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Infection of humans by Toxoplasma gondii leads to an acute systemic phase, in which tachyzoites disseminate throughout the body, followed by a chronic phase characterized by the presence of tissue cysts, containing bradyzoites, in brain, heart and skeletal muscles. This work focused on studying the antigenic regions of bradyzoite-specific proteins involved in human B- and T-cell responses. To this aim, we constructed a phage-display library of DNA fragments derived from the bradyzoite-specific genes BAG1, MAG1, SAG2D, SAG4, BSR4, LDH2, ENO1 and p-ATPase. Challenge of the bradyzoite library with sera of infected individuals led to the identification of antigenic regions within BAG1 and MAG1 gene products. Analysis of the humoral and lymphoproliferative responses to recombinant antigens demonstrated that the BAG1 fragment induced T-cell proliferation in 34% of T. gondii-exposed individuals, while 50% of them had specific IgG. In the same subjects, the MAG1 fragment was recognized by T cells from 17% of the exposed donors and by antibodies from 73% of them. A detailed analysis of the antibody response against BAG1 and MAG1 antigen fragments demonstrated that the immune response against bradyzoites occurs early after infection in humans. Finally, we provide evidence that the T-cell response against BAG1 is associated with the production of interferon-gamma, suggesting that bradyzoite antigens should be considered in the design of potential vaccines in humans.

The objective of this work was to develop an antibody-specific immunoglobulin G (IgG) avidity assay to discriminate between acute and latent phases ofToxoplasma gondiiinfection by using recombinant antigens. One hundred twenty-one serum samples from women who developed IgG antibodies againstToxoplasmaduring pregnancy were used. The IgG avidities of antibodies directed against epitopes carried by fragments of GRA3, GRA7, MIC3, and SAG1 antigens were measured by performing parallel enzyme immunoassays. The avidity index forToxoplasma-specific antibodies against a homogeneous mixture of recombinant GRA3, GRA7, MIC3, and SAG1 antigens correlated closely with the IgG avidity of antibodies against lysed whole-cellT. gondiiantigen. The avidity assay performed with the recombinant MIC3 antigen highlighted the presence of avidity low-antibodies IgG exclusively in sera collected within 2 months after primary infection. The presence ofT. gondii-specific, low-avidity IgG antibodies against recombinant MIC3 antigen can be used to determine the point of infection withT. gondiiwithin a 2-month time frame after infection.

The role of innate immune response mediated by Toll-like receptors in HCV infection, is not yet well understood and there is a lack of data regarding liver tissue expression of these molecules in chronic hepatitis C (CHC). Our study is aimed to investigate ex vivo, liver expression of TLR2, TLR3 and TLR7, which are more involved in the immune-pathogenesis of CHC, and to explore possible correlations with features of disease. We obtained liver biopsies and collected peripheral blood mononuclear cells (PBMC) from 23 consecutive patients with CHC and from 6 patients of control, without liver disease, undergoing surgery for cholecystectomy. The levels of TLRs mRNA in the samples were determined using a real-time reverse transcription quantitative PCR (RT-qPCR). We found a significant high expression of TLR3 in the liver of CHC patients respect to controls (also higher than expression in the PBMC). Conversely no differences emerged in the TLR2 and TLR7 levels between cases and controls. Also we found a correlation of TLR2 and TLR7 levels with the grade of necro-inflammation in the liver. Furthermore TLR7 hepatic levels resulted related to a more advanced stage of liver fibrosis. Ours is the first study to provide data on tissue expression of TLRs during chronic hepatitis C and we believe that it could lead to a better understanding of the role of these molecules in the HCV-mediated liver damage.

