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Studio di fase i del satraplatino, composto orale del platino, in combinazione sequenziale con capecitabina nel trattamento di pazienti affetti da tumore solido avanzato.
No full textObiettivo Definire la dose massima tollerata (MTD) della combinazione di satraplatino, composto orale del platino (S) e capecitabina (C) in pazienti affetti da tumore solido avanzato. Obiettivo secondario è la valutazione della risposta tumorale.
Metodi Sono stati esplorati quattro livelli di dose: S 60 mg/m2/giorno and C 1650 mg/m2/giorno, S80 and C 1650, S60 and C 2000, S70 and C 2000. Un ciclo corrisponde a 28 giorni di somministrazione sequenziale di S (giorni 1-5) seguito da C (giorni 8-21) e da una settimana di pausa di una settimana.
Risultati Trentasette pazienti sono stati trattati, 24 nella fase di aumento di dose e 13 nella fase di espansione; alla MTD definita a S 70 mg/m2/giorno and C 2000 mg/m2/giorno due pazienti hanno presentato le tossicità limitanti la dose (DLTs): mancato recupero dalla neutropenia entro il 42°giorno e nausea con mancata assunzione della dose di C. Altre tossicità frequenti sono state: nausea (57%), diarrea (51%), neutropenia (46%), anoressia, fatica, vomito (38% ciascuno).
In 2 pazienti affetti da tumore ovarico platino sensibile e un paziente affetto da tumore prostatico sono state osservate riposte parziali confermate.
Conclusione Alla dose di S 70 mg/m2/giorno and C 2000 mg/m2/giorno la combinazione sequenziale di S e C è ben tollerata con tossicità accettabile; è auspicabile l’uso della combinazione in caso di pazienti affetti da tumore noto per la sensibilità al derivati del platino e fluoro uracile soprattutto per la mancanza di nefrotossicità e neurotossicità e la facilità di somministrazione senza necessità di ricovero
Valutazione del danno da ischemia e riperfusione epatica in corso di laparoscopia mediante microdialisi intra-parenchimale: studio sperimentale e clinico.
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Molecular cloning and expression analysis of genes involved in the compensatory growth of sea bass (Dicentrarchus labrax).
No full textIn natural aquatic systems, many fish species experience periods of fasting during their life cycle because of the high variation in food availability. Although fish are highly tolerant to relatively long periods of food deprivation, energy storage and growth rate is reduced in those that experience different degrees of starvation, redirecting this energy towards maintenance metabolism. Fasting-associated growth retardation is completely overcome, or at least reduced, if an abundant food supply becomes available after a prolonged period of food shortage. Then, fish display an exceptionally rapid growth rate known as “compensatory growth”.
There are several descriptions of compensatory growth in fishes, but the mechanisms involved in such rapid recovery from fasting are still not fully understood. Such mechanisms have principally been searched for at the level of total fish growth where a number of circulating hormones are thought to be involved. Some attention has also been paid to muscle growth and to locally produced paracrine/autocrine factors, which can profoundly affect tissue growth and development. Only little information is available at the molecular level.
Accordingly, this research focused on identifying candidate genes whose expression contributes to the compensatory growth induced by refeeding in sea bass (Dicentrarchus labrax), a marine fish of great interest for Mediterranean aquaculture. In the course of the study, we firstly isolated the complete cDNAs encoding diverse physiologically relevant proteins involved in lipid and protein metabolism, and then assessed the impact of chronic feed deprivation and subsequent refeeding on their mRNA abundance levels in different tissues, with the aim to relate these expression levels to fish feeding status.
The molecular cloning and sequencing strategy resulted in the isolation of cDNAs that encode the following proteins:
- Δ6 desaturase, which has a recognized capacity to desaturate the fatty acids with 18 carbon atoms, into highly unsaturated fatty acids (HUFA) with 20 and 22 carbon atoms.
- Lipin, an enzyme required for triacylglycerol and phospholipid biosynthesis, and a transcriptional coactivator in regulating lipid metabolism genes.
- Peroxisome proliferator-activated receptor (PPARγ), a central factor in the control of the expression of genes involved in lipid homeostasis.
