STAT3 mutation impacts biological and clinical features of T-LGL leukemia

STAT3 mutations have been described in 30-40% of T-large granular lymphocyte (T-LGL) leukemia patients, leading to STAT3 pathway activation. Considering the heterogeneity of the disease and the several immunophenotypes that LGL clone may express, the aim of this work was to evaluate whether STAT3 mutations might be associated with a distinctive LGL immunophenotype and/or might be indicative for specific clinical features.Our series of cases included a pilot cohort of 101 T-LGL leukemia patients (68 CD8+/CD4- and 33 CD4+/CD8\ub1) from Padua Hematology Unit (Italy) and a validation cohort of additional 20 patients from Rennes Hematology Unit (France).Our results indicate that i) CD8+ T-LGL leukemia patients with CD16+/CD56- immunophenotype identify a subset of patients characterized by the presence of STAT3 mutations and neutropenia, ii) CD4+/CD8\ub1 T-LGL leukemia are devoid of STAT3 mutations but characterized by STAT5b mutations, and iii) a correlation exists between STAT3 activation and presence of Fas ligand, this molecule resulting highly expressed in CD8+/CD16+/CD56- patients. Experiments with stimulation and inhibition of STAT3 phosphorylation confirmed this relationship. In conclusion, our data show that T-LGL leukemia with specific molecular and phenotypic patterns is associated with discrete clinical features contributing to get insights into molecular bases accounting for the development of Fas ligand-mediated neutropenia

Procalcific Phenotypic Drift of Circulating Progenitor Cells in Type 2 Diabetes with Coronary Artery Disease

Diabetes mellitus (DM) alters circulating progenitor cells relevant for the pathophysiology of coronary artery disease (CAD). While endothelial progenitor cells (EPCs) are reduced, there is no data on procalcific polarization of circulating progenitors, which may contribute to vascular calcification in these patients. In a cohort of 107 subjects with and without DM and CAD, we analyzed the pro-calcific versus endothelial differentiation status of circulating CD34+ progenitor cells. Endothelial commitment was determined by expression of VEGFR-2 (KDR) and pro-calcific polarization by expression of osteocalcin (OC) and bone alkaline phosphatase (BAP). We found that DM patients had significantly higher expression of OC and BAP on circulating CD34+ cells than control subjects, especially in the presence of CAD. In patients with DM and CAD, the ratio of OC/KDR, BAP/KDR, and OC+BAP/KDR was about 3-fold increased than in other groups. EPCs cultured from DM patients with CAD occasionally formed structures highly suggestive of calcified nodules, and the expression of osteogenic markers by EPCs from control subjects was significantly increased in response to the toll-like receptor agonist LPS. In conclusion, circulating progenitor cells of diabetic patients show a phenotypic drift toward a pro-calcific phenotype that may be driven by inflammatory signals

The Oral Dipeptidyl Peptidase-4 Inhibitor Sitagliptin Increases Circulating Endothelial Progenitor Cells in Patients With Type 2 Diabetes: Possible role of stromal-derived factor-1α

OBJECTIVE: Vasculoprotective endothelial progenitor cells (EPCs) are regulated by stromal-derived factor-1alpha (SDF-1alpha) and are reduced in type 2 diabetes. Because SDF-1alpha is a substrate of dipeptidyl-peptidase-4 (DPP-4), we investigated whether the DPP-4 inhibitor sitagliptin modulates EPC levels in type 2 diabetic patients. RESEARCH DESIGN AND METHODS: This was a controlled, nonrandomized clinical trial comparing 4-week sitagliptin (n = 16) versus no additional treatment (n = 16) in addition to metformin and/or secretagogues in type 2 diabetic patients. We determined circulating EPC levels and plasma concentrations of SDF-1alpha, monocyte chemoattractant protein-1 (MCP-1), vascular endothelial growth factor (VEGF), and nitrites/nitrates. RESULTS: There was no difference in clinical baseline data between the sitagliptin and control arms. After 4 weeks, as compared with control subjects, patients receiving sitagliptin showed a significant increase in EPCs and SDF-1alpha and a decrease in MCP-1. CONCLUSIONS: Sitagliptin increases circulating EPCs in type 2 diabetic patients with concomitant upregulation of SDF-1alpha. This ancillary effect of DPP-4 inhibition might have potential favorable cardiovascular implications