Several lines of evidence demonstrated that organisms ranging from bacteria to higher animals possess a regulated endonucleolytic cleavage pathway producing half-tRNA fragments. In the present study, we investigated the occurrence of this phenomenon in two distantly related apicomplexan parasites, Toxoplasma gondii, the agent of toxoplasmosis, and the rodent malaria parasite Plasmodium berghei. A low-scale molecular characterization of the small RNA fraction of T. gondii revealed the endonucleolytic processing of 10 distinct tRNA species, with cleavage in the anticodon loop and upstream of the 3'-terminal CCA sequence yielding 5'- or 3'-end half-tRNAs. T. gondii and P. berghei exhibited variable rates of tRNA cleavage upon egress from host cells and in response to stage differentiation, amino acid starvation and heat-shock. Moreover, avirulent isolates of T. gondii and attenuated P. berghei parasites showed a higher rate of tRNA cleavage than virulent strains. Interestingly, half-tRNA production was significantly higher in the metabolically quiescent bradyzoite and sporozoite stages of T. gondii, compared to the fast-growing tachyzoite. Collectively, our findings shed light for the first time on the occurrence of tRNA cleavage in apicomplexan parasites and suggest a relationship between half-tRNA production and growth rate in this important group of organisms.

The saliva of blood-feeding arthropods contains an apyrase that facilitates hematophagy by inhibiting the ADP-induced aggregation of the host platelets. We report here the isolation of a salivary gland-specific cDNA encoding a secreted protein that likely represents the Anopheles gambiae apyrase. We describe also two additional members of the apyrase/5′-nucleotidase family. The cDNA corresponding to the AgApyL1 gene encodes a secreted protein that is closely related in sequence to the apyrase of the yellow fever mosquito, Aedes aegypti, and whose expression appears enriched in, but not restricted to, female salivary glands. The AgApyL2 gene was found searching anA. gambiae data base, and its expression is restricted to larval stages. We isolated the gene encoding the presumed A. gambiae apyrase (AgApy) and we tested its putative promoter for the tissue-specific expression of the LacZgene from Escherichia coli in transgenic Drosophila melanogaster. All the transgenic lines analyzed showed a weak but unambiguous staining of the adult glands, indicating that some of the salivary gland-specific transcriptional regulatory elements are conserved between the malaria mosquito and the fruit fly. The availability of salivary gland-specific promoters may be useful both for studies on vector-parasite interactions and, potentially, for the targeted tissue-specific expression of anti-parasite genes in the mosquito.

Background Genotype 1 (G1) chronic hepatitis C (CHC) patients achieving a rapid virological response (RVR) on pegylated interferon (PEG-IFN) plus ribavirin have a high chance of sustained virological response (SVR), influenced by IL28B status, viral load, fibrosis and insulin resistance. We assessed whether 25-hydroxyvitamin D (25[OH]D) serum levels are linked to RVR and can be used together with IL28B to construct a pretreatment model to predict RVR. Methods A total of 117 consecutive patients with G1 CHC were evaluated by biopsy and anthropometric and metabolic measurements. 25(OH)D serum levels were measured by HPLC. IL28B rs12979860 and rs8099917 polymorphisms were also evaluated. All patients underwent antiviral therapy with PEG-IFN-α2a plus ribavirin. HCV RNA was assessed at baseline, week 4, week 12, at the end of therapy and after 6 months of follow-up. Results Mean ±sd 25(OH)D serum levels were 26.3 ±10.6 μg/l (range 8.0–58.0) and 31 (26.5%) patients had the rs12979860 CC polymorphism. RVR was achieved in 35 (29.9%) patients, and 32 (91.4%) of them had an SVR, compared to 26 of 82 (31.7%) without RVR. The rs12979860 CC polymorphism (OR 4.575, 95% CI 1.761, 11.889; P=0.002) and higher 25(OH)D levels (OR 1.055, 95% CI 1.010, 1.101; P=0.01) were independently associated with the achievement of RVR by multivariate analysis. The likelihood of RVR progressively increased from patients in the worst class (vitamin DConclusions In patients with G1 CHC, 25(OH)D serum levels and IL28B status are independently associated with the likelihood to achieve RVR and SVR. When incorporated into a pretreatment predictive model they can assist in further discriminating patients with a high likelihood of achieving RVR and SVR.

Blood stage malaria parasites causing a mild and self limited infection in mice have been obtained with either radiation or chemical mutagenesis showing the possibility of developing an attenuated malaria vaccine. Targeted disruption of plasmepsin-4 (pm4) or the merozoite surface protein-7 (msp7) genes also induces a virulence-attenuated phenotype in terms of absence of experimental cerebral malaria (ECM), delayed increase of parasitemia and reduced mortality rate. The decrease in virulence in parasites lacking either pm4 or msp7 is however incomplete and dependent on the parasite and mouse strain combination. The sequential disruption of both genes induced remarkable virulence-attenuated blood-stage parasites characterized by a self-resolving infection with low levels of parasitemia and no ECM. Furthermore, convalescent mice were protected against the challenge with P. berghei or P. yoelii parasites for several months. These observations provide a proof-of-concept step for the development of human malaria vaccines based on genetically attenuated blood-stage parasites.

