Cytokine secretion responsiveness of lymphomonocytes following cortisol cell exposure: Sex differences

The stress hormone cortisol has been recognized as a coordinator of immune response. However, its different ability to modulate the release of inflammatory mediators in males and females has not been clarified yet. Indeed, the dissection of cortisol specific actions may be difficult due to the complex hormonal and physio-pathological individual status. Herein, the release of inflammatory mediators following increasing cortisol concentrations was investigated in an in vitro model of primary human male and female lymphomonocytes. The use of a defined cellular model to assess sex differences in inflammatory cytokine secretion could be useful to exclude the effects of divergent and fluctuating sex hormone levels occurring in vivo. Herein, the cells were challenged with cortisol concentrations resembling the plasma levels achieving in physiological and stressful conditions. The production of cytokines and other molecules involved in inflammatory process was determined. In basal conditions, male cells presented higher levels of some pro-inflammatory molecules (NF-kB and IDO-1 mRNAs, IL-6 and kynurenine) than female cells. Following cortisol exposure, the levels of the pro-inflammatory cytokines, IL-6 and IL-8, were increased in male cells. Conversely, in female cells IL-6 release was unchanged and IL-8 levels were decreased. Anti-inflammatory cytokines, IL-4 and IL-10, did not change in male cells and increased in female cells. Interestingly, kynurenine levels were higher in female cells than in male cells following cortisol stimulus. These results highlighted that cortisol differently affects male and female lymphomonocytes, shifting the cytokine release in favour of a pro-inflammatory pattern in male cells and an anti-inflammatory secretion profile in female cells, opening the way to study the influences of other stressful factors involved in the neurohumoral changes occurring in the response to stress conditions

Traslocatore Proteico (TSPO,18 kDa): Analisi quantitativa del “Residence Time” di ligandi sintetici e valutazione dell’effetto antitumorale in vitro esercitato dal ligando irreversibile irDE-MPIGA

Il Traslocatore Proteico (TSPO, 18kDa) è una proteina ubiquitaria che svolge un ruolo cruciale nella regolazione di processi fondamentali alla base della vita/morte cellulare, quali proliferazione, differenziamento, respirazione mitocondriale e sintesi degli steroidi [1]. A livello subcellulare, TSPO è localizzato nella membrana mitocondriale esterna e prende parte alla regolazione del poro multiproteico implicato nella transizione di permeabilità della membrana mitocondriale (MPT) [1], fenomeno consistente in un improvviso aumento di permeabilità della membrana interna. In conseguenza a MPT si verifica dissipazione del potenziale di membrana, “swelling” osmotico della matrice mitocondriale e permeabilizzazione della membrana esterna, che causa il rilascio di potenti segnali pro-apoptotici [2]. Il ruolo modulatorio di TSPO in tale processo e il ritrovamento della sua sovraespressione nei tumori lo hanno reso un promettente target farmacologico nella cura del cancro. Nell’ambito della ricerca di farmaci indirizzati a TSPO come potenziali antitumorali, numerosi studi hanno dimostrato che ligandi con alta affinità di legame a TSPO sono capaci di causare efficacemente, attraverso induzione di MPT, morte apoptotica in differenti tipi di cellule maligne [3-6]. Tuttavia, la mancanza di correlazione tra la affinità di legame dei ligandi e la dose di efficacia ha spesso messo in discussione la specificità degli effetti osservati [7]. Recentemente è stato suggerito che gli effetti benefici di farmaci indirizzati verso altri target biologici e comunemente in uso per la cura di determinati stati patologici, possono essere attribuiti, più che all’alta affinità di legame del farmaco, alla lunga durata del complesso farmaco-bersaglio, parametro noto come “Residence-time” [8]. Il Residence-time (RT) è un parametro cinetico che può essere determinato mediante saggi di binding radioattivo e corrisponde al reciproco della costante di dissociazione (Koff) del ligando dal suo target (RT=1/Koff). Queste evidenze ci hanno spinto ad investigare, per la prima volta, se il “Residence-time” sia un parametro in grado di influenzare l’efficacia di un ligando, anche nel caso dei ligandi al TSPO. A tale scopo la presente tesi è stata finalizzata al raggiungimento di due obiettivi principali: 1) determinazione del parametro RT per ligandi al TSPO; 2) studio degli effetti causati dalla stabile interazione del ligando irreversibile irDE-MPIGA con TSPO in un modello di cancro in vitro e confronto dei risultati con i dati ottenuti usando un ligando reversibile. Per il raggiungimento dell’obiettivo 1 è stata effettuata la messa a punto di un metodo, noto come “metodo di associazione competitiva”, capace di quantificare i parametri cinetici di legame di ligandi non radiomarcati [9]. Mediante tale sperimentazione è stato possibile determinare il valore di RT di ligandi classici al TSPO, quali PK11195 e Ro5-4864 e ligandi a struttura indolilgliossilammidica. Nel caso dell’obiettivo 2, i risultati più rilevanti riguardano la capacità di una dose nanomolare di irDE-MPIGA di sensibilizzare il poro MPT al Ca2+ e attivare rapidamente apoptosi nelle cellule tumorali. In parallelo, per ottenere effetti simili è risultata necessaria una dose micromolare di ligando reversibile al TSPO. L' efficacie effetto antitumorale osservato durante la stabile interazione irDE-MPIGA-TSPO suggerisce che il Residence-time dovrebbe essere preso in considerazione come parametro importante nei processi di selezione di composti indirizzati al TSPO con potenziale attività antitumorale

