Brain Derived Neurotrophic Factor (BDNF) e Sviluppo delle Alterazioni Neuropsichiatriche in Corso di Insufficienza Renale Cronica.

Negli ultimi venti anni sono stai fatti notevoli progressi nel trattamento dell’insufficienza renale cronica sia predialitica che terminale, tuttavia il tasso di mortalità resta significativamente elevato rispetto alla popolazione generale e la qualità della vita rimane insoddisfacente per l’insorgenza di alterazioni psico-fisiche di varia natura. In particolare dal punto di vista neurologico sono state descritte alterazioni sia a carico del sistema nervoso periferico (neuropatia uremica) sia del sistema nervoso centrale (encefalopatia uremica). Nell’encefalopatia uremica le prime manifestazioni consistono in alterazioni della personalità, irritabilità, sindrome ansiosa, difficoltà all’attenzione, e perdita della memoria. Sono inoltre presenti turbe del sonno, con sonnolenza durante il giorno e insonnia notturna, fino allo sviluppo di una vera e propria Depressione maggiore, soprattutto nei pazienti che vanno incontro a trattamento sostitutivo. Recentemente lo studio DOPPS (Dialysis Outcomes and Practice Patterns Study) ha evidenziato come la Depressione di per sé rappresenti un fattore predittivo indipendente di mortalità e ospedalizzazione nei pazienti dializzati. Proprio sulla base di questa osservazione abbiamo deciso di investigare i meccanismi fisiopatologici che portano allo sviluppo di questa patologia nell’insufficienza renale cronica. Negli ultimi anni la depressione è stata sempre più considerata come una patologia neurodegenerativa, caratterizzata da atrofia e morte cellulare a carico delle cellule della glia e dei neuroni nella corteccia e nell’ippocampo. Questa teoria si basa sul fatto che in soggetti depressi si osserva, oltre alla ben nota riduzione dei neurotrasmettitori noradrenalina e serotonina, una diminuzione dei livelli di neurotrofine, in particolare del Brain Derived Neurotrophic Factor (BDNF); questa famiglia di molecole svolge un ruolo importante nel trofismo cellulare, in quanto promuove meccanismi di differenziazione, sopravvivenza cellulare, plasticità sinaptica e neurogenesi. A conferma del ruolo cruciale di questi mediatori, è stato dimostrato che la terapia con antidepressivi determina un aumento dei livelli di neurotrofine associato ad un recupero morfo-anatomico oltre che sintomatologico. Per quanto riguarda l’insufficienza renale cronica, non esistono in letteratura dati riguardanti le eventuali modificazioni di tali fattori. Abbiamo pertanto deciso di disegnare un progetto di ricerca atto ad indagare il comportamento dei fattori neurotrofici in questa particolare condizione. Materiali e metodi La fase sperimentale è stata condotta su 30 ratti Wistar femmine, che sono stati randommizzati in tre gruppi: ratti controllo (C), ratti nefrectomizzati (Nx) e ratti nefrectomizzati trattati con Fluoxetina (Nx-F). I ratti nefrectomizzati sono stati sottoposti a procedura chirurgica di nefrectomia 5/6 con asportazione del rene dx e successiva resezione dei poli superiore e inferiore del rene sx (corrispondente a circa 2/3 del tessuto). Tutti gli animali sono stati sacrificati alla dodicesima settimana. Gli animali del gruppo Nx-F hanno ricevuto il farmaco alla dose di 15 mg/kg per un periodo di trattamento di due settimane, durante le quali sono stati registrati i pesi corporei e sono state raccolte le urine. Al momento del sacrificio sono stati prelevati anche campioni di