Valutazione di marker diagnostici, epidemiologia e nuove prospettive terapeutiche nell' avvelenamento da rodenticidi anticoagulanti nel cane.

In Medicina Veterinaria l'avvelenamento da rodenticidi anticoagulanti è conosciuto e studiato ormai da anni, essendo una delle intossicazioni più comunemente riscontrate nelle specie non target. In letteratura si rinvengono numerose pubblicazioni ma alcuni aspetti sono rimasti ancora inesplorati.Questo studio si propone di valutare il processo infiammatorio, mediante le proteine di fase acuta (APPs), in corso di fenomeni emorragici, prendendo come modello reale un gruppo di soggetti accidentalmente avvelenati da rodenticidi anticoagulanti. I 102 soggetti avvelenati presentano un valore più elevato di proteina C reattiva (CRP)con una mediana di 4.77 mg/dl statisticamente significativo rispetto alla mediana delle due popolazioni di controllo di pari entità numerica create con cross match di sesso, razza ed età; rispettivamente 0.02 mg/dl dei soggetti sani e 0.37 mg/dl dei soggetti malati di altre patologie. Inoltre all'interno del gruppo dei soggetti avvelenati un valore di CRP elevato all'ammissione può predisporre al decesso. La proteina C reattiva assume quindi un ruolo diagnostico e prognostico in questo avvelenamento. Un'altra finalità, di non inferiore importanza, è quella di definire una linea guida terapeutica con l'ausilio di biomarker coagulativi e di valutare la sicurezza della vitamina K per via endovenosa: in 73 cani, non in terapia con vitamina k, intossicati da rodenticidi anticoagulanti, i tempi della coagulazione (PT ed aPTT) ritornano nel range di normalità dopo 4 ore dalla prima somministrazione di 5 mg/kg di vitamina k per via endovenosa e nessun soggetto durante e dopo il trattamento ha manifestato reazioni anafilattiche, nessuno dei pazienti ha necessitato trasfusione ematica e tutti sono sopravvissuti. Infine si è valutata l'epidemiologia dell'ingestione dei prodotti rodenticidi nella specie oggetto di studio e la determinazione dei principi attivi mediante cromatografia liquida abbinata a spettrofotometria di massa (UPLC-MS/MS).

Genetic and environmental factors associated with the risk of cognitive decline and dementia

AD is the most common age related neurodegenerative disease in the industrialized world. Clinically AD is defined as a progressing decline of cognitive functions. Neuropathologically, AD is characterized by the aggregation of b-amyloid (Ab) peptide in the form of extracellular senile plaques, and hyperphosphorlylated tau protein in the form of intracellular neurofibrillary tangles. These neuropathological hallmarks are often accompanied by abundant microvascular damage and pronounced inflammation of the affected brain regions. In this thesis we investigated several aspects of AD focusing on the genetic aspect. We confirmed that Alpha 1 antichymotrypsin (ACT), an acute phase protein, was associated to AD subjects, being plasma levels higher in AD cases than controls. In addition, in a GWA study we demonstrated that two different gene, Clusterin and CR1 were strongly associated to AD. A single gene association not explain such a complex disease like AD. The goal should be to created a network of genetic, phenotypic and clinical data associated to AD. We used a new algorithm, the ANNs, aimed to map variables and search for connectivity among variables. We found specific variables associated to AD like cholesterol levels, the presence of variation in HMGCR enzyme and the age. Other factors such as the BMI, the amount of HDL and blood folate levels were also associated with AD. Pathogen infections, above all viral infections, have been previously associated to AD. The hypothesis suggests that virus and in particular herpes virus could enter the brain when an individual becomes older, perhaps because of a decline in the immune system. Our new hypothesis is that the presence of SNPs in our GWA gene study results in a genetic signature that might affect individual brain susceptibility to infection by herpes virus family during aging.

