Role of ascorbic acid in the inhibition of polyphenol oxidase and the prevention of browning in different browning-sensitive Lactuca sativa var. capitata (L.) and Eruca sativa (Mill.) stored as fresh-cut produce.

BACKGROUND: Polyphenol oxidase (PPO) and, to a minor extent, peroxidase (POD) represent the key enzymes involved in enzymatic browning, a negative process induced by cutting fresh-cut produce such as lettuce (Lactuca sativa) and rocket salad (Eruca sativa). Although ascorbic acid is frequently utilised as an anti-browning agent, its mechanism in the prevention of the browning phenomenon is not clearly understood. RESULTS: The activity of PPO and POD and their isoforms in lettuce (a high-browning and low-ascorbic acid species) and rocket salad (a low-browning and high-ascorbic species) was characterised. The kinetic parameters of PPO and in vitro ascorbic acid-PPO inhibition were also investigated. In rocket salad, PPO activity was much lower than that in lettuce and cutting induced an increase in PPO activity only in lettuce. Exogenous ascorbic acid (5 mmol L(-1)) reduced PPO activity by about 90% in lettuce. POD did not appear to be closely related to browning in lettuce. CONCLUSION: PPO is the main enzyme involved in the browning phenomenon; POD appears to play a minor role. The concentration of endogenous ascorbic acid in rocket salad was related to its low-browning sensitivity after cutting. In lettuce, the addition of ascorbic acid directly inhibited PPO activity. The results suggest that the high ascorbic acid content found in rocket salad plays an effective role in reducing PPO activity.

Requirement of Mucosa-Associated Lymphoid Tissue Lymphoma Translocation Protein 1 Protease Activity for Fcγ Receptor-Induced Arthritis, but Not Fcγ Receptor-Mediated Platelet Elimination, in Mice.

OBJECTIVE: Fcγ receptors (FcγR) play important roles in both protective and pathogenic immune responses. The assembly of the CBM signalosome encompassing caspase recruitment domain-containing protein 9, B cell CLL/lymphoma 10, and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT-1) is required for optimal FcγR-induced canonical NF-κB activation and proinflammatory cytokine release. This study was undertaken to clarify the relevance of MALT-1 protease activity in FcγR-driven events and evaluate the therapeutic potential of selective MALT-1 protease inhibitors in FcγR-mediated diseases. METHODS: Using genetic and pharmacologic disruption of MALT-1 scaffolding and enzymatic activity, we assessed the relevance of MALT-1 function in murine and human primary myeloid cells upon stimulation with immune complexes (ICs) and in murine models of autoantibody-driven arthritis and immune thrombocytopenic purpura (ITP). RESULTS: MALT-1 protease function is essential for optimal FcγR-induced production of proinflammatory cytokines by various murine and human myeloid cells stimulated with ICs. In contrast, MALT-1 protease inhibition did not affect the Syk-dependent, FcγR-mediated production of reactive oxygen species or leukotriene B4 . Notably, pharmacologic MALT-1 protease inhibition in vivo reduced joint inflammation in the murine K/BxN serum-induced arthritis model (mean area under the curve for paw swelling of 45.42% versus 100% in control mice; P = 0.0007) but did not affect platelet depletion in a passive model of ITP. CONCLUSION: Our findings indicate a specific contribution of MALT-1 protease activity to FcγR-mediated events and suggest that MALT-1 protease inhibitors have therapeutic potential in a subset of FcγR-driven inflammatory disorders.

Antioxidant defences and oxidative damage in salt-treated olive plants under contrasting sunlight irradiance.

