is characterized by a progressive decrease in CD4+ T-cell count, and it could seem paradoxical that psoriasis exacerbations are more frequent in this subset of patients than the general population, even though it is commonly observed at any stage of infection. For a long time, there have been limited therapeutic choices for PLWH affected by psoriasis. The introduction of the combined antiretroviral therapy dramatically changed the natural course of both HIV and psoriasis in PLWH, leading to an improvement of quality and duration of life. However, the clinical severity of psoriasis in PLWH often requires the use of immunosuppressant drugs. Knowledge about their safety and efficacy are limited to case-reports, small case-series and studies, therefore their use has not yet entered the routine. Further studies are needed to determine if immunosuppressive drugs can be safely and effectively used in PLWH affected by psoriasis and other autoimmune disorders.

Bacteremic meningitis due to Pasteurella multocida resistant to first line antibiotic therapy.

Pasteurella species reside in the gastrointestinal tract of many animals, especially in pets such as cats or dogs. Zoonotic transmission of Pasteurella to human is documented. We describe a case of meningitis in a 66-year-old woman with positive blood culture for Pasteurella multocida. Meningitis caused by zoonosis agents is a rare event, but it should be suspected in patients that have recreational or professional exposure to animals. In this case, not only the etiologic agent was rare, but the microorganism was also resistant to firstline antibiotic drugs.

Outbreak of Brucella melitensis infection in Eastern Sicily: risk factors, clinical characteristics and complication rate.

Brucellosis is one of the most common zoonoses in the world, especially in Southern Italy, where many cases are still recorded every year. 128 cases of brucellosis were observed in Messina (Sicily) in 2016, representing a tenfold increase in the number of cases of brucellosis expected. The aim of this multicenter retrospective study was to analyze clinical and microbiological aspects of a brucellosis outbreak in the province of Messina in 2016, the incidence of its complications and the treatment combinations applied. The principal transmission route was through the ingestion of unpasteurized fresh cheese. The mean latency period between the onset of the symptoms and diagnosis was 35.58 ± 42.75 days. A late diagnosis increases the risk of developing complications. Drug-resistant strains of B. melitensis to Trimethoprim/Sulfamethoxazole and Ciprofloxacin were found in blood cultures of 58.4% patients. Brucellosis is still present in Sicily. A diagnostic delay predisposes to complications requiring prolonged therapies. The finding of Brucella melitensis strains resistant to the most widespread treatments is worrisome and needs further investigation. Moreover, the use of alternative combination antibiotic therapy is recommended.

Unusual Signs and Symptoms in HIV-Positive Patients Coinfected with Leishmania spp: The Importance of Neglected Tropical Disease in Differential Diagnosis.

BACKGROUND: Leishmaniasis is a parasitic disease affecting both animals and humans, acquired with the bite of sand flies or, in Injection Drug Users (IDUs), with contaminated needles, still hypoendemic in Sicily and the Mediterranean basin. Even though it is responsible for 20,000 to 40,000 deaths per year, this parasitic infection is still considered a neglected tropical disease. People Living with HIV (PLWH) are considered at high-risk of developing Leishmaniasis and, despite the introduction of Highly Active Anti-Retroviral Therapy (HAART), mortality rate and relapses prevalence are still high in coinfected people. CASE REPORT: We present a case of HIV-Leishmania coinfection, posing the attention on the atypical signs and symptoms and the importance of thinking about other causes than the HIV infection progression when the patient presents with a worsening of his immune status during HAART. CONCLUSION: This parasitic disease has a high mortality rate, so it is mandatory to think about it in all the patients having a low CD4+ T-cell count and an inverted CD4/CD8 ratio under HAART.

Head and neck squamous cell carcinoma and its correlation with human papillomavirus in people living with HIV: a systematic review.

Over the last 20 years we assisted to an increase in the mean age of People Living with HIV and their comorbidities. Especially, there was an increase in Human Papillomavirus-related head and neck squamous cell carcinomas. Despite their increasing incidence in HIV-positive people, mechanisms that lead to their development and progression are only partially understood. The aim of this review is to identify key data and factors about HPV-related head and neck squamous cell carcinoma in HIV-seropositive patients. Systematic search and review of the relevant literature-peer-reviewed and grey-was conducted using the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) guidelines. We included in our review only the 35 full-text articles we considered the most substantial. It is mandatory to improve our knowledge about the interactions existing between HPV and HIV, and about their actions on oral mucosa immune system.

Celecoxib inhibits proliferation and survival of chronic myelogeous leukemia (CML) cells via AMPK-dependent regulation of ß-catenin and mTORC1/2.

