Enzymes and activation of intracellular signalling in cancer and neurodegenerative diseases

In recent years, many studies have shown evidence that glia play a central role in the central nervous system (CNS), creating many new questions. What is the normal function of glia and what is their role in disease of the CNS? Might glia be important drug targets? If so, this has the opportunity to open a new avenue of drug therapies for patients suffering from brain injuries and neurological disease. In my thesis I described a number of studies of different compounds that exert effects on astroglial cells and on the associated neurological diseases, including inflammation and cancer. In chapter 2, the biological characterization of a series of 2-phenylindol-3-ylglyoxylamides, and the test of their validity as new TSPO probes was reported. All of these compounds were characterized by the presence of a chemoreactive isothiocyanate group, able to bind the receptor protein irreversibly and covalently. Moreover, compound 18 featuring the NBD-fluorescent moiety was synthesized to develop an irreversible fluorescent probe. The presence of these two chemical groups (a chemoreactive group and a fluorescent chromophore) on a single molecule may offer numerous advantages, both in protein purification/characterization, and in protein cell visualization/density determination. In chapter 3, I described the experiments performed using small molecule activators of Nrf2 mediated transcription on a reporter cell line. Two libraries were screened by the ‘fast track’ process and 6 hits were identified. In particular two of these molecules were able to induce the activation of the Nrf2-ARE pathway at a 10x times lower concentration than the reference compound. Further investigations, such as in silico assays, are necessary in order to determine chemical and physical characteristics of some of the selected molecules and to investigate their ability to activate the Nrf2-ARE pathway on human cells. In chapters 4 and 5, the biological characterization of novel promising molecules acting on different interesting targets for glioma therapy is reported. In chapter 4, the biological characterization of new MMP-inhibitors is reported. In the first part of this chapter, the attention has been directed towards a new class of MMP-2 inhibitors belonging to the recently identified family of N-O-isopropyl sulfonamide hydroxamates. Two of these MMP inhibitors (compound 1 and 2) were tested at nanomolar concentrations to investigate their potential efficacy in inhibiting the invasiveness and the proliferation of the human glioma cell line, U87MG. As a first step, the two compounds at nanomolar concentrations were assayed on their ability to inhibit MMP-2 cleavage of gelatin. Moreover, the MMP-2 expression was assessed following MMP inhibitor cell treatments. Of particular novelty was the effect of the inhibitors on the mRNA levels of MMP2 and in the future it would be interesting to look at the mechanism behind this observation. In order to help the development of multitarget therapies against GBM, the combined treatment of the U87MG glioma cells with the MMP inhibitors and the conventional chemotherapeutic drug TMZ was evaluated. In the second half of chapter 4 the biological evaluation of a new series of 4-butylphenyl(ethynylthiophene)sulfonamido-based hydroxamates was described. As well, the effects of substitutions of different chemical groups on MMP-2 and MMP-9 activity and selectivity have been described. First, the reported compounds were examined in vitro by fluorometric assay on isolated enzymes. The compounds 5a-d, 6, 7 were then tested for their ability to inhibit in vitro invasion of U87MG glioma cells through matrigel and to inhibit cell growth. Chapter 5, looked at glioma cell p53 reactivation by small molecule MDM2 inhibitors and their impact on cell viability and growth. The effect on p53 target genes was analysed at the mRNA level, as well as cell senescence and apoptosis. In particular, I investigated whether a novel small-molecule MDM2 inhibitor, named ISA27, affected the growth of the U87MG human GBM cell line and compared its efficacy with that of Nutlin-3. In this chapter I showed that ISA27 activated the p53 pathway in U87MG cells and elicited a dose- and time dependent inhibition of cell growth by the induction of permanent cell cycle arrest and apoptosis. Compared to Nutlin-3, ISA27 was effective at a lower dose and caused a faster antiproliferative response. If confirmed in vivo, the use of this MDM2 inhibitor could offer a novel therapy concept for the treatment of GBM patients by inducing tumor growth inhibition and regression

Real-world clinical and psychosocial outcomes among people with mild or moderate haemophilia A treated on-demand in the Italian CHESS II cohort: a real-world data analysis