siero, liquor, corteccia prefrontale, ippocampo e tessuto renale. I reni sono stati inclusi in paraffina per l’allestimento di preparati istologici con PAS. I campioni di corteccia ed ippocampo sono stati omogenati per valutare i livelli di BDNF centrale. Il BDNF è stato dosato anche nei campioni di liquor, siero, plasma e urine. Per la valutazione del BDNF è stato utilizzata la metodica immunoenzimatica ELISA impiegando appositi kit (BDNF Emax Immunoassay-Promega, USA ) seguendo i protocolli elaborati dal costruttore.. Risultati I livelli di BDNF nel liquor di Nx (73,4±24,3 pg BDNF/ml) risultano significativamente ridotti rispetto al gruppo C (128,7±35,3 pg BDNF/ml), mentre nel gruppo trattato con Fluoxetina risultano aumentati (Nx-F: 95,6±23,1 pg BDNF/ml)in modo significativo rispetto al gruppo non trattato, ma non rispetto al controllo. Nella corteccia prefrontale i livelli di BDNF sono significativamente ridotti nel gruppo Nx rispetto al gruppo C (Nx: 28,7±7,3 pg BDNF/mg prot vs C: 66,2±10,2 pg BDNF/mg prot; p<0.05) e mostrano un parziale recupero nel gruppo Nx-F (48,3±8,2 pg BDNF/mg prot). A livello ippocampale, i livelli di BDNF presentano differenze significative tra i tre gruppi, con un aumento nel gruppo Nx (Nx: 238±54,3 pg BDNF/mg prot; C: 90,1±29,4 pg BDNF/mg prot; Nx-F: 112,5±55,1 pg BDNF/mg prot). L’espressione tissutale di ERK 1/2 totale rispecchia l’andamento dei livelli tissutali di BDNF, sia a livello di cortex sia di ippocampo, così come quello della forma fosforilata (forma attiva). Per quanto riguarda i livelli periferici di BDNF nei ratti del gruppo Nx, nelle urine si ha una riduzione significativa (29,2±12,5 pg/mg creat. vs 90,3±99,3 pg/mg creat. nel controllo) ed analogamente nel plasma (228,7±90,7 pg/ml vs 429,7±137,4 pg/ml nel controllo), mentre nel gruppo Nx-F si assiste ad un parziale recupero (54,6±25,8 pg/mg creat e 364,6±112,4 pg/ml, rispettivamente). Conclusioni Il nostro studio dimostra che nell’IRC si ha una riduzione dei livelli plasmatici di BDNF totale che si accompagna ad una riduzione della sua escrezione urinaria. Tale risultato sembra confermare che questa neurotrofina sia filtrata a livello glomerulare: un calo nei livelli circolanti si accompagna ad una riduzione nei livelli urinari negli animali con IRC. Questo fenomeno può accadere se si ha una ridotta liberazione di BDNF nel torrente circolatorio: se così non fosse dovremmo assistere ad suo incremento plasmatico, come avviene per la creatinina. Questi risultati rispecchiano tuttavia parzialmente la situazione evidenziata a livello del sistema nervoso centrale: mentre a livello del liquor e della corteccia si registra una diminuzione di BDNF, a livello ippocampale si assiste esattamente all’opposto: nei ratti Nx i livelli di neurotrofina sono significativamente più elevati. Tale risultato è confermato dall’espressione tissutale di ERK 1/ 2 totale ed attivato, indicatori di attivazione della via di traduzione del segnale attivata dal BDNF. Questa ultima osservazione potrebbe essere spiegata dal fatto che mentre a livello corticale si ha un danno ormai instaurato, a livello ippocampale potrebbe rappresentare un tentativo di riparazione del danno causato dall’IRC. Infine la somministrazione di fluoxetina ai dosaggi comunemente impiegati per la terapia delle sindromi depressive, dimostra una efficacia parziale nel ripristinare i livelli di BDNF, inducendo ad ipotizzare l’esistenza di meccanismi di danno a livello del SNC specifici dell’IRC