Clinicopathologic findings in thoroughbred horses during a high speed – short term competition

The aim of this study was to examine whether a real high speed-short term competition influences clinicopathological data focusing on muscle enzymes, iron profile and Acute Phase Proteins. 30 Thoroughbred racing horses (15 geldings and 15 females) aged between 4-12 years (mean 7 years), were used for the study. All the animals performed a high speed-short term competition for a total distance of 154 m in about 12 seconds, repeated 8 times, within approximately one hour (Niballo Horse Race). Blood samples were obtained 24 hours before and within 30 minutes after the end of the races. On all samples were performed a complete blood count (CBC), biochemical and haemostatic profiles. The post-race concentrations for the single parameter were corrected using an estimation of the plasma volume contraction according to the individual Alb concentration. Data were analysed with descriptive statistics and the percentage of variation from the baseline values were recorded. Pre- and post-race results were compared with non-parametric statistics (Mann Whitney U test). A difference was considered significant at p<0.05. A significant plasma volume contraction after the race was detected (Hct, Alb; p<0.01). Other relevant findings were increased concentrations of muscular enzymes (CK, LDH; p<0.01), Crt (p<0.01), significant increased uric acid (p<0.01), a significant decrease of haptoglobin (p<0.01) associated to an increase of ferritin concentrations (p<0.01), significant decrease of fibrinogen (p<0.05) accompanied by a non-significant increase of D-Dimers concentrations (p=0.08). This competition produced relevant abnormalities on clinical pathology in galloping horses. This study confirms a significant muscular damage, oxidative stress, intravascular haemolysis and subclinical hemostatic alterations. Further studies are needed to better understand the pathogenesis, the medical relevance and the impact on performance of these alterations in equine sport medicine.

Fattori prognostici in corso di malattia renale cronica (ckd) nel cane

Negli ultimi anni si è assistito ad un miglioramento della qualità di vita dei piccoli animali che, oltre ad aumentarne l'aspettativa di vita, ha determinato un aumento della frequenza di patologie associate all'età medio-avanzata, quali le patologie renali croniche. Il presente studio si fonda sulla necessità, sempre più sentita nella pratica clinica veterinaria, di poter fornire al proprietario del paziente affetto da CKD, una serie di parametri che, oltre a fungere da target terapeutico, possano aiutare a comprenderne la prognosi. Lo studio ha valutato una popolazione di cani affetti da CKD e ne ha seguito o ricostruito il follow-up, per tutto il periodo di sopravvivenza fino al momento dell’exitus. Di tali soggetti sono stati raccolti dati relativi ad anamnesi, esame clinico, misurazione della pressione arteriosa, diagnostica per immagini, esami ematochimici, analisi delle urine ed eventuale esame istologico renale. È stato possibile individuare alcuni importanti fattori prognostici per la sopravvivenza in pazienti con CKD. Oltre a fattori ben noti in letteratura, come ad esempio elevati valori di creatinina e fosforo, o la presenza di proteinuria, è stato possibile anche evidenziare il ruolo prognostico negativo di alcuni parametri meno noti, ed in particolare delle proteine di fase acuta positive e negative, e del rapporto albumina/globuline. Una possibile spiegazione del valore prognostico di tali parametri risiede nel ruolo prognostico negativo dell’infiammazione nel paziente con CKD: tale ruolo è stato suggerito e dimostrato nell’uomo e avrebbe alla base numerosi possibili meccanismi (sviluppo di anemia, complicazioni gastroenteriche, neoplasie, etc.), ma dati analoghi sono mancanti in medicina veterinaria. Una seconda possibile spiegazione risiede nel fatto che potenzialmente i livelli delle proteine di fase acuta possono essere influenzati dalla presenza di proteinuria nel paziente con CKD e di conseguenza potrebbero essere una conferma di come la proteinuria influenzi negativamente l'outcome.

L'associazione Tipifarnib-Bortezomib nel trattamento delle leucemie acute mieloidi: risultati di uno studio multicentrico di fase I/II e validazione di un profilo genico predittivo di risposta

