Parameters and algorithms to evaluate cardiac mechanics by conductance catheter

This work is structured as follows: In Section 1 we discuss the clinical problem of heart failure. In particular, we present the phenomenon known as ventricular mechanical dyssynchrony: its impact on cardiac function, the therapy for its treatment and the methods for its quantification. Specifically, we describe the conductance catheter and its use for the measurement of dyssynchrony. At the end of the Section 1, we propose a new set of indexes to quantify the dyssynchrony that are studied and validated thereafter. In Section 2 we describe the studies carried out in this work: we report the experimental protocols, we present and discuss the results obtained. Finally, we report the overall conclusions drawn from this work and we try to envisage future works and possible clinical applications of our results. Ancillary studies that were carried out during this work mainly to investigate several aspects of cardiac resynchronization therapy (CRT) are mentioned in Appendix. -------- Ventricular mechanical dyssynchrony plays a regulating role already in normal physiology but is especially important in pathological conditions, such as hypertrophy, ischemia, infarction, or heart failure (Chapter 1,2.). Several prospective randomized controlled trials supported the clinical efficacy and safety of cardiac resynchronization therapy (CRT) in patients with moderate or severe heart failure and ventricular dyssynchrony. CRT resynchronizes ventricular contraction by simultaneous pacing of both left and right ventricle (biventricular pacing) (Chapter 1.). Currently, the conductance catheter method has been used extensively to assess global systolic and diastolic ventricular function and, more recently, the ability of this instrument to pick-up multiple segmental volume signals has been used to quantify mechanical ventricular dyssynchrony. Specifically, novel indexes based on volume signals acquired with the conductance catheter were introduced to quantify dyssynchrony (Chapter 3,4.). Present work was aimed to describe the characteristics of the conductancevolume signals, to investigate the performance of the indexes of ventricular dyssynchrony described in literature and to introduce and validate improved dyssynchrony indexes. Morevoer, using the conductance catheter method and the new indexes, the clinical problem of the ventricular pacing site optimization was addressed and the measurement protocol to adopt for hemodynamic tests on cardiac pacing was investigated. In accordance to the aims of the work, in addition to the classical time-domain parameters, a new set of indexes has been extracted, based on coherent averaging procedure and on spectral and cross-spectral analysis (Chapter 4.). Our analyses were carried out on patients with indications for electrophysiologic study or device implantation (Chapter 5.). For the first time, besides patients with heart failure, indexes of mechanical dyssynchrony based on conductance catheter were extracted and studied in a population of patients with preserved ventricular function, providing information on the normal range of such a kind of values. By performing a frequency domain analysis and by applying an optimized coherent averaging procedure (Chapter 6.a.), we were able to describe some characteristics of the conductance-volume signals (Chapter 6.b.). We unmasked the presence of considerable beat-to-beat variations in dyssynchrony that seemed more frequent in patients with ventricular dysfunction and to play a role in discriminating patients. These non-recurrent mechanical ventricular non-uniformities are probably the expression of the substantial beat-to-beat hemodynamic variations, often associated with heart failure and due to cardiopulmonary interaction and conduction disturbances. We investigated how the coherent averaging procedure may affect or refine the conductance based indexes; in addition, we proposed and tested a new set of indexes which quantify the non-periodic components of the volume signals. Using the new set of indexes we studied the acute effects of the CRT and the right ventricular pacing, in patients with heart failure and patients with preserved ventricular function. In the overall population we observed a correlation between the hemodynamic changes induced by the pacing and the indexes of dyssynchrony, and this may have practical implications for hemodynamic-guided device implantation. The optimal ventricular pacing site for patients with conventional indications for pacing remains controversial. The majority of them do not meet current clinical indications for CRT pacing. Thus, we carried out an analysis to compare the impact of several ventricular pacing sites on global and regional ventricular function and dyssynchrony (Chapter 6.c.). We observed that right ventricular pacing worsens cardiac function in patients with and without ventricular dysfunction unless the pacing site is optimized. CRT preserves left ventricular function in patients with normal ejection fraction and improves function in patients with poor ejection fraction despite no clinical indication for CRT. Moreover, the analysis of the results obtained using new indexes of regional dyssynchrony, suggests that pacing site may influence overall global ventricular function depending on its relative effects on regional function and synchrony. Another clinical problem that has been investigated in this work is the optimal right ventricular lead location for CRT (Chapter 6.d.). Similarly to the previous analysis, using novel parameters describing local synchrony and efficiency, we tested the hypothesis and we demonstrated that biventricular pacing with alternative right ventricular pacing sites produces acute improvement of ventricular systolic function and improves mechanical synchrony when compared to standard right ventricular pacing. Although no specific right ventricular location was shown to be superior during CRT, the right ventricular pacing site that produced the optimal acute hemodynamic response varied between patients. Acute hemodynamic effects of cardiac pacing are conventionally evaluated after stabilization episodes. The applied duration of stabilization periods in most cardiac pacing studies varied considerably. With an ad hoc protocol (Chapter 6.e.) and indexes of mechanical dyssynchrony derived by conductance catheter we demonstrated that the usage of stabilization periods during evaluation of cardiac pacing may mask early changes in systolic and diastolic intra-ventricular dyssynchrony. In fact, at the onset of ventricular pacing, the main dyssynchrony and ventricular performance changes occur within a 10s time span, initiated by the changes in ventricular mechanical dyssynchrony induced by aberrant conduction and followed by a partial or even complete recovery. It was already demonstrated in normal animals that ventricular mechanical dyssynchrony may act as a physiologic modulator of cardiac performance together with heart rate, contractile state, preload and afterload. The present observation, which shows the compensatory mechanism of mechanical dyssynchrony, suggests that ventricular dyssynchrony may be regarded as an intrinsic cardiac property, with baseline dyssynchrony at increased level in heart failure patients. To make available an independent system for cardiac output estimation, in order to confirm the results obtained with conductance volume method, we developed and validated a novel technique to apply the Modelflow method (a method that derives an aortic flow waveform from arterial pressure by simulation of a non-linear three-element aortic input impedance model, Wesseling et al. 1993) to the left ventricular pressure signal, instead of the arterial pressure used in the classical approach (Chapter 7.). The results confirmed that in patients without valve abnormalities, undergoing conductance catheter evaluations, the continuous monitoring of cardiac output using the intra-ventricular pressure signal is reliable. Thus, cardiac output can be monitored quantitatively and continuously with a simple and low-cost method. During this work, additional studies were carried out to investigate several areas of uncertainty of CRT. The results of these studies are briefly presented in Appendix: the long-term survival in patients treated with CRT in clinical practice, the effects of CRT in patients with mild symptoms of heart failure and in very old patients, the limited thoracotomy as a second choice alternative to transvenous implant for CRT delivery, the evolution and prognostic significance of diastolic filling pattern in CRT, the selection of candidates to CRT with echocardiographic criteria and the prediction of response to the therapy.