Biomarkers of Chronic Inflammatory State in Uremia and Cardiovascular Disease

Cardiovascular disease is the leading cause of death in the general population; traditional risk factors seem inadequate to explain completely the remarkable prevalence of cardiovascular mortality and morbidity observed in the uremic population. A role for chronic inflammation has been well established in the development of atherosclerotic disease, and, on the basis of these observations, atherosclerosis might be considered an inflammatory disease. Inflammation has been implicated in the etiology of coronary artery disease in the general population, and traditional inflammatory biomarkers such as C-reactive protein (CRP) and interleukin-6 (IL-6) have been shown to predict cardiovascular events in both symptomatic and asymptomatic individuals as well as those in the uremic population. Later on, new nontraditional markers were related to the risk of cardiovascular morbidity and mortality in general and in uremic population. As a consequence of the expanding research base and availability of assays, the number of inflammatory marker tests ordered by clinicians for cardiovascular disease (CVD) risk prediction has grown rapidly and several commercial assays have become available. So, up to now we can consider that several new nontraditional markers as CD40-CD40 ligand system and pentraxin-3 seem to be significant features of cardiovascular disease in general and in ESRD population

Treatment with 1,25-dihydroxyvitamin D3 preserves glomerular slit diaphragm-associated protein expression in experimental glomerulonephritis

In this study, we investigated the effect of 1,25(OH)2D3on proteinuria and on the alteration of slit diaphragm-associated proteins induced by anti-Thy 1.1 in Wistar rats. Four groups of animals were studied: group I, anti-Thy 1.1 treated rats; group II, anti-Thy1.1 treated group that at day 2, after the onset of overt proteinuria, started the treatment with 1,25(OH)2D3; group III, normal control rats injected with vehicle alone; group IV, rats that received only 1,25(OH)2D3. At day 2, in group I and II, before the administration of 1,25(OH)2D3, protein excretion was significantly increased when compared to controls. Overt proteinuria was maintained until day 14 in group I whereas in group II protein excretion was significantly reduced from day 3 to day 14. Moreover, treatment with 1,25(OH)2D3abrogated podocytes injury, detected as desmin expression and loss of nephrin and zonula occludens-1 (ZO-1), two slit diaphragm-associated proteins, and glomerular polyanion staining, that were observed in group I. In conclusion, these results suggest that 1,25(OH)2D3administrated with a therapeutic regiment may revert proteinuria, counteracting glomerular podocyte injury

Improved overall survival in dendritic cell vaccination-induced immunoreactive subgroup of advanced melanoma patients

BACKGROUND: We present our experience of therapeutic vaccination using dendritic cells (DC) pulsed with autologous tumor antigens in patients with advanced melanoma. METHODS: Twenty-one pretreated advanced melanoma patients were vaccinated with autologous DC pulsed with 100 μg/ml of autologous-tumor-lysate (ATL) or – homogenate (ATH) and 50 μg/ml of keyhole limpet hemocyanin (KLH). The first 8 patients were treated subcutaneously or intradermally with immature-DC (iDC) (range 4.5 – 82 × 10(6)) and the remaining 13 intradermally with in vitro matured DC (mDC) (range 1.2–26 × 10(6)). Subcutaneous interleukin-2 (3 × 10(6 )IU) was administered from days 3 to 7 of each treatment cycle. RESULTS: Three of the 8 iDC patients obtained stabilizations (SD), each of 6 months' duration. The 13 mDC patients showed 1 complete response (8 months), 1 partial response (3 months), 2 mixed responses (6 and 12 months) and 3 SD (9, 7+, and 3+ months). Overall responses (OR) were observed in 4/21 (19%) patients, or 4/13 (30.7%) considering mDC treatment only. 10/21 (47.6%) patients showed non progressive disease (NPD), with 7/13 (53.8%) cases of NPD for mDC-treated patients. No major toxicities were observed. The positive delayed-type hypersensitivity (DTH) test to ATL/ATH and/or KLH correlated with increased overall survival (OS). Median OS was 24 months (range 3 – 45) for the 10 DTH-positive (1 iDC and 9 mDC) and 5 months (range 3–14) for the 11 DTH-negative patients (P < 0.001). The in vitro evaluation of gamma IFN-secreting T-cells in 10 patients showed good correlation with both DTH (75%) and clinical outcome (70%). CONCLUSION: Vaccination using DC pulsed with ATL/ATH and KLH in advanced melanoma patients is well tolerated and can induce a clinical response, especially when mDC are used. Successful immunization, verified by positive DTH, leads to longer survival