Time Course and Mechanisms of Circulating Progenitor Cell Reduction in the Natural History of Type 2 Diabetes

OBJECTIVE: Reduction of bone marrow-derived circulating progenitor cells has been proposed as a novel mechanism of cardiovascular disease in type 2 diabetes. The present study was designed to describe the extent and potential mechanisms of progenitor cell reduction during the natural history of type 2 diabetes. RESEARCH DESIGN AND METHODS: We identified 425 individuals, divided into seven categories according to carbohydrate metabolism status (normal glucose tolerance [NGT], impaired fasting glucose, impaired glucose tolerance [IGT], and newly diagnosed type 2 diabetes) and diabetes duration (0-9, 10-19, and >or=20 years). These categories were examined as ideally describing the natural history of type 2 diabetes development and progression. We measured CD34+ and CD34+KDR+ progenitor cells by flow cytometry. We also evaluated progenitor cells in 20 coupled bone marrow and peripheral blood samples and examined progenitor cell apoptosis in 34 subjects. RESULTS: In comparison to NGT, CD34+ cells were significantly reduced in IGT and had a first nadir in newly diagnosed type 2 diabetes and a second nadir after 20 years of diabetes. Statistical adjustment for possible confounders confirmed that CD34+ cell counts are deeply reduced at time of diagnosis, that they partially recover during the subsequent 0-19 years, and that they dip again after >or=20 years. A similar, but less consistent, trend was detected for CD34+KDR+ cells. Peripheral blood CD34+ cells were directly correlated with bone marrow CD34+ cells and inversely correlated with CD34+ cell apoptosis. CONCLUSIONS: Circulating progenitor cell reduction marks the clinical onset of type 2 diabetes. Both defective mobilization and increased apoptosis may account for this phenomenon. While a partial recovery occurs during subsequent years, bone marrow reserve seems exhausted in the long term

Effects of androgens on endothelial progenitor cells in vitro and in vivo

The beneficial or detrimental effects of androgens on the cardiovascular system are debated. Endothelial progenitor cells are bone-marrow-derived cells involved in endothelial healing and angiogenesis, which promote cardiovascular health. Oestrogens are potent stimulators of endothelial progenitor cells, and previous findings have indicated that androgens may improve the biology of these cells as well. In the present study, we show that testosterone and its active metabolite dihydrotestosterone exert no effects on the expansion and function of late endothelial progenitors isolated from the peripheral blood of healthy human adult males, whereas they positively modulate early ‘monocytic’ endothelial progenitor cells. In parallel, we show that castration in rats is followed by a decrease in circulating endothelial progenitor cells, but that testosterone and dihydrotestosterone replacement fails to restore endothelial progenitor cells towards normal levels. This is associated with persistently low oestrogen levels after androgen replacement in castrated rats. In a sample of 62 healthy middle-aged men, we show that circulating endothelial progenitor cell levels are more directly associated with oestradiol, rather than with testosterone, concentrations. In conclusion, our results collectively demonstrate that androgens exert no direct effects on endothelial progenitor cell biology in vitro and in vivo

Valutazione dell’impatto delle azioni intraprese attraverso un programma regionale di supporto alla salute mentale materna