The interactive effects of root-zone salinity and sunlight on leaf biochemistry, with special emphasis on antioxidant defences, were analysed in Olea europaea L. cv. Allora, during the summer period. Plants were grown outside under 15% (shade plants) or 100% sunlight (sun plants) and supplied with 0 or 125 mM NaCl. The following measurements were performed: (1) the contribution of ions and soluble carbohydrates to osmotic potentials; (2) the photosystem II (PSII) photochemistry and the photosynthetic pigment concentration; (3) the concentration and the tissue-specific distribution of leaf flavonoids; (4) the activity of antioxidant enzymes; and (5) the leaf oxidative damage. The concentrations of Na(+) and Cl(-) were significantly greater in sun than in shade leaves, as also observed for the concentration of the 'antioxidant' sugar-alcohol mannitol. The de-epoxidation state of violaxanthin-cycle pigments increased in response to salinity stress in sun leaves. This finding agrees with a greater maximal PSII photochemistry (F(v)/F(m)) at midday, detected in salt-treated than in control plants, growing in full sunshine. By contrast, salt-treated plants in the shade suffered from midday depression in F(v)/F(m) to a greater degree than that observed in control plants. The high concentration of violaxanthin-cycle pigments in sun leaves suggests that zeaxanthin may protect the chloroplast from photo-oxidative damage, rather than dissipating excess excitation energy via non-photochemical quenching mechanisms. Dihydroxy B-ring-substituted flavonoid glycosides accumulate greatly in the mesophyll, not only in the epidermal cells, in response to high sunlight. The activity of antioxidant enzymes varied little because of sunlight irradiance, but declined sharply in response to high salinity in shade leaves. Interestingly, control and particularly salt-treated plants in the shade underwent greater oxidative damage than their sunny counterparts. These findings, which conform to the evolution of O. europaea in sunny environments, suggest that under partial shading, the antioxidant defence system may be ineffective to counter salt-induced oxidative damage.

Effects of ozone exposure or fungal pathogen on white lupin leaves as determined by imaging of chlorophyll a fluorescence.

Chlorophyll fluorescence has been used routinely to investigate photosynthetic activity in plants subjected to both biotic and abiotic stresses. The aim of this work was to compare the perturbations in photosynthesis induced by ozone and by a pathogen. By using a conventional fluorometer a similar response pattern was observed in inoculated and O(3)-fumigated leaves. The application of chlorophyll fluorescence imaging provided further detailed information on the spatial-temporal heterogeneity of the response of white lupin leaves to fungal pathogen or to ozone fumigation. In particular, 48 h after artificial inoculation with the necrotrophic fungal pathogen Pleiochaeta setosa, the leaves showed a remarkable alteration in PSII operating efficiency (Phi(PSII)), which affected the whole surface. Afterwards, the infection site was surrounded by a ring of increased photosynthetic activity. The response of ozonated leaves was quite different. The reduction in Phi(PSII) was already evident 24h after fumigation; moreover, a distinct heterogeneity of the fluorescence yield was observed and the major veins displayed a lowered Phi(PSII).

Critical impact of the kinetics of dendritic cells activation on the in vivo induction of tumor-specific T lymphocytes.

Dendritic cells (DCs) need activation for the priming of antigen-specific immune responses. Recently activated DCs were described to prime in vitro strong T helper cell type 1 (Th(1)) responses, whereas at later time points, the same cells preferentially prime Th(2) cells [Langenkemp, A. et al., Nat. Immunol. 1: 311-316, 2000]. Because the immune response against cancer strongly depends on CTLs of Th(1)-like phenotype (Tc(1)), we verified here whether the kinetics of DCs activation also impacted on in vivo priming of tumor-specific CTLs. After pulsing with the CTL epitope TRP-2(180-188), bone-marrow-derived DCs, exposed to lipopolysaccharide (LPS) for 8 h (8hDC), elicited a more powerful Tc(1) response in C57BL/6 mice than did untreated DCs, or DCs exposed to LPS for 48 h (48hDC). Indeed, 8hDCs were the most potent protective and therapeutic vaccine against B16 melanoma. Despite a higher expression of MHC and costimulatory molecules by 48hDCs, 8hDCs and 48hDCs showed comparable allostimulatory and migration potential, and susceptibility to CTL-mediated apoptosis. However, 8hDCs exhibited a significantly higher interleukin (IL)-12 production potential. Release of IL-12 was necessary to induce potent Tc(1) cells, because DCs from IL-12p40(-/-) mice, irrespective of their maturation level, generated low CTL responses, comparable with 48hDCs and 0hDCs from wild-type animals. Our data are relevant for the design of DC-based vaccines.