The mouse monoclonal antibody (mAb) technology still represents a key source of reagents for research and clinical diagnosis, although it is relatively inefficient and expensive and therefore unsuitable for high-throughput production against a vast repertoire of antigens. In this article, we describe a protocol that combines the immunization of individual mice with complex mixtures of influenza virus strains and a microarray-based immunoassay procedure to perform a parallel screening against the viral antigens. The protocol involves testing the supernatants of somatic cell hybrids against a capture substratum containing an array of different antigens. For each fusion experiment, we carried out more than 25,000 antigen-antibody reactivity tests in less than a week, a throughput that is two orders of magnitude higher than that of traditional antibody detection assays such as enzyme-linked immunosorbent assays and immunofluorescence. Using a limited number of mice, we can develop a vast repertoire of mAbs directed against nuclear and surface proteins of several human and avian influenza virus strains.

The adhesion of microalgae to living substrata is well known in marine and freshwater environments (Wahl, 1989; Prieur, 1991; Carman and Dobbs, 1997; Di Camillo et al., 2005; Romagnoli et al., 2007). Therefore, a number of phyla of marine organisms commonly host on their surface a complex community of microorganisms composed of bacteria, fungi, and protists.

Solid-state (23)Na and (31)P magic-angle spinning nuclear magnetic resonance spectroscopy and X-ray crystallography have been used to study the structures of the chain metaphosphates NaCa(PO(3))(3) and NaSr(PO(3))(3). The compounds are isostructural and crystallise in space group P(-1) with the following parameters: NaCa(PO(3))(3), a = 6.711 A, b = 6.934 A, c = 7.619 A, alpha = 83.44 degrees , beta = 81.41 degrees , gamma = 82.80 degrees ; NaSr(PO(3))(3)a = 6.805 A, b = 7.133 A, c = 7.720 A and alpha = 83.71 degrees , beta = 80.48 degrees , gamma = 82.87 degrees . Both structures contain anionic metaphosphate chains of (PO(3))(n) (n) with ionic contacts to Na(+) ions in distorted octahedral sites and Ca(2+) (or Sr(2+)) in distorted dodecahedral sites. (31)P and (23)Na NMR are entirely consistent with the crystallographic data and an empirical method for assigning (31)P resonances to particular crystallographically unique P atoms is described.

During Toxoplasma gondii infection, a fraction of the multiplying parasites, the tachyzoites, converts into bradyzoites, a dormant stage, which form tissue cysts localized mainly in brain, heart, and skeletal muscles that persist for several years after infection. At this stage the parasite is protected from the immune system, and it is believed to be inaccessible to drugs. While the long persistence of tissue cysts does not represent a medical problem for healthy individuals, this condition represents a major risk for patients with a compromised immune system, who can develop recrudescent life-threatening T. gondii infections. We have investigated for the first time the dynamics and the kinetics of tachyzoite-to-bradyzoite interconversion and cyst formation in vivo by using stage-specific bioluminescent parasites in a mouse model. Our findings provide a new framework for understanding the process of bradyzoite differentiation in vivo. We have also demonstrated that complex molecules such as d -luciferin have access to tissue cysts and are metabolically processed, thus providing a rationale for developing drugs that attack the parasite at this developmental stage.