La salute mentale materna in epoca perinatale è un tema di salute pubblica che deve avere un’attenzione prioritaria. Un mancato riconoscimento di un disturbo mentale perinatale può avere conseguenze infauste sul neonato, sulla donna e sulla loro famiglia. Purtroppo la patologia è decisamente poco riconosciuta, sia per una scarsa identificazione da parte dei servizi, sia per una scarsa ricerca di aiuto da parte delle donne, dovuta allo stigma nell’ambito della patologia mentale oltre che alla mancanza di informazione sul tema. In letteratura sono noti diversi fattori di rischio per lo sviluppo di depressione perinatale e sono, inoltre, disponibili test di screening, come ad esempio l’EPDS - Edinburgh Postnatal Depression Scale, per individuare precocemente le donne più a rischio di sviluppare depressione perinatale. Infatti, ci sono evidenze che dimostrano l’efficacia di diversi trattamenti, da applicare in base alla complessità del singolo caso, che sono tanto più efficaci quanto più precocemente vengono intrapresi; questi vanno da interventi psicosociali quali visite domiciliari, terapie psicoterapiche, fin’ anche a terapie farmacologiche che si sono dimostrate, laddove necessarie e assunte sotto lo stretto monitoraggio di specialisti, sicure e vantaggiose nella valutazione rischio/benefici per la donna e il bambino. In regione Veneto è stato attivato uno specifico programma di screening nell’ambito del Progetto sulla Depressione Perinatale, universale, cioè proposto a tutte le puerpere ricoverate per il parto, effettuato con un test combinato (EPDS + questionario sui fattori di rischio), supportato da un sistema informativo basato su un algoritmo per il calcolo del rischio di sviluppare depressione e utile strumento di raccolta dati. Le donne grazie allo screening vengono suddivise in tre gruppi di rischio, alto, medio e basso, in base ai quali si differenziano i percorsi di presa in carico. E’ stata realizzata un’ampia formazione degli operatori coinvolti su base regionale. Scopo di questa tesi è valutare l’impatto delle azioni intraprese nell’ambito del progetto regionale, dalla formazione degli operatori, all’andamento dello screening nei primi quattro mesi di attività, all’efficacia del test combinato. Materiali e metodi: sono state realizzate diverse analisi descrittive e multivariate utilizzando i dati raccolti durante la formazione, riguardanti i partecipanti ai corsi; inoltre sono stati analizzati i dati raccolti tramite il sistema informativo, relativi allo screening. Per verificare la presenza di patologia psichiatrica nei mesi successivi allo screening, nei flussi correnti regionali sono state rintracciate eventuali diagnosi psichiatriche a carico delle donne sottoposte a screening o uso di psicofarmaci. Si è condotta, infine, una sensitivity analysis del test utilizzato per condurre lo screening. Risultati: Hanno partecipato alla formazione 1723 operatori sanitari operanti nel sistema sanitario del Veneto. Il 98% dei corsisti esprime un buon gradimento dei corsi. Il gruppo più numeroso è quello delle ostetriche, operatori maggiormente coinvolti nel progetto, che si sono dimostrate interessate all’argomento e hanno completato nel 93% dei casi la formazione. Sono stati eseguiti 6.233 screening su 10.829 parti avvenuti da settembre a dicembre 2020. L’aderenza allo screening è del 70% dopo i primi 4 mesi di implementazione (con trend crescente da settembre a dicembre). A livello regionale, il 7.6% delle donne sono state classificate ad alto rischio di sviluppare una depressione perinatale, mentre il 7.5% risultano essere a medio rischio. Il 2.2% delle donne sottoposte a screening si sono rivelate patologiche. Mediante la sensitivity analysis, dal confronto delle curve ROC, si è potuto dimostrare che l’utilizzo del test combinato è più efficace rispetto all’uso del solo EPDS, perché permette di ottimizzare sensibilità