Ex vivo analysis of human memory B lymphocytes specific for A and B influenza hemagglutinin by polychromatic flow-cytometry.

Understanding the impact that human memory B-cells (MBC), primed by previous infections or vaccination, exert on neutralizing antibody responses against drifted influenza hemagglutinin (HA) is key to design best protective vaccines. A major obstacle to these studies is the lack of practical tools to analyze HA-specific MBCs in human PBMCs ex vivo. We report here an efficient method to identify MBCs carrying HA-specific BCR in frozen PBMC samples. By using fluorochrome-tagged recombinant HA baits, and vaccine antigens from mismatched influenza strains to block BCR-independent binding, we developed a protocol suitable for quantitative, functional and molecular analysis of single MBCs specific for HA from up to two different influenza strains in the same tube. This approach will permit to identify the naive and MBC precursors of plasmablasts and novel MBCs appearing in the blood following infection or vaccination, thus clarifying the actual contribution of pre-existing MBCs in antibody responses against novel influenza viruses. Finally, this protocol can allow applying high throughput deep sequencing to analyze changes in the repertoire of HA⁺ B-cells in longitudinal samples from large cohorts of vaccinees and infected subjects with the ultimate goal of understanding the in vivo B-cell dynamics driving the evolution of broadly cross-protective antibody responses.

Ozone tolerance in Phaseolus vulgaris depends on more than one mechanism.

Two bean cultivars with different sensitivity to ozone, i.e. the O3-sensitive Cannellino and the O3-tolerant Top Crop, were exposed to acute O3-stress (165 nL L(-1)) with the aim of evaluating physiological and biochemical traits that may confer O3-tolerance. Stomatal conductance was smaller and the ability to dissipate excess energy, via regulated and unregulated nonphotochemical quenching mechanisms was greater in Top Crop than in Cannellino. These morphological and physiological-traits allowed the O3-tolerant cultivar to compensate for the light-induced declines in PhiPSII, to preserve photosystem II from excitation-energy, and likely to prevent the generation of ROS to a superior degree than the O3-sensitive cultivar. Furthermore, the potential capacities to reducing the superoxide anion and H2O2 were significantly greater in Top Crop than in Cannellino. These findings are consistent with the early accumulation of H2O2, the almost complete disruption of cell structure, and irreversible damages to the photosynthetic apparatus observed in the O3-sensitive cultivar.

Concomitant tumor and minor histocompatibility antigen-specific immunity initiate rejection and maintain remission from established spontaneous solid tumors.

Nonmyeloablative hematopoietic cell transplantation can cure patients with hematologic malignancies but has reported limited success against solid tumors. This is possibly because of profound peripheral tolerance mechanisms and/or suboptimal tumor recognition by effector T lymphocytes. We report that in mice developing spontaneous prostate cancer, nonmyeloablative minor histocompatibility mismatched hematopoietic stem cell transplantation, and donor lymphocyte infusion of unmanipulated lymphocytes combined with posttransplant tumor-specific vaccination circumvents tumor-specific tolerance, allowing acute tumor rejection and the establishment of protective immunosurveillance. Although donor-derived tumor-specific T cells readily differentiated into effector cells and infiltrated the tumor soon after infusion, they were alone insufficient for tumor eradication, which instead required the concomitance of minor histocompatibiltiy antigen-specific CD8(+) T-cell responses. The establishment of protective immunosurveillance was best induced by posttransplant tumor-specific vaccination. Hence, these results provide the proof of principle that tumor-specific T-cell responses have to be harnessed together with minor histocompatibility responses and sustained by posttransplant tumor-specific vaccination to improve the efficacy of allotransplantion for the cure of solid tumors.

The effect of salinity on photosynthetic activity in potassium-deficient barley species.