CML is effectively treated with tyrosine kinase inhibitors (TKIs). However, the efficacy of these drugs is confined to the chronic phase of the disease and development of resistance to TKIs remains a pressing issue. The anti-inflammatory COX2 inhibitor celecoxib has been utilized as anti-tumour drug due to its anti-proliferative activity. However, its effects in hematological malignancies, in particular CML, have not been investigated yet. Thus, we tested biological effects and mechanisms of action of celecoxib in Philadelphia-positive (Ph+) CML and ALL cells.We show here that celecoxib suppresses the growth of Ph+ cell lines by increasing G1-phase and apoptotic cells and reducing S- and G2-phase cells. These effects were independent of COX2 inhibition but required the rapid activation of AMP-activated protein kinase (AMPK) and the consequent inhibition mTORC1 and 2. Treatment with celecoxib also restored GSK3ß function and led to down-regulation of ß-catenin activity through transcriptional and post-translational mechanisms, two effects likely to contribute to Ph+ cell growth suppression by celecoxib.Celecoxib inhibited colony formation of TKI-resistant Ph+ cell lines including those with the T315I BCR-ABL mutation and acted synergistically with imatinib in suppressing colony formation of TKI-sensitive Ph+ cell lines. Finally, it suppressed colony formation of CD34+ cells from CML patients, while sparing most CD34+ progenitors from healthy donors, and induced apoptosis of primary Ph+ ALL cells.Together, these findings indicate that celecoxib may serve as a COX2-independent lead compound to simultaneously target the mTOR and ß-catenin pathways, key players in the resistance of CML stem cells to TKIs.

Triazole-curcuminoids: A new class of derivatives for 'tuning' curcumin bioactivities?

Curcumin is a unique blend of pharmacophores responsible for the pleiotropy of this natural pigment. In the present study we have replaced the 1,3-dicarbonyl moiety with a 1,2,3-triazole ring to furnish a new class of triazole-curcuminoids as a possible strategy to generate new compounds with different potency and selectivity compared to curcumin. We obtained a proof-of-principle library of 28 compounds tested for their cytotoxicity (SY-SY5Y and HeLa cells) and for their ability to inhibit NF-κB. Furthermore, we also generated 1,3-dicarbonyl curcuminoids of selected click compounds. Triazole-curcuminoids lost their ability to be Michael's acceptors, yet maintained some of the features of the parent compounds and disclosed new ones. In particular, we found that some compounds were able to inhibit NF-κB without showing cytotoxicity, while others, unlike curcumin, activated NF-κB signalling. This validates the hypothesis that click libraries can be used to investigate the biological activities of curcumin as well as generate analogs with selected features.

Sirtuin-1 and HIV-1: an overview.

Sirtuins are a family of NAD+-dependent protein deacetylases, which regulate cell survival and energy metabolism, inflammation and cancer. Recent studies have shown that sirtuin-1 (SIRT1) modulates Human Immunodeficiency Virus (HIV)-1 transcription. The HIV-1 Tat protein is a substrate for the deacetylase activity of SIRT1; SIRT1 recycles Tat to its unacetylated form, catalyzing a fundamental step to start new cycles of viral transcription. Moreover, Tat has been reported to promote T-cell hyperactivation by suppressing SIRT1 activity. In fact, Tat blocks the ability of SIRT1 to deacetylate lysine 310 in the p65 subunit of nuclear factor- κB (NF- κB) by interacting with the deacetylase domain of SIRT1. This mechanism leads therefore to the hyperactivation of NF- κB proinflammatory pathway and may likely contribute to the chronic immune activation state of HIV-infected individuals. In the present review we first briefly describe the biological functions of sirtuins, then we delineate the interplay between SIRT1 and HIV-1 and discuss the potential role of SIRT1 as a pharmacological target of HIV-1 replication.

Dasatinib: is it all in the dose?

Dasatinib is approved for the treatment of chronic myeloid leukemia (CML) in patients with resistance or intolerance to imatinib. This article reviews pharmacokinetic, pharmacodynamic, and clinical data on dasatinib, and highlights some of the most important issues that need to be addressed. Imatinib and dasatinib both target the tyrosine kinase activity of the BCR/ABL oncogenic fusion protein. In terms of pharmacodynamics, the two agents differ in several ways: (i) dasatinib is >300-fold more potent than imatinib in inhibiting BCR/ABL activity; (ii) inhibition profiles on other tyrosine kinases differ between imatinib and dasatinib; and (iii) dasatinib has other peculiar effects on the leukemogenic signaling, including activation of p38 mitogen-activated protein kinase (MAPK) and reduction of the apoptotic-inactive form of the BCL2-associated agonist of cell death (BAD) protein. Recent pharmacodynamic data suggested combination therapy with dasatinib and signaling inhibitors (e.g. flavopiridol, farnesyl transferase inhibitors, or histone deacetylase inhibitors) may be beneficial. In contrast to other tyrosine kinase inhibitors (TKIs), dasatinib has a reduced half-life and no active metabolites. In a randomized, open-label, phase III trial, dasatinib 100 mg once daily demonstrated similar efficacy and a better tolerability profile than 70 mg twice daily. This unexpected result has been confirmed in recent studies, in which a dose of dasatinib 100 mg once daily was sufficient to trigger apoptosis in leukemic cells. Furthermore, cytogenetic responses correlate with BCR/ABL inhibition. Data suggest dasatinib 100 mg once daily achieves oncogenic shock and chronic inhibition of BCR/ABL activity, suggesting that in the future, pulse therapy with TKIs may be an option in some specific patients with CML.