- Oligopeptide transporter (PepT1), an integral plasma membrane protein responsible for the uptake of dietary di- and tri-peptides from the intestinal lumen into the enterocytes.
The isolated sequences were then deposited in GenBank databases with the following accession numbers: EU647692 for Δ6 desaturase; EU644089 for lipin; FJ237043 for PepT1. The evolutionary relationship of all target genes with respect to other publicly available related genes in other teleosts, amphibian, avian, and mammalian species, was also studied.
After the sequence isolation, for the quantification by real-time RT-PCR of each gene transcript levels, in response to the nutritional status of the animals, a fasting and refeeding trial was conducted at the marine water recirculating system at our department in Varese. Briefly, 140 sea bass were stocked into four tanks of 2 m3
each, with 35 fish per tank. At the start of the experiment, all the fish were weighed, and two of the tanks were randomly assigned to each of two treatments.
Fish in these two tanks were fed to apparent satiety (control), whereas fish in the other two tanks were deprived of food for 35 days and then refed to apparent
satiety for 21 days with the same type of feed utilized before fasting. Five fish from each of the experimental groups were sampled at the following time points: before fasting (day 0), 4 days after fasting, at the end of fasting, and then sequentially at 4, 14, and 21 days following refeeding. For the molecular biology analysis, the whole digestive tract, liver, gill, heart, kidney, ovary, brain, muscle and spleen were dissected out.
After 35 days of fasting body weight of fasted fish were lower than the fed controls, whereas during the subsequent refeeding period, previously unfed fish were able to increase body weight sufficiently to overcome weight loss imposed by the 35 days feed restriction. This is the evidence that the compensatory growth was in play.
The results of the real-time RT-PCR absolute quantification using the mRNA standard curve method, revealed that the nutritional status significantly influenced E6 desaturase, lipin and PPARγ mRNA copy number in the liver of sea bass, inducing an up-regulation during prolonged fasting (35 days) and a downregulation during the recovery from fasting (21 days of refeeding).
In the proximal intestine, 35 days of fasting contributed to a significant decrease in E6 desaturase transcript levels, whereas the subsequent recovery from fasting was associated with an increase in E6 desaturase mRNA copy number, which return to normal (control) levels after 21 days of refeeding.
The abundance of lipin and PPARγ mRNA levels in the proximal intestine were not affected by the availability of food, as they remained similar between fed, fasted and refed fish.
The nutritional status significantly influenced PepT1 mRNA copy number in the proximal intestine of sea bass, inducing a down-regulation during 35 days of fasting and an up-regulation during the subsequent 21 days of refeeding. This pattern of expression seems to be in complete support of the compensatory growth. The increase in the transcripts of the intestinal oligopeptide transporter Pept1 might be correlated to the great quantity of protein taken in with the food in the initial days of refeeding. In fact, refeeding of sea bass after a long starvation period was marked by hyperphagia, as early as the first day.
In conclusion, we have isolated the cDNA sequences encoding Δ6 desaturase, lipin, PPARγ and Pept1in sea bass, and also demonstrated that the nutritional state of the animal influences their levels of expression. The present study is the first one to investigate the behavior of these transcripts over a long period of fasting and subsequent refeeding of fish. We recognize that mRNA levels in our study do not measure physiological effects produced by the proteins. Due to this, our hypothesis that the aforementioned genes are important triggers of the fasting response in the sea bass, will have to be confirmed by future studies
Sindrome delle apnee ostruttive, sindrome metabolica e rimodellamento cardiaco nell’obesita’ addominale: studio trasversale e longitudinale durante terapia con cpap.
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New anti-tumor vaccine and immunotherapeutic strategies based on optimal stimulation of CD4+ T Helper cells.
