Accuratezza diagnostica e prognostica del dosaggio del BNP in pazienti pediatrici con difetti cardiaci congeniti.

RIASSUNTO: I Cardiomiociti producono e secernono una famiglia di ormoni peptidici denominati Peptidi Natriuretici Cardiaci (PNC), che possiede una potente azione diuretica, natriuretica, vasodilatante, ed effettua complesse interazioni sia con il sistema neuro-ormonale che immunologico. Ad oggi, il dosaggio del Brain Natriuretic Peptide (BNP) e del suo relativo pro-peptide N-terminale (NT-proBNP) sono raccomandati dalle Linee Guida Internazionali come marcatori per la diagnosi, la prognosi ed il monitoraggio terapeutico nei pazienti adulti con patologia cardiaca. Al contrario, il dosaggio del BNP, soltanto recentemente ha iniziato ad essere preso in considerazione nei soggetti pediatrici con difetti cardiaci congeniti (CHD). Tuttavia, alcune limitazioni hanno ritardato l'introduzione del BNP nella pratica clinica pediatrica, come la mancanza di valori di riferimento specifici per l’età ed il numero piuttosto limitato di studi clinici riportati in letteratura, riguardanti soprattutto le cardiopatie congenite. La prima parte dell’attività sperimentale, pertanto, si è rivolta alla determinazione dei valori di riferimento per il BNP in soggetti pediatrici sani utilizzando una piattaforma completamente automatizzata (Triage BNP, Access® Systems Immunoassay, REF 98200, Beckman Coulter, Fullerton, CA, USA). Il BNP plasmatico, è stato dosato in 188 neonati sani entro la prima settimana di vita extra uterina ed in campioni di plasma appartenenti ad un gruppo di 245 bambini sani con età compresa tra sette giorni di vita e dodici anni. Le concentrazioni del BNP sono risultate più elevate durante i primi 2 giorni di vita mentre, poi, sono progressivamente diminuite. Inoltre, i valori di BNP nella prima settimana di vita, sono risultati significativamente più alti (p < 0,0001) rispetto ai valori osservati nei periodi successivi. Nella seconda parte della tesi, l’obiettivo principale è stato quello di caratterizzare in dettaglio l’andamento temporale del BNP nei primi giorni di vita in un consistente numero di neonati e bambini affetti da cardiopatie congenite (n = 218), allo scopo di aumentare l’accuratezza diagnostica del BNP nei pazienti pediatrici. Inoltre, 222 bambini sani, appaiati per età e sesso, sono stati arruolati come gruppo di controllo. I valori di BNP sono risultati significativamente più elevati nel gruppo dei pazienti con CHD (mediana 1029,8 ng/L, intervallo 25-20152 ng/L) rispetto ai controlli (mediana 1495 ng/L, intervallo 9-866 ng/L). Si è evidenziato un differente comportamento tra i valori di BNP rispettivamente osservati nei neonati sani e in pazienti con CHD. Infatti, nei pazienti con CHD, dopo un progressivo aumento iniziale riscontrato nei primi quattro giorni di vita, i valori di BNP, nei giorni seguenti, tendono a stabilizzarsi su livelli elevati. Al contrario, nei neonati sani si osserva un picco del valore di BNP nel secondo-terzo giorno di vita, seguito da un progressivo calo nelle settimane seguenti. Considerando questo differente andamento temporale del biomarcatore nei soggetti sani e nei pazienti con CHD, l'accuratezza diagnostica del BNP nei campioni raccolti nei primi 4 giorni di vita (AUC dell'analisi ROC 0,8626; IC al 95% = 0,8253-0,8999) è risultata significativamente peggiore (p < 0,0001) rispetto a quella dei campioni raccolti dal quinto al trentesimo giorno di vita (AUC 0,9710; IC al 95% = 0,9509-0,9910). Il livello ottimale di cut-off per il BNP, calcolato con l’analisi delle curve ROC, è risultato anche strettamente dipendente dall'età del soggetto con un valore ottimale per i primi 4 giorni di vita di 363,5 ng/L, mentre quello dal quinto al trentesimo giorno è risultato essere di 109,5 ng/L. In conculsione, questo studio dimostra che l’accuratezza diagnostica ed i valori decisionali del BNP variano moltissimo nei primi giorni di vita. Quindi, i clinici devono prestare grande attenzione all’età dei neonati ed infanti nel valutare i risultati del dosaggio del BNP. ABSTRACT: Cardiomyocytes produce and secrete a family of related peptide hormones (cardiac natriuretic hormones, CNH), which have potent diuretic, natriuretic and vascular smooth muscle- relaxing effects, and also share complex interactions with the neurohormonal system. In particular, the measurement of circulating brain natriuretic peptide (BNP) and its related peptide, the N-terminal fragment of proBNP (NT-proBNP), is now recommended by international guidelines as a biomarker for diagnosis, prognosis and therapeutic monitoring in adult patients with cardiac disease, especially those with acute chronic heart failure. Conversely, the use of BNP assay in pediatric patients with congenital heart disease (CHD) has only recently gained consensus. However, some limitations have delayed the introduction of BNP assay in clinical pediatric practice, including the lack of age-specific references values and the very limitated results reported in literature on complex CHD. Moreover, the first part of experimental work has turned to the determination of reference values for the BNP in pediatric subjects healthy, using the fully automated platform (Triage BNP, Access® Immunoassay Systems, REF 98200, Beckman Coulter, Fullerton, CA, USA). Plasma BNP, was measured in 188 apparently healthy newborns and infants throughout the first month of extra-uterine life, as well as in 245 healthy infants ranging from 1 month to 12 years of age. BNP showed the highest in the first 2 days of life, with a progressive decline afterwards. Moreover, BNP values in the first week of life were significantly higher (p<0.0001) than values observed in the next periods. In the second part of the thesis, the main objective was to characterize in detail the time course of BNP in the first days of life in larger populations of neonate and infant with congenital heart diseases (CHD) (n=218) in order to increase the diagnostic accuracy of BNP assay in pediatric patients; moreover, 222 healthy children, matched for age, served as controls. BNP values were significantly higher (P<0.001) in the whole group of CHD patients (median 1029,8 ng/L, range 25-20152 ng/L) than in controls (median 149,5 ng/L, range 9-866 ng/L). A different trend between BNP values and age was observed in healthy subjects and CHD patients. After an initial increase within the first 4 days of life, BNP values in CHD patients tended to stabilize to high values in the following days. On the contrary, in control subjects a peak of BNP levels was observed in the second or third day, followed by a progressive decrease. Therefore, the diagnostic accuracy of BNP assay, calculated in the samples collected in the first four days of life (AUC of ROC analysis 0,86, 95% CI 0,83-0,90) was significantly lower (P<0.0001) compared to samples collected from 5 days to 30 days of life (AUC 0,97, 95% CI 0,95-0,99). Optimal cut-off values for BNP assay, as calculated by ROC analysis, were also age-dependent (cutoff for the first 4 days of life: 363,5 ng/L; cut off values from 5 to 30 days of life: 109,5 ng/L). This study demonstrates that differences in time-courses of BNP values between newborns with and without CHD throughout the first days of life clearly affect the diagnostic accuracy of BNP assay. Indeed, the diagnostic accuracy of BNP assay in discriminating between healthy newborns and CHD patients progressively increases after the 4th day of life. As a result, also cut-off values of BNP assay greatly change throughout the first days of life. However, decision values of BNP assay are strongly method-dependent, consequently clinicians should give great care to compare results obtained by different laboratories, especially when different methods are use