Biological action of two tomato cysteine-rich miniproteins

Fin dal passato i prodotti naturali sono stati utilizzati come rimedio alle malattie. In tempi recenti questi prodotti naturali hanno ricevuto grande attenzione nella prevenzione delle malattie grazie ai loro effetti benefici sulla salute, come ad esempio la bassa tossicit\ue0 e effetti collaterali minimi. Le miniproteine cystine-knot (CKMs) appartengono ad una grande famiglia di piccole proteine ricche in cisteine diffuse nelle piante e nelle specie animali. CKMs mostrano un vasto spettro di attivit\ue0 biologiche naturali utili da un punto di vista terapeutico, infatti molte di queste CKMs sono commercializzate come agenti terapeutici o sono in vari fasi di sviluppo clinico. CKMs constano tipicamente di meno di 50 aminoacidi e hanno una struttura tridimensionale unica caratterizzata da tre legami disolfuro intramolecolari che formano un \u201ccystine-knot\u201d (nodo di cisteine) e da un piccolo foglietto beta. Il nodo di cisteine agisce come impalcatura per la proteina conferendo a tutte le miniproteine cystine-knot una struttura compatta e sorprendentemente stabile a vari fattori, come pH estremi, denaturazioni chimiche o termiche e attacchi proteolitici. Questa struttura \ue8 stata individuata per la prima volta nel 1982 in un inibitore di carbossipeptidasi di patata (PCI). PCI inibisce la carbossipeptidasi pancreatica dei mammiferi e pu\uf2 agire come fattore antitrombotico; inoltre \ue8 stato dimostrato che PCI agisce come inibitore della crescita di linee cellulari di adenocarcinoma pancreatico funzionando come antagonista del fattore di crescita umano dell\u2019epidermide legandosi al recettore del fattore di crescita dell\u2019epidermide (EGFR). \uc8 stato visto che l\u2019attivit\ue0 dell\u2019EGFR, che \ue8 un recettore tirosin-kinasico appartenente alla famiglia ErbB, \ue8 aumentata in maniera anomala nella maggior parte dei tumori solidi umani e questa attivit\ue0 anomala \ue8 associata con la progressione del tumore e con una prognosi sfavorevole. Oggigiorno \ue8 diventato evidente che la via di segnale del EGFR, oltre a un effetto sulla progressione, gioca un ruolo importante anche nell\u2019angiogenesi, ovvero nella formazione di nuovi vasi sanguigni partendo da vasi preesistenti per formare una rete capillare. L\u2019angiogenesi \ue8 un evento fondamentale nei processi fisiologici come l\u2019organogenesi, la guarigione delle ferite e la crescita dei muscoli, ma avviene anche in situazioni patologiche dove promuove la crescita e la progressione tumorale, la formazione di metastasi cos\uec come la retinopatia diabetica e la psoriasi. Alla luce di ci\uf2 \ue8 facile intuire che il trattamento anti-angiogenico \ue8 considerato una strategia efficace nella terapia tumorale e l\u2019inibizione di EGFR sembra rappresentare un nuovo e promettente approccio. Questa ricerca mira ad investigare l\u2019azione biologica su cellule umane di due miniproteine di pomodoro che sono componenti consueti della nostra dieta. La motivazione di questa ricerca scaturisce dalla scoperta che due miniproteine cystine-knot di pomodoro (TCMPs), di cui una espressa nel fiore (TCMP-1) e una nel frutto maturo (TCMP-2), mostrano similarit\ue0 di sequenza e struttura con un inibitore di carbossipeptidasi di patata (PCI) che agisce come antagonista di EGF. Poich\ue9 EGF \ue8 una delle molecole segnale che partecipa alla formazione di nuovi vasi, abbiamo investigato se TCMP-1 e TMCP-2, che sono capaci di legarsi alla superficie di cellule umane, possono esercitare una attivit\ue0 anti-angiogenica avendo contemporaneamente una bassa tossicit\ue0 cellulare. In questo lavoro abbiamo dimostrato che gli inibitori di carbossipeptidasi cystine-knot di pomodoro, sia in forma naturale che ricombinante, possiedono attivit\ue0 anti-angiogenica in vitro e che questa attivit\ue0 \ue8 svolta ad una concentrazione in un range nano molare, range che \ue8 di particolare rilevanza biologica. Inoltre i nostri esperimenti hanno provato che le due miniproteine non esercitano alcun effetto tossico sulla proliferazione e vitalit\ue0 delle cellule endoteliali e che la loro attivit\ue0 anti-angiogenica \ue8 associata con l\u2019inibizione dell\u2019attivazione di ERK1/2, suggerendo che TCMPs interferiscano con la via di segnale delle MAPK. In conclusione possiamo dire che poich\ue9 gli inibitori di carbossipeptidasi cystine-knot di pomodoro sono presenti nel frutto commestibile, questi composti