© 2024 Giancarlo Castaman et al., published by SciendoBackground: The burden of severe haemophilia A (HA) has been studied extensively owing to the higher bleeding frequency and associated treatment requirements, leaving a clear unmet need for research focused on the burden of mild and moderate HA. Aims: This study sought to characterise the clinical and psychosocial burden of mild and moderate HA in the Italian cohort of the CHESS II study. Methods: This was a retrospective analysis of clinical and psychosocial outcomes in a cohort of male adults (≥18 years old) with mild or moderate HA who participated in the cross-sectional CHESS II study (October 2019-November 2020). Treatment patterns, acute and chronic clinical outcomes and mental health indicators were collected via physician-completed forms. Psychosocial outcomes related to impact of HA on social activities, exercise, opportunities, and lifestyle were collected via a participant self-complete questionnaire. All results were reported descriptively. Results: A total of 113 people with haemophilia A (PwHA) were included, 79 (70%) with moderate HA and 34 (30%) with mild HA, with mean age of 41.4 and 36.6 years, respectively. No one in the sample was receiving a prophylaxis at the time of data capture, with factor VIII use in the 12 months prior reported in 30% and 29% of moderate and mild PwHA, respectively. Ninety-one PwHA (81%) experienced ≥1 bleeding event in the preceding 12 months. People with moderate HA had higher mean annual bleed rate (2.9 vs. 1.1, respectively) and higher prevalence of chronic pain (74% vs. 35%), anxiety (20% vs. 12%), and/or depression (15% vs. 3%). Target joints were reported in 22% and 12% of moderate and mild PwHA, and problem joints in 51% and 12%, respectively. Of 113 participants, 44 (39%) completed the self-complete form (moderate HA, 57%; mild HA, 43%). Overall, 40% vs. 10% of those with moderate vs mild HA reported reducing or giving up social activities, 44% vs. 21% reducing or giving up exercise, 36% vs. 26% missing out on opportunities, and 48% vs. 26% reported HA impacted their lifestyle. Conclusion: Moderate PwHA from the Italian CHESS II cohort appeared to have greater clinical morbidity and lifestyle impact than mild PwHA. Psychosocial outcomes were also worse among moderate PwHA, but significant burden was also observed among mild PwHA. These findings, and the absence of prophylactic treatment in the sample examined, highlight that improving management for potentially undertreated mild/moderate PwHA may aid the avoidance long-term clinical morbidity and negative psychosocial impact.Acknowledgements: This paper reports a retrospective study in which no human participants or animals are directly involved. Medical writing/editorial support was provided by Jeff Frimpter, MPH, funded by HCD Economics. This analysis was funded by Roche SpA, Rozzano, Italy. Roche were involved in the analysis conception, design and interpretation of results, as well as drafting and submission of the manuscript. EFG, TB1, and TB2 are employees of HCD Economics. LS, RT and SB are employees of Roche SpA

associations between general and abdominal adiposity and mortality in individuals with diabetes mellitus

Individuals with diabetes mellitus are advised to achieve a healthy weight to prevent complications. However, fat mass distribution has hardly been investigated as a risk factor for diabetes complications. The authors studied associations between body mass index, waist circumference, waist/hip ratio, and waist/height ratio and mortality among individuals with diabetes mellitus. Within the European Prospective Investigation into Cancer and Nutrition, a subcohort was defined as 5,435 individuals with a confirmed self-report of diabetes mellitus at baseline in 1992-2000. Participants were aged 57.3 (standard deviation, 6.3) years, 54% were men, the median diabetes duration was 4.6 (interquartile range, 2.0-9.8) years, and 22% of the participants used insulin. Body mass index, as indicator of general obesity, was not associated with higher mortality, whereas all measurements of abdominal obesity showed a positive association. Associations generally were slightly weaker in women. The strongest association was observed for waist/height ratio: In the fifth quintile, the hazard rate ratio was 1.88 (95% confidence interval: 1.33, 2.65) for men and 2.46 (95% confidence interval: 1.46, 4.14) for women. Measurements of abdominal, but not general, adiposity were associated with higher mortality in diabetic individuals. The waist/height ratio showed the strongest association. Respective indicators might be investigated in risk prediction models

TSPO ligand residence time influences human glioblastoma multiforme cell death/life balance

Abstract Ligands addressed to the mitochondrial Translocator Protein (TSPO) have been suggested as cell death/life and steroidogenesis modulators. Thus, TSPO ligands have been proposed as drug candidates in several diseases; nevertheless, a correlation between their binding affinity and in vitro efficacy has not been demonstrated yet, questioning the specificity of the observed effects. Since drug-target residence time is an emerging parameter able to influence drug pharmacological features, herein, the interaction between TSPO and irDE-MPIGA, a covalent TSPO ligand, was investigated in order to explore TSPO control on death/life processes in a standardized glioblastoma cell setting. After 90 min irDE-MPIGA cell treatment, 25 nM ligand concentration saturated irreversibly all TSPO binding sites; after 24 h, TSPO de-novo synthesis occurred and about 40 % TSPO binding sites resulted covalently bound to irDE-MPIGA. During cell culture treatments, several dynamic events were observed: (a) early apoptotic markers appeared, such as mitochondrial membrane potential collapse (at 3 h) and externalization of phosphatidylserine (at 6 h); (b) cell viability was reduced (at 6 h), without cell cycle arrest. After digitonin-permeabilized cell suspension treatment, a modulation of mitochondrial permeability transition pore was evidenced. Similar effects were elicited by the reversible TSPO ligand PIGA only when applied at micromolar dose. Interestingly, after 6 h, irDE-MPIGA cell exposure restored cell survival parameters. These results highlighted the ligand-target residence time and the cellular setting are crucial parameters that should be taken into account to understand the drug binding affinity and efficacy correlation and, above all, to translate efficiently cellular drug responses from bench to bedside