e specificità dello screening.Perinatal mental health is a public health issue, which deserves priority attention. Failure to identify perinatal mental disorders can result in dire consequences for the newborn, the woman, and their family. Unfortunately, such diseases are not detected as often as they should be. Causes are manifold: surely women seldom ask for help because of the stigma in the field of mental pathology, as well as for lack of information on the subject. In literature, a lot of papers point to the risk factors for the development of perinatal depression, and screening tests are also available, such as the EPDS - Edinburgh Postnatal Depression Scale, to identify the women most at risk of developing perinatal depression at an early stage. In fact, the earlier any therapy is undertaken, the more effective it will be. There is evidence that demonstrates the effectiveness of different treatments. Treatment choice depends on the severity of the symptoms; possible treatments range from psychosocial interventions, such as home visits for monitoring and support, psychotherapeutic therapies for individuals or groups, up to pharmacological treatment. That last has proven to be safe and advantageous in risk/benefit assessment for women and children. In Veneto region, a specific screening program has been activated as part of the Perinatal Depression Project. Screening is universal, that is, proposed to all mothers hospitalized for childbirth; it is carried out with a combined test (EPDS + questionnaire on risk factors), supported by an information system. The web based information system is based on an algorithm calculating the risk of developing depression and it is an useful data collection tool. Screening scores divide women according to their risk of developing depression into three risk groups: high, medium and low. For each group the care pathways differ. Perinatal Depression Project included extensive training of the staff involved. The aim of this thesis is to evaluate the impact of the actions taken within the regional project, from the training of operators, to the progress of the screening in the first four months of activity, to the effectiveness of the combined test. Materials and methods: many descriptive and multivariate analysis were carried out using the data collected during the training, regarding the participants in the courses; in addition, the data collected through the information system relating to screening were analysed. To verify the presence of psychiatric pathology in the months following the screening, any psychiatric diagnoses of women undergoing screening or their use of psychiatric drugs were traced in the current regional data flows. Finally, a sensitivity analysis of the test used to conduct the screening was conducted. Results: 1723 health workers operating in the Veneto health system participated in the training. 98% of the learners express a good satisfaction with the courses. Midwives accounted for the largest group, as the most involved operators in the project, showing keen interest in the subject and having completed their training in 93% of cases. 6,233 women completed the screening test, on 10,829 deliveries from September to December 2020. Adherence to screening is 70% after the first 4 months of implementation (with an increasing trend from September to December). The algorithm classified 7.6% of women as at high risk of developing perinatal depression, while 7.5% were classified as at medium risk. In the months following delivery, psychiatric pathologies have been diagnosed for 2.2% of the screened women. Through the sensitivity analysis, from the comparison of the ROC curves, it was possible to demonstrate that the use of the combined test is more effective than the use of the EPDS alone, as it allows optimization of the sensitivity and specificity of the screening