Marcatura di molecole biologiche a funzione antigenica per lo studio e la caratterizzazione di protocolli di vaccinoterapia in oncologia medica

Dendritic Cells (DCs) derived from human blood monocytes that have been nurtured in GM-CSF and IL-4, followed by maturation in a monocyte-conditioned medium, are the most potent APCs known. These DCs have many features of primary DCs, including the expression of molecules that enhance antigen capture and selective receptors that guide DCs to and from several sites in the body, where they elicit the T cell mediated immune response. For these features, immature DCs (iDC) loaded with tumor antigen and matured (mDC) with a standard cytokine cocktail, are used for therapeutic vaccination in clinical trials of different cancers. However, the efficacy of DCs in the development of immunocompetence is critically influenced by the type (whole lysate, proteins, peptides, mRNA), the amount and the time of exposure of the tumor antigens used for loading in the presentation phase. The aim of the present study was to create instruments to acquire more information about DC antigen uptake and presentation mechanisms to improve the clinical efficacy of DCbased vaccine. In particular, two different tumor antigen were studied: the monoclonal immunoglobulin (IgG or IgA) produced in Myeloma Multiple, and the whole lysate obtained from melanoma tissues. These proteins were conjugated with fluorescent probe (FITC) to evaluate the kinetic of tumor antigen capturing process and its localization into DCs, by cytofluorimetric and fluorescence microscopy analysis, respectively. iDC pulsed with 100μg of IgG-FITC/106 cells were monitored from 2 to 22 hours after loading. By the cytofluorimetric analysis it was observed that the monoclonal antibody was completely captured after 2 hours from pulsing, and was decreased into mDC in 5 hours after maturation stimulus. To monitor the lysate uptake, iDC were pulsed with 80μg of tumor lysate/106 cells, then were monitored in the 2h to 22 hours interval time after loading. Then, to reveal difference between increasing lysate concentration, iDC were loaded with 20-40-80-100-200-400μg of tumor lysate/106 cells and monitored at 2-4-8-13h from pulsing. By the cytofluorimetric analysis, it was observed that, the 20-40-80-100μg uptake, after 8 hours loading was completed reaching a plateau phase. For 200 and 400μg the mean fluorescence of cells increased until 13h from pulsing. The lysate localization into iDC was evaluated with conventional and confocal fluorescence microscopy analysis. In the 2h to 8h time interval from loading an intensive and diffuse fluorescence was observed within the cytoplasmic compartment. Moreover, after 8h, the lysate fluorescence appeared to be organized in a restricted cloudy-shaded area with a typical polarized aspect. In addition, small fluorescent spots clearly appeared with an increment in the number and fluorescence intensity. The nature of these spot-like formations and cloudy area is now being investigated detecting the colocalization of the fluorescence lysate and specific markers for lysosomes, autophagosomes, endoplasmic reticulum and MHCII positive vesicles.