Background. Outcome of elderly acute myeloid leukemia (AML) patients is dismal. Targeted-therapies might improve current results by overcoming drug-resistance and reducing toxicity. Aim. We conduced a phase II study aiming to assess efficacy and toxicity of Tipifarnib (Zarnestra®) and Bortezomib (Velcade®) association in AML patients >18 years, unfit for conventional therapy, or >60 years, in relapse. Furthermore, we aimed to evaluated the predictive value of the RASGRP1/APTX ratio, which was previously found to be associated to treatment sensitivity in patients receiving Zarnestra alone. Methods. Velcade (1.0 mg/m2) was administered as weekly infusion for 3 weeks (days 1, 8, 15). Zarnestra was administered at dose of 300-600 mg BID for 21 consecutive days. Real-time quantitative-PCR (q-PCR) was used for RASGRP1/APTX quantification. Results. 50 patients were enrolled. Median age was 71 years (56-89). 3 patients achieved complete remission (CR) and 1 partial response (PR). 2 patients obtained an hematological improvement (HI), and 3 died during marrow aplasia. 10 had progressive disease (PD) and the remaining showed stable disease (SD). RASGRP1/APTX was evaluated before treatment initiation on bone marrow (BM) and/or peripheral blood (PB). The median RASGRP/APTX value on BM was higher in responder (R) patients than in non responders (NR) ones, respectively (p=0.006). Interestingly, no marrow responses were recorded in patients with BM RASGRP1/APTX ratio <12, while the response rate was 50% in patients with ratio >12. Toxicity was overall mild, the most common being febrile neutropenia. Conclusion. We conclude that the clinical efficacy of the combination Zarnestra-Velcade was similar to what reported for Zarnestra alone. However we could confirm that the RASGPR1/APTX level is an effective predictor of response. Though higher RASGRP1/APTX is relatively rare (~10% of cases), Zarnestra (±Velcade) may represent an important option in a subset of high risk/frail AML patients.

adaptive capabilities of the pi3k/akt/mtor pathway in acute myeloid leukemia revealed by the use of selective inhibitors

Because of its aberrant activation, the PI3K/AKT/mTOR signaling pathway represents a pharmacological target in blast cells from patients with acute myelogenous leukemia (AML). Using Reverse Phase Protein Microarrays (RPMA), we have analyzed 20 phosphorylated epitopes of the PI3K/Akt/mTor signal pathway of peripheral blood and bone marrow specimens of 84 patients with newly diagnosed AML. Fresh blast cells were grown for 2 h, 4 h or 20 h untreated or treated with a panel of phase I or phase II Akt allosteric inhibitors, either alone or in combination with the mTOR kinase inhibitor Torin1 or the broad RTK inhibitor Sunitinib. By unsupervised hierarchical clustering a strong phosphorylation/activity of most of the sampled members of the PI3K/Akt/mTOR pathway was observed in 70% of samples from AML patients. Remarkably, however, we observed that inhibition of Akt phosphorylation, as well as of its substrates, was transient, and recovered or even increased far above basal level after 20 h in 60% samples. We demonstrated that inhibition of Akt induces FOXO-dependent insulin receptor expression and IRS-1 activation, attenuating the effect of drug treatment by reactivation of PI3K/Akt. Consistent with this model we found that combined inhibition of Akt and RTKs is much more effective than either alone, revealing the adaptive capabilities of signaling networks in blast cells and highliting the limations of these drugs if used as monotherapy.

Pre-clinical and clinical development of leukemia stem cell inhibitors in acute leukemias

In Leukemias, recent developments have demonstrated that the Hedgehog pathway plays a key-role in the peculiar ability of self renewal of leukemia stem cells. The aim of this research activity was to investigate, through a first in man, Phase I, open label, clinical trial, the role and the impact, mainly in terms of safety profile, adverse events and pharmacokinetics, of a Sonic Hedgehog inhibitor compound on a population of heavely pretreated patients affected by AML, CML, MF, or MDS, resistant or refractory to standard chemotherapy. Thirty-five patients have been enrolled. The drug was administered orally, in 28 days cycles, without rest periods. The compound showed a good safety profile. The half life was of 17-35 hours, justifying the daily administration. Significant signs of activity, in terms of reduction of bone marrow blast cell amount were seen in most of the patients enrolled. Interestingly, correlative biological studies demonstrated that, comparing the gene expression profyiling signature of separated CD34+ cells before and after one cycle of treatment, the most variably expressed genes were involved in the Hh pathway. Moreover, we observed that many genes involved in MDR (multidrug resistance)were significantly down regulated after treatment. These data might lead to future clinical trials based on combinatory approaches, including, for instance, Hh inhibitors and conventional chemotherapy.