Fattori prognostici nella leucemia linfatica cronica di tipo B

B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in adults and it is characterized by the accumulation of monoclonal small CD5+ B lymphocytes, in peripheral blood, bone marrow and lymphoid organs. The clinical course encompasses indolent to aggressive disorders, the latter requiring intensive therapeutic intervention. In the past, several clinical and biological prognostic factors have been proposed, including clinical stage, lymphocyte doubling time, bone marrow infiltration, percentage of prolymphocytes, serum β2 microglobulin, tymidine kinase and soluble CD23 levels. Even if these factors correlate with the clinical outcome of the CLL, they were not able to predict the evolution of the disease in patients at the earliest stages of disease. For this reason novel prognostic factors able to predict at diagnosis the clinical evolution of the disease and stratifying patients into groups with a different risk degree have been recently identified. During the three years of this project we collected pheripheral blood samples from 247 patients affected by B-CLL referring to our Hematology and Clinical Immunology Unit and we evaluated the prognostic role of some new factors, including: somatic hypermutations of the Ig variable region genes (SHM), BCR VH repertoire, expression of CD38, CD305 and ZAP-70 in leukemic cells. By flow cytometry, we compared two techniques for ZAP-70 analysis: the isotype control and the ratiometric method. Between the two methods we selected the second one because it was indipendent of the operator and more reproducible. Then we standardized this method for our laboratory instruments. We optimized threshold value for ZAP-70 expression and we selected the cut-off that had greatest sensitivity and specificity. Finally we compared the results obtained from 4 different monoclonal antibodies (MoAb) anti-ZAP-70, identifying two MoAb for the detection of ZAP-70: the anti-ZAP70 Alexa Fluor 488, Caltag Laboratories and anti-ZAP70 FITC, Upstate Cell Signaling Solution. The analysis of LAIR-1 showed that it was reduced in CLL patients (41% ± 32) with respect to in healthy subjects (84% ± 1) and, in particular high-risk patients (stage 3 and 4) had less protein expression than lower-risk group (stage 0-2). Our data confirmed the prognostic role of SHM in CLL: the median survival times for mutated and unmutated patients were 260 and 99 months, respectively (p<0,001). The evaluation of the VH repertoire highlighted a prevalent expression of the gene families VH3 (58% of the patients), VH1 (18% of the sample) and VH4 (18%). The expression of VH1 genes was associated with an unmutated IgVH status (58% of CLL patients); the expression of VH4 family was associated to a mutated status (SHM≥2%) and to a median global survival of 220 months, significantly higher when compared to the average of the whole sample (p<0,001). We also demostrated a prognostic role for CD38 and ZAP-70 expression: the median overall survival for CD38 negative and positive patients was 123 and 250 months, respectively (p<0,01); the median survival for the ZAP-70 positive and negative patients was 135 and 220 months (p<0,01). We analyzed the correlations between these prognostic factors. Statistical analysis showed a significant correlation between CD38 expression and the unmutated IgVH status (p<0,01) and between ZAP-70 expression and the absence of hypersomatic mutation (p<0,05). LAIR-1 was less expressed in both SHM negative patients and in CD38 positive patients. Our data confirm the utility of new prognostic factors as far as they may predict the clinical evolution of the disease. However, the mutational status of variable heavy chain Ig genes at present represents the most reliable and mandatory prognostic factor. ZAP-70 evaluation by flow cytometry tecnology is promising but every laboratory should standardize appropriate methods according to the instruments and reagents available in their setting.La leucemia linfatica cronica di tipo B (LLC-B) è la forma più comune di leucemia dell'adulto ed è caratterizzata dall’accumulo nel sangue periferico, nel midollo osseo e negli organi linfatici di piccoli linfociti B monoclonali esprimenti il marcatore CD5. È una patologia eterogenea, la cui evoluzione varia da un decorso clinico indolente, che non necessita di alcuna terapia, ad una rapida progressione che richiede un trattamento. L’identificazione di fattori che permettano di stratificare pazienti a prognosi differente fin dalle fasi iniziali della malattia è uno dei principali obiettivi degli studi riguardanti la LLC-B. Negli anni sono stati definiti fattori di prognosi classici (il tempo di raddoppiamento linfocitario, l’infiltrazione del midollo osseo, la percentuale di prolinfociti, i livelli di β2 microglobulina, di timidina chinasi e di CD23 solubile), e, più recentemente, fattori prognostici correlati a caratteristiche molecolari del clone leucemico, tra i quali la presenza di alterazioni citogenetiche, lo stato mutazionale dei geni della catena pesante delle immunoglobuline (SHM), l’espressione dell’enzima telomerasi e di molecole quali CD38 e ZAP-70. Un possibile fattore prognostico, ancora in fase di valutazione, è infine il leucocyte-associated Ig-like receptor-1 (LAIR-1 o CD305), un recettore inibitorio, espresso sulla superficie delle cellule B, che può indurre la defosforilazione di diverse chinasi. Il progetto