GENERATION OF TUMOR-SPECIFIC CYTOTOXIC T-LYMPHOCYTES FROM PEROPHERAL BLOOD OF COLORECTAL CANCER PATIENTS FOR ADOPTIVE T-CELL TRANSFER

Colorectal cancer (CRC) is the third most common cancer worldwide and the fourth most common cause of death in the developed Western countries. Adoptive T-cell transfer (ACT) refers to an immunotherapeutic approach in which anti-tumor T lymphocytes, usually the tumor infiltrating lymphocytes (TIL), are identified, grown ex vivo and then re-infused into the cancer patient. ACT of EBV-specific T-cell lines and T Cytotoxic Lymphocytes (CTLs) for the therapy of EBV-induced lymphomas is the best demonstration of clinically efficacious ACT, but there are many evidences also for leukemia and multiple myeloma. As regards to the solid tumors, ACT using autologous TIL, grown ex-vivo and then re-infused into the cancer patient, has emerged as an effective treatment for metastatic melanoma and renal cell carcinoma (RCC), that are the most immunogenic tumors in humans. Randomized clinical trials are ongoing for gastric cancer, hepatocellular carcinoma and lung cancer. These approaches mainly use the TIL and the definition of tumor associated antigen (TAA), tumor specific antigen (TSA) or cancer testis antigen (CTA), that are generally correlated with tumor progression and immunogenicity in various types of cancer. However these antigens are often found to be poorly expressed in CRC, and few is known about their relationship with this type of neoplasia. In addition, although a clear association between TIL and clinical outcome of CRC has been documented, active and adoptive immunotherapy do not play yet an important role in the treatment of advanced CRC. In order to develop an ACT protocol for CRC treatment, we designed an experimental approach that does not require neither the definition of molecular defined tumor antigens, nor the availability of TIL. Our strategy was based on the in vitro stimulation of patient\u2019s CD8+-enriched T-cells from peripheral blood mononuclear cells (PBMCs) with dendritic cells (DCs), pulsed with apoptotic tumor cells as a source of tumor antigens, in order to generate autologous CTLs with strong anti-tumor activity. In this study, 78 CRC patients were enrolled. Tumor biopsies were obtained at surgery, together with 100 ml of heparinized peripheral blood (PB). Tumors were mechanically dissociated to a single-cell suspension and cultured to obtain tumor cell line from each patient. DCs were generated from previously separated PBMCs, using a magnetic positive selection of CD14+ monocytes, cultured in presence of recombinant human Interleukin-4 (rh IL-4) and recombinant human Granulocyte-Macrophage Colony-Stimulating Factor (rh GM-CSF). Anti-tumor CTLs were elicited in co/micro-culture using DCs as antigen-presenting cells, autologous apoptotic tumor cells as source of antigens and T CD8+ lymphocytes enriched effectors, with weekly stimulation. CTLs Interferon-\u3b3 (IFN-\u3b3) secretion was assessed by ELISpot assay to evaluate their activation in response to autologous tumor. Tumor cell lines were obtained from 20 out of 78 patients (25,6%), because gut intestinal flora had adversly affected the establishment of primary tumor cell line and a loss of expansion of tumor cells was observed. DCs were generated from 26 patients, but only 6 patients had the corresponding tumor cell line, indispensable for the co-culture setting up. This was the reason why co/micro-cultures were set up only for 6 patients. ELISpot assay was performed at the end of co/micro-culture stimulations to evaluate effectors IFN-\u3b3 secretion. ELISpot results showed that strong and significant IFN-\u3b3 secretion was detected at the third, fourth and fifth stimulations for one patient and at the second for another patient, whereas for three patients a weak secretion was detected during the second and third stimulations. Although our immunological study must be performed on an increased number of CRC patients, and the CTLs expansion, together with CTLs lytic ability against autologous tumor cells, must be still performed, our results suggested that the generation of tumor-specific CTLs could be useful for supporting an ACT approach in CRC

Cryotolerance of equine spermatozoa correlates with specific fatty acid pattern: A pilot study