Studio degli effetti dell'inquinamento atmosferico in bambini in eta' scolare nella citta' di Pisa

L'inquinamento atmosferico è responsabile di molti effetti nocivi sulla salute della popolazione. In particolare i bambini in età scolare risultano essere più suscettibili agli effetti degli agenti aerodispersi in virtù di una maggiore quantità di aria inalata per unità di peso, dell'elevato numero di ore trascorse all'aria aperta e dell' incompleta maturità dei sistemi di riparazione cellulare e di alcuni organi, tra cui i polmoni. La popolazione infantile rappresenta quindi un gruppo ad alto rischio rispetto agli effetti dell'inquinamento atmosferico sulla salute ed alcuni studi epidemiologici sembrano suggerire come l'esposizione precoce ad inquinanti atmosferici possa avere un ruolo di rilievo nello sviluppo di malattie croniche e degenerative in età adulta. Diviene quindi interessante indagare la possibile presenza di danni genotossici e mutageni in bambini sottoposti ad inquinamento atmosferico attraverso lo studio di particolari indicatori biologici; nel nostro studio abbiamo cercato di valutare in un gruppo di bambini la distribuzione di biomarcatori di danno precoce al DNA in relazione ai livelli di inquinanti aerodispersi e all'attività mutagena dell'aria di Pisa, confrontando poi i risultati con i dati ottenuti da altre quattro unità di ricerca (Brescia, Torino, Perugia e Lecce) che hanno realizzato il medesimo studio nelle proprie città di riferimento. Durante questo lavoro di tesi abbiamo condotto due campagne di campionamento (invernale: Gennaio 2015 - Marzo 2015 e primaverile: Aprile - Giugno 2015) in quattro scuole di Pisa tramite le quali abbiamo prelevato campioni biologici di saliva e cellule epiteliali su un totale di 200 bambini tra i 6 e gli 8 anni. Per valutare i danni al DNA ed evidenziare la presenza di biomarcatori di effetto biologico precoce abbiamo effettuato due test genetici: il comet test sui leucociti presenti nella saliva ed il test del micronucleo sulle cellule epiteliali della mucosa orale. In contemporanea al prelievo biologico abbiamo realizzato un campionamento ambientale dell'aria per 72h consecutive tramite un campionatore ad alto volume nei pressi delle scuole selezionate in modo da valutare la qualità dell'aria quotidianamente respirata dai bambini e la concentrazione del particolato inquinante (PM0,5 e PM10). Nel periodo del campionamento sono stati anche ricavati i dati ARPAT relativi alle misurazioni giornaliere di PM10 nella città di Pisa. Inoltre ai genitori dei bambini coinvolti nello studio è stato consegnato un questionario riguardante le abitudini alimentari e gli stili di vita della famiglia al fine di stimare il ruolo, protettivo o meno, di vari fattori (variabili demografiche e socio-economiche, fumo passivo, esposizione ad inquinamento indoor, attività fisica, dieta ) in relazione al danno biologico nella popolazione infantile. In questo modo si prevede di valutare l'associazione tra l'inquinamento atmosferico e la presenza di marcatori di effetto biologico precoce nei campioni esaminati, al fine di ottenere dei dati di popolazione sull'esposizione ad inquinanti dannosi per la salute. Questo lavoro di tesi è legato al progetto europeo MAPEC-Life+ (Monitoring of Air Pollution Effects on Children), uno studio epidemiologico a coorte che si propone di monitorare gli effetti dell'inquinamento atmosferico su 1000 bambini residenti in cinque città italiane (Brescia, Pisa, Torino, Perugia e Lecce) caratterizzate da diversi livelli di inquinamento. Gli obiettivi finali della ricerca sono quelli di valutare la correlazione tra i livelli di inquinamento atmosferico e la presenza di biomarcatori di danno biologico precoce, come il danno al DNA e la presenza di micronuclei, e di costruire un modello globale per stimare il rischio di avere effetti biologici precoci causati da inquinanti atmosferici ed in relazione ad altri fattori quali alimentazione e stili di vita dei bambini; i risultati della ricerca permetteranno in ultima analisi di fornire informazioni utili per interventi di prevenzione e sanità pubblica volti a proteggere la salute della collettività, con particolare riguardo a quella della popolazione infantile

