Transfer of efficient anti-melanocyte T cells from vitiligo donors to melanoma patients as a novel immunotherapeutical strategy

Background

Vitiligo is a relatively common progressive depigmentary condition that is believed to be due to the autoimmune-mediated loss of epidermal melanocytes. High frequencies of self-reactive T lymphocytes directed toward melanocyte differentiation antigens are found in vitiligo patients and might be directly responsible for the pathogenesis of the disease. An interesting aspect of vitiligo is its relation to melanoma: cytotoxic T lymphocytes directed to self antigens shared by normal melanocytes and melanoma cells are found in both conditions, but the resulting immune reactions are completely different. From this standpoint, the selective destruction of pigment cells that occurs in cases of vitiligo is the therapeutic goal sought in melanoma research.

Presentation of the hypothesis

Our working hypothesis is that vitiligo patients might represent a unique source of therapeutic cells to be used in allo-transfer for HLA-matched melanoma patients. The adoptive transfer of ex-vivo generated autologous tumor-specific T cells is a therapy that has met with only limited success, essentially because of inability to isolate therapeutically valuable T cells from the majority of tumor patients. Ideally, model systems where strong and efficient responses against the same (tumor) antigens are achieved would represent a better source of therapeutic cells. We believe it is possible to identify one such model in the melanoma-vitiligo dichotomy: T lymphocytes specific for different melanocyte differentiation antigens are found in vitiligo and represent the effective anti-melanocyte reactivity that is often ineffective in melanoma.

Testing the hypothesis

Melanocyte-specific T cell clones can be isolated from the peripheral blood of vitiligo patients and tested for their capacity to efficiently expand in vitro without loosing their cytotoxic activity and to migrate to the skin. Cytotoxicity against melanoma patients' non-tumor cells can also be tested. In addition, it would be interesting to attempt an in vivo animal model. If the results obtained from these validation steps will be satisfactory, it might be possible to plan the clinical grade preparation of relevant clones for transfer.

Implications of the hypothesis

When translated into a clinical trial, the possibility of in vitro selecting few effective tumor-specific T cell clones for infusion, inherent with this approach, could enhance the therapeutic graft-versus-tumor effect while possibly decreasing the risk of graft-versus-host disease.

     

Protein Characterization of Protein Bodies from Cotyledons of Mucuna pruriens (L.) DC

Seeds of Mucuna pruriens (L.) DC. (Fabaceae) were analyzed for protein composition of protein bodies isolated from cotyledons. Protein bodies were successfully separated by Lympholyte and those of dry seeds, observed by scanning electron microscope, were elliptical or spherical in shape with a diameter of 5–12 μm. Protein content in dry seed protein bodies was 10.6 mg/g dry weight. Globulin was the largest protein fraction isolated (62.5 %), followed by albumin (18.3 %), glutelin (15.8 %) and prolamin (3.4 %). The prolamin fraction and high glutelin content are uncommon in legumes. SDS-PAGE of albumins, globulins, prolamins and glutelins provided different band numbers and molecular weights under reducing and non reducing conditions and suggested that the albumin fraction is rich in disulphide bonds.     

Photic and non-photic entrainment on daily rhythm of locomotor activity in goats

We studied the photic (L/D cycle) and non-photic (restricted feeding) entrainment on the patterns of daily rhythm of total locomotor activity in goats. Six female Maltese goats were subjected to three different artificial L/D cycles: 12/12 L/D, 12/12 D/L and constant light. During the 12/12 L/D and 12/12 D/L, food and water were available ad libitum . During constant light, animals were subjected to a restricted feeding treatment. Total activity was recorded by means of an actigraphy-based data logger (Actiwatch-Mini^®). Our results showed that goats exhibited clear daily rhythms of activity in 12/12 L/D cycle, 12/12 D/L cycle and constant light, although they showed FAA prior the feeding time during the restricted feeding treatment. Goats were diurnal, with activity consistently beginning promptly following the onset of light. Even when the L/D cycle was delayed by 12 h on some days, to the daily rhythm was re-established. During the constant light period, the onset of activity was linked to the time of food administration. Our study evidences two factors for the rhythm of total locomotor activity in goats: light stimuli (photic) and food access (non photic), strongly coupled to permit organisms the adaptive temporal coordination of behaviour with stable and unstable environmental periodicities.     