No full textVaccination with a poorly or not immunogenic tumor fails to protect the host from a subsequent challenge with the same tumor. The mechanisms underlying the failure of these tumors to sensitize therapeutic T cells are not clearly understood, but the inability of host T cells to recognize tumor has been implicated. Previous works from our laboratory have shown that the highly aggressive BALB/c mammary adenocarcinoma TS/A, can be rejected if genetically modified to express MHC class II genes upon transfection of the MHC class II transactivator CIITA. MHC class II molecules are required for presenting antigenic peptides to T helper cell and thus activate the cascade of events leading to immune effector functions such as antibody production and cytolytic T cell activity. The purpose of my thesis was to investigate whether the above approach could be generalized and thus extended to tumors of distinct histological origin. Moreover, it was assessed whether anti-tumor lymphocytes generated by this approach could be used as an immunotherapeutic tool for established cancers. Beside the previously described TS/A-CIITA breast carcinoma cells, stable CIITA-transfectants of colon adenocarcinoma C51, renal adenocarcinoma RENCA, and sarcoma WEHI-164, were generated. Tumor cells transfectants were injected in vivo and their grow kinetics and recipient’s immune response were analyzed. Tumor rejection and/or retardation of growth was found for CIITA-transfected C51 and RENCA adenocarcinomas, as well as WEHI-164 sarcoma. As for TS/A, this tumor rejection correlated mostly with the stability and the amount of CIITA-induced MHC class II expression. Interestingly, mice rejecting CIITA-transfected tumors acquired specific immunological memory, as demonstrated by resistance to challenge with untransfected parental tumors. Adoptive cell transfer experiments demonstrated that tumor immunity correlates with CD4+ and CD8+ T lymphocytes. In this thesis, it is also shown that CIITA-induced MHC class II expression on tumor cells is able to generate antitumor therapeutic T cells. Indeed, T cells from TS/A-vaccinated mice were used in an adoptive cell immunotherapy (ACT) model of established tumors. The results showed the cure at early stages and a significantly prolonged survival at later stages of tumor progression. Importantly, CD4+ T cells were clearly superior to CD8+ T cells in anti-tumor protective function. Interestingly, the protective phenotype was associated to both a Th1 and Th2 polarization of the immune effectors. Of great importance, the T cells obtained from vaccinated mice were therapeutic also without other in vitro activation passages, like treatment with anti-CD3 and IL-2, differently to some other approaches of vaccination and ACT. Furthermore, ACT induced proliferation of tumor-specific immune splenocytes in receiving mice, leading to rejection of subsequent tumor challenge or to protection from metastasis in animals having established tumors. These data support our original idea that CIITA-expressing tumor cells can act as antigen-presenting cells (APCs) for their own tumor-associated antigens to CD4+ T cells, to induce specific and potent anti-tumor responses. These results establish the general application of our tumor vaccine model. This approach let us to envisage additional applications of this strategy for producing better lymphocyte effectors for adoptive anti-tumor cell immunotherapy, particularly in those cases of tumors non-responsive to existing therapies
Electrophysiological characterization and drug interaction in glycine and GABA transporters.
No full textNeurotransmitter transporters are involved in various neurological disorders and are believed to be involved in the etiology of some mood disorders. Furthermore many of these molecules represent the target of therapeutical agents used in neurological pathologies or of drugs of abuse. In contrast to their medical and social relevance, the study of neurotransmitter transporters is not as advanced as the study of channels and other molecules involved in the synaptic transmission. Due to the coupling of the uptake of the substrate with transmembrane movement of the driver ion(s), whose electrochemical gradient represents the energy source of the process, most cotransporters generate electrical currents during their activity. For this reason it is possible to express the transporter RNA in heterologous systems and investigate their electric properties with electrophysiological techniques.
The electrophysiological and biophysical properties of glycine transporters GlyT1b and GlyT2a expressed in Xenopus oocytes were investigated with two electrode voltage clamp.
Integration of the transients isolated by subtraction of the traces in presence from those in absence of saturating glycine confirmed the intramembrane nature of the charge movement underlying the process. The sigmoidal Q/V relationships is much steeper for GlyT2a compared to GlyT1b, in agreement with different Na: Cl: glycine stoichiometry of the two transporters. The transient currents are best fitted by the sum of two exponentials, with the slow time constant (τslow) being larger for GlyT2a and in the order of tens of milliseconds. The apparent affinity for glycine is in the micromolar range and voltage-dependent, decreasing at positive potentials for both transporters. A simple, three-state model, is sufficient to explain the main features of GlyT1b operation, including the voltage-dependent affinity, while for GlyT2a a more complex scheme is required.