i dovrebbero essere dotati di bassa tossicit\ue0 quando testati su modelli animali e sull\u2019uomo; inoltre l\u2019angiogenesi tumorale pu\uf2 rappresentare un target per l\u2019uso terapeutico delle miniproteine di pomodoro e la loro attivit\ue0 anti-angiogenica pu\uf2 essere testata anche per la prevenzione di alter malattie. In base a questi dati e considerazioni le miniproteine di pomodoro possono avere un potenziale interesse farmacologico che merita successivi approfondimenti.Natural products are known since the past as disease remedies. In recent years, natural products have received great attention for disease prevention owing to their various health benefits, including low toxicity and diminished side effects. Cystine-knot miniproteins (CKMs) are members of a large family of small cysteine-rich proteins widespread in plant and animal species. CKMs display a broad spectrum of therapeutically useful natural biological activities and several CKMs are marketed as therapeutics or are in clinical development. CKMs typically consist of less than 50 amino acids and have a unique three-dimensional structure characterized by three intra-molecular disulfide bonds forming a cystine-knot and a small tripled stranded beta-sheet. The cystine-knot acts as structural scaffold for the protein conferring to the cystine-knot miniproteins a compact and remarkably stable structure towards extreme pH, chemical and thermal denaturation, and proteolytic attack. This scaffold was first discovered in 1982 in a carboxypeptidase inhibitor from potato (PCI). PCI inhibits mammalian pancreatic carboxypeptidase and can act as antithrombotic agent. Moreover, it has been shown that PCI also function as inhibitor of pancreatic adenocarcinoma cell lines growth acting as antagonist of the human epidermal growth factor by binding to the epidermal growth factor receptor (EGFR). The activity of the EGFR, that is a tyrosine kinase receptor of the ErbB family, is abnormally elevated in most human solid tumors and has been associated with progression and poor prognosis. Nowadays, it has become clear that the EGFR signaling pathway also plays a key role in angiogenesis, the formation of new blood vessels from pre-existing vessels to form capillary networks. Angiogenesis is a fundamental event of physiological processes, like organogenesis, wound healing and muscle growth, but it also takes place in pathological situations and promotes tumor growth and progression, metastasis as well as diabetic retinopathy and psoriasis. Therefore, anti-angiogenic treatment is considered a very effective strategy in cancer therapy and inhibition of EGFR seems to represent a novel and promising approach. This research aims to investigate the biological action on human cells of two tomato miniproteins which are normal components of our diet. The motivation for this research stems from the discovery that two tomato cystine-knot miniproteins (TCMPs), one expressed in the flower (TCMP-1) and the other in the mature fruit (TCMP-2), are similar in sequence and structure to a potato carboxypeptidase inhibitor (PCI) that acts as EGF antagonist. Since EGF is one of the signalling molecules participating in the formation of new vessels, we have investigated whether, TCMP-1 and TCMP-2, that can bind to the surface of human cells, could exert anti-angiogenic activity with limited cell toxicity. We have demonstrated that tomato cystine-knot carboxypeptidase inhibitors both native and recombinant, possess anti-angiogenic activity in vitro. The tomato miniproteins act as an anti-angiogenic factor in the nanomolar concentration range, which is of particular biological relevance. Furthermore, our experiments have proved that the two miniproteins do not exert toxic effects on endothelial cell proliferation and viability. Their anti-angiogenic activity is associated with an inhibitory effect on ERK1/2 activation, suggesting that TCMPs interfere with MAPK signaling pathway. Since tomato cystine-knot carboxypeptidase inhibitors are present in edible fruits, these compounds should be endowed with low toxicity when tested in animal models of disease and in humans. Tumorigenic angiogenesis could represent a target for therapeutic intervention with tomato miniproteins, and their anti-angiogenic activity could be tested as well for the prevention of other diseases. On these bases, miniproteins from tomato may be of potential pharmacological interest and deserve further investigation

