Angiogenesi e popolazioni mieloidi CD33 positive nelle complicanze agli arti inferiori nel diabete di tipo 2

Type 2 Diabetes Mellitus is a chronic weakening disease involving about 400 million people worldwide, in particular in the civilized countries. It is associated with many chronic complications, both macrovascular and microvascular: nephropathy, retinopathy, cardio-vascular disease and erectile dysfunction. In particular, one important long-term complication involved the vascular district and, together with diabetic neuropathy, leads to permanent nerve damage and augmented risk of diabetic ulcers or wounds in legs and foot. One process that controls vascular regeneration is angiogenesis, that is reported to be dysregulated in type 2 diabetes. Indeed, new vessels formation in diabetes is augmented on the retina and reduced in peripheral limbs, in particular in foot lesions. S100B is a low molecular weight protein expressed in several cell types, of both neural and vascular origin. It exerts both intracellular and extracellular effects, that are mediated by its interaction with RAGE transmembrane receptor. S100B can regulate different steps of the angiogenic process, in particular cell proliferation and migration, but these effects are not fully explained. The first chapter of the thesis aims to investigate in vitro and in vivo the role of the protein in different steps of the angiogenic process, and to verify the relevance of S100B-RAGE ligation and the involvement of ERK1/2 Kinase pathway. A good immune system is also necessary to prevent infections on diabetic foot. Blood myeloid cell population is actively involved in defensive mechanisms by the recruitment of circulating monocytes and their interaction with endothelial cells. Dendritic cells instead are essential to process and present the antigen to lymphocytes, promoting the immune response. A recurrent marker expressed on almost all myeloid cells at different degree of intensity is CD33, that is considered a marker of immunity but its role is still unclear. CD33 contains ITIM inhibitory motifs that can control the inflammatory response, preventing cytokine signalling and monocytes activation, but there are no informations about the connection with the diabetic disease. The second chapter of the thesis aims to evaluate the presence of CD33 on peripheral blood cells in type 2 diabetic patients with or without complications and to mimic the in vivo hyperglycemic environment on cultured mononuclear cell to assess the behavior of CD33 in monocytes and dendritic cells in healthy controls and diabetic patients with foot lesions.Il diabete di tipo 2 rientra tra le patologie di maggior impatto sanitario nel mondo occidentale, con costi elevati sia in termini di spesa sanitaria sia in termini di benessere e sopravvivenza del paziente. La malattia infatti è caratterizzata da complicanze a carico di diversi distretti, in particolar modo neurologico e vascolare. La compromissione a livello dei vasi può causare deficit in organi importanti quali il rene e l’occhio ed essere parte integrante dei meccanismi che causano il fenomeno del piede diabetico, insieme a neuropatia ed infezione. Tra i processi che controllano la rigenerazione vascolare e che sono disregolati nel diabete rientra l’angiogenesi. La formazione di nuovi vasi infatti è accentuata sulla retina mentre si riduce drasticamente a livello degli arti inferiori, con una marcata deplezione di precursori endoteliali in pazienti con lesioni ai piedi. S100B è una proteina a basso peso molecolare espressa in cellule di diversa origine, sia neuronale che muscolare che vascolare. La sua azione dipende dalla concentrazione di proteina e dal legame con il recettore RAGE, esercitando effetti sia di tipo intracellulare che extracellulare. È stato evidenziato un possibile ruolo di S100B nel regolare alcuni processi chiave nell’angiogenesi, ad esempio proliferazione e migrazione cellulare, ma il ruolo esatto, la concentrazione di utilizzo e i pathway coinvolti ancora non sono stati pienamente dimostrati. La prima parte di questa tesi si propone pertanto di mettere in luce il ruolo della proteina in diverse tappe del processo angiogenico quali proliferazione, migrazione e capacità di formare strutture capillaro-simili in vitro su cellule endoteliali da vena di cordone ombelicale e in vivo, verificando la necessità del legame S100B-RAGE e valutando il coinvolgimento del pathway mediato dalla chinasi ERK1/2. Una componente importante della complicanza agli arti inferiori è la presenza di un corretto funzionamento del sistema immunitario, per prevenire e debellare le eventuali infezioni in corso.La popolazione cellulare mieloide è attivamente coinvolta a livello sanguigno nel promuovere meccanismi di difesa di tipo infiammatorio, in particolar modo con l’ausilio di monociti circolanti e la loro interazione con le cellule endoteliali vascolari.5 La popolazione dendritica invece si rivela indispensabile nei processi di presentazione dell’antigene batterico o virale ai linfociti, inducendo una successiva risposta cellulo-mediata. Un marcatore di origine midollare presente sulla superficie delle cellule di linea mieloide è CD33, molecola utilizzata come marcatore di immaturità ma dal ruolo non ancora chiaro. La proteina infatti contiene dei motivi detti ITIM, con funzione inibitoria, ed è stato riportato come possa controllare la risposta infiammatoria prevenendo il signaling dato dalle citochine e l’attivazione dei monociti, ma non sono ancora disponibili sufficienti evidenze per quanto riguarda il link con la patologia diabetica. La seconda parte di tesi si propone pertanto di evidenziare la presenza di CD33 su sangue periferico di pazienti con diabete di tipo 2 non complicato o con complicanze per capire se può essere considerato un marcatore di interesse nella progressione della lesione. La ricerca si propone inoltre di valutare il comportamento di CD33 su diverse classi di popolazioni cellulari mieloidi isolate da controlli sani o da pazienti diabetici con lesioni agli arti inferiori, in particolar modo monociti classici e non classici e cellule dendritiche, mimando in vitro l’ambiente iperglicemico protratto nel diabete