Ω3 Supplementation and Intermittent Hypobaric Hypoxia Induce Cardioprotection Enhancing Antioxidant Mechanisms in Adult Rats

Intermittent hypobaric hypoxia (IH) is linked with oxidative stress, impairing cardiac function. However, early IH also activate cardio-protective mechanisms. Omega 3 fatty acids (Ω3) induce cardioprotection by reducing infarct size and reinforcing antioxidant defenses. The aim of this work was to determine the combined effects of IH and Ω3 on cardiac function; oxidative balance and inflammatory state. Twenty-eight rats were randomly divided into four groups: normobaric normoxia (N); N + Ω3 (0.3 g·kg⁻¹·day⁻¹); IH; and IH + Ω3. IH was induced by 4 intercalate periods of hypoxia (4 days)—normoxia (4 days) in a hypobaric chamber during 32 days. At the end of the exposure, hearts were mounted in a Langendorff system and subjected to 30 min of ischemia followed by 120 min of reperfusion. In addition, we determined HIF-1α and ATP levels, as well as oxidative stress by malondialdehyde and nitrotyrosine quantification. Further, the expression of the antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase was determined. NF-kappaB and myeloperoxidase levels were assessed in the hearts. Relative to N hearts, IH improved left ventricular function (Left ventricular developed pressure: N; 21.8 ± 3.4 vs. IH; 42.8 ± 7.1 mmHg; p < 0.05); reduced oxidative stress (Malondialdehyde: N; 14.4 ± 1.8 vs. IH; 7.3 ± 2.1 μmol/mg prot.; p < 0.05); and increased antioxidant enzymes expression. Supplementation with Ω3 induces similar responses as IH group. Our findings suggest that both, IH and Ω3 in an independent manner, induce functional improvement by antioxidant and anti-inflammatory mechanisms, establishing cardio-protection.     

Effects of water stress on emission of volatile organic compounds by <Emphasis Type="Italic">Vicia faba</Emphasis>, and consequences for attraction of the egg parasitoid <Emphasis Type="Italic">Trissolcus basalis</Emphasis>

When plants are damaged by herbivorous insects, blends of volatile organic compounds (VOCs) are induced and released and can also be used by parasitoids to locate hosts. The aim was to determine whether VOCs induced by water stress affect the plant–herbivore–parasitoid system represented by broad bean (Vicia faba; Fabales: Fabaceae) stink bug (Nezara viridula; Heteroptera: Pentatomidae) egg parasitoid (Trissolcus basalis; Hymenoptera: Platygastridae). The effects of water stress (expressed as the percentage fraction of transpirable soil water [FTSW] supplied) alone and in combination with N. viridula damage (feeding plus oviposition) were determined according to: (1) the behavioural response of the egg parasitoid in a Y-tube olfactometer and (2) the plant VOCs collected and analysed by thermal desorption–gas chromatography–mass spectrometry. With pot water capacity as FTSW₁₀₀, water stress was applied as mild (FTSW₈₀), moderate (FTSW₅₀) and severe (FTSW₁₀). Bioassays with plants under abiotic stress alone showed that egg parasitoids are more attracted by FTSW₁₀ plants than by well-watered plants. When plants were under abiotic and biotic stress interactions, the egg parasitoids are more attracted by FTSW₁₀ and FTSW₅₀ plants than by well-watered plants infested with N. viridula. Considering VOCs emissions, projection to latent structures discriminant analysis (PLS-DA) separated treatments according to egg parasitoid responses. Water stress alone and in combination with biotic stress induced changes in VOC emissions of V. faba plants that attract egg parasitoids. These findings contribute to our understanding of how water stress affects the interactions between plants, insect pests and egg parasitoids.     