Sperm cryopreservation represents a powerful tool for horse breeding. To improve the efficiency of artificial insemination in the horse using cryopreserved spermatozoa, an adequate understanding of the underlying biophysical properties that affect sperm cryosurvival needs to be reached yet. In this pilot study, we described isolation and analysis of the main fatty acids from sperms of stallions classified as good and poor freezers (7 GF and 5 PF, according to sperm motility and viability, before and after cryopreservation). Fatty acid profiles were only assessed in pre-thaw sperms. Eight main fatty acids were identified, using gas chromatography, and their contents were expressed as percentage of the total lipid content. We found that lauric, myristic and oleic acid (C12:0, C14:0 and C18:1n9c) turned out to be about 2-fold more abundant in the sperm cells of the GFs compared with PFs. Moreover, we described for the first time the presence of a very high amount of a trans geometrical isomer of linoleic acid, linolelaidic acid (C18:2n6t), in pre-thaw PF spermatozoa. Notably, we found in fresh sperms of PF stallions a ratio of unsaturated fatty acids to saturated fatty acids which was twice that of those of GF group, suggesting a positive effect of a high saturated-to-unsaturated fatty acid ratio for the “freezability” of equine spermatozoa. Finally, principal component analysis (PCA) confirmed the relationships between specific fatty acids and cryotolerance of equine spermatozoa, also providing a graphical classification and additional information about the dominant variables governing the classification process

Development of L-Lysine-Loaded PLGA Microparticles as a Controlled Release System for Angiogenesis Enhancement

Vascularization is a highly conserved and considerably complex and precise process that is finely driven by endogenous regulatory processes at the tissue and systemic levels. However, it can reveal itself to be slow and inadequate for tissue repair and regeneration consequent to severe lesions/damages. Several biomaterial-based strategies were developed to support and enhance vasculogenesis by supplying pro-angiogenic agents. Several approaches were adopted to develop effective drug delivery systems for the controlled release of a huge variety of compounds. In this work, a microparticulate system was chosen to be loaded with the essential amino acid L-lysine, a molecule that has recently gained interest due to its involvement in pro-angiogenic, pro-regenerative, and anti-inflammatory mechanisms. Poly (lactic-co-glycolic acid), the most widely used FDA-approved biodegradable synthetic polymer for the development of drug delivery systems, was chosen due to its versatility and ability to promote neovascularization and wound healing. This study dealt with the development and the effectiveness evaluation of a PLGA-based microparticulate system for the controlled release of L-lysine. Therefore, in order to maximize L-lysine encapsulation efficiency and tune its release kinetics, the microparticle synthesis protocol was optimized by varying some processing parameters. All developed formulations were characterized from a morphological and physicochemical point of view. The optimized formulation was further characterized via the evaluation of its preliminary biological efficacy in vitro. The cellular and molecular studies revealed that the L-lysine-loaded PLGA microparticles were non-toxic, biocompatible, and supported cell proliferation and angiogenesis well by stimulating the expression of pro-angiogenic genes such as metalloproteinase-9, focal adhesion kinases, and different growth factors. Thus, this work showed the potential of delivering L-lysine encapsulated in PLGA microparticles as a cost-effective promoter system for angiogenesis enhancement and rapid healing

Effect of oleic acid supplementation on prostaglandin production in maternal endometrial and fetal allantochorion cells isolated from late gestation ewes

Elevated circulating non-esterified fatty acids including oleic acid (OA) are associated with many pregnancy related complications. Prostaglandins (PGs) play crucial roles during parturition. We investigated the effect of OA supplementation on PG production using an in vitro model of ovine placenta

Imaging the structural style of an active normal fault through multidisciplinary geophysical investigation: a case study from the Mw 6.1, 2009 L'Aquila earthquake region (central Italy)

The normal fault-system responsible of the 2009 Mw 6.1 L'Aquila earthquake (Paganica-San Demetrio fault-system) comprises several narrow, fault-parallel valleys of controversial origin. We investigated a key section of the southeastern portion of this fault network along the small Verupola Valley. In order to characterize its nature and possible tectonic activity, we applied multiple-geosciences techniques able to image at depth the structure associated to this peculiar landform. We integrated magnetometry, 2-D P wave and resistivity tomography, surface waves and seismic noise analysis coupled with field mapping, shallow boreholes and trenching. According to our results, the Verupola Valley is a ∼30–40-m-deep graben controlled by a SW-dipping master fault and synthetic splays paired with an antithetic NE-dipping fault. The SW-dipping splays are active and cut very shallow (<2 m deep) Late Pleistocene sediments. The small amount of cumulated vertical offset (∼15 m) across the conjugated system may indicate a young fault inception or very low Quaternary slip-rates. Due to its structural continuity with the adjacent mapped strands of the Paganica–San Demetrio fault network, we relate the Verupola Valley to the recent activity of the southeastern segment of this fault system. We also suggest that other fault-parallel valleys can have the same tectonic origin and setting of the Verupola Valley. This latter represents a scale-independent analogue from metric scale (exposed in the palaeoseismological trenches) to the Middle Aterno Basin scale (seen from seismic profiles and fault mapping). Overall, the imaged structural style is coherent with the regional tectonic setting due to Quaternary crustal extension