Pulsology Reloaded

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Phospho-Specific Flow Cytometry Reveals Signaling Heterogeneity in T-Cell Acute Lymphoblastic Leukemia Cell Lines

Several signaling pathways are aberrantly activated in T-ALL due to genetic alterations of their components and in response to external microenvironmental cues. To functionally characterize elements of the signaling network in T-ALL, here we analyzed ten signaling proteins that are frequently altered in T-ALL -namely Akt, Erk1/2, JNK, Lck, NF-κB p65, p38, STAT3, STAT5, ZAP70, Rb- in Jurkat, CEM and MOLT4 cell lines, using phospho-specific flow cytometry. Phosphorylation statuses of signaling proteins were measured in the basal condition or under modulation with H2O2, PMA, CXCL12 or IL7. Signaling profiles are characterized by a high variability across the analyzed T-ALL cell lines. Hierarchical clustering analysis documents that higher intrinsic phosphorylation of Erk1/2, Lck, ZAP70, and Akt, together with ZAP70 phosphorylation induced by H2O2, identifies Jurkat cells. In contrast, CEM are characterized by higher intrinsic phosphorylation of JNK and Rb and higher responsiveness of Akt to external stimuli. MOLT4 cells are characterized by higher basal STAT3 phosphorylation. These data document that phospho-specific flow cytometry reveals a high variability in intrinsic as well as modulated signaling networks across different T-ALL cell lines. Characterizing signaling network profiles across individual leukemia could provide the basis to identify molecular targets for personalized T-ALL therapy