Beyond the anticoagulant activity: different effect of glyco-anticoagulants and oligosaccharides on angiogenesis and vasculogenesis

Purpose: Anticoagulants are used to reduce complications after acute coronary syndrome. The effects of these drugs on angiogenesis and vasculogenesis, with an important role in plaque destabilization, are not known. Our aim was to test the effect of Bemiparin (B), the low molecular weight heparin with the lowest MW (3600 Da) and the highest anti-FXa/anti-FIIa activity ratio (8:1), and of the synthetic pentasaccharide Fondaparinux (F), a FXa inhibitor, on vasculogenesis and angiogenesis, mediated by endothelial progenitor cells (EPC) and mature endothelial cells (HUVEC), as compared to unfractionated heparin (UFH). Methods: HUVEC or EPC were treated for 24 h with B (0.01-5 I.U./ml), UFH (0.01-5 I.U./ml) or F (0.0005-0.05 mg/ml) before assay for cell viability (MTS), proliferation (BrdU) and in vitro angiogenesis (matrigel) and vasculogenesis (EPC incorporation on matrigel). Drug doses were chosen as the ones used in clinical practice. For angiogenesis, HUVEC were detached and seeded on matrigel. After 20 h, tubule network was quantified. For vasculogenesis, EPC were detached, stained with DiI and seeded on matrigel together with HUVEC. Incorporation of EPC in the tubules was quantified after 20 h. Results: Viability was significantly reduced by the three anticoagulants only at the highest concentration..

Anti-hypertensive drugs and skin cancer risk: a review of the literature and meta-analysis

Introduction Several anti-hypertensive drugs have photosensitizing properties, however it remains unclear whether long-term users of these drugs are also at increased risk of skin malignancies. We conducted a literature review and meta-analysis on the association between use of anti-hypertensive drugs and the risk of cutaneous melanoma and non-melanoma skin cancer (NMSC). Methods We searched PubMed, EMBASE, Google Scholar and the Cochrane Library, and included observational and experimental epidemiological studies published until February 28th, 2017. We calculated summary relative risk (SRR) and 95% confidence intervals (95% CI) through random effect models to estimate the risk of skin malignancies among users of the following classes of anti-hypertensive drugs: thiazide diuretics, angiotensin converting enzyme inhibitors (ACEi), angiotensin receptor blockers (ARB), calcium channel blockers (CCB) and β-blockers. We conducted sub-group and sensitivity analysis to explore causes of between-studies heterogeneity, and assessed publication bias using a funnel-plot based approach. Results Nineteen independent studies were included in the meta-analysis. CCB users were at increased skin cancer risk (SRR 1.14, 95% CI 1.07â\u80\u931.21), and β-blockers users were at increased risk of developing cutaneous melanoma (SRR 1.21, 95% CI 1.05â\u80\u931.40), with acceptable between-studies heterogeneity (I2< 50%). There was no association between thiazide diuretics, ACEi or ARB use and skin cancer risk. We found no evidence of publication bias affecting the results. Conclusion Family doctors and clinicians should inform their patients about the increased risk of skin cancer associated with the use of CCB and β-blockers and instruct them to perform periodic skin self-examination. Further studies are warranted to elucidate the observed associations

Micro-RNA carrying exosomes in motor neuron disease patients

Overexpression of miR-206 and miR-29 could delay the muscle weakness observed in ALS/MND patients, thus providing a potential therapeutic target

Erratum to: Coffee, tea and caffeine intake and the risk of non-melanoma skin cancer: a review of the literature and meta-analysis

In the original publication of the article, the author group has been published incorrectly. The correct author group is as follows: Saverio Caini, Maria Sofia Cattaruzza, Benedetta Bendinelli, Giulio Tosti, Giovanna Masala, Patrizia Gnagnarella, Melania Assedi, Ignazio Stanganelli, Domenico Palli, Sara Gandini

Blood inflammatory markers in motor neuron disease patients: pattern changes over time along disease progression

In ALS patients, we observed a direct correlation between the ALSFRS scores and the levels of some blood analytes (CD3, CD16, CD25, IL17, CD44, sTNF-R2, VCAM, Eselectin) and an inverse correlation with others (CD19,sIL6-R, sTNF-R1, TGF, IGF, PDGF, VEGF, P-selectin);whereas, in l-MND patients, a direct correlation was found with some analytes (CD19, IFNbeta), but an inverse one with others (EPO, IL6, sTNF-R1, PDGF, E and P selectins). We found that CD3, CD4, CD8, CD16, CD25, sIL6-R, IL5 and VCAM are differentially expressed in ALS and in l-MND patients. Conclusion: Our findings are useful as prognostic and diagnostic tools for ALS/MND patients. DOI: 10.1080/21678421.2017.1371525/001