The effects of the tricyclic antidepressant imipramine on the activity of the GABA cotransporter GAT-1 have been studied with electrophysiological and confocal microscopy experiments on Xenopus oocytes transfected with GAT-1 cRNA. Imipramine at concentrations 10 – 1000 μM progressively inhibits the transient (presteady-state) currents in absence of GABA, as well as the currents associated to the neurotransmitter translocation. In contrast, the GAT-1-related uncoupled current, best seen in presence of Li+, appears to be unaffected by the drug. Redistribution studies using GAT1-GFP constructs on transfected HEK293 cells indicate no evident internalization of the transporter upon imipramine exposure. The drug also appears to inhibit a K+ M-like current endogenously present in the oocytes.
The effect of TCA on GAT-1 mutant K448E were investigated also. Lysine 448 of GAT-1 corresponds to aspartate 401 of LeuT, a residue found to be involved in the TCA binding. Desipramine is the antidepressant that shows the most powerful inhibition of transport and presteady state currents on the mutant compared to the wild type transporter. The result confirms the ability of TCAs to act on multiple targets, and the involvement of specific aminoacidic residues in substrate and inhibitor binding
Synthesis, characterisation and catalytic activity of metal complex of pthalocyanine with benz (b-f) azepine frame.
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Role of MeCP2 (Meyhyl CpG-Binding Protein) during replication, epigenetic inheritance and chromocenter organization of pericentric heterochromatin.
No full text“Epigenetic” is a term used to describe mitotically and meiotically heritable states of gene expression that are not due to changes in DNA sequence.
Epigenetic events are important in all aspects of biology such as cell proliferation, development and differentiation. The maintenance of a correct epigenome is fundamental in the proper progression of silencing effect, pivotal during cellular differentiation. Multi-protein complexes required to enable the heterochromatic stable epigenetic inheritance are associated to the structural organization of heterochromatin that is determined at the time of replication, in mid-late S phase.
MeCP2 is a Methyl CpG-binding protein that preferentially binds methylated DNA, localizes with pericentric heterochromatin and it has a key role to mediate large-scale chromatin organization and compaction.
While the impact of loss and mutations of MeCP2 has been extensively studied as cause of RTT and other neurodevelopmental and autismspectrum diseases little is known about its function in pericentric heterochromatin replication, structure and epigenetic inheritance. To test whether MeCP2 has a role in these processes, I used proliferating cells to study the effect of MeCP2 functional ablation during cell-cycle S-phase. I found that MeCP2 is not involved in heterochromatin replication, chromocenter organization and epigenetic modifications (H3K9me3, H4K20me3 and DNA methylation). Interestingly, MeCP2 influences cell cycle progression without triggering a strong apoptotic effect. Intriguingly, low levels of LaminB, LBR and LaminA/C proteins were found, suggesting that MeCP2 could be involved in nuclear lamina organization and/or in the expression of these genes. Besides, MeCP2 silencing determines low levels of EZH2 (component of PRC2, one of the two Polycomb-Repressive Complexes). In the light of this finding is intriguing to investigate the link between MeCP2 and EZH2 due the fact that a large number of neuronal differentiation genes which are required for neuronal development cells are bound by Polycomb complexes, whereas MeCP2 is a transcriptional regulator implicated in development of the brain that is required to interpret the DNA methylation signal in neurons.
In parallel, using different biochemical tools (TAP, gel filtration, mass spectrometry) I investigated the ability of MeCP2 to bind proteins involved in heterochromatin organization. A new interactor Np95, component of pHDBs (pericentric heterochromatin duplication bodies), was found.
My results show that MeCP2 in addition to play a role in maturation of neurons and synaptic plasticity it is implicated in cell proliferation. Besides, MeCP2 might be involved in nuclear envelope stability and/or in the expression of lamins, and in Polycomb proteins pathway. On the basis of this work further experiments should be done to better investigate the new functions of MeCP2