The interactive effects of salinity and potassium deficiency on the growth, mineral elements and photosynthetic performance were investigated in wild (Hordeum maritimum L.) and cultivated barley (Hordeum vulgare L. var. Manel). At 28 d of growth, plants were treated with 3 mM K and 0 mM NaCl (3-0); 3 mM K and 100 mM NaCl (3-100); 0 mM K and 0 mM NaCl (0-0), 0 mM K and 100 mM NaCl (0-100) for 14 d. In both species, biomass production decreased considerably when the two constraints were applied simultaneously. Salinity affected shoots more than roots, whereas for potassium deficiency, the reverse occurred. Generally, potassium uptake was more affected in wild than in cultivated barley and, independent of potassium availability, 100 mM NaCl increased Na+ content in both species, whereas K+ deprivation increased Na(+) content only in H. maritimum shoots (0-0). Potassium-use efficiency (KUE) increased in all treated plants. Potassium deficiency increased the negative effects induced by salt in the photosynthetic process of H. vulgare, and this species seemed to be unable to counteract the negative effects of salinity. H. maritimum showed limitation in CO2 photoassimilation, but this species displayed mechanisms that play a role in avoiding PSII photodamage aimed to dissipate the excess energy.

Bioactive compounds during storage of fresh-cut spinach: the role of endogenous ascorbic acid in the improvement of product quality.

Spinach is rich in bioactive constituents such as vitamin C, flavonoids and phenolic acids. In this work, the biochemical modifications occurring during one week of storage at 4 degrees C were evaluated both in intact and in fresh-cut spinach. Results showed that vitamin C concentration is less affected by storage in fresh-cut spinach with respect to intact spinach. MS/MS analysis showed that the main flavonoids are not modified during storage in intact leaves, while some of them increased significantly during storage in the fresh-cut samples. Fresh-cut spinach did not show color alteration even if PPO activity increased significantly during storage. This finding was related to the high ascorbic acid content, which delays the subsequent polymerization events. This finding was confirmed by the unaltered concentration of phenolic compounds in fresh-cut spinach during storage. In conclusion, data about nutritional content and visual performance concurrently suggest that spinach is a suitable species for marketing as a fresh-cut product.

CO2 fixation and chlorophyll a fluorescence in leaves of Ramonda serbica during a dehydration-rehydration cycle.

Changes in CO(2) photo-assimilation and PSII photochemical efficiency in Ramonda serbica leaves during a dehydration-rehydration cycle were examined. The rate of CO(2) photo-assimilation was greatly reduced during dehydration, but recovery was complete with rehydration when the relative water content of leaves reached values similar to those of well-hydrated, control leaves. The results showed that the response of R. serbica leaves to severe water stress involves two different mechanisms. In the first, CO(2) assimilation is limited by stomata closure that creates an excess proton concentration in the lumen and activates non-photochemical quenching. This plays an important role in the mechanism of photoprotection by dissipation of excitation energy. When dehydration became severe and leaf RWC reached very low values, the electron transport rate (ETR) decreased markedly, while the capacity for regulatory mechanisms such as q(NP) (non-photochemical quenching) was greatly reduced. For severely dehydrated leaves of R. serbica, it appears that reactive oxygen species (ROS) formation is better prevented by mechanisms that quench chlorophyll triplet formation via lutein.

Arabidopsis thaliana MYB75/PAP1 transcription factor induces anthocyanin production in transgenic tomato plants.

Tomato (Solanum lycopersicum L.) cv. Micro-Tom plants were transformed with the Arabidopsis thaliana (L.)Heyhn. MYB75/PAP1 (PRODUCTION OF ANTHOCYANIN PIGMENT 1) gene. This gene encodes for a well known transcription factor, which is involved in anthocyanin production and is modulated by light and sucrose. Transgenic tomato plants expressing AtMYB75 were characterised by a significantly higher anthocyanin production in leaves, stems, roots and flowers under normal growth conditions. Further, they also exhibited anthocyanins in fruits. Anthocyanin accumulation was not widespread but took place in specific groups of cells located in epidermal or cortical regions or in proximity of vascular bundles. In all the organs of the transgenic plants, where AtMYB75 overexpression was determined, a clear increase in the accumulation of DFR (DIHYDROFLAVONOL 4-REDUCTASE) transcript was also detected. The expression of the tomato MYB-gene ANT1 (ANTHOCYANIN1), which had previously been identified as a transcriptional endogenous regulator of anthocyanin biosynthesis, was not altered. The higher basal content of anthocyanins in the leaves of the transgenic plants could be further increased in the presence of high light conditions and contributed to mitigate photobleaching damages under high irradiance.

