Role of rabbit prostate granules on sperm viability and acrosome reaction evaluated with different methods

In the present study, the role of rabbit seminal granules was observed. Their influence on motility, capacitation and acrosome reaction, as well as the presence of apoptosis and the morphology of rabbit sperm, were compared in different conditions. Ejaculated sperm from five mature New Zealand White rabbit bucks during three series of collections were studied, comparing raw semen, Percoll-selected sperm and Percoll-selected sperm plus prostate granules. We observed sperm motility kinetic traits by computer-assisted sperm analyzer (CASA) analysis in each sample. Acrosome status was evaluated by FITC-labeled Pisum sativum Agglutinin staining and chlortetracycline fluorescence assay, phosphatidylserine translocation was determined by AnnexinV/Propidium iodide assay and sperm morphology was studied using transmission electron microscopy (TEM). All traits were observed after 30 min incubation at 37 °C in 5% CO₂. Data showed that sperm motility and viability markedly improved in the presence of prostate granules, whereas capacitation, acrosome reaction and phosphatidylserine translocation were lowered. TEM confirmed these results. In conclusion, the role of granules was confirmed in synchronizing sperm capacitation and acrosome reaction with egg availability; indeed, rabbit ovulation occurs only 6 to 10 h after mating.     

Bone marrow-infiltrating human neuroblastoma cells express high levels of calprotectin and HLA-G proteins

Metastases in the bone marrow (BM) are grim prognostic factors in patients with neuroblastoma (NB). In spite of extensive analysis of primary tumor cells from high- and low-risk NB patients, a characterization of freshly isolated BM-infiltrating metastatic NB cells is still lacking. Our aim was to identify proteins specifically expressed by metastatic NB cells, that may be relevant for prognostic and therapeutic purposes. Sixty-six Italian children over 18 months of age, diagnosed with stage 4 NB, were included in the study. Metastatic NB cells were freshly isolated from patients' BM by positive immunomagnetic bead manipulation using anti-GD2 monoclonal antibody. Gene expression profiles were compared with those obtained from archived NB primary tumors from patients with 5 y-follow-up. After validation by RT-qPCR, expression/secretion of the proteins encoded by the up-regulated genes in the BM-infiltrating NB cells was evaluated by flow cytometry and ELISA. Compared to primary tumor cells, BM-infiltrating NB cells down-modulated the expression of CX3CL1, AGT, ATP1A2 mRNAs, whereas they up-regulated several genes commonly expressed by various lineages of BM resident cells. BM-infiltrating NB cells expressed indeed the proteins encoded by the top-ranked genes, S100A8 and A9 (calprotectin), CD177 and CD3, and secreted the CXCL7 chemokine. BM-infiltrating NB cells also expressed CD271 and HLA-G. We have identified proteins specifically expressed by BM-infiltrating NB cells. Among them, calprotectin, a potent inflammatory protein, and HLA-G, endowed with tolerogenic properties facilitating tumor escape from host immune response, may represent novel biomarkers and/or targets for therapeutic intervention in high-risk NB patients.     

Occurrence and assemblage composition of millipedes (Myriapoda, Diplopoda) and terrestrial isopods (Crustacea, Isopoda, Oniscidea) in urban areas of Switzerland

Terrestrial isopods and millipedes, members of the invertebrate macro-decomposer guild, were collected through pitfall traps in three Swiss cities (Zurich, Lucerne, Lugano). A total of 7,198 individuals of 17 isopod species (7093 ind.), and 10 millipede species (105 ind.) were captured. Besides the Alpine endemic isopod (Trichoniscus alemannicus) and millipede (Cylindroiulus verhoeffi), urban assemblages were mainly composed of widespread, native European and even cosmopolitan species, which are frequent in anthropogenic areas. Overall species richness (isopods and millipedes combined) was similar in Zurich (17 species) and Lucerne (16), while only 13 species were sampled in Lugano. According to the Sørensen index of similarity, species composition of Zurich and Lucerne were more alike, while the one of Lugano was more distinct from the other two cities. This result can be explained by the spatial proximity of Zurich and Lucerne in the north of the Alps compared to Lugano, which is located more distantly and in the south of the Alps. Dominant isopods and millipedes in Zurich and Lucerne were found to be widespread synanthropic species in temperate Europe(Porcellio scaber, Trachelipus rathkii and Ophyiulus pilosus) while the dominant isopod in Lugano (Trachelipus razzautii) is a species with a north-eastern Mediterranean distribution. Our study reveals that the urban millipede and isopod fauna in Swiss cities mainly consists of widespread species, but species of narrower distribution (e.g. Trichoniscus alemannicus, Cylindroiulus verhoeffi) may also find suitable habitats in cities. Despite some signs of biotic homogenization, our study also found compositional differences of millipede and isopod assemblages between northern and southern cities that suggest geographical effects of the regional species pool.     

