Evaluating the efficacy of restoration plantings through DNA barcoding of frugivorous bird diets.

Frugivores are critical components of restoration programs because they are seed dispersers. Thus, knowledge about bird-plant trophic relationships is essential in the evaluation of the efficacy of restoration processes. Traditionally, the diet of frugivores is characterized by microscopically identifying plant residues in droppings, which is time-consuming, requires botanical knowledge, and cannot be used for fragments lacking detectable morphological characteristics (e.g., fragmented seeds and skins). We examined whether DNA barcoding can be used as a universal tool to rapidly characterize the diet of a frugivorous bird, Eurasian blackcap (Sylvia atricapilla). We used the DNA barcoding results to assess restoration efforts and monitor the diversity of potentially dispersed plants in a protected area in northern Italy. We collected 642 Eurasian Blackcap droppings at the restored site during the autumn migration over 3 years. Intact seeds and fragmented plant material were analyzed at 2 plastidial barcode loci (rbcL and trnH-psbA), and the resulting plant identifications were validated by comparison with a reference molecular data set of local flora. At least 17 plant species, including 7 of the 11 newly transplanted taxa, were found. Our results demonstrate the potential for DNA barcoding to be used to monitor the effectiveness of restoration plantings and to obtain information about fruit consumption and dispersal of invasive or unexpected plant species. Such an approach provides valuable information that could be used to study local plant biodiversity and to survey its evolution over time.

Conservation genetics of two island endemic Ribes spp. (Grossulariaceae) of Sardinia: survival or extinction?

Measuring levels of population genetic diversity is an important step for assessing the conservation status of rare or endangered plant species and implementing appropriate conservation strategies. Populations of Ribes multiflorum subsp. sandalioticum and R. sardoum, two endangered endemic species from Sardinia, representing the whole genus on the island, were investigated using ISSR and SSR markers to determine levels and structure of genetic variability in their natural populations. Results indicated medium to low genetic diversity at the population level: Nei's gene diversity for ISSR markers ranged from 0.0840 to 0.1316; the expected heterozygosity (HE ) for SSR ranged from 0.4281 to 0.7012. In addition, only one remnant population of R. sardoum showed a high level of inbreeding, in accordance with its very small size. Regarding the structure of the six R. sandalioticum populations, both principal coordinates analysis (PCoA) and STRUCTURE analysis of ISSR and SSR data highlighted low population structure, although two populations appeared to be clearly distinct from the others. The genetic pattern of the two taxa associated with their different ecological positions indicated resilience of R. sandalioticum populations in fresh and humid habitats and uncertain future resistance for the residual R. sardoum population in xeric calcareous stands. Hence, this study highlights the importance of an integrated conservation approach (genetic plus in situ and ex situ conservation studies/measures) for activating management programmes in these endemic and threatened taxa that can be considered as crop wild relatives of cultivated Ribes species.

A DNA barcoding approach to identify plant species in multiflower honey.

The purpose of this study was to test the ability of DNA barcoding to identify the plant origins of processed honey. Four multifloral honeys produced at different sites in a floristically rich area in the northern Italian Alps were examined by using the rbcL and trnH-psbA plastid regions as barcode markers. An extensive reference database of barcode sequences was generated for the local flora to determine the taxonomic composition of honey. Thirty-nine plant species were identified in the four honey samples, each of which originated from a mix of common plants belonging to Castanea, Quercus, Fagus and several herbaceous taxa. Interestingly, at least one endemic plant was found in all four honey samples, providing a clear signature for the geographic identity of these products. DNA of the toxic plant Atropa belladonna was detected in one sample, illustrating the usefulness of DNA barcoding for evaluating the safety of honey.

The vaccines consistency approach project: an EPAA initiative.

The consistency approach for release testing of established vaccines promotes the use of in vitro, analytical, non-animal based systems allowing the monitoring of quality parameters during the whole production process. By using highly sensitive non-animal methods, the consistency approach has the potential to improve the quality of testing and to foster the 3Rs (replacement, refinement and reduction of animal use) for quality control of established vaccines. This concept offers an alternative to the current quality control strategy which often requires large numbers of laboratory animals. In order to facilitate the introduction of the consistency approach for established human and veterinary vaccine quality control, the European Partnership for Alternatives to Animal Testing (EPAA) initiated a project, the "Vaccines Consistency Approach Project", aiming at developing and validating the consistency approach with stakeholders from academia, regulators, OMCLs, EDQM, European Commission and industry. This report summarises progress since the project's inception.

Wild grapevine: silvestris, hybrids or cultivars that escaped from vineyards? Molecular evidence in Sardinia.

Vitis vinifera ssp. silvestris, the spontaneous subspecies of V. vinifera L., is believed to be the ancestor of present grapevine cultivars. In this work, polymorphism at 13 SSR loci was investigated to answer the following key question: are wild plants (i) true silvestris, (ii) hybrids between wild and cultivated plants or (iii) or 'escapes' from vineyards? In particular, the objective of the present study was to identify truly wild individuals and to search for possible hybridization events. The study was performed in Sardinia, the second largest island in the Mediterranean Sea, which is characterized by a large and well-described number of both grape cultivars and wild populations. This region was ideal for the study because of its spatial isolation and, consequently, limited contamination from outside material. The results of this study show that domesticated and wild grapevine germplasms are genetically divergent and thus are real silvestris. Pure lineages (both domesticated and wild) show very high average posterior probabilities of assignment to their own clusters, with a low level of introgression.