Angiogenesi e popolazioni mieloidi CD33 positive nelle complicanze agli arti inferiori nel diabete di tipo 2

Type 2 Diabetes Mellitus is a chronic weakening disease involving about 400 million people worldwide, in particular in the civilized countries. It is associated with many chronic complications, both macrovascular and microvascular: nephropathy, retinopathy, cardio-vascular disease and erectile dysfunction. In particular, one important long-term complication involved the vascular district and, together with diabetic neuropathy, leads to permanent nerve damage and augmented risk of diabetic ulcers or wounds in legs and foot. One process that controls vascular regeneration is angiogenesis, that is reported to be dysregulated in type 2 diabetes. Indeed, new vessels formation in diabetes is augmented on the retina and reduced in peripheral limbs, in particular in foot lesions. S100B is a low molecular weight protein expressed in several cell types, of both neural and vascular origin. It exerts both intracellular and extracellular effects, that are mediated by its interaction with RAGE transmembrane receptor. S100B can regulate different steps of the angiogenic process, in particular cell proliferation and migration, but these effects are not fully explained. The first chapter of the thesis aims to investigate in vitro and in vivo the role of the protein in different steps of the angiogenic process, and to verify the relevance of S100B-RAGE ligation and the involvement of ERK1/2 Kinase pathway. A good immune system is also necessary to prevent infections on diabetic foot. Blood myeloid cell population is actively involved in defensive mechanisms by the recruitment of circulating monocytes and their interaction with endothelial cells. Dendritic cells instead are essential to process and present the antigen to lymphocytes, promoting the immune response. A recurrent marker expressed on almost all myeloid cells at different degree of intensity is CD33, that is considered a marker of immunity but its role is still unclear. CD33 contains ITIM inhibitory motifs that can control the inflammatory response, preventing cytokine signalling and monocytes activation, but there are no informations about the connection with the diabetic disease. The second chapter of the thesis aims to evaluate the presence of CD33 on peripheral blood cells in type 2 diabetic patients with or without complications and to mimic the in vivo hyperglycemic environment on cultured mononuclear cell to assess the behavior of CD33 in monocytes and dendritic cells in healthy controls and diabetic patients with foot lesions

Proinsulin-expressing dendritic cells in type 2 neuropathic diabetic patients with and without foot lesions

Diabetic neuropathy is the most common complication of diabetes and is frequently associated with foot ischemia and infection, but its pathogenesis is controversial. We hypothesized that proinsulin expression in peripheral blood mononuclear cells is a process relevant to this condition and could represent a link among hyperglycemia, nerve susceptibility, and diabetic foot lesions. We assessed proinsulin expression by using flow cytometry in dendritic cells from control participants and patients with type 2 diabetic with or without peripheral neuropathy or accompanied by diabetic foot. Among 32 non-neuropathic and 120 neuropathic patients with type 2 diabetic, we performed leg electromyography and found average sensory sural nerve conduction velocities of 48 \ub1 4 and 30 \ub1 4 m/s, respectively ( P < 0.03). Of those with neuropathy, 42 were without lesions, 39 had foot lesions, and 39 had neuroischemic foot lesions (allux oximetry <30 mmHg). In this well-defined diabetic population, but not in nondiabetic participants, a progressively increasing level of peripheral blood dendritic cell proinsulin expression was detected, which directly correlated with circulating TNF-\u3b1 levels ( P < 0.002) and multiple conduction velocities of leg nerves ( P < 0.05). These results are consistent with the hypothesis that, in type 2 diabetes, proinsulin-expressing blood cells, possibly via their involvement in innate immunity, may play a role in diabetic peripheral neuropathy and foot lesions.-Sambataro, M., Sambado, L., Trevisiol, E., Cacciatore, M., Furlan, A., Stefani, P. M., Seganfreddo, E., Durante, E., Conte, S., Della Bella, S., Paccagnella, A., dei Tos, A. P. Proinsulin-expressing dendritic cells in type 2 neuropathic diabetic patients with and without foot lesions

Enteral nutrition at home and in nursing homes: an 11-year (2002-2012) epidemiological analysis

Home enteral nutrition (HEN) is a well-established extra-hospital therapy that can reduce the risk of malnutrition, ensure the rapid discharge of patients from hospital and significantly reduce health care expenditure. The data reported in this study allow us to understand the relationships between mortality, the place of treatment either at patients' homes (PH) or in nursing homes (NHR) and nutritional status

Enteral nutrition at home and in nursing homes: an 11-year (2002-2012) epidemiological analysis

Home enteral nutrition (HEN) is a well-established extra-hospital therapy that can reduce the risk of malnutrition, ensure the rapid discharge of patients from hospital and significantly reduce health care expenditure. The data reported in this study allow us to understand the relationships between mortality, the place of treatment either at patients' homes (PH) or in nursing homes (NHR) and nutritional status