1 Biotechnology and Biological Control Agency, Via del Bosco 10, 00060 Sacrofano, Rome, Italy. 2 Ataturk University, Faculty of Agriculture, Plant Protection Department, 25240 TR Erzurum, Turkey. 3 ENEA C.R. Casaccia BIOTEC, Via Anguillarese 301, 00123 S. Maria di Galeria, Rome, Italy. 4 USDA-ARS, 800 Buchanan Street, Albany, CA 94710, USA. Corresponding author: A. Paolini . © CAB International 2008 A lace bug as biological control agent of yellow starthistle, Centaurea solstitialis L. (Asteraceae): an unusual choice

Summary Russianthistle, Salsola tragus L.,�(Chenopodiaceae),�atroublesomeweedcomplexinthedrierregions� ofwesternNorthAmerica,�isnativetoCentralAsiaandwidelydistributedthroughoutthePalaearctic� Region.�Since�2003,�severalexplorationandsurveytripstodiscovernewpotentialbiologicalcontrol� agentsfortheweedwerecarriedoutinItaly,�Russia,�Turkey,�Tunisia,�Kazakhstan,�Greece,�Egyptand� Morocco. Twenty-five arthropod species (one gall midge, one heteropteran, two flea beetles, three mothsand�18�weevils)�andtwofungiwerepreliminarilyselectedduringthesurveys.�Amongthem,�four� arthropod species (two weevils and two moths) were selected for further biology and host-specificity � studies.�Atthemoment,�themostpromisingamongtheseisthestem-boringweevilAnthypurinusbi­ impressusBrisout.�Preliminaryhostrangeno-choicetestsandlifecycleobservationsshowedthatthis� speciesisrestrictedforfeeding,�ovipositionanddevelopmenttoRussianthistle.

The “Spina” of Turin is about planning renewal of the urban settlement. Its purpose is to virtuously redraw the city as a whole: a heir of the great; complex regeneration plans for the 19th century metropolis. The “Spina” program, in its complexity and capacity of involving the entire city, is completely different from the most of recent plans and is intended to modernize Italian cities, historically cities that were unable to be improved., Macramè, macramè 1–2007

To improve tumor targeting in a subset of patients, where tumor cells do not express the well-known tumor antigens widely used in immunotherapy, we have developed a novel biotechnological tool. It is useful for tumors of various origins for the identification of tumor-associated proteins, which are differentially expressed in tumor cells with respect to normal tissue, and exposed on the cell surface. For this purpose, a combination of techniques, such as "suppression subtractive hybridization" and "transmembrane trapping," was employed. In applying this novel approach to breast cancer, we identified a large panel of cDNA fragments encoding for the well-known tumor-associated surface antigens, such as erb-B2, erbB3 and the urokinase receptor and, more importantly, for several clones overexpressed in breast cancer, whose cDNA fragments match the sequences of hypothetical transmembrane proteins with unknown function. The latter may represent novel tumor-specific targets.

Infection of the host by Toxoplasma gondii leads to an acute systemic dissemination of tachyzoites, followed by a chronic phase, in which bradyzoites, enclosed in cysts, persist in the brain, the heart, and other tissues. Among putative vaccine candidates, the bradyzoite antigens BAG1 and MAG1 look promising since they are preferentially expressed during the chronic stage of the parasite. This work focused on studying the immunogenicity of bradyzoite antigens in a mouse model of chronic toxoplasmosis. A mixture of plasmids directing the cytoplasmic expression of MAG1 and BAG1 in mammalian cells was used to immunize mice. We show here that immunized mice developed, preferentially, specific anti-MAG1 and anti-BAG1 IgG2a subclass antibodies, indicating a shift towards a Th1-like response after DNA immunization. We then demonstrated that DNA immunization followed by challenge infection elicited effective protection in mice, suggesting that bradyzoite antigens should be considered in the design of vaccines against toxoplasmosis.

Si conclude dicendo in generale che una parte dell’architettura contemporanea si sta rifondando come ricerca sperimentale di geometrie topologiche in vista di un’architettura plastica e spaziale. La tecnica, la poetica ed il ruolo concettuale della trasformazione in architettura sembrano emergere sempre piu frequentemente come il risultato dell’incontro tra topologia e tecnologia digitale; attraverso le crescenti possibilita di modellazione al computer l’impiego della geometria topologica produce un effetto liberatorio sulle forme architettoniche, permettendo agli architetti di progettare con livelli di complessita sempre maggiori. Ma bisogna dire che, nonostante l’aiuto straordinario del computer, la cosi detta architettura topologica e in una condizione ancora di evoluzione; il suo sviluppo e la sua diffusione, una volta raggiunta una maggiore maturita, dovranno, poi, inevitabilmente, confrontarsi sempre di piu con l’industria e l’economia delle costruzioni e, naturalmente, con il giudizio dei fruitori.