Study of novel natural compounds as potential drugs in the treatment of acute leukemias

Introduction: Among all cancer types leukemia represents the leading cause of cancer death in man younger than 40 years. Single-target drug therapy has generally been highly ineffective in treating complex diseases such as cancer. A growing interest has been directed toward multi-target drugs able to hit multiple targets. In this context, plant products, based on their intrinsic complexity, could represent an interesting and promising approach. Aim of the research followed during my PhD was to indentify and study novel natural compounds for the treatment of acute leukemias. Two potential multi-target drugs were identified in Hemidesmus indicus and piperlongumine. Methodology/Principal Findings: A variety of cellular assays and flow cytometry were performed on different cell lines. We demonstrated that Hemidesmus modulates many components of intracellular signaling pathways involved in cell viability and proliferation and alters gene and protein expression, eventually leading to tumor cell death, mediated by a loss of mitochondrial transmembrane potential, raise of [Ca2+]i, inhibition of Mcl-1, increasing Bax/Bcl-2 ratio, and ROS formation. Moreover, we proved that the decoction causes differentiation of HL-60 and regulates angiogenesis of HUVECs in hypoxia and normoxia, by the inhibition of new vessel formation and the processes of migration/invasion. Clinically relevant observations are that its cytotoxic activity was also recorded in primary cells from acute myeloid leukemia (AML) patients. Moreover, both Hemidesmus and piperlongumine showed a selective action toward leukemic stem cell (LSC). Conclusions: Our results indicate the molecular basis of the anti-leukemic effects of Hemidesmus indicus and indentify the mitochondrial pathways, [Ca2+]i, cytodifferentiation and angiogenesis inhibition as crucial actors in its anticancer activity. The ability to selectively hit LSC showed by Hemidesmus and piperlongumine enriched the knowledge of their anti-leukemic activity. On these bases, we conclude that Hemidesmus and piperlongumine can represent a valuable strategy in the anticancer pharmacology.

Study of PI3K/Akt signaling pathway as potential molecular target for T-cell acute lymphoblastic leukemia (T-ALL) treatment: pan-inhibition of PI3K catalitic isoforms as better therapeutic approach

Class I phosphatidylinositol 3-kinases (PI3Ks) are heterodimeric lipid kinases consisting of a regulatory subunit and one of four catalytic subunits (p110α, p110β, p110γ or p110δ). p110γ/p110δ PI3Ks are highly enriched in leukocytes. In general, PI3Ks regulate a variety of cellular processes including cell proliferation, survival and metabolism, by generating the second messenger phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3). Their activity is tightly regulated by the phosphatase and tensin homolog (PTEN) lipid phosphatase. PI3Ks are widely implicated in human cancers, and in particular are upregulated in T-cell acute lymphoblastic leukemia (T-ALL), mainly due to loss of PTEN function. These observations lend compelling weight to the application of PI3K inhibitors in the therapy of T-ALL. At present different compounds which target single or multiple PI3K isoforms have entered clinical trials. In the present research, it has been analyzed the therapeutic potential of the pan-PI3K inhibitor BKM120, an orally bioavailable 2,6-dimorpholino pyrimidine derivative, which has entered clinical trials for solid tumors, on both T-ALL cell lines and patient samples. BKM120 treatment resulted in cell cycle arrest and apoptosis, being cytotoxic to a panel of T-ALL cell lines and patient T-lymphoblasts. Remarkably, BKM120 synergized with chemotherapeutic agents currently used for treating T-ALL patients. BKM120 efficacy was confirmed in in vivo studies to a subcutaneous xenotransplant model of human T-ALL. Because it is still unclear which agents among isoform-specific or pan inhibitors can achieve the greater efficacy, further analyses have been conducted to investigate the effects of PI3K inhibition, in order to elucidate the mechanisms responsible for the proliferative impairment of T-ALL. Overall, these results indicated that BKM120 may be an efficient treatment for T-ALLs that have aberrant up-regulation of the PI3K signaling pathway and strongly support clinical application of pan-class I PI3K rather than single-isoform inhibitors in T-ALL treatment.