Vasculature-targeted tumor necrosis factor-alpha increases the therapeutic index of doxorubicin against prostate cancer.

BACKGROUND: Poor penetration and uneven distribution of doxorubicin in tumors limits the efficacy of this drug in patients with prostate cancer (PC). Aim of the study was to investigate whether pre-treatment with NGR-TNF, a tumor necrosis factor-alpha derivative able to target tumor vessels and alter vessel permeability, increases the penetration and the efficacy of doxorubicin in pre-clinical models of PC. METHODS: Wild type C57BL/6 mice bearing androgen-independent TRAMP-C1 PC and transgenic adenocarcinoma of the mouse prostate (TRAMP) mice, which spontaneously develop PC and metastasis, were treated with repeated cycles of doxorubicin, administered either alone or following NGR-TNF. Tumor growth and drug uptake by cancer cells was evaluated. RESULTS: Doxorubicin as a single agent blocked the growth of TRAMP-C1 cells in vitro but not in vivo. Pre-treatment of mice bearing subcutaneous TRAMP-C1 tumors with NGR-TNF favored doxorubicin penetration into the tumor mass, and in both TRAMP-C1 and TRAMP models significantly delayed tumor growth without increasing drug-related toxicity. CONCLUSIONS: Pre-treatment with NGR-TNF significantly expanded the therapeutic index of doxorubicin in mouse models of hormone-dependent and -independent PC.

Peripheral T-cell tolerance associated with prostate cancer is independent from CD4+CD25+ regulatory T cells.

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) are thought to suppress the natural and vaccine-induced immune response against tumor-associated antigens (TAA). Here, we show that Treg accumulate in tumors and tumor-draining lymph nodes of aging transgenic adenocarcinoma of the mouse prostate (TRAMP) male mice, which spontaneously develop prostate cancer. TAA overexpression and disease progression associate also with induction of TAA-specific tolerance. TAA-specific T cells were found in the lymphoid organs of tumor-bearing mice. However, they had lost the ability to release IFN-gamma and kill relevant targets. Neither in vivo depletion of Treg by PC61 monoclonal antibody followed by repeated vaccinations with antigen-pulsed dendritic cells nor the combined treatment with 1-methyl-L-tryptophan inhibitor of the enzyme indoleamine 2,3-dyoxigenase, PC61 antibody, and dendritic cell vaccination restored the TAA-specific immune response. Treg did not seem to control the early phases of tolerance induction, as well. Indeed, depletion of Treg, starting at week 6, the age at which TRAMP mice are not yet tolerant, and prolonged up to week 12, did not avoid tolerance induction. A similar accumulation of Treg was found in the lymph nodes draining the site of dendritic cell vaccination both in TRAMP and wild-type animals. Hence, we conclude that Treg accrual is a phenomenon common to the sites of an ongoing immune response, and in TRAMP mice in particular, Treg are dispensable for induction of tumor-specific tolerance.

Prolonged exposure of dendritic cells to maturation stimuli favors the induction of type-2 cytotoxic T lymphocytes.

Dendritic cell (DC) maturation influences the priming and polarization of T lymphocytes. We recently found that early activated DC (i.e. DC exposed to pro-maturation stimuli for 8 h) were more prone to prime in vivo a type-1 cytotoxic T cell (Tc1) response than DC exposed to pro-maturation stimuli for 48 h (48h-DC). We investigated whether 48h-DC, conversely, allowed the induction of Tc2 cells. Antigen-pulsed mouse bone-marrow-derived DC at any maturation stage, in the presence of exogenous IL-12, skewed in vitro naive CD8(+) T cells towards Tc1 cells, but 48h-DC most potently, in the presence of exogenous IL-4, favored the induction of Tc2 cells. In vivo, full maturation of DC promoted expansion of Tc2 and fall of Tc1 cells. Tc2 cells maintained a high cytolytic activity and produced significant amounts of IL-4, IL-5, IL-10 and TGF-beta. Our results indicate that polarization of naive CD8(+) T cells to Tc2 cells is dependent on the amount of time DC have been exposed to maturation stimuli, and might be favored in late and/or chronic phases of an immune response.

