Detection of Listeria monocytogenes in Italian-style soft cheeses

AIMS: A rapid detection system specific for Listeria monocytogenes and based on the polymerase chain reaction (PCR) was developed. METHODS AND RESULTS: Primers annealing to the coding region of the actA gene, critically involved in virulence and capable of discrimination between two different alleles naturally occurring in L. monocytogenes, have been utilized. The procedure was applied to recover L. monocytogenes cells in artificially contaminated fresh Italian soft cheeses (mozzarella, crescenza and ricotta). Low levels of L. monocytogenes were detected in mozzarella and crescenza homogenates (0.04-0.4 and 4 CFU g(-1), respectively) whereas in ricotta the detection limit was higher (40 CFU g(-1)). CONCLUSIONS: This PCR-based assay is highly specific as primers used recognize the DNA from different L. monocytogenes strains of clinical and food origin, while no amplification products result with any other Listeria spp. strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlighted a low-cost and rapid procedure that can be appropriated for the detection in real time of low L. monocytogenes levels in soft cheese.     

High-affinity HLA-A(*)02.01 peptides from parathyroid hormone-related protein generate in vitro and in vivo antitumor CTL response without autoimmune side effects

Parathyroid hormone-related protein (PTH-rP), a protein produced by prostate carcinoma and other epithelial cancers, is a key agent in the development of bone metastases. We investigated whether the protein follows the self-tolerance paradigm or can be used as a target Ag for anticancer immunotherapy by investigating the immunogenicity of two HLA-A(*)02.01-binding PTH-rP-derived peptides (PTR-2 and -4) with different affinity qualities. PTH-rP peptide-specific CTL lines were generated from the PBMC of two HLA-A(*)02.01(+) healthy individuals, stimulated in vitro with PTH-rP peptide-loaded autologous dendritic cells and IL-2. The peptide-specific CTLs were able to kill PTH-rP(+)HLA-A(*)02.01(+) breast and prostate carcinoma cell lines. The two peptides were also able to elicit a strong antitumor PTH-rP-specific CTL response in HLA-A(*)02.01 (HHD) transgenic mice. The vaccinated mice did not show any sign of side effects due to cell-mediated autoimmunity or toxicity. In this study we describe two immunogenic and toxic-free PTH-rP peptides as valid candidates for the design of peptide-based vaccination strategies against prostate cancer and bone metastases from the most common epithelial malignancies.     

The impact of fisheries on vulnerable habitats: the case of trawling on circa-littoral grounds in the Strait of Sicily (central Mediterranean Sea)

The Council Regulation (EC) no. 1967/2006 introduced the adoption of the 40 mm square-mesh or alternatively a 50 mm diamond-mesh codend for trawlers of EU Member States. In this context, the main aim of the current study was to compare the catch composition of Lampedusa’s shallow-water trawl fishery, in terms of retained and discarded fraction, using the 24 mm square-mesh codend (illegal and supposed unselective) and the 50 mm current diamond-mesh codend (legal and supposed selective). During 2012, 21 hauls were performed, under commercial conditions, with the 24 mm square mesh codend and 27 with the 50 mm diamond mesh codend in an area of the Strait of Sicily at depths from 40 to 80 m. The introduction of a 50 mm diamond-mesh, although not significantly, reduced the CPUEs of retained and discarded fraction compared with the 24 mm mesh. Moreover, for most target species and their size classes (especially immature individuals), the legal mesh recorded a lower mean number of specimens than the banned mesh size. All these advantages aside, the discard of the shallow-water trawl fishery, in Lampedusa, represented a very important part of the total catch (more than three-quarters) by both mesh sizes and a large amount of this was represented by species and habitat of conservation concern. Our results suggest that spatial restrictions, such as no-take zones, could be a more effective management tool to protect this sensitive ecosystem in the area than the technical measures (e.g. reduction of mesh size) imposed today by the EU.     

Betulaceae, Corylaceae, Cupressaceae, Fagaceae and Salicaceae around Parma (Northern Italy): Pollen calendars from 1995 to 1997

Many seasonal respiratory allergies are caused byairborne pollens. There is an evident correlationbetween allergic attacks and the amount of pollens inthe atmosphere at any time. The airbornetree pollen concentration and the relevantrepercussions on pollinotics hasnot yet been extensively reported for all Italian regions.We present the results of a 3-year, weekly, tree pollen count byBurkard spore trap from the atmosphere of Parma,Italy. Annual pollen calendars were made from theresults. The study has been conducted because, in Europe,an increasing frequency of allergic sensitisation to thesepollens has been observed.     

Polyfunctional Specific Response to Echinococcus Granulosus Associates to the Biological Activity of the Cysts

Background

Cystic echinococcosis (CE) is a complex disease caused by Echinococcus granulosus (E.granulosus), and its immunophatogenesis is still not clearly defined. A peculiar feature of chronic CE is the coexistence of Th1 and Th2 responses. It has been suggested that Th1 cytokines are related to disease resistance, whereas Th2 cytokines are related to disease susceptibility and chronicity. The aim of this study was to evaluate, by multi-parametric flow cytometry (FACS), the presence of CE specific immune signatures.