Eosinophilia, granuloma formation, migratory behaviour of second stage larvae in murine Toxocara canis infection. Effect of the inoculum size

Experimental infection of mice with Toxocara canis provides one of the best models for immunological and pathological studies of the visceral larva migrans syndrome. Blood eosinophilia, the migratory behaviour of second stage larvae and granuloma formation were studied in Swiss mice infected with Toxocara canis. Eosinophilia, spleen, liver and lung indexes were followed during a primary infection with different inoculum sizes (500 and 1500 eggs) while the migratory behaviour of larvae was studied in a primary infection with 1500 eggs over a period of 4 months. In mice infected with three challenges of 1500 eggs in order to elicit a strong inflammatory reaction in the tissues, a histopathological study was carried out. The results showed that eosinophilia, spleen and lung indexes (but not the liver index) were influenced by the parasite inoculum size. The migratory behaviour study showed that larval recovery was maximal three days post-infection, from the liver and lungs; the peak recovery from the skeletal muscles and brain being on days 15 and 30 post-infection, respectively. The histopathological study revealed the formation of granulomas in all the tissues examined (liver, lungs, kidneys, spleen, lymph nodes, myocardium etc.) but not in nervous tissue or in the retina of the eye. Granulomas in the lungs were larger than those found in the liver. The implications of these results are discussed considering host-parasite inter-relations.     

Fluorocytometric study of proliferation antigens, nuclear proteins and ploidy in acute leukosis]

P53 protein, Ki67 proliferative associated antigen and DNA content have been studied by flow cytometry in the blood blastic cells from 41 patients with acute leukemia. The results were compared with the F.A.B. classification. Cells were permeabilised and fixed by PLP solution before using the FITC conjugated Ki67 MoAb and the p53 MoAb (clone 1801). Propidium iodide and RNAase has been used in order to determine ploidy. Ki67 and p53 protein were found to be expressed at higher level in leukemia cells than in normal bone marrow cells; however there was no correlation between Ki67 and p53 expression and F.A.B. subtype. In acute leukemia patients the range of positivity of Ki67 was 1.1-52.1% while it ranged from 1.8% to 80.1% for the p53 protein. On the basis of these findings we conclude that the flow cytometry evaluation of Ki67 and p53 represents a useful tool for the study of the biologic characteristics of the leukemic cells in patients with acute leukemia.     

Toxoplasma gondii Chitinase Induces Macrophage Activation

Toxoplasma gondii is an obligate intracellular protozoan parasite found worldwide that is able to chronically infect almost all vertebrate species, especially birds and mammalians. Chitinases are essential to various biological processes, and some pathogens rely on chitinases for successful parasitization. Here, we purified and characterized a chitinase from T. gondii. The enzyme, provisionally named Tg_chitinase, has a molecular mass of 13.7 kDa and exhibits a Km of 0.34 mM and a Vmax of 2.64. The optimal environmental conditions for enzymatic function were at pH 4.0 and 50°C. Tg_chitinase was immunolocalized in the cytoplasm of highly virulent T. gondii RH strain tachyzoites, mainly at the apical extremity. Tg_chitinase induced macrophage activation as manifested by the production of high levels of pro-inflammatory cytokines, a pathogenic hallmark of T. gondii infection. In conclusion, to our knowledge, we describe for the first time a chitinase of T. gondii tachyzoites and provide evidence that this enzyme might influence the pathogenesis of T. gondii infection.     

Scanning electron microscopy studies of cuticle micromorphology in Cycas L. (Cycadaceae)

Abstract

A scanning electron microscopy (SEM) study of Cycas cuticle characteristics was undertaken in order to expand our knowledge of microscopic characters not observable under light microscopy and to clarify unresolved affinitites among some species within the genus. Whole leaf and isolated cuticle specimens from the middle region of leaflets of greenhouse-grown plants of Cycas revoluta, Cycas rumphii, Cycas circinalis, Cycas media, and Cycas normanbyana were examined using SEM for interior and exterior features. Characteristics in common include hypostomy, hair bases on abaxial and adaxial surfaces, adaxial cells randomly arranged, adaxial exterior cuticle smooth, and stomata sunken to various degrees but stomatal pit always formed by two layers of epidermal cells. Stomatal complex is of the polyperigenous type. Stomata randomly dispersed and oriented, and except C. revoluta, are not contiguous. Stomata deeply sunken in C. revoluta, intermediate in C. rumphii and C. normanbyana, and less sunken in C. circinalis and C. media. Aperture between guard cells extends the entire stomatal length in C. rumphii and C. normanbyana, ～80% in C. circinalis and C. media, and ～50% in C. revoluta. Cuticular features of C. revoluta show the greatest difference from the other species in complex relief of exterior cuticle and interior cuticular structure of subsidiary cells; C. media and C. circinalis show close similarity to each other and their stomatal complex dimensions fall within the same unique cluster using principal component analysis under normalized variables. C. normanbyana and C. rumphii show the most similarity to each other in cuticular micromorphology. Stomatal complex dimensions of these two species fall into a second cluster that also includes C. revoluta. These data contrast with current taxonomy placing C. normanbyana synonymous to C. media.