A New Bioassay Platform Design for the Discovery of Small Molecules with Anticancer Immunotherapeutic Activity

Immunotherapy takes advantage of the immune system to prevent, control, and eliminate neoplastic cells. The research in the field has already led to major breakthroughs to treat cancer. In this work, we describe a platform that integrates in vitro bioassays to test the immune response and direct antitumor effects for the preclinical discovery of anticancer candidates. The platform relies on the use of dendritic cells that are professional antigen-presenting cells (APC) able to activate T cells and trigger a primary adaptive immune response. The experimental procedure is based on two phenotypic assays for the selection of chemical leads by both a panel of nine tumor cell lines and growth factor-dependent immature mouse dendritic cells (D1). The positive hits are then validated by a secondary test on human monocyte-derived dendritic cells (MoDCs). The aim of this approach is the selection of potential immunotherapeutic small molecules from natural extracts or chemical libraries.     

Kiwellin, a modular protein from green and gold kiwi fruits: evidence of in vivo and in vitro processing and IgE binding

Kiwellin, an allergenic protein formerly isolated from green kiwi fruit, has been identified as the most abundant component of the gold kiwi species. A protein named KiTH, showing a 20 kDa band on reducing SDS-PAGE and 100% identity with the C-terminal region of kiwellin, has been identified in the extract of the ripe green species. In vitro treatment of purified kiwellin with the protease actinidin from green kiwi fruit originated KiTH and kissper, a recently described pore-forming peptide. Primary structure analysis and experimental evidence suggest that kiwellin is a modular protein with two domains. It may undergo in vivo proteolytic processing by actinidin, thus producing KiTH and kissper. When probed with sera recognizing kiwellin from green kiwi fruit, KiTH showed IgE binding, with reactivity levels sometimes different from those of kiwellin. The IgE-binding capacity of kiwellin from gold kiwi fruit appears to be similar to that of the green species.     

Varietal differences among the phenolic profiles and antioxidant activities of seven table grape cultivars grown in the south of Italy based on chemometrics

Seven table grape cultivars grown in Apulia region were considered: Italia, Baresana, Pizzutello, Red Globe, Michele Palieri, Crimson Seedless, and Thompson Seedless. Seeds, skins and pulps were extracted and analyzed for their phenolic profiles and antioxidant activities. The hierarchy in the phenolic contents was seeds, skins, and pulps. These results indicate that the intake of the whole berries (seeds included) must be strongly recommended. The highest phenolic contents were detected on Italia and Michele Palieri cv., respectively within the white and the red/black table grapes. Seeds gave a high contribution to the berry antioxidant activity, as they had higher phenolic content than skins and contained high quantities of proanthocyanidines, but the strongest antioxidant activity was shown by the pulp juices due to their content in hydroxycinnamyl acids. The principal component analysis applied to the phenolic composition and antioxidant activity of skins, pulps, and seeds allowed a good separation of Italia and Michele Palieri cultivars. According to the cluster analysis, cultivars were grouped into two clusters, one including Michele Palieri and the other one including Italia, Baresana, Pizzutello, and Thompson Seedless.     

Inhibition of inducible nitric oxide synthase expression by an acetonic extract from Feijoa sellowiana Berg. fruits

Feijoa sellowiana Berg. fruits and especially the acetonic extract have been shown to possess biological activities, although the responsible compounds have never been identified. The present study was designed to evaluate the anti-inflammatory activity of an acetonic extract from F. sellowiana Berg. fruits on the nitric oxide (NO) pathway, which plays an important role in inflammation. To this aim the J774 cell line, which expresses inducible nitric oxide synthase (iNOS) following stimulation with lipopolysaccharide (LPS), has been utilized, and the effects of this extract and its fractions on NO production, iNOS protein expression, and signal pathways involved in its regulation have been evaluated. This study demonstrates that at least some part of the anti-inflammatory activity of the acetonic extract is due to the suppression of NO production by flavone and stearic acid. The mechanism of this inhibition seems to be related to an action on the expression of the enzyme iNOS through the attenuation of nuclear factor kappaB (NF-kappaB) and/or mitogen-activated protein kinase (MAPK) activation.     