Chloroplast microsatellites markers to assess genetic diversity in wild and cultivated grapevines of Iran.

To assess the haplotype diversity and genetic relationship between them, A set of 69 Iranian cultivated accessions, six European cultivars and an accession of Vitis labrusca along with 63 wild grapevine individuals were studied using chloroplast microsatellite markers. Results showed that among analyzed cpssr loci only ccmp 3 and ccmp10 were polymorphic within cultivars and only ccmp3 was polymorphic in wild grape individuals. The size variants of both loci combine in a total of 4 different haplotypes. All the 4 haplotype are displayed in the cultivars while only 2 are presented in wild grapes. Sultani or keshmeshi Bidane cultivar has the haplotype III that there is not this haplotype among the wild grapes of studied regions. Concerning to existence of both haplotypes I and II in the number of Iranian cultivated and wild grapes, it is possible to consider that the wild grapes are ancestor of some of our native cultivars.

Antigen-experienced T cells undergo a transient phase of unresponsiveness following optimal stimulation.

Interaction of the Ag-specific receptor of T lymphocytes with its Ag/MHC ligand can lead either to cell activation or to a state of unresponsiveness often referred to as anergy. It has been generally assumed that anergy develops as a consequence of inadequate stimulation, such as in response to altered peptide ligands or to agonists presented by costimulatory-deficient accessory cells. The present study uncovers an alternative way of inducing an unresponsive state in T cells. Indeed, we demonstrate herein that Ag-stimulation of murine CD4+ Th clones induces cellular activation, characterized by cytokine production and cell proliferation, followed by a state of transient (lasting up to 6 days) unresponsiveness to further antigenic stimulation. This state of activation-induced unresponsiveness 1) is not a consequence of inadequate costimulation, as it occurs when cells are stimulated in the presence of dendritic cells or anti-CD28 Abs; 2) develops after an optimal response to Ag; 3) is not due to cell death/apoptosis or CTLA-4 engagement; 4) down-regulates the proliferation and cytokine production of both Th1- and Th2-like clones; and 5) does not affect the early steps of signal transduction. Finally, naive T cells are not sensitive to this novel form of unresponsiveness, but become gradually susceptible to activation-induced unresponsiveness upon Ag stimulation. Collectively, these data suggest that activation-induced T cell unresponsiveness may represent a regulatory mechanism limiting the clonal expansion and effector cell function of Ag-experienced T cells, thus contributing to the homeostasis of an immune response.

Regulation of T helper cell differentiation in vivo by soluble and membrane proteins provided by antigen-presenting cells.

The aim of this study was to test whether the nature of the antigen-presenting cell (APC) can influence the Th1/Th2 balance in vivo. Our data show that dendritic cells (DC), pulsed extracorporeally with antigen, induced the development of cells secreting IL-2, IFN-gamma and IL-4 upon antigen rechallenge in vitro. Priming with peritoneal macrophages sensitized cells that produced IL-4 but not IFN-gamma. To identify the factors involved in T helper development, mice were primed with APC with or without treatment with neutralizing antibodies to costimulatory molecules or cytokines. Our results indicate that priming with DC or macrophages is strictly dependent on the CD28-CTLA4/B7 interaction. Of note, CD86 provides the initial signal to induce naive T cells to become IL-4 producers, whereas CD80 is a more neutral differentiation signal. IL-12, released by the DC, appears as a potent and obligatory inducer of differentiation for IFN-gamma-producing cells. IL-6, although produced by both APC populations, is necessary to direct activation of the Th2-type response by macrophages but not by DC.

Induction of T cell unresponsiveness by anti-CD3 antibodies occurs independently of co-stimulatory functions.

Antibodies to the T cell receptor (TcR)-associated CD3 molecules represent potent immunosuppressive agents in vivo in both human and animals models, in spite of their well-characterized mitogenic properties. We demonstrate in this report that antibodies to the B7.2 molecule inhibit IL-2 production in vivo caused by anti-CD3 administration, suggesting that anti-CD3 monoclonal antibodies (mAb) stimulate naive T cells in vivo in a co-stimulation-dependent fashion. To characterize better the mechanisms by which antibodies to CD3 induce antigen unresponsiveness in naive T cells, we developed a model of activation-induced T cell unresponsiveness in vitro. Our data indicate that following interaction with mitogenic anti-CD3 mAb in vitro, naive purified CD4+ T cells become refractory to a further stimulus. This unresponsive state develops independently of co-stimulatory functions, as neither B7-expressing antigen-presenting cells nor anti-CD28 mAb are able to prevent anergy induction in this model. We therefore conclude that induction of unresponsiveness in naive T cells by anti-CD3 mAb is not a consequence of co-stimulus-deficient stimulation, but may develop following a productive response both in vivo and in vitro. Unresponsive T cells display a defective calcium mobilization upon TcR triggering, suggesting that anergy is maintained in these cells through receptor desensitization. The potential role of co-stimulation-independent TcR desensitization in the down-regulation of immune responses in vivo is briefly discussed.