Adhesion and growth of osteoblast-like cells on laser-engineered porous titanium surface: expression and localization of N-cadherin and beta-catenin.

Response of different types of cells on biomaterials is crucial for the applications of tissue engineering and regenerative medicine. It is recognized that cell behaviour depends largely on material surface characteristics. The purpose of this study was to define the biologic response of MG63 cells to the innovative patented surface SYNTHEGRA. MG63 morphology and distribution on the three different titanium disk surfaces (sandblasted, smooth, and laser-treated) were evaluated by microscopy analysis after staining with hematoxylin and eosin. Cell adhesion was determined by crystal violet assay at 48 h while proliferation and cytotoxicity were performed by MTT assay at 24, 48, 72 and 240 h. The expression and localization of N-cadherin and beta-catenin were studied by immunofluorescence and confocal microscopy. At 48 h the adhesion was similar in all titanium surfaces, no difference in cell viability were observed in all titanium disks when compared with controls, while the cell growth on laser-treated disks was significantly higher at 240 h than at 24 and 72 h. Morphological analysis show that cells are aligned along the grooves and inside the cavities. beta-catenin signal appeared more diffuse and localized underneath the cell membrane, while N-cadherin signal was fainter in cells grown on SYNTHEGRA surface. This work put into evidence the performance of newly designed laser-micromachined surface for adhesion, growth and distribution of human osteoblast-like cells. SYNTHEGRA surface inducing modification of N-cadherin and beta-catenin expression and localization, are suggestive of cells undergoing differentiation towards osteocytes and could be particularly suited for immediate load implant procedures.

Comparison between three adjuvants for a vaccine against canine leishmaniasis: In vitro evaluation of macrophage killing ability.

The aim of this study was to evaluate, in terms of dog macrophage killing ability in vitro, a vaccine based on Leishmania infantum promastigote soluble antigen (LSA) formulated with three different adjuvants (BCG, AdjuPrime, MPL/TDM/CWS). A significant increase of the macrophage killing ability was observed in dogs vaccinated with LSA+MPL/TDM/CWS after 1 month from vaccination. A similar increase of macrophage parasitocidal ability was present only after 5 months in dogs vaccinated with LSA+BCG or LSA+AdjuPrime. In all dogs the augmented killing percentage was still present after 12 months from vaccination. Therefore, in particular LSA+MPL/TDM/CWS vaccine seems promising for further studies in dogs.

Polymorphisms in genes involved in autoimmune disease and the risk of FVIII inhibitor development in Italian patients with haemophilia A.

One of the most severe and important complication in the treatment of patients with haemophilia A is the formation of neutralizing antibodies (FVIII inhibitors) that inhibit the clotting activity of substituted FVIII. Both genetic and environmental factors influence the susceptibility of patients to develop inhibitors. The objective of this study was to evaluate whether polymorphisms in different genes involved in the regulation of the immune system may confer susceptibility to inhibitor development in patients with HA. We analysed the distribution of polymorphisms in the CTLA4, PTPN22, IL10, TNFalpha, FOXP3 and IRF5 genes that have been reported to be associated with a number of autoimmune disease. In addition, we evaluated the distribution of IL10 haplotypes in haemophilic patients and healthy controls to assess whether specific polymorphisms in IL10 gene were associated to the risk of inhibitor development. We focused on a cohort of Italian unrelated haemophilic patients with and without a history of inhibitors. Genotyping was carried out with standard methods including RFLP, real time PCR and direct DNA sequencing. Our data show that, considering single nucleotide variations, genotype frequencies in patients with inhibitors were not significantly different from those observed in patients without inhibitors, suggesting a lack of association between these polymorphisms and the development of inhibitors. Moreover, no relationship was found between specific combinations of IL10 alleles and the antibody production. Previous contradictory association studies may depend on the different genetic background of the population examined. Further studies may contribute to a clearer understanding of this process.

PCR assay to detect Bacillus anthracis spores in heat-treated specimens.

Recent interest in anthrax is due to its potential use in bioterrorism and as a biowarfare agent against civilian populations. The development of rapid and sensitive techniques to detect anthrax spores in suspicious specimens is the most important aim for public health. With a view to preventing exposure of laboratory workers to viable Bacillus anthracis spores, this study evaluated the suitability of PCR assays for detecting anthrax spores previously inactivated at 121 degrees C for 45 min. The results indicate that heat treatment ensures the complete inactivation of B. anthracis spores without significantly affecting the efficiency of PCR assays.

Inducible nitric oxide synthase expression in Leishmania-infected dog macrophages.