The link of biocompatibility to cytokine production

The link of biocompatibility to cytokine production. Recent studies suggest that chronic inflammation plays a role in the pathogenesis of cardiovascular disease. Cytokines released from jeopardized tissues stimulate the liver to synthesize acute phase proteins, including C-reactive protein (CRP). Baseline levels of CRP in apparently healthy persons or in persons with unstable angina constitute an independent risk factor for cardiovascular events. More recently, it has been suggested that CRP is useful not only as a marker of the acute phase response, but is also involved in the pathogenesis of the disease. CRP may, in fact, directly interact with the atherosclerotic vessels or ischemic myocardium by activation of the complement system, thereby promoting inflammation and thrombosis. Several studies in uremic patients have implicated CRP as a marker of malnutrition, resistance to erythropoietin, and chronic stimulation in hemodialysis. An increased cytokine production secondary to blood interaction with bioincompatible dialysis components has been reported by several studies; interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), and mainly IL-6 are the three proinflammatory cytokines involved in the pathogenesis of hemodialysis-related disease. We have provided evidence for the occurrence of high CRP and IL-6 levels in chronic dialytic patients exposed to contaminate dialysate and suggest that backfiltration may induce a chronic, slowly developing inflammatory state that may be abrogated by avoiding backfiltration of contaminate dialysate. Therefore, CRP is implicated as a marker linking bioincompatibility associated with backfiltration and increased cytokine production with a clinical state of chronic inflammation

Extracellular Vesicles Derived from Endothelial Progenitor Cells Protect Human Glomerular Endothelial Cells and Podocytes from Complement- and Cytokine-Mediated Injury

Glomerulonephritis are renal inflammatory processes characterized by increased permeability of the Glomerular Filtration Barrier (GFB) with consequent hematuria and proteinuria. Glomerular endothelial cells (GEC) and podocytes are part of the GFB and contribute to the maintenance of its structural and functional integrity through the release of paracrine mediators. Activation of the complement cascade and pro-inflammatory cytokines (CK) such as Tumor Necrosis Factor α (TNF-α) and Interleukin-6 (IL-6) can alter GFB function, causing acute glomerular injury and progression toward chronic kidney disease. Endothelial Progenitor Cells (EPC) are bone-marrow-derived hematopoietic stem cells circulating in peripheral blood and able to induce angiogenesis and to repair injured endothelium by releasing paracrine mediators including Extracellular Vesicles (EVs), microparticles involved in intercellular communication by transferring proteins, lipids, and genetic material (mRNA, microRNA, lncRNA) to target cells. We have previously demonstrated that EPC-derived EVs activate an angiogenic program in quiescent endothelial cells and renoprotection in different experimental models. The aim of the present study was to evaluate in vitro the protective effect of EPC-derived EVs on GECs and podocytes cultured in detrimental conditions with CKs (TNF-α/IL-6) and the complement protein C5a. EVs were internalized in both GECs and podocytes mainly through a L-selectin-based mechanism. In GECs, EVs enhanced the formation of capillary-like structures and cell migration by modulating gene expression and inducing the release of growth factors such as VEGF-A and HGF. In the presence of CKs, and C5a, EPC-derived EVs protected GECs from apoptosis by decreasing oxidative stress and prevented leukocyte adhesion by inhibiting the expression of adhesion molecules (ICAM-1, VCAM-1, E-selectin). On podocytes, EVs inhibited apoptosis and prevented nephrin shedding induced by CKs and C5a. In a co-culture model of GECs/podocytes that mimicked GFB, EPC-derived EVs protected cell function and permeselectivity from inflammatory-mediated damage. Moreover, RNase pre-treatment of EVs abrogated their protective effects, suggesting the crucial role of RNA transfer from EVs to damaged glomerular cells. In conclusion, EPC-derived EVs preserved GFB integrity from complement- and cytokine-induced damage, suggesting their potential role as therapeutic agents for drug-resistant glomerulonephritis