di ricerca sviluppato nei tre anni di dottorato mirava a definire il valore di alcuni fattori prognostici di recente definizione (CD38, CD305, ZAP-70 e SHM) e le possibili correlazioni esistenti tra essi. In particolare, poiché le modalità di determinazione dell’espressione della chinasi ZAP-70 sono oggetto di discussione a livello internazionale, una parte rilevante del triennio di questo dottorato di ricerca è stata dedicata alla valutazione ed alla comparazione di diversi metodi sperimentali, al fine di identificare un procedimento affidabile e ripetibile per la quantificazione di questa proteina. L’individuazione di un metodo affidabile e riproducibile per l’analisi di ZAP-70 mediante analisi citofluorimetrica ha portato alla scelta del metodo raziometrico, che valuta l’intensità media di fluorescenza di ZAP-70 nei linfociti B patologici in rapporto all’intensità media di fluorescenza della proteina nei linfociti T. Il metodo si è rivelato infatti più indipendente dall’operatore rispetto alle altre metodiche analizzate. Una volta stabilito il metodo più appropriato, abbiamo adeguato la metodica alla strumentazione del nostro laboratorio di Ematologia e Immunologia Clinica. Abbiamo quindi stabilito il valore soglia che meglio distingueva tra pazienti positivi e negativi e che ci permetteva di ottenere le maggiori specificità e sensibilità e abbiamo infine confrontato i dati ottenuti dall’utilizzo di diversi anticorpi monoclonali in grado di riconoscere la proteina ZAP-70 dimostrando che i due anticorpi che davano risultati maggiormente riproducibili e più simili tra loro erano l’anticorpo anti-ZAP70 Alexa Fluor 488, Caltag Laboratories e l’anticorpo anti-ZAP70 FITC, Upstate cell signaling solution. Per quanto riguarda gli altri fattori prognostici esaminati, abbiamo innanzitutto confermano il ruolo prognostico delle SHM nei 247 pazienti da noi analizzati e afferenti all’Unità operativa di Ematologia e Immunologia Clinica. Infatti il valore medio di sopravvivenza globale per i pazienti con SHM≥2% rispetto alle sequenze germline è risultato pari a 260 mesi e 99 mesi rispettivamente (p<0,001). La valutazione del repertorio VH ha evidenziato una prevalente espressione della famiglia VH3 (58% del campione). Le altre famiglie VH più rappresentate erano la famiglia VH1 (18% del campione) e VH4 (18%). L’espressione della famiglia VH1, era associata ad un’elevata probabilità di avere uno stato mutazionale <2% delle IgVH (58%). L’espressione della famiglia VH4, invece, si associa ad uno stato mutato (≥2%) delle IgVH (67%) e ad una sopravvivenza globale media di 220 mesi, significativamente superiore rispetto alla media dell’intero campione (p<0,001). Anche CD38 e ZAP-70 hanno dimostrato un ruolo prognostico importante: la sopravvivenza globale media per i pazienti CD38 positivi e negativi era pari rispettivamente a 123 mesi e 250 mesi (p=0,002); la sopravvivenza globale media per i pazienti ZAP-70 positivi e negativi era pari a 135 e 220 mesi (p=0,009). Lo studio dell’espressione di LAIR-1 ha dimostrato che questo recettore è espresso mediamente in quantità minore (41%±32) rispetto ai soggetti sani (84%±1), ed in particolare i pazienti ad alto rischio (stadio 3 e 4) avevano un’espressione minore della proteina rispetto al gruppo a minor rischio (stadio 0-2). Abbiamo valutato l’esistenza di correlazioni tra i diversi fattori prognostici. Abbiamo così rilevato una correlazione statisticamente significativa tra l’espressione di CD38 e l’assenza di ipermutazioni somatiche (p<0,01). Analogamente, abbiamo osservato una correlazione tra la positività di espressione di ZAP-70 e la mancanza di SHM (p<0,05). Per quanto riguarda LAIR-1, il recettore risulta espresso in quantità minore sia nei pazienti con SHM<2% sia in quelli CD38 positivi, mentre non è emersa alcuna differenza quando si sono considerati i pazienti per l’espressione di ZAP-70. I risultati ottenuti confermano l’efficacia dei fattori prognostici innovativi nel predire fin dal momento della diagnosi il possibile decorso clinico della malattia. Lo stato mutazionale rimane il fattore prognostico di riferimento e attualmente non sostituibile. Per quanto riguarda la proteina ZAP-70, promettente per la praticità e la rapidità della metodica impiegata per la sua valutazione, va sottolineato che ogni laboratorio deve standardizzare la metodica adeguandola agli strumenti ed ai reagenti in dotazione

Mechanisms and significance of progenitor cell reduction in the metabolic syndrome.

Bone marrow-derived progenitor cells are involved in the homeostasis of the cardiovascular system through differentiation into endothelium, smooth muscle, and cardiomyocytes. Alterations of these extremely plastic cells have been recognized as both markers of cardiovascular risk and pathophysiological links between risk factors and development of atherosclerosis. Metabolic syndrome, as a cluster of well-defined cardiovascular risk factors, represents a strong predictor of cardiovascular events and death. Moreover, components of the syndrome interact with one another and synergistically increase this risk. Here we describe all metabolic syndrome components as being characterized by alterations in circulating progenitor cells, especially endothelial cells. We also highlight how endothelial progenitors may mediate the interactions between cardiometabolic risk factors in a complex interplay and discuss potential implications for prevention and therapy