Development of real-time PCR systems based on SYBR? Green I and TaqMan? technologies for specific quantitative detection of Phoma tracheiphila in infected Citrus

Real-time PCR assays based on SYBR? Green I and TaqMan? technologies were developed for in planta detection and quantification of Phoma tracheiphila, the mitosporic fungus causing ‘mal secco’ disease on citrus. Primers and a hybridization probe were designed on the basis of the internal transcribed spacer (ITS) region of the nuclear rRNA genes. The real-time PCR assays were compared with a classic isolation method in two separate experiments carried out on 6 and 24 month-old sour orange seedlings, artificially inoculated with a conidial suspension of the pathogen. Both technologies made it possible to follow the progression of infection by P. tracheiphila, enabling detection and quantification of the target fungus prior to the development of symptoms. The detection limit was 10 copies of the cloned target sequence and 15 pg of genomic DNA extracted from fungal spores. The values of the cycle threshold (Ct) were linearly correlated with the concentration of the target DNA, indicating that the method is suitable as a qualitative and quantitative assay. The presence of non-target fungal DNA had no effect on the specificity of the assay, but resulted in a 10-fold reduction of sensitivity. Total inhibition of the reaction occurred when conidia of the target pathogen were mixed with an organic soil substrate before extracting DNA using the standard protocol, while an alternative purification kit resulted in a significant decrease in sensitivity. Compared to classic methods, real-time PCR proved faster and easier to perform and showed a higher sensitivity. These results suggest that real-time PCR, based on both chemistries, has a great potential for early diagnosis of ‘mal secco’ disease and for quantitative estimation of fungal growth within host tissue.     

Litter-fall and litter decomposition in a low Mediterranean shrubland

Annual production of litter by Cistus incanus (L.) and Myrtus communis (L.) and decomposition dynamics of leaf litter of these species was studied in a Mediterranean shrubland. Myrtus and Cistus produced 472 and 429 g dry weight litter m^-2 year^-1, respectively. Leaves were the predominant litter component for both species. The average decay constant of Myrtus and Cistus litters enclosed in litter bags, calculated over the whole study period (38 months), was 0.71 year^-1 and 0.31 year^-1 respectively. In green leaves the N content differed during growth seasons for both species, whereas the content of Ca, Mg, P, K, and Na did not show significant changes. Abscised leaves had lower N, P and K contents than green leaves, evidencing that a nutrient translocation before abscission occurred from senescent leaves. The nutrient contents of the leaves at abscission time, generally higher in Cistus than in Myrtus, allowed us to estimate the annual nutrient input to the soil. Phosphorus and K more than N were rapidly released by the decomposing litters after exposure. Nutrient limitation, in particular P, might be considered the main growth limiting factor for Myrtus and Cistus. Both species were adapted to recovery and rapidly recycle P more than N and K in the living biomass through retranslocation from green leaves before abscission and/or a high release rate from the decomposing litter. The former strategy was better used by Cistus, the latter by Myrtus.     

Evaluation of intravenous T-61 as a euthanasia method for birds

The use of T-61 as a sole euthanasia agent for birds was investigated. Nine broiler chickens (Gallus gallus domesticus) were euthanized by intravenous T-61 and assessed for insensibility [brainstem reflexes: nictitating membrane reflex (NIC), palpebral blink reflex (PAL)], brain death [isoelectric electroencephalogram activity (EEG)], cessation of audible heartbeat, and abnormal electrocardiogram. Birds were considered dead when the heart rate was less than 180 beats/minute with an isoelectric EEG. No vocalization or wing flapping occurred. Both NIC and PAL were lost 10.5 s from start of injection and audible heartbeat ceased at 24.5 s. Latency to isoelectric activity was 16.6 s. All but 1 bird died within 60 s. Rapid induction of insensibility meant birds did not experience pain and distress within 10.5 s from start of injection and birds were not conscious during cardiac and circulatory arrest. Intravenous injection of T-61 is an effective and efficient euthanasia method for birds.     