Diabetic Foot Infections:The Diagnostic Challenges

Diabetic foot infections (DFIs) are severe complications of long-standing diabetes, and they represent a diagnostic challenge, since the differentiation between osteomyelitis (OM), soft tissue infection (STI), and Charcot's osteoarthropathy is very difficult to achieve. Nevertheless, such differential diagnosis is mandatory in order to plan the most appropriate treatment for the patient. The isolation of the pathogen from bone or soft tissues is still the gold standard for diagnosis; however, it would be desirable to have a non-invasive test that is able to detect, localize, and evaluate the extent of the infection with high accuracy. A multidisciplinary approach is the key for the correct management of diabetic patients dealing with infective complications, but at the moment, no definite diagnostic flow charts still exist. This review aims at providing an overview on multimodality imaging for the diagnosis of DFI and to address evidence-based answers to the clinicians when they appeal to radiologists or nuclear medicine (NM) physicians for studying their patients

Unveiling the morphogenetic code: A new path at the intersection of physical energies and chemical signaling

In this editorial, we discuss the remarkable role of physical energies in the control of cell signaling networks and in the specification of the architectural plan of both somatic and stem cells. In particular, we focus on the biological relevance of bioelectricity in the pattern control that orchestrates both developmental and regenerative pathways. To this end, the narrative starts from the dawn of the first studies on animal electricity, reconsidering the pioneer work of Harold Saxton Burr in the light of the current achievements. We finally discuss the most recent evidence showing that bioelectric signaling is an essential component of the informational processes that control pattern specification during embryogenesis, regeneration, or even malignant transformation. We conclude that there is now mounting evidence for the existence of a Morphogenetic Code, and that deciphering this code may lead to unprecedented opportunities for the development of novel paradigms of cure in regenerative and precision medicine

Monitoring Chemical Changes of Coffee Beans During Roasting Using Real-time NIR Spectroscopy and Chemometrics

Variations occurring in coffee beans during roasting are ascribable to several chemical-physical phenomena: to quickly track the whole process and to ensure its reproducibility, a process analytical technology (PAT) approach is needed. In this study, a method combining in-line Fourier transform near-infrared (FT-NIR) spectroscopy and chemometric modelling was investigated to get real-time and practical knowledge about the roasting effects on coffee’s chemical-physical composition. In-line spectra were acquired by inserting a NIR probe into a laboratory coffee roaster, running twenty-four roasting experiments, planned spanning different coffee species (Arabica and Robusta), four roasting temperature settings (TS1–TS4) and times (650–1580 s). Multivariate curve resolution-alternate least squares (MCR-ALS) was used to model the chemical-physical changes occurring during the roasting process, and information about maximum rate, acceleration and deceleration of the process was obtained, also highlighting potential effects due to the different roasting temperatures and coffee varieties. The proposed approach provides the groundwork for direct real-time implementation of rapid, non-invasive automated monitoring of the roasting process at industrial scale

The Citrus Flavanone Naringenin Protects Myocardial Cells against Age-Associated Damage

In recent years, the health-promoting effects of the citrus flavanone naringenin have been examined. The results have provided evidence for the modulation of some key mechanisms involved in cellular damage by this compound. In particular, naringenin has been revealed to have protective properties such as an antioxidant effect in cardiometabolic disorders. Very recently, beneficial effects of naringenin have been demonstrated in old rats. Because aging has been demonstrated to be directly related to the occurrence of cardiac disorders, in the present study, the ability of naringenin to prevent cardiac cell senescence was investigated. For this purpose, a cellular model of senescent myocardial cells was set up and evaluated using colorimetric, fluorimetric, and immunometric techniques. Relevant cellular senescence markers, such as X-gal staining, cell cycle regulator levels, and the percentage of cell cycle-arrested cells, were found to be reduced in the presence of naringenin. In addition, cardiac markers of aging-induced damage, including radical oxidative species levels, mitochondrial metabolic activity, mitochondrial calcium buffer capacity, and estrogenic signaling functions, were also modulated by the compound. These results suggested that naringenin has antiaging effects on myocardial cells