Comparative study on Insulin-Like Growth Factor I (IGF-I) plasma concentrations in new-born horse foals, donkey foals and calves

During the postnatal period, the most important growth regulator is represented by the insulin-like growth factor-I (IGF-I). Horses, donkeys, and cattle are monotocous species with considerable gestational lengths. The birth of a live, viable new born at the end of a healthy pregnancy, and the survival of the single newborn, represent essential prerequisites for a successful reproductive success. Therefore, the full knowledge about neonatal biology of these species is mandatory. The aim of this study was to investigate the IGF-I plasma profiles in the neonates of these three species during the first 14 days of life. Six horse foals, 6 donkey foals and 6 calves, healthy and mature, were enrolled, and blood samples collected at 30 minutes, 3, 12, 24 hours, and 3, 7, 10, 14 days after birth. IGF-I plasma concentrations were analysed by RIA. Horse foals showed higher (p<0.05) IGF-I plasma concentrations at 10 and 14 days of age in comparison to the values observed at 24 hours of age, while higher (p<0.05) IGF-I plasma concentrations were detected at 14 days of age in comparison to all the previous sampling times Donkey foals showed a significantly higher (p<0.05) value at 10 days compared to the first 24 hours. Calves showed the highest value at 30 minutes, followed by a significant (p<0.05) decrease at 3 hours and a further significant (p<0.05) decrease at 7 days of age. Comparing the profiles of the three species, no differences between horse and donkey foals were observed, while calves showed significant (p<0.05) lower levels compared to donkeys at 7,10 and 14 days and to horse foals at 14 days. This study confirm the important role of IGF-I n the early postnatal life in these three species, but also underlines the need of additional species-specific studies for a better understanding of the possible different role played by IGF-I in the different species neonatal physiology

Association between Mediterranean lifestyle and perception of well-being and distress in a sample population of university Italian students

We investigated the extent to which adherence to the Mediterranean diet (MD) in combination with Mediterranean lifestyle factors influenced students’ perceptions of subjective well-being (SWB) and distress. 939 undergraduates completed a survey to assess sociodemographic and lifestyle characteristics, including adherence to the MD, depression, anxiety, stress, and SWB. Data were analysed with correlation, logistic, and multiple linear regression models. Higher adherence to MD correlated with better SWB. Fruit, red meat, sweet and caffeinated beverages contributed significantly. However, it was the combination of adherence to MD with other factors, including quality of social relationships, income, smoking, sleep, and physical activity that better predicted SWB. Our results confirm the positive influence of MD on SWB. However, they also suggest the need to consider perceptions of well-being by a more holistic approach that considers physical and social factors simultaneously to improve the development of more effective educational and motivational programmes.info:eu-repo/semantics/publishedVersio

Ochratoxin A affects oocyte maturation and subsequent embryo developmental dynamics in the juvenile sheep model

The genotoxic and nephrotoxic mycotoxin Ochratoxin A (OTA) has also been reported to have adverse effects on oocyte maturation and embryo development. Previous studies on the effects of OTA on female fertility have used micromolar concentrations, but no information is available to date on effects in a more relevant nanomolar range. This study used a juvenile sheep model to evaluate the effects of oocyte exposure to low levels of OTA on maturation, fertilization, and embryo development. Further, it was investigated whether different mechanisms of action of OTA could be responsible for varying toxic effects at different levels of exposure. Cumulus-oocyte-complexes (COCs) were exposed to 10&nbsp;μmol/L–0.1&nbsp;nmol/L OTA during in vitro maturation and evaluated for cumulus viability, oocyte maturation, and bioenergetic/oxidative status. COCs were subjected to in vitro fertilization, embryo culture, and embryo quality assessment via morphology, viability, bioenergetic/oxidative status, and time-lapse monitoring. At micromolar concentrations, OTA induced cytotoxic effects, by reducing cumulus expansion and oocyte maturation. OTA altered temporospatial dynamics of zygote pronuclear formation and embryo morphokinetics. Blastocysts, even morphologically normal, were found to undergo collapse events, which were probably related to boosted blastocyst mitochondrial activity. At nanomolar concentrations, OTA did not affect COC morpho-functional parameters, but impaired oocyte ability to prevent polyspermy and increased blastocyst apoptosis. In conclusion, in the female germ cell, cytotoxic nonspecific effects characterize OTA-induced toxicity at high exposure levels, whereas fine tuning-mode effects, not associated with altered cell viability and integrity, characterize OTA toxic action at low levels