TFE3 transcription factor regulates the expression of MAFB during macrophage differentiation.

Transcription Factor for Immunoglobulin Heavy-Chain Enhancer 3 (Tfe3) is a transactivator of metabolic genes that are regulated through an EBox located in their promoters. It is involved in physiological processes such as osteoclast and macrophage differentiation, as well as in pathological processes such as translocations underlying different cancer diseases. MAFB is a basic region/leucine zipper transcription factor that affects transcription by binding specific DNA regions known as MARE. It plays a pivotal role in regulating lineage-specific hematopoiesis by repressing transcription of erythroid specific genes in myeloid cells and enhancing expression of macrophage and megakaryocytic genes. Here we have shown MAFB to be highly induced in human hematopoietic cells undergoing macrophage differentiation following Tfe3 ectopic expression, and to be down regulated, compared to the controls, in the same cell population following Phorbol Esters (PMA) dependent differentiation coupled to Tfe3 gene silencing. Electrophoretic mobility shift assays identified a Tfe3-binding site (EBox) in the MAFB promoter region that is conserved in different mammalian species. MAFB promoter was transactivated by co-expression of Tfe3 in reporter gene assays while deletion or mutation of the MAFB EBox prevented transactivation by Tfe3. Both of these genes were previously included in the group of transcription factors able to drive macrophage differentiation. The observation that MAFB belongs to the Tfe3 regulon suggests the existence of a pathway where these two gene families act synergistically to determine differentiation.

Solution-phase parallel synthesis and biological evaluation of combretatriazoles.

Combretastatin A-4 is an antitumoral and antitubulin agent that is active only in its cis configuration. In the present manuscript, we have synthesized cis-locked combretastatins containing a triazole ring (combretatriazoles). To achieve this, we have developed a column chromatography-free parallel solution-phase synthesis that exploits the reaction between azides and alpha-keto phosphorus ylids, which is known to regioselectively generate the 1,5-disubstituted triazoles. The prepared compounds were screened as antitubulinic agents, allowing us to identify three new compounds with high potency, two of which show a new mechanism of action that induces cells to appear multinucleated and display a high number of mitotic spindles.

NAD depletion by FK866 induces autophagy.

NAD is a multifunctional molecule involved in both metabolic processes and signaling pathways. Such signalling pathways consume NAD which is replenished via one of several biosynthesis pathways. We show that influx of NAD across the plasma membrane may be able to contribute to the homeostasis of intracellular NAD levels. Indeed, extracellular application of NAD was able to replete NAD levels that had been lowered pharmacologically using the novel drug FK866 and was also able to rescue cells from FK866-induced cell death. A marked lag between the drop in NAD levels and cell death prompted us to investigate the mechanism of cell death. We were unable to find evidence of apoptosis as assessed by immunoblotting for the Caspase 3 activation fragment and immunostaining for cytochrome C and AIF translocation. We, therefore, investigated whether autophagy was initiated by FK866. Indeed, we were able to observe the formation of LC3-positive vesicles that had fused with lysosomes in FK866-treated but not control cells. Furthermore, this autophagic phenotype could be reverted by the addition of NAD to the extracellular medium.

Characterization of NAD uptake in mammalian cells.

Recent evidence has shown that NAD(P) plays a variety of roles in cell-signaling processes. Surprisingly, the presence of NAD(P) utilizing ectoenzymes suggests that NAD(P) is present extracellularly. Indeed, nanomolar concentrations of NAD have been found in plasma and other body fluids. Although very high concentrations of NAD have been shown to enter cells, it is not known whether lower, more physiological concentrations are able to be taken up. Here we show that two mammalian cell types are able to transport low NAD concentrations effectively. Furthermore, extracellular application of NAD was able to counteract FK866-induced cell death and restore intracellular NAD(P) levels. We propose that NAD uptake may play a role in physiological NAD homeostasis.