Type 2 cytotoxic T lymphocytes modulate the activity of dendritic cells toward type 2 immune responses.

Activated CD8+ T cells can differentiate into type 1 (Tc1) cells, producing mainly IFN-gamma, and type 2 (Tc2) cells, producing mostly IL-4, IL-5, and IL-10. Tc1 cells are potent CTL involved in the defense against intracellular pathogens and cancer cells. The role of Tc2 cells in the immune response is largely unknown, although their presence in chronic infections, cancer, and autoimmune diseases is associated with disease severity and progression. Here, we show that mouse Tc2 cells modify, through a cell-to-cell contact mechanism, the function of bone marrow-derived dendritic cells (DC). Indeed, Tc2-conditioned DC displayed a reduced expression of MHC class II and costimulatory molecules, produced IL-10 instead of IL-12, and favored the differentiation of both naive CD4+ and CD8+ T cells toward type 2 cells in the absence of added polarizing cytokines. The novel function for Tc2 cells suggests a type 2 loop in which Tc2 cells modify DC function and favor differentiation of naive T cells to type 2 cells. The type 2 loop may at least in part explain the unexpected high frequency of type 2 cells during a chronic exposure to the Ag.

Peripheral T cell tolerance occurs early during spontaneous prostate cancer development and can be rescued by dendritic cell immunization.

In the tumor-prone transgenic adenocarcinoma mouse prostate (TRAMP) mouse model we followed the fate of the immune response against the SV40 large T antigen (Tag) selectively expressed in the prostate epithelium during the endogenous transformation from normal cells to tumors. Young (5-7-week-old) male TRAMP mice, despite a dim and patchy expression of Tag overlapping foci of mouse prostate intraepithelial neoplasia, displayed a strong Tag-specific cytotoxic T lymphocyte (CTL) response after an intradermal injection of peptide-pulsed dendritic cells (DC). This response was weaker than the one found in vaccinated wild-type littermates, and was characterized by a reduced frequency and avidity of Tag-specific CTL. Early DC vaccination also subverted the profound state of peripheral tolerance typically found in TRAMP mice older than 9-10 weeks. The DC-induced CTL response indeed was still detectable in TRAMP mice of 16 weeks, and was associated with histology evidence of reduced disease progression. Our findings suggest that tumor antigens are handled as self antigens, and peripheral tolerance is associated with in situ antigen overexpression and cancer progression. Our data also support a relevant role for DC-based vaccines in controlling the induction of peripheral tolerance to tumor antigens.

On the role of flavonoids in the integrated mechanisms of response of Ligustrum vulgare and Phillyrea latifolia to high solar radiation.

The role of flavonoids in mechanisms of acclimation to high solar radiation was analysed in Ligustrum vulgare and Phillyrea latifolia, two Mediterranean shrubs that have the same flavonoid composition but differ strikingly in their leaf morpho-anatomical traits. In plants exposed to 12 or 100% solar radiation, measurements were made for surface morphology and leaf anatomy; optical properties, photosynthetic pigments, and photosystem II efficiency; antioxidant enzymes, lipid peroxidation and phenylalanine ammonia lyase; synthesis of hydroxycinnamates and flavonoids; and the tissue-specific distribution of flavonoid aglycones and ortho-dihydroxylated B-ring flavonoid glycosides. A denser indumentum of glandular trichomes, coupled with both a thicker cuticle and a larger amount of cuticular flavonoids, allowed P. latifolia to prevent highly damaging solar wavelengths from reaching sensitive targets to a greater degree than L. vulgare. Antioxidant enzymes in P. latifolia were also more effective in countering light-induced oxidative load than those in L. vulgare. Consistently, light-induced accumulation of flavonoids in L. vulgare, particularly ortho-dihydroxylated flavonoids in the leaf mesophyll, greatly exceeded that in P. latifolia. We conclude that the accumulation of flavonoid glycosides associated with high solar radiation-induced oxidative stress and, hence, biosynthesis of flavonoids appear to be unrelated to 'tolerance' to high solar radiation in the species examined.