Nitric oxide (NO) production by the inducible NO synthase (iNOS or NOS2) represents one of the main microbicidal mechanisms of murine macrophages, but its role in other animal models is poorly investigated. Therefore, the aim of this work was to evaluate NOS2 expression in dog macrophages infected with Leishmania infantum. Macrophages obtained from peripheral blood of healthy dogs were activated with recombinant human interferon (rhIFN)-gamma and bacterial lipopolysaccharide (LPS) and then infected with L. infantum promastigotes. zymodeme MONI. For the immunofluorescence assay fixed macrophages were incubated with polyclonal rabbit anti-NOS2 and then with rhodamine F(ab')2 goat anti-rabbit IgG. For immunoblotting, cell lysates were submitted to SDS-PAGE and blots were incubated with polyclonal rabbit anti-NOS2 and then with horseradish peroxidase-conjugated goat anti-rabbit IgG. Results demonstrated that L. infantum-infected cells, after stimulation with rhIFN-gamma and LPS, displayed high levels of fluorescence for the NOS2 in their cytoplasm, unlike unstimulated uninfected macrophages. In western blotting, polyclonal anti-NOS2 reacted specifically with a protein band corresponding to 130 kDa. The signal produced in Leishmania-infected cells stimulated with rhIFN-gamma and LPS was higher than that produced in Leishmania-infected unstimulated cells. No band was detected in cellular lysates from uninfected unstimulated cells. These results indicate that dog macrophages can express NOS2, and suggest a role for IFN-gamma and LPS in NOS2 induction also in this animal model.

Detection of anthrax vaccine virulence factors by polymerase chain reaction.

In Italy, an attenuated Bacillus anthracis strain, named 'Carbosap', is used for immunization against ovine and bovine anthrax. Analysis on 'Carbosap', Sterne vaccine strain F34 and Pasteur vaccine strain SS104, were performed using primers specific for the sequences, encoding the toxic factors, located on plasmids pXO1 and pXO2 and primers specific for the chromosome. The results obtained from polymerase chain reaction (PCR) assay revealed the presence of both plasmids pXO1 and pXO2 in 'Carbosap' strain. This study showed that the 'Carbosap' vaccine strain has a different plasmid pattern in comparison to Pasteur vaccine strain SS104 and Sterne vaccine strain F34.

Nitric oxide production by macrophages of dogs vaccinated with killed Leishmania infantum promastigotes.

Human visceral leishmaniosis is endemic in Southern Italy, where the dog is the main reservoir of viscerotropic strains of Leishmania infantum. The release of nitric oxide (NO) by interferon (IFN)-gamma-activated macrophages is an important leishmanicidal mechanism in several animal species. In this work NO production, phagocytosis and killing capacity of monocyte-derived dog macrophages were evaluated in vitro before and after administration of a vaccine composed of killed Leishmania infantum promastigotes. Moreover, IFN-gamma content was measured in concanavalin A-activated dog peripheral blood mononuclear cell (PBMC) supernatants employed for macrophage stimulation. Phagocytosis, killing capacity and NO production by canine macrophages increased significantly 1 month after vaccine administration, and the increase also persisted 5 months later. In addition, the amount of IFN-gamma in PBMC supernatants was significantly higher after vaccination. Overall, our results suggest the usefulness of evaluating the in vivo protective role of this promastigote preparation in dogs.

Inducible nitric oxide synthase and nitric oxide production in Leishmania infantum-infected human macrophages stimulated with interferon-gamma and bacterial lipopolysaccharide.

Nitric oxide produced by an inducible nitric oxide synthase constitutes one of the main microbicidal mechanisms of murine macrophages and its importance is now being recognized for human macrophages. In this study we evaluated inducible nitric oxide synthase expression, nitric oxide release, and parasitocidal ability of Leishmania infantum-infected monocyte-derived human macrophages. The inducible nitric oxide synthase was detected by immunofluorescence and western blotting and nitric oxide production was measured by the Griess reaction for nitrites. Parasite killing was microscopically evaluated by fluorescent dyes. Experiments were performed on macrophages with or without previous stimulation with recombinant human interferon-gamma and bacterial lipopolysaccharide. Inducible nitric oxide synthase expression and nitric oxide release were higher in Leishmania-infected stimulated macrophages than in uninfected cells or infected cells without previous stimulation. Nitric oxide production and parasitocidal activity against Leishmania infantum were reduced in macrophages treated with the nitric oxide synthase inhibitor L-N(G) monomethylarginine. These results suggest a microbicidal role for nitric oxide in human leishmaniasis, with the possible practical application of immunological or pharmacological regulation of nitric oxide synthesis in the treatment of this infection.