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.

Rationale for an adjunctive therapy with fenofibrate in pharmacoresistant nocturnal frontal lobe epilepsy (NFLE).

Nocturnal Frontal Lobe Epilepsy (NFLE) is characterized by onset during infancy or childhood with persistence in adulthood, family history of similar nocturnal episodes simulating non-REM parasomnias (sleep terrors or sleepwalking), general absence of morphological substrates, often by normal interictal electroencephalographical recordings (EEGs) during wakefulness. A family history of epilepsy may be present with Mendelian autosomal dominant inheritance has been described in some families. Recent studies indicate the involvement of neuronal nicotinic acetylcholine receptors (nAChRs) in the molecular mechanisms of NFLE. Mutations in the genes encoding for the α4 (CHRNA4) and ß2 (CHRNB2) subunits of the nAChR induce changes in the biophysical properties of nAChR, resulting generally in a “gain of function”. Preclinical studies report that activation of a nuclear receptor called type peroxisome proliferator-activated receptor (PPAR-α) by endogenous molecules or by medications (e.g. fenofibrate) reduces the activity of the nAChR and, therefore, may decrease the frequency of seizures. Thus, we hypothesize that negative modulation of nAChRs might represent a therapeutic strategy to be explored for pharmacological treatment of this form of epilepsy, which only partially responds to conventional antiepileptic drugs. In fact, carbamazepine, the current medication for NFLE, abolishes the seizures only in one third of the patients. The aim of the project is: 1)_to verify the clinical efficacy of adjunctive therapy with fenofibrate in pharmacoresistant NFLE and ADNFLE patients; focousing on the analysis of the polysomnographic action of the PPAR- agonist (fenofibrate). 2)_to demonstrate the subtended mechanism of efficacy by means of electrophysiological and behavioral experiments in an animal model of the disease: particularly, transgenic mice carrying the mutation in the nAChR 4 subunit (Chrna4S252F) homologous to that found in the humans. Given that a PPAR-α agonist, FENOFIBRATE, already clinically utilized for lipid metabolism disorders, provides a promising therapeutic avenue in the treatment of NFLE\ADNFLE.

Clinical outcome and biological characteristics of Chronic Myeloid Leukemia patients treated with nilotinib front-line

The present work reports the outcome of the GIMEMA CML WP study CML0811, an independent trial investigating nilotinib as front-line treatment in chronic phase chronic myeloid leukemia (CML). Moreover, the results of the proteomic analysis of the CD34+ cells collected at CML diagnosis, compared to the counterpart from healthy donors, are reported. Our study confirmed that nilotinib is highly effective in the prevention of the progression to accelerated/blast phase, a condition that today is still associated with high mortality rates. Despite the relatively short follow-up, cardiovascular issues, particularly atherosclerotic adverse events (AE), have emerged, and the frequency of these AEs may counterbalance the anti-leukemic efficacy. The deep molecular response rates in our study compare favorably to those obtained with imatinib, in historic cohorts, and confirm the findings of the Company-sponsored ENESTnd study. Considering the increasing rates of deep MR over time we observed, a significant proportion of patients will be candidate to treatment discontinuation in the next years, with higher probability of remaining disease-free in the long term. The presence of the additional and complex changes we found at the proteomic level in CML CD34+ cells should be taken into account for the investigation on novel targeted therapies, aimed at the eradication of the disease.