Advanced Cell Culture Models Illuminate the Interplay between Mammary Tumor Cells and Activated Fibroblasts

The interaction between tumor cells and activated fibroblasts determines malignant features of desmoplastic carcinomas such as rapid growth, progression towards a metastatic phenotype, and resistance to chemotherapy. On one hand, tumor cells can activate normal fibroblasts and even reprogram them into CAFs through complex mechanisms that also involve soluble factors. Among them, transforming growth factor beta (TGF-β) and Platelet-Derived Growth Factor (PDGF) have an established role in the acquisition of pro-tumorigenic phenotypes by fibroblasts. On the other hand, activated fibroblasts release Interleukin-6 (IL-6), which increases tumor-cell invasiveness and chemoresistance. However, the interplay between breast cancer cells and fibroblasts, as well as the modes of action of TGF-β, PDGF, and IL-6, are difficult to investigate in vivo. Here, we validated the usage of advanced cell culture models as tools to study the interplay between mammary tumor cells and fibroblasts, taking mouse and human triple-negative tumor cells and fibroblasts as a case study. We employed two different settings, one permitting only paracrine signaling, the other both paracrine and cell-contact-based signaling. These co-culture systems allowed us to unmask how TGF-β, PDGF and IL-6 mediate the interplay between mammary tumor cells and fibroblasts. We found that the fibroblasts underwent activation induced by the TGF-β and the PDGF produced by the tumor cells, which increased their proliferation and IL-6 secretion. The IL-6 secreted by activated fibroblasts enhanced tumor-cell proliferation and chemoresistance. These results show that these breast cancer avatars possess an unexpected high level of complexity, which resembles that observed in vivo. As such, advanced co-cultures provide a pathologically relevant tractable system to study the role of the TME in breast cancer progression with a reductionist approach

Continuous vs intermittent Non-Invasive blood pressure MONitoring in preventing postoperative organ failure (niMON): study protocol for an open-label, multicenter randomized trial

Background: Blood pressure has become one of the most important vital signs to monitor in the perioperative setting. Recently, the Italian Society of Anesthesia Analgesia Resuscitation and Intensive Care (SIAARTI) recommended, with low level of evidence, continuous monitoring of blood pressure during the intraoperative period. Continuous monitoring allows for early detection of hypotension, which may potentially lead to a timely treatment. Whether the ability to detect more hypotension events by continuous noninvasive blood pressure (C-NiBP) monitoring can improve patient outcomes is still unclear. Here, we report the rationale, study design, and statistical analysis plan of the niMON trial, which aims to evaluate the effect of intraoperative C-NiBP compared with intermittent (I-NiBP) monitoring on postoperative myocardial and renal injury. Methods: The niMon trial is an investigator-initiated, multicenter, international, open-label, parallel-group, randomized clinical trial. Eligible patients will be randomized in a 1:1 ratio to receive C-NiBP or I-NiBP as an intraoperative monitoring strategy. The proportion of patients who develop myocardial injury in the first postoperative week is the primary outcome; the secondary outcomes are the proportions of patients who develop postoperative AKI, in-hospital mortality rate, and 30 and 90 postoperative days events. A sample size of 1265 patients will provide a power of 80% to detect a 4% absolute reduction in the rate of the primary outcome. Conclusions: The niMON data will provide evidence to guide the choice of the most appropriate intraoperative blood pressure monitoring strategy. Clinical trial registration: Clinical Trial Registration: NCT05496322, registered on the 5th of August 2023