Methodology/Principal Findings

We enrolled 54 subjects with suspected CE; 42 of them had a confirmed diagnosis, whereas 12 were classified as NO-CE. Based on the ultrasonography images, CE patients were further categorized as being in "active stages" (25) and "inactive stages" (17). The ability of CD4⁺ T-cells to produce IFN-γ, IL-2, TNF-α, Th2 cytokines or IL-10 was assessed by FACS on antigen-specific T-cells after overnight stimulation with Antigen B (AgB) of E.granulosus. Cytokine profiles were evaluated in all the enrolled subjects. The results show that none of the NO-CE subjects had a detectable AgB-specific response. Among the CE patients, the frequency and proportions of AgB-specific CD4⁺ T-cells producing IL-2⁺TNF-α⁺Th2⁺ or TNF-α⁺Th2⁺ were significantly increased in the “active stages” group compared to the “inactive stages” group. Moreover, an increased proportion of the total polyfunctional subsets, as triple-and double-functional CD4 T-cells, was found in CE patients with active disease. The response to the mitogen, used as a control stimulus to evaluate the immune competence status, was characterized by the same cytokine subsets in all the subjects enrolled, independent of CE.

Conclusions

We demonstrate, for the first time to our knowledge, that polyfunctional T-cell subsets as IL-2⁺TNF-α⁺Th2⁺ triple-positive and TNF-α⁺Th2⁺ double-positive specific T-cells associate with cyst biological activity. These results contribute to increase knowledge of CE immunophatogenesis and the disease outcome in terms of control and persistence.     

Preparation of a glycosynthase from the β-glycosidase of the Archaeon Pyrococcus horikoshii

The β-glycosidase from the hyperthermophilic Archaeon Pyrococcus horikoshii (Phoβ-gly) is a monomeric enzyme with wide substrate specificity belonging to family 1 of glycoside hydrolases classification. Inspection of the three-dimensional structure of the enzyme, recently resolved, showed that Phoβ-gly is membrane bound and that the residues putatively involved in the catalytic activity are Glu155 and Glu324 working as the general acid/base and the nucleophile of the reaction, respectively. We show here that mutation of the latter completely eliminated the activity of the enzyme and that it could be reactivated in the presence of sodium formate. Analysis of the products obtained in the presence of sodium formate buffer pH 4.0 at 75°C showed that the Glu324Gly mutant acts as a hyperthermophilic glycosynthase.     

The characterization of <Emphasis Type="Italic">Thermotoga maritima</Emphasis> ferritin reveals an unusual subunit dissociation behavior and efficient DNA protection from iron-mediated oxidative stress

Ferritin from the hyperthermophilic anaerobe Thermotoga maritima, a bacterium of ancient phylogenetic origin, is structurally similar to known bacterial and eukaryotic ferritins: 24 identical subunits assemble into a shell having octahedral symmetry and a Mr of about 460 kDa. T. maritima ferritin (TmFtn), purified to homogeneity as a recombinant protein, contains approximately 2–3 iron atoms and can incorporate efficiently up to 3,500 atoms in the form of a ferric oxy-hydroxide mineral at 80°C, the optimal growth temperature of the bacterium. The 24-mer unexpectedly dissociates reversibly into dimers at low ionic strengths. In turn, dimers re-associate into the native 24-mer assembly at high protein concentrations and upon incorporation of iron micelles containing at least 500 Fe(III). TmFtn uses O₂ as efficient iron oxidant. The reaction stoichiometry is 3–4 O₂:Fe(II) as in all bacterial ferritins. Accordingly no H₂O₂ is released into solution, a feature reflected in the in vitro ability of TmFtn to reduce significantly iron-mediated oxidative damage to DNA at 80°C. A similar TmFtn-mediated ROS detoxifying role likely occurs in the bacterium which lacks the SOD/catalase defense systems of the aerobic world.     

Correlation between surface chemistry and settlement behaviour in barnacle cyprids (Balanus improvisus)

In laboratory-based biofouling assays, the influence of physico-chemical surface characteristics on barnacle settlement has been tested most frequently using the model organism Balanus amphitrite (= Amphibalanus amphitrite). Very few studies have addressed the settlement preferences of other barnacle species, such as Balanus improvisus (= Amphibalanus improvisus). This study aimed to unravel the effects of surface physico-chemical cues, in particular surface-free energy (SFE) and surface charge, on the settlement of cyprids of B. improvisus. The use of well-defined surfaces under controlled conditions further facilitates comparison of the results with recent similar data for B. amphitrite. Zero-day-old cyprids of B. improvisus were exposed to a series of model surfaces, namely self-assembled monolayers (SAMs) of alkanethiols with varying end-groups, homogenously applied to gold-coated polystyrene (PS) Petri dishes. As with B. amphitrite, settlement of cyprids of B. improvisus was influenced by both SFE and charge, with higher settlement on low-energy (hydrophobic) surfaces and negatively charged SAMs. Positively charged SAMs resulted in low settlement, with intermediate settlement on neutral SAMs of similar SFE. In conclusion, it is demonstrated that despite previous suggestions to the contrary, these two species of barnacle show similar preferences in response to SFE; they also respond similarly to charge. These findings have positive implications for the development of novel antifouling (AF) coatings and support the importance of consistency in substratum choice for assays designed to compare surface preferences of fouling organisms.