Evaluation of in vivo labelled dendritic cell migration in cancer patients

BACKGROUND: Dendritic Cell (DC) vaccination is a very promising therapeutic strategy in cancer patients. The immunizing ability of DC is critically influenced by their migration activity to lymphatic tissues, where they have the task of priming naïve T-cells. In the present study in vivo DC migration was investigated within the context of a clinical trial of antitumor vaccination. In particular, we compared the migration activity of mature Dendritic Cells (mDC) with that of immature Dendritic Cells (iDC) and also assessed intradermal versus subcutaneous administration. METHODS: DC were labelled with (99m)Tc-HMPAO or (111)In-Oxine, and the presence of labelled DC in regional lymph nodes was evaluated at pre-set times up to a maximum of 72 h after inoculation. Determinations were carried out in 8 patients (7 melanoma and 1 renal cell carcinoma). RESULTS: It was verified that intradermal administration resulted in about a threefold higher migration to lymph nodes than subcutaneous administration, while mDC showed, on average, a six-to eightfold higher migration than iDC. The first DC were detected in lymph nodes 20–60 min after inoculation and the maximum concentration was reached after 48–72 h. CONCLUSIONS: These data obtained in vivo provide preliminary basic information on DC with respect to their antitumor immunization activity. Further research is needed to optimize the therapeutic potential of vaccination with DC

Citrate anion improves chronic dialysis efficacy, reduces systemic inflammation and prevents Chemerin-mediated microvascular injury

Systemic inflammation and uremic toxins (UT) determine the increased cardiovascular mortality observed in chronic hemodialysis (HD) patients. Among UT, the adipokine Chemerin induces vascular dysfunction by targeting both endothelial and vascular smooth muscular cells (EC and VSMC). As Citrate anion modulates oxidative metabolism, systemic inflammation and vascular function, we evaluated whether citrate-buffered dialysis improves HD efficiency, inflammatory parameters and chemerin-mediated microvascular injury. 45 patients were treated in sequence with acetate, citrate and, again, acetate-buffered dialysis solution (3 months per interval). At study admission and after each treatment switch, we evaluated dialysis efficacy and circulating levels of chemerin and different inflammatory biomarkers. In vitro, we stimulated EC and VSMC with patients' plasma and we investigated the role of chemerin as UT. Citrate dialysis increased HD efficacy and reduced plasma levels of CRP, fibrinogen, IL6 and chemerin. In vitro, patients' plasma induced EC and VSMC dysfunction. These effects were reduced by citrate-buffered solutions and paralleled by the decrease of chemerin levels. Consistently, chemerin receptor knockdown reduced EC and VSMC dysfunction. In conclusion, Switching from acetate to citrate improved dialysis efficacy and inflammatory parameters; in vitro, chemerin-induced EC and VSMC injury were decreased by using citrate as dialysis buffer

Unexpected High Response Rate to Traditional Therapy after Dendritic Cell-Based Vaccine in Advanced Melanoma: Update of Clinical Outcome and Subgroup Analysis

We reviewed the clinical results of a dendritic cell-based phase II clinical vaccine trial in stage IV melanoma and analyzed a patient subgroup treated with standard therapies after stopping vaccination. From 2003 to 2009, 24 metastatic melanoma patients were treated with mature dendritic cells pulsed with autologous tumor lysate and keyhole limpet hemocyanin and low-dose interleukin-2. Overall response (OR) to vaccination was 37.5% with a clinical benefit of 54.1%. All 14 responders showed delayed type hypersensitivity positivity. Median overall survival (OS) was 15 months (95% CI, 8–33). Eleven patients underwent other treatments (3 surgery, 2 biotherapy, 2 radiotherapy, 2 chemotherapy, and 4 biochemotherapy) after stopping vaccination. Of these, 2 patients had a complete response and 5 a partial response, with an OR of 63.6%. Median OS was 34 months (range 16–61). Our results suggest that therapeutic DC vaccination could favor clinical response in patients after more than one line of therapy