Initial cooling time before freezing affects post‐thaw quality and reproductive performance of rabbit semen

The aim of this study was to investigate the effect of initial cooling time at 5°C during semen cryopreservation on post‐thaw quality and reproductive performance of rabbit semen. Pooled semen samples (n = 6) were divided into two subsamples and cooled at 5°C for 45 or 90 min. After cooling, the semen samples were diluted to a ratio of 1:1 (v:v) with a freezing extender composed of Tris‐citrate‐glucose (TCG) containing 16% of dimethylsulfoxide and 0.1 mol/L sucrose. The semen was subsequently loaded in 0.25 ml straws, equilibrated at 5°C and frozen in liquid nitrogen vapor. After thawing, sperm motility, viability, osmotic resistance, acrosome and DNA integrity were assessed. Our results indicate that the longer cooling time, that is, 90 min before cryopreservation significantly improves sperm post‐thaw viability, motility and fertility. In fact, reproductive performances obtained with semen frozen after a 90 min cooling time were similar to those produced by fresh semen insemination. Hence, the present research provides an effective freezing protocol for rabbit semen that will allow for the creation of a sperm cryobank for the conservation of Italian rabbit genetic resources, as well as the use of frozen semen doses in commercial farms.     

Response of an agricultural soil to pentachlorophenol (PCP) contamination and the addition of compost or dissolved organic matter

The response of a fresh, agricultural soil when contaminated with pentachlorophenol (PCP) and supplemented with compost (C) or dissolved organic matter (DOM) was studied in the laboratory. The concentration of PCP and the changes in various functionally related properties (i.e. microbial biomass, basal respiration, soil hydrolase and oxidoreductase activity) were measured over 150 d. Variations in the main physical and chemical properties of the soils were also monitored. Two different doses of compost (C1 = 0.27% and C2 = 0.83%, corresponding to 10 and 30 t ha⁻¹, respectively) or DOM (D1 = 0.07% and D2 = 0.2%) equivalent to the carbon content of the two compost doses C1 and C2 were used and the following five systems were investigated: soil (S), soil–compost (S-C1 and S-C2) and soil–DOM (S-D1 and S-D2). PCP concentrations declined progressively and significantly with time. This effect was most pronounced for the soils amended with the lower compost dose C1 (S-C1) and with the two DOM (S-D1 and S-D2) amounts. Significantly reduced amounts of PCP were extracted after its 500-d residence in the various systems. Higher amounts of the residual PCP were extracted from the humic acids (HA), fulvic acids (FA) and humin–mineral (HU) fractions of the 500 d aged samples than from the same unfractionated samples, indicating that the residual PCP preferentially accumulated in the organic fractions of soil. The soil showed an endogenous microbial activity as indicated by basal respiration, microbial biomass and all the enzymatic activities tested (dehydrogenase, glucosidase, phosphatase, arylsulphatase and urease). Addition of the PCP severely depressed some of the tested biochemical properties suggesting an inhibitory effect on microbial activity. Conversely, higher basal respiration, and similar β-glucosidase and phosphatase activities were measured in comparison with the controls. No significant effects were observed following the addition of two doses of the compost or the DOM. Fungal colonies belonging to the taxonomic group of Ascomycetes and identified as Byssochlamys fulva developed with time in all the PCP-contaminated samples. Growth of B. fulva in vitro in the presence of PCP showed that the isolate was tolerant to 12.5 and 25 mg l⁻¹ PCP and degraded 20% of its initial concentration in 8 d. Overall, the results indicate that many complex processes occurred in the contaminated soil and combinations of these determined the response to PCP contamination. The sorption of PCP to the soil matrix (which increased with time) and its degradation/transformation by indigenous soil microbial activity were likely involved. Both the processes appeared to be favoured by the presence of dissolved organic matter.