Determination of bisphenol a and bisphenol B residues in canned peeled tomatoes by reversed-phase liquid chromatography

Bisphenol A (BPA) and bisphenol B (BPB) concentrations were determined in peeled canned tomatoes of different brands bought in Italian supermarkets. Tomato samples analyzed were packaged in cans coated with either epoxyphenolic lacquer or low BADGE enamel. A solid phase extraction (SPE) was performed on C-18 Strata E cartridge followed by a step on Florisil cartridge. Detection and quantitation were performed by a reversed phase high-performance liquid chromatography (RP-HPLC) method with both UV and fluorescence detection (FD). On the total of 42 tested tomato samples, BPA was detected in 22 samples (52.4%), while BPB was detected in 9 samples (21.4%). BPA and BPB were simultaneously present in 8 of the analyzed samples. The levels of BPA found in this study are much lower than the European Union migration limits of 3 mg/kg food and reasonably unable to produce a daily intake exceeding the limit of 0.05 mg/kg body weight, established by European Food Safety Authority.     

Benefits of Biochars and NPK Fertilizers for Soil Quality and Growth of Cowpea (Vigna unguiculata L. Walp.) in an Acid Arenosol

Fertilization is required for optimum plant growth, particularly in unfertile soils, while optimizing nutrient use efficiency is an alternative to reduce inorganic fertilizer needs and reduce environmental problems caused by nutrient leaching. This study investigated soil properties and cowpea yield responses to biochars (BCs) made from different feedstocks, baby corn peel biochar (BC1), branches of mango tree biochar (BC2), and rice husk biochar (BC3), applied in combination with nitrogen-phosphorus-potassium (NPK) fertilizers. The experiment was conducted in a greenhouse, using an acid sandy soil (Arenosol) that was submitted for 70 d to the following eight treatments:i) control; ii) full dose of NPK (a commercial compound fertilizer (12-24-12 of N-P₂O₅-K₂O) + urea (46% N)); iii) BC1 + half dose of NPK; iv) BC1 + full dose of NPK; v) BC2 + half dose of NPK; vi) BC2 + full dose of NPK; vii) BC3 + half dose of NPK; and viii) BC3 + full dose of NPK. All biochars were applied at a rate of 0.9% (weight/weight), and each type of biochar was combined with half and full doses of NPK fertilizers. Soil pH increased significantly (P < 0.05) in treatments with BC1 and BC2, while cation exchange capacity (CEC) and available P were higher in the treatments with BC1; BC1 and BC2 also induced higher activity of enzymes related to the P cycle and higher cowpea yield. Similar soil properties and cowpea yield parameters were obtained with the full and half doses of NPK fertilizers for each type of biochar used. In conclusion, biochars in the combination with NPK fertilizers improved soil chemistry and enzymatic activities, allowing reduced fertilizer application and food production costs in the acid soil studied.     

Rapid and Specific Detection of Virulent Pseudomonas avellanae Strains by PCR Amplification

Specific oligonucleotides, based on hrpW (hypersensitive response and pathogenicity) gene sequences encoding harpin protein in phytopathogenic bacteria, were designed to detect and identify virulent strains of Pseudomonas avellanae by polymerase chain reaction (PCR). A population of virulent P. avellanae strains, isolated in central Italy (Viterbo region), was assessed with hrpW-derived primers, producing a specific band of about 350 base pairs in length. This target was successfully amplified from purified genomic DNA, from bacterial culture and from hazelnut bark tissue. No amplification was obtained when the PCR assay was performed on other plant-pathogenic species from the following genera Agrobacterium, Erwinia, Brenneria, Pseudomonas, Ralstonia, Xanthomonas or from hazelnut-associated bacteria, indicating the specificity of these primers. Moreover DNA from strain ISPaVe-MCB-596, isolated from north Italy (Piedmont region) and belonging to the less aggressive population of P. avellanae, did not amplify in PCR. The PCR assay with the primers described here provides a rapid, specific and sensitive diagnostic method for virulent P. avellanae strains and a useful tool to evaluate the progress of sanitation of the area.     

The degradation of methyl bromide in some natural fresh waters. influence of temperature,pH and light

The influence of some chemical and physical factors (pH, temperature and light) on degradation (hydrolysis) and volatilisation of methyl bromide (MB) from natural fresh waters was investigated. The complexity of the processes involved in MB persistence was shown. Under static and air-free conditions the half-life is quite long (30-50 days) but is decreased by an increase in pH and temperature (9-10 days), by UV irradiation (10-12 days) and, most of all, by exposure to air and